New bioanalytical methods to assure food safety and ... · to assure food safety and quality Neue bioanalytische Methoden ... Effect of food quality on GIT eubiosis for Agricultural

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

[email protected]


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, …)


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


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


Hans-Eisenmann-Zentrumfor Agricultural ScienceImmune response in the GIT

food ingredients

secondary plant components => healthy intestine

prebiotics & probiotics

NFB


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


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



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)


growth promoter IGF-1

IL-10 in the colon

in stomach


pro-inflammatory vs. anti-inflammatorymarker genes marker genes



Estrogens => development of primary andsecondary sexual characteristics

Prolactin => breast development, milk production

Gynecomastia: development of abnormally largemammary glands



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


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


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



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


Hans-Eisenmann-Zentrumfor Agricultural ScienceFunctional target gene groups

• steroid receptors

• steroid hormone metabolism

• proliferation / apoptosis

• angiogenesis

• tissue remodelling

• immune relevant factors



mRNA expressionfrom heifers ovary samples



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


Hans-Eisenmann-Zentrumfor Agricultural ScienceWBC expression data analysis

Significantly regulated genes in heiferblood (16 days after treatment)

PCA & HCA



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


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



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



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


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


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


Hans-Eisenmann-Zentrumfor Agricultural ScienceAcknowledgements

Documents

New bioanalytical methods to assure food safety and ... · to assure food safety and quality Neue bioanalytische Methoden ... Effect of food quality on GIT eubiosis for Agricultural