http://www.agroscope.admin.ch

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Federal Department of Economic Affairs, Education and Research EAER Agroscope

Flavour analysis in dairy and meat products

Dr. Katharina Breme Food Analytics

Agroscope Liebefeld-Posieux Research Station ALP-Haras

katharina.breme@agroscope.admin.ch

http://www.agroscope.admin.ch

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Topics

• Agroscope Liebefeld-Posieux Research Station ALP-Haras

• Flavour formation in dairy products

• Flavour analytics at Agroscope

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3

EAER

Fed. Dep of Economic Affairs, Education&Research (Eidg. Dep. Wirtschaft, Bildung&Forschung WBF)

Agroscope

Agroscope Reckenholz-

Tänikon ART Agroscope

Liebefeld- Posieux

ALP

Agroscope Changins- Wädenswil

ACW FOAG Fed. Office for Agriculture

SECO

State Secretariat for Economic

Affairs

SERI State Secretariat

for Education, Research and Innovation

FONES Fed. Office for

National Economic

Supply

FHO Fed. Housing

Office

Swiss National Stud Farm

Avenches Haras

ALP-Haras since 01.01.2008

Agroscope ALP-Haras within the Federal

Administration

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Agroscope

• research, teaching, consulting and enforcement

• managed according to MPM (management by performance mandate&global budget)

• differs from universities of applied sciences (FH) and Swiss Institute of Technology (ETH)

• longtime effect oriented research in chosen topics

• unique infrastructure, strongly used by universities and institutes

• important reorganisation over the last few years

• reduction of sites, enforced collaborations (national, international)

• less research funding from federal department, more third-party funds

• member of the R&D-pool Swiss Food Research (SFR)

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-

Processing -

Production

ALP-Haras: Expertise throughout the whole value chain

From Animal Feed to Comestible Food

Feedstuffs

Meat and meat processing

Milk and milk processing

Honey

Nutrition Livestock

Cattle, pigs, sheep, bees, horses

Horse

Keeping, education, biodiversity,

health, ethology

The horse as a product in

society

Research along the value-added chain for

milk and meat

Healthy & sustainable

nutrition

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• launch of systematic collection in the 1920ies

• lactic acid bacteria (LAB) from milk, cheese, whey with focus on thermophilic species

• propionic acid bacteria, few mesophilic bacteria

• 1980, 1990

• NSLAB: facultatively heterofermentative lactobacilli → prevention of late blowing in Emmental cheese

• microorganisms from cheese surface smear

• over the last years

• potentially probiotic microorganisms

• more mesophilic LAB (lactococci)

• microorganisms from fermented sausages

• biodiversity: most likely due to use of individual „home flora“ of each cheese production unit during collection period of strains

Strain collection

S. Orla-Jensen, microbiologist, worked at the research station in the beginning of the 20

^th

century

Strain collection contact: Dr. Noam Shani noam.shani@agroscope.admin.ch or Dr. Elisabeth Eugster-Meier elisabeth.eugster@agroscope.admin.ch

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• started over 100 years ago

• today, 35 commercial starter, secondary and adjunct cultures (liquid and freeze-dried)

• approx. 95‘000 bottles (100 mL) sold in 2012

• exclusively for the Swiss market: traditional Swiss cheeses

Agroscope culture production

Excellent scores for traditional Swiss cheeses at the Cheese World Championship

Culture production contact Christoph Kohn: christoph.kohn@agroscope.admin.ch

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Flavour formation in dairy products

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Strecker aldehydes

sulphur compounds

phenylacetaldehyde, -ethanol

Acetaldehyde, phenol

….

Milk

Lactose citrate

Pyruvate α-acetolactate casein

peptides

Amino acids AA triglycerides

Free fatty acids

FFA

Flavour formation in dairy products: degradation of milk constituents

A.E.M. Boelrijk et al. Flavour generation in dairy products, In Dairy processing, G. Smit: CRC Press Boca Raton USA, 2003, 130–148 B. d’Acampora et al. Flavour formation, In Handbook of dairy food analysis, L.M.L. Nollet&F. Toldra: CRC Press Boca Raton USA, 2010, 277–288

W.J.M. Engels et al. Flavour formation in cheese, In Dairy processing, G. Smit: CRC Press Boca Raton USA, 2003, 492–507 degradation of

milk fat (lipolysis)

degradation of milk proteins (proteolysis)

methyl ketones sec. alcohols

lactones aldehydes

alcohols

… esters

Diacetyl Acetoin Acetaldehyde

Acetic acid

Lactic acid, lactate:

mild-sour taste

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• fermented dairy products: e.g. yoghurt, cheese…

• fermentation mainly by lactid acid bacteria (LAB)

• main purpose: conservation

• rapid conversion of lactose into lactic acid through LAB

• reduction of pH, lowering of redox potential

• growth inhibition of undesired bacteria, e.g. pathogenes

• volatile organic flavour compounds are generated during/through ripening and fermentation

• hard- and semi-hard cheeses: important microbial flavour formation through degradation of milk proteins (catabolism of AA)

Microbial flavour formation in dairy products

W.J.M. Engels et al. Flavour formation in cheese, In Dairy processing, G. Smit: CRC Press Boca Raton USA, 2003, 492–507

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Flavour analytics at Agroscope

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Source: Switzerland Cheese Marketing AG

• how do we get the flavour out of the product and into an analytical instrument?

• and how can an analytical intrument evaluate a sensory perception such as smell?

Source: Stern.de

http://www.stern.de/wissen/natur/duftrekorde r-japaner-zeichnen-gerueche-auf-

564399.html

Flavour analysis....

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1. sampling & sample preparation (extraction, concentration....)

2. separation of target analytes

• odorant compounds => volatile organic compounds (VOC) => gas chromatography GC

3. detection, identification and data processing

Sample

Solution

Sample preparation

(classical extraction, headspace HS techniques)

Extract

(sample)

Analysis GC

Flavour analysis: three steps

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Extraction of volatile organic compounds, for example by headspace solid-phase microextraction HS-SPME

• dairy and meat products: extraction in HS

• aqeous samples: immersion possible

• use of different polymer coatings depending on application

• important parameters: fibre coating, equilibration, temperature, time, agitation, addition of salt…

Stop for manual application

Piston

Fibre with polymer coating Needle

plunger

Support

Breme K. Analyse d'extraits naturels parfumants par GC/Olfactométrie: Application à des extraits de Brassicales et d'Astérales (French Edition), ISBN 978-6131501517, Éditions Universitaires Européennes: Saarbrücken, Germany, 2010, 280 pp. Images: CTC CombiPAL

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1. sampling & sample preparation (extraction, concentration....)

2. separation of target analytes

• odorant compounds => volatile organic compounds (VOC) => gas chromatography GC

3. detection, identification and data processing

Sample

Solution

Sample preparation

(classical extraction, headspace HS techniques)

Extract

(sample)

Analysis GC

Flavour analysis: three steps

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GC

physical detection

GC-MS

GC/FID GC/element specific detection

Identification . MS data bases, LRI, co-inj

Quantification EC, AC, IC

Heteroatom. comp.

N-, S-compounds (VNC, VSC)

Detector

(FID, MS…) Injector

Capillary separative column

Analysis GC

(1D, 2D)

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17 Structure Name / Odor description Occurence

Perception threshold in

water (ppt)

1-p-menthene-8-thiol

grapefruit

0.1 Furfurylthiol

roasted coffee 6

Dimethyl trisulphide

rotten cabbage, onion 10

Dimethyl disulphide

cabbage

160 Methional

baked potatoe

200 Dimethyl sulphide

cooked cabbage

onion, garlic

300 Diacetyl

butter

6500

Volatile sulphur compound (VSC) analytics

The GC-MS/PFPD experimental setup at Agroscope ALP-Haras:

A GC (Thermo Finnigan Trace GC) is equipped with a mass spectrometer (Thermo Scientific DSQ II Single Quadrupole) and a PFPD (O.I. Analytical, detector model 5380) to simultaneously measure MS trace and the sulphur specific response

chromatogramme

• VSCs: essential to the characteristic flavour of many foods

• often key flavour compounds: low odour thresholds, trace compounds

• detection by human nose, but not always by classical physical detectors (MS)

• element specific detection: e.g. pulsed-flame photometric detection PFPD

Fuchsmann P., Irmler S., Breme K. Volatile sulphur compounds in cheeses – an odorous analytical challenge, Chimia: Highlights of Analytical Sciences in Switzerland 2013, submitted.

http://www.leffingwell.com/odorthre.htm, 11.11.2012

O

SH

S S

S

SH

O

S S

S

S H

O

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GC

physical detection

GC physiological detection:

Olfactometry Analysis

GC

(1D, 2D)

GC-MS

GC/FID GC/element specific detection

.

Identification

Known compound

Unknown compound

Isolation

NMR 1D, 2D, HRMS,

IR Synthesis

Identification

Identification MS data bases, LRI, co-inj

Quantification EC, AC, IC

Heteroatom. comp.

N-, S-compounds (VNC, VSC)

odorant compounds

odour quality&impact

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Water

Joystick

Joystick Air

Capillary column Injector

Gas chromatograph

Physical detector FID, PFPD…

Splitter

Mass spectrometer

Air

Y

Water

GC-Olfactometry (GC-O)

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Odour-active compounds Volatile compounds Chromatogramme (physical detector)

Aromagram (human nose)

GC-Olfactometry (GC-O)

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1. sampling & sample preparation (extraction, concentration....)

2. separation of target analytes

• odorant compounds => volatile organic compounds (VOC) => gas chromatography GC

3. detection, identification and data processing

Sample

Solution

Sample preparation

(classical extraction, headspace HS techniques)

Extract

(sample)

Analysis GC

Flavour analysis: three steps

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• volatile and odorant compound profiles (GC-MS/PFPD, GC- Olfactometry, panel) to distinguish samples

• identification of new odour compounds, determination of off- flavours

• development and optimisation of extraction and enrichment procedures

• target molecule quantification (certified lab, validated methods)

• analysis of volatile sulphur compounds

• focus on milk and meat products and honey

Flavour analytic inquiries at Agroscope

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23

07-32-72 1. Versuchstag

0 1 2 3 4 5 6

Kontrolle FAM1233 FAM18098 FAM8407 FAM18108 &

FAM18110

Menge in mmol / kg

Serin Threonin α-Aminobuttersäure

• investigation of bacterial metabolic pathways

• search for biomarkers coding for specific aroma

• defining and improving the characteristic flavour of dairy and meat products

• flavour analytics: characterisation/quantification of target aroma compounds

• sensory analyses: preferences

• effect of culture addition in end product

• flavour analytics: GC-MS/PFPD, GC-O

• sensory analyses: analytical tests, hedonic consumer tests

Flavour research & strain collection:

enhancement of natural flavours

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GC-O aroma profile comparison of differently fabricated butter samples 5.00 10.00 15.00 20.00 25.00 30.00 35.00

creamy sour

cooked milk

cooked milk greasy

coconut

peach

t (min) creamy

greasy

HS-SPME-GC-MS-Olfactometry results: butter

Coconut, peach:

lactones

• lactones: potent flavour compounds for nutty and fruity notes

• investigation of potential of LAB to produce lactones in cream as a raw material for butter production

• supplementation with supposed precursors: hydroxy fatty acids

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25 Lucchetti et al. Formation of lactones by Lactococcus lactis subsp. lactis biovar. diacetylactis during fermentation in cream, 7^th International Conference on Chemical

Reactions in Foods, 14–16 Nov 2012, Prague, Czech Republic. The authors thank the Bioflavour COST Action FA0907 for financial support

Microbial enhancement of fruity-nutty flavour in cream

0.05

0.05 0.03

0.03 0.01

0.01

0.1 0.1 0.3 0.5 0.7

0.7 0.5 0.3

peak area lactone/peak area ISpeak area lactone/peak area IS

LAB strain FAM18027 from the Agroscope strain collection seems to form lactones during fermentation

Trial n 1

• incubation of cream

• formation of octa- and decalactone

• HS-SPME-GC-MS

Trial n 2

• fermentation of cream

• chosen LAB strain

• formation of C8- and C10-lactone

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GC-MS-Olfactometry results

• odour-active zones in chromatogramme

• classification of employed vocabulary into odour families for data processing

Odour family Examples of vocabulary used by the olfactometry judges Meaty-sulphury

Bouillon, Popcorn, roasted, Basmati rice, meat, burnt, potatoes, garlic, salami, coffee, vegetables, cabbage, onions

Fatty Grass, hay, fried fat, oxidised, citrus, soapy, green Spicy Pepper, mint, terpenes, pine

Fruity Sweet, ester, fruity, peach, banana, apple Metallic Mushroom, earthy, woody, pilzig

Buttery Milk, butter, cream, caramell, malt

Animcalic-cheesy Cheesy, rancid, moldy, sweaty, animal, urine, feces Chemical Solvent, chemical, artificial, plastic

Flowery Honey, rosy, flowery, almond

Odour-active compounds

Volatile compounds

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e.g. α-pinene e.g. 2-methyl-3-furanethiol

greasy

spicy

fruity

metallic

e.g. 1-octene-3-ol

buttery animalic-rancid

e.g. butyric acid e.g. phenylethanol

chemical floral

meaty-sulphury

GC-O spiderweb with odour family frequency of a salami sample GC-O spiderweb with odour family frequency of a cheese sample

e.g. hexanal

e.g. 2,3-butanedione (diacetyl) e.g. 3-methylbutanal

Meaty-sulphury greasy

spicy

fruity

metallic buttery

animalic-rancid chemical

floral roasted

e.g.

2-methylpropylethanoate e.g.

2-acetyl-1-pyrroline

HS-SPME-GC-MS-Olfactometry results

Vocabulary and odour families are matrix-dependent!

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Odour profiles of different salami by HS-SPME-GC-MS-O

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Monitoring of target aroma-active key VSCs in Cheddar during ripening by GC/PFPD

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Square root (Area compound/Area ISTD)

MS signal PFPD signal

Me₂SO₂

DMSO

DMTS DMDS

ISTD2 ISTD1 MeSH DMS

H₂S

CS₂

Acetic acid Butanoic acid Hexanoic acid Octanoic acid

Acetoin

Comparison of the MS-TIC (total ion count) signal vs. PFPD signal of cheese headspace

C H

H H

S H

Dimethyl sulfide Methanethiol

Hydrogen sulfide

…..

Evolution of target VSCs (H

₂

S, MeSH, DMS, DMDS and DMTS) during ripening Over 24 hours, 30, 60 and 90 days

1: FAM4067, 2: FAM18101, 3: FAM18108, 4: FAM18110 (reference), 5: FAM18149, 6: Reference sampled after 24 hours of cheese ripening and stored at –40C for 90d.

Total amount of target VSCs in model Cheddar cheese

30 days 60 days

1 2 3 4 5 6

90 days

1 day

Bogicevic, B. et al. A preliminary study on the effect of Lactobacillus casei expressing ctl1/ctl2 on Cheddar cheese and the formation of sulphur-containing compounds, Int. Dairy J., 2013, accepted manuscript: DOI information 10.1016/j.idairyj.2013.05.005

Fuchsmann P. et al. GC/PFPD monitoring of volatile sulfur compounds produced by metabolism of sulfur-containing amino acids in model cheddar cheeses during ripening, 6^th IDF Cheese Ripening and Technology Symposium, 21 24 May 2012, Madison/Wisconsin, USA.

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Volatile sulphur compounds (VSC) in cheese by HS-SPME-GC/PFPD

Fuchsmann P., Irmler S., Breme K. Volatile sulphur compounds in cheeses – an odorous analytical challenge, Chimia: Highlights of Analytical Sciences in Switzerland 2013, submitted.

Pulsed-flame photometric detector sulphur specific trace comparison between three different cheeses produced

in Switzerland

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Target aroma-active key VSCs and their odour impact in Swiss Tilsit by GC/PFPD and GC-O

C H

H H

S H

Dimethyl sulfide Methanethiol

Hydrogen sulfide

…..

Fuchsmann P., Brügger Y.-A., Breme K., Identification, quantification and odor impact of selected volatile sulfur compounds in Swiss Tilsit cheese by headspace- GC/pulsed flame photometric detection and GC-Olfactometry, 10^th Wartburg Symposium on Flavor Chemistry & Biology, April 16^th–19^th, 2013, Eisenach, Germany

Acquisniff® software: developed and provided by INRA, distributed by INRA Transfert, F-75015 Paris, France. AcquiSniff@clermont.inra.fr

GC/PFPD signal of Tilsit headspace extracted with a CAR/PDMS 85 μm

1cm SPME fibre

GC-Olfactometry aromagram of Tilsit cheese indicating identified odorant VSCs and odour descriptors

Data processing by taking into account detection frequency and odour intensity using the Acquisniff

^®

software

12 14 16 18 20 22 24

0 5000000 10000000 15000000 20000000 25000000 30000000 35000000 40000000 45000000 50000000 55000000 60000000 65000000 70000000

Counts

Time (min)

S O

S O O S

S S

S S S S

O S

C S

S C H O

S H C H 3 S H

0 2 4 6 8 10

Time (s)

960 940 920 900 880 860 840 820 800 780 760 740 720 700 680 660 640 620 600 580 560 540 520 500 480 460 440 420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 1 1 1 0.9 0.9 0.9 0.9 0.9 0.8 0.8 0.8 0.8 0.8 0.7 0.7 0.7 0.7 0.7 0.6 0.6 0.6 0.6 0.6 0.5 0.5 0.5 0.5 0.5 0.4 0.4 0.4 0.4 0.4 0.3 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.1 0.0 0.0 0

Total olfactive signal (TOS) Mean detection frequency x mean intensity

Identified odorant VSCs Other odorant compounds

1 cheesy, sulfury Methanethiol

(MeSH)

3 sulfury, onion, cooked Dimethyl disulfide

(DMDS)

4 onion, sulfury, cabbage Dimethyl trisulfide

(DMTS) 2 sulfury,

cooked Dimethyl sulfide

(DMS)

1

2 3

4

480 940

0.9

0.5

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Acknowledgements

• Colleagues at Agroscope ALP-Haras

Team Flavour Analytics Team Sensory Analyses

Group Biochemistry of Milk and Microorganisms Group Ingredients

Group Fermentation Organisms, Team Strain Collection Group Biotechnology

Group Management of Cultures and Infrastructure Research Group Cultures, Biodiversity and Terroir Research Group Quality of Milk and Meat Products Sensory panellists