Analytical techniques of flavour

1. Techniques in Flavour
Analysis
Measuring the Beauty Of Food

2. S.Thiruchenduran (FST/2014-018)
FST 507 Techniques in Food Analysis
MSc Food Technology, Department of Food and Nutrition
PG&RCPJTSAU

3. We Will See
 Teaser
 Flavour
 Importance and Applications
 Analysis
 Summary
 Acknowledgement

4. TEASER

5. Question?

6. IDENTIFY THE FLAVOURS…

7. ANSWERS
• Orange
• Strawberry
• Chocalate
• Vanila
• Blackcurrent
• Blueberry
FLAVOURS
COLOURS

8. Whats Inside This?

9. DEFINITION

10. “Flavour”
• The word is not found in many languages.
• Scientifically used to indicate two meanings
1. Sensation (e.g: mmmm Yummy!!!)
- Flavour, Sight, Mouthfeel – 3 Important sensation of food
2. Components (e.g: Flavour compounds in Strawberry Ice
Cream)
- Flavour, Appearance, Texture – 3 Important quality
parameters.

11. Flavour (Sensation)
Definition. Complex combination
of the olfactory, gustatory and
trigeminal sensations
perceived during tasting
(ISO 5492:2008)

12. Flavour (Component)
Flavour is the result of the correct
balance and concentration of a wide
variety of volatile flavour compounds
Mulder (1952)
Kosikowski and Mocquot (1958)

13. Flavour Detection
PC:- Wine Tasting Class, Chicago Life Long Learning Centre

14. Flavour Detection

15. Flavour Detection

16. Olfaction

17. Gustatory

18. Trigeminal
Trigeminal

19. Overall Flavour Sensation
Trigeminal

22. Flavour Sensation and Components
Flavour
Sensation
70% Volatile
Aroma
Compounds
30% Taste
Compounds,
Texture, Temp,
Kinesthetic Effect,
SoundAnne Thierry
STLO, INRA Rennes
John Hannon
Teagasc Food Research Centre, Moorepark, Cork

23. Why Analysis Flvour Compounds?
Flavour
Compoun
ds
Sensory
attributes
Customer
Delight
Success

24. APPLICATIONS AND
IMPORTANCE

25. APPLICATIONS AND IMPORTANCE
Tegasc Food Research Center
1. Flavour Profiling

26. APPLICATIONS AND IMPORTANCE
Tegasc Food Research Center
1. Flavour Profiling

27. More…APPLICATIONS AND
IMPORTANCE
2. Identification of key olfactory components – To
synthesize artificial flavours.
Impact Substances or Top Notes
Flavour mixtures or Flavour building blocks
Octanal – Orange, pungent
R-limoene – Citrus like
Neral/geranial – lemon like

28. More…APPLICATIONS AND
IMPORTANCE
3. Off Flavour Analysis – To shelf life determination online
and off line, Quality Control Parameter.
Milk - TNA , Rancid, Soapy flavour
Beer – Taintness
Potato chips – Rancid Flavour

29. More…APPLICATIONS AND
IMPORTANCE
4. Design New Productse.g: 7UP, Avas, Kurkure, RTE
Payasam
PC:-The New York Times

30. More…APPLICATIONS AND
IMPORTANCE
5. Product Optimization ???
Dr. Howard Moskowitz
Pc:- www.wordpress.com

31. More…APPLICATIONS AND
IMPORTANCE
6. Packaging Quality Check – Permeation of volatiles
7. Check Adulteration – e.g: Artificial Flavour
8. Check Authenticity e.g: “Ceylon Tea” – Made in India
, Ceylon tea cis-hexanol is present India Tea no
cis-hexanol, Organic Vs Commercial
9. Assessing the suitability of individual processing steps

32. Non Food Applications
1. Fragrance Profiling in Consumer Products.. E.g: Pears
honey Soap, Milk White Soap, Flavoured Condumer
goods
Annick
Goutal's Eau
d'Hadrien:
Price $1,500

33. Non-Food Application
2. Bad-Breadth Analysis – Smoking, Bacterial Infection
3. Aroma therapy – Relaxation and Stress Relief

34. Analysis

35. 2. Concentration
Extract
Simplified Analysis
.
.
.

36. Analysis
• Objectives
• Analyte
• Analytical Steps

37. Objectives (General)
1. To obtain aroma isolate to accurately identify and
quantify every aroma constituent in food.
2. To identify only key component of an aroma profile.
3. To identify an off note in a food product.
4. To monitor aroma changes with time.
5. To predict sensory attributes.
Based on the objective the methodology of analysis will
change

38. The Analyte
• Volatile compounds
• Low boiling point
• Trace amounts
• Highly reactive
• Varying concentration with time
• Varying threshold detection level ( odour activity )
• Varying functional moieties
• Varying molecular mass (up to 300)

39. The Analyte
• These characteristics of the volatile flavour compounds
makes it difficult to analyze.
• The methods of sample preparation and analysis will
vary vastly with the compound or food of consideration.

40. Physical Characteristics of Flavour
Compounds
Flavour
Compounds
B.P.(0C) 760
mm-Hg
Solubility in
H2O g/100
ml
Sense of smell
(ppm)
n-propanol 61 20 0.17
n-butanol 75.7 4 0.07
n-hexanal 131 0.5 0.03
acetone 56 20 500
2-butanone 79.6 3.7 50
Ref:- Dr. David B. Min., Flavor Chemistry.,Ohio State University., Lecture., 2008

41. The Analyte e.g:
• Tea :- Linalool. Geraniol,
2,5-dimethylpyrazine,
linalool oxide, benzyl alcohol,
jasmone, 2-phenylethanol,
coumarin,
• Pine apple :- Methyl butyrate
• Wine :- Ethyl acetate
• Orange :- Ethyl butyrate
• Banana :- Isomyl acetate
• Pear :- Pentyl butyrate
• Fish :- TMA
• Strawberry :- Benzyl acetate ,
Furaneol (>1000 volatile
compounds)
• Coffe :- 3-methylbutanol,
furfural, ethyl nononoate
• Clove :- Eugeneol
• Almond :- Benzaldehyde
• Cinnamon :-
Cinnamaldehyde
• Coconut :- gamma-
Nonalactone

42. Discovery of Flavour Compounds
1970 – 1,000
2000- 7,000
>10,000
John. C. Lefingwell, Leffingwel and Associates, 50th anniversary presentation
Why?

44. Analytical Steps
1. Isolation of Volatile Flavor Compounds (VFC)
2. Extraction
3. Fractionation
4. Identification

45. Isolation
Analyte is Volatile Flavour Compounds

46. Isolation
1. Objective
2. Methods

47. Objectives of Isolation
1. To Remove Interfering Compounds As Much as
Possible from Analyte (Volatile Flavour Compouunds)
2. To Get a Representative Sample. A near complete
flavour profile of the food matrix.
3. To avoid artifacts formation

48. Methods of Isolation
1. Headspace Extraction
2. Distillation and Extraction

49. Headspace Extraction
1. Static Head Space Method
1. Headspace Condensation Method
2. Syringe Method
2. Dynamic Head Space Method
3. Solid Phase Micro Extraction (SPME)

50. Headspace Condensation Method of
Isolation
Captured From:-Morton I.D, Maceleod A.J, “Developments in Food Sciences”, Food
Flavours, Elsevier, NYP, 1982
Introduced by Schreyen et al
in 1976

51. Syringe Method
Ref:- Dr. David B. Min., Flavor Chemistry.,Ohio State University., Lecture., 2008

52. Dynamic Head Space Method
Ref:- Dr. David B. Min., Flavor Chemistry.,Ohio State University., Lecture., 2008

53. Extraction

54. Extraction
1. Steam distillation (SD)
2. Solvent extraction (SE),
3. Supercritical fluid extraction (SFE)
4. Simultaneous distillation–extraction(SDE)
5. Pressurized-fluid extraction
6. Solvent assisted flavour evaporation (SAFE)
7. Microwave-assisted hydro distillation (MAHD),
8. Cryofocussing

55. 1. SD
1. Steam (H2O(g))
2. Continuous Stirring
3. Cold trap
1. Ice Water
2. Dry Ice/Acetone
3. Liquid Nitrogen
4. Low temperature 30oC - 60oC
5. Vacuum 8 psi
6. Falling film evaporators/Climbing film evaporators
Distillate of very dilute solution of Flavour compounds

56. SD cont…
1. Extract the distillate with organic solvent.
1. E.g. pentane, diethyl ether, dichloro methane
2. Dry the isolate with MgSO3 or other drying agents.
3. Filtered and Concentrated for instrumental analysis.

57. 2. SE
• Pure solvents. 99.9% (a blank solvent also should be run
to monitor solvent artefact detection)
• Diethyl ether, hexane, dichloro methane,
• Need further processing to separate aroma components
from lipids. – molecular distillation, steam distillation,
purge and trap or dialysis.
• Simple procedure
• Fat free foods – wines, breads, fruit and berry juices,
some vegetables and alcoholic beverages.

58. Solvent selection
Solvent selection
1. Polarity
1. Non-polar solvent for alcoholic beverages (Schreir 1980;
Shreir et al 1979; Williams and Tucknott 1973)
• Pentane, hexane, isopentane, trichlorofluoromethane
2. Polar solvents for Non-alcoholic food matices
• Diethyl ether (low BP)

59. Solvent selection
Solvent selection...
2. Analyte
1. Low boiling point flavour
1. Freon 11 (23.7oC)
2. Freon 12 (-29.7oC)
3. CO2 (-78oC)
2. High boiling-point flavours
1. Hexane
2. Chloroform (Kazeniak and Hall 1980)

60. Simple SE Procedure
1. Add solvent (diethyl ether ) with
food sample (apple juice) in a
sepratory funnel.
2. Shake well
3. Collect the solvent
4. Dry it with an anhydrous salt
(anhydrous Na2SO4)
5. The concentrate is used in instrumental analysis.

61. 3. SFE
1. Same procedure as in SE
2. Supercritical CO2 is used
3. Pressurized chamber (80-100 bar)
4. Very low boiling point (35-40 oC)
5. No residues or left overs of the solvent
6. Penetrates food matrices effectively
7. Solvent properties controlled through T and P or
chemical modifiers (methanol)

62. SFE
Drawbacks
• Expensive
• Intrumentation
• Non-polar nature
• Small sample size

63. 3. SDE
1. Both distillation and Solvent extraction done
simultaneously.
2. First introduced by Lickens and Nickerson in 1964.
3. Efficient flavour extraction by intimate molecular mixing
of steam and solvent vapour.
4. Two types
1. Atmospheric SDE
2. Reduced pressure SDE

64. SDE
Captured From:-Morton I.D, Maceleod A.J, “Developments in Food Sciences”, Food
Flavours, Elsevier, NYP, 1982
Apparatus
introduced by
Lickens and
Nickerson (1964)

65. SDE
Captured From:-Morton I.D, Maceleod A.J, “Developments in Food Sciences”, Food
Flavours, Elsevier, NYP, 1982
Apparatus
modified by
McLeod and Cave
(1975)
Advantage is it
reduce artifact
formation by
reducing the
temperature.

66. SDE
Further SDE combined with
1. Tenax or Charcoal trap (Sugisawa et al. (1984))
• Flavour molecules adsorbed to the trap and then
desorbed with solvent
2. Purged with innert gas (Umano and Shibamoto (1987))
• Sample is purged with innert gas and then the flavour
compounds are trapped in water which is continously
refluxed with the solvent.

67. 4. SAFE
• Vacuum Low T distillation
• Nitrogen(l) Trapping. – Little changes to extract
• Volatiles and Non-Volatiles
• GCO

68. 5. SPME
• Solvent Free Micro Extraction
• Principle – Equilibrium adsorption and desorption
process
• Stationary Phase - Organics Coated on a Silica Fibre
• Solid Sample - Head Space Equilibrate to 15–30 min
• Liquid Samples - Salting out of Aroma Compounds From
Food to Head space

69. SPME Technique

70. Injecting and Running a Sample on GC
This is where you
inject your SPME
needle on the GC-MS

71. SPME
Advantages
• SPME is a solvent-free micro extraction technique
• Cost efficient
• Simple to understand and use
• High sensitivity
• Low detection limits
• Can be used to sample analytes of many types
• Used in many areas of industry

72. Problems in Isolation
1. Artifacts formation
2. Loss of volatiles
3. Binding of Volatiles to the solid food matrix
4. Low concetration

73. Artifacts Formation
Enzymatic
1. Hydrolysis of esters
2. Oxidative cleavage of unsaturated fatty acids
3. Hydrogenation of aldehydes

74. Artifacts Formation Cont…
Non-enzymatic
1. Hydrolysis of glycosides
2. Lactones from hydroxy acids
3. Cyclization of di-, tri-, and polyols
4. Dehydration and rearrangement of tert-allyl alcohols
5. Reactions of thiols, amines, and aldehydes in the aroma
concentrate
6. Reduction of disulfides by reductones from the Maillard reaction
7. Fragmentation of hydroperoxides

75. Methods to control artifacts formation
1. Addition of CaCl2 (Enzyme inhibitor)
2. Addition of methanol or ethanol (Some times mask the
Volatiles)
3. Rapid sample preparation
4. Temperature must be controlled in accordance with the type
of food stuff.
1. <50oC to prevent Maillard reaction
2. < 40oC to prevent hydroperoxides fragmentation in fats and oils.

76. Fractionation

77. Fractionation
1. Preliminary fractionation
1. Acid, Neutral and Basic compounds
2. Final fractionation
1. Gas-Liquid Chromatography

78. GC Animation
PC:- Sam Houston State University, Texas,
US. Link:-

79. GC/MS Schematic Movie
PC:- Sam Houston State University, Texas,
US. Link:-
http://www.shsu.edu/chm_tgc/sounds/push
movies/chrom.html

80. GC/MS
Picture Courtesy: Polimerek, Wikipedia.

81. Identification

82. Identification
1. FID
2. Mass Spectrometry
3. GC-MS/Olfactometry

83. GC-MS/O
• GC/MS
• Sensorry Evaluation

84. GC-O Schematic
PC:- Willey online Science Library

85. Graphs
GC
Olfactometry
Volatile Concentration
Odour Active Component

86. ACKNOWLEDGEMENT
For their kind help

87. Thank you!!!
• Dr.K.Uma Maheshwari, HOD – Dept of Food and Nutrition,
Programme Director Food Science and Technology and
Professor, PG&RC, PJTSAU.
• Socialist Democratic Secular Govt of India and ICCR, For their
courtesy of Sponsoring me to study here.
• PJTSAU, Central Library. For lending Books and Internet
• My Class Mates.
• Ms. Vishnupriya, Ms. Supta Sarkar and Ms. Ekta Belwal for
sharing few books.
• All who are present here.

88. The Oldest Reference.

89. References
1. A Taste For Flavour Characterization, Laboratory
Newshttp://www.labnews.co.uk/features/a-taste-for-flavour-
characterisation
2. Andrew J. Taylor, Robert Linforth., Food Flavour Technology.,
John Wiley & Sons,2009.
3. Arie J. Haggen Smit., The Chemistry Of Flavour., Engineering
and Science Monthly., 1949.
4. Arielle J. Johnson, Gregory D. Hirson, Susan E. Ebeler.,
Perceptual Characterization and Analysis of Aroma Mixtures
Using Gas Chromatography Recomposition-Olfactometry., PLoS
ONE. 2012

90. References
6. Barbara d’Acampora Zellner , Paola Dugo, Giovanni Dugo,
Luigi Mondello, Gas chromatography–olfactometry in food
flavour analysis, Journal of Chromatography A, Science
Direct, 2007.
7. Bethany J. Hausch., Flavor Chemistry Of Lemon-Lime
Carbonated Beverages., University Of Illinois, Urbana
Campaign, 2010
8. Dr. David B. Min., Flavor Chemistry.,Ohio State University.,
Lecture., 2008
9. Factors affecting retention and release of flavour compounds
in food carbohydrates., Naknean, P. and Meenune, M.,
International Food Research Journal, 2010.

91. References
10. Fatma A. M. Hassan, Mona A. M. Abd El- Gawad, A. K. Enab.,
Flavour Compounds in Cheese (Review)., Research on
Precision Instrument and Machinery., 2013.
11. Gary Reineccius., Sourcebook of Flavors.,Springer Science &
Business Media, 1998. H.-D. Belitz · W. Grosch · P. Schieberle,
Food Chemistry, Springer 2009
12. Identification of Potent Odorants in a Novel Nonalcoholic beverage
Produced by Fermentation of Wort with Shiitake (Lentinula edodes)
13. Jida Zhang, Gang Cao, Yunhua Xia, Chengping Wen, Yongsheng
Fan, Fast analysis of principal volatile compounds in crude and
processed Atractylodes macrocephala by an automated static
headspace gas chromatography-mass spectrometry, Pharmaconosy
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92. References
14. Jon G. Wilkes, Eric D. Conte, Yongkyoung Kim, Manuel Holcomb, John B.
Sutherland, Dwight W., Miller., Sample preparation for the analysis of flavors
and off-flavors in foods, Journal of Chromatography A, Elsevier, 2000.
15. Katherine A Thompson Witrick., Characterization of aroma and flavor
compounds present in lambic (gueuze) beer., Virginia Polytechnic Institute
and State University., 2012
16. Kathrin Ohla, Ulrike Toepe, Johannes le Coutre, Julie Hudry., Visual-
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93. References
17. Malika Auvray and Charles Spence., The multisensory
perception of flavor., Conciousness and Cognition., Elsevier
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18. Maria E.O. Mamede a, Gla´ucia M. Pastore; Study of
methods for the extraction of volatile compounds from
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in Vanilla Extracts and Model Vanilla Ice Cream Mixes Using
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94. References
21. Michael H. Tunick , Susan K. Iandola and Diane L. Van Hekken.,
Comparison of SPME Methods for Determining Volatile Compounds
in Milk, Cheese, and Whey Powder., Foods., 2013.
22. Michael Moss, The Newyork Times, The Extraordinary Science of
Addictive Junk Food,
http://www.nytimes.com/2013/02/24/magazine/the-extraordinary-
science-of-junk-food.html?pagewanted=all&_r=0
23. Naknean, P. and Meenune, M., Factors affecting retention and
release of flavour compounds in food carbohydrates., International
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24. O.Bensebia , D.Barth, A.Dahmani Supercritical Carbon Dioxide
Extraction Of Rosemary Comparison With Steam Distillation And
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95. References
25. Perrut M., Nunes Da Ponte M., The Extraction Of Aromas
From Fermented And Distilled Beverages., Proceedings Of
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96. References
29. Qian M., and Reineccius G., Identification of Aroma Compounds in
Parmigiano-Reggiano Cheese by Gas
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