Flavor Chemistry

FST 820

Flavor Chemistry Winter quarter. 3 credits.

Course Description Chemical properties, isolation, separation, identification, formation and interaction mechanisms, and application of flavor compounds. Instructor: Dr. David B. Min

Telephone

292-7801(O), 436-9289 (H)

e-Mail

min.2@osu.edu

General Objective
The objective of this course is to teach students the role of flavor chemistry in food quality. Chemical structures and formation of flavor compounds, organic, bio, and analytical chemistries involved in flavor research, the effects of processing, packaging and storage conditions on the flavor quality and stability of foods, and current research related to flavor are covered. Upon completion of this course, students should be able to: 1 Understand Chemical reactions involved in flavor compounds formation in natural and processed food. 2 Comprehend the effects of food components, processing parameters and storage conditions on flavor quality of foods. 3 Understand principles, techniques and applications of analytical instruments involved in flavor analysis. 4 Optimize ingredient concentration, processing parameters, packing materials and storage conditions for optimum quality and stability. 5 Develop simple research programs of flavor chemistry. 6 Specify the flavor qualities of raw ingredients.

Evaluation
Midterm Examinations (2) Final Examination Home Work and Class Participation 40% 30% 30%

1.

INTRODUCTION
I. II. III. Definition of Flavor Classification of Food Flavor Scope of Flavor Chemistry
1. 2. 3. 4. 5. 6. Chemical compounds responsible for food flavor Flavor of foods Reconstitution of flavor compounds Precursors of the flavor compounds Mechanism for the formation of flavor compounds and precursors in foods Relationship between physical properties and its flavor

IV.

Objectives of Flavor Chemistry

2. ISOLATION AND SEPARATION OF FLAVOR COMPOUNDS

I. II. III. Objective Prerequisites Apparatus for Isolation
1. 2. 3. Headspace analysis Continuous solvent extraction Steam distillation and continuous solvent extraction

IV. V. VI. VII.

Extraction and Concentration Preliminary and Final Fractionation Dynamic Headspace analyzer Solid Phase Microextraction Analysis

3. FLAVOR IDENTIFICATION BY SPECTROMETRIC METHODS

I. II. III. IV. Introduction of Spectrometric Analyses Ultra Violet Spectrometry Infrared Spectrometry Nuclear Magnetic Resonance Spectrometry

V.

Mass Spectrometry
1. 2. 3. 4. 5. Furans Pyrroles Thiophenes Pyridines Pyrazines

4. MANUFACTURE OF FOOD FLAVOR

I. II. III. IV. V. Natural or Imitation Flavor Problems of Using Natural Flavor Disadvantages of Using Imitation Flavor Advantages of Imitation Flavor Methods in Synthetic Flavor Reconstitution

5. CHEMISTRY OF FLAVOR PRECURSORS

I. Flavor Compounds from Carbohydrates and Proteins
1. 2. 3. 4. 5. Maillard reaction Strecker degradation Pyrazine formation Oxazole formation Thiazole formation

II.

Thermal Degradation of Vitamin B1

1. 2. 3. 4. Basic condition Acidic condition Thiazole compounds Furan compounds

III.

Lipid Oxidation
1. 2. 3. 4. Chemistry of triplet oxygen General mechanisms of autoxidation Chemistry of singlet oxygen Enzymatic lipid oxidation (Lipoxygenase)

IV.

Flavor Generated from Enzymatic Method, Microbiological Reaction, and Biogenesis

1. 2. 3. 4. 5. 6. 7. 8.

Free fatty acids by lipase Generation of diacetyl in butter Fresh banana flavor Onion and garlic flavor Tomato flavor Asparagusic acid in Asparagus Mushroom volatiles Flavor formation by Neurospora

6.

DAIRY PRODUCTS FLAVOR CHEMISTRY

I. Milk Flavor
1. 2. 3. 4. 5. 6. Oxidized flavor Rancid flavor Heated flavor Microbiological flavor Absorbed flavor Sunlight flavor

II.

Cheese Flavor
1. 2. 3. 4. 5. 6. 7. 8. Isolation, separation and identification of cheese flavor Biological pathways of fat in cheese flavor Reaction products of methionine Biochemical pathways of cheese flavor formation from protein 2-Butanone and 2-Butanol formation from diacetyl and acetone Biochemical pathways of cheese flavor formation from lactose Lactone formation Mechanisms of methyl ketone formation

7. MEAT FLAVOR CHEMISTRY

I. Effect of Psychrotropic Bacteria on the Volatile Compounds of Raw Beef
1. 2. 3. Introduction Effects of light and dark storage on the volatile compounds of asceptic raw ground beef Effects of psychrotropic bacteria on the volatile compounds of aseptic raw ground beef

II. III.

Isolation, Separation, and Identification of Roast Beef Flavor Simulated Meat Flavor Formation

8.

ORANGE FLAVOR STUDY BY PULSED ELECTRIC FIELD PROCESS

9. INTERACTION OF FLAVOR COMPOUNDS WITH FOODS

I. II. III. IV. Physical and Chemical Stability of Flavor Effects and Interactions of Lipids with Flavor Compounds Effects and Interactions of Carbohydrates with Flavor Compounds Effects and Interactions of Proteins with Flavor Compounds

10. PACKAGING AND FLAVOR COMPOUNDS INTERACTION

I. II. Effects of Packaging Materials on the Flavor Quality of Food Sorption of Orange Flavor Compounds by Packaging Materials

11. FAVOR COMPOUNDS AND SOLVENT INTERACTION

I. II. Commercial Cherry Flavor and Solvent Interaction Acetal Formation

Reference
Acree, T. E., Teranishi, R. Flavor Science: Sensible Principles and Techniques. American Chemical Society, Washington, D.C., 1993. Ashurst P. R. Food Flavorings. AVI, New York, 1991. Bellanca, Furia. Fenaroli Handbook of Flavor Ingredients. The Chemical Rubber Company. 1972. Bills, D. D., Mussinan, C. J. Characterization and Measurement of Flavor Compounds. American Chemical Society, Washington, D.C., 1985. Charalambous, G. Flavors and Off-flavors '89. Elsevier Science Publishing Company INC, New York, 1989. Charalambous, G. Food Science and Human Nutrition. Elsevier Science Publishing Company INC, New York, 1992. Charalambous, G. Frontier of Flavor. Elsevier Science Publishing Company INC, New York, 1988. Charalambous, G. Off-flavors in Foods and Beverages. Elsevier Science Publishing Company INC, New York, 1992. Charalambous, G. Shelf Life Studies of Foods and Beverages. Elsevier Science Publishing Company INC, New York, 1993. Department of Army, Advisory Board of Quartermaster Research and Development. Chemistry of Natural Food Flavors. 1957. Gabelman, A. Bioprocess Production of Flavor, Fragrance, and Color Ingredients. John Wiley & Sons, New York, 1994. Ho, C. T., Hartman, T. G. Lipids in Food Flavors. American Chemical Society, Washington, D.C., 1994. Ho, C. T., Manley C. H. Flavor Measurement. Marcel Dekker, INC., New York, 1993.

Hornstein, Irwin. Flavor Chemistry, A Symposium. American Chemical Society, Washington, D.C. 1966. Ikan, R. The Maillard Reaction: Consequences for the Chemical and Life Sciences. John Wiley & Sons, New York, 1996. Labuza, T. P., Reineccius, G. A., Monnier, V., O'Brien, J., Baynes, J. Maillard Reactions in Chemistry, Food, and Health. The Royal Society of Chemistry, Cambridge, 1994. Min, D. B. Akoh C. C. Food Lipids. Marcel Dekker, Inc. New York, NY,1998. Min, D. B. McDonald R. E. Food Lipids and Health. IFT. Marcel Dekker, Inc. New York, NY,1996. Min, D. B., Smouse, T. H. Flavor Chemistry of Fats and Oils. The American Oil Chemists' Society, Champaign, Illinois, 1985. Min, D. B., Smouse, T. H. Flavor Chemistry of Lipid Foods. The American Oil Chemists' Society, Champaign, Illinois, 1989. Morton, I. D., Macleod A. J. Food Flavor: Part A. Introduction. Elsevier Science Publishing Company INC, New York, 1982. Morton, I. D., Macleod A. J. Food Flavor: Part C. The Flavor of Fruit. Elsevier Science Publishing Company INC, New York, 1990. Ohloff, G. and A. F. Thomas. Gustation and Olfaction. Academic Press. New York. 1971. Parliment, T. H., Morello, M. J., McGorrin, R. J. Thermally Generated Flavors: Maillard, Microwave, and Extrusion Processes. American Chemical Society, Washington, D.C., 1994. Piggott, J. R., Paterson, A. Understanding Natural Flavors. Blackie Academic & Professional, New York, 1994. Reineccius, G. Source Book of Flavors, 2nd Edition. Chapman & Hall, New York, 1992.

Scanlan, R. A. Flavor Quality: Objective Measurement. American Chemical Society, Washington, D.C., 1977. Schultz, H. W., E. A. Day, and R. V. Sinnhuber. Lipids and Their Oxidation. AVI Publishing Company, Inc., Westport, Connecticut. 1962. Shahidi, F. Flavor of Meat and Meat Products. Blackie Academic & Professional, New York, 1994. Spanier, A. M., Okai, H., Tamura, M. Food Flavor and Safety: Molecular Analysis and Design. American Chemical Society, Washington, D.C., 1993. Supran, M. K. Lipids as a Source of Flavor. American Chemical Society, Washington, D.C., 1978. Teranishi, Roy, Phillip Issenberg, Irwin Hornstein, and Emily L. Wick. Flavor Research, Principles and Techniques. Marcel Dekker. 1971. Vernin, G. Chemistry of Heterocyclic Compounds in Flavors and Aromas. John Wiley & Sons, New York, 1982.

1. INTRODUCTION
I. Definition of Flavor 1. Flavor is the sensation produced by a material taken in the mouth, perceived principally by the senses of taste and smell, and also by the general pain, tactile, and temperature receptors in the mouth. Flavor also denotes the sum of the characteristics of the material which produces that sensation. 2. Flavor is one of the three main sensory properties which are decisive in the selection, acceptance, and ingestion of a food.

Stimulus

Man Senses

Response (sensory property)

sight taste

appearance

flavor odor food hearing touch kinesthesis texture

10

II. Classification of Food Flavors

Flavor Class
Fruit flavor Vegetable flavors Spice flavors

Subdivision
citrus-type flavors (terpeny) berry-type flavors (non-terpeny) aromatic lachrymogenic hot unfermented flavors fermented flavors compounded flavors mammal flavors sea food flavors

Representative Example
grapefruit, orange apple, raspberry, banana lettuce, celery cinnamon, peppermint onion, garlic pepper, ginger juices, milk wine, beer, tea soft drinks lean beef fish, clams olive oil, coconut fat, pork fat, butter fat beef bouillon legume, potatoes marmalade ham processed meat products coffee, snack foods, processed cereals cheese

Beverage flavors

Meat flavors Fat flavors Cooked flavors

Processed flavors

broth vegetable fruit smoky flavors broiled, fried flavors roasted, toasted, baked flavors

Stench flavors

III. Scope of Flavor Chemistry 1. Chemical compounds responsible for food flavor 1) Even distribution: Brandy 2) Star compound: A star compound can not be identical to the total true flavor but is close and can not produce the true flavor without the star compound.

11

Almond: benzoaldehyde

CHO

Green pepper:

2-methoxy-3-isobutyl-pyrazine

N N

OCH3 CH2CH CH3 CH3

Both pyrazin and thiazol are important flavor compound groups

N N
pyrazine

4 5

N S
1

thiazol

12

Vanilla: 4-hydroxy-3-methoxy-benzolaldehyde

CHO

OCH3 OH

Cucumber: 2-trans-6-cis-nonadienal

H CH 2 CH 2 CH 3 CH 2 C C H H

C C

CHO H

Reversion flavor of soybean oil: 2-pentylfuran and 2-pentenylfuran

(CH 2)4 CH 3

13

2. Flavor of foods 1) Desirable flavor orange juice potato chip roast beef 2) Undesirable flavor (off-flavor) oxidized stale rancid warmed-over

3. Reconstitution of flavor compounds GC composition

4. Precursors of flavor compounds linoleate 1) Non-enzymatic reaction Precursor of beef flavor can be isolated as a white fluffy powder. White fluffy powder
Oil Water

2-pentylfuran

broil stew

beef broth

Amino acid + Sugar

Maillard reaction
14

2) Enzymatic reaction Processed banana no fresh banana flavor

enzyme extracted from banana peel Fresh banana flavor

5. Mechanisms for the formation of flavor compounds and precursors in foods 1) Volatile flavors developed in most food plants mainly at the ripening stage - the result of plant metabolism through enzymatic reaction. 2) Raw meat must be heated before it develops any organoleptically acceptable flavor. meat flavor (boiled beef)

S H3 C
5

S
3

S
4

CH 3

3, 5-dimethyl-1,2,4-trithiolane

15

Model studies: S S S

CH 3 CHO + H 2 S

H2S + CH3CHO

(S)

CH3 CH S CH CH 3 SH SH (O) H3C S S S CH 3

Therefore,CH3CHO, H2S are precursors HS C C NH2

Apply the knowledge we gained from the mechanism and precursor studies to processed food. a. Enhance the desirable food flavor. b. Elimination of the undesirable food flavor. c. Application of heated model system to processed foods.

COOH

B e e f fla v o r (re a c tio n fla v o r)

16

6. Relationship between physical properties of a compound and its flavor B.P.(0C) 760 mm-Hg n-propanol n-butanol n-hexanal acetone 2-butanone CH3-S-CH3 Threshold (ppm) 2-t-pentenal 2-t-hexa(e)nal 2-t-hepta(e)nal 2-t-octenal 2-t-nonenal 2-t-decenal 2-t-undecenal odor 2.3 10.0 14.0 7.0 3.2 33.8 150.0 61.0 75.7 131.0 56.0 79.6 37.5 Solubility in H2O g/100 ml 20 4 0.5 20 3.7 insoluble Sense of smell (ppm) 0.17 0.07 0.03 500 50 0.012

The series has an increase b.p. and decreased solubility in H2O

The vapor compositions of flavor compounds are effected by the medium. head space analysis compound (conc. 200ppm) aq. System ( peak area ) acetone 2-butanone 2-pentanone 2-hexanone 2-heptanone 10 14 22 29 24 corn oil system ( peak area ) 47 11 5.7 2.7 0.7

17

IV. Objectives of Flavor Chemistry 1. To understand the chemical composition of natural flavors and the mechanism of their formation. 2. To retard or prevent the development of the off-flavors in foods. reversion flavor in soybean oil hexenal, 2-pentyl furan ( they are resulted from polyunsaturated triglycerides, i.e.: linolenate, linoleate ) 3. To restore the fresh flavor to a processed food 4. To improve the flavor of food by the addition of synthetic flavor. 5. To produce new foods with special flavor such as potato chip flavor. 6. To improve flavor by the acceleration of reactions which produce desirable flavor compound (onion flavor: pH 5~7). 7. To assist geneticist to breed food raw material with improved flavor compounds or flavor precursors. 8. To specify raw material and to control quality of food products. The price of tea can be correlated with GLC peak of linalool.
OH CH 3 C CH 3 CH CH 2 CH 2 C CH 3 CH CH 2

Ceylon tea contains cis-hexenol, India tea doesnt contain cis-hexenol

18

2. ISOLATION AND SEPARATION OF FLAVOR COMPOUNDS

I. Objectives Produce volatile flavor compounds of the true flavor of the original with minimum artifact.

1. 2. 3. 4. 5. 6. 7. 8.

Selection of Good flavor sample Isolation of Volatile Flavor Compounds (VFC) Extraction and Concentration Fractionation Preparation of pure compound Identification Synthesis Reconstitution of the flavor

II. Prerequisites 1. Selection of sample 2. No alternation of the original flavor 3. No artifacts due to : decomposition autooxidation

19

III. Apparatus for Isolation 1. Headspace analysis 1) Without enrichment silicone rubber stopper syringe

can

2) With Enrichment Using inert gas

20

Apparatus for the isolation of trace volatile constituents from relatively large amount of food.

21

2. Continuous Solvent Extraction

Beverage sample

Continuous Liquid-liquid extractor for use with solvents lighter-than-water

22

3. Steam Distillation and Continuous Solvent Extraction

Modified Likens-Nickerson simultaneous steam distillation-solvent extraction distillation assembly

23

IV. Extraction & Concentration 1. Extraction Simple Extraction solvent used: diethyl ether, pentene, freone, etc.. salting out.

ether NaCl

24

2. Concentration Oldershaw column

thermometer

ether

ether

Concentrated to 50~100 ml

25

Kuderna-Danish assembly for the evaporation of solvent from flavor extracts

26

V. Preliminary and Final Fractionation 1. Preliminary fractionation Acid, Neutral and Basic compounds Total flavor isolate in ether (200 ml )
10% Na2CO3

H2O layer
Acidified with 10% HCl Ext. with ether

Et2O layer
+ 10% HCl Extraction

ether layer
Dried with Anhy. Na2SO4 Filter

aq. Layer ether layer ( basic compounds ) ( neutral compound )

+ 10% NaOH Ext. with ether Dried with anhy. Na2SO4

acidic compounds

basic compounds Earthy, nutty aroma

neutral compound Meaty flavor

concentration

G.C.

27

2. Final fractionation Gas-Liquid Chromatography Sample: as concentrate as possible

GC-Mass: Use capillary column Identification of the important peaks by mass spectrometry

28

Comparison of GC separation of oak leaves extract achieved using standard film thickness and thick film fused silica glass capillary column

29

VII. Solid Phase Microextracion Analysis

Instrumental Analysis of Volatile Compounds

Static headspace analysis Dynamic headspace analysis Solid phase microextraction

Detection Limits and Precision of Organic Volatile in Water

Technique Detection Limit with FID ( ppb ) 0.05-0.3 1- 2 0.003-0.005 17-240 Precision (% rsd )

SPME Static Headspace Dynamic Headspace Direct Injection

1-3 1-3 1-8 2-13

30

Solid Phase Microextraction

Solid Phase Microextraction has been commercially available for 5 years and new applications are being developed for flavor and food analyses rapidly

Objectives of Solid Phase Microextraction

Conventional Sample Preparation Time and Labor Intensive Multiple Steps Loss of Sample Errors in each steps Contamination To produce sample with highest compound concentration, lowest level contamination and shortest sample preparation time

31

Solid Phase Microextraction

A technique that uses a short, thin, solid rod of fused silica, coated with absorbent polymer for extraction of volatile compounds Equilibrium partitioning of the compounds between the coating fiber and sample or headspace.

Diagram of SPME Extraction

Direct sampling SPME

Headspace SPME

32

Principles of Headspace SPME n : Number of compounds in solid phase

f

nf=

KfhVfVsCo KfhVf+Khs Vh+Vs

K:

Partition coefficient

Kfh=

Concentration of coating Concentration of headspace

Vf,Vs,Vh: Volume of solid phase,

solution, and headspace, respectively

Co: Initial concentration of compounds

in the solution

SPME Analysis of Volatile Compounds

Plunger Gauge

Barrel

Solid Phase Water bath

33

Types of Solid Phases

CB/PDMS:Carboxen/Polydimethylsiloxane PDMS: Polydimethylsiloxane CW/DVB: Carbowax/Divinylbenzene PA: Polyacrylate.

Effects of Different Solid Phases on the Hexanal Analysis in Soybean Oil

Hexanal Peak in Electronic Count
Mean CB/PDMS PA PDMS CW/DVB 499 739 966 1,520 CV (%) 4.2 7.2 3.2 2.9 (10.7)

CV: Coefficient Variation (%) for n =5

Significant difference (P<0.05)

34

SPME Reproducibility of Major Flavor Compounds in Orange Juice

Replicates
1 2 3 4 5 6 SD ave CV(%)

Ethyl butyrate (ppm)

0.432 0.400 0.391 0.380 0.403 0.397 0.017 0.400 4.36

-Pinene (ppm)
1.378 1.391 1.343 1.389 1.402 1.470 0.042 1.395 3.00

Octanal (ppm)
1.089 1.050 1.054 1.059 1.020 1.010 0.029 1.047 2.71

Limonene (ppm)
251.05 254.28 248.26 256.25 255.71 260.01 4.130 254.26 1.63

Decanal (ppm)
1.005 0.925 0.987 0.995 1.015 1.007 0.033 1.989 3.32

Effect of G.C. Injection Temperature on Soybean Oil Volatile Compound Analysis

230 C

250 C

35

Effect of Coating Thickness on the Absorption for the Extraction of 0.1 ppm Benzene
100 80 60 40 20 0 0 200 Time (S) 400 600

100 m

Mass (ng)

56 m 15 m

Effect of Distribution Constant on the Absorption Profile of 0.1 ppm Analyte

30 25 Mass (ng) 20 15 10 5 0 0 1000 time (S) 2000 3000
K fs= 294 ( Toluene) K fs= 125 ( Benzene) K fs= 831 (p-Xylene)

36

Effect on Sample Temperature on the GC Chromatogram of Compounds

Extracted at 25 C

Extracted at 130 C

Extracted at 200 C

Effect of Water and Microwave Heating on the chromatograms of Headspace Polyaromatic Compounds
Mass Extracted (ng) 100 80 60 40 20 0 1 2 3 4 5 Compound Number
1, naphthalene: 2, acenaphthylene: 3, acenaphthalene: 4, fluorene: 5,anthracene

Water Heating Microwave Heating

37

Effect of Stirring Rate on the Extraction of 1 ppm Benzene in Water

40 Mass (ng) 30 20
0 rpm 2,500 rpm 400 rpm

10 0 0 200 Time (S) 400 600

Effect of Agitation Method on the Extraction of 1 ppm Benzene in Water

40 Mass (ng) 30
Sonication, Magnetic Stirring

20
No stirring

10 0 0 200 Time (S) 400 600

38

Effect of Benzene Concentration on Extraction by SPME

1000 Mass (ng) 100 10 1 0.1 0 100 200 300 Time (S) 400 500 600
C s = 10 ppm C s = 1 ppm Cs = 0.1 ppm

Effect of Salts on the Extraction of Volatile Compounds by SPME

Normalized FID Response

No Salt Sodium Chloride Sodium Sulfate Potassium Carbonate

Benzene

Dioxane

39

Matrix Effect on the Extraction of Alcohols by SPME

Water water-salt 12% Ethanol 12% Ethanol-salt

Detector Response

Cltronellol

Geranlol

Gas Chromatogram of Orange Juice Flavor by SPME Headspace Sampling

40

Regression Equations between Flavor Compounds (ppm) and GC Peak Areas

Concentration range (ppm) 0.1-1.2 0.1-1.3 0.1-1.1 0.2-2.0 20-50

Compounds Ethyl butyrate n-Octanal Decanal -Pinene Limonene

Regression Eq Y=0.2891X+0.015 Y=0.4913X+0.003 Y=0.2010X+0.066 Y=0.3428X+0.092 Y=17.922X+9.462

R2 0.99 1.00 0.99 0.99 0.99

Y: Compound part per million, X:Electronic counts of GC peak area

Effects of Temperature and Time on the Equilibrium of Flavor Compounds Between the SPME Coating and the Headspace of Orange Juice
30

25

25C 40C

FID r esponse

20

50C
15

60C
10

80C

0 0 10 20 30 40 Adsorption Time (minutes) 50 60

41

Isolation Time Effect on Soybean Oil Volatile Compounds by SPME

40
60 C

30

45 C 35 C

Relative Peak Size

20

10

0 0 30 60 90 120 150

Isolation Time (min)

Isolation Temperature Effect on Soybean Oil Volatile Compounds by SPME

30 PV 1 25 20 15 10 5 0 35 45 60

Relative Peak Size

PV 50

Isolation Temperature (C)

42

Chromatograms of Volatile Compounds of Soybean Oil by SPME

Volatile Compounds in the Headspace of Soybean Oil by SPME-GC-MS

Compounds Retention Time (min) 1.38 2.06 3.84 3.97 5.90 6.45 8.40 10.99 11.53 14.00 14.29 18.69 Relative (%) 3.65 5.31 23.5 9.09 2.70 4.76 4.77 5.04 3.37 2.86 0.55 34.3

Pentane Pentanal Hexanal 2-Butanone Heptanal 2-Heptenal 2-Pentylfuran 2,4-Heptadienal t-2-Octenal Nonanal t-2-Nonenal 2-Decenal

43

Effect of Isolation Temperature on Corn Oil Volatile Compounds by SPME

25C 45C

35C

60C

Volatile Compounds in the Headspace of Corn Oil by SPME-GC-MS

Compounds Retention Time (min) 1.29 1.88 3.62 5.36 6.21 8.59 10.88 11.51 13.88 14.23 18.61 20.20 20.70 Relative (%) 13.03 5.52 5.39 1.83 29.52 2.53 7.69 18.07 6.27 1.33 4.93 1.17 2.71

Pentane Pentanal Hexanal Heptanal 2-Heptenal 2-Pentylfuran 2,4-Heptadienal t-2-Octenal Nonanal t-2-Nonenal 2-Decenal t,t-2,4-Decadienal t,c-2,4-Decadienal

44

Chromatograms of Soybean Oil and Corn Oil

Soybean Oil

Corn Oil

Improving Sensitivity of Solid Phase Microextraction

Solid Phase Thickness Extraction Temperature and Time Sample Agitation and Concentration Direct sampling versus Headspace Sampling Selection of Proper Solid Phases Saturation of Sample with Proper Salts Maximum Ratio of Sample to Headspace Volume Large Sampling Vial

45

Conclusion
The SPME-GC is a Reproducible Economic Simple Sensitive for the analysis of volatile compounds in most foods.

46

3.
I. II. III. IV. V.

FLAVOR IDENTIFICATION BY SPECTROMETRIC METHODS

Introduction of Spectrometric Analyses Ultra Violet Spectrometry Infrared Spectrometry Nuclear Magnetic Resonance Spectrometry Mass Spectrometry

47

I. Introduction of Spectrometric Analyses

The study how the sample interacts with different wavelenghts in a given region of electromagnetic radiation is called spectroscopy or spectrochemical analysis. The collection of measurements signals (absorbance) as a function of electromagnetic radiation is called a spectrum.

Energy Absorption
The mechanism of absorption energy is different in the Ultraviolet, Infrared, and Nuclear magnetic resonance regions. However, the fundamental process is the absorption of certain amount of energy. The energy required for the transition from a state of lower energy to a state of higher energy is directly related to the frequency of electromagnetic radiation that causes the transition.

Spectral Distribution of Radiant Energy

Wave number (cycles/cm) X- ray 200 U.V. Visible 400 800 Wavelength (nm) I.R. Microwave

V' C V

= Wave number (cm -1) = Wave length (nm) = Velocity of Radiation (constant) 3 1010 cm/sec = Frequency of Radiation (cycles/sec) V 1 V' = = C (The energy of photon) E = Vh (Planck's Constant 6.62 10-27 erg - sec) C C = V C V =

E = Vh = h

48

The Electromagnetic Spectrum.

10
8 -10

10

-8

10

-6

Wavelength, , 10-4 10-2 1 microwave infrared

102 radio

104

106

1020

1018

1016

ultraviolet visible

- ray

- ray

1014

1012

1010 108 106 frequency, , (cycles/sec) red visible region 800

104

102

400

500

600 Wavelength, , nm

orange
49

yellow

violet

green

blue

700

50

II. Ultra Violet Spectrometry

The absorption of ultraviolet radiation by molecules is dependent upon the electronic structure of the molecule. So the ultraviolet spectrum is called electronic spectrum.

Electronic Excitation
The absorption of light energy by organic compounds in the visible and ultraviolet region involves the promotion of electrons in , , and n-orbitals from the ground state to higher energy states (This is also called Energy Transition). These higher energy states are molecular orbitals called antibonding. * * Energy n * n * * * n Antibonding Antibonding Nonbonding Bonding Bonding

51

Electronic Molecular Energy Levels The higher energy transitions ( *) occur a shorter wavelength and the low energy transitions (*, n *) occur at longer wavelength.
* Energy hv *

3 2 hv 1 * * hv

3 2 1

n * hv n

Ground Electronic State

Exited Electronic State

52

Chromophore is a functional group which absorbs a characteristic ultraviolet or visible region.

UV

210 nm 233 nm 268 nm 315 nm

Double Bonds Conjugated Diene Conjugated Triene Conjugated Tetraene

and * orbitals

and * orbitals

53

III. Infrared Spectrometry

Radiation energy in the infrared region is absorbed by the organic compound and converted into energy of molecular vibration. The energy absorption pattern thus obtained is commonly referred to as an infrared spectrum which has the plot of intensity of radiation absorption versus wavelength of absorption.

Some Molecular Vibrations

Symmetrical bend H H

H C Stretch C

O Unsymmetrical bend O

54

Atom, Group, and Molecular Rotations

Z H atom rotation H CH3 group rotation H H C C O O

Y COOH group rotation

X Molecular rotation H

OH group rotation

Center of gravity of the molecule is at the origin

IR 3.4 m 6.0 m 10.3 m 5.8 m 3.7 m 2.9 m Alkane cis-Double Bond trans-Double Bond Carbonyl Hydroxyl Stretching of Acid Group Hydroxyl

55

IV. Nuclear Magnetic Resonance Spectrometry

Nuclear magnetic dipole Precessional orbit

Spining proton

Ho Spinning charge in proton generates magnetic dipole Proton precessing in a magnetic field Ho

Precessional orbit Low energy spin state (1/2) Rotation component of

Nuclear magnetic dipole axis of nuclear rotation

High energy precession

Nuclear Spin

Low energy precession Reference axis

Oscillator Coil

Precessional orbit High energy spin state Ho Oscillator generates rotating component of magnetic field H1 Precession -Energy Relationship

56

H1 (Magnetic component of radio frequency from oscillator coil): oscillator frequency H1 can be resolved into 2 components rotating in opposite directions. (1) Rotating in the same direction in the precessional orbit of the molecular magnetic dipole (2) Rotating in the opposite direction as the precessional orbit of the nuclear magnetic dipole ; disregard

Magnetic Properties of Nuclei

Nuclei of certain atoms posses a mechanical spin or angular momentum. The total angular momentum depends on the nuclear spin or spin number (spin quantum number) I. The numerical value of the spin number ( I ) is related to the mass number and the atomic number. Each proton and neutron has its own spin and I is a result of these spins. Mass Number Odd Even Even Atomic Number Even or odd Even Odd Spin Number 1/2, 3/2, 5/2,---0 1, 2, 3, ---

The magnetic nucleus may assume any one of ( 2 I + 1) orientations with respect to the directions of the applied magnetic field. Therefore, a proton (1/2) will be able to assume only one of two possible orientations that correspond to energy levels of + or - H in an applied magnetic field, where H is the strength of the external magnetic field. If proper v is introduced, the Wo will be resonance with the properly applied radio frequency (Hi) and the proton will absorb the applied frequency and will be raised to the high spin (energy) state. Even though the external magnetic field strength (Ho) applied to the molecule is the same, the actual magnetic field strength exerted to the protons of the molecule are different if the protons are in the different electronic chemical environment.

57

Fundamental NMR Equation of Radio Frequency and Magnetic Field Strength

The energy difference between the two states is Ho V= 2 : (Magnetogyric Ratio) : Constant and a fundamental nuclear constant. V : Electromagnetic frequency in radio frequency Ho : An external magnetic field Wo = Ho Ho = 2V Therefore Wo = 2V = 2 / hI = Magnetic Moment (Magnetic Dipole Moment) h = Planck's Constant I = Spin Number

Relationship between Radio Frequency and Magnetic Field Strength for Proton Radio Frequency (Mega Hertz) 60 100 300 500 Magnetic Field (Gauss) 14,100 23,500 70,500 117,500

E = hv

1.4 T 60 MHz

2.35 T 100 MHz

4.7 T 200 MHz

7.0 T 300 MHz

58

Schematic Diagram of an NMR Spectrometer

Sweep coils Sample R-F transmitter R-F receiver and

Sweep generator

Transmitter coil

Receiver coil

Recorder

Magnet

Chemical Shift
The difference in the absorption frequency of a particular proton of the sample from the absorption frequency (position) of a reference proton. The protons at the electron rich environments (strong electonegaticve molecules such as oxygen and halogens) will feel less external magnetic field strength because the magnetic field strength generated by electrons surrounding the proton will counteract the applied magnetic field strength (Ho), which can be said deshielded proton.

Therefor, the Wo of the protons in the electron rich chemical environments will be less and require less radio frequency to be resonance with the applied radio frequency compared to the protons in the electron poor chemical environments.

ppm = (reference frequency - sample frequency) 106 Operating instrument frequency

59

The Reference Compounds : TetraMethylSilane (TMS)

H H H H C H Si C H H C H H

C H

General Regions of Chemical Shifts

Aliphatic alicyclic -Substituted aliphatic Acetylenic -Monosubstituted aliphatic -Disubstitutid aliphatic Olefinic Aromatic and heteroaromatic Aldehydic 10 9 8 7 6 5 4 3 2 1 0

60

R CH C H C H2 C H CH C H2
5.3 2.7 2.0

O C O C H3
3.6

Rest of the protons on CH3 and CH2 absorb at 0.8 - 2.0 broad, big peak

very crowded area, usually see a

Spin-Spin Coupling (Spin-Spin Splitting)

Spin-Spin Coupling is the indirect coupling of proton spins through the intervening bonding electrons. It occurs because there is some tendency for a bonding electron to pair its spins with the spin of the nearest protons. The spin of a bonding electron having been thus influenced. Coupling is ordinarily not important beyond 3 bonds unless there is ring strains as in small rings or bridged systems, or bond delocalizaion as in aromatic or unsaturated systems

61

Signal a is split into a doublet by coupling with one proton; signal b is split into a triplet by two protons. Spacing in both sets is same (Jab). a b Jab b a

C H2Br C HBr2

Jab Jab

Information from NMR Spectrum

The Number of signals The Position of signals The Intensity of signals The Splitting of signals

62

NMR of Fatty Acid Methyl - Ester

C 19H32O 2

Methly linolenate

CH3 CH2 CH CH (CH2 CH CH)2 CH2 (CH2)5 CH2

Chemical shift (ppm) a b c d 0.97 1.33 2.80 3.67 e ca.5.38

O C OMe
d

63

V. Mass Spectrometry

Definition
A mass spectrometer bombards a substance under investigation with an electron beam and quantitatively records the result as a spectrum of positive ion fragments. This record is a Mass Spectrum. A mass spectrum is a presentation of the masses of the positively charged fragments vs. their relative concentration. Separation of the positive charge ion fragment is on the basis of mass. (Mass/Charge)

Essential Features of Mass Spectrometer

(1) Sample Inlet System

64

a) GC inlet system - The samples separated by gas chromatography are introduced into the ion source of mass spectrometer. b) Heated expansion reservoir - Pure liquid and gas samples are conveniently injected by syringe into the all glass heated expansion reservoir and leaked into the ion source of mass spectrometer through a vernier value - Temp. 250C at 10-2 Torr. c) Direct Introduction Probe (DIP) - Solids and viscous liquids are introduced directly into the ion source of the mass spectrometer by the direct introduction probe. The sample is placed in a glass capillary and gently heated to produce the required vapor pressure without thermal decomposition. (2) Ion Source (Ionization Chamber) The stream of vaporized sample molecules from sample injection (Inlet) system entering the ion source interact with the beam of electrons to form positive ions. The electron beam is emitted from a hot filament.

(3) Accelerating Chambers

The positive ions are pushed out of the source by relatively small "repeller" potential, and then accelerated by a large potential difference (1 to 10KV - a strong electrostatic field) between the first and second accelerating slits. Small potentials can be applied to the repeller and ion focus slit to produce a defined beam of positive ion.

(4) Analyzer (Ion Separation)

The collimated ion beam for the ion source can be separated according to the respective masses of the ions by a variety of techniques such as magnetic deflection in a magnetic field by varying either the magnetic field applied to the analyzer tube or the accelerating voltage between the first and second ion slits. The mass which passes through the exit slit is dependent upon the radius (4

65

cm) of the ion path in the magnetic field, the magnetic field strength (B, gauss) and the ion accelerating potential (V, volt) is defined by the fundamental equation: m/e = 4.82 x 10-5 B2 r2 /v

Changing the magnetic field changes the amount of ion deflection, bringing a different m/e into focus on the collector slit, continuously changing the magnetic field while recording the ion signals on a strip chart and then producing a mass spectrum.

(5) Ion Collector The positive ions striking the collector produce a flow of ions proportional to the ion abundance. The ions are amplified by an ion multiplier.

(6) Recorder The amplified ion currents (signals) are measured on a photographic paper.

66

Fatty Acids
Molecular ion peak of a straight chain monocarboxylic acid is weak but usually discernible. The most characteristic peak (sometimes the base peak) is at m/e 60 due to McLafferty rearrangement .

H O HO C C H2
+ O H O +

CH R CH2 H

H 2C

C HR

H
O HO C +

HO C

CH2

HO C CH2

CH2

Methyl - Ester of Fatty Acids

The mass spectrum of a methyl - ester is very similar to that of corresponding carboxylic acid. The methyl ester is more volatile than the free fatty acids and therefore the easier to examine. m/e 74; Corresponding to the m/e 60 peak of fatty acid is usually base peak or predominant

McLafferty Rearrangement

H
O +

C R2 CH2 C H2

R 2C CH2

CH3O

C H

H
O +

H
O

O +

C H3O

C CH2

C H3O

C CH2

CH3O

C+ CH2

67

68

C H2O H

OH

-H
+

-CO

m/e 108

m/e 107 + H H

-H2
+ H H m/e 79 [C6H7]+ m/e 77 [C6H5]+ H

69

CH3 + CH3 +

H H

- CH3

CH3

m/e 91

H H

CH2 CH2 H CHR H CH2 CHR

H H
+

+
H H

CH2 H H

H H

70

71

72

73

74

1. Furans Furan is an example of a 6-electron heteroaromatic system. Its stability is evidenced by an intense molecular ion in the mass spectrum accounting for 25% of the total ion current. Theoretical considerations indicate that the most energetically favored bondcleavage in the furan molecular ion is that of a carbon-oxygen bond, and it results in the ring-opened molecular ion 1a, which may then undergo electronic rearrangement to 1b. Homolytic cleavage of the C 4 - C 5 bond in 1b results in elimination is the base peak in the mass spectrum and is best formulated as the cyclopropenyl ion (1c), a stable 2 -electron aromatic system. Heterolytic cleavage of the C4-C5 bond in 1b would result in elimination of the cyclopropenyl radical and formation of the formyl ion 1d.

39 68 (M+) 29 40

42

75

4 ( 5 O 1

3 2 )+ O + (1a) - CO H + H ( m/z 40 H H + ) -H ( O (1b)

+ )

C3H3

HC O

m/z 29 (1d) -CHO +

M+ m/z 68

m/z 39 (base peak ) (1c)

In 2-methylfuran cleavage of the O-C2 or the O-C5 bond may occur, resulting in two different ring-opened molecular ions (2a and 2b, respectively). These fragments by the progresses described for furan, giving the intense cyclopropenyl and methylcyclopropenyl ions as well as a weaker acetyl ion.

( O (2b)

+ ) CH3 - C3H3 + H3C C O

( O (15.9% ) (2)

+ ) CH3

( O (2a)

+ ) - CHO CH3

CH3

m/z 53 (21.6% ) (base peak)

- C2H3O

m/z 39 (20% )

m/z 43 (4.4% )

76

With larger 2-substituents ring fragmentation with resultant formation of cyclopropenyl or acyl ions is unimportant, and B-fission becomes the dominant fragmentation process.

O + CH2 CH2

- C2H5 CH3 + O m/z 81 (43.1 % )

m/z 110 (11.9% )

Cleavage to the furan ring with loss of the alkyl group is insignificant as it leads to an unfavored vinyl or diradical ion.

O +

or

77

If the 2-site-chain is n-propyl or longer, a McLafferty rearrangement can occur. Thus with 2-n-butyl- and 2-n-pentylfuran the loss of propene and butene, respectively, results in m/z 82 as the most intense ion in both spectra.
- CH2= CHR

O + H

CH2 CH R CH2

+ O H

CH2

+ O H

CH2

n-propylfuran m/z 82 3.4% m/z 81 43.1%

n-butylfuran 10.6% 46.1%

n-pentylfuran 11.9% 41%

With 2-n-propenylfuran loss of H is favored relative to ring-opening since it gives the fully conjugated oxonium ion. Loss of CO occurs as the second step, forming the intense benzonium ion which further loses a molecule of hydrogen to give the phenyl ion.
-H O + M
+

CH CH CH2 H m/z 108

O +

CH CH CH2 m/z 107 (3.5% )

- CO

(16.7% )

+ H H m/z 79 (15.1% )

- H2

C6H5 + m/z 77 (8.1 % )

78

In the mass spectrum of 2-(1-pentenyl)furan, a character-impact compound of reversion flavor of soybean oil, the base ion observed at m/z 107 may be produced by the loss of CO from the parent ion with recyclization to form the cyclopentadiene radical ion which further loses a hydrogen atom forming the stable cyclopentadienyl ion (m/z 107). Alternatively, loss of CHO from the parent ion also leads to the cyclopentadienyl ion. The metastable ion observed at m/z 84.2 confirms that the m/z 107 ion is the daughter ion of m/z 136. The fragmentation mechanism for the observation of metastable peaks at 65 and 58.3 confirms the following transitions:

and

136+ + 107

94+ + CH3 + 79 + CH2

CH CH2

CH2

cis-2-(1-pentenyl)furan

107

94 81 77 39 50

135

m/z

79

H -CO

CH2CH2CH3

m/z 108

CH CHCH2CH2CH3 m/z 136 -CHO - H2C CHCH3 H CH CH2 H H

-H

CH2CH2CH3

m/z 107

O m/z 94 136

94

+ CH3CH=CH2

80

O + m/z 81 -CH CCH2CH3 - CH=CHCH2CH3

O +

CHCH=CHCH2CH3 -H -CO

CH=CHCH2CH2CH3 m/z 136

+ H CH2CH3 m/z 107 - CH2=CH2

+ H H

- H2

C6H5

m/z 79

m/z 77

107

79+ +CH2=CH2

81

The mass spectra of 2-furanaldehydes are characterized by an abundant parent ion and an abundant M-1 ion, the resonance-stabilized furoyl cation. This further fragments by loss of two molecules of carbon monoxide, forming a cyclopropenyl ion.

( O M
+

) CHO

_H O _ CO C + O m/z 95 (21.2% ) O + C O

, m/z 96 (21.8% )

+ _ CO O m/z 67 (1.6% ) + m/z 39 (27.6% )

An intense furoyl ion is also observed in the spectra of 2-furyl alkyl ketones. If the side chain is n-butyryl or longer, the McLafferty rearrangement involving the carbonyl group becomes an important process. Thus, it gives the base peak of the spectrum of 2-n-valerylfuran, competing favorably with formation of the furoyl ion.

- C4 H9 O C + O O

C
+

_C H 3 6 CH2 CH2 CH3 O C

+O

CH2

O H CH

82

2. Pyrroles N- and C- alkylated pyrroles show marked differences in fragmentation. The mass spectrum of 1-methylpyrrole is shown below.
N CH3 80 81 (M +. )

39 53 42 55 78

m/z

It is noted that the chief feature of the spectrum is the strong M-1 ion which may be the ring-expanded species.
_C H 3 3 N m/e 42 M
+

CH3

CH

-H N+ CH2 m/e 80 _C H N 2 4 (strong peak) _HCN C4H5 m/e 39

+

CH3

N+ H

m/e 81 CH3 N

CH

m/e 53

83

The fragmentations of certain long-chain N-alkylpyrroles have been studied in some detail by means of labeling and high-resolution techniques. The best peak (m/z 81) of the mass spectrum of N-butylpyrrole was initially thought to result from transfer of the terminal methyl group to nitrogen.

_C H N H2C CH2 or CH3 CH2

3 6

CH3

N H2C

H CH2

CH2 CH2 H2C

N H

84

In the mass spectra of C-alkylpyrroles, the -cleavage is the predominant fragmentation.

_H N H M m/e 81
+

- CH3 + N H m/e 80 base peak CH2

+

CH2 H

N H M

+ N H
+

CH2 CH3

m/e 80

m/e 95

The spectra of 2-formyl and 2-acetylpyrroles show the expected fragmentation with the intense acylium cation being presumably well-stabilized by resonance.

-R N H M
+ +

C O

N H

N H

m/e 94

85

3. Thiophenes The mass spectra of 2- and 3- alkylthiophenes have been studied, and in all cases the base peak is the ion C5H5S+, m/z 97, resulting from fission of the bond in the alkyl group between the carbon atoms in position and B relative to the ring.

CH2 S

_R S + CH2

m/e 97

CH2 or S S
+

Thiopyrilium ion m/e 97

86

The close resemblance to the fragmentation of toluene is immediately apparent, and the thiopyrilium ion has been suggested for the species m/z 97. For disubstituted thiophenes, the stability of the neutral fragment controls the major mode of fragmentation.

_ S
+

H H3C M
+

_C H 2 5 S
+

CH2 CH2 CH3

H3C

S
+

m/e 139 (10%)

m/e 140

m/e 111 (100%)

_ CH S
+

_C H 2 5 S S + m/e 125 (100%)

m/e 139 (10%)

87

4. Pyridines In pyridine and methyl derivatives molecular ions are the base peaks as expected for aromatic rings. Mass spectra of the methylpyridine isomers show three important primary processes arising from the molecular ions.
+ M

(i) (ii) (iii)

_H _ CH
3

m/z 92 m/z 78 m/z 66

M
+ M

_ HCN

The cleavage processes of pyridines substituted with higher alkyl groups can be classified in three categories. (1) -Cleavage in ethyl derivatives is easier in the 3 position than in other positions. This is attributed to the relatively high electron density at this position. Thus the resulting fragment is the base peak in 3-ethylpyridine.
+

CH2CH3 ( N )
+

_CH

CH2

m/z 92

m/z 65 +

_ HCN

+ N

88

These fragments undergo further elimination of hydrogen cyanide leading to the peak at m/z 65.
(2) -cleavage is especially favored in 2-alkylpyridines. The relative intensity of the

resulting fragment ion depends on the nature of the radical lost.

( N

_ R N
+

CH2 CH2 R

CH2 CH2

(3) The McLafferty rearrangement takes place when the adjacent position to the

heteroatom bears a side-chain with at least three carbon atoms.

- C4H8 N + CH2 CH2 CH2 CH2 CH3 N H m/z 93 (100%) base peak
+

CH2

89

5. Pyrazines The mass spectrum of parent pyrazine is dominated by the loss of HCN molecules. The fragmentation of 2-methylpyrazine involves losses of HCN and CH3CN from the molecular ion.

CH CH N + m/z 53 _ HCN ( HC + HC CH ) +

_ CH CN 3 a

N N +

CH3 _ HCN b HC N +

CH3

m/z 67 _ HCN

H2C

CH
+

_H

H3C
H + H

CH )+
H ( H )+

CH

m/z 26

m/z 39

m/z 40

90

Pyrazines which possess an n-propyl or longer side chain (containing -hydrogen) undergo McLafferty rearrangement. In general, this gives the base peak for most pyrazines containing long side chain. The fragmentation of 2-n-pentyl-5,6dimethylpyrazine is shown below.
CH2 CH3

N N m
+ 178

N N H

HC H2 C

N N H CH2

m/z 122 (100%) - C3H7

- C2H5

- CH3

N + N m/z 135 (50%)

N + N m/z 149 (36%)

N + N m/z 163 (10%)

91

4.

MANUFACTURE OF FOOD FLAVOR

I. Natural or Imitation Flavor 1) Price 2) Availability of raw material 3) Permissibility under current legislation (toxicity test) 4) Type of end product in which the flavoring is to be used II. Problems of Using Natural Flavors 1) Many natural flavor have low intensity, it should be used at a high dosage which results in an unsatisfactory texture and poor stability. 2) Concentration of natural flavors is usually accompanied by significant changes in the flavor profile. 3) Natural flavors exhibit variations in strength and quality. 4) The supply of natural materials is becoming uncertain. 5) Most natural flavors are unstable and undergo changes during postharvest handling, processing or storage. 6) Many natural products contain enzyme systems which may result in the formation of off-notes. 7) The toxicity of many natural products has yet to be established. III. Disadvantages of Using Imitation Flavors 1) Original natural flavor more subtle imitation flavor maybe described as chemical 2) Difficulties in labeling 3) Many natural flavors have a built in reservoir of flavor precursors which can result in the generation of additional flavor imitation flavors are not. 4) Imitation flavor generally require the use of either a solvent or a carrier 5) Restriction by legislation 6) Problems with texture in the end product

92

IV. Advantage of Imitation Flavor 1) Cheaper than natural flavor 2) Stable 3) Can be design to withstand severe processing condition 4) Can be produced in a variety of forms ( e.g., alcohol-based, oil-based, or encapsulated powders ) 5) Generally readily available 6) Consistency of quality

93

V. Methods in Synthetic Flavor Reconstitution 1) Scientific Approach Isolation of flavor concentrate

Separation of components

Identification Quantitative GC analysis

Synthesis

Scientifically reconstituted formulation (correct until GC identical ) Organoleptically adjusted formulation

Process and product development 1) Application 2) Physical formulation 3) Synthetic process development Manufacture and end use in consumer product

94

Limitations a. Some compounds decompose or do not come out of GC b. Wide variety of flavor threshold (Some compounds can not be identified. 2) Organoleptic Approach Example Smell-taste analysis of food or flavor concentrate Blue cheese

Resolution into subjective arbitrary quality components

Buttery, fatty, moldy

1 buttery, 5 fatty, 3 moldy Assigning of rough intensity value to each quality component Diacetyl, methyl nonyl ketone, methyl amyl ketone

Association of quality components with known flavor

Formulation of reconstituted flavor

0.3% diacetyl 5% methyl nonyl ketone 1% methyl amyl ketone

Same steps as in scientific reconstitution

Limitations a. labeling b. toxicity c. no precursor d. an artistic craft rather than science

95

5.

CHEMISTRY OF FLAVOR PRECURSORS

I. Flavor derived from carbohydrate and proteins (Browning Reaction, Maillard Reaction) Reducing Sugars and -amino acids N-glycosylamine or N-fructosylamine 1-Amino-1-deoxy-2-ketose (Amadori intermediate) or 2-Amino-2-deoxy-1-aldose (Heynes intermediate) Reductones and dehydroreductones H2S NH3 Amino acids Strecker degradation

Retroaldol condensation

Furans Thiophenes Pyrroles

Hydroxyacetone Hydroxyacetylaldehyde Acetoin Acetylaldehyde

Glyoxal Pyruvaldehyde Glycerolaldehyde

Aldehydes + -aminoketone (Methional, NH3, H2S)

Heterocyclizaion Pyrazines Pyridines Oxazoles Thiazoles Pyrroles

96

1. Maillard Reaction

CH2OH C O CHOH R + H2NR

CH2OH OH C NHR CHOH R

H2O

CO H H C NHR CHOH R

2-AMINO-2-DEOXY-1-ALDOSE

HEYNES REARRANGEMENT

97

NHR H2C C O CHOH CHOH R 1-AMINO-1-DEOXY-2-KETOSE Ketoenolization

NHR H2C CHOH C OH C OH R 2,3-ENEDIOL - NH2R

CH3 C O C O CHOH R DEHYDROREDUCTONE Ketoenolization

C H3 C O C OH C OH R REDUCTONE

TRANSFORAMTION OF AMADORI INTERMEDIATE TO FORM REDUCTONES AND DEHYDROREDUCTONES

98

CH3 C O C O CHOH CHOH CH2OH DEHYDROREDUCTONE FROM AMADORI _ H2O

CH3 C O C O CH COH CH2OH KETO FROM

CH3 C O C O CH2 C O CH2O H

CH3 C O ENOL C O CH2 C OH CHOH

CH3 C O C O CH2 HC OH HC O 1,4 DIDEOXYHEXOSONE

1,4 DIDEOXYHEXONE FROM AMADORI PRODUCT

99

HC O HC O C O CH2 CHOH CHOH CH2OH 3-DEOXYHEXOSONE C O CH3 PYRUVIC ALDEHYDE + CHO CHOH CH2OH GLYCERALDEHYDE _HO 2 CHO C O CH3 PYRUVIC ALDEHYDE CH2OH + H2O C O CH2OH DIHYDROXYACETONE

RETRO-ALDOL CONDENSATION

CH3 CH3 C O C O CH2 CHOH COH 1, 4 DIDEOXYHEXOSONE CO CO CH3 DIACETYL + CHO CHO GLYOXAL

RETRO-ALDOL CONDENSATION OF DEOXYHEXOSONES

100

ALDOL

101

CHO C O CH CH CHOH CH2OH DEHYDROREDUCTONE FROM HEXOSE CHO C O CH CH CH2OH DEHYDROREDUCTONE FROM PENTOSE FURFURAL O OH CHO _ HO 2 O CHO 5-HYDROXYMETHYLFURFURAL H2COH O OH CHO _ HO 2 H2COH O CHO

HYDROXYMETHYLFURAL AND FURFURAL FORMATION

102

CH3 C O COH COH CH2OH REDUCTONE FROM PENTOSE O

3 5 2

CH3 Ketonization C O CHOH C O CH2OH CYCLIZATION

3 4

O H O

OH OH CH3

- H2O

OH HO2HC
2

OH

OH COOH

CH3

OH

5-METHYL-4-HYDROXY-3-(2H)-FURANONE (NOR-FURANEOL)

5-KETOGLUCONIC ACID

FORMATION OF 5-METHYL-4-HYDROXY-3(2H)-FURANONE

103

N R'

CHO

Formyl Pyrrol H H H

C C C C H C R

O O R'NH 2 H - H2O H OH

C C C C H C R

O O H H NHR'

Basic Condition

C C C C H C R

O O H H N R

- H2O R CHO

N R'

104

1 2 3 4 5 6

CH3 C O COH COH CHOH CH3

CH3 C O CHOH C O CHOH CH3 ISOMALTOL CH3

5 2

OH - H2O OH CH3 CH3

OH

CH3

REDUCTONE (RHAMNOSE)

2,5-DIMETHYL-4-HYDROXY-3(2H)-FURANONE

CH3 CH2 C

C O HO KETOBUTYRIC ACID ALDOL

+

CH3 CH C

O - H2O

CH3 HOOC CH3

HOOC C C O CH3 OH HO

O O

HOOC C O CH3 CH3 O CH3

OH

CH3 O

CH3 O

A MAPLE LACTONE

105

2. Strecker Degradation Mechanism

O O H3C C C H pyruvaldehyde

H O H2N C C R H amino acid

O H O H3C C C N C C H R OH

Keto form of Schiff's base

OH O H3C C C N C C H R OH

Enol form of Schiff's base

OH H H3C C C N C H R

CO2

eneaminol 1) self condensation 2) condensation with other eneaminols 3) hydrolyze to amino acetone + aldehyde or ketone

106

R2 C O C O R3 DICARBONYL AMINO ACID + H2N CH COOH R1

R2 HO C R3 HN CH COOH R1 C O

R2 R2
-H2O

H N
+

C R3

C H CO O H R1
-

CO2

-C

CH R1

C O R3

C O

SCHIFF BASE (IMINE) R2 H C N CH C O R1 R3 + H2O R1 CHO

+

R2 H C NH2 C O R3 AMINOCARBONYL

STRECKER DEGRADATION

107

O O CH3 C C CH3 CH3

O O C C CH2CH3

DIACETYL

2,3- PENTANEDIONE CH3 O O OH HO HO L DEOXYHEXOSONE (FROM AMADORI)

CH CH2OH O OH

DEHYDROASCORBIC ACID

DICARBONYL COMPOUNDS IN FOODS FOR STRECKER DEGRADATION

108

2) Methionine:

H3C

CH2 CH2 CH NH2

COOH

H3C

CH2 CH2 CHO

H3C

SH

+ CH2

CH

CHO

2 H3C

CH3

H3C

CH3

H3C

CH3

H3C

CH3

109

COOH NH2

O O R C C R'

S H2O

CHO

CH3SH

HOH2C CH2

CHO

Strecker aldehyde

METHYLMERCAPTAN

METHIONINE BREAKDOWN

110

NH2 HS O O R C C R' CO O H

HS CH2

CHO

H O NH2 R C C R' ENAMINOL

H2S + CH3CHO + O C R

C NH R'

Mercapto Acetaldehyde

H2S FORMATION FROM CYSTEINE

111

3. Pyrazines formation Cocoa, coffee, French fry etc. roasted beef. pathway 1: sugar + amino acid

C H2C

O NH2 +

H2N O

C C

CH2OH H

- H 2O

N N

CH2OH

N N

CH3 H OH -

N N

CH2

pathway 2:
H H C C O O NH3 HO H C C NH2 + O H2N O HC CH2

,dicarbonyl

Cyclization
H N N H

N N

-3H2O

HO HO

OH H

112

4.

Oxazole formation Trimethyl-oxazoline in beef stew N 2,4,5-trimethyl oxazole O

Possible mechanism for the formation of trimethyloxazole from diacetyl, CH3CHO, NH3.

H3C C C CH3 O O

+ H2O

OH H3C C C CH3 O O H

OH H3C C C CH3 O H

+
O H3C CH + NH3 NH H3C C H

-H2O

H3C C

- H2O
H3C H3C O

-H2O
H3C C C CH3 N CH3 O

H3C C

Possible mechanism for the formation of trimethyl-oxazoline

113

5.

Thiazole formation Trimethyl thiazole (less nutty, sulfur )

H3C H3C S N CH3 ( identified in potato, beef, coffee, tea, cocoa bean ) OH H3C C C CH3 S O H
OH H3C C C CH3 SH N

+ H3C C C CH3 H2S O O

+
O H3C CH + NH3 NH H3C C H

- H2O

H3C C

- H2O
H3C H3C S

-H2O
H3C C C CH3 N CH3 S

H3C C

114

II. Thermal Degradation of Vitamin B1 1. Basic condition

H2N Cl+ N HO S N N - OH+ H2O HO S N + CH3 N no odor H2N N

has found some use in the flavor industry ( identified in coffee aroma with meaty note )

2. Acidic condition
H 2N H 2N N N N HO S H+ N H N H HO S CHO

H+

+ H2O

+ H2O

O HO (C H 2 ) 2 C H C C H3 SH + HCOOH + H 2N

H 2N N N

coffee
no odor

115

3. Thiazole compounds
N HO S -H2O N S Formed in cocoa reduction N S ( cocoa, beef )

methyl-vinyl-thiazole

methyl, ethyl-thiazole

4. Furan compounds

O H 3C C CH CH 2 C H 2 OH SH

-SH + H+

O H 3C C CH 2 CH 2 CH 2 OH

cyclization

Reduction

OH O CH3

-H2O

-H2

( coffee, tea ) H 3C O

116

Cyclization

Cyclization

117

III. Lipid Oxidation 1. Chemistry of triplet oxygen

Molecular Atomic
* 2Px 2Py 2Pz *

Atomic
* 2Pz 2Py 2Px

E
2S

* 2S

* 1S 1S

Molecular Orbital of Triplet Oxygen

118

2. General Mechanisms of Autoxidation

14

13

12

11

10

C H3

(C H2)3 CH2 C H CH CH2 CH CH

INITIATION (METAL) 12 11

C H2 R

- H
10 9

CH3

(CH2)4 CH C H CH CH C H + O2
12 11 10 9

CH2 R

(C H2)4 CH C H CH C H CH O O PROPAGATION + H
12 11 10

C H3

CH2 R

(CH2)4 CH C H C H CH CH HYDROPEROXIDE O DECOMPOSION O - OH H

12 11 10 9

C H3

C H2

C H3

(C H2)4 CH C H CH C H CH O

CH2 R

O CH3 (C H2)3 C H2
TERMINATION

+ H

C C H CH CH CH CH2 R + H

C H3

(C H2)3 C H3 (PENTANE)

119

Mechanisms of Oxidation

1. Initiation RH R + O2 R + H

ROO

2. Propagation RO O + R1H 3. Termination R

R OO H

R1

+ R

RR ROOR ROR ROOR 2 ROOR + O2 + O2

R OO + RO O RO + R ROO + R 2 RO + 2R OO

120

Oxidation of Mono-Olefines Oleic acid - 4 Hydroperoxides Double bond shifts to

11

10

11

10

C C C C C C O O H

C C C C C C O O H

12

11

10

12

11

10

C C C C C C O O H

11

C C C C C C O O H

10

121

Hydroperoxides from Linolenate

16

15

14

13

12

11

10

C C C C C C C C O O H

16

15

14

13

12

11

10

C C C C C C C C O O H

12

16

15

14

13

12

11

10

C C C C C C C C O O H

13

16

15

14

13

12

11

10

C C C C C C C C O O H

16

122

Peroxide Decomposition General H R C R1 O O H H C O

R1

OH

+ R' R R'H + O C R1 + R' H

O R + R1
or

H1

O R C H +

R1

H C OH

R1

R '

R' + OH Effects of Metal on Peroxide Decomposition Cu+ + C u++ + RO OH RO + O H+ +

ROH

Cu++ C u+ H+ + O HH2O

ROOH 2RO OH

R O O + H+ RO +

R O O +

123

H H H H H H C C C C C C C R H H H

H H H H H H C C C C C C C R H H O O B A H O C C H2 C O C C H3 H H C H2 O H C H2 C H C H R O

O H

H H H H C C C C C R

H H H C C C C R H H

124

Ethyl vinyl ketone isolated and identified in raw soybean

O C CH

H 3C C H

CH

( raw beany, grasoy )

CH CH C O O H COOH

H 3 C C H 2C H

CHCH2

H 3 C C H 2C H

CH CH2

H 3C C H 2 C H C H

CH2

O 2 , RH H 3C C H 2 C H C H O O H CH2

H 3C C H 2 C O

CH

CH2

125

Lactones in butter flavor

Important lactones in butter are decalactone dodecalactone tetradecalactone 5-20 ppm The lactones have coconut-like flavor which is desirable in molten butter, undesirable in fresh butter and dry whole milk.

fresh butter heated butter

content of lactones is low lactone increases

126

Lactones come from hydroxy acids in milk

H 2C HC H 2C

O O O

O C O C O C

R R' (C H 2 ) 3 C H ( C H 2 ) n C H 3 OH 100~150 o C

O C

HO

( C H 2 ) 3 C H (C H 2 ) n C H 3 OH
- H2O

O O

CH2

CH2

CH2 CH (C H 2)n C H 3

R CH (CH2)2 O

COOH CH C O

carboxyl-()lactone

odorless, well crystalized at 80~120 oC, decarboxylated to lactone

127

3.Chemistry of singlet oxygen

Molecular Atomic
* 2Px 2Py 2Pz *

Atomic
* 2Pz 2Py 2Px

* 2S 2S

* 1S 1S

Molecular Orbital of Singlet Oxygen

128

Excitation and Deactivation of Photosensitizer

Excited state

Sen* k = 1- 20 108 /sec ISC

k = 2 10 /sec
8

h k = 10 - 104 /sec
1

Sen*

+ O2 k = 1 - 3 109 /sec

Ground state

Sen Singlet oxygen formation

h Sen
1

ISC Sen*

Sen*

+ RH R + Sen H + 3O2 ROOH O2- + Sen+ K2 + 3O2

K1
1

+ 3O2 O2 + RH

ROOH

Photooxidation process (RH : Substrate ; K1 = 1 - 3 109 /M sec. ; K2 < 107 /M sec)

129

(11) RCOO + RCOO (10) ENDOPEROXIDES

PRODUCT S

(12) ENZYMES

(1) 3 O2 + SENSITIZER

RC O + RCOH

SENSITIZER H2O2 + OCl

(2)

H2O + Cl1

(9) OZONIDES

PRODUCT S

O2
OH- + OH

(3) H2O2 + O2-

H2O + OH(8) H2O2 + HO22H+ O2- + O2(7) H2O2 Y eO2(5) (4) OH OH

+ O2

O2- + Y+ (6)

Production of 1O2 by Photochemical, Chemical, and Biological Systems

130

O + O Endoperoxide

1,4- Cycloaddition:

O +

O O

ENE Reaction :

Hydroperoxide Allyl

CH2 + CH2

1,2 Cycloaddition:
O

O O Dioxetane

Reactions of Singlet Oxygen with Various Types of Double Bonds

R H O I

O R'

O O II

R' H

131

Conjugated and Nonconjugated Hydroperoxides Arising via the 6-Centered Transition state

OOH R R' + R

O OH

R'

h/sensitizer/ O

R'

isomerization OOH R R'

132

Reversion Flavor

CH3 CH2

O CH CH CH2 C H CH CH2 CH CH CH2 (CH2)6 COH

15 14 13 12 11 10 9 8
1

O2
11 10 9 8

15

14

13

12

CH3 CH2 CH CH CH2 CH CH CH2 CH CH CH (CH2)6 COH O O H CH3 CH2 O CH CH CH2 CH CH CH2 CH CH CH (CH2)6 COH O
15 14 13 12 11 10 9 8

CH3 CH2 CH CH CH2 CH CH CH2 CH O

1

O2

CH3 CH2 CH CH CH2 CH CH CH2 CH O O O H CH3 CH2 CH C H CH2 CH CH CH2 CH O O

CH3 C H2 CH CH CH2 CH CH CH2 CH O O

4-Keto-6-nonenal Formation from Linolenic Acid

133

Mechanism for 4-Keto-5-nonenal Formation from 4-Keto-6-nonenal

CH3 CH2 CH CH C H2 C CH2 CH2 CH O O - H CH3 CH2 CHCH CH C CH2 CH2 C H O O

+ H C H3 CH2 CH2 CH CH C CH CH2 CH 2 O O

4-keto-5-nonenal

134

Formation of 2-(1-Pentenyl)furan from 4-Keto-5-nonenal and 2-(2Pentenyl)furan from 4-Keto-6-nonenal

C H3 (C H2) 2 C H CH C CH2 C H 2 C H O O

C H3 C H2 C H C H C H2 C C H 2 C H2 C H O O

C H3 (C H2)2 C H CH C C H C H C H OH OH
- H2O

C H3 C H2 C H C H C H2 C C H OH
- H2O

CH C H OH

C H3 (C H2 )2 C H C H

C H3 CH2 C H C H C H2 2-(2-pentenyl)-furan

2-(1-pentenyl)-furan

135

CH3

(CH2)4

Formation of 2- Pentyl furan CH CH CH2 CH C H CH2 (C H2)6 COO H

1

O2 (CH2)6 CO O H

CH3

(C H2)4 CH CH CH2 C H CH CH O OH CH3

(C H2)4 CH C H CH2 CHO

1

O2

CH3

(C H2)4 CH C H CH2 C HO O O (C H2)4 CH C H CH2 C HO O O + 2RH (CH2)4 CH C H2 CH2 CHO + 2R O OH

CH3

CH3

CH3

(CH2)4 CH CH2 CH2 C HO O

CH3

(C H2)4 C CH2 CH2 C HO O (CH2)4 C CH CH CH O O H H H2O C H CH (C H2)4 C O

136

CH3

CH3

CH

4. Enzymatic lipid oxidation (Lipoxygenase)

Detrimental effects. a. Destruction of the essential fatty acids. b. The free radicals produced damage other compounds including vitamins and proteins. c. Development of off-flavor and odor in beans and peas ! a hay-like flavor.

Specificity of Lipoxygenase - a cis, cis-Penta-1,4-Diene Unit (-CH=CH-CH2-CH=CH-) - Methylene group of the Penta-1,4-Diene Unit to be in the -8 position. Mechanism of Action 1. The enzyme forms a stereospecific complex with the unsaturated fatty acid. 2. The enzyme abstracts either an electron or a hydrogen atom stereospecifically from the -8 position producing a free radical at -8 of the fatty acid. 3. While still attached to the enzyme, the fatty acid free radical isomerizes to place the unshared electron at -8 causing conjugation and isomerization of the double bond. 4. O2 reacts with the free radical at -6 to give a peroxy free radical. 5. A hydrogen from the medium forms the hydroperoxide which then dissociates from the enzyme.

137

H C CH 3 (CH 2)4 C

H H C H

H C C

H H C H

H C cis C

(C H )

26

COOH

-H

H C CH3 (CH2)4

cis

H C H C

H C cis C

H H C H

H C cis C

H (CH2)6

COOH

H H CH3 (CH2)4 H C trans C C C H + 3O2 H H CH3 (CH2)4 C O O tr ans C C C H cis C H cis C

H H C H

H C cis C

(CH2)6

CO OH

H H C H C cis C

H (CH2)6

COOH

.
H H trans C C C H cis C H H C H H C cis C (CH2)6 COOH H C O OH

CH3

(CH2)4

138

Linolenic Acid C14 O2 + Lipoxygenase

H trans cis cis C C C C C C C C C C OOH O (C)7 C OH

O
C C C C C C H cis-3-hexenal

O H+ C C C C C C H trans-2-hexenal AOR

AOR* O C H C C C C C C OH H cis - 3 - hexenol + C C C C H C C OH H n - hexanol O C C C C C C H AOR AOR C C C C C H C C C C C H C OH H

n-hexanal

H C C C C C C OH H n - hexanol
C

trans - 2 - hexenol + H C C C C C OH H n-hexanol

n - hexanal H C (C)3 C C O cis C C (C)7 C OH

trans C C

OOH *AOR: Alcohol Oxidoreductase O2 + Lipoxygenase

Linoleic Acid C14

Aldehyde and Alcohol Formation in Tomato from Linolenic and Linoleic Acid.

139

IV.

Flavor Generated from Enzymatic Method, and Microbiological Reaction, and Biognesis

1. Free fatty acids by lipase Optimum temperature. 15~40 oC lipids

lipase

free fatty acid

2. Generation of diacetyl in butter lactose

S. lactis

lactic acid

diacetyl ( creamy flavor, 1ppm )

3. Fresh banana flavor fresh banana

processing lost flavor

processed banana

banana peel processed banana

extraction
flavorase 3~4 hrs

enzyme ( flavorase ) fresh banana flavor

If pyruvate, acetate, amino acid, unsaturated fatty acid are added, then the time for flavor production will be shortened to 30 min.

140

Lactose S. Lactis Lactic acid CH3 H COH COOH [H] Oxalacetic acid COOH C O CH2 COOH -CO 2 Citric acid COOH -acetate CH2 HO C COOH CH2 COOH OH OH H3C C C CH3 H H 2.3.-butylane glycol ( odorless )

[O]

Pyruvic acid CH3 C O COOH O CH3 H3C C C OH COOH Acetyl lactic acid -CO 2

[H] in absence of oxygen O OH H3C C C CH3 H Acetoin [o] in presence of oxygen

-CO 2 Acetaldehyde CH3 C O H

O O H3C C C CH3 Diacetyl

141

4. Onion and Garlic Flavor Enzymatic reaction of cysteine derivative

HS CH2CH COOH NH2 O R S CH2CH COOH NH2 Allin R: CH3 CH3 -CH =CH - ( propenyl alliin CH2=CH-CH2 - ( allyl alliin ) H 2O H R S O sulfenic acid when R: CH3-CH=CHH3C CH H CH S O onion more prodominate) gallic

Alliinase O + H3C C COOH pyruvic acid

NH3

H3C CH2CH S O thiopropenal oxide lachrymator in onion

1 min

propenyl cysteine sulfoxide + onion enzyme after reacted for 1 hr. m/e = 90 became weak m/e = 58 m/e = 98 appears m/e = 58 propanal m/e = 98 2-methyl-2-pentenal m/e = 90 disappears after 2 hrs.
142

product (m/e = 90) (this is thiopropenal oxide )

H3C CH2CH S O

H3C CH CH SHO -S H3C CH CH OH

( m/e = 90 )

( m/e = 58 )

H3C CH2CH O

( m/e = 58 )

Aldol condensation

H3C C CHO H3C CH2 CH O R S H sulfenic acid

( m/e = 98 )

O R S S R H3C CH

thiosulfinate ( responsible for fresh flavor of onion and garlic ) CH CH3 fresh onion odor

O CH S S CH

H2C

O CH CH2 S S CH2 CH

CH2

fresh pleasant garlic-like odor

143

O R S S R
Fresh Flavor

(Thiosulfinate)

O R S S R O
Aged Flavor

(Thiosulfonate)

R S

O O

Bitter Flavor (Off Flavor)

H2C

CHCH2 S S CH2CH O S NH

CH2

typical garlic-like odor isolated and identified

H2C H3C HC

CH2 CH COOH

- H 2O O HC CHCH2 S CH2CH NH2 OH COOH

cycloalliin ( no favor contribution ) +H2O O CH S CH CH NH2 COOH

H3C CH

144

5. Biogenesis of Flavor Compounds in Tomato Important volatile flavor compounds in tomato 3-cis-hexenol green note isovalervaldehyde hexanol contribute green or grassy odor hexanal 2-trans-hexenal 2-cis-hexenal 2-isobutylthiazole --- strong green leaf odor 3-methyl-1-butanol 1) amino acid precursors COOH H2N CH CH2 H3C CH CH3 L-leucine COOH C O CO2 CH2 H3C CH CH3 CHO CH2 H3C CH CH3
NADH + H+ [ADH] NAD+

CH2OH CH2 H3C CH CH3

2-Keto-4-methylpentanoic acid

3-methyl-butanal

3-methyl-1butanol

[ADH] - alcohol: NAD + oxidoreductase alcohol dehydrogenase add to use l-[ C] leucine add to boiled extract of tomato --- no reaction indicates the enzymatic nature of this reaction
14

crude extract of fresh tomato --- get 14C label 3-methyl-1-butanol

145

2) Fatty acid precursors

linolenic acid CH3

16 15 13 12 10 9

O OH

CH2 CH CH CH2 CH CH CH2 CH CH (CH2)7 C cis cis cis O2 + lipoxygenase O

CH3

CH2 CH CH CH2 CH OOH

16

15

14

13

C CH CH CH (CH2)7 C trans cis

12

11

10

OH

13-hydroperoxide

O CH3 CH2 CH CH CH2 3-cis-hexenal C H

146

O CH3 AOR CH3 CH2 CH CH CH2 3-cis-hexenal CH2OH CH3 CH2 CH CH CH2 hexenal CH2 CH CH CH2 3-cis-hexenal C H CH3 CH2 CH2 CH CH 2-trans-hexenal AOR H CH3 CH2 CH2 CH CH trans-2-hexenol

O C H

O C

CH2OH

CH3

CH2 CH CH CH2 n-hexenal

CH2OH

AOR: alcohol oxidoreductase

147

6. Asparagusic Acid in Asparagus asparagusic acid: 1,2-dithiolane-4-carboxylic acid

S S

COOH

asparagusic acid , its methyl and ethyl esters and several other sulfur compounds were synthesized in the intact plant cells of asparagus. This is an exceptional case of formation of sulfur-containing flavor components. Sulfur compounds in vegetables are normally formed by enzymic or chemical cleavage of nonvolatile precursors such as S-alkylcysteine sulfoxides and glucosinolates during the crushing of the plant material.

NH2 COOH valine

O COOH COOH 2-methyl propanoic acid CH3

S COOH COOH CH3 C O SH CH3 C S O SH SH

COOH

SH

SH

COOH

COOH

COOH

COOH

COOH

148

7. Mushroom Volatiles Edible mushroom like Agaricus Bisporus produce 1-octen-3-ol, 3-octanol, 2-octen-1ol and 1-octen-3one as volatile constituents.1-octen-3-ol possesses a mushroom-like aroma and is known as mushroom alcohol. Tressel et al. investigated the enzymic conversion of linoleic and liolenic acids into C8 and C10 components by mushrooms. They proposed the presence of ahydroperoxide cleavage enzyme for the cleavage of 13- and 9-hydroperoxide into C8 and C10 components. Following figure shows the scheme proposed by Tressl for the formation of mushroom volatiles.

149

150

8. Flavor formation by Neurospora Production of Fruity Aroma by Various Strains of Neurospora

Neurospora Species
Neurospora sitophila ATTC46892 Neurospora No 1 Neurospora No. 2 Neurospora No. 3 Neurospora No 4 Neurospora No 5 Neurospora No 6 Neurospora No 7 Neurospora tetrasperma NRRA2164 Neurospora crassa NRRA 2223 Neurospora sitophila NRRA 2884 Neurospora intermedia NRRA 5506

Aroma
Fruity Fruity Fruity Fruity Fruity Fruity Fruity Fruity No aroma No aroma No aroma No aroma

Neurospora sitophila ATTC46892, Neurospora No.1,2,3,4,5,6, and 7 were isolated from beiju. Tweenty strains of Neurospora sp.isolated from the state of Sao Paulo did not produce fruity aroma

151

152

Volatile Compounds (ppm) produced by Neurospora sp. Isolated from beiju Ethyl Acetate 4.8 9.0 0.9 2.8 Ethanol 128 111 111 99 3-Methyl1-butanol 318 ND 117 208 Ethyl hexanoate 59 ND 10 20 1-Octen3-ol 40 ND 50 ND

Neurospora sitophila Neurospora Sp. 1 Neurospora Sp. 5 Neurospora Sp. 6

ND: Not detected

153

6. DAIRY PRODUCTS FLAVOR

1. Milk Flavor 1. Oxidized flavor Cardboard: due to some lactones Metallic: vinyl methyl ketone Oily: oct-1-ene-3-one Tallowy: 2t, 6t-nonadienal Preventive method a. Avoid cupric iron and ferric ion b. Elimination of oxygen pack under vacuum or nitrogen c. Avoid light Better quality milk, less bacteria, more susceptible to oxidized flavor. The bacteria can either using up the available oxygen or generate antioxidant compounds. 2. Rancid flavor Hydrolysis of triglycerides by lipase. The lipase are present in the aqueous phase of the milk at the time of secretion. Any process which alter the membrane, such as homogenization, agitation, and warming and cooling will accelerate the rancidity. 3. Heated flavor 1) General Pasteurization induces heated flavor. Now people are used to Pasteurization and consider it as the flavor of normal milk. Cooked flavor is the off-flavor induced by temp. above 75 oC beyond the pasteurization. Too much heat will develop caramelized flavor.

154

2) Origin a. Cooked flavor: protein b. caramelized flavor H2S

CH2 CHO

from phenylalanine

CH2 CH COOH NH2 O H3C C COOH pyruvic acid

( Strecker degradation )

CH2

CHO + H3C CH COOH NH2

155

4. Microbiological flavor 1) Ggeneral Molds, yeast, bacteria can all grow in milk and effect flavor. 2) Origin a. Psychrophilic bacteria : Bitter, fruity, stale, putrid flavor b. Moldy flavor H3C H3C CH CH2CH COOH NH2 leucine 2H2 S. latics var. maltigens

H3C H3C

CH CH2CH2 CHO

+ NH3 + H 2O

threshold 0.5 ppm

156

5. Absorbed flavor Feed flavor Weed flavor Barney flavor 1) Nose or mouth 2) Digestive tract lung blood blood udder cell udder cell milk milk

157

6. Sunlight flavor Sunlight will induce oxidized flavor and sunlight flavor and hay-like flavor. Oxidized flavor Sunlight flavor: burnt cabbage Burnt and cabbage flavor: Riboflavin is a catalyst for production of the sunlight flavor. 1) milk protein and riboflavin sunlight sunlight flavor 2) riboflavin increase in milk will increase the sunlight flavor 3) riboflavin removal prevent the sunlight flavor

158

Fig. 1. Effect of time of exposure to fluorescent light on headspace volatile compounds and dimethyl disulfide of skim milk. Peak A,B,C,D and E are 2butanene, ehtanol, diacethyl, dimethyl disulfide, and n-butanol, respectively

Fig.2 Mass Spectrum of peak D (top) of Fig.1 and standard dimethyl disulfide (bottom)

159

Postulated mechanism of dimethyl disulfide formation by singlet oxygen oxidation of methionine

Effect of ascorbic acid concentration on dimethyl disulfide (Peak D) content in skim milk during light exposure for 1 hour.

160

II.

Cheese Flavor

1. Isolation, separation, and identification of cheese flavor

Dynamic headspace analyzer, gas chromatographer, and mass spectrometer arrangement

161

Reproducibility of gas chromatograms of headspace volatile compounds of Brewster Cheddar cheese after one week of storage

162

163

164

Changes of total headspace volatile compounds of Cheddar cheese at 11C, and Swiss cheeses at 21 C during ripening

165

2. Biochemical pathways of fats in cheese flavor formation

Fats

Amides Aldehydes Primary Alcohols Methyl Ketones Secondary Alcohols Easters Lactones

Fatty Acids

166

3. Reaction products of methionine CH3SCH2CH2CH(NH2)COOH Methionine CH3SSCH3 Dimethyl disulphide

[O]

CH3SCH2CH2CHO Methional
H2O H2O

CH3SH + Methanethiol

CH2CHCHO Acrolein

+ CH3SCH2CH3 Ethylmethyl sulphide

HCOOH Formic Acid

+ CH2CH2 CH3SH Methanethiol Ethylene

+ HCOOH Formic Acid

167

4. Biochemical pathways of cheese flavor formation from protein Products = Caseins (+trace of whey) Amines a-keto acids Acids Phenols H2S NH3

Peptides

Alcohols

Amino Acids

168

5. Formation of 2-butanone and 2-butanol from diacetyl CH3COCOCH3 Diacetyl CH3CHOHCOCH3 Acetoin

[H2]

CH3COCH2CH3 2-Butanone
[H2]

CH3COH=COHCH3 2,3-Butyleneglycol

CH3CHOHCH2CH3 2-Butanol

6. Biochemical pathways of cheese flavor formation from lactose Lactose Lactic Acid Diacetyl Pyruvic Acid Acetaldehyde Acetic Acid CO2 Ethanol

169

7. Lactone formation
O H2C O C O HC O C O H2C O C (CH2)3 CH OH (CH2)4 CH3 R1 R

+ H2O DG O HO C (CH2)3 CH OH (CH2)4 CH3

- H2O O C (CH2)3 O CH (CH2)4 CH3

170

8. Mechanism of Methylketone Formation

O H2C O C O HC O C O H2C O C (CH2)3 CH OH (CH2)4 CH3 R1 R

+ H2O DG O HO C (CH2)3 CH OH (CH2)4 CH3

- CO2 O H3C C (CH2)n CH3

171

7. MEAT FLAVOR CHEMISTRY

I. Effects of Psychrotropic Bacteria on the Volatile Compounds of Raw Beef
1. Introduction 1) Meat palatability a. Volatile flavor compounds b. Appearance c. Juiciness d. Tenderness 2) Factors affecting flavor or raw beef a. Breed, Sex, Diet, Age b. Fat, Microorganisms

Sample preparation for isolation and separation of volatile compounds Ground beef: 5 g ground beef was transferred into 30 ml serum bottle and sealed air tightly. Analysis of volatile compounds a. Dynamic headspace sampler (DHS) b. Capillary-Gas chromatography (GC)

172

2. Effects of light and dark storage on the volatile compounds of asceptic raw ground beef 1) Storage condition a. Aseptic ground beef stored under light at 5oC b. Aseptic ground beef stored under dark at 5oC 2) Evaluations a. Dynamic headspace sample/gas chromatography b. TBA c. Panel Evaluation for off-odor 3. Effects of psychrotropic bacteria on the volatile compounds of aseptic raw ground beef 1) Samples a. Aseptic ground beef b. Aseptic ground beef + Pseudomonas putrifaciens c. Aseptic ground beef + Acinetobacter spp. 2) Evaluations a. b. c. d. Dynamic headspace sample/gas chromatography/mass selective TBA value Total bacteria count Panel evaluation for off-odor

173

3) Identification of volatile compounds of aseptic raw beef by DHS/GC/MSD Condition of Mass Selective Detector Column Carrier gas Ion source temp. Ionization voltage Mass scan range Scan rate DB-5, 30m symbol 180 \f "Symbol" \s 12} 0.25mm, 1.0symbol 109 \f "Symbol" \s 12m film thickness Helium gas (99.999%) at 1 ml/min 170oC 70eV 25-250 a.m.u. 1.0 scan/sec

174

Diagram of Dynamic Headspace Sampler/Gas Chromatograph

175

176

177

Chromatogram of 0 day storage

Chromatogram of 8 day under the dark storage

Chromatogram of 8 day under the light storage

178

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Total ion chromatogram of volatile compounds of (a) aseptic ground beef, (b) aseptic ground beef with Pseudomonas putrifaciens or (c) Acinetobacter spp.
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II. Isolation, Separation, and Identification of Roast Beef Flavor

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III. Simulated Meat Flavor Compounds Formation

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8.

ORANGE FLAVOR STUDY BY PULSED ELECTRIC FIELD

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9. INTERACTIONS OF FLAVOR COMPOUND WITH FOODS

I. Physical and Chemical Stability of Flavor Compounds 1. Mechanisms of flavor perception 1) Flavor compounds interact with olfactory and lingual receptors 2) Volatile compounds are generally responsible for odor perception and nonvolatile compounds for taste. 2. Concentration of flavor compounds in the receptors 1) 2) 3) 4) 5) 6) The rates of flavor compounds release from foods. The concentration and disposition of flavor compounds in the food. The components of the food. The particle size of food components. The extend of mixing. The temperature of foods.

3. Factors affecting partition and release of flavor compounds in the mouth 1) 2) 3) 4) 5) Hydration Dispersion Reduction of Particle Size Homogenization Emulsification

4. Rate of volatilization 1) The partition coefficient of flavor compounds. 2) Molecular interaction between flavor compounds and food components. 3) The viscosity of food material.

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5. Physical and chemical states of flavor compounds in foods Flavor compounds may be dissolved, adsorbed, absorbed, or entrapped in food components depending upon functional groups, molecular size, shape and volatility, and chemical properties of the components in the food. 6. Importance of binding behavior of flavor compounds Knowledge of the binding behavior of flavor compounds to food components is: 1) Important in the flavor perception and the determination of relative retention of flavor compounds during processing, storage and mastication. 2) Critical in a. the determination of appropriate flavor blend added to food b. the choice of methods for dispersing flavor compounds c. the selection of appropriate flavor compounds carriers d. the determination of improved conditions for efficient drying of flavored foods e. the minimization of flavor compounds loss. 3) Important in the determination of how to maximize flavor impact and minimize cost. 7. Effects of selective binding on flavor perception The selective binding of one flavor compound of a blend to food components or packaging material can markedly alter the overall flavor impact. Binding limits its volatilization and diffusion and hence impairs its immediate perception as a components of an overall flavor when food is taken into the mouth. 8. Factors affecting partition coefficients 1) 2) 3) 4) Temperature The presence of soluble solutes and nonsoluble materials Diffusion rates in the aqueous phase Physical retention of flavor compound

Air-Water Partition Coefficients for Homologous Series of Ketones and Aldehydes at 25oC

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Compounds Acetone Butan-2-one Pentan-2-one Heptan-2-one Octan-2-one Nonan-2-one Undecan-2-one

Coefficients 1.6x10-3 1.9x10-3 2.6x10-3 5.9x10-3 7.7x10-3 15x10-3 26x10-3

Compounds Acetaldehyde Propanol Butanal Pentanal Hexanal Heptanal Octanal Nonanal

Coefficients 2.7x10-3 3.0x10-3 4.7x10-3 6.0x10-3 8.7x10-3 11x10-3 21x10-3 30x10-3

Types of Possible Interactions between Flavor Compounds and Food Components. Component Lipids; Possible Interaction -solution -dispersion -adsorption -entrapment Carbohydrates; -adsorption -entrapment -complexation Proteins; -specific binding -adsorption -absorption -entrapment

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II. Effects and Interactions of Lipids with Flavor Compounds 1) Increase flavor compounds adsorption and retention 2) Decrease the partition coefficients 3) Increase the flavor threshold concentration Effects of Physical Phase on Perception of Flavor Compounds Compounds Octanoic acid -decalactone Pentanal Hexanal 2,4-Decadienal Threshold Concentration (ppm) Water 5.8 0.05 0.07 0.03 0.5x10-3

Oil 350 3.0 0.3 0.05 0.3

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III. Effects and Interactions of Carbohydrates with Flavor Compounds 1. Soluble sugars increase the vapor pressures of volatile compounds. 2. Polysaccharides stabilize flavor compounds in foods during processing due to entrapment, adsorption, reduced mass transport effects due to increased viscosity. 3. Cellulose adsorbs flavor compounds in intramolecular region. 4. Amylose forms inclusion complexes with aliphatic flavor compounds which fit inside the amylose helix. 5. The association constants with starch were 383, 930 and 2277 for limonene, methanol and decanal, respectively.

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Adsorption and Desorption of Volatile Compounds to Polysaccharides (mol/kg) Polysaccharide Ethyl Acetate Ethanol Butylamine A B A B A B Cellulose 0.1 trace 2.2 0.2 11 0.3 Pectin 0.2 0.1 2.1 trace 46 4.0 Starch 0.2 0.1 4.5 1.0 27 2.2 A maximum adsorption; B vacuum desorption (Maier, 1975)

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IV. Effects and Interactions of Proteins with Flavor Compounds 1. The binding capacity of protein depends upon the surface topography, porosity, and bulk density. 2. Proteins bind aldehydes and ketones to differing extents, indicating differences in intrinsic binding affinities, structural features of the protein, differences in available surface area. 3. The Mechanisms of Flavor Compounds Interaction with Protein 1) Scatchard equation v/[L] = nK-vK v is the number of moles of flavor compounds bound per mole of protein. L is the molar concentration of flavor compounds. n is the total number of binding sites. K is the intrinsic binding constant. Plot of v/L vs. v gives a slope of -K and intercept on nK.

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2) Klotz equation 1/v = 1/n+1/nK[L] A plot of 1/v vs. 1/[L] Intercept = 1/n Slope = 1/nK 3) Determinations of Thermodynamic Parameters G = -RT ln K H = -R(dln/d(1/T)) S = -R(Ho-Go)/T

Binding and Thermodynamic Data for the Interactions of Carbonyl Compounds with Soy Protein, b-Lactoglobulin and Bovine Serum Albumin Compounds 2-Heptanone 2-Octanone 2-Nonanone 2-Heptanone 2-Octanone 2-Nonanone 2-Heptanone 2-Nonanone Protein Soy Protein Soy Protein Soy Protein -Lactoglobulin -Lactoglobulin -Lactoglobulin Serum Albumin Serum Albumin n 4 4 4 1 1 1 6 6 Keq/M 110 310 930 152 481 2439 600 1800 -G(Cal/M) 2.78 3.39 4.04 2.98 3.66 4.62 --4.90

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Binding and Thermodynamic Data for the Interactions of Carbonyl Compounds with soybean Protein, b-lactoglobulin an d Bovine Serum Albumin Ligand Soy Protein 2-Heptanone 2-Octanone 2-Nonanone 2-Nonanone 2-Nonanone 2-Nonanone -lactoglobulin 2-Heptanone 2-Octanone 2-Nonanone Bovine Serum Albumin 2-Heptanone 2-Nonanone Protein Native Native Succinylated Native (25C) Native (5C) Heated (90C) Native Native Native n 4 4 2 4 2 4 1 1 1 Keq/M 110 310 850 930 2000 1240 152 481 2439 -G(Kcal/M) 2.781 3.395 3.992 4.045 4.221 4.215 2.980 3.660 4.620

Native Native

6 6

500 1800

--4.900

Effects of Temperature and Modification on the Binding and Thermodynamic Data for Interactions of Carbonyl Compounds with Soy Protein Compounds 2-Heptanone 2-Octanone 2-Nonanone 2-Nonanone Temperature 5C 25C 90C Succinylated-25C n 4 4 4 2 Keq/M 2000 930 1240 850 -G(Cal/M) 4.22 4.06 4.21 3.99

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360 Fluorescence (nm) ( )

2500 2000

350

K,M-1 ( )

1500 1000 500

340

330 0 2 4 6 Urea Concentration (M)

Effects of urea induced conformational change s reflected in fluorescence on the binding affinity of 2-nonanone for b-lactoglobulin

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Adsorption and Desorption of 2-Pentanone onto Whey Protein Adsorption P/Pv Rel. Mass Gain (Flavor) (x103) 0.000 0.000 0.085 1.273 0.131 1.367 0.216 1.599 0.307 2.438 0.431 5.199 0.490 6.103 0.611 9.985 0.752 12.19 0.876 13.51 Desorption P/Pv Rel. Mass Gain (Flavor) (x103) 0.0876 13.51 0.739 12.80 0.575 12.40 0.490 12.12 0.432 11.81 0.307 10.50 0.167 9.131 0.072 6.830 0.000 3.000

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Summary 1. Several mechanisms are involved in interaction of flavor compounds with food components. 2. In lipid system, solubilization and rates of partitioning control the interactions and partition coefficients, thus determine-s the rates of release. 3. In polysaccharide system, polysaccharides interact with flavor compounds by nonspecific adsorption and formation of inclusion compounds. 4. In protein system, protein involves adsorption, specific binding, entrapment, covalent binding and these mechanisms may account for the retention of flavor compounds. 5. Moisture affects diffusion and partition coefficients and macromolecular structures in the case of protein and polysaccharides and thereby affect the rate of release of flavor compound.

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10. PACKAGING AND FLAVOR COMPOUNDS INTERACTION

I. II. Effects of Packaging Materials on the Flavor Quality of Food Sorption of Orange Flavor Compounds by Packaging Materials

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11. FLAVOR COMPOUNDS AND SOLVENT INTERACTION

I. II. Commercial Cherry Flavor and Solvent Interaction Acetal Formation

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