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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 [email protected]
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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) 40% Final Examination 30% Home Work and Class Participation 30%
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1. INTRODUCTION
I. Definition of Flavor II. Classification of Food Flavor III. Scope of Flavor Chemistry
1. Chemical compounds responsible for food flavor 2. Flavor of foods 3. Reconstitution of flavor compounds 4. Precursors of the flavor compounds 5. Mechanism for the formation of flavor compounds and precursors in
foods 6. Relationship between physical properties and its flavor
IV. Objectives of Flavor Chemistry
2. ISOLATION AND SEPARATION OF FLAVOR COMPOUNDS
I. Objective II. Prerequisites III. Apparatus for Isolation
1. Headspace analysis 2. Continuous solvent extraction 3. Steam distillation and continuous solvent extraction
IV. Extraction and Concentration V. Preliminary and Final Fractionation VI. Dynamic Headspace analyzer VII. Solid Phase Microextraction Analysis
3. FLAVOR IDENTIFICATION BY SPECTROMETRIC METHODS
I. Introduction of Spectrometric Analyses II. Ultra Violet Spectrometry III. Infrared Spectrometry IV. Nuclear Magnetic Resonance Spectrometry
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V. Mass Spectrometry 1. Furans 2. Pyrroles 3. Thiophenes 4. Pyridines 5. Pyrazines
4. MANUFACTURE OF FOOD FLAVOR
I. Natural or Imitation Flavor II. Problems of Using Natural Flavor III. Disadvantages of Using Imitation Flavor IV. Advantages of Imitation Flavor V. Methods in Synthetic Flavor Reconstitution
5. CHEMISTRY OF FLAVOR PRECURSORS I. Flavor Compounds from Carbohydrates and Proteins
1. Maillard reaction 2. Strecker degradation 3. Pyrazine formation 4. Oxazole formation 5. Thiazole formation
II. Thermal Degradation of Vitamin B1
1. Basic condition 2. Acidic condition 3. Thiazole compounds 4. Furan compounds
III. Lipid Oxidation
1. Chemistry of triplet oxygen 2. General mechanisms of autoxidation 3. Chemistry of singlet oxygen 4. Enzymatic lipid oxidation (Lipoxygenase)
IV. Flavor Generated from Enzymatic Method, Microbiological Reaction,
and Biogenesis
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1. Free fatty acids by lipase 2. Generation of diacetyl in butter 3. Fresh banana flavor 4. Onion and garlic flavor
5. Tomato flavor 6. Asparagusic acid in Asparagus 7. Mushroom volatiles 8. Flavor formation by Neurospora
6. DAIRY PRODUCTS FLAVOR CHEMISTRY
I. Milk Flavor 1. Oxidized flavor 2. Rancid flavor 3. Heated flavor 4. Microbiological flavor
5. Absorbed flavor 6. Sunlight flavor
II. Cheese Flavor
1. Isolation, separation and identification of cheese flavor 2. Biological pathways of fat in cheese flavor 3. Reaction products of methionine 4. Biochemical pathways of cheese flavor formation from protein 5. 2-Butanone and 2-Butanol formation from diacetyl and acetone 6. Biochemical pathways of cheese flavor formation from lactose
7. Lactone formation 8. Mechanisms of methyl ketone formation
7. MEAT FLAVOR CHEMISTRY
I. Effect of Psychrotropic Bacteria on the Volatile Compounds of Raw Beef
1. Introduction 2. Effects of light and dark storage on the volatile compounds of asceptic
raw ground beef 3. Effects of psychrotropic bacteria on the volatile compounds of aseptic
raw ground beef
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II. Isolation, Separation, and Identification of Roast Beef Flavor III. Simulated Meat Flavor Formation
8. ORANGE FLAVOR STUDY BY PULSED ELECTRIC FIELD PROCESS
9. INTERACTION OF FLAVOR COMPOUNDS WITH
FOODS
I. Physical and Chemical Stability of Flavor II. Effects and Interactions of Lipids with Flavor Compounds III. Effects and Interactions of Carbohydrates with Flavor Compounds IV. Effects and Interactions of Proteins with Flavor Compounds
10. PACKAGING AND FLAVOR COMPOUNDS
INTERACTION
I. Effects of Packaging Materials on the Flavor Quality of Food II. Sorption of Orange Flavor Compounds by Packaging Materials
11. FAVOR COMPOUNDS AND SOLVENT INTERACTION
I. Commercial Cherry Flavor and Solvent Interaction II. Acetal Formation
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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.
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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.
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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.
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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 appearance
taste flavor
odor food
hearing
touch texture
kinesthesis
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II. Classification of Food Flavors
Flavor Class Subdivision Representative Example Fruit flavor citrus-type flavors (terpeny) grapefruit, orange berry-type flavors (non-terpeny) apple, raspberry, banana Vegetable flavors lettuce, celery Spice flavors aromatic cinnamon, peppermint lachrymogenic onion, garlic hot pepper, ginger Beverage flavors unfermented flavors juices, milk fermented flavors wine, beer, tea compounded flavors soft drinks Meat flavors mammal flavors lean beef sea food flavors fish, clams
Fat flavors olive oil, coconut fat, pork fat, butter fat
Cooked flavors broth beef bouillon vegetable legume, potatoes fruit marmalade Processed flavors smoky flavors ham broiled, fried flavors processed meat products
roasted, toasted, baked flavors coffee, snack foods, processed cereals
Stench flavors cheese
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.
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Almond: benzoaldehyde
C H O
Green pepper: 2-methoxy-3-isobutyl-pyrazine
N
N
OCH3
CH2CHCH3
CH3
Both pyrazin and thiazol are important flavor compound groups
N
S1
2
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5
N
N
pyrazine thiazol
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Vanilla: 4-hydroxy-3-methoxy-benzolaldehyde
CHO
OHOCH3
Cucumber: 2-trans-6-cis-nonadienal
CH 3 CH2 C CH H
CH2 CH2C C CHOH
H
Reversion flavor of soybean oil: 2-pentylfuran and 2-pentenylfuran
O (CH2)4 CH3
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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 2-pentylfuran 1) Non-enzymatic reaction Precursor of beef flavor can be isolated as a white fluffy powder. White fluffy powder Oil Water broil stew beef broth Amino acid + Sugar Maillard reaction
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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 S
S CH3H3C4
3
1 2
5
3, 5-dimethyl-1,2,4-trithiolane
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Model studies:
CH3CHO + H 2SSS
S
H2S + CH3CHO CH3 CH S CH CH3
SH SH
( S )
( O )
S S
S CH3H3C
HS C C COOHNH2
are precursorsCH3CHO, H2S Therefore,
B e e f f la v o r ( r e a c t io n f l a v o r )
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.
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6. Relationship between physical properties of a compound and its flavor B.P.(0C) 760 mm-Hg Solubility in H2O
g/100 ml Sense of smell (ppm)
n-propanol 61.0 20 0.17 n-butanol 75.7 4 0.07 n-hexanal 131.0 0.5 0.03 acetone 56.0 20 500 2-butanone 79.6 3.7 50 CH3-S-CH3 37.5 insoluble 0.012 Threshold (ppm)
odor 2-t-pentenal 2.3 2-t-hexa(e)nal 10.0 2-t-hepta(e)nal 14.0 2-t-octenal 7.0 2-t-nonenal 3.2 2-t-decenal 33.8 2-t-undecenal 150.0
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 ) corn oil system
( peak area ) acetone 10 47
2-butanone 14 11 2-pentanone 22 5.7 2-hexanone 29 2.7 2-heptanone 24 0.7
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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.
CH 3 C
CH3
CH CH2 CH2 C CH CH2
CH3
OH
Ceylon tea contains cis-hexenol, India tea doesn’t contain cis-hexenol
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2. ISOLATION AND SEPARATION OF FLAVOR COMPOUNDS
I. Objectives Produce volatile flavor compounds of the true flavor of the original with minimum artifact. 1. Selection of “Good flavor sample” 2. Isolation of Volatile Flavor Compounds (VFC) 3. Extraction and Concentration 4. Fractionation 5. Preparation of pure compound 6. Identification 7. Synthesis 8. Reconstitution of the flavor II. Prerequisites 1. Selection of sample 2. No alternation of the original flavor 3. No artifacts due to : decomposition autooxidation
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III. Apparatus for Isolation 1. Headspace analysis 1) Without enrichment
can
siliconerubberstopper
syringe
2) With Enrichment
Using inert gas
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Apparatus for the isolation of trace volatile constituents from relatively large amount of food.
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2. Continuous Solvent Extraction
Continuous Liquid-liquid extractor for use with solvents lighter-than-water
Beverage sample
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3. Steam Distillation and Continuous Solvent Extraction
Modified Likens-Nickerson simultaneous steam distillation-solvent extraction distillation assembly
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IV. Extraction & Concentration 1. Extraction Simple Extraction solvent used: diethyl ether, pentene, freone, … etc.. salting out.
ether
NaCl
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2. Concentration Oldershaw column
ether
thermometer
ether
Concentrated to 50~100 ml
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Kuderna-Danish assembly for the evaporation of solvent from flavor extracts
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V. Preliminary and Final Fractionation 1. Preliminary fractionation Acid, Neutral and Basic compounds
Total flavor isolate in ether (200 ml )
H2O layer Et2O layer Acidified with 10% HCl Ext. with ether aq. Layer ether layer ether layer ( basic compounds ) ( neutral compound ) Dried with + 10% NaOH Dried Anhy. Na2SO4 Ext. with ether with Filter anhy. Na2SO4
acidic compounds basic compounds neutral compound concentration G.C.
10% Na2CO3
+ 10% HCl Extraction
Earthy, nutty aroma Meaty flavor
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2. Final fractionation Gas-Liquid Chromatography
Sample: as concentrate as possible
GC-Mass:
Use capillary column
Identification of the important peaks by mass spectrometry
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Comparison of GC separation of oak leaves extract achieved using standard film thickness and thick film fused silica glass capillary column
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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 )
Precision (% rsd )
SPME
Static Headspace
Dynamic Headspace
0.05-0.3
1- 2
0.003-0.005
1-3
1-3
1-8
Direct Injection 17-240 2-13
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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 compoundconcentration, lowest level contamination andshortest sample preparation time
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Solid Phase Microextraction
Equilibrium partitioning of the compoundsbetween the coating fiber and sample orheadspace.
A technique that uses a short, thin, solid rodof fused silica, coated with absorbent polymerfor extraction of volatile compounds
Diagram of SPME Extraction
Direct sampling SPME Headspace SPME
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Principles of Headspace SPME
KfhVfVsCo
KfhVf+KhsVh+Vs
nf=
nf: Number of compounds in solid phase
K : Partition coefficient
Kfh=
Vf,Vs,Vh: Volume of solid phase,
solution, and headspace, respectively
Co: Initial concentration of compoundsin the solution
Concentration of coatingConcentration of headspace
Plunger
Barrel
Gauge
Water bath
Solid Phase
SPME Analysis of Volatile Compounds
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Types of Solid Phases
• CB/PDMS:Carboxen/Polydimethylsiloxane
• PDMS: Polydimethylsiloxane
• CW/DVB: Carbowax/Divinylbenzene
• PA: Polyacrylate.
Effects of Different Solid Phases on theHexanal Analysis in Soybean Oil
Mean CV (%)
CB/PDMS 499 4.2PA 739 7.2PDMS 966 3.2CW/DVB 1,520 2.9 (10.7)
CV: Coefficient Variation (%) for n =5Significant difference (P<0.05)
Hexanal Peak in Electronic Count
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SPME Reproducibility of Major Flavor Compounds inOrange Juice
ReplicatesEthyl butyrate
(ppm)α-Pinene(ppm)
Octanal(ppm)
Limonene(ppm)
Decanal(ppm)
1 0.432 1.378 1.089 251.05 1.0052 0.400 1.391 1.050 254.28 0.925
3 0.391 1.343 1.054 248.26 0.987
4 0.380 1.389 1.059 256.25 0.995
5 0.403 1.402 1.020 255.71 1.015
6 0.397 1.470 1.010 260.01 1.007
SD 0.017 0.042 0.029 4.130 0.033
CV(%) 4.36 3.00 2.71 1.63 3.32
ave 0.400 1.395 1.047 254.26 1.989
Effect of G.C. Injection Temperature on SoybeanOil Volatile Compound Analysis
230 °C
250 °C
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Effect of Coating Thickness on the Absorptionfor the Extraction of 0.1 ppm Benzene
0
20
40
60
80
100
0 200 400 600
Time (S)
Mas
s (n
g)100 µm
56 µm
15 µm
0
510
15
2025
30
0 1000 2000 3000time (S)
Mas
s (n
g)
Effect of Distribution Constant on theAbsorption Profile of 0.1 ppm Analyte
Kfs= 831 (p-Xylene)
Kfs= 294 ( Toluene)
Kfs= 125 ( Benzene)
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Effect on Sample Temperature on the GC Chromatogramof Compounds
Extracted at 25 °C
Extracted at 130 °C
Extracted at 200 °C
Effect of Water and Microwave Heating on thechromatograms of Headspace Polyaromatic Compounds
1, naphthalene: 2, acenaphthylene: 3, acenaphthalene: 4, fluorene: 5,anthracene
0
20
40
60
80
100
1 2 3 4 5
Compound Number
Mas
s Ex
trac
ted
(ng) Water Heating
Microwave Heating
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Effect of Stirring Rate on the Extraction of1 ppm Benzene in Water
0
10
20
30
40
0 200 400 600Time (S)
Mas
s (n
g) 400 rpm
0 rpm
2,500 rpm
Effect of Agitation Method on the Extractionof 1 ppm Benzene in Water
0
10
20
30
40
0 200 400 600Time (S)
Mas
s (n
g)
No stirring
Sonication,
Magnetic Stirring
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Effect of Benzene Concentration onExtraction by SPME
0.1
1
10
100
1000
0 100 200 300 400 500 600Time (S)
Mas
s (n
g)
Cs = 0.1 ppm
Cs = 10 ppm
Cs = 1 ppm
Benzene Dioxane
Norm
aliz
ed F
ID R
espo
nse
No SaltSodium ChlorideSodium SulfatePotassium Carbonate
Effect of Salts on the Extraction of VolatileCompounds by SPME
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Matrix Effect on the Extraction ofAlcohols by SPME
Cltronellol Geranlol
Det
ecto
r Res
pons
e
Waterwater-salt12% Ethanol12% Ethanol-salt
Gas Chromatogram of Orange Juice Flavorby SPME Headspace Sampling
41
Regression Equations between Flavor Compounds(ppm) and GC Peak Areas
Compounds
Ethyl butyrate
α-Pinene
n-Octanal
Limonene
Decanal
Regression Eq R2Concentrationrange (ppm)
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
0.99
1.00
0.99
0.99
0.99
0.1-1.2
0.1-1.3
0.1-1.1
0.2-2.0
20-50
Y: Compound part per million, X:Electronic counts of GC peak area
Effects of Temperature and Time on the Equilibriumof Flavor Compounds Between the SPME Coating
and the Headspace of Orange Juice
0
5
10
15
20
25
30
0 10 20 30 40 50 60
Adsorption Time (minutes)
FID
res
pons
e
80°C
60°C
50°C
40°C
25°C
42
Isolation Time Effect on Soybean OilVolatile Compounds by SPME
40
Isolation Time (min)
Rel
ativ
e P
eak
Size
0
10
20
30
0 30 60 90 120 150
60
45
35
°C
°C
°C
Isolation Temperature Effect on SoybeanOil Volatile Compounds by SPME
Isolation Temperature (C)
Rel
ativ
e P
eak
Size
0
5
10
15
20
25
30
35 45 60
PV 1
PV 50
43
Chromatograms of Volatile Compounds ofSoybean Oil by SPME
Volatile Compounds in the Headspace of SoybeanOil by SPME-GC-MS
Pentane 1.38 3.65Pentanal 2.06 5.31Hexanal 3.84 23.52-Butanone 3.97 9.09Heptanal 5.90 2.702-Heptenal 6.45 4.762-Pentylfuran 8.40 4.772,4-Heptadienal 10.99 5.04t-2-Octenal 11.53 3.37Nonanal 14.00 2.86t-2-Nonenal 14.29 0.552-Decenal 18.69 34.3
Compounds Retention Time (min) Relative (%)
44
Effect of Isolation Temperature on Corn OilVolatile Compounds by SPME
25°C 45°C
60°C35°C
Volatile Compounds in the Headspace ofCorn Oil by SPME-GC-MS
Pentane 1.29 13.03Pentanal 1.88 5.52Hexanal 3.62 5.39Heptanal 5.36 1.832-Heptenal 6.21 29.522-Pentylfuran 8.59 2.532,4-Heptadienal 10.88 7.69t-2-Octenal 11.51 18.07Nonanal 13.88 6.27t-2-Nonenal 14.23 1.332-Decenal 18.61 4.93t,t-2,4-Decadienal 20.20 1.17t,c-2,4-Decadienal 20.70 2.71
Compounds Retention Time (min) Relative (%)
45
Chromatograms of Soybean Oil and Corn Oil
Soybean Oil
Corn Oil
Improving Sensitivity of Solid PhaseMicroextraction
• 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
46
Conclusion
• Reproducible
• Economic
• Simple
• Sensitive
The SPME-GC is a
for the analysis of volatile compounds inmost foods.
47
3. FLAVOR IDENTIFICATION BY SPECTROMETRIC
METHODS I. Introduction of Spectrometric Analyses II. Ultra Violet Spectrometry III. Infrared Spectrometry IV. Nuclear Magnetic Resonance Spectrometry V. Mass Spectrometry
48
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
X- ray U.V. Visible I.R. Microwave V' = Wave number (cm -1) λ = Wave length (nm) C = Velocity of Radiation (constant) 3× 1010 cm/sec V = Frequency of Radiation (cycles/sec) V' = = (The energy of photon) E = Vh (Planck's Constant 6.62× 10-27 erg - sec) E = Vh = h
C = V λ V =
200 400 800
C
V
λ
1
λ
C
λ
C
Wavelength (nm)
Wave number (cycles/cm)
The Electromagnetic Spectrum.
γ- ra
y χ-
ray
ul
travi
olet
visi
ble
viol
et
bl
ue
gr
een
yello
w
400 500
1020 1018 1016 1
Wavelength, λ,
49
in
frar
ed
m
icro
wav
e
radi
o
or
ange
014 1012 10 8 6 104 102
m
frequency, ν, (cycles/sec)10 10 10
ed
visible region
10-10 10-8 10-6 10-4 10-2 1 102 104 106
8
r
700 800
600 Wavelength, λ, n
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 σ, π, 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.
Ener
gy
* Antibonding
σ* * * * Antibonding
π*n
σ →
σ
π→
π
n →
σ
n →
πNonbonding Bonding
π
Bonding
σ
51
Electronic Molecular Energy Levels The higher energy transitions (σ →σ*) occur a shorter wavelength and the low energy transitions (π→π*, n →π*) occur at longer wavelength.
Energy
σ* σ*
hv
h
σ
π2
hv
π3
π1
π*
nπ
hvv
σ
π3
π2
π1
Ground Electronic Stateπ→π*
n →π*Exited Electronic State
52
53
Chromophore is a functional group which absorbs a characteristic ultraviolet or visible region. UV
210 nm Double Bonds 233 nm Conjugated Diene 268 nm Conjugated Triene 315 nm Conjugated Tetraene
• • • •
σ and σ* orbitals π and π* orbitals
54
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
C C
O
O HH
H
H
Stretch
Unsymmetrical bend
Symmetrical bend
55
Atom, Group, and Molecular Rotations
IR
3.4 µm Alkane 6.0 µm cis-Double Bond 10.3 µm trans-Double Bond 5.8 µm Carbonyl 3.7 µm Hydroxyl Stretching of Acid Group 2.9 µm Hydroxyl
C C
O
O HH
H
H
X
YZ
OH group rotation
H atom rotation COOH group rotation
CH3 group rotation Molecular rotation
Center of gravity of the molecule is at the origin
IV. Nuclear Magnetic Resonance Spectrometry
Spinning charge in proton generates magnetic dipole
Proton precessing in a magnetic field Ho
Om
Ho
Precessional orbit
Nuclear magnetic dipole µ
Spining proton
Oscillator
axis of nuclear rotation
Low energy precession Nuclear Spin
Nuclear magnetic dipole µ
Rotation component of
56
Precession -Energy Rscillator generates rotating component of
agnetic field H1
Ho
Coil Re
High energy precession
Precessional orbit Low energy spin state (-1/2)
ference axis
Precessional orbit High energy spin state
elationship
57
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 Atomic Number Spin Number
Odd Even or odd 1/2, 3/2, 5/2,---- Even Even 0 Even Odd 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 -µ 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.
Fundamental NMR Equation of Radio Frequency and Magnetic Field Strength The energy difference between the two states is
V =
γ : (Magnetogyric Ratio) : CV : Electromagnetic frequencHo : An external magnetic fieWo = γHo γHo = 2πV Therefore Wo = 2πV γ = 2πµ / hI µ = Magnetic Moment (Magnh = Planck's Constant I = Spin Number
Relationship between Radio Frequen
Radio Frequency (Mega Hertz) 60 100 300 500
1.4 T 60 MHz
2.35 T 100 MHz
4. 20
2π
γHo
58
onstant and a fundamental nuclear constant. y in radio frequency ld
etic Dipole Moment)
cy and Magnetic Field Strength for Proton
Magnetic Field (Gauss) 14,100 23,500 70,500 117,500
7 T
0 MHz
∆E = hv
7.0 T 300 MHz
59
Schematic Diagram of an NMR Spectrometer
Chemical Shift The difference in the absorption frequency of a particular proton of the samp
absorption frequency (position) of a reference proton.
The protons at the electron rich environments (strong electonegaticve molecu
oxygen and halogens) will feel less external magnetic field strength because
strength generated by electrons surrounding the proton will counteract the ap
field strength (Ho), which can be said deshielded proton.
Therefor, the Wo of the protons in the electron rich chemical environments w
require less radio frequency to be resonance with the applied radio frequency
protons in the electron poor chemical environments.
δ ppm = (reference frequency - sample frequency) × 106
R-F ° ° transmitter
Sweep ° °generator
Magnet
Transmitter coil Receiver coil
Sweep coils
Sample
Operating instrument frequency
° ° R-F receiver
and
le from the
les such as
the magnetic field
plied magnetic
ill be less and
compared to the
° ° Recorder
The Reference Compounds : TetraMethylSilane (TMS)
General Regions of Chemical Shifts
56 10 7 8 9
Aldehydic
Aromatic and heteroaromatic
Olefin
α-Disu
Acetylenic
β-Substituted aliphatic
c
S i C
C
C
C H H
H
H H
H
H
H H
H H
H
α-Monosubstituted aliphatic
60
3 4 2
ic
bstitutid aliphatic
Aliphatic alicycli
0 δ 1
61
Rest of the protons on CH3 and CH2 absorb at 0.8 - 2
broad, big peak
Spin-Spin Coupling (
Spin-Spin Coupling is the indirect coupling of proto
It occurs because there is some tendency for a bondi
nearest protons. The spin of a bonding electron havi
Coupling is ordinarily not important beyond 3 bonds
bridged systems, or bond delocalizaion as in aromati
•
R C H C H C H 2 C H C H C H C
O
O C H
• • •2 3
5.3 δ 2.7 δ.0 δ very crowde
Spin-Spin Splitting)
n spins through the inter
ng electron to pair its sp
ng been thus influenced.
unless there is ring stra
c or unsaturated systems
3.6 δ
2.0 δd area, usually see a
vening bonding electrons.
ins with the spin of the
ins as in small rings or
62
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).
Information from NMR Spectrum
The Number of signals
The Position of signals
The Intensity of signals
The Splitting of signals
a b
Jab
Jab
Jab
b
a C H 2 B r C H B r 2
63
NMR of Fatty Acid Methyl - Ester
CH3 CH2 CH CH (CH2 CH CH)2 CH2 (CH2)5 CH2 CO
OMe
Methly linolenate C 1 9 H 3 2 O 2
a e e c e e b
Chemical shift (ppm) a 0.97 e ca.5.38 b 1.33 c 2.80 d 3.67
d
64
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
65
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. 250°C 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
66
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.
67
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 .
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
O
C O H C H 2
C H 2
C H R
H
H 2 C C H R
McLafferty Rearrangement
C H O C H 2
O H
H O C
O
H
C H 2
•
•
+ + •
• • •
H O C
O
H
C H 2
• • • • +
•
• + • •
• • + • • • +
• • •
+
O
C C H 3 O C H 2
C H 2
C R 2
H R 2 C C H 2
O
H
C C H 3 O
C H 2
O
H
C C H 3 O C H 2
O
H
C C H 3 O
C H 2 •
•
•
68
+
+ •
m/e 108
m/e 79 [C6H7]+
C H 2 O H O H
H H
•
-H69
+
-H2
m/e 107
m/e 77[C6H5]+
H
H
H
+
-COH
H
70
m/e 91
+ • + + •
H
H
C H 3
+ • + •
H C H 2
H H
H
H C H 2
C H R H
C H 2 C H R
H
H
H H
H H
C H 2
CH3
CH3
- CH3 •
71
72
73
74
75
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 bond-cleavage 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.
O
42
40
39
29
68 (M+)
76
O1
2
34
5( )O+
O
C3H3-HC O+
m/z 29
HH
m/z 40 m/z 39 (base peak )
-CHO
-H)( +
M+ m/z 68 (1a)(1b)
(1d)
(1c)
+
- CO
HH
+
+
)(+
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 CH3
( )+
O CH3O CH3
-CH3
(2a)
CHO
H3C C O+
m/z 43m/z 39(20% Σ) (4.4% Σ)
(2b)(15.9% Σ)
(2)
m/z 53(21.6% Σ)
(base peak)
+)(
- C3H3- C2H3O
+)(
77
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 CH3
β γ
O
m/z 81
(43.1 % Σ)
C2H5-
β
m/z 110 (11.9% Σ)
+ +
Cleavage to the furan ring with loss of the alkyl group is insignificant as it leads to an unfavored vinyl or diradical ion.
O R O O or
+
+
α
78
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.
+O CH2O CH2
OH
CHCH2
CH2
R
+ H+
n-propylfuran n-butylfuran n-pentylfuran
m/z 82 3.4% Σ 10.6% Σ 11.9% Σm/z 81 43.1% Σ 46.1% Σ 41% Σ
H
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.
O CH CH CH2 H+ O CH CH CH2+
H H
+ C6H5 +H2-
m/z 77(8.1 % Σ)
m/z 79(15.1% Σ)
CO-
m/z 107 (3.5% Σ)
H-
M+m/z 108
(16.7% Σ)
- CH2= CHR
79
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:
136+ 94+ + CH3 CH CH2
and 107+ 79+ + CH2 CH2
cis-2-(1-pentenyl)furan
m/z
39 50 77
81
94
107
135
80
H H
H CH2CH2CH3HH
m/z 108
H H
HCH2CH2CH3H
- H
+
m/z 107
OCH CHCH2CH2CH3
m/z 136
-CO
-CHOH2C CHCH3-
OCH CH2
m/z 94
136 94+ CH3CH=CH2+
81
O+
m/z 81
OCHCH=CHCH2CH3
+
-CH CCH2CH3
OCH=CHCH2CH2CH3
-Hm/z 136
-CO
CH2CH3H
+
m/z 79 m/z 77
C6H5+
107 79+ +CH2=CH2
+
HH
- H2
- CH2=CH2
m/z 107
- CH=CHCH2CH3
82
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 CHO( )
+
O C O+ O+
CO
O +
+
M+, m/z 96 (21.8% Σ)
H_
CO_m/z 95 (21.2% Σ)
m/z 67(1.6% Σ)
CO_
m/z 39
(27.6% Σ) 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.
O C O+
O C
HCHCH2
CH2
CH3O+
αO C
CH2
OH
+
C3H6_- C4 H9
•
83
2. Pyrroles N- and C- alkylated pyrroles show marked differences in fragmentation. The mass spectrum of 1-methylpyrrole is shown below.
NCH3 81 (M +. )
80
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.
CH3 N CH+
N
CH3
N +
CH2
NH
+
m/e 80
(strong peak)
C4H5+
m/e 53
HCN_
M+
m/e 81
m/e 39
C2H4N_
m/e 42
C3H3_ - H
+
CH3 N CH•
84
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.
N
H 2 C C H 2
C H 2
C H 3 N
C H 3
N
H 2 C C H 2
C H 2
H N
H 2 C H
+
+ C H 2
or
C 3 H 6 _
•
•
•
85
In the mass spectra of C-alkylpyrroles, the β-cleavage is the predominant fragmentation.
N CH2 HH
N CH2
H
+NH
+ NH
CH2 CH3+
M+
m/e 95m/e 80
base peak
H_
M+m/e 81 m/e 80
- CH3
The spectra of 2-formyl and 2-acetylpyrroles show the expected fragmentation with the intense acylium cation being presumably well-stabilized by resonance.
N CO
R N C O N COH H
+ + +
H
M+ m/e 94
- R
86
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.
S
R C H 2
S C H 2
S S
C H 2
+
+ β α
R _
or
Thiopyrilium ion
m/e 97
m/e 97
87
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 CC
C+
S CH2 CH2 CH3H3C + SH3C+
H_C2H5
_
m/e 139 (10%)M+
m/e 140m/e 111 (100%)
S+
S S+
m/e 125 (100%)
C2H5_CH3
_
m/e 139 (10%)
88
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.
(i) M+ m/z 92
(ii) M+m/z 78
(iii) M+ m/z 66
H_
CH3_
HCN_
•
•
• The cleavage processes of pyridines substituted with higher alkyl groups can be classified in three categories. (1) β-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.
N
C H 2 C H 3
N
C H 2
N
+
+
C H 3 _
( ) +
+
HCN _
m/z 92
m/z 65
•
•
89
These fragments undergo further elimination of hydrogen cyanide leading to the peak at m/z 65. (2) γ-cleavage is especially favored in 2-alkylpyridines. The relative intensity of the
resulting fragment ion depends on the nature of the radical lost.
N C H 2 C H 2 R
( ) +
N C H 2 C H 2
+
R _
N +
• •
•
•
(3) The McLafferty rearrangement takes place when the adjacent position to the
heteroatom bears a side-chain with at least three carbon atoms.
N CH2 CH2 CH2 CH2 CH3+N CH2
H
+
m/z 93 (100%)
base peak
- C4H8
90
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. a b
CH
NCH
N
N CH3
+ +
CH3CN_ HCN_
a b HCN
CH3
+
H3C C CH( )+H2C C CH+
H_
m/z 39
HC CH( ) +
m/z 26
HCN_
m/z 67m/z 53
HCN_
HC CH+ + HH
)( +
HH
+
m/z 40
91
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-5,6-dimethylpyrazine is shown below.
N
N
N
N
HN
N
CH2H
CH2HC
H2 C CH3
-
m/z 122 (100%)m+
178
- C3H7
N
N
+ +N
N
+N
N
- C2H5- CH3
m/z 135 (50%) m/z 149 (36%) m/z 163 (10%)
92
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
93
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
94
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
95
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 Buttery, fatty, moldy quality components 1 buttery, 5 fatty, 3 moldy Assigning of rough intensity value to each quality component Diacetyl, methyl nonyl Association of quality components ketone, methyl amyl ketone 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
96
5. CHEMISTRY OF FLAVOR PRECURSORS I. Flavor derived from carbohydrate and proteins (Browning Reaction, Maillard Reaction)
Reducing Sugars and α-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
Retroaldol condensation
Furans Thiophenes Pyrroles
Hydroxyacetone Hydroxyacetylaldehyde Acetoin Acetylaldehyde
Glyoxal Pyruvaldehyde Glycerolaldehyde
Aldehydes + α-aminoketone (Methional, NH3, H2S)
Pyrazines Pyridines Oxazoles
Heterocyclizaion
Thiazoles Pyrroles
Strecker degradation
Amino acids
H2S NH3
97
1. Maillard Reaction
C H 2 O H
C O
C H O H
R
H 2 N R +
C H 2 O H
C
C H O H R
N H R O H H
C O H
C
C H O H R
N H R _ H 2 O
2-AMINO-2-DEOXY-1-ALDOSE
HEYNES REARRANGEMENT
98
TRANSFORAMTION OF AMADORI INTERMEDIATE TO FORM REDUCTONES AND DEHYDROREDUCTONES
1-AMINO-1-DEOXY-2-KETOSE
H 2 C
C H O H
C H O H
C
N H R
O
R
H 2 C
C O H
C O H
C H O H
N H R
R
2,3-ENEDIOL
- N H 2 R
C H O H
C
C
C H 3 O
O
R
C O H
C O H
C
C H 3 O
R
DEHYDROREDUCTONE REDUCTONE
Ketoenolization
Ketoenolization
99
DEHYDROREDUCTONE
C H O H
C
C
C H 3 O
O
C H O H
C H 2 O H
FROM AMADORI
_ H 2 O
C H
C
C
C H 3 O
O
C O H
C H 2 O H
C H 2
C
C
C H 3 O
O
C
C H 2 O H
O
KETO FROM
C H 2
C
C
C H 3 O
O
C
C H O H
O H
C H 2
C
C
C H 3 O
O
H C
C
O H
O
ENOL
1,4 DIDEOXYHEXOSONE
1,4 DIDEOXYHEXONE FROM AMADORI PRODUCT
H
100
H C
C
C H 2
O
O
C H O H
C H O H
C H 2 O H
3-DEOXYHEXOSONE
H C
C
C H 3
O
O
C H O
C H O H
C H 2 O H
+
PYRUVIC ALDEHYDE
GLYCER- ALDEHYDE
C
C H 2 O H
O
C H 2 O H
H 2 O
H 2 O
C H O
C
C H 3
O
+
_
PYRUVIC ALDEHYDE
DIHYDROXYACETONE
RETRO-ALDOL CONDENSATION
1, 4 DIDEOXYHEXOSONE
C H 3
C
C
C H 2 C H O H
C O H
O
O
C H O
C H O
C H 3 C O
C O
C H 3
G L Y O X A L
DIACETYL
+
RETRO-ALDOL CONDENSATION OF DEOXYHEXOSONES
101
ALDOL
102
HYDROXYMETHYLFURAL AND FURFURAL FORMATION
C H O
C O
C H
C H
C H O H
C H 2 O H
O C H O
O H
H 2 C O H H 2 C O H O C H O
DEHYDROREDUCTONE FROM HEXOSE
_ H 2 O
C H O
C O
C H
C H
C H 2 O H
H 2 O _
O C H O O C H O
O H
DEHYDROREDUCTONE FROM PENTOSE
5-HYDROXYMETHYLFURFURAL
FURFURAL
103
C H 3 C O
C O H
C O H
C H 2 O H
Ketonization
C H 3 C O
C H O H
C
C H 2 O H
O O C H 3
O H O
O H H 2
3
5
3
2 4
- H 2 O 2
3
O C H 3
O H O
CYCLIZATION
5-METHYL-4-HYDROXY-3-(2H)-FURANONE (NOR-FURANEOL)
H O 2 H C C O O H
O
O H
O H
O H
5-KETOGLUCONIC ACID
FORMATION OF 5-METHYL-4-HYDROXY-3(2H)-FURANONE
R E D U C T O N E F R O M P E N T O S E
104
N
R '
C H O R
Formyl Pyrrol
C
C O
H C
H C
H C
R
O
O H
H
R'NH 2
C
C O
H C
H C
H C
R
O
N H R '
H C
C O
H C
H C
H C
R
O
N - R’
H
Basic Condition
N C H O
R '
R
- H2O
- H2O
105
REDUCTONE(RHAMNOSE)
ISOMALTOL
-
O
OH
CH3
O
CH3
H2O
2
O
OH
OHO
CH3
CH3
C O
C
C
CHOH
CH3
O
6
5
4
2
3
1 CH3
C O
C
COH
CHOH
CH3
OH HOHCH3
34
5
2,5-DIMETHYL-4-HYDROXY-3(2H)-FURANONE
C H 3 C H 2 C
C H O
O
O
H O O C C O
C H 3
+ ALDOL
C H 3 C H C
C H O
O
O
C H O O C C H 3 O H
- H 2 O
O O
O C H 3
C H 3
H O O C
O O
O C H 3
C H 3 O O
O H C H 3
C H 3
A MAPLE LACTONE
KETOBUTYRIC ACID
106
2. Strecker Degradation Mechanism
eneaminol
1) self condensation2) condensation with other eneaminols3) hydrolyze to amino acetone + aldehyde or ketone
CO2+H3C C CH
OHCR
NH
Enol form of Schiff's baseH3C C CH
OHC CR OH
ON
Keto form of Schiff's baseH3C C C N C CR
H
OH
O
H
O
pyruvaldehyde amino acid
H2N C CO
H
H
R+H3C C C
OO
H
107
R
C O
C O
R
α D I C A R B O N Y L
+ H N C H C O O H
R 1
2
3 3
2 R
C
C O
R
H O H 2 N C H C O O H
R 1
AMINO ACID
- H 2 O R
C
C O
R
2
3
C H C O O H
R 1
N
S C H I F F B A S E ( I M I N E )
C O 2 N
H C H
R 1 3
2 R
C
C O
R
- +
-
R
C
C O
R
N C H
R 1
H 2
3
+ H 2 O R 1 C H O
3
2 R
C
C O
R
H N H 2
α A M I N O C A R B O N Y L
+
STRECKER DEGRADATION
·
108
C H 3 C
O
C
O
C H 3 C H 3 C
O
C
O
C H 2 C H 3
O
O O
O C H
O H
C H 2 O H
D E H Y D R O A S C O R B I C A C I D H O
C H 3 O
O O H
H O
L D E O X Y H E X O S O N E
DIACETYL 2,3- PENTANEDIONE
DICARBONYL COMPOUNDS IN FOODS
FOR STRECKER DEGRADATION
(FROM AMADORI)
109
2) Methionine:
H 3 C S C H 2 C H 2 C H C O O H
N H 2
H 3 C S C H 2 C H 2 C H O
C H 2 C H C H O H 3 C S H +
H 3 C S S C H 3
H 3 C S C H 3 + H 3 C S S S C H 3
H 3 C S S S S C H 3
2
110
S C O O H
N H 2
R C O
C O
R '
S C H O
H 2 O
C H 3 S H C H 2 C H O H O H 2 C +
METHYLMERCAPTAN
METHIONINE BREAKDOWN
Strecker aldehyde
111
H S C O O H
N H 2
R C O
C O
R '
H S C H 2 C H O R C O
C R '
N H 2 +
H
H 2 S + C H 3 C H O + C O
R
C N H
R ' ENAMINOL
H 2 S F O RM A T IO N F RO M CY S T E IN E
Mercapto Acetaldehyde
112
3. Pyrazines formation Cocoa, coffee, French fry etc. roasted beef. pathway 1: sugar + amino acid
CH2C
O
NH2
H
+CC
O
CH2OH
H
H2N - H2O
N
N CH2OH
N
N
CH2
H
OH-
N
N
CH3
pathway 2:
CC
O
OH
HNH3 C
CO
HO
H
NH2
α,β−dicarbonyl
+ HC
CH2H2N
O
N
NH
H
OH
HHO
HON
N
-3H2O
Cyclization
113
4. Oxazole formation
Trimethyl-oxazoline in beef stew
N
O2,4,5-trimethyl oxazole
Possible mechanism for the formation of trimethyloxazole from diacetyl, CH3CHO, NH3.
Possible mechanism for the formation of trimethyl-oxazoline
H3C C
O
C
O
CH3H2O
H3C COH
C CH3
OH
O
H3C CH
O NH3
H3C C H
NH+
·
H2O-
H3C COH
C CH3
OH N
H3C C·
H2O-
H3C C
H3C C C CH3
O N
·
··
·N
O CH3H3C
H3C
+
+
H2O-
114
5. Thiazole formation
Trimethyl thiazole (less nutty, sulfur )
N
S
H3C
H3C CH3( identified in potato, beef, coffee, tea, cocoa bean )
H3C CO
C
O
CH3H2S
H3C COH
C CH3
SH
O
H3C CH
O NH3
H3C C H
NH+
·
H2O-
H3C COH
C CH3
SH N
H3C C·
H2O-
H3C C
H3C C C CH3
S N
·
··
·N
S CH3H3C
H3C
+
+
-H2O
115
II. Thermal Degradation of Vitamin B1 1. Basic condition
2. Acidic condition
N
SH O
N
N
H 2N
H +
+N
N
C H O
H O S
NH +
HO
O
S H
C H3CC H(C H 2)2 + HCOOH +
H 2N
H 2NN
N
no odor
H 2N
coffee
H2O H2O+ +
H
H
Cl-+N
SHON
NH2N
N
SHO+
N
NH2N
no odor
has found some use in the flavor industry( identified in coffee aroma with meaty note )
- OH-
+ H2O CH3
116
3. Thiazole compounds
S
NHO
-H2O
S
N
Formed in cocoa
methyl-vinyl-thiazole
reduction
S
N
( cocoa, beef )
methyl, ethyl-thiazole 4. Furan compounds
H3C C CH CH 2 CH 2 OHO
S H
-SH
+ H+
H3C C CH2 CH2 CH2 OHO
O OH3C( coffee, tea )
cyclization
-H2O
Reduction
-H2
OOH
CH3
117
Cyclization Cyclization
118
III. Lipid Oxidation
1. Chemistry of triplet oxygen
Molecular Atomic Atomic
2Px 2Py 2Pz
*
2S
1S
Molecular Orbital o
σ
2Pz 2Py 2Px
*
*
f
σ
Triple
π
ππ*
π*2S
σ1S
σ
σ
σ
Et Oxygen
119
2. General Mechanisms of Autoxidation
14 13 12 11 10 9
12 11
•
•
12 11
12 11
HYDROPEROXIDE DECOMPOSION
•
12 11
TERMINATION
•
C H 2 C H C H C H 2 C H C H C H 2 R ( C H 2 ) 3 C H 3
INITIATION (METAL)
( C H 2 ) 4 C H 3 C H CC H
( C H 2 ) 4 C H 3 C H C HC H
O
O PROPAGATION
C H C H C H( C H 2 ) 4 C H 3 O
O
H
( C H 2 ) 4 C H 3 C H C HC H
O
C H 3 ( C H 2 ) 3 C H 2
C H 3 ( C H 2 ) 3 C H
O C
H
•
- H 10 9H C H C H C H 2 R
+ O2
10 9
•
C H C H C H 2 R
+ H
10
C H C H C H 2 R
- OH•
10 9
•
C H C H C H 2 R
C H C H C H C H C H 2 R
+ H
+
(PENTANE) 3
120
Mechanisms of Oxidation 1. Initiation 2. Propagation 3. Termination
• • +
• • +
• + + •
• • +
+ + • •
R O R R O R • •
•
+
• • +
R O R O O R O O R O 2 + + • 2 2 2
R H R H
R O 2 R O O
R O O R 1 H R O O H R1
R R R R
R O O R O O R O O R
R O O R R O O R
O 2
121
Oxidation of Mono-Olefines Oleic acid - 4 Hydroperoxides
12 11 10 9 8 7 12 11 10 9 8 7
11 10 9 8 7 9
8
11 10
C C C C C C
O
O
H
11 10 9 8 7
C C C C C C
O
O
H
C C C C C
O
O
H
C
C C C C
O
O
H
C C
Double bond shifts to
Hydroperoxides from Linolenate
9 C C C C C C
O
O
H
C C
16 15 14 13 12 11 10 9
16 15 14 13 12 11 10 9
12 C C C C C C C C O
O
H
16 15 14 13 12 11 10 913 C C C C C C C C
O
O
H
16 15 14 13 12 11 10 9122
16 C C C C C C
O
O
H
C C
123
Peroxide Decomposition
General
Effects of Metal on Peroxide Decomposition
O H +
•
•
• +
or
R C
O
H R 1 + • • +
R ' O H R O H • +
+
R '
R ' H +
+ •
R C R 1
H
O
O
H
R C R 1
H
O
R C R 1
O
R ' H
R C R 1
H
O H
R 1 C H 1
O
R
R '
•
C u + R O O H R O O H - C u + +
C u + H + R O O R O O H C u + +
R O O H R O R O O H + O H -
H 2 O
+ + +
+ + +
+ + + 2
•
•
• •
124
C C C C
O
H C C C
H
H H H H
H
H H
R
C C C C
H H H H
R
H H
H H
O
C C C
O
O
H
C C C
O
H
H H H H
R C C C C
O
H
H
H
H H
R C C
C H 2 C H 2 C H C H R
O C
H
C H 3
O C
H
C H 2 C O
H
A B
125
Ethyl vinyl ketone isolated and identified in raw soybean
H 3 C C H 2 C C HO
C H 2 ( raw beany, grasoy )
H 3 C C H 2 C H C H C H 2 C H C H C C O O H O O H
H 3 C C H 2 C H C H C H 2
C H 2 C H H 3 C C H 2 C H
O 2 , RH
C H 2 C H C H 2 C H H 3 C O O H
C H 2 C H C H 2 C H 3 C O
126
Lactones in butter flavor Important lactones in butter are δ−decalactone δ−dodecalactone δ−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 content of lactones is low heated butter lactone increases
127
Lactones come from δ−hydroxy acids in milk
H 2 C
H C
H 2 C
O CO
O CO
O CO
R
R '
(C H 2 ) 3 C H (C H 2 )n C H 3O H
100~150oC∆
H O CO
(C H 2 ) 3 C H (C H 2 )n C H 3O H
OC H
C H 2
C H 2C H 2O
(C H 2 )n C H 3
(CH2)2 CH CCOOH
OO
CHR
α−carboxyl-γ−(δ)−lactone
odorless, well crystalized
at 80~120 oC, decarboxylated to lactone
- H2O
128
3.Chemistry of singlet oxygen
Molecular Atomic Atomic
2Px 2Py 2Pz
2S
1S
*
*
Molecular Orbital of Si
σ*
2Pz 2Py 2Px
σ
nglet O
π
ππ*
π*2S
σ1S
σ
σ
σ
E
xygen
129
Excitation and Deactivation of Photosensitizer
Photooxidation process (RH : Substrate ; K1 = 1 - 3 × 109 /M •sec. ; K2 < 107 /M •sec)
Excited state
Ground state
k = 2 × 108 /sec
k = 10 - 104 /sec
k = 1- 20 × 108 /sec
k = 1 - 3 × 109 /sec
hν
1Sen*
1Sen
3Sen*
Singlet oxygen formation
Sen hν
1Sen*
ISC
ISC 3Sen*
+ RH
+ RH
+ 3O2
1O2 + 3O2
O2- + Sen+
+ 3O2
ROOH ROOH
K2
K1
R + Sen H
+ 3O2
Production of 1O2 by Ph
R C O O R C O O • • +
(1)
(7)
(8)
(9)
(10)
O 2 - O 2
- +
+ H 2 O 2 H O2
OZONIDES
ENDOPEROXIDES
2H+
H 2 O 2
R C+
R CPRODUCTS
PRODUCTS
H 2 O + O H -
(12) ENZYMES
130
otochemical, Chemical, and Biological S
(6)
1O2
O 2 + 3SENSITIZER
(2) H 2 O 2
+
O C l -
H 2 O 2 + O 2 -
(3)
O H
(5) O 2
-
O 2 - + Y+
SENSITIZER
H 2 O C l-
O H - + O H
O H -
e- Y
O
O H +
•
•
(4)+ O 2 -
-
(11)
ystems
131
1,4- Cycloaddition:
ENE Reaction :
1,2 – Cycloaddition:
Reactions of Singlet Oxygen with Various Types of Double Bonds
O
O
O
OEndoperoxide
Allyl Hydroperoxide
O
O
H
O
O
O
O
O
O Dioxetane
R R ' H O
O
I
R R' H O
O
II
CH2
CH2
+
+
+
Conjugated and Nonconjugated Hydroperoxides Arising via the 6-Centered Transition state
R R'R R'
OOH O O H
R R ' +
hν/sensitizer/O
132
RR'
OOH
isomerization
133
Reversion Flavor
C H 3 C H 2 C H C H C H 2 C H C H C H 2 C H C H C H 2 ( C H 2 ) 6 C O H O
C H 3 C H 2 C H C H C H 2 C H C H C H 2 C H O C O H ( C H 2 ) 6 C H C H
O O H
C H 3 C H 2 C H C H C H 2 C H C H C H 2 C H O C O H ( C H 2 ) 6 C H C H
C H 3 C H 2 C H C H C H 2 C H
C H 3 C H 2 C H C H C H 2 C HO O H
C H 3 C H 2 C H C H C H 2 C HO
C H 3 C H 2 C H C H C H 2 C HO
1O2
15 14 13 12 11 10 9 8
15 14 13 12 11 10 9 8
1O2
•
15 14 13 12 11 10 9 8
4-Keto-6-nonenal Formation
O •
C H C H 2 C H O
O C H C H 2 C H
O C H C H 2 C H
O C H C H 2 C H
•
•
from Linolenic Acid
134
Mechanism for 4-Keto-5-nonenal Formation from 4-Keto-6-nonenal
4-keto-5-nonenal
C H 3 C H 2 C H 2 C H C H 2 C H O O
C C H C H
- H•
• C H 3 C H 2 C H C H C H 2 C H O O
C C H C H ••
+ H•
C H 3 C H 2 C H C H C H 2 C O O
C H C H 2 C H 2
2
2
135
Formation of 2-(1-Pentenyl)furan from 4-Keto-5-nonenal and 2-(2-Pentenyl)furan from 4-Keto-6-nonenal
2-(1-pentenyl)-furan
2-(2-pentenyl)-furan
C H3 (C H2)2 C H CH C CH2O
C H2 C HO
C HC C HO H O H
O
C H
O
O HC H C H
OH
C C H
C H2 C HO
C C H2 O
H C 3 C H2 C H C H C H2
C H3 (C H2)2 C H CH
C H3 (C H2)2 C H C H
H C 3 C H2 C H C H C H2
H C 3 C H2 C H C H C H2
- H2O - H2O
136
C H 3 ( C H 2 ) 4 C H C H C H 2 C H C H C H 2 ( C H 2 ) 6 C O O H
C H 3 ( C H 2 ) 4 C H C H C H 2 C H O
C H 3 ( C H 2 ) 4 C H
O
C H C H 2 C H O
O
C H 3 ( C H 2 ) 4 C H
O
C H 2 C H 2 C H O
O H
C H 3 ( C H 2 ) 4 C H
O
C H 2 C H 2 C H O
C H 3 ( C H 2 ) 4 C
O
C H 2 C H 2 C H O
C H 3 ( C H 2 ) 4 C
H
O
C H C H C H
H
O
C H 3 ( C H 2 ) 4 C
O C H
C H C H
1O2
C H 3 ( C H 2 ) 4 C H C H C H 2 C H
O
O H
C O O H ( C H 2 ) 6 C H C H
1O2
C H 3 ( C H 2 ) 4 C H
O
C H C H 2 C H O
O •
•
•
+ 2RH
+ 2R •
- H2O
Formation of 2- Pentyl furan
137
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 ω-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 ω-8 position producing a free radical at ω-8 of the fatty acid.
3. While still attached to the enzyme, the fatty acid free radical isomerizes to place
the unshared electron at ω-8 causing conjugation and isomerization of the double
bond.
4. O2 reacts with the free radical at ω-6 to give a peroxy free radical.
5. A hydrogen from the medium forms the hydroperoxide which then dissociates
from the enzyme.
138
H
H cis
C O O H ( C H 2 ) 6
H
C
H
C C
H H
C H C C
H
H H C C
(CH 2 ) 4 CH 3
cis cis
CH 3 (CH 2 ) 4
C C H H
C C H C
H H
C C
H
C
H
( C H 2 ) 6 C O O H
cis H
H
. C
trans
H
cis
H
H cis
C O O H ( C H 2 ) 6
H
C
H
C C
H H
C C H C C
(CH 2 ) 4 CH 3
.
C
O
O
tr a n s
H
cis
CH 3 (CH 2 ) 4
C C H C C
H H
C C
H
C
H
( C H 2 ) 6 C O O H
cis H
H
C
O O H
trans
H
cis
H
H cis
C O O H ( C H 2 ) 6
H
C
H
C C
H H
C C H C
(CH 2 ) 4 CH 3
C
H
.
- H .
+ 3O2
139
Aldehyde and Alcohol Formation in Tomato from Linolenic and Linoleic Acid.
Linolenic Acid – C14
O2 + Lipoxygenase
C C C C C C C C C C (C)7 C OH
OH
OOH
cis trans cis
C C C C C C H
cis-3-hexenal
C C C C C C
O
H
trans-2-hexenal
C C C C C C
H
OH
Htrans - 2 - hexenol
+
C C C C C C OH
H
H
n-hexanol
C C C C C C OHH
Hn - hexanol
O2 + Lipoxygenase*AOR: AlcoholOxidoreductase
C (C)3 C C C C C C (C)7 C OH
OH
OOH
trans cis
C C C C C C H
O
n - hexanal
AOR
AOR
C C C C C C H
O
n-hexanal
AOR
H+
AOR*
C C C C C C OH
H
H
cis - 3 - hexenol
+
C C C C C C OH
H
H
n - hexanol
O
Linoleic Acid – C14
140
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 processed banana lost flavor
banana peel extraction enzyme ( flavorase ) processed banana flavorase fresh banana flavor 3~4 hrs
If pyruvate, acetate, amino acid, unsaturated fatty acid are added, then the time for flavor production will be shortened to 30 min.
141
Lactose
S. Lactis
Lactic acid Oxalacetic acid Citric acidCH3COHCOOH
H
[H][O]
COOHCCH2COOH
O
-CO2
Pyruvic acid
-acetateCOOHCH2CCH2
COOHHO
COOH
CH3CCOOH
O
-CO2
Acetaldehyde
CH3CH
O
H3C C C OHCH3O
COOH
Acetyl lactic acid Acetoin
-CO2 H3C C C CH3
O
H
OH
[o] in presence of oxygen
H3C C C CH3
O O
Diacetyl
[H] in absence of oxygen
H3C C C CH3H
OH
H
OH
2.3.-butylane glycol ( odorless )
142
4. Onion and Garlic Flavor Enzymatic reaction of cysteine derivative
CH2CH COOHHSNH2
CH2CH COOHSNH2
RO
Allin
R: CH3
CH3-CH =CH - ( propenyl alliin onion more prodominate)
CH2=CH-CH2 (- allyl alliin ) gallic
H2O Alliinase
R S OH
+ NH3 + H3C C COOHO
sulfenic acid pyruvic acid
when R:CH3-CH=CH-
CH S OH
CHH3C
CH S OCH2H3C
thiopropenal oxide
lachrymator in onion 1 min propenyl cysteine sulfoxide + onion enzyme product (m/e = 90) (this is thiopropenal oxide ) 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.
143
H 3 C C H 2 C H S O H 3 C C H C H S H O
H 3 C C H C H O H ( m/e = 58 )
H 3 C C H 2 C H O ( m/e = 58 )
Aldol condensation
H 3 C C C H O C H C H 2 H 3 C ( m/e = 98 )
- S
( m/e = 90 )
R S HO
sulfenic acid
R S S RO
thiosulfinate ( responsible for fresh flavor of onion and garlic )
CH CH S S CH CHO
H3C CH3 fresh onion odor
CH2 S S CH2
OCH CH2CHH2C fresh pleasant
garlic-like odor
144
S C H 2 C H C H 2 S C H 2 C H H 2 C typical garlic-like odor
H 2 C
H C N H
C H
C H 2 S O
C O O H H 3 C
cycloalliin ( no favor contribution )
isolated and identified
H C C H C H 2 S C H 2 C H N H 2 O H
O
C O O H
+H 2 O
C H S C H C H N H 2 C O O H
O C H H 3 C
R S S
O
R
R S S
O
R
O
R S O
O
(Thiosulfinate)
(Thiosulfonate)
Aged Flavor
Fresh Flavor
Bitter Flavor (Off Flavor)
- H 2 O
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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
[ADH] - alcohol: NAD + oxidoreductase alcohol dehydrogenase add to use l-[14C] leucine crude extract of fresh tomato --- get 14C label 3-methyl-1-butanol add to boiled extract of tomato --- no reaction indicates the enzymatic nature of this reaction
L-leucine 2-Keto-4-methyl-pentanoic acid
3-methyl-butanal 3-methyl-1-butanol
COOHCHH2NCH2CHH3CCH3
COOHCCH2CHH3CCH3
OCHOCH2CHH3CCH3
CH2OHCH2CHH3CCH3
CO2NADH + H+
NAD+
[ADH]
146
2) Fatty acid precursors
3-cis-hexenal
CH2 CH CH CH2 C HCH3
O
13-hydroperoxide
trans cisCH3 CH2 CH CH CH2 CH
O
OHC(CH2)7CHCHCHC
OOH
16 15 14 13 12 11 10 9
+ lipoxygenase O2
cis cis cis
16 15 13 12 10 9CH3 CH2 CH CH CH2 CH CH CH2 CH CH (CH2)7 C OH
Olinolenic acid
147
3-cis-hexenal
CH2 CH CH CH2 C HCH3
O
hexenal
CH2 CH CH CH2 C HCH3
O
n-hexenal
CH2 CH CH CH2 CH2OHCH3
3-cis-hexenal
CH2 CH CH CH2 CH2OHCH3
AOR 2-trans-hexenal
CH2 CH2CH3
O
HCCHCH
AOR
trans-2-hexenol
CH2 CH2CH3 CH2OHCHCH
AOR: alcohol oxidoreductase
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6. Asparagusic Acid in Asparagus asparagusic acid: 1,2-dithiolane-4-carboxylic acid
SS
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.
COOH
NH2
COOH
OCOOH
valine 2-methyl propanoic acid
COOH
S
COOH
CH3
SH
COOH
SH
COOH
S
COOH
C
CH3
O
S
COOH
C
CH3
O
SHSH
COOH
SH
SS
COOH
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7. Mushroom Volatiles Edible mushroom like Agaricus Bisporus produce 1-octen-3-ol, 3-octanol, 2-octen-1-ol 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.
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8. Flavor formation by Neurospora
Production of Fruity Aroma by Various Strains of Neurospora Neurospora Species Aroma Neurospora sitophila ATTC46892 Fruity Neurospora No 1 Fruity Neurospora No. 2 Fruity Neurospora No. 3 Fruity Neurospora No 4 Fruity Neurospora No 5 Fruity Neurospora No 6 Fruity Neurospora No 7 Fruity Neurospora tetrasperma NRRA2164 No aroma Neurospora crassa NRRA 2223 No aroma Neurospora sitophila NRRA 2884 No aroma Neurospora intermedia NRRA 5506 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
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Volatile Compounds (ppm) produced by Neurospora sp. Isolated from beiju
Ethyl Acetate
Ethanol 3-Methyl-1-butanol
Ethyl hexanoate
1-Octen- 3-ol
Neurospora sitophila
4.8 128 318 59 40
Neurospora Sp. 1
9.0 111 ND ND ND
Neurospora Sp. 5
0.9 111 117 10 50
Neurospora Sp. 6
2.8 99 208 20 ND
ND: Not detected
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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.
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2) Origin a. Cooked flavor: protein H2S b. caramelized flavor
CH2 CHO from phenylalanine
H 3 C C H C O O H
N H 2
C H 2 C H O
( Strecker degradation )
pyruvic acid H 3 C C C O O H
O
C H 2 C H C O O H
N H 2
+
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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
C H C H 2 C H C O O H
N H 2
H 3 C
H 3 C
leucine
S. latics var. maltigens
C H C H 2 C H 2 C H O H 3 C
H 3 C + NH 3
threshold 0.5 ppm
2H2
2 + H O
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5. Absorbed flavor Feed flavor Weed flavor Barney flavor 1) Nose or mouth lung blood udder cell milk 2) Digestive tract blood udder cell milk
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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
Fig.2 Mass Spectrum of peak D (top) of Fig.1 and standard dimethyl disulfide (bottom)
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 2-butanene, ehtanol, diacethyl, dimethyl disulfide, and n-butanol, respectively
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160
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.
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II. Cheese Flavor 1. Isolation, separation, and identification of cheese flavor
Dynamic headspace analyzer, gas chromatographer, and mass spectrometer arrangement
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Reproducibility of gas chromatograms of headspace volatile compounds of Brewster Cheddar cheese after one week of storage
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164
165
Changes of total headspace volatile compounds of Cheddar cheese at 11°C, and Swiss cheeses at 21 °C during ripening
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2. Biochemical pathways of fats in cheese flavor formation Fats Amides Aldehydes Primary Alcohols Methyl Ketones Fatty Acids Secondary Alcohols Easters Lactones
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3. Reaction products of methionine CH3SCH2CH2CH(NH2)COOH CH3SSCH3
Methionine Dimethyl disulphide CH3SCH2CH2CHO CH3SH + CH2CHCHO Methional Methanethiol Acrolein CH3SCH2CH3 + Ethylmethyl sulphide Formic Acid CH3SH + CH2CH2 + HCOOH Methanethiol Ethylene Formic Acid
[O]
H2O
H2O HCOOH
168
4. Biochemical pathways of cheese flavor formation from protein Products = Caseins (+trace of whey) Amines Peptides a-keto acids Acids Alcohols Phenols Amino Acids H2S NH3
169
5. Formation of 2-butanone and 2-butanol from diacetyl CH3COCOCH3 CH3CHOHCOCH3 Diacetyl Acetoin CH3COCH2CH3 CH3COH=COHCH3 2-Butanone 2,3-Butyleneglycol CH3CHOHCH2CH3 2-Butanol 6. Biochemical pathways of cheese flavor formation from lactose Lactose Lactic Acid Diacetyl Pyruvic Acid Ethanol Acetaldehyde Acetic Acid CO2
[H2]
[H2]
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7. Lactone formation
H2C O C R
O
HC O C R1
H2C O C (CH2)3
O
O
CH
OH
(CH2)4 CH3
DG
HO C
O
(CH2)3 CH
OH
(CH2)4 CH3
-H2O
C (CH2)3 CH (CH2)4 CH3
O
O
+ H2O
171
8. Mechanism of Methylketone Formation
H2C O C R
O
HC O C R1
H2C O C (CH2)3
O
O
CH
OH
(CH2)4 CH3
DG
HO C
O
(CH2)3 CH
OH
(CH2)4 CH3
-CO2
+ H2O
C (CH2)n
O
H3C CH3
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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)
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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. Dynamic headspace sample/gas chromatography/mass selective b. TBA value c. Total bacteria count d. Panel evaluation for off-odor
174
3) Identification of volatile compounds of aseptic raw beef by DHS/GC/MSD Condition of Mass Selective Detector Column DB-5, 30m symbol 180 \f "Symbol" \s 12×} 0.25mm,
1.0symbol 109 \f "Symbol" \s 12µm film thickness Carrier gas Helium gas (99.999%) at 1 ml/min Ion source temp. 170oC Ionization voltage 70eV Mass scan range 25-250 a.m.u. Scan rate 1.0 scan/sec
175
Diagram of Dynamic Headspace Sampler/Gas Chromatograph
176
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178
Chromatogram of 0 day storage
Chromatogram of 8 day under the dark storage
Chromatogram of 8 day under the light storage
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180
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182
183
184
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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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189
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II. Isolation, Separation, and Identification of Roast Beef Flavor
191
192
193
194
195
III. Simulated Meat Flavor Compounds Formation
196
197
8. ORANGE FLAVOR STUDY BY PULSED ELECTRIC FIELD
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200
201
202
203
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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) The rates of flavor compounds release from foods. 2) The concentration and disposition of flavor compounds in the food. 3) The components of the food. 4) The particle size of food components. 5) The extend of mixing. 6) The temperature of foods. 3. Factors affecting partition and release of flavor compounds in the mouth 1) Hydration 2) Dispersion 3) Reduction of Particle Size 4) Homogenization 5) 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) Temperature 2) The presence of soluble solutes and nonsoluble materials 3) Diffusion rates in the aqueous phase 4) Physical retention of flavor compound Air-Water Partition Coefficients for Homologous Series of Ketones and Aldehydes at 25oC
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Compounds Coefficients Compounds Coefficients Acetone 1.6x10-3 Acetaldehyde 2.7x10-3 Butan-2-one 1.9x10-3 Propanol 3.0x10-3 Pentan-2-one 2.6x10-3 Butanal 4.7x10-3 Heptan-2-one 5.9x10-3 Pentanal 6.0x10-3 Octan-2-one 7.7x10-3 Hexanal 8.7x10-3 Nonan-2-one 15x10-3 Heptanal 11x10-3 Undecan-2-one 26x10-3 Octanal 21x10-3 Nonanal 30x10-3 Types of Possible Interactions between Flavor Compounds and Food Components. Component Possible Interaction Lipids;
-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 Threshold Concentration (ppm) Water Oil Octanoic acid 5.8 350 γ-decalactone 0.05 3.0 Pentanal 0.07 0.3 Hexanal 0.03 0.05 2,4-Decadienal 0.5x10-3 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 Protein n Keq/M -G(Cal/M) 2-Heptanone Soy Protein 4 110 2.78 2-Octanone Soy Protein 4 310 3.39 2-Nonanone Soy Protein 4 930 4.04 2-Heptanone β-Lactoglobulin 1 152 2.98 2-Octanone β-Lactoglobulin 1 481 3.66 2-Nonanone β-Lactoglobulin 1 2439 4.62 2-Heptanone Serum Albumin 6 600 --- 2-Nonanone Serum Albumin 6 1800 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 Protein n Keq/M -G(Kcal/M) Soy Protein 2-Heptanone Native 4 110 2.781 2-Octanone Native 4 310 3.395 2-Nonanone Succinylated 2 850 3.992 2-Nonanone Native (25C) 4 930 4.045 2-Nonanone Native (5C) 2 2000 4.221 2-Nonanone Heated (90C) 4 1240 4.215 β-lactoglobulin
2-Heptanone Native 1 152 2.980 2-Octanone Native 1 481 3.660 2-Nonanone Native 1 2439 4.620 Bovine Serum Albumin
2-Heptanone Native 6 500 --- 2-Nonanone Native 6 1800 4.900 Effects of Temperature and Modification on the Binding and Thermodynamic Data for Interactions of Carbonyl Compounds with Soy Protein Compounds Temperature n Keq/M -G(Cal/M) 2-Heptanone 5C 4 2000 4.22 2-Octanone 25C 4 930 4.06 2-Nonanone 90C 4 1240 4.21 2-Nonanone Succinylated-25C 2 850 3.99
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215
330
340
350
360
0 2 4 6
Urea Concentration (M)
Fluo
resc
ence
(nm
) (
)
0
500
1000
1500
2000
2500
K,M
-1 ( )
Effects of urea induced conformational change s reflected in fluorescence on the binding affinity of 2-nonanone for b-lactoglobulin
216
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218
Adsorption and Desorption of 2-Pentanone onto Whey Protein
Adsorption Desorption P/Pv Rel. Mass Gain P/Pv Rel. Mass Gain
(Flavor) (x103) (Flavor) (x103) 0.000 0.000 0.0876 13.51 0.085 1.273 0.739 12.80 0.131 1.367 0.575 12.40 0.216 1.599 0.490 12.12 0.307 2.438 0.432 11.81 0.431 5.199 0.307 10.50 0.490 6.103 0.167 9.131 0.611 9.985 0.072 6.830 0.752 12.19 0.000 3.000 0.876 13.51
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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. Effects of Packaging Materials on the Flavor Quality of Food II. Sorption of Orange Flavor Compounds by Packaging Materials
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11. FLAVOR COMPOUNDS AND SOLVENT INTERACTION
I. Commercial Cherry Flavor and Solvent Interaction II. Acetal Formation
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