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Applying Chemistry to Solve Protein Flavoring Issues Robert J. McGorrin, Ph.D., CFS Department Head and Jacobs-Root Professor Food Science & Technology Oregon State University 2014 Protein Trends & Technologies Seminar April 8-9, 2014 • Arlington Heights, IL, USA

Overview  A basic understanding of flavor and flavor chemistry  Impacts of processing on flavor stability  Sources of protein off-flavors and flavor-protein interactions

 An overview of the chemistry of protein systems and causes of flavor change

 Applications of off-flavor masking agents

Food Products are Complex Systems! Water Fat

Protein Carbohydrates Minerals Emulsifiers Gums

Antioxidants Vitamins Phytonutrients / botanicals Color Flavor

The Significance of Flavor



Flavor quality is a major driver of consumer acceptance for food products



Commercial success of a newly launched food product is directly linked to flavor



While flavors are present at only trace levels – they exert high impact!

What is Flavor? The Flavor Experience = Aroma + Taste + Chemesthesis

 Aroma:     



Aromatics

Volatile Primarily fat soluble Over 7,000 known aroma chemicals Organic (carbon) molecules with oxygen, nitrogen, sulfur Perceived ortho-nasal (smell) and retro-nasal (mouth)

Taste: Tastants  Non-volatile  Water soluble (saliva)  Sweet, sour, salty, bitter, umami

 Chemical Feeling: Chemesthesis (Trigeminal nerve)  

Skin response to chemical irritation; not only in mouth Examples: Pepper burn, menthol cooling, cranberry astringency

Chemistry of Flavors Volatile compounds (Aromatics)

 

Typical molecular weight range between 34 – 300



A natural flavor can contain 200 – 1,000 volatile constituents



Individual components are typically present at partsper-million to parts-per trillion concentrations



Some aroma chemicals provide unique flavor characters or sensory impressions (so-called “character-impact compounds”)

Boiling points:  -60°C Hydrogen sulfide (egg)  20°C Acetaldehyde (orange juice)  131°C Hexanal (green; rancid)  320°C d-Dodecanal (coconut; cream)

Chemistry of Flavors Examples of volatile Character-Impact compounds O CH=O

OCH3

OH

NH2

Benzaldehyde cherry, almond

Methyl anthranilate Concord grape

Menthol peppermint CH=O

O

N N

OCH3

OCH3 OH

Nootkatone grapefruit

2-Methoxy-3-isobutyl pyrazine green pepper

Vanillin vanilla

Chemistry of Flavors Non-volatile compounds (Tastants)

     

Typical molecular weight range between 40 – 1,000 Sweet: sucrose, fructose, aspartame, sucralose Bitter: caffeine, quinine Salty: sodium chloride, potassium chloride Sour: citric acid (citrus sour), butyric acid, lactic acid (dairy); acetic acid (vinegar) Savory: monosodium glutamate, amino acids

Causes of Flavor Deterioration  Heating      

 High temperature processing  Volatile flash-off pH Metal ions  Iron  Copper Oxidation of fats – Air / light Oxidation of fats – Enzymes  Lipoxygenase + fatty acids in soybean oil Maillard browning Interactions of flavors with food ingredients  Cherry flavor with Aspartame  Vanilla flavor with whey protein concentrate

Flavors and Proteins The addition of protein to a food product may alter flavor by: 1. Imparting undesirable off-flavors “Beany” flavors; astringency; chalky mouthfeel 2. Changing the food’s flavor profile due to: - Flavor interactions - Flavor binding - Flavor release Depending on the specific protein, and how they interact with it, flavors come across as either “brighter” or “muted”. We’re just beginning to understand the chemistry behind the flavor changes

Flavor Changes from Proteins

1. Imparting undesirable off-flavors • Proteins generally should not impart flavor characteristics or contribute flavor However . . . • Typical ingredient processing and storage conditions can produce undesirable off-flavors: - Volatile compounds produced from amino acids or protein fragments - Oxidation of trace amounts of fat - Maillard browning reactions

Flavor Changes from Proteins

1. Imparting undesirable off-flavors • Soy protein - Beany, green, bitter • Pea protein - Earthy, grassy, nutty, savory; grainy mouthfeel • Whey protein concentrate (WPC) - Grassy, hay, cheesy, astringent • Whey protein isolate (WPI) - Cardboard, wet dog, cucumber, cooked milk, cabbage, bitter, astringent • Casein (milk protein) - Stale milk, gluey, cheesy, musty, sour • Protein hydrolysates - Astringency

Protein Off-Flavors 200 volatile chemical compounds have been identified in whey (dry and liquid) that may influence/ contribute to their flavor and aroma in finished product

Fatty acids Acetic acid Vinegar Hexanoic acid Sweaty Butanoic acid Cheesy/rancid

Amino Acid breakdown Cysteine, methionine, tryptophan, phenylalanine Dimethyl sulfide Garlic/rubbery Dimethyl trisulfide Cabbage o-Aminoacetophenone Grape Methional Potato 2-Methoxy phenol Smoky Adapted from: Carunchia Whetstine, Croissant, Drake (2005).

Protein Off-Flavors Fat oxidation Hexanal Nonanal Octanal Decanal

Green grass Fatty/citrus Citrus/green Fatty

(E)-2-Nonenal Cucumber/old books (E,Z)-2,6-Nonadienal Cucumber (E,E)-2,4-Decadienal Fatty/oxidized γ-Nonalactone Coconut

Maillard reactions 2-Methyl-3-furanthiol Brothy/burnt 2-Acetyl-1-pyrroline Popcorn 3-Hydroxy-4,5-dimethyl-2(5H) furanone 2,5-Dimethyl-4-hydroxy-3(2H) furanone

Maple/spicy Burnt sugar

Adapted from: Carunchia Whetstine, Croissant, Drake (2005).

Maillard Reactions and Flavor BAKED

TOASTED POPPED

BROWNED

GRILLED Broiled Roasted

 Maillard Reactions / Thermally-processed foods:  Roasted peanuts  Toasted bread  Fried chicken  Baked potatoes  Grilled flavors

The Maillard Reaction + Reducing Sugars

Amino Acids

“Brown” aromas and colors

Early Maillard Reaction Amadori Rearrangement

Advanced Maillard Reaction

Fission Dicarbonyls

Dehydration Strecker Degradation Dicarbonyls, amino cmpds. Aldehydes, creatinine

MELANOIDINS (brown polymeric pigments)

Thermally-Generated Flavors

COOKING OF FOODS

FOOD STORAGE (Staling over shelf-life)

Positive Flavors - Roasted peanuts - Baked potatoes - Toasted bread - Grilled steak - Fried chicken

Flavor Defects - Dried / UHT milk - Whey powder - Dried sour cream - Cheese powder - Soy milk

Chemical Processes: Maillard Reactions, Caramelization, Strecker degradation

Maillard Reaction Parameters Heating Temperature Heating Time pH Water Activity

Rate Influence by Temperature 5000

SH

O 4000

Reaction Rate

N 3000

N 2000

O

1000

CHO

0 0

50

100

150

200

250

Temperature (°C) G. A. Reineccius, Flavor Chemistry and Technology, 2006

pH Effects Reaction with D-Glucose

1.2

Absorbance, 420 nm

1 L-Lysine 0.8

L-Alanine

0.6 0.4

L-Arginine 0.2

0 6

7

8

9

pH

10

11

12

Heat Treatment / Shelf-Life Storage

Off-Flavor Formation in Proteins Maillard reaction chemistry UHT Milk

Valero, Food Chem., 2000

Milk powder

Renner, J. Dairy Res., 1988; Preininger & Ullrich, 2001

Whey powder

Morr, Int. Dairy J., 1991

Dried sour cream

Marsili, ACS Symp. Ser. 971, 2007

Cheese powder

Marsili, ACS Symp. Ser. 971, 2007

Soy milk

Kwok, Food Sci. Technol., 1995

Thermally Generated Off-Flavors SPRAY-DRIED CREAM Diacetyl

+

Arginine

N

Maillard Rxn.

2,4,5-Trimethyloxazole (“melon”, “ripe kiwi”)

O R. Marsili, in Flavor of Dairy Products, ACS Symposium Series 971, K. R. Cadwallader, M. A. Drake, and R. J. McGorrin, Eds., ACS Books, Washington, D.C., 2007, 79-91.

O

SPRAY-DRIED MILK POWDER

N

Maillard Rxn.

Tryptophane

S Benzothiazole (“sulfuric, quinoline”)

+

NH2

2-Aminoacetophenone (“musty, stale”)

H. Shiratsuchi, et al., J. Jpn. Soc. Food Sci. Technol., 43, 7 (1996). M. Preininger and F. Ullrich in Gas Chromatography-Olfactometry, ACS Symp Series 782, 2001, p. 46.

Thermally Generated Off-Flavors ULTRA-HIGH TEMPERATURE (UHT) MILK Dicarbonyls

+

Amino acids

Maillard Reaction

2,6-Dimethylpyrazine (“nutty”)

Strecker Degradation

2-Ethyl-3-methylpyrazine (“nutty, earthy”) 2-Ethylpyrazine (“nutty”) Methional (“boiled potato”)

K. Iwatsuki et al., J. Jpn. Soc. Food Sci., 46, 587 (1999) E. Valero et al., Food Chem., 72, 51-58 (2000)

P. A. Vazquez-Landaverde, M. Qian, et al., J. Dairy Sci., 88, 3764 (2005)

2. Changing the food’s flavor profile due to: ● Flavor interactions ●Flavor binding ●Flavor release 

Flavor perception in food systems is governed by complex multiple interactions with proteins, as well as carbohydrate and fat components



Food systems contain multiple phases and structures which can substantially influence flavor interactions:  Phases: Emulsions, dispersions  Structures: Membranes, interfaces



The relative balance of different flavor-ingredient combinations ultimately influences overall flavor perception

Flavor Interactions with Proteins Definitions of some flavor interaction terms:

 Flavor Absorption 

Trapping of volatile flavor compounds onto non-volatile food constituents (e.g., proteins)

 Flavor Binding 

Covalent bond formation; hydrogen bonding; or hydrophobic interactions between flavor and protein

 Flavor Release 

Aroma Availability of aroma compounds to be freed from the bulk of the food into the gas phase for sensory perception



Taste Availability of non-volatile compounds to be freed from the bulk of the food into the aqueous phase for sensory perception http://chubbylemonscience.blogspot.com

Protein-Flavor Interactions 

Proteins in food can interact with flavor compounds



Flavor–protein binding interactions: The most studied are the binding of flavors to soy protein and casein (milk protein)



Flavor binding – retention or absorption of volatiles onto non-volatile protein



Forms of interactions - Hydrogen bonding: oxygen, nitrogen, sulfur reversible - Covalent bond formation irreversible

Flavor Interactions with Proteins

+ Food Protein (α-Helix)

Flavor Chemical Mixture

Protein-Flavor Complex

Protein-Flavor Interactions 

In general, alcohols and ketone-containing flavors reversibly bind through hydrophobic interactions and hydrogen bonding



Aldehyde flavors may chemically react with amino groups of proteins, forming irreversible covalent bonds (Schiff bases)



Binding capacity depends on pH, temperature, moisture content, salt level, degree of denaturation. Protein denaturation can increase flavor absorption, through greater exposure of hydrophobic regions



Result: Flavor fade (reduction of flavor intensity)

Flavor-Food Interactions:

Reactions: Flavors + Amino Groups O H2 N

N

H C

H

Aspartame

+ Benzaldehyde (Cherry)

H

HO2 C

O

CO2 CH3

N

X

CH3 H3C CH3

HO2C

N

CO2 CH3

H

“Schiff base”

O HN

HO2 C

O

N

CO2CH3

H

Neotame See also: Chobpattana, W. et al. J. Agric Food Chem. 48, 3885-3889 (2000)

Flavor Binding and Protein Structure     

Protein binding properties are influenced by its 3-D structure Hydrogen bonds between amino acids Disulfide bridges between amino acids Hydrophobic “pockets” Ionic complexes

Hydrogen bond formation

Hydrogen bonds

http://www.chemguide.co.uk/organicprops/aminoacids/proteinstruct.html

Flavor Binding and Protein Structure Cys

CH2SH

Cys

CH2SH

Disulfide bond formation

Sulfur bridge formed

CH2S CH2S

Note: Sulfur flavors (mercaptans, thiols, etc.) also form disulfide bonds with proteins

Sulfur Amino Acids – Off-Flavor Contributors

Methionine

Cysteine

Cystine

Flavor Binding and Protein Structure Leu

Hydrophobic pockets

Flavor Phe C

O

H

NH3+

Flavor

Arg, Gln, Lys

Ionic regions

Glu

COO-

Flavor

Binding/Interaction Related to the Type of Protein

Soy > Whey > Gelatin > Casein > Corn

Flavor-Protein Interactions:

Vanilla Binding with Dairy Proteins H

Vanillin Intensity

0.6

C=O

0.5

OCH3

0.4

OH 0.3

Sodium Caseinate

0.2

Whey Protein Conc.

0.1

0 0

0.125

0.25

0.5

Protein Concentration (%) Hansen and Booker in Flavor-Food Interactions, McGorrin, R.J. and Leland, J. V. ACS Sym Series #633, 1996, 75-89.

Flavor-Protein Interactions:

Vanilla Binding with Dairy Proteins H

100

Sodium Caseinate (3%)

90

% Free Vanillin

C=O

OCH3

Whey Protein Isolate (3%)

OH

80

Bovine Serum Albumin (3%) 70

60 0

20

40

60

80

Reaction Time (min.) Source: Chobpattana, W.; Jeon, I. J.; Smith, J. S.; Loughin, T. M. J. Food Science, 67, 973-977 (2002).

100

120

Flavor-Protein Interactions:

Ketone Binding with Whey Proteins -Lactoglobulin

O’Neill , T. E. in Flavor-Food Interactions, McGorrin, R.J. and Leland, J. V. ACS Sym. Series #633, 1996, 59-74.

Flavor-Protein Interactions:

Effect of Heat Treatment (75°C) 2-Nonanone Binding with Whey Protein

O’Neill , T. E. in Flavor-Food Interactions, McGorrin, R.J. and Leland, J. V. ACS Sym. Series #633, 1996, 59-74.

Protein - Flavor Applications

Flavor Challenges High-Protein Beverages

March, 2007 pp. 20-26



Difficult to select/choose appropriate flavors



Challenges to control the proper level of flavoring

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Challenges to achieve the desired flavor intensity in the finished product



Continued opportunity for taste improvement in nutritional food and drink products

Protein Sources North America Food & Beverages 2007–2011 Launches

Mintel GNPD

Challenges with Flavoring High-Protein Foods • Flavors are challenged by adding nutritional ingredients! Proteins (Soy, whey, casein, pea, rice) + HTST, UHT (Burnt, caramelized, nutty, beany, sulfuric, bitter) Amino acids, minerals (Bitterness, metallic off-flavors) • Manufacturers use many combinations / blends of proteins  Soy, whey  Soy, whey, caseinate, rice  Whey, pea, rice • To achieve optimum protein value / PDCAAS / PER • Concentrates, isolates, hydrolyzed • Minimize inherent off-flavor characteristics of an individual protein • Soy and whey proteins complement each other • Soy manages sulfide and eggy notes from whey • Net: A fairly complicated process from a flavorist’s perspective

Flavor Development - Proteins • Need to use flavor by the “bucket-load” (4-10 X more) • Proteins are good at binding / absorbing flavor

• Proteins contribute: Bitterness, astringency, chalkiness (particularly true if beverage is acidic, pH 3.5) • Optimum pH 6-7 to avoid gritty texture / astringent taste • However, pH 3.5 works best with citrus flavors (orange, lemon); actually enhances flavor; makes flavors “pop”

Flavor Development - Proteins Hydrolyzed proteins: • Clear beverages – flavor issues • Hydrolyzed proteins contribute off-tastes • Sulfur amino acids: Rubbery (cysteine, methionine) • Flavors not muted as much as intact proteins; less binding, so don’t need to add as much flavor Flavor Rebalancing: • Added flavor is initially unbalanced; (need to wait 5 days before evaluating) • Formulate flavor to increase top notes, middle notes • Compensates for expected losses during shelf-life, retort heating, etc. • Will be balanced in finished product

Challenges with Flavoring High-Protein Foods

Protein Bars Low moisture aW = 0.2 Non-thermal process Flavor system is “immobile” RT shelf life temperature swings

Protein Beverages High moisture aW = 1.0 Thermal process Flavors more reactive Flavor scalping Refrigerated

Appropriate Flavor Types Protein Bars Browned / roasted Flavors Chocolate Double fudge Mocha/coffee Chocolate/peanut butter Caramel/peanut Cookies & creme

Protein Beverages Chocolate; Fruit flavors Chocolate Banana creme Peach mango Cookies & crème Challenges Vanilla Strawberry Citrus flavors

Flavor Suppliers • Optimum to involve flavor house early in the process! • Provide as much information as possible: - Moisture content, pH - Heat process / upper temperature - Room temperature, refrigerated, frozen - % protein - Vitamins, natural/high-potency sweeteners

• Cuts development time tremendously!

Protein Milk • Milk protein concentrate • 25g protein/ serving • Shelf-life: 100 days Flavors: Chocolate, Vanilla, Strawberry, Cookies n’ Cream

Protein Ice Cream • Organic, pasture-fed cows • Soy milk, whey protein concentrate • 14-28 g of protein/serving

Flavors: Chocolate bliss, Java Gym Coffee, Chai green tea, Berry Burst l

Repositioned Products

Beyond Meat: Chicken-Free Strips

“Looks, feels, tastes and acts like chicken – without the cluck”

Soy Protein Isolate, Pea Protein Isolate

Flavor system: Chicken flavor (yeast extract), hickory smoke, spices

Flavor Masking Situations frequently occur where it is necessary to add other flavors to “mask” or cover-up sensory defects Flavor defect

Food product / ingredient

Beany, grassy Harshness, bitterness Astringency Vitamin B off-flavor Metallic

Soy beverages, bars Artificial sweeteners Low pH, whey ingredients Vitamin fortification Mineral fortification

Eckert, M.; Riker, P. Overcoming challenges in functional beverages, Food Technology, 20-26, March 2007.

Flavor “Masking” Example #1 Protein Off-flavor • Flavor Congruency – “Systems approach” – Select a flavor system which also contains the inherent off-flavor aspects of a particular protein • Example: “Earthy” notes – pea protein; “beany” notes – soy • Complement with use of peanut or nut flavors to mask

• Flavor Completion / Insertion – Instead of masking undesirable notes, utilize them as part of the flavor system • Example: “Green” notes from soy protein • Additive effect with “jammy” strawberry flavor that lacks green notes

Flavor “Masking” Example #2 Soymilk off-taste • Taste – Soy protein isolates tend to become increasingly bitter as pH is lowered

– Vanilla and peach flavors are useful to mask bitter off-notes (and the “beany” flavor of soy) – Nanoprocessing (nanoshear) may produce creamier taste; flavor emulsion stability – Benefit: less flavor is used for same taste effect

Flavor “Masking”Example #3 Bitterness off-flavor • Bitterness is typically modulated by: (1) increasing sweetness (2) blocking the bitter taste receptors • Bitterness blockers (“B-blocker”) – Sodium chloride – Monosodium glutamate – Adenosine monophosphate S. J. Keast, P. A. Breslin, Pharm. Res. 19, 1019 (2002)

Na+

Flavor “Masking”Example #4 Astringency • Not a flavor, but a mouth drying sensation • Biggest challenge in whey beverages • pH level: Increasing the pH above pH 3.5 decreases astringency, but heat stability becomes more challenging and clarity decreases. • Flavor selection: Tropical flavors (mango, pineapple, coconut) and citrus, peach, apple work well with whey protein ingredients; mask whey off-flavor and aroma. • Berry flavors (strawberry, raspberry, blueberry, etc.) are a challenge to use with whey protein ingredients; do not mask whey flavor and aroma as well. • Complementary ingredients: Adding larger carbohydrates such as soluble fiber also may decrease astringency

Summary Comments 

Consider flavor functionality early in the formulation / development process! Involve your flavor supplier ASAP!  Analytical tools can often measure and diagnose potential causes of flavor-food interactions:  Degradation during processing  Cross-reactivity with matrix components

 The need continues for practical alternatives to measure flavor interactions with total food system components.  Screen ingredients for their flavor effects using realistic model flavors (appropriate functional types and levels)