Browning

Browning as a food process

1. BROWNING As a food process BROWNING

2. Browning is a process that produces a brown color in food.

3. Importance of browning reactions in food systems Browning may increase the acceptability of food by developing appropriate flavor and color in food. For example, flavor development in tea, flavor and color development in figs and raisins, toast production in bread. Browning may cause food quality deterioration and thus a decline in the market value of food. Since color is the first sensory quality by which food is judged, browning after peeling/slicing fruit and vegetables, mushroom discoloration, and blackspots in shrimps and lobsters have great economic cost.

4. Two Types of Browning Enzymatic browning Non-enzymatic browning

5. A chemical process involving phenolases and other enzymes that produce benzoquinones and melanins from natural phenols, resulting in a brown color in fruit and vegetables. Enzymatic B rowning phenolases quinones phenols

6. Phenolases - enzymes that act on the substrates to produce the brown products Phenols - substrates acted upon by the enzymes to produce the brown products Quinones – the brown products produced by the enzymes from the substrates Phenolases Phenols Quinones

7. E B is one of the most important color reactions affecting fruit and vegetables, and seafoods .

8. Desirable effects of E B Flavor and color development in tea, coffee, cocoa, and dried fruits Production of melanoidins , which may exhibit antibacterial, antifungal, anticancer, and antioxidant properties. W ound healing and post-molting hardening of the shell ( sclerotization ) in insects and crustaceans such as shrimp and lobsters. F ermentation

9. Undesirable effects of E B Hinders ease of processing fruit slices and juices Unwanted color changes in lettuce and other green leafy vegetables; potatoes and other starchy staples such as sweet potato, breadfruit, and yam; mushrooms; fruits such as apples , avocados, bananas, grapes, olives, and peaches; and crustaceans

10. The Enzymes

11. Enzymes involved in E B are collectively called PHENOLASE and, since their discovery, their international nomenclatures have undergone marked changes.

12. Phenolase An  oxidoreductase enzyme   that catalyzes the oxidation of phenols and other related substances . This enzyme is found in many plants, fungi and microorganisms. It catalyzes the oxidation of certain molecules such as tyrosine. The action of this enzyme is evident in fruit   browning when fruits such as apples and bananas are cut or bruised.

13. PHENOLASES Polyphenol oxidase initiates browning Catechol oxidase syn. diphenol oxidase) Laccase

14. Polyphenol oxidase ( PPO) Systematic name: 1,2-Benzenediol:oxygen oxidoreductase (EC 1.10.3.2) Synonyms: phenoloxidase , phenolase , monophenol oxidase, diphenol oxidase, tyrosinase , catechol oxidase An oxidoreductase, with oxygen as the hydrogen acceptor, that acts on phenols Requires the presence of BOTH a copper prosthetic group and oxygen for its activity

15. T wo basic reactions catalyzed by PPO o- Hydroxylation of monophenols to o - diphenols ( cresolase or monophenol oxidase activity) E.g., oxidation of catechol to o -benzoquinone Oxidation of diphenol to o -benzoquinones ( catecholase or diphenol oxidase activity ) E.g., oxidation of L -tyrosine to 3,4- dihydroxyphenylalanine (occurs in potatoes)

16. PPO-Catalyzed Reactions

17. Catechol Oxidase (CO) Systematic name: 1,2-Benzenediol:oxygen oxidoreductase (EC 1.10.3.1) Synonym: Diphenol o xidase Copper-containing enzyme with a similar activity to tyrosinase (EC 1.14.18.1) Catalyzes, using dioxygen , the oxidation of a broad range of o - diphenols such as catechol to the corresponding o - quinones coupled with the reduction of oxygen to water. The yellow  compound produced (benzoquinone) is) then oxidized in air to form dark-brown  melanin. Found in fruits (e.g., banana, apple, and pear

18. Laccase (LAC or DPO) p - Diphenol oxidase or urushiol oxidase (EC 1.10.3.2 ) ( DPO or LAC ) C opper-containing oxidase that catalyzes the oxidation of p - diphenols and o - diphenols Also oxidizes 4-benzenediol to 4-benzosemiquinone O ccur in many phytopathogenic fungi, higher plants, peaches and apricots I s involved in  lignin  degradation, pigment biosynthesis and detoxification of lignin-derived products.

19. PPO and CO oxidize ONLY o - diphenols , whereas LAC oxidizes BOTH o -phenols and p -phenols.

20. PEROXIDASE (POD) A hemoprotein that catalyzes the oxidation of phenolic substrates through the associated reduction of hydrogen peroxide in the peroxidative cycle that produces reactive oxygen species such as a superoxide anion (O 2 -● ) or a hydroxyl radical (OH ● )

21. Peroxidase Oxidoreductase acting on phenols in the presence of H 2 O 2

22. Reactions C atalyzed by PPO and POD

23. Common Phenolic Compounds in Food Material 1. Simple phenols - include mono-, o -di- and tri-phenols E.g., L -tyrosine, catechol, and gallic acid 2. Cinnamic acid derivatives E.g., chlorogenic acid, p- coumaric acid, caffeic acid, ferulic acid, sinapic acid 3. Flavonoids - structurally related to flavones E.g., catechins , leucoanthocyanidins (food tannins), anthocyanins , and flavonols

24. Structures of Phenolic Compounds

25. F ood sources of Phenolic Compounds Source Phenolic Compounds Apple Chlorogenic acid (flesh), catechin (peel), catechol , DOPA Avocado 4-Methyl catechol, dopamine, pyrogallol Cacao Catechins , anthocyanins Coffee beans Chlorogenic acid, caffeic acid Lobster, shrimp Tyrosine Tea Flavonols , tannins, cinnamic acid

26. E B in Schematic Summary Phenolase Quinones Phenolics

27. How do you control E B ? Answer: Inhibit PPO activity

28. Inhibition of PPO Activity 1. Exclusion of reactants such as oxygen Denaturation of enzyme Interaction of copper prosthetic group Interaction with phenolics or quinones 1. Exclusion of reactants such as oxygen

29. (1) Oxygen Exclusion Oxygen exposure prevention, the simplest method of which is water immersion. The method is limited for fruit and vegetables as they will brown upon air re-exposure or via oxygen occurring naturally in plant tissue. May lead to anaerobiosis in case of extended storage of fruits and vegetables, which in turn may lead to tissue breakdown

30. Inhibition of PPO Activity 1. Exclusion of reactants such as oxygen Denaturation of enzyme Interaction of copper prosthetic group Interaction with phenolics or quinones

31. (2) Enzyme Denaturation Heat treatment PPOs work at room temperature.

32. Optimal Temperatures for A ctivity of PPOs from D ifferent S ources Source Optimal Temp ( o C ) Apricot 25 Banana 37 Apple 25-30 Grape 25-30 Potato 22

33. (2) Enzyme Denaturation Heat treatment PPOs work at room temperature. pH reduction Optimum pH range of most PPOs: 4-7 Application of powerful PPO inhibitors

34. PPO Inhibitors Cinnamon acid, benzoic acid, ascorbic acid in apple juice Carbon monoxide in mushrooms 4-Hexylresorcinol in shrimp Inorganic halides Sodium chloride Zinc chloride + calcium chloride, ascorbic acid, citric acid

35. Inhibition of PPO Activity 1. Exclusion of reactants such as oxygen Denaturation of enzyme Interaction of copper prosthetic group Interaction with phenolics or quinones

36. (3) Binding of Copper Prosthetic Group Addition of c omplexing agents that binds to the copper prosthetic group E .g., ethylenediaminetetraacetate ( EDTA) diethyldithiocarbamate (DIECA), sodium azide , potassium ethylxanthate , sodium acid pyrophosphate, and citric acid Cu 2+ is essential for PPO activity. Thus, chelating Cu 2+ will inhibit PPO activity

37. Inhibition of PPO Activity 1. Exclusion of reactants such as oxygen Denaturation of enzyme Interaction of copper prosthetic group Interaction with phenolics or quinones

38. (4) Prevention of Action on Polyphenol Addition of polyvinylpolypyrollidone (PVPP), which bind polyphenols, thereby eliminating substrate of PPO, preventing browning Addition of compounds with similar chemical structures to o - diphenols but are not PPO substrates Guaiacol Resorcinol Phloroglucinol

39. Methods of E B prevention used in the Food Industry? Oxygen exclusion Heat treatment Acid treatment Application of sulfur dioxide and sulfites

40. Oxygen Exclusion Water immersion Use of O 2 -impermeable packages Use of edible films such as sulfated polysaccharides, e.g., carrageenan, amylose sulfate, and xylan sulfate as wrappers Prevention of mechanical bruising during shipping of fresh fruits to prevent O 2 exposure of fruit flesh

41. 5. Use of N 2 headspace in packaging 6. Reduced oxygen packaging (i.e., vacuum packaging, modified atmosphere packaging, controlled atmosphere packaging) But not too low an O 2 concentration: Off-flavor production by anaerobic glycolysis Risk of Clostridium botulinum growth

42. Heat Treatment Blanching Water immersion at 93 o C for 2 min. Steaming and water immersion at 70-105 o C Pasteurization at 60-85 o C Refrigeration, e.g., freezing at - 18 o C High-temperature inactivates PPO. Low temperature retards PPO activity. The heat inactivation of PPO is dependent on time and pH.

43. Acid Treatment 1. Addition of acids occurring naturally in plants, e.g., citric, malic, phosphoric and ascorbic acids [ Ascorbic acid is a very effective PPO inhibitor. At its level used in the industry, it has no detectable flavor or corrosive action on metals .] Used extensively in the food industry Based on the fact that lowering plant tissue pH will retard E B Sodium acid pyrophosphate has been suggested as an alternative to organic acids. SSAP is less sour than most organic acids and minimizes after-cooking blackening in potatoes.

44. S ulfur Dioxide and S ulfite Application Powerful reducing agents and PPO inhibitors that can be applied in gaseous or solution form. Applicable in cases where heating will cause undesirable effects, e.g., textural changes and off-flavor Disadvantages: Off-flavor and off-odor development, bleaching of natural food pigments, hastening of can corrosion, degradation of Vitamin B1, and toxicity at high levels Advantages: High effectivity , preservation of Vitamin C, and low cost

45. A chemical process involving __________ and other enzymes that produce ____________ and melanins from natural _______, resulting in a brown color in fruit and vegetables. Fill in the blanks: Enzymatic B rowning

46. NON-ENZYMATIC BROWNING BROWNING

47. NON-ENZYMATIC BROWNING (NEB) A chemical process that produces a brown color in food without involving enzymes without

48. TYPES OF NEB Maillard reaction Caramelization Ascorbic acid oxidation Lipid browning Maillard reaction

49. MAILLARD REACTION A chemical reaction between a free amino group from amino acids, peptides, or proteins and the carbonyl group of a reducing sugar A NEB reaction, caused by the condensation of an amino group and a reducing sugar, resulting in complex changes in biological and food systems A chemical reaction between a free amino group from amino acids, peptides, or proteins and the carbonyl group of a reducing sugar A NEB reaction, caused by the condensation of an amino group and a reducing sugar , resulting in complex changes in biological and food systems

50. FEATURES OF MAILLARD REACTION First described by Louis Maillard in 1912 Generally requires heat addition, BUT also occurs during storage Favored under alkaline condition

51. ADVANTAGES OF MAILLARD REACTION Development of caramel aroma Development of golden brown color Formation of antioxidative Maillard reaction products (e.g., in honey and tomato puree manufacture)

52. DISADVANTAGES OF MAILLARD REACTION Dark pigmentation Off-flavor development Deterioration of proteins during food processing and storage Reduction in protein digestibility Loss of nutritional quality Formation of mutagenic and carcinogenic compounds during frying, grilling , and baking of meat ( e.g., acrylamide)

53. IUPAC name is  prop-2-enamide (C 3 H 5 NO). Considered a carcinogen D iscovered accidentally in food in April 2002 by scientists in Sweden Forms at moderate levels (5–50 g/kg) during heating of protein-rich food and at higher levels (150–4000 g/kg) during heating of carbohydrate-rich food ACRYLAMIDE

54. Asparagine + reducing sugar Strecker aldehyde Acrylamide Mutagen from the Maillard Reaction

55. Aldose sugar + amino compound N -substituted glycosylamine + H 2 O Amadori rearrangement 1-amino-1-deoxy-2-ketose S ugar dehydration Sugar fragmentation Strecker degradation Volatile and nonvolatile monomers Schiff base of HMF or furfural Reductones Fission products Aldehydes Others Highly colored products Aldols and N -free products Melanoidins Aldol condensation Carbonyl amine polymerization MAILLARD REACTION

56. THREE STAGES OF THE MAILLARD REACTION 1. Early stage 2. Intermediate stage 3. Final stage

57. C ondensation of primary amino groups of amino acids, peptides, or proteins with the carbonyl group of reducing sugars (aldoses) -- T he carbonylamino reaction Formation of Amadori products via Schiff’s base formation and Amadori rearrangement EARLY STAGE

58. amino acid or protein + glucose Schiff’s base b - pyranosyl Amadori rearragement Amadori products EARLY STAGE

59. AMADORI PRODUCTS Alanine-fructose and leucine - fructose (precursors of numerous compounds important in the formation of characteristic flavors, aromas, and brown polymers) Hydroxyproline - fructose and tryptophan-fructose These are formed before the occurrence of sensory changes. Alanine-fructose Tryptophan-fructose

60. INTERMEDIATE STAGE B reakdown of Amadori compounds (or other products related to Schiff’s base) F ormation of degradation products, reactive intermediates (3-deoxyglucosone), and volatile compounds (flavor compounds)

61. Flavor compounds in processed foods, such as beef products, soy products, processed cheese, coffee, tea, potatoes Include p yrazines , p yrroles , oxazoles , oxazolines , and thiazoles (formed from sulfur amino acids) ALSO formed from Strecker degradation HETEROCYCLIC COMPOUNDS

62. Oxidative degradation of amino acids into aldehydes in the presence of α - dicarbonyls or other conjugated dibarbonyls formed from Amadori compounds. NOT directly involved in pigment formation Aldehydes formed contribute to flavor development STRECKER DEGRADATION

63. STRECKER DEGRADATION PATHWAY

64. FINAL STAGE P roduction of nitrogen-containing brown polymers and copolymers known as melanoidins Series of aldol condensation and polymerization reactions

65. TWO MAJOR PATHWAYS FROM AMADORI COMPOUNDS TO MELANOIDINS Amadori compounds

66. Aromas and volatile compounds produced from L -amino acids in the Maillard reaction system Amino acid Volatile compound Aroma Alanine Acetaldehyde Roasted barley Cysteine Thiol , H 2 S Meaty Valine 2-Methylpropanal Leucine 3-Methylbutanal Cheesy Lysine Breadlike Methionine Methional

67. pH Type of reducing sugar Type of amino acid Temperature Concentration and ratio of reducing sugar to amino acid Water activity (Aw ) Metals FACTORS AFFECTING THE MAILLARD REACTION

68. An increase in pH enhances Maillard reaction Therefore, h igh acidity (that is, low pH) makes food less susceptible to the reaction. Has a less dramatic effect on aroma than temperature, time or water content The most desirable meaty and pot-roasted aroma has been obtained at pH 4.7 NOTE B uffer concentration also affects the reaction: A higher buffer concentration leads to a higher reaction rate. pH

69. Pentoses (e.g., ribose) react more readily than hexoses (e.g., glucose) Hexoses are more reactive than disaccharides (e.g., lactose) Glucose is more reactive than fructose And therefore pentoses , hexoses and glucose enhance Maillard reaction compared with their counterparts. TYPE OF REDUCING SUGAR

70. TYPE OF AMINO ACID

71. An increase in temperature increases the rate of browning. The temperature of a chemical reaction is often expressed as the activation energy ( Ea ), which is highly dependent on pH and the participating reactants; thus, it is difficult to isolate the effect of temperature as a single variable. TEMPERATURE

72. Browning reaction rate increases with increasing glycine:glucose ratio in the range from 0.1:1 to 5:1 ( Wolfrom et al., 1974). Using a model system of intermediate moisture (aw, 0.52), Warmbier et al. (1976) observed an increase in browning reaction rate when the molar ratio of glucose to lysine increased from 0.5:1 to 3.0:1. CONCENTRATION AND RATIO OF REDUCING SUGAR TO AMINO ACID

73. The Maillard reaction occurs less readily in food with high a w . At a high a w , the reactants are diluted. BUT note that a low a w does not translate to a high reaction rate . Note that, at a low a w , the mobility of the reactants is limited, despite their increased concentrations. The browning reaction rate is maximum in the a w range from 0.5 to 0.8 in dried and intermediate-moisture foods. WATER ACTIVITY (a w )

74. Metals form metal complexes with amino acids and for yet unexplained reasons such complex formation hastens Maillard reaction For instance, browning is accelerated by Cu 2+ and Fe 3+ . METALS

75. TYPES OF NEB Maillard reaction Caramelization Ascorbic acid oxidation Lipid browning Maillard reaction Caramelization

76. CARAMELIZATION Pyrolysis of food carbohydrates by heat treatment above the melting point of the sugar under alkaline or acidic condition Involves only carbohydrates, not amines Occurs when surfaces are heated strongly, when processing foods with high sugar content, or in wine processing

77. DESIRABLE EFFECTS OF CARAMELIZATION Pleasant caramel flavor Enticing brown color in some foods

78. Formation of mutagenic compounds Excessive changes in sensory attributes UNDESIRABLE EFFECTS OF CARAMELIZATION

79. STEPS IN CARAMELIZATION OF REDUCING SUGARS 1. Ring opening of the hemiacetal ring 2. Enolization via acid-base catalysis 3. Formation of isomers (aldose to ketose interconversion ; rate increases with increasing pH)

80. ISOMER FORMATION STEP

81. STEPS IN CARAMELIZATION OF REDUCING SUGARS 1. Ring opening of the hemiacetal ring 2. Enolization via acid-base catalysis 3. Formation of isomers (aldose to ketose interconversion ; rate increases with increasing pH) 4. Dehydration - leads to the formation of furaldehydes (e.g., hydroxymethylfurfural )

82. DEHYDRATION STEP

83. 5. Formation of fragmentation products such as acetol , acetoin , and diacetylformic and oxidation products such as acetic, and other organic acids 6. Reaction of these products, forming brown pigments and flavor compounds STEPS IN CARAMELIZATION OF REDUCING SUGARS

84. FACTORS AFFECTING CARAMELIZATION pH 2. Temperature 3 . Water activity 4. Type of sugar

85. pH Reaction occurs faster under alkaline condition than under neutral or acid condition. The optimum pH for the reaction is 10. Temperature Reaction is favored at > 120 o C. Reaction rate increases from 80 to 110 o C. Aw Faster browning at Aw approaching 1 than at Aw=0.75 Type of sugar Faster reaction with fructose than with sucrose as well as with glucose than with starch Sugars with more reducing groups hastens reaction .

86. TYPES OF NEB Maillard reaction Caramelization Ascorbic acid oxidation Lipid browning Maillard reaction Caramelization Ascorbic acid oxidation

87. ASCORBIC ACID OXIDATION (AAO) Ascorbic acid browning Spontaneous thermal decomposition of ascorbic acid under both aerobic and anaerobic conditions, by oxidative or nonoxidative mechanisms, in either the presence or absence of amino compounds Observed in citrus , asparagus, broccoli, cauliflower, peas, potatoes, spinach, apples, green beans, apricots, melons, strawberries, corn, and dehydrated fruits

88. FACTORS AFFECTING AAO Temperature Salt and sugar concentration pH Oxygen Enzyme ( ascorbic acid oxidase) Metal catalysts (Cu 2+ and Fe 2+ ) A mino acids O xidants or reductants Initial concentration of ascorbic acid R atio of ascorbic acid to dehydroascorbic acid

89. Aerobic Anaerobic

90. TYPES OF NEB Maillard reaction Caramelization Ascorbic acid oxidation Lipid browning Maillard reaction Caramelization Ascorbic acid oxidation Lipid browning

91. LIPID BROWNING O xidative deterioration of unsaturated glycerides followed by polymerization accelerated by ammonia , amines or proteins Protein browning caused by reaction of acetaldehyde (derived from unsaturated lipids) with protein-free amino groups, by repeated aldol condensations Protein-oxidized fatty acid reactions

92. First observed in discoloration of white fish muscle during frozen storage May be non-enzymatic or enzymatic Its first stage is lipid oxidation, which produces hydroperoxides as the initial products Via polymerization, brown oxypolymers can be produced subsequently from lipid oxidation derivatives LIPID BROWNING

93. Oxidized products can also interact with free amino groups of amino acids, peptides, proteins Observed during storage and processing of some fatty foods, salted sun-dried fish, boiled and dried anchovy, smoked tuna, meat and meat products, and rancid oils and fats with amino acids or proteins LIPID BROWNING

94. Comparison of Mechanisms of Nonenzymatic Browning Mechanism Requires O 2 Requires amino group ph optimum in initial reaction Maillard reaction − + Alkaline Caramelization − − Alkaline, acid Ascorbic acid + − Slightly acid oxidation

95. Reaction of 4,5-epoxy-2-alkenals (formed during lipid peroxidation) with the amino group of amino acids or proteins Always accompanied by the production of N -substituted pyrroles (II ), which are stable N -Substituted 2-(1-hydroxyalkyl) pyrroles are also formed, but are unstable ; they polymerize rapidly and spontaneously to produce brown macromolecules with fluorescent melanoidin -like characteristics LIPID BROWNING

96. DESIRABLE EFFECT OF LIPID BROWNING Produces lipid-amino acid reaction products that exert antioxidant properties when added to vegetable oils

97. L oss of nutritional quality due to the destruction of essential amino acids such as tryptophan, lysine, and methionine and of essential fatty acids. D ecrease in digestibility and inhibition of proteolytic and glycolytic enzymes UNDESIRABLE EFFECT OF LIPID BROWNING

98. CONTROL OF NONENZYMATIC BROWNING Addition of sulfites, thiol compounds, maltitol , sugars, and sorbitol Modified atmosphere packaging Microwave heating Ultrasound assisted thermal processing Pulsed electric field processing Carbon dioxide-assisted high-pressure processing

99. END