Food Chemistry 3 FCHE30

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Food Chemistry 3FCHE30

• MODULE 8
• Enzymic Browning

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General overview of enzymatic browning

• Appearance, flavour, texture and nutritional
  value are four attributes considered by consumers
  when making food choices.
• Appearance which is significantly impacted by
  colour is one of the first attributes used by
  consumers in evaluating food quality.
• Colour may be influenced by naturally occurring
  pigments such as chlorophylls, carotenoids and
  anthocyanins in food, or by pigments resulting
  from both enzymatic and non-enzymatic reactions.

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General overview of enzymatic browning

• Enzymatic browning is one of the most important
  colour reactions that affects fruits, vegetables
  and seafoods. It is catalysed by the enzyme
  polyphenol oxidase, which is also referred to as
  phenoloxidase, phenolase, monophenol oxidase,
  diphenol oxidase and tyrosinase.
• Enzymatic browning is one of the most studied
  reactions in fruits, vegetables and seafoods.

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Economic benefits of browning in fruits and
vegetables

• Some enzymatic browning reactions are however
  very beneficial to the overall acceptability of
  foods. Tea, coffee, and cocoa are important
  commodities for many developing countries.
• Colour development in cocoa is facilitated by
  polyphenol oxidase activity during fermentation
  and drying. Polyphenol oxidases are also
  responsible for development of the characteristic
  golden brown colour in dried fruits such as
  raisins, prunes, dates and figs.

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Economic benefits of browning in fruits and
vegetables

• Blanching is generally required for inactivation
  of the enzyme after colour development, in order
  to minimize discolouration
• Polyphenol oxidases are believed to play key
  physiological roles both in preventing insects
  and microorganisms from attacking plants and as
  part of the wound response of plants and plant
  products to insects, microorganisms and bruising.
• As fruits and vegetables ripen, their
  susceptibility to disease and infestation is
  increased due to a decline in their phenolic
  content.
• Phenoloxidase enzymes endogenous to fruits and
  vegetables, catalyse the production of quinones
  from their phenolic constituents.

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Economic benefits of browning in fruits and
vegetables

• Once formed, these quinones undergo
  polymerization reactions, leading to the
  production of melanins, which exhibit both
  antibacterial and antifungal activity and assist
  in keeping the fruit and/or vegetable
  physiologically wholesome.
• Research describing the antibacterial, anticancer
  and antioxidant nature of melanins has triggered
  considerable interest in enzymatic browning

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Economic losses due to browning in fruits and
vegetables

• Increases in fruit and vegetable markets
  projected for the future will not occur if
  enzymatic browning is not understood and
  controlled.
• Enzymatic browning is one of the most devastating
  reactions for many exotic fruits and vegetables,
  in particular tropical and subtropical varieties.
  
• It is estimated that over 50 percent losses in
  fruit occur as a result of enzymatic browning

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Economic losses due to browning in fruits and
vegetables

• Such losses have prompted considerable interest
  in understanding and controlling phenoloxidase
  enzymes in foods.
• Lettuce, other green leafy vegetables, potatoes
  and other starchy staples, such as sweet potato,
  breadfruit, yam, mushrooms, apples, avocados,
  bananas, grapes, peaches, and a variety of other
  tropical and subtropical fruits and vegetables,
  are susceptible to browning and therefore cause
  economic losses for the agriculturist.

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Economic losses due to browning in fruits and
vegetables

• These losses are greater if browning occurs
  closer to the consumer in the processing scheme,
  due to storage and handling costs prior to this
  point.
• The control of browning from harvest to consumer
  is therefore very critical for minimizing losses
  and maintaining economic value to the
  agriculturist and food processor. Browning can
  also adversely affect flavour and nutritional
  value.

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Figure 1 shows examples of enzymatic browning in
fruits and vegetables.
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Enzymatic browning in fruits and vegetables
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Enzymatic browning in fruits and vegetables
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Enzymatic browning in fruits and vegetables
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Enzymatic browning in fruits and vegetables
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Enzymatic browning in fruits and vegetables
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PHYSIOLOGICAL ROLE OF ENZYMATIC BROWNING

• In order for the food scientist to better
  understand how to prevent enzymatic browning, it
  is important to understand why polyphenol oxidase
  is present in plant and animal tissues.
• Despite knowing and hypothesizing some functions
  of polyphenol oxidase in these tissues,
  researchers are still trying to piece together
  the functional puzzle of this enzyme in both
  plant and animal systems

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Plants

• Polyphenol oxidases were first discovered in
  mushrooms and are widely distributed in nature.
• They appear to reside in the plastids and
  chloroplasts of plants, although freely existing
  in the cytoplasm of senescing or ripening plants
• Cloning and sequencing studies of the copper A
  binding region of these enzymes shows high
  conservation between polyphenol oxidases from
  plants, microorganisms and animals.

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Plants

• Polyphenol oxidase is thought to play an
  important role in the resistance of plants to
  microbial and viral infections and to adverse
  climatic conditions.
• Phenolics, such as chlorogenic acid, caffeic acid
  and scopolin, etc., which are substrates of this
  enzyme have been shown to exhibit fungicidal
  properties.

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Plants

• Polyphenol oxidase catalyses the initial step in
  the polymerization of phenolics to produce
  quinones, which undergo further polymerization to
  yield dark, insoluble polymers referred to as
  melanins.
• These melanins form barriers and have
  antimicrobial properties which prevent the spread
  of infection or bruising in plant tissues.
• Plants, which exhibit comparably high resistance
  to climatic stress, have been shown to posses
  relatively higher polyphenol oxidase levels than
  susceptible varieties.

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Plants

• Other enzyme systems in plants, such as
  chitinase, peroxidase, lipoxygenase,
  phenylalanine ammonia lyase, b-1,3-glucanase,
  etc., also show increased activity when subjected
  to stress

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Characteristics of polyphenol oxidase

• Polyphenol oxidase catalyses two basic reactions
  
• hydroxylation to the o-position adjacent to an
  existing hydroxyl group of the phenolic substrate
  (monophenol oxidase activity),
• and oxidation of diphenol to o-benzoquinones
  (dipehnol oxidase activity).
• Both reactions utilize molecular oxygen as a
  co-substrate.
• when both monophenol- and diphenol oxidases are
  present in plants, the ratio of monophenol to
  diphenol oxidase activity is usually 110 or as
  low as 140.

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Monophenol oxidase

• Monophenol oxidase catalyses the hydroxylation of
  monophenols to o-diphenols.
• The enzyme is referred to as tyrosinase in
  animals, since L-tyrosine is the major
  monophenolic substrate.
• In plants, the enzyme is sometimes referred to as
  cresolase owing to the ability of the enzyme to
  utilize the monophenolic substrate, cresol.
• Tyrosinase activity is also used to describe
  monophenol and diphenol oxidases in plant
  systems, although L-tyrosine is probably not a
  major substrate for the enzyme in plant systems,
  considering the rich abundance of phenolics in
  plant systems.

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Monophenol oxidase

• This rich abundance of phenolics in plants is
  also the probable reason for referring to the
  enzyme as a polyphenol oxidase.
• Monophenol oxidase pathway producing the
  diphenol.

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Phenolic substrates

• Phenolic compounds are widely distributed in the
  plant kingdom and are considered to be secondary
  metabolites.
• Structurally they contain an aromatic ring
  bearing one or more hydroxyl groups, together
  with a number of other substituents.
• Plants provide nearly all the phenols found in
  higher animals, since higher animals are
  incapable of synthesizing compounds with
  benzonoid rings from aliphatic precursors.

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Phenolic substrates

• The polyphenolic composition of fruits varies in
  accordance with species, cultivar, degree of
  ripening and environmental conditions of growth
  and storage.
• Phenolics also contribute to colour, astringency,
  bitterness, and flavour in fruits.
• Phenolic compounds occurring in food materials
  are mostly of the flavonoid type. Of the
  naturally occurring flavonoid compounds,
  anthocyanidins, flavonols, and cinnamic acid
  derivatives occur most frequently in foods.

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Phenolic substrates

• Catechins are also naturally occurring compounds,
  which are structurally related to other
  flavonoids having the basic nucleus of
  1,3-diphenylpropane
• Flavonols, together with flavones and flavanones
  are light yellow in colour, and are collectively
  termed anthoxanthin pigments.
• Quercetin, myricetin, and kaempferol are the most
  commonly occurring flavonols and are generally
  glycosylated.
• The majority of naturally occurring flavonols
  possess a B-ring hydroxylation pattern similar to
  catechol.

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Phenolic substrates

• Catechol can therefore be considered to be the
  significant o-dihydroxyphenol.
• It becomes extremely important as a model
  substrate in enzymatic oxidation studies.
• Tyrosine on the other hand which is a monohydroxy
  phenol, is an important amino acid.
• Hydroxylation of tyrosine leads to the formation
  of dihydroxyphenylalanine (DOPA).

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Phenolic substrates

• Relatively few of the phenolic compounds in
  fruits and vegetables serve as substrates for
  polyphenol oxidase.
• Catechins, cinnamic acid esters, 3,4-dihydroxy
  phenylalanine (DOPA), and tyrosine are the most
  important natural substrates of polyphenol
  oxidase in fruits and vegetables.
• The main substrates of polyphenol oxidase in
  certain fruits and vegetables do not however
  commonly occur as phenolic constituents of plant
  material.

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CONTROL OF BROWNING

• Enzymatic browning does not occur in intact plant
  cells since phenolic compounds in cell vacuoles
  are separated from the polyphenol oxidase which
  is present in the cytoplasm.
• Once tissue is damaged by slicing, cutting or
  pulping, however, the formation of brown pigments
  occurs.
• Both the organoleptic and biochemical
  characteristics of fruits and vegetables are
  altered by pigment formation.

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CONTROL OF BROWNING

• The rate of enzymatic browning in fruit and
  vegetables is governed by the active polyphenol
  oxidase content of the tissues, the phenolic
  content of the tissue, pH, temperature and oxygen
  availability within the tissue.

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CONTROL OF BROWNING

• polyphenol oxidase catalyses the oxidation of
  phenols to o-quinones, which are highly reactive
  compounds.
• O-quinones thus formed undergo spontaneous
  polymerization to produce high-molecular-weight
  compounds or brown pigments (melanins).
• These melanins may in turn react with amino acids
  and proteins leading to enhancement of the brown
  colour produced.

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CONTROL OF BROWNING

• Many studies have focused on either inhibiting or
  preventing polyphenol oxidase activity in foods.
• Various techniques and mechanisms have been
  developed over the years for the control of these
  undesirable enzyme activities.
• These techniques attempt to eliminate one or more
  of the essential components (oxygen, enzyme,
  copper, or substrate) from the reaction.

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CONTROL OF BROWNING

• i) The elimination of oxygen from the cut surface
  of fruits or vegetables greatly retards the
  browning reaction. Browning however occurs
  rapidly upon exposure to oxygen. Exclusion of
  oxygen is possible by immersion in water, syrup,
  brine, or by vacuum treatment.

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CONTROL OF BROWNING

• ii) This copper prosthetic group of polyphenol
  oxidases must be present for the enzymatic
  browning reaction to occur. Chelating agents are
  effective in removing copper.

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CONTROL OF BROWNING

• iii) Inactivation of the polyphenol oxidases by
  heat treatments such as steam blanching is
  effectively applied for the control of browning
  in fruits and vegetables to be canned or frozen.
  Heat treatments are not however practically
  applicable in the storage of fresh produce.

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CONTROL OF BROWNING

• iv) Polyphenol oxidase catalyses the oxidation of
  phenolic substrates such as caffeic acid,
  protocatechuic acid, chlorogenic acid, and
  tyrosine. Chemical modification of these
  substrates can however prevent oxidation.

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CONTROL OF BROWNING

• v) Certain chemical compounds react with the
  products of polyphenol oxidase activity and
  inhibit the formation of the coloured compounds
  produced in the secondary, non-enzymatic reaction
  steps, which lead to the formation of melanin.

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CONTROL OF BROWNING

• Many techniques are applied in the prevention of
  enzymatic browning.
• Relatively new techniques, such as the use of
  killer enzymes, naturally occurring enzyme
  inhibitors and ionizing radiation, have been
  explored and exploited as alternatives to heat
  treatment and the health risks associated with
  certain chemical treatments.
• Processing technologies applied in the control of
  enzymatic browning in fruits and vegetables are
  now reviewed.

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CONTROL OF BROWNING - Inhibitors

• Enzymatic browning can be inhibited by targeting
  the enzyme, the substrates (oxygen and
  polyphenols) or the products of the reaction.
• i) Inhibition targeted toward the enzyme
• ii) Inhibition targeted toward the substrate
• iii) Inhibition targeted toward the products

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Inhibitors i) Inhibition targeted toward the
enzyme

• Classified the inhibitors which act directly on
  polyphenol oxidase into two groups.
• The first group, which consists of metal ion
  chelators, such as azide, cyanide, carbon
  monoxide, halide ions and tropolone, is well
  documented for the inhibition of polyphenol
  oxidase from various sources.
• The chloride ion was shown to be noncompetitive
  for apple polyphenol oxidase, while other halide
  ions were observed to have a competitive
  inhibitory effect

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Inhibitors i) Inhibition targeted toward the
enzyme

• The second group of inhibitors, which consists of
  aromatic carboxylic acids of the benzoic and
  cinnamic series, has been widely studied
• Compounds of this group behave as competitive
  inhibitors of polyphenol oxidase, owing to their
  structural similarity with phenolic substrates.

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Inhibitors ii) Inhibition targeted toward the
substrate

• Enzymatic browning can be controlled by removal
  of either the oxygen or phenolic substrates, from
  the reaction medium.
• Elimination of oxygen is perhaps the most
  satisfactory methodology for preventing phenol
  oxidase catalysed phenolic oxidation.
• The removal of oxygen can however result in
  metabolic deviations since excessive reduction of
  oxygen induces anaerobic metabolism, leading to
  breakdown and off flavour development in foods

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Inhibitors ii) Inhibition targeted toward the
substrate

• Vacuum packaging of pre-peeled potatoes to
  exclude oxygen, was observed to extend their
  shelf life.
• Vacuum packaged products however rapidly undergo
  browning upon exposure to air. Anaerobic
  conditions created by vacuum packaging are a
  cause for safety concern in that they are
  potentially capable of supporting the growth of
  Clostridium botulinum and the production of its
  toxin

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Inhibitors ii) Inhibition targeted toward the
substrate

• Specific adsorbents, which undergo complexation
  with the phenolic substrate may be applied in the
  physical elimination of phenolic compounds from
  food systems.
• The use of cyclodextrins for the removal of
  phenolic compounds from raw fruit and vegetable
  juices has been patented in the United States
• Cyclodextrins are thought to inhibit polyphenol
  oxidase activity through the formation of
  inclusion complexes with polyphenols

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Inhibitors ii) Inhibition targeted toward the
substrate

• Sulphated polysaccharides also have an inhibitory
  effect on browning
• Apart from possible complexation, sulphate groups
  are believed to exert their inhibitory effect
  through chelation of the copper prosthetic group
  of the polyphenol oxidase

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Inhibitors ii) Inhibition targeted toward the
substrate

• Enzymatic modification of phenolic substrates may
  serve to inhibit polyphenol oxidase activity.
• O-methyltransferase for example converts
  o-dihydroxy phenolics to the corresponding
  methoxy derivatives, which do not serve as
  substrates for polyphenol oxidase
• Similarly, protocatechuate 3,4-dioxigenase
  purified from Pseudomonas aeruginosa prevents the
  browning of Gravenstein apple juice due to
  substrate modification

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Inhibitors ii) Inhibition targeted toward the
substrate

• These enzyme modification techniques are not
  however feasible in commercial use, owing to the
  high cost of these enzymes.

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Inhibitorsiii) Inhibition targeted toward the
products

• O-quinones, which are the products of diphenol
  oxidation, are capable of reacting with each
  other, resulting in the formation of dimers of
  the original phenol.
• These dimers, which possess an o-diphenolic
  structure, undergo re-oxidation resulting in the
  formation of larger oligomers of varying colour
  intensities. Ascorbic acid, thiol compounds,
  sulphites, and amino acids are however capable of
  inhibiting dimer formation and re-oxidation
  either by reducing o-quinones to o-diphenols, or
  through the formation of colourless addition
  products.

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Classification of Inhibitors

• The use of browning inhibitors in food processing
  is restricted by considerations relevant to
  toxicity, wholesomeness, and effect on taste,
  flavour, texture, and cost.
• Browning inhibitors may be classified in
  accordance with their primary mode of action. Six
  categories of polyphenol oxidase inhibitors are
  applicable in the prevention of enzymatic browning

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Classification of Inhibitors

• These include (1) reducing agents (2)
  acidulants (3) chelating agents (4) complexing
  agents (5) enzyme inhibitors (6) enzyme
  treatments. Each category of inhibitor is now
  discussed

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Representative inhibitors of enzymatic browning

• Reducing agents
• sulphiting agents
• ascorbic acid and analogs
• cysteine
• glutathione
• Chelating agents
• phosphates
• EDTA
• organic acids

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Representative inhibitors of enzymatic browning

• Acidulants
• citric acid
• phosphoric acid
• Enzyme inhibitors
• aromatic carboxylic acids
• aliphatic alcohol
• anions
• peptides
• substituted resorcinols

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Representative inhibitors of enzymatic browning

• Enzyme treatments
• oxygenases
• o-methyl transferase
• proteases
• Complexing agents
• cyclodextrins

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Sulphiting agents

• Sulphites are the most widely used inhibitors of
  enzymatic browning. Sulphiting agents include
  sulphur dioxide (SO) and several forms of
  inorganic sulphite that liberate SO2 under the
  conditions of their use.
• SO2 sulphur dioxide
• SO32- sulphite
• HSO3- bisulphite
• S2O52- metabisulphite

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Sulphiting agents

• SO2 and sulphite salts form sulphurous acid
  (H2SO3) and exist as a mixture of the ionic
  species, bisulphite (HSO3-) and sulphite (SO32-)
  anions in aqueous solution.
• The predominant ionic species varies in
  accordance with pH, ionic environment, water
  activity, presence of non-electrolytes, and
  concentration of the medium in which they are
  dissolved.
• Maximum HOS3- concentrations exist at pH 4, while
  at pH 7, both SO32- and HSO3- exist in
  approximately equivalent concentrations

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Sulphiting agents

• Increased concentrations of sulfite at pHs of
  less than 5 were observed to enhance the
  inhibition of polyphenol oxidase-catalysed
  browning
• The dibasic acid undergoes ionization according
  to the following reaction scheme
• SO2.H2O -gt (H2SO3)- -gt HSO3- H
• HSO3- -gtSO32- H

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Sulphiting agents

• Sulphites serve a multifunctional role in foods.
• They possess antimicrobial activity and inhibit
  both enzymatic and non-enzymatic browning
  reactions.
• proposed that bisulphite exerted a competitive
  inhibitory effect on polyphenol oxidase, by
  binding a sulphydryl group at the active site of
  the enzyme.

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Sulphiting agents

• on the other hand, proposed that bisulphate
  inhibition was due to the reaction of sulphites
  with intermediate quinones, resulting in the
  formation of sulphoquinones, which irreversibly
  inhibited polyphenol oxidase, causing complete
  inactivation.
• Mechanisms involved in the control of enzymatic
  browning by sulphites are shown in Figure
• The primary role of reducing agents such as
  sulphiting agents in the inhibition of enzymatic
  browning is to reduce the pigment precursors
  (quinones) to colourless, less-reactive
  diphenols.

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 .
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Sulphiting agents

• Although sulphites are very effective in
  controlling browning, they are subject to
  regulatory restrictions owing to their
  potentially adverse effects on health.
• Many reports have described allergic reactions
  in humans, following the ingestion of
  sulphite-treated foods by hypersensitive
  asthmatics.
• The use of sulphiting agents in food processing
  is based on sulfur dioxide equivalence

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Sulphiting agents

• Food and Agriculture Organization (FAO) recommend
  an acceptable sulphite daily intake of 0-0.7 mg
  sulphur dioxide per kg of body weight.

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Sulphiting agents

• Sulphites are currently applied for the
  inhibition of melanosis (blackspot) in shrimp,
  potatoes, mushrooms, apples, and other fruits and
  vegetables.
• Sulphites are also applied in stabilizing the
  flavour and colour of wines.
• Sulphite concentrations necessary for controlling
  enzymatic browning vary widely in accordance with
  the food material and the time required for
  inhibition of the browning reaction

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Sulphiting agents

• Where only monophenolic substrates, such as
  tyrosine are present, as in the case of potatoes,
  relatively low levels of sulphite are effective
  in inhibiting browning.
• On the other hand, where diphenols are present,
  as is the case in avocados, much higher sulfite
  concentrations are required for the control of
  browning.

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Sulphiting agents

• Sulphites no longer have "Generally Required as
  Safe Status" (GRAS) status for use on fruits and
  vegetables served raw, sold raw or presented to
  the consumer as raw in the United States.
• According to the United States Federal Register
  (1988) foods containing detectable levels of a
  sulphiting agent, at 10 ppm regardless of source,
  must declare the sulfite and its content on the
  ingredient label.

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Sulphiting agents

• More regulatory restrictions are likely to be
  globally applied to the use of sulphites in foods
  since sulphite allergies pose a health risk in
  many populations.
• Regulations enacted by the United States Food and
  Drug Administration (FDA) in 1995 prohibit the
  use of sulphites in salad bars. As a result,
  there has been a considerable focus on
  identifying appropriate sulphite substitutes for
  use in foods.

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Sulphiting agents

• The FDA has proposed maximum residual sulphur
  dioxide levels for certain foods. In accordance
  with these proposed limits,
• residual sulphur dioxide levels for fruit juices,
  dehydrated potatoes, and dried fruit, are 300,
  500, and 2000 ppm respectively (Federal Register,
  1988).
• Shrimp products having residual sulphite levels
  in excess of 100 ppm are considered adulterated,
  since these levels are considered unsafe (Federal
  Register, 1985).

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L-Ascorbic acid

• Ascorbic acid is a moderately strong reducing
  compound, which is acidic in nature, forms
  neutral salts with bases, and is highly
  water-soluble.
• L-ascorbic acid (vitamin C) and its various
  neutral salts and other derivatives have been the
  leading GRAS antioxidants for use on fruits and
  vegetables and in fruit juices, for the
  prevention of browning and other oxidative
  reactions

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L-Ascorbic acid

• Ascorbic acid also acts as an oxygen scavenger
  for the removal of molecular oxygen in polyphenol
  oxidase reactions.
• Polyphenol oxidase inhibition by ascorbic acid
  has been attributed to the reduction of
  enzymatically formed o-quinones to their
  precursor diphenols
• Ascorbic acid is however irreversibly oxidized
  to dehydroascorbic acid during the reduction
  process, thus allowing browning to occur upon its
  depletion (Figure slide 72).

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L-Ascorbic acid

• More stable forms of ascorbic acid derivatives,
  such as erythrobic acid, 2- and 3-phosphate
  derivatives of ascorbic acid, phosphinate esters
  of ascorbic acid, and ascorbyl-6-fatty acid
  esters of ascorbic acid, have however been
  developed to overcome these problems Ascorbic
  acid esters release ascorbic acid upon hydrolysis
  by acid phosphatases

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L-Ascorbic acid

• Their relative effectiveness as browning
  inhibitors varies in accordance with the food
  product Compounds containing reactive amino or
  thiol groups can greatly affect the reactivity of
  o-quinones.

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Mechanism of prevention of colour formation by
ascorbic acid.
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L-Ascorbic acid

• Ascorbic acid causes a distinct yellow
  off-colour, when used in the prevention of
  melanosis in shrimp
• It is usually applied in conjunction with citric
  acid in order to maintain a more acidic pH level.
  In addition, it is also believed to have a
  chelating effect on the copper prosthetic group
  of polyphenol oxidase

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