Enzymatic Hydrolysis Hydrolases

- ubiquitous presence of hydrolases in foodstuffs - extremely important mechanism of introducing sensory alteration in food why?

- acyl migration from position 2 to 1 is thermodinamically favoured and normally precedes enzymatic hydrolysis of that acyl residue

Hydrolases

-microorganisms also use hydrolases for food digestion -actively involved in changing food properties -Examples of hydrolysis -changes from soapy notes imparted upon blue cheeses by hydrolases originating from the mould (desireable) , -unpleasant high butyric notes in butter -unpleasant lauric acidity in coconut oil

Hydrolases

Lipases are active on a water/lipid interface.

Esterase enzymes cleave only water-soluble esters, such as triacetylglycerol.

Applications of Lipases

Lipases with high specificity may be used to taylor food ingredients. -production of CBS, which are replacements for cocoa butter, -production of high polyunsaturate oils which may have nutritional advantages - the use of unspecific lipases when randomization of acyl groups is to be obtained (enzyme mediated interesterification for the production of margarines and spreads)

Phospholipases

Phospholipases A1, A2, B occur in many mammals.

Phospholipase C does not ocurr in mammals, only in bacteria, and in some snake venoms.

Phospholipase D is widespread in cereals and oil seeds.

Lipoxygenase

Lipoxygenases (linoleic acid oxygen oxidoreductase) are ubiquitous. Present in many vegetable and animal cells. Lipoxygenases catalyse the oxidation of 1-cis-4cis unsaturated fatty acids to the hydroperoxides.

Lipoxygenases are metaloproteins with an Fe atom in its active center.

Oxidation of Unsaturated Acyl Lipids

The oxidative deterioration of fats and oils proceeds through the peroxidation of unsaturated components in fats and oils .

Peroxidation reaction is favoured by light, transition metal ions, high temperature and low water activity.

free radical chain-reaction mechanism

Write down correct mechanism

Comparing abstracting a hydrogen atom from: - an allylic group (DR-H=322 kJ/mole) - a bi-allylic position as in linoleic or linolenic moieties (DR-H=272 kJ/mole) - an alkyl radical (dissociation energies above the 400 kJ/mole) Explain the difference in

reaction rates

and

induction periods

observed at moderate temperatures when linolenic or linoleic are suffering autoxidation, relative to that shown by oleic or even to the stability of stearic moieties.

An oleic substrate will actually yield, as results of the first autoxidation stage, a mixture of 8-, 9-, 10-, and 11 hydroperoxides.

If both geometrical (cis-trans) and stereochemical (R-S) isomerism is taken into acount. This means that ??? different hydroperoxides are produced for linoleic acid.

autoxidation ( 3 O 2 ) and photoxidation ( 1 O 2 )

Two different types of reactions have been recognized as important in this process:

photosensitised oxidation (photooxidation)

and

lipid oxidation by lipoxygenase catalysis.

Photooxidation - the oxygen ground state is a triplet - an excited singlet is available with an energy only 92 kJ/mole above that of the ground state Triplet ground state oxygen tends to react as a diradical, using its semioccupied orbitals for the purpose of building new bonds and preferring other radicals as substrates.

The excited singlet oxygen can use wholy occupied and/or empty orbitals for the same purpose, hence behaving as an

nucleophile/electrophile

and participating in electrocyclic reactions and reacting with other molecular entities.

Light can trigger lipid oxidation in two different ways, both mediated by small amounts of compounds called sensitisers.

Type I sensitisers (S), once activated by light (S*), reacts directly with a substrate, generating radicals which are the initiators of the oxidation process.

Type II photosensitisers are those which activate the ground state of oxygen to the first singlet excited state.

Chlorophylls, pheophytins, and riboflavin, commonly present in food items = Type II photosensitizer.

Reaction of singlet oxygen ( 1 O 2 ) with double bonds is inhibited by carotenoids present. How??

Quenching effect is very fast (k = 3 x 1010 mole -1 s -1 ).

Explain chlorophyll and carotenoids presents??

Heavy metal ions

Fats, oils and foods always contain traces of heavy metals (Fe, Cu and Co), the complete removal of which in a refining step is not industrially performable.

-catalysing the decomposition of hydroperoxides into radicals -initiate new radical chains in the oxidation process alkoxide radicals hydroxyl radical

Vegetable oils of the linoleic acid type, such as sunflower and corn germ oil, which are easily oxidisable, should contain less than 0.03 ppm Fe or 0.01 ppm Cu to ensure acceptable stability.

Both very low and very high water activity values seem to promote oxidation, the minima occuring at intermediate values of

0.25-0.3.

- at high water activity prooxidant mobility (including enzymes) is the probable reason for more oxidation - at very low water activity, increased oxygen mobility permited through positions left vacant by removed water molecules.

Water phase antioxidants such as ascorbic acid can prove to have a prooxidant effect for they tend to reduce oxidised metal ions to their lower oxidations states, hence permitting more of the decomposition of hydroperoxide to take place.

Heme compounds •Proteins exhibiting heme-like prosthetic groups are ubiquitous and an indispensable part of the electron transport chain. •They can chelate peroxides cleaving them into an alkoxy and a hydroxy radical and then releasing them.

•This can occur especially after having been denatured in order to expose the heme groups. •This effect is not dependent on pH or added ascorbic acid as it does not involve an alteration of the oxidation state of the metal ion.

•Denaturation of lipoxygenase which is the main enzymatic culprit for oxidation of vegetable foods must be done with care not to denature catalase or peroxidase too strongly, as this may expose their heme groups which will become oxidation catalysts.

Superoxide Anion one electron reduction of dioxygen, O 2 behaves as a nucleophile its protonated form = hydroperoxide radical .OOH

generated by the flavin enzymes

Main volatile carbonyl compounds from unsaturated fatty acids after uptake of 12 mole oxygen, (in ppm).

The autoxidation of a-linolenic acid produces especially strong odours, and it is therefore extremely easy to spot early.

3-cis-hexenal and 2-trans,6-cis-nonadienal

hydroperoxide-epidioxides

Polymerization of oils

Polymerization of oils

Protection of lipids against deterioration

citric acid, phosphoric acid and some of its derivatives phenolic compounds, tocols