Introduction

The action of alliinase on flavour precursors

Properties and Stability of Alliinase

Alliinase activity

Formation of thiosulphinates

Stability of thiosulphinates and the formation of secondary flavour compounds

Contributions of Non-Volatile Flavour Precursors of Garlic to Flavour Generation

Conclusions



Introduction

Like other members of the onion family, garlic actually creates the chemicals that give it its sharp flavor when the plant's cells are damaged. When a cell of a garlic clove is broken by chopping, chewing, or crushing, enzymes stored in cell vacuoles activate the breakdown of several sulfur-containing compounds stored in the cell fluids. The resultant compounds are responsible for the sharp-hot taste and strong smell of garlic. Some of the compounds are unstable and continue to evolve over time. Among the members of the onion family, garlic has by far the highest concentrations of initial reaction products, making garlic much more potent than onions, shallots, or leeks. 1 In this section the formation of primary and secondary flavour compounds derived from crushed garlic tissue is studied.





Figure 1. Subcellular location of biosynthetic intermediates, flavour precursors and alliinase in garlic

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The action of alliinase on flavour precursors

The key compounds in intact garlic tissue which serve as precursors of the flavour and odour compounds are the alkyl- and alkenylcysteine sulphoxides. The major compound in garlic is (+)-S-allyl-L-cysteine sulphoxide (allylcysteine sulphoxide or alliin) with smaller amounts of (+)-S-methyl-L-cysteine sulphoxide (methylcysteine sulphoxide) and (+)-S-trans-(1-propenyl)-L-cysteine sulphoxide (trans-1-propenylcysteine sulphoxide or isoalliin).

3D structure of Alliin (f)


The enzyme alliinase is a pyridoxal 5’-phosphate dependent α,β-eliminating lyase which acts upon the S-substituted-L-cysteine sulphoxides when intact tissue is disrupted. The enzymatic reaction gives rise initially to a group of flavour intermediates, the sulphenic acids, which are very unstable and undergo condensation to form thiosulphinates, the primary flavour compounds of garlic. The co-factor, pyridoxal phosphate, has been shown to act on the alkyl- and alkenylcysteine sulphoxide substrates so that a complex is formed with the enzyme.2

Figure 2. Active site of allinase and its co-factor pyridoxal 5’-phosphate

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Properties and Stability of Alliinase

Alliinase in garlic has been studied extensively and its properties are summarised in Figure 3. A 3Dstructure of this enzyme is also reported.

Purified alliinase loses its activity after 14 days storage at 3°C (pH 6.4) and cannot be stored at all at 10°C, particularly in diluted concentration. However the addition of glycerol to dilute concentrations (final concentration 10% v/v) stabilises activity completely for at least 30 days.60

Figure 3. General properties of Alliinase
Alliinase (4.4.1.4) (g)

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Alliinase activity

It has been suggested that there are two different alliinase activities for garlic, one being specific for substrate (+)-S-2-propenyl-L-cysteine sulphoxide and (+)-S-1-propenyl-L-cysteine sulphoxide at an optimum pH of 4.5 and the other specific for substrate (+)-S-methyl-L-cysteine sulphoxide at an optimum pH of 6.5. Both of these alliinases are irreversibly deactivated at pH 1.5-3 and >9. Although alliinase can act on both (+)and (-) sulphoxides, it shows a preference for the (+) isomers. 3

The transformation rate of thiosulphinates from their relative precursors is also different while maximum formation of diallyl thiosulphinate (allicin) can reach 100% within 1 minute, it takes more than 5 minutes for allyl methyl thiosulphinate to reach 100%. The activity of alliinase is also pH dependent. Below pH 4, alliinase is partially deactivated whilst at pH 4.5-7, alliinase has very high activity. The effects of pH adjustment during the blending of garlic cloves on the formation of flavour compounds were studied by Yu and Wu 1 who showed that maximum allicin formation occurred around pH 6.5 (Figure 4.).

Figure 4. Graphic describing the influence of pH on allinase activity

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Formation of thiosulphinates

Thiosulphinates, the primary flavour compounds in garlic and other alliums are pungent-smellingn and volatile compounds. A general scheme for the enzymatic reaction and the formation of tlavour intermediates and thiosulphinates in garlic and other related alliums is shown in Figure 5.

Figure 5. General scheme of production of thiosulphinate(alliinase-catalised reaction)

When intact garlic tissue is disrupted , alliinase acts upon the flavour precursors to generate sulphenic acids, the flavour intermediates. Being very unstable, the sulphenic acids undergo rapid condensation to form thiosulphinates, the principal flavour compounds in garlic, pyruvic acid and ammonia. Being an enzymatic decomposition product of the cysteine sulphoxides, pyruvic acid has been used as a measure of garlic flavour and onion pungency.

Figure 6. Reaction describing the enzymatic decomposition of cysteine sulphoxides

Symmetrical thiosulphinates are considered the result from bimolecular condensation of sulphenic acids with the same alk(en)yl radicals; among them, allicin is the main compound responsible of garlic flavour.

3D structure of Allicin (h)


Thiosulphinates with unsymmetrical alk(en)yl groups also occur by:

  1. the condensation of two different sulphenic acids
  2. mixing two different thiosulphinates
  3. the catalytic effect of thiols

as shown in Figure 7.

Figure 7. Formation of unsymmetrical alk(en)yl thiosulphinates

Fresh homogenates of garlic contain between 3-6 mg/g total thiosulphinates, 4 a typical composition being given in Figure 8.

Figure 8. Thiosulphinate content of garlic clove homogenates

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Stability of thiosulphinates and the formation of secondary flavour compounds

Thiosulphinates are in themselves unstable and tend to transform spontaneously at room temperature to thiosulphonates and disulphides as shown in equation (1) of Figure 9. Thiosulphonates are considered to be less significant as intermediate compounds than the corresponding thiosulphinates. Derived from thiosulphinates, disulphides can also undergo transformation to form trisulphides and monosulphides according to equation (2) in Figure 9.

Figure 9. Disproportionation of thiosulphinate and disulphide

A study on the stability of allicin at room temperature showed that the transformation half-life of allicin and the transformation products from allicin were both pH and solvent dependent. Its half-life in water was 96 hours which increased to 240 hours in mild acid (pH 2- 3); the main transformation products being diallyl trisulphide and diallyl disulphide in both cases.

In a strong base (pH ≥ 10) the main transformation product is diallyl disulphide. It is worth noting that because of the stabilising effect of water through hydrogen bonding on the thiosulphinate, mildly acidic conditions (e.g. pH 2-4) also seem to stabilise thiosulphinates. The transformation of allicin in organic solvents has been the basis of a number of studies primarily due to the importance of the transformation products, i.e. methyl ajoene ( (E)-2,6,7-trithiadeca-4,9-diene 2-oxide) as potent antithrombotic agent (Figure 10).

Figure 10. Formation of methyl ajoene from allicin

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Contributions of Non-Volatile Flavour Precursors of Garlic to Flavour Generation

It has been found that the non-volatile flavour precursors in garlic can contribute to the thermal flavour generation of thermally degraded garlic. Both the alk(en)yl cysteine sulphoxides and γ-glutamyl alk(en)yl cysteines are available as non-volatile precursors within garlic cloves and when cloves are heated in boiling water to deactivate the enzyme alliinase, γ-glutamyl allyl cysteine is converted to S-allylcysteine (deoxyalliin), γ-glutamyl-(E)-1-propenylcysteine to (E)-1 and (Z)-1-propenyl cysteines, and alliin is completely lost over time to various compounds. The predominant volatile compounds generated from the further thermal degradation of ‘inactivated’ garlic in water are 2-formylthiophene, 2-acetylthiazole and ethyl acetate.

In a further study 5 on the decomposition of alliin in aqueous solution (variable pH) other compounds were discovered. This work proposes a mechanism for the transformations of alliin caused by the thermal treatment in aqueous solution:

  1. a decomposition of alliin into principally allyl alcohol and cysteine (Figure 11.)
  2. cysteine then decomposes further into acetaldehyde, hydrogen sulphide and ammonia (Figure 12.)
  3. these small volatiles then react with each other to form methyl sulphides, thiazoles, thrithiolanes and cyclic sulphur-containing volatiles (Figure 12.-13.-14.)

Figure 11. Proposed mechanism for the formation of allyl alcohol and cysteine from alliin.

Figure 12. Proposed mechanisms for the formation of several volatile compounds from cysteine.

Figure 13. Proposed mechanism for the formation of 2-acetylthiazole and 2-methylthiazole from cysteine

Figure 14. Proposed mechanisms for the formation of some of the oxygen-containing volatile compounds from acetaldehyde

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Conclusions

The flavour compounds in qarlic are derived from the components of complex metabolic pathways that control the growth of the plant. The chemistry of the primary flavour compounds is well understood and based on several decades of research, yet the complexities of the biochemical pathways and in particular their role in overall plant metabolism remains unresolved. Because, as a result of plant sterility, genetic variations low, the basic principle of genetics which states that,

GENOTYPE I + ENVIRONMENT I = PHENOTYPE I

GENOTYPE I + ENVIRONMENT 2 = PHENOTYPE 2

has great relevance. Although information is limited it would appear that plant characteristics are influenced disproportionately by environmental conditions and that flavour compounds should be considered phenotypic traits just like bulb colour or clove number.

It is therefore the effect of external influences on the complex, sulphur-based metabolic pathways that ultimately affect flavour profile and strength and as the priorities within the plant change (shoot or bulb initiation, leaf growth, dormancy, flowering response, etc) so the balance of sulphur-based compounds change. Although many ‘cause and effect’ experiments have been undertaken, the controlled manipulation of garlic to produce the compounds required will only come as a result of a more detailed understanding of the sulphur biochemistry.

Given the availability of the primary flavour compounds and their precursors, even if in varying quantities and proportions, a significant body of work exists on transformation products and the identification of end-product volatiles. This work tends to based on chemical identification and, with notably few exceptions, little has been attempted to translate the flavour compounds to sensory descriptions.

The overall picture of garlic research shows a need for a combined physiological/biochemical approach to the understanding of the complexities of the sulphur-based metabolic pathways and a requirement for a “translation” of the chemistry of transformation products to a language that applies to the use of garlic as a flavouring in food. 1

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