The quantitative and qualitative assessment of the γ-glutamyl peptide and alk(en)yl cysteine sulphoxide content of garlic is made difficult by the problems of natural variation associated with variety, sulphate nutrition, water regime, temperature, and storage. 1



Variety

Sulphate nutrition

Water regime

Temperature

Storage

Variety

The variation in allicin yield (and therefore its precursors) changes from bulb to bulb within a field by 7% and from farm to farm in the same location by 25%. These variations however, are small when compared to variation among garlic varieties grown around the world which can be up to five-fold (0.13% - 0.6%)

Of forty varieties analysed by Block, 1 about 80% fell within the 2.5-fold range of a 0.21% - 0.54% allicin yield, however it is likely that soil and climate conditions have a far greater effect on allicin yield than variety. Thirty-four varieties of garlic, representing both major sub-species (Allium sativum var. sativum and Allium sativum var. ophioscorodon) were grown on the same area of land and analysed for allicin and other thiosulphinates. No difference in levels between the two sub-species was evident and there was only a 1.6-fold range of variation between all thirty-four varieties. It was noted however that Allium sativum var. sativum contained nearly twice as much γ-glutamyl-S-allylcysteine as Allium sativum var. ophioscorodon but had the same amount of γ-glutamyl-S-trans-1-propenylcysteine.

In studies on other alliums it has been shown that genotype determines the potential for flavour production although that potential may be modified by the environment. Since the cultivated forms of garlic are all sterile and can only be propagated vegetatively, little intraspecific variation would be expected. Whilst, morphophysiologically, there are many strikingly different clones, genetic variability, as measured by isozyme diversity is very low. Work to assess whether the potential for flavour production varies significantly within these clonal groups has yet to be undertaken.

Compound
Content (mg/g)
S-(+)-Alkyl-L-cysteine sulphoxides
 
Allylcysteine sulphoxide (alliin)
Methylcysteine sulphoxide
trans-1-Propenylcysteine sulphoxide (isoalliin)

7 - 14
0.5 - 2.0
0.1 - 2.0

γ-L-Glutamyl-S-alkyl-L-cysteines  
γ-Glutamyl-S-trans-1-propenylcysteine
γ-Glutamyl-S-allylcysteine
γ-Glutamyl-S-methylcysteine

3 - 9
2 - 6
0.1 - 0.4

Table 1. Data represent the range obtained from eight varieties of garlic less than two months after harvesting.

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Sulphate nutrition

Freeman and Mossadeghi undertook work in the 1970s to assess the relationship between the sulphate nutrition of garlic and flavour strength. 2 Plants were grown in sand culture at two concentrations of sulphate (0 and 3 mequiv./l) in the nutrient medium and their subsequent flavour strengths determined by sensory, biochemical and chromatographic methods. Field grown samples were used for comparison. The results of the tests are shown in Table 2. In the triangular taste test, 10 out of 11 judges (91%) distinguished between the sand-grown samples (S0 and S3), being almost unanimous in ranking the flavour strength of S3, as much stronger than S0. Both samples were said to have the characteristic garlic-like odours and tastes which were qualitatively indistinguishable from the field-grown sample.

Table 2. Influence of sulphate nutrition on growth and flavour strength of garlic grown in sand culture as compared with the field-grown plant.

Using pyruvate values as a measure of flavour it should be noted that the range of flavour strengths investigated is much less (~ 5-fold) for garlic than those obtained for onion, Allium cepa L. (~16-fold) and wild onion, Allium vineale L. (~13-fold). Other experiments indicate that sulphur is required for both growth and flavour. Available sulphur is preferentially used for growth and when this requirement has been met sulphur becomes available for incorporation into the biosynthetic pathway leading to the production and accumulation of flavour precursors. In his studies on sulphur deficiency in onions, Allium cepa L., they concluded that concentration and ratio of flavour precursors changed in response to sulphur fertility and that when bulbs were stressed for sulphur the methyl- and propylcysteine sulphoxides acted as very strong sinks for the available sulphur, even et the expense of growth and development.

The peptides in the flavour pathway were also shown to be affected by sulphur fertility, with those that occur late in the pathway accumulating at higher concentrations. Interestingly, at low sulphur fertility (0.1 meq S), over90% of the bulb sulphur could be accounted for by the peptides and flavour precursors measured, while at high sulphur fertility (3.1 meq S) only 40% of the bulb sulphur could be attributed to these measured compounds suggesting that sulphur was being stored in non-flavour compounds as sulphur fertility increased. Although conducted on onion, these experimental findings on changes in pungency (as measured by pyruvic acid) in response to increasing sulphur fertility serve to show the dose similarities to be found within the Allium group and to support Freeman & Mossadeghis work 2 on garlic.

Figure 4. Mean pungency of onion in response of increasing sulphur fertility

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Water regime

Another factor that has a pronounced influence on the flavour of vegetables is the water content of the soil in which they are grown. For many years it has been appreciated that some vegetables grown with an abundant water supply tend to be large and lush but relatively lacking in flavour, whereas those grown with restricted amounts of water may be smaller and less attractive in appearance, but are often richer in flavour. These observations have been confirmed by Freeman and Mossadeghi 2 for onions and other vegetables in studies based on modem methods of flavour assay. The underlying mechanism appears the depend upon changes in the amounts of the flavour precursors caused by stresses imposed by restricted water availability.

There are many examples in the literature of accumulation of low molecular metabolites (e.g. sugars, amino acids and organic acids) in plants subjected to stresses, such as nutrient deficiency, water stress or low temperatures, and it is postulated that the accumulation of flavour precursors in response to water stress offers a probable mechanism of the observed effects. In early work by Platenius 3 it was shown that onions, Allium cepa L. grown under natural rainfall (240 mm.) were stronger (as measured by volatile sulphur content) than onions grown under irrigation (430 mm.). Although stronger, the onions grown under natural rainfall were correspondingly lower in weight than those grown under irrigation. Using headspace analysis, pyruvate measurement and sensory evaluation, Freeman and Mossadeghi 2 confirmed these results and in their experiments fiavour was correlated with reduction in yield. It has been proposed that flavour intensity is caused by a dilution effect from high irrigation rates - high irrigation rates produce less concentrated flavour precursors in larger cells. Since investigations into the effect of sulphur fertility on flavour intensity indicate that plant water usage and sulphur uptake are unrelated this provides additional evidence in support of the dilution theory.

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Temperature

Cold storage, growth temperature and photoperiod are the main environmental factors affecting the ontogeny of garlic, but the response to these factors depend on the phenological stage. For example, in the absence of dormancy sprout emergence is controlled mainly by temperature, whereas bulb initiation is promoted by previous exposure of cloves to low temperatures and growth temperature and photoperiod. Since bulbs grown at higher temperatures bulb more rapidly than those grown at lower temperatures it is difficult to separate a temperature effect per se from an ontogenetic effect.

In studies on onions Platenius 3 grew plants at 10-15C, 15-21C and 21 -27C with a resulting three-fold increase in volatile sulphur at the higher temperature compared to the lower one. Although the onions grown at 21- 27C were more developmentally advanced, and thus had a higher flavour content, the differences obtained were so significant as to prompt further work. In a subsequent two part study, Randle et al 1 investigated the effects of temperature on flavour intensity by growing plants at different temperatures for a given length of time (maturity varied), and by growing plants at different temperatures until maturity was reached (time varied). When plants were grown for a specific period of time and maturity was allowed to vary, plant sulphur concentration and pungency increased with increased temperature (Figure 5.).

Figure 5. Variation of onion bulb sulphur concentration and pungency (μMols pyruuic Acid) with growth temperature

When plants were grown to maturity and time varied, bulb sulphur concentration ranged from 0.2% to 0.4% and pungency ranged from 2.2 mol-1 g f.w. to 4.4 mol-1 g f.w. at 10C to 31C respectively. Bulbs grown at 31C matured 6 weeks after the initiation of a bulbing photoperiod, in 8 weeks at 24C, in 10 weeks at 17C and in 15 weeks at 10C. In both cases flavour intensity increased as temperature increased.

Whilst garlic and onion differ in their response to low temperature bulb induction, both show dependence on photoperiod and temperature. Although the above data describes the effect of temperature on the flavour content of onion, such are the similarities in environmental response between onion (Allium cepa L.) and garlic (Allium sativum L.) that one would expect the findings to at least provide an indication of the effect in garlic.

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Storage

While studying the γ-qlutamyl-S-alkylcysteine content of garlic, Lawson et al 3 observed that when garlic cloves were stored in a refrigerator they began to sprout sooner than cloves stored at room temperature. Subsequent analysis of the refrigerated cloves revealed an increase in the trans-1-propenyl/alIyl thiosulphinates, the primary flavour compounds in garlic, and a decrease in the γ-glutamyl-S-alkylcysteines. This phenomenon was studied in greater detail using freshly picked garlic bulbs stored at 4C and room temperature. The results of this experiment are shown in Figure 6.



Figure 6. Effect of storage at 4C and 22C on the homogenate content of γ-glutamyl-S-trans-1-propenyl cysteine (GTPC), γ-glutamyl-S-allylcysteine (GAC), trans-1 -propenyl/allyl thiosulphinates (x l0) (TP/AT), and allicin offreshly picked cloves (ALLICIN). The moisture content remained constant throughout the experiment.

After 10 weeks at 4C, the γ-glutamyl-S-trans-1-propenyl cysteine and γ-glutamyl-S-allylcysteine content decreased by 70% each, while over the same period the primary flavour compounds, trans-1-propenyl-allyl thiosulphinate, allyl-trans1-propenyl thiosulphinate, trans-1-propenyl-methyl thiosulphinate and methyl-trans-1-propenyl thiosulphinate increased 16-fold although the allicin (diallyl thiosulphinate) content remained constant. At room temperature these effects were much diminished to only a quarter of the extent, and again the allicin content remained stable.

Virtanen has reported that the γ-glutamyl peptides of onions disappear when the bulbs begin to sprout and that there is a corresponding increase in y-glutamyl transpeptidase activity. This increase in γ-glutamyl transpeptidase activity also occurs as garlic begins to germinate, a process that is accelerated by cool temperatures which mimic wintering. Unlike onion however, Lawson states that the disappearance of the γ-glutamyl peptides of garlic was near maximal long before external sprouting was visible. Internal inspection of the cloves however did reveal that the sprouts were nearly ready to protrude.

Twenty four γ-glutamyl peptides have been isolated from Allium species, nine of which occur as intermediates in the biosynthesis of S-alk(en)yI-L-cysteine sulphoxides (flavour precursors) including γ-glutamyl trans-(+)-S-(1-propenyl)-L- cysteine sulphoxide and S-2-carboxypropyl glutathione. The enzyme γ-glutamyl transpeptidase has been shown to hydrolyse γ -glutamyl trans-(+)-S-(1-propenyl)-L- cysteine sulphoxide to S-(1-propenyl)-L-cysteine sulphoxide in the final step of the biosynthetic pathway while S-2-carboxypropyl glutathione has been shown to be an early intermediate in the biosynthetic pathway from glutathione to flavour precursors. Lancaster 4 has shown that sprouting in onions is characterised by increased γ-glutamyl transpeptidase activity and decreased levels of γ-glutamyl peptides and that this accumulation of intermediate peptides during bulbing and remobilization during sprouting is a reflection of the cessation and later resumption of enzyme activity in the biosynthetic pathway.

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