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The Chemistry of Garlic

The Determination of the Pyruvic Acid Content of Garlic Tissue Homogenates

Introduction   Download this report
 

The principal compounds of intact garlic tissue which serve as precursors of the typical flavour and odour compounds are the alkyl- and alkenylcysteine sulphoxides. The major compound found 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). When garlic tissue is disrupted the vacuolar enzyme alliinase or alliin lyase (EC 4.4.1.4), rapidly lyases the cytosolic alk(en)yl cysteine sulphoxides to form sulphenic acids  (R-SOH); these immediately condense to form the alkyl alkanethiosulphinates (R1-SS(O)-R2), the principal flavour compounds of garlic.
The formation of thiosulphinates in disrupted garlic tissue is extremely rapid with most workers agreeing that all reactions are complete within 10 minutes. All possible combinations of 2-propene-, 1-propene-, and methanesulphenic acids result in thiosulphinates and these rearrangements are accompanied by the production of pyruvic acid and ammonia.
The large amounts of endogenous ammonia in garlic tissue render its determination unattractive, however the measurement of pyruvic acid as an indicator of pungency and flavour in both onions and garlic has been employed by a number of workers. The experimental method employed here is a modification of the technique described by Schwimmer et al in which filtered onion juice is reacted with 2,4-dinitrophenylhydrazine and where the resulting dinitrophenylhydrazones are measured colorimetrically.

Since this procedure actually measures total carbonyl content it was necessary to compare results obtained by this method with those of a method specific for pyruvic acid. In this latter method, reduced diphosphopyridine nucleotide is oxidised by pyruvic acid in the presence of excess lactic dehydrogenase and the decrease of reduced nucleotide is used as measure of pyruvic acid. Since the two methods are found to yield approximately the same values it can be concluded that the increase in 2,4-dinitrophenylhydrazone, as measured colorimetrically, can be largely attributed to the production of pyruvic acid.

 
 


Materials and Method
 

Preparation of garlic homogenates: Randomly chosen cloves from a number of garlic bulbs were selected, peeled and coarsely chopped with a knife. Each 100 g sample of chopped garlic was mixed by hand and 5 g samples removed for assessment. Each 5 g sample of chopped garlic was homogenised for 2 min with 20 ml distilled water in a food blender and then made up to 1 l with distilled water. The sample was allowed to stand at room temperature for 15 min before being filtered through Whatman No. 4 filter paper.

Controls: Garlic tissue contains a low level of pyruvic acid that is not a product of the alliin → allicin reaction. In order to account for this, control samples were prepared in which the enzyme alliinase had been heat deactivated. Unpeeled garlic samples, each weighing approximately 10 g, were heated in a microwave oven for 30 sec (Matsui model 170TC, λ=2450 MHz, O/P = 650W) before being processed in the normal way.

Determination of pyruvic acid: A 0.0125% 2,4-dinitrophenylhydrazine (DNPH) solution was prepared by dissolving 0.1625 g of wet DNPH powder (~30% water) in 1000 ml 2N HCl. A 2 ml sample of the diluted, filtered homogenate was added to 1 ml of a 0.0125% solution of 2,4-dinitrophenylhydrazine in 2N HCl. After 15 min in a water bath at 37C, 5 ml of 0.6N NaOH was added and the absorbance measured immediately on a Shimadzu model UV-160A spectrophotometer (420 nm filter, set at zero absorbance with reagent blank). The difference between the pyruvic acid content of the homogenates from unheated (PT) and heated garlic samples (PC) is defined as mmoles of enzymatically produced pyruvic acid (PE) per gram garlic.

Calibration: The method was calibrated using sodium pyruvate as standard. Pyruvic acid standards were prepared using sodium pyruvate. A 10μM/ml solution was prepared by dissolving 1.1g sodium pyruvate (m.w. = 110.0g) in 1000 ml of distilled water. A subsequent ten-fold dilution gave a 1μM/ml standard solution which was further diluted to prepare calibration standards.

Dry weight determination: Garlic cloves were peeled, sliced laterally into approximately 1 mm slices and then dried in an oven at 105C for 10 h.

 


Results: The results obtained using sodium pyruvate as standard are shown in Table 1.

Standard Solution
(c) μM/ml

Absorbance 1
A1

Absorbance 2
A2
Mean Absorbance
AM
0.00 0.000 0.000 0.000
0.05 0.118 0.120 0.119
0.10 0.212 0.212 0.212
0.15 0.337 0.344 0.340
0.20 0.400 0.410 0.405
0.25 0.531 0.495 0.513
0.30 0.552 0.552 0.552
0.35 0.662 0.595 0.628
0.40 0.680 0.661 0.670
0.50 0.762 0.729 0.745

Table 1. Absorbance values for sodium pyruvate standard.

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