SHORT COMMUNIGITZON PROBLEMS ASSOCIATED WITH THE ESTIMATION OF CYANIDE IN RUBBER SEED KERNEL

INTRODUCTION The cyanogenic glucosides of cassava (mainly linamarin) have bem reliably and reproducibly estimated using an exogenous enzyme (linamarase) assay for almost two decades.lq2 Rubber seed has the same cyanogenic glucoside394 but previous work with this material had either used trichloroacetic acid as a cyanide liberator3 or yielded very variable results with a ~ t o l ~ s i s . ~ This study is an attempt to understand the lack of reproducibility in total cyanide determinations and to validate the techniques used previously to estimate total cyanide content in rubber seed.


INTRODUCTION
The cyanogenic glucosides of cassava (mainly linamarin) have bem reliably and reproducibly estimated using an exogenous enzyme (linamarase) assay for almost two decades.lq2 Rubber seed has the same cyanogenic glucoside394 but previous work with this material had either used trichloroacetic acid as a cyanide liberator3 or yielded very variable results with a~t o l~s i s .~ This study is an attempt to understand the lack of reproducibility in total cyanide determinations and to validate the techniques used previously to estimate total cyanide content in rubber seed.

METHODS AND MATERIALS
Details are given in the legends of tables.

Sampling :
Collection of random samples of rubber seed and analysis by the autolysis techniques yielded total cyanide content varying from 100 -4000 mgkg-' . These variations can be attributed to (i) inherent differences in the sample and (ii) experimental error.
Differences in the sample may be due to among others (a) genetic variation (b) effect of maturity and (c) the history of seeds after seed fall. G. I/. Mallika et 01.
To limit such variations samples seeds were collected from the same field one day after seed fall. Results from six such samples gave 1512, 1177, 2214, 2072, 2176 and 1763 mgkg" respectively i.e. 1819 * 414 (mean + standard deviation). Analysis of seeds from the same fruit collected from a tree gave surprisingly wide disparities (Table I), indicating that in addition to the factors listed above, other factors possibly affecting the cyanophoric system such as autolytic cyanide release, could be operating even while the fruit is still on the tree. To an analyst this has serious implications especially since rubber has a relatively large seed and therefore might require a relatively large sample weight if variations were to be averaged out to give an analytically significant result. However analysis of large samples present other problems, viz., loss of cyanide during handling (experimental errors) due to the seed having large quantities of cyanide liberating enzymes.' Table 1 : Variation of total cyanide in the seeds of the same fruit Fruits (lrilocular) No's 1 lo 6 wen collected from the tree and each or the lhree seeds analysed ,scpara(c~y.~ Total cyanlde was determined by the plcrate method2" In individual seeds of the samb 5 trul1'by homogenlzallon and sutolp$ (24h).

Sample No.
Fruit 1 Fruit 2 Fruit 3 Fruit 4 Fruit 5 Fruit 6 In practice it has been found that least deviations were obtained by using 6 -8 g samples at 0 -9 C while minimising handling time. However a coefficient of variation of 15 -25 % can yet be observed.

Hydrolysis of cyanogenic glucosides
When trichloroacetic acid was used, release was less than 3% of the available total cyanide. Results obtained previously using this method are therefore grossly mi~leadin~.~ Use of 2M H2S04 at room temperature also released very little cyanide. This acid required 30 h refluxing to completely release total cyanide from rubber seed. These experiments also show that low yields obtained with short reflux times did not reflect a hydrolysis of the C i N triple bond as previously theorized as the full complement of total cyanide could be released from this reaction mixture by the action of added linamarase (exogenous) after pH adjustment (Table 2). Two hours of autolysis was sufficient to completely release total cyanide.. Longer' autolysis times could be used for release, provided cyanide utilizing systems (e.g. rhodanese) are not active. Results of this study show that 24h incubation at ambient temperature resulted in a loss of approximately 10% cyanide (Table 3).
Addition of exogenous linamarase to the distilled autolysate residue did not yield significant additional cyanide in processed material Like rubber seed meal, indicating that the activity of endogenous enzyme was adequate for the release of cyanide even after processing.