At cellular and subcellular levels, the localization of β-glucosidase can be clarified using techniques such as immunolocalization; similarly, the cellular localization of glucovanillin remains to be determined.
Accumulation of Glucovanillin and β-Glucosidase During Vanilla Bean Development
Another area where very little research has been carried out is the evolution of glucovanillin and of β-glucosidase activity during bean development, with even less research been done on their evolution by tissue type.
The only point of agreement that has emerged from the various research studies on the evolution of glucovanillin (Ranadive et al., 1983; Sagrero-Nieves and Schwartz, 1988; Kanisawa et al., 1994; Brodélius, 1994; Havkin-Frenkel et al., 1999) is that the accumulation of vanillin or glucovanillin in the fruit starts from the 15th week after pollination and continues until around the 30th week.
However, the form in which vanillin is accumulated (free or glucosyl form) may lead to confusion. Ranadive et al. (1983) and Sagrero-Nieves and Schwartz (1988) show the evolution of free vanillin without prior hydrolysis; Ranadive’s results even show that free vanillin represents between 50% and 90% of the potential total vanillin. Brodélius (1994) considers that most of the vanillin is in glucosyl form with the free form not exceeding 15% of the potential total amount. Kanisawa et al. (1994) do not report the presence of vanillin in its free form during the development of the green fruit, and Havkin-Frenkel et al. (1999) indicate that vanillin is only accumulated in its glucosyl form. The latter point is confirmed by Leong (1991), who does not find the free form in the green beans. Arana (1943) had already found that vanillin is present almost exclusively in glucosyl form. Except in certain exceptional cases, our own analyses have always shown that in mature green fruits, the glucosyl form is predominant (at around 95% of the total), if we prevent any accidental hydrolysis during glucovanillin extraction (e.g., by conducting extraction in pure methanol at −18°C). However, a more timely study (unpublished results) on the evolution of glucovanillin during the development of the fruit showed that for beans around 3, 5, 7, and 9 months of development after pollination, the percentage of free vanillin in relation to the total (glucosyl plus free forms) was 33, 6, 1.5, and 0.2%, respectively. It would be interesting to get a confirmation of this evolution, which raises questions on the role of glucosylation of vanillin in the vanilla bean.
According to different researchers who monitored the evolution of glucosidase activity during bean development on the vine (Wild-Altamirano, 1969, Ranadive et al., 1983; Kanisawa et al., 1994), it would appear that this activity is measurable at all stages of fruit growth. However, the enzyme activity increases considerably between the third and the fourth month after pollination, reaching a maximum at around the fifth month. Therefore, the evolution of β-glucosidase activity during bean growth is on par with that of glucovanillin. The assays (unpublished results) conducted by the authors for glucosidase activity in fruits harvested during February 2005 in Madagascar at a developmental stage estimated at less than two months after pollination (highly asymmetrical fruit shape with a floral part that is far more rounded than the peduncular part) showed a glucosidase activity of around 650 nkatal/g of fresh weight for the floral part, compared to 230 nkatal/g of fresh weight for the peduncular part. These activities were already high and suggested an activity gradient in phase with the fruit development. For fruits from the same batch that had reached full size, but had not developed for more than 5 months after pollination, the glucosi-dase activities for the floral parts and peduncles were almost identical, at around 1100 nkatal/g of fresh weight, or the mean value obtained for fruits at the usual harvesting time (see below).
Glucovanillin contents obtained for the green fruit after eight months of development differ greatly from one research study to the other. If we convert the different values given in the literature into grams of glucovanillin per 100 g of dry weight, they range between 2% (Sagrero-Nieves and Schwartz, 1988) and 12% (Havkin-Frenkel et al., 1999). Further research studies have confirmed that mature green beans could comprise of glucovanillin around 10–15% of the dry weight (Ansaldi et al., 1988; Leong, 1991; Brunerie, 1993; Odoux, 2000; Havkin-Frenkel et al., 2005). Determination of glucovanillin from 70 green beans of seven different batches during the year 2000 in Madagascar (unpublished results) showed that glucovanillin contents could vary from 1.5% to 12% of dry weight depending on the fruit, with the majority of individual beans presenting a glucovanillin content of around 10%. Other determinations conducted in 2006 on batches from Papua New Guinea even showed maximum glucovanillin contents of more than 20% of dry weight for certain fruits, confirming the extreme variability that may exist in the glucovanillin content of V. planifolia fruits.
For β-glucosidase activities, it is impossible to compare the values given in the bibliography because of the means of expression (units) used, the protocols for obtaining enzyme extracts, the nature of the buffers used (pH, ionic strength, etc.), the molarity of the substrate (usually pNPG), and so on. Our own experience in this field has shown that in mature green beans with a physiologically healthy appearance and with a standardized, accurate protocol (Odoux, 2004), this activity could also vary considerably. Based on around 100 fruits from the Madagascan harvest in 2000 (unpublished results), the β-glucosidase activities ranged from around 100 to 2000 nkatal/g of fresh weight, with the majority of individual beans presenting an activity of around 1000 nkatal/g of fresh weight.
More systematic studies on the evolution of glucovanillin (and other glucosides) and of glucosidase activity during bean development are essential in order to confirm or refute the research already published. In the case of the aroma components, very strict analytical protocols must be established in order to remove any ambiguity regarding the form in which they are present at the different stages of development; it would also be useful to standardize the assays for glucosidase activity, for which the results can almost never be compared from one study to another.
These evolutions should also be measured by tissue type; in the case of gluco-sides, this could make it possible to obtain additional information on the biosynthetic pathways and sites for these components (see the following section).
Biosynthetic Site and Pathway for Glucovanillin
In his work on biosynthesis of vanillin, Lecomte (1901, 1913) concluded that it involved “coniferoside” (coniferin) that produced coniferyl alcohol through enzymatic hydrolysis, which was then turned into vanillin through the action of an “ oxidase.” Goris, who isolated “vanilloside” (glucovanillin) in 1924, suggested that a second possible pathway consisted of imagining the action of the “oxidase” before that of the “hydrolase” (Figure 10.12). Unable to isolate either the “coniferoside” or the coniferyl alcohol, he finally concluded that these hypotheses were unconfirmed (Goris, 1947). However, they were later resumed by Anwar (1963).
FIGURE 10.12 Biosynthetic pathway of vanillin proposed by Lecomte (1901, 1913) and Goris (1947).
It is now accepted that vanillin is a product of the biosynthetic pathway of shikimic acid, via phenylalanine, which leads to the phenylpropane compounds through enzymatic deamination, and primarily to cinnamic acid (Figure 10.13). Successive enzymatic hydroxylations and methylations then lead to the formation of p-hydroxycinnamic acids and, notably, coumaric, caffeic, ferulic, and sinapic acids.