Involvement of Colonizing Bacillus Isolates in Glucovanillin Hydrolysis during the Curing of Vanilla planifolia Andrews

Abstract

Vanilla beans were analyzed using biochemical methods, which revealed that glucovanillin disperses from the inner part to the outer part of the vanilla bean during the curing process and is simultaneously hydrolyzed by ␤-D-glucosidase. Enzymatic hydrolysis was found to occur on the surface of the vanilla beans. Transcripts of the ␤-D-glucosidase gene (bgl) of colonizing microorganisms were detected. The results directly indicate that colonizing microorganisms are involved in glucovanillin hydrolysis. Phylogenetic analysis based on 16S rRNA gene sequences showed that the colonizing microorganisms mainly belonged to the Bacillus genus. bgl was detected in all the isolates and presented clustering similar to that of the isolate taxonomy. Furthermore, inoculation of green fluorescent protein-tagged isolates showed that the Bacillus isolates can colonize vanilla beans. Glucovanillin was metabolized as the sole source of carbon in a culture of the isolates within 24 h. These isolates presented unique glucovanillin degradation capabilities. Vanillin was the major volatile compound in the culture. Other compounds, such as ␣-cubebene, ␤-pinene, and guaiacol, were detected in some isolate cultures. Colonizing Bacillus isolates were found to hydrolyze glucovanillin in culture, indirectly demonstrating the involvement of colonizing Bacillus isolates in glucovanillin hydrolysis during the vanilla curing process. Based on these results, we conclude that colonizing Bacillus isolates produce ␤-D-glucosidase, which mediates glucovanillin hydrolysis and influences flavor formation. V anilla flavoring obtained from cured Vanilla planifolia beans is widely used in food, beverages, and cosmetics, such that the total worldwide consumption is markedly increasing (1, 2). The characteristics of the vanilla flavor are formed only during a careful curing process that yields the main aromatic constituent, vanillin, and over 200 other volatile compounds with delicate sweet fragrances (3).The conventional curing process starts with a blanching step in which the mature green vanilla bean is immersed in hot water for 3 to 5 min. The vanilla beans are then subjected to a process that involves periodic sweating and drying. During the remaining part of the day, the vanilla beans are allowed to acclimate on wooden racks in a well-ventilated room and are then stored in small bundles in plastic vacuum bags at room temperature (4, 5).In fresh vanilla beans, vanillin is exclusively present in a conjugated form, principally as glucovanillin. At this stage, the beans display no trace of vanilla flavor (6, 7). One of the most important aspects of curing is when glucovanillin comes into contact with ␤-D-glucosidase, thus releasing free vanillin (8). Thermal treatment, plant enzyme reactions, and microbial activity are all important in vanillin flavor generation (9). Röling et al. (10) reported that differences in microbial abundance, communities, and strain characteristics result in variations in vanilla flavor. These results show that colonizing microorg...

Distinct Roles for Bacterial and Fungal Communities During the Curing of Vanilla

“…The microorganism involved in the curing of vanilla beans was supported by many researches ( Röling et al, 2001 ; General et al, 2009 ; Chen et al, 2015 ). In this study, Bacillus and Aspergillus were found changed during the curing process.…”

“…It was found that the structure of bacteria and fungi were closely related to the vanillin content in this study. Colonizing Bacillus isolates produced β-D-glucosidase, which mediated the glucovanillin hydrolysis and influenced the vanillin formation (Chen et al, 2015). Furthermore, it was reported that Bacillus siamensis XY18 and Bacillus subtilis XY20 isolated from vanilla beans could increase the vanillin content (Gu et al, 2015).…”

“…Gu et al (2017) reported that the biosynthesis of vanillin by glucose, cresol, capsaicin, and vanillyl alcohol was widely distributed in the microbial metabolism. Chen et al (2015) indicated that the Bacillus colonized on vanilla beans produced β-D-glucosidase, which mediated the hydrolysis of glucovanillin. These studies have shown that microorganisms may play an important role in the formation of the vanilla flavor.…”

Tahitian Vanilla (Vanilla ×tahitensis): A Vanilla Species with Unique Features

“…Another consequence is that there is no need for any initial heat treatment in hot water or in an oven, as it is performed commonly in the Indian Ocean region, in Central America or in New Guinea. During curing, many biotransformations take place due to the presence of intrinsic enzymes located in the pods and/or the action of colonizing microorganisms on the surface as evidenced by the latest findings on V. planifolia [32]. Even if the different biotransformations involved are still unclear, they will definitely contribute to the development of the final vanilla flavour that one can experience when smelling cured vanilla pods.…”

Contribution of Bacillus Isolates to the Flavor Profiles of Vanilla Beans Assessed through Aroma Analysis and Chemometrics

“…We have recently found that colonizing Bacillus in vanilla beans is involved in glucovanillin hydrolysis and vanillin formation during conventional curing [14]. Therefore, we speculated that Bacillus isolates can be used to develop a new, cheap, and simple method to increase vanillin production.…”