Handbook of Vanilla Science and Technology

Biotechnologically produced vanillin is not to replace the vanilla extract. It is to replace the synthetic vanillin with a natural flavor at an affordable price. Rhovanil^R, marketed by Rhodia, is the first such vanillin manufactured through biotechnology. Given customers’ growing resistance towards genetically manipulated foods, rDNA modified strains (GMOs) have been used very sparingly in the food and flavor industry. At the same time, genetic manipulation offers tremendous possibilities and opportunities for strain and productivity improvements.

Moreover, fermentation products can be certified as organic if they comply with the regulations for organic foods. In manufacturing of organic foods, only non-GMO ingredients are allowed. In addition, the requirements for kosher status need to be considered.

19.1.2 How?

Biotechnologically produced vanillin can be formed by microorganisms, enzymes, and cell culture processes. The microorganisms can be also adapted for the formation of other vanillin related flavorings, when they present either economic advantage or distinctive end products.

In general, biological processes are performed under gentle processing conditions and tend to have lower yields than chemical reactions. Typical hurdles are the initial concentration of precursor, its solubility, toxicity, and the accumulation of the finished product. To make good yields economically feasible, the engineering of the process must be coupled with a detailed understanding of the metabolic pathways. More advanced processing technologies such as whole-cell biocatalysis in biphasic systems, cofactor regeneration for in vitro oxidation, immobilization, membrane separation of the product, etc., are being introduced to improve the product yields.

Microbial or enzymatic processes to produce vanillin have used any of the following precursors: lignin, curcumin, siam benzoin resin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, aromatic amino acids, and glucose via de novo biosynthesis (Figure 19.1). The most attention was given to the biotransformation of ferulic acid and eugenol, as they are readily available precursors. A number of fungi and bacteria have the capacity to metabolize the above-mentioned precursors. Some microbial metabolic pathways have been elucidated with the help of genetic engineering.

Fig. 19.1 Different microbial routes to vanillin.

Over the past two decades, a number of excellent papers and reviews have been written (Rosazza et al. 1995; Walton et al. 2000; Priefert et al. 2001; Xu et al. 2007). Considerable progress has been made in characterizing the biochemistry and molecular genetics of the microbial catabolic and anabolic pathways leading to vanillin. Accumulated knowledge presents an opportunity for direct evolution of specific genes, improvement of fermentation conditions, and targeted selection.

19.2 SUBSTRATES

19.2.1 Ferulic acid (4-hydroxy-3-methoxycinnamic acid)

This phenolic acid has been considered to be the most attractive precursor for microbial formation of vanillin. Ferulic acid has a similar chemical structure and is abundant in nature. It is one of the hydroxycinnamates that are major constituents of plants such as grains, woods, grasses, fruits, vegetables, etc., bound as esters of polysaccharides, flavonoids, amides, lipids, and long chains alcohols. Ferulic acid forms a covalent bond with carbohydrates. Almost all ferulic acid in cereals is esterified with arabinose, glucose, xylose, or galactose residues in the pectic or hemicellulosic component of cell walls. The ester bond is between the carboxylic group of the acid and the arabinoxylan. The molecular weight of these esters goes up to more than 50,000 daltons. These esters behave as antioxidants.

Crystalline ferulic acid is relatively insoluble in water at pH lower than 9. Therefore, some studies suggested using a solution of ferulic acid dissolved in 0.2 N sodium hydroxide. The higher pH helps with biotransformation since phenolic compounds tend to oxidize at the high pH to their corresponding quinoid. Quinoid, being an electrophilic intermediate, reacts immediately with nucleophiles. The pH determines the solubility of ferulic acid and thus its availability to the biotransformation.

Highly purified ferulic acid is essential for elucidation of metabolic pathways and for determination of the fundamentals; however, it is not a viable substrate for commercial processes. To make ferulic acid a more cost effective substrate, a manufacturing process needs to be developed and optimized. Chemical or enzymatic hydrolysis of agricultural products such as sugar beet pulp, brewer’s spent grain, rice bran, wheat bran, or maize bran have been identified as potential sources of a cost effective ferulic acid (Cheetham et al. 1996, 2005).