Terpenoids

Terpenoids, constitute one of the largest groups of natural products and impart a wide variety of pleasant and floral scents. These compounds contain one, or more, basic isoprene units which are joined head to tail. Depending on the number of units, terpenes are classified as monoterpenes (2 units), sesquiterpenes (3 units), diterpenes (4 units), triterpenes (6 units), and polyterpenes (higher units). Terpenoids, produced from filamentous fungi are used by various food industries. Readily available monoterpenes, such as a-pinene and limonene, are used as substrates for conversion into flavoring compounds (Berger et al. 1992; Van der Werf et al. 1997).

2.1.1 Monoterpenes a. Limonene Conversion. Limonene can be converted to major products like carveol and carvone by Penicillium italicum and P. digitatum (Bowen 1975). When limonene concentration was increased there was a decrease in the quantity of end-products produced. Addition of sucrose increased microbial growth but the conversion was low. The conversion of limonene to carveol seems to be a single-step reaction, involving addition of a hydroxyl group at C-3 (Figure 1). Limonene biotransformation was first carried out by Rama Devi and Bhattacharyya (1978) who studied the oxygenative and prototropic molecular rearrangements during terpene transformation by A. niger.

Abraham et al. (1985) investigated the biotransformation of (R)-(+) limonene to a-terpineol by P. digitatum. DSM 62840. Abraham et al. (1986) studied microbial transformation of terpenoids with 1-p-menthene; they used Corynespora cassiicola and Diplodia gossypina to convert (S)-(—) and (R)-(+) limonene, a-terpinene, g-terpinene, and

Figure 1 Biotransformation of limonene.

terpinolene to 1,2-trans-diols. They found that the intermediary epoxide could be cleaved by the hydroxyl groups present in the substrates. Noma et al. (1992) employed Aspergillus cellulosae to transform limonene to major products like carveol, perillyl alcohol, and a-terpineol. They reported the possibility of introducing an oxygen functional group into limonene at the C-3 position, utilizing citrus peel oil as C source. Demyttenaere et al. (2001) used a solid phase micro-extraction technique to study the conversion of limonene by P. digitatum, to obtain a-terpineol as the major product. For details refer to the review by Van der Werf et al. (1997), which deals with terpene biotransformation, and with the problems related to commercialization of terpenoid products. Rensburg et al. (1997) have shown the possibility of limonene biotransformation in yeasts.

b. Pinenes. The biotransformation of a-pinene has considerable commercial importance. Hydroxylation of a-pinene by A. niger has been reported by several workers (Bhattacharyya et al. 1960); as also the references in their papers. Verbenol, verbenone, and sobrerol were obtained on biotransformation of a-pinene (Bhattacharyya et al. 1960). Verbenone was probably a product of auto-oxidation, whereas verbenol was formed by microbial oxidation (Figure 2; Bhattacharyya and Ganapathy 1965). Rama Devi and Bhattacharyya (1978) studied the conversion of a-pinene to 1-p-menthane, involving rupture of the cyclobutane ring in the bicyclic system.

Agrawal (1999) used an UV auxotroph of A. niger to increase the yield of verbenol from 10% obtained with a wild type to 25%. The enzyme a-pinene hydroxylase involved in a-pinene conversion to verbenol was NADPH dependent, and could be stabilized for 3 days using sorbitol along with DTT (Nazhat-ul-Ainn and Agrawal 2002). No biotransformed products from a-pinene were found under nitrate reducing conditions (Pavlostathis and Misra 1999). Optimization of growth conditions and media conditions can enhance the yield of verbenone (Agrawal and Joseph (2000a)) from a-pinene using Penicillium sp. Major biotransformations of some terpene substrates to flavoring compounds are given in Table 1.

2.1.2 Other Compounds

Many commercially important flavouring constituents, like esters, lactones, aldehydes, ketones, tobacco flavorings, and alcohols are produced with the help of fungi.

Figure 2 Biotransformation of pinenes.

Table 1 Fungal biotransformation of terpene substrates to high valued flavoring compounds

Fungi Substrate Products References

P. italicum Limonene Carveol, carvone Bowen (1975)

A. cellulosae Limonene Carveol, perillyl alcohol, and a-terpineol. Noma et al. (1992)

Y. lipolytica Limonene Perillic acid, 7-hydroxy piperitone Rensburg et al. (1997)

P. digitatum Limonene a-Terpineol Demyttenaere et al. (2001)

A. niger a-Pinene Verbenol, verbenone Prema and Bhattacharyya (1962)

Penicillium sp. a-Pinene Verbenol Agrawal et al. (1999)

c. Ester. Various aliphatic esters could be obtained from lyophilized, whole cells of Rhizopus oryzae; such cells can tolerate high substrate concentrations, and hence allow the production of large amounts by semi-continuous or continuous addition of the substrate, e.g., geranyl butyrate (Molinari et al. 1995). Lyophilized whole cells of R. Delemer were utilized to catalyze direct esterification of primary alcohols (n-hexanol) to give a very high yield (98%) of hexyl caprylate (Molinari et al. 1998). Agrawal et al. (2000) have demonstrated the production of dihydrocarvyl acetate from nerol, using Mucor sp. Although the metabolic pathway was not studied, it appears that ring closure through geranyl pyrophosphate led to the formation of dihydrocarvone, which may then be reduced to form dihydrocarveol and then acetylated to form dihydrocarvyl acetate. Regio-specific esters were obtained by using A. niger to form acetates of citronellol, geraniol, and linalool (Madhyastha 1988). Patel et al. (1992) described a simple method of utilizing Geotrichum candidum to improve the optical purity of (S)-(— )-4-chloro-3-hydroxy butanoic acid methyl ester by converting it to 4-chloro-3-oxobutanoic acid methyl ester. Cell extracts contained a single enzyme that catalyzed the reduction to the hydroxy product. Farbood et al. (1987) demonstrated the synthesis of terpene esters through an amino acid precursor in G. fragrans. Gatfield (1988) worked out the possibility of ester synthesis from lipase enzyme in Mucor michei; this process could improve upon the isolation and purification steps, when compared to the aqueous fermentation systems.

d. Lactones, Aldehydes, and Ketones. Biogeneration of volatile lactones from fungi has proved to be industrially successful (Cardillo et al. 1990). Lactone formation appears to be the result of a metabolic overflow. When the regular 3-hydroxylation of fatty acids is expanded to 4- or 5-hydroxylations, lactone formation takes place. These compounds impart fruit-like, buttery, sweet, or nutty odors.

Macrocyclic musks were produced from Ustilago zeae, using ustilagic acids as precursors (Gatfield 1988). Using octanoic acid, Gregory and Eilerman (1989) accomplished the bioconversion in Mucor sp. to delta-gamma octalactone. Farbood et al. (1990) obtained a mixture of saturated and unsaturated g-decalactones using g-keto acid as the substrate; the acid is reduced to g-hydroxy acid, which ultimately cyclizes to g-lactones. Serrano-Carreon et al. (1992) studied the formation of g-8- and 8-hydroxy acids in Trichoderma harzianum. Sporobolomyces odorus culture was able to produce an intense peach (g-decalactone) and mutton (cis-6-dodecen-4-olide) odors (Lee and Chou 1994), P. roqueforti was employed for producing lactones, mainly 4-dodecanolide from hydrolyzed oils like soybean and copra (Chalier and Crouzet 1992). The compound, 4-dodecanolide is formed by g-hydroxylation of the corresponding saturated acid, or by b-oxidation of oleic acid into 3-dodecanoic acid, followed by lactonization of the mentioned acids. The biosynthetic pathway in S. odorus, which results in the formation of 4-hexanolide as an oxidation product of linoleic acid, was studied by Taylor and Mottram 1996. It is possible that lactone is the result of b-oxidation of linoleic acid to 3,6-dodecadienoic acid, which is later hydrated and lactonized.

Production of 6-pentyl-a-pyrone by Trichoderma viride has been reported (Prapulla and Karanth 1992). The addition of amberlite XAD-2 resin to the medium overcomes product inhibition. Kalyani et al. (2000) studied the formation of 6-pentyl-a-pyrone in surface cultures of T. harzianum, under submerged conditions. Characterization of 6-pentyl-a-pyrone also has been done using T. koningii (Benoni et al. 1990).

Aldehydes are usually formed via a Strecker degradation of amino acids. This involves oxidative deamination and decarboxylation of a-amino acids, leading to the formation of an aldehyde containing one carbon atom less than the original amino acid (Mottram 1994). Fungi have shown great potential in producing high yields of these flavoring compounds. Among aldehydes, benzaldehyde (almond aroma) and vanillin, are widely used by food industries. Cis/trans

Cis/trans iwieugenol Vanillin

Figure 3 Biotransformation of isoeugenol.

Cis/trans iwieugenol Vanillin

Figure 3 Biotransformation of isoeugenol.

isoeugenol (1:4) was converted by A. niger into vanillin (Figure 3; Robenhorst and Hopp 1991).

Berger et al. (1987) studied the formation of a methoxy benzaldehyde in Ischnoderma benzoicum. Casey and Dobb (1992) reported the formation of aromatic aldehydes from aromatic amino acids, via a phenylpyruvic acid like benzaldehyde, using Trichosporon beiglii. Ketones are characterized by the presence of a carbonyl group, and are classified as aliphatic, aromatic, or phenol derivatives. They are synthesized by fungi in response to the presence of short-chain fatty acids, or as a means of recycling of COA. Mestri (1994) has discussed the production of nootkatone from valencene by an oxidation process involving P. camemberti and A. niger (Figure 4).

Biotransformation of nerol, geraniol, and citral, in surface cultures of A. niger and P. digitatum, to 6-methyl-hept-5-en-2-one (92-99%) has been studied by Demyttenaere and DePooter (1996) and Demyttenaere et al. (2000). These workers indicated an oxidative pathway wherein the alcohol is oxidized to aldehyde, and then to 6-methyl-hept-5-en-2-one, with no intermediary products. The pathway of this biotransformation of geraniol into 6-methylhept-5-en-2-one by P. digitatum has been elucidated recently by Wolken and Van' der Werf (2001), who also point out that citral is converted into geranic acid in this process. Furthermore, they also detected a novel enzymatic activity, wherein citral lyase converts citral to methylheptenone and acetaldehyde, independently of cofactors.

e. Ionone. Tobacco flavor is obtained by the transformation of ionone compounds. These compounds are widely distributed in nature, and are the constituents of many essential oils. Fungi, such as A. niger, converted ionone to a-cyclohomo-geraniol, 3-oxo-a-cyclohomo-geraniol, and benzofuran, as depicted in Figure 5 (Krasnobajew and Helmlinger 1982).

Figure 4 Biotransformation of valencene.

Larroche et al. (1995) have reported the formation of hydroxy and exo derivatives from b-ionone by A. niger. The recovery of the products was 100%, after 230 h of cultivation. This work paved the way for a possible fed-batch procedure, without replacement of the medium. When b-ionone was the only carbon source in the medium it stopped fungal growth, and was converted into hydroxy metabolites, probably by the action of a hydroxylase system. Grivel et al. (1999) made a dynamic model for biotransformation of b-ionone in A. niger. As the precursor is less soluble in water, it gave rise to a two-phase liquid system with high volatility and poor chemical stability. The products were 5,6-epoxy-5,6-dihydro-b-ionone, dihydro actinidiolide, and 4-oxo-b-ionone, with a molar yield of 32%; a high loss by stripping is a serious drawback of this process.

f. Acid Formation. The yields of acid produced by fungi are found to be commercially feasible, and are being utilized by industries. Armstrong et al. (1989) reported high yields of citric acid, which is perhaps the best known flavor compound produced by A. niger. Fabritius et al. (1998) studied the conversion of palmitic acid to (R)-(Z)-3-hydroxy-9-octadecendioic acid in Candida tropicalis; when ricinoleic acid was utilized as the sole carbon, optically pure (R)-(Z)-7-hydroxy-9-octadecenedioic acid was obtained. As only one regio-isomer was obtained, the hydroxylation was regiospecific. Fatty acids are, therefore of great interest to the chemical industry, as they provide a new avenue for commercial exploitation.

g. Alcohols. Various alcohols, which are utilized widely as flavors are produced by fungal conversions. The formation of sclareol, a labdoane diterpene used in foods, has been studied in C. albidus (Farbood et al. 1986). Bioconversion of citronellol, leading to the formation of 2,6-dimethyl-1,8-octanediol and (E)-2,6-dimethyl-2-octene-1,8-diol was studied with four strains of Botrytis cinerea in grape must (Brunerie et al. 1987). The substrate was metabolized to the v-hydroxylation product (E)-2,6 dimethyl-2-octene-1,8-diol and 2,6-dimethyl-1,8-octanediol. Regiospecific esters were produced by A. niger from acetates of citronellol, geraniol and, linalool (Madhyastha 1988).The major product formed from citronellol acetate, was citronellol. Geraniol was produced from geranyl acetate while linallol and 8-hydroxy linalool were the major compounds from linalyl acetate. A bioreactor, based on an aqueous/organic two-phase system, was designed by Doig et al. (1998) for the biotransformation of baker's yeast to geraniol. Uzura et al. (2001), using resting cells of Fusarium moniliforme, and propyl benzene as substrate, was able to form 1-phenylpropenol with high regio-and stereo-specificity. Lomascolo et al. (2001) worked out the possibility of producing 2-phenylethanol (rose flavor) from A. niger, using as precursor phenyl alanine, which was synthesized as the sole aromatic product. Some examples of nonterpene fungal flavoring compounds are given in Table 2.

Figure S Biotransformation of ionone.

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