Immobilized cell cultures of Capsicum and biotransformation to produce secondary metabolites of importance

101 Toxic Food Ingredients

101 Toxic Food Ingredients

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Biotransformation is a growing field of biotechnology and encompasses both enzymatic and plant/microbial biocatalysis. It is a process through which the functional groups of organic compounds, for example, substrates or precursors or-intermediates, are modified either stereo- or regio- specifically by living cultures, entrapped cells or enzymes or permeabilized cells to a chemically different product. Biotransformations can produce novel compounds, and it is possible to enhance the productivity of a desired compound. It also overcomes the problems associated with chemical synthesis.

The production of high value food metabolites, fine chemicals and pharmaceuticals can be achieved by biotransformations using biological catalysts in the form of enzymes and whole cells (Armstrong et al., 1993; Rhodes et al., 1994; Berger, 1995; Cheetham, 1995; Dornenburg and Knorr, 1996 a,b; Meyer et al., 1997; Scragg, 1997; Krings and Berger, 1998; Ramachandra Rao and Ravishankar, 2000a; Ravishankar and Ramachandra Rao, 2000). Consumer sensitivity about synthetic food additives has stimulated interest in the manufacture of natural and nature identical food ingredients using novel biotechnological methods. From an industrial point of view, biotransformations performed by plant cell culture systems can be desirable when a given reaction is unique to plant cells and the product of the reaction has a high value.

The production of capsaicin in cell cultures of the Capsicum species, C. frutescens and C. annuum was proposed by Yeoman et al. (1980). They described the formation of /raas-cinnamic acid from phenlyalanine, an aromatic amino acid, by PAL, which is further converted into /¿-coumaric acid by cinnamic acid 4-hydroxylase. The conversion of /¿-coumaric acid into caffeic acid and ferulic acids is initiated by two enzymes, /¿-coumaric acid 3-hydroxylase and caffeic acid O-methyltransferase, respectively. Ferulic acid can divert into lignin pathway or can be converted into vanillin and vanillylamine by oxidation and oxidative deamination reactions, respectively. Vanillylamine condenses with /3-methylnonenoic acid, obtained from valine, and the pungent principle, capsaicin is synthesized by capsaicin synthetase.

Enhancement of capsaicin production by media manipulation

A careful analysis of secondary metabolite profiles during the culture of plant cells indicate that most of the secondary compounds are produced during the post-exponential or stationary phase of growth (Lindsey and Yeoman, 1985). The biochemical factors underlying this phenomena are the channeling of precursors from growth related processes to secondary metabolism. This observation for capsaicin production in immobilized cell culture system has already been reported (Ravishankar et al., 1988). The above situation could be induced by subjecting the cells to nutrient stress. We found that nitrates and phosphates stress enhances the capsaicin production in immobilized cells by 13- and 5-fold, respectively, in comparison with free cell culture systems. Mathematical modelling of capsaicin production in immobilized cells of Capsicum was studied by Suvarnalatha et al. (1993) to optimize physical parameters, such as the bead strength of calcium alginate used for immobilization and the medium constituents for enhanced yield.

Influence of elicitors on secondary metabolite production

Secondary metabolite production in plant cell cultures can be elicited using a range of elicitors (Di Cosmo and Talleri, 1985). Elicitation is envisaged to overcome the problem of low productivity of plant cells for industrial applications (Knorr et al., 1993). Treatment of immobilized cells and placental tissues with various elicitors, such as fungal extracts (A. niger and Rhizopus oligosporus) and bacterial polysaccharides, curdlan and xanthan were performed. It was found that curdlan was most effective in eliciting capsaicin synthesis (Johnson et al., 1991). Immobilized cells responded more effectively than placental tissues for curdlan treatment. Curdlan and xanthan in combination enhanced capsaicin production by nearly 8-fold for curdlan treatment. Suvarnalatha et al. (1993) showed the optimization for capsaicinoid formation of immobilized C. frutescens cells using Response Surface Methodology.

Attempts have been made to increase the production of capsaicin in both freely suspended and immobilized cell cultures of C. frutescens by the feeding of phenylpropanoid intermediates. A 6- to 7-fold increase was found in the capsaicin accumulation upon precursor biotransformation. The feeding of intermediate precursors to Capsicum cell cultures not only increased the capsaicin accumulation (Figure 6.4) but also shortened the time required to produce high amounts of capsaicin (Johnson et al., 1990, 1991). Immobilized placenta, which is the site of synthesis of capsaicin in the fruit, was administered with intermediates of capsaicin pathway which resulted in large amounts of capsaicin accumulation, which is similar to the content of pungent variety of Capsicum fruit (Johnson and Ravishankar, 1996). During biotransformation studies to increase capsaicin yields, it was found that low capsaicin producing Capsicum cell cultures formed vanillin when fed with phenylpropanoid compounds — protocatechuic acid, caffeic acid, ferulic acid, vanillylamine, coniferyl aldehyde and veratraldehyde.

"Capsaicin czzaDihydrocapsaicin

Phe Cinn Coum Caff Fer Van Valine

Precursor treatment

Figure 6.4 Influence of feeding intermediate metabolites (added individually at 2.5 mM final conc.) on capsaicin and dihydrocapsaicin formation in immobilized placenta of C. frutescens. Abbreviations for the compounds used, Phe: phenylalanine; Cinn: cinnamic acid; Coum:/»-coumaric acid; Caff: caffeic acid; Fer: ferulic acid; Van: vanillylamine; Cultures were treated with precursors at the 2.5 mM level on day zero and analysis was done after five days of culture.

Phe Cinn Coum Caff Fer Van Valine

Precursor treatment

Figure 6.4 Influence of feeding intermediate metabolites (added individually at 2.5 mM final conc.) on capsaicin and dihydrocapsaicin formation in immobilized placenta of C. frutescens. Abbreviations for the compounds used, Phe: phenylalanine; Cinn: cinnamic acid; Coum:/»-coumaric acid; Caff: caffeic acid; Fer: ferulic acid; Van: vanillylamine; Cultures were treated with precursors at the 2.5 mM level on day zero and analysis was done after five days of culture.

Protocatechuic aldehyde and caffeic acid biotransformation leading to vanillin and capsaicin production

Freely suspended cells and immobilized cell cultures of C. frutescens biotransform externally-fed protocatechuic aldehyde to vanillin, whereas caffeic acid treated cultures accumulate more capsaicin than vanillin. The addition of S-adenosyl-L-methionine (SAM), a methyl donor, to protocatechuic aldehyde-treated immobilized C. frutescens cell cultures results in 14.08 mg/L vanillin accumulation, which is 2.5-fold higher than that in cultures with protocatechuic aldehyde alone. This result suggests that the influence of SAM on O-methylation of protocatechuic aldehyde results in a higher accumulation of vanillin. The increase in vanillin accumulation correlates well with an increase in the specific activity of caffeic acid O-methyltransferase in protocatechuic aldehyde and SAM-treated immobilized C. frutescens cell cultures. Capsaicin accumulation is also increased in protocatechuic acid and caffeic acid-fed immobilized C. frutescens cell cultures (Table 6.6). The formation of vanillin from protocatechuic aldehyde involves O-methylation at the meta position; further oxidation of vanillin leads to the formation of vanillic acid, which upon further demethylation or oxidation of protocatechuic aldehyde yields protocatechuic acid (Figure 6.5). (Ramachandra Rao and Ravishankar, 2000b).

Ferulic acid, vanillylamine and coniferyl aldehyde biotransformation leading to vanillin and capsaicin production

Other phenylpropanoid compounds, ferulic acid, vanillylamine and coniferyl aldehyde were also tested for the biotransformation to capsaicin and vanillin in free cell and immobilized cell cultures of C. frutescens (Figure 6.6).

Table 6.6 Influence of intermittent feeding of protocatechuic aldehyde and caffeic acid together (1.25 mM each) to immobilized cell cultures of C. frutescens

Addition of precursor (day)

j >y) day

6th day

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