Upgrading of the monooligomeric components

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As stated previously, wastes from fruit and vegetable transformation contain mainly plant cell wall which is a very complex structure. Some of the cell wall components are usable in their monomeric or oligomeric forms. Hydrolysis of the cell wall is therefore required and enzymes are often used in this purpose. However, due to the complex structure of cell wall components, many enzymes are required to achieve their degradation. These enzymes are produced by several microorganisms and are the subject of numerous studies and reviews (Kulkarni et al., 1999; de Vries and Visser, 2001; Benen et al., 2002).

16.5.1 Bioactive oligosaccharides

Enzymatic hydrolysis of cell-wall-rich residues releases soluble oligosac-charides, some of which, called oligosaccharins, are biologically active. Oligogalacturonides, which are linear molecules of 2 to about 20 (1^4)-a-d-GalpA units arising from pectic 'smooth' regions (see Section 16.4.1,

Smooth regions), were the first plant oligosaccharins to be discovered (Bishop et al., 1981 and Hahn et al., 1981, reviewed by Ridley et al., 2001).

Oligosaccharins exhibit a variety of regulatory effects in plants, including the elicitation of defence responses, regulation of growth and development, and induction of rapid responses at the cell surfaces (Ridley et al., 2001). They could form the basis of a new non-toxic crop protection system that would be environmentally safe, would enable plants to respond to infection faster than normal and prevent potential pathogens from successfully colonising the host.

16.5.2 Antioxidants

Radical oxygen species (such as superoxide O2-, hydroxyl (OH), peroxyl (LOO), alkoxyl (LO), nitric oxide (NO)) and some strong oxidants (such as hypochlorous acid HOCl, hydrogen peroxide H2O2) can interact with a number of biomolecules (DNA, lipids, carbohydrates, proteins) causing some transient or irreversible damage which has been associated with serious diseases such as cancer or artherosclerosis.

Living cells have developed antioxidant systems to protect themselves against the reactive oxygen species. These antioxidants are defined as 'any substance that, when present at low concentration compared to the one of an oxidizable molecule, significantly delays or prevents oxidation of that molecule'. Antioxidants are also of interest in the industry as they minimise the oxidation of lipids in food and non-food matrices. Amongst the anti-oxidants of biological or industrial interest, plant-derived phenolic compounds are the most recently discovered and studied.

Hydroxycinnamic acids

Hydroxycinnamic acids include ferulic acid, caffeic acid and p-coumaric acid. Many of these compounds exhibit potential health benefits such as inhibitory effects on tumor promotion (Huang et al., 1988) or cardioprotective properties (Huang et al., 1998). They can also block the formation of mutagenic compounds such as nitrosamines (Kuenzig et al., 1984) and may protect against photooxydative skin damage (Saija et al., 1999). Their capacity to protect lipid systems is reported by several authors (Sharma, 1976; Yagi and Ohishi, 1979; Cuvelier et al., 1992; Scott et al., 1993; Castellucio et al., 1995; Meyer et al., 1998; Natella et al., 1999).

The structural explanation for the antioxidant properties of these molecules is far from clear. It appears to be partly linked to the ability of hydroxycinnamic acids to donate H+ to form stable phenoxy radicals and to the presence of substitutions on the phenolic ring. The number and position of the hydroxyl and methoxyl groups and the presence of an unsat-urated conjugation system would influence the stability of the molecule (Graf, 1992).

Ferulic acid (3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid) is the most abundant hydroxycinnamic acid in the plant kingdom. It was described for the first time in 1866 as a component of Ferula foetida Reg (Umbelliferae) (von Hlasiwetz and Barth, 1866, cited by Rosazza et al., 1995). In plants, ferulic acid is rarely found in the free form and it is differently located according to the source. In monocots, arabinose residues in arabinoxylans are esterified on position O-5 by ferulic acid (Saulnier and Thibault, 1999). In dicots, phenolic acids are generally not found except in a few plants such as the Chenopodiaceae family. In sugar beet for example, ferulic acid is present as part of the pectin. It is linked on O-2 of arabinose residues and on O-6 of galactose residues (Ralet et al., 1994b). The chemical and biological properties of ferulic acid were exhaustively reviewed by Graf (1992).

Ferulic acid is also found in various dimeric forms in the plant cell wall. Indeed, the phenoxyradicals may react together to form different dehy-drodimer isomers: 5-5', 8-O-4', 8-5' and 8-8' (Ralph et al., 1994). As the monomer, ferulic acid dehydrodimers have in vitro antioxidant capacities (Garcia-Conesa et al., 1997). Dimerisation modifies the antioxidant capacity but the final effect depends on a combination of factors such as structure (type of linkage, number of hydroxyl groups and conjugation of the molecule), lipophilicity and interaction between the test compounds and other compounds in the reaction mixture.

Removal of the ferulates may be carried out by alkalis. However, many studies have discussed the enzymatic release of ferulic acid (Graf, 1992; Cheetham, 1993; Micard et al., 1994). The first publications detailing the isolation and characterisation of feruloyl esterases are now more than 15 years old (Faulds and Williamson, 1991; Tenkanen et al., 1991). Since then, feruloyl esterases have been isolated from a large number of microorganisms and the related protein sequences elucidated (Crepin et al., 2004). Based on substrate utilisation data and supported by primary sequence identity, four sub-classes have been characterised and termed type-A, -B, -C and -D.

At present, the lack of highly conserved sequences within the sequenced esterases does not permit further classification for the feruloyl esterases, other than that their primary amino acid sequences place them in family 1 (Coutinho and Henrissat, 1999) of the carbohydrate esterase classification (http://afmb.cnrs-mrs.fr/~cazy/CAZY/). They exhibit different specificities according to the nature of the sugar and the linkage between sugar and ferulic acid as well as according to the length of the oligosaccharide moiety (Table 16.3 from Crepin et al., 2004). Two of these sub-classes release 5-5' diferulic acid (type-A and -D). After the enzymatic release of ferulic acid, it is necessary to purify it from the reaction medium at minimal cost, for further uses. This was investigated using polystyrenic resins (Couteau and Mathaly, 1997) and activated carbon (Couteau and Mathaly, 1998).

Table 16.3 Ferulate esterase specificities (from Crepin et al., 2004)

Enzyme

Preferred substrate

Sequence similarity

FAE A

O-5 feruloylated arabinose

Lipase

FAE B

O-2 feruloylated arabinose

CE 1 acetyl xylan esterase

O-6 feruloylated galactose

FAE C

Chlorogenate esterase

Tannase

FAE D

Xylanase

Hydroxytyrosol: a new potent antioxidant

Olive oil contains natural antioxidants such as tocopherols, carotenoids, sterols and phenolic compounds (Boskou, 1996). The main phenolics identified in olive are: tyrosol (p-hydroxyphenyl ethanol); hydroxytyrosol (3,4-dihydroxyphenyl ethanol); gallic, caffeic, vanillic, p-coumaric, syringic, ferulic, homovanillic, p-hydroxybenzoic and protacateuric acids; and oleu-ropein (Montedoro et al., 1992). They are found in olive oil but also in liquid and solid by-products generated by oil extraction. Commercial enzyme preparations, added during olive oil extraction, improve the release of phenolic compounds in olive oil and its by-products (Montedoro et al., 1993). The enzymatic release of simple phenolic compounds, and especially hydroxytyrosol, from olive oil by-products was enhanced by using culture broths of Aspergillus niger enriched in cinnamoyl esterases. The antioxidant activity of hydroxytyrosol is higher than that of synthetic antioxidants widely used in the food industry (ascorbic acid or butylhy-droxytoluene) (Bouzid et al., 2005). Hydroxytyrosol is believed to be the antioxidant with the highest free radical scavenging capacity (O'Dowd et al., 2004).

Olive pulp also contains polyphenols that have potentially significant antioxidant effects in vivo and have the same health-promoting properties as other polyphenols: prevention of atherosclerosis, promotion of intestinal and respiratory health, and prevention of cancer and heart disease (Uccella, 2001). For all these reasons, the polyphenol content in olive oil should be increased. In this way, phenolic compounds contained in olive mill waste-water are tentatively being transformed into valuable products using Lactobacillus plantarum during olive oil processing in order to favour their transportation to olive oil (Kachouri and Hamdi, 2004). Moreover, olive polyphenols have been demonstrated in vitro to inhibit or delay the rate of growth of bacteria such as Salmonella, Cholerae, Staphylococcus, Pseudomonas and Influenza. These data suggest a potential role for olive water polyphenol antioxidants in promoting intestinal and respiratory human and animal wellness, and as an antimicrobial food additive in pest management programmes.

16.5.3 Aromas

Flavour compounds, substances stimulating taste and smell, are extremely important for the food, animal-feed, cosmetics and pharmaceutical industries, as they represent more than 25% of the total food-additive market. In the past, they were mainly extracted from plants. However, they are most often present at very low concentrations and their extraction is thus difficult and expensive. Moreover, their availability is highly dependent on agricultural variations, plant diseases or sociopolitical stability of the producing countries. In order to bypass these difficulties, chemical synthesis of most of the flavours of industrial interest has been performed leading to cheap molecules being widely available.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most universally used flavours in the food, pharmaceutical, cosmetic and even detergent industries. Natural vanillin is extracted from the fermented pods of vanilla orchids (mainly Vanilla planifolia). The major producers are Mexico, Madagascar, Tahiti and Indonesia. Vanillin is absent from the green pod and is released during curing of the pods after harvest. Cured pods contain 2-3% by weight of vanillin, it occurs as vanillin-P-d-glucoside and is associated with many other flavouring compounds. Approximately 12 000 tonnes/ year of vanillin are consumed, essentially as synthetic vanillin at around US$ 15/kg; in contrast the natural vanilla extract can be estimated at US$ 4000/kg (Lomascolo et al., 1999).

Even though many synthetic flavours are available at very low prices, recent years have seen an increasing consumer demand for natural compounds. Fermentation and enzymatic reactions, together forming biotechnology, can be employed for the production of so-called 'natural' aromas (Krings and Berger, 1998). Several authors have described the use of enzyme preparations containing P-glucosidase to achieve vanillin release from vanilla pods, as an alternative to conventional curing (Dignum et al., 2001). An enzymatic route for vanillin synthesis - from the widely available principle of red pepper, capsaicin - was also reported (van den Heuvel et al., 2001). More interestingly, microorganisms can be exploited as they have rapid growth rates, they produced flavours de novo as secondary metabolites (i.e. not linked to cell house-keeping) and they can be genetically modified quite easily to produce new molecules from a selected precursor. In the case of vanillin, several potential precursors have been suggested -including curcumin, siam benzoin resin, phenolic stilbenes, eugenol and ferulic acid. The biotransformation of these molecules in vanillin by various microorganisms has been extensively studied, patented and/or reviewed (Falconnier et al., 1994; Lesage-Meessen et al., 1996; Narbad et al., 1997; Rabenhorst and Hopp, 1997; Muheim et al., 1998; Steinbuchel et al., 1998; Thibault et al., 1998; Lesage-Meessen et al., 1999; Walton et al., 2000; Topakas et al., 2003). In any case, vanillin has posed an intriguing biosyn-thetic problem for many years. In vanilla, vanillin P-glucoside formation appears to be much more complex than initially envisaged. Due to the commercial importance of vanilla-type flavourings, it is still necessary to learn more about the molecular genetic characterisation of vanillin formation that might open up the possibilities to introduce new or enhanced biosynthetic capacities in plants (Walton et al., 2003).

In addition to vanillin, other aromas can be produced from other mono-/oligomeric components originating from plant co-products. This is particularly the case for sugar monomers present in cell wall polysaccharides. l-Rhamnose is mainly found in the pectic fraction of the cell wall. Commercially available rhamnose is produced by chemical hydrolysis of arabic and karaya gums, or from rutin or citrus fruits which contain by weight 10-30% rhamnose. Rhamnose is a raw material for the production of furaneol (2,5-dimethyl-4-hydroxy-3(2H)-furanone), a strawberry flavour used in caramel and fruit flavour applications (Haleva-Toledo et al., 1999). Arabinose, another pectic monomer, is a precursor of l-fructose and l-glucose that would be excellent as low-calorie sweeteners (Vogel, 1991).

16.5.4 Ethanol

Fruit and vegetable by-products are rich in fermentescible sugars, and as such they can be used as raw material for ethanol production. Production of ethanol from agricultural and forestry residues or other sources of ligno-cellulosic biomass is of both economic and environmental interest. It could be a way to counter the inevitable depletion of the world's petroleum supply and to decrease air pollution. Ethanol can be produced from glucose and xylose fermentation, both originating from cell wall polysaccharide degradation. Sugar cane bagasse is the main raw material used for this purpose but other biomass is also used, such as hardwood and grasses. Some other wastes have been studied, e.g. chicory roots (Leplus, 2004). The biomass can be treated by a concentrated acid process that uses sulphuric acid (Fig. 16.4). In that case, very efficient acid recycling is required for the process to be economically acceptable. The second possibility to recover the monomers is to degrade the wastes using enzymatic tools. This environmentally friendly process needs to overcome the natural resistance of lignocellulosic biomass to enzymatic breakdown. It was thus necessary to develop concomitant physical pretreatment to alter biomass structure and thus enhance the biodegradability of the waste, so that hydrolysis of the carbohydrate to monomeric sugars can be achieved more rapidly and with greater yields. A number of pretreatment options have been investigated, including steam-explosion, ammonia fibre explosion, organosolvents, supercritical extraction and dilute acid pretreatment (Wyman, 1994; Mosier et al., 2005).

Consequently, processing of plant waste to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation and product separation/purification. Plant wastes are enzymatically degraded by a combi-

Fig. 16.4 Simplified diagram showing possible processes for ethanol production (from Mielenz, 2001). Top, concentrated acid process; bottom, enzymatic production.

Fig. 16.4 Simplified diagram showing possible processes for ethanol production (from Mielenz, 2001). Top, concentrated acid process; bottom, enzymatic production.

nation of cellulases, hemicellulases and pectinases. The hexoses released (mainly glucose and galactose) are readily fermented to ethanol by many naturally occurring microorganisms, whereas pentoses (mainly xylose and arabinose) are fermented to ethanol by few native strains with most often relatively low yields (Mosier et al., 2005). Both yeasts (such as Saccharomyces and Pichia species) and bacteria (such as Escherichia coli, Klebsiella and Zymomonas) have been genetically modified to ferment hexoses and pen-toses (reviewed by Mielenz, 2001). The ideal bioethanol-producing strain would ferment all sugars simultaneously whilst being resistant to inhibitory by-products. This situation has been approached by cloning endoglucanase genes from Erwinia into Klebsiella species (Zhou and Ingram, 1999).

16.5.5 Organic acids

Since the early 1990s, food and food ingredient markets have been characterised by a dynamic development not seen before. The requirements for food processing are increasing. The ageing population, predominantly in Western countries, also adds to an increased demand for high-quality foods. Consequently, the demand for specific ingredients used in preparing high-quality and preserved foods also increases (BCC, 2002). Of outstanding importance in this sector are ascorbic acid (l-3-ketothreohexuronic acid lactone), isoascorbic acid (d-erythro-Hex-2-enonic acid y-lactone) and citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid). Ascorbic acid, in addition to its role in preservation is vitamin C, which is of increasing importance as scientific data support its beneficial effect on human health. Isoascorbic acid is the isomer of ascorbic acid and is very popular for the preservation of meat and fish products. Citric acid is an organic acid of diverse economic uses. It is a standard acidulant and the food and beverage industries use it extensively as a food additive. It is also able to complex heavy metal ions, like iron and copper, and is therefore applied in the stabilisation of oils and fats.

Citric acid is produced by fermentation and Aspergillus niger is the most popular strain for citric acid production on an industrial scale. The worldwide estimate of production in 2000 was 9 million tonnes (Karaffa and Kubicek, 2003), with an annual 2-3% increase. Carbon sources used should be cheap and available such as agricultural waste and waste papers. The basic substrates for citric acid fermentation using a submerged technique of fermentation are beet or cane-molasses (Pazouki et al., 2000). The biochemical pathway leading to citric acid hyperproduction was discovered 50 years ago when Cleland and Johnson (1954, reviewed by Karaffa and Kubicek, 2003) demonstrated that citric acid biosynthesis involves the gly-colytic catabolism of glucose to two moles of pyruvate, of which one is converted to acetyl-CoA (by releasing one mole of carbon dioxide) and the other one to oxaloacetate (by fixing this one mole of carbon dioxide onto the second pyruvate) and then finally condensing these two precursors to citric acid. Genetic engineering of Aspergillus niger primary metabolism is employed to improve citric acid production by this fungus (Haq et al., 2001), but the nutritional status of the organism and basic fermentation parameters have also been studied (Ali et al., 2002).

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