Recovery of functional biopolymers

The polysaccharides of the fruit and vegetable trimmings (pectins, cellulose and hemicelluloses), whose relative proportions vary with respect to the plant source, can be more or less selectively recovered from fruit and vegetable wastes for multiple food and non-food uses.

16.4.1 Pectins

Chemical structure

Pectin is an extremely complex polysaccharide, composed of as many as 17 different monosaccharides which can be envisioned as a multiblock co-biopolymer (Figure 16.1). The simplest of these blocks is homogalacturo-nan (HG), an unbranched polymer of (1^4)-a-d-GalpA, the so-called pectic 'smooth' region. Other minor types of galacturonans (so-called 'substituted galacturonans' (Ridley et al., 2001)) can be distinguished, for example, rhamnogalacturonan II (RG-II) and xylogalacturonan (XGA) (Vincken et al., 2003). A second major block, rhamnogalacturonan I (RG-I), is composed of a repeating disaccharide unit [^2)-a-l-Rhap-(1^4)-a-d-GalpA-(1^]n. RG-I is decorated primarily with other blocks, namely arabinan and arabinogalactan side chains. Complexes of RG-I, arabinan and arabinogalactan are often referred to as pectic 'hairy' regions in which arabinans and arabinogalactans are the 'hairs'.

'Smooth' regions

'Smooth' regions were selectively isolated and their macromolecular parameters (molar mass, degree of polymerisation and polydispersity) were

Arabinan and/or arabinogalactan side chains

Homogalacturonan

Homogalacturonan

Rhamnogalacturona

'Smooth' region

'Hairy' region

Fig. 16.1 Schematic representation of the structure of pectin.

investigated. In some studies (Powell et al., 1982; Thibault et al., 1993), differences in the susceptibility to acid hydrolysis of the glycosidic linkages were exploited: under mild acid conditions the linkages between adjacent GalA residues (HG domains) are much more stable than linkages between GalA and Rha (RG-I domains). Using this chemical approach, Thibault et al. (1993) isolated almost pure HG from apple, beet and citrus pectins. A weight-average degree of polymerisation of 108-138 was estimated by HPSEC-MALLS (high-performance size exclusion chromatography coupled on-line with multiple angle laser light scattering). Similar values were recently obtained for HG isolated by enzymatic means (Bonnin et al., 2002; Hellin et al., 2005). 'Smooth' regions isolated by chemical or enzymatic means were all characterised by a very low polydispersity index, revealing a high homogeneity with respect to molar mass (Hellin et al., 2005).

GalpA residue carboxylic functions are naturally partly methyl-esterified (Pilnik and Voragen, 1970). The degree of methylation (DM) is defined as the percentage of GalA units esterified with methanol. The methyl-esteri-fication of HG has been the subject of several investigations since it determines to a large extent the industrial applicability of pectin and their interaction ability in muro (Rolin et al., 1998; Ralet et al., 2001). Not only the DM, but also the distribution of methyl groups on the HG has a deep impact on pectin gelation properties (Kohn et al., 1983; Thibault and Rinaudo, 1985; Ralet et al., 2001).

Secondary alcoholic functions of GalpA residues can also be partly acetyl-esterified. The degree of acetylation (DAc) is defined as the number of acetyl groups for 100 GalA units. The DAc is generally low but pectins with high DAc - such as sugar beet, pear, carrot and potato - have been reported (Voragen et al., 1995). Recently, the sugar beet HG acetylation pattern was assessed (Ralet et al., 2005).

'Hairy' regions

As in 'smooth' regions, GalpA residues in RG-I regions may be O-acetylated. In contrast, there is no conclusive chemical evidence that GalpA residues are methyl-esterified in RG-I regions.

Depending on the plant source and method of isolation, 20-80% of the Rhap residues in RG-I are substituted, mainly at C-4 but sometimes at C-3, with oligomeric or polymeric side chains (O'Neill et al., 1990). The predominant side chains contain linear and branched Ara/, and/or Galp residues, although their relative proportions and chain lengths differ a lot depending on the plant source (Lerouge et al., 1993). Among the side chains of neutral sugars, arabinans and arabinogalactans (type I and II) can be distinguished. Arabinans consist of a 1,5-linked a-l-Ara/ backbone, which can be substituted a-l-Ara/-(1^2), a-l-Ara/-(1^3), and/or a-l-Ara/-(1^3)-a-l-Ara/-(1^3) side chains. Pectins with arabinans attached have been isolated from several sources including sugar beet (Guillon and Thibault, 1989), carrots (Massiot et al., 1988), cabbage (Stevens and Selvendran, 1984) and onion (Ishii, 1982). In sugar beet, arabinans are present as long chains (backbone length of 60-70 residues) with 45-65% of the Ara residues substituted mainly by single Ara residues (Oosterveld et al., 2002). Arabinogalactans occur in two structurally different forms. Type I arabinogalactans are composed of a 1,4-linked-P-d-Galp backbone more or less substituted at O-3 with short 1,5-linked-a-l-Ara/ chains (Voragen et al., 1995). Pectins with type I arabinogalactans attached are commonly found in several fruits and vegetables, e.g. citrus (Labavitch et al., 1976), potato (Jarvis et al., 1981) and tomato (Seymour et al., 1990). Type II arabinogalactan is a highly branched polysaccharide with ramified chains of P-d-Galp residues joined by 1^3 and 1^6 linkages. 1,4-linked-P-d-Galp are generally terminated by l-Ara/ and to some extent by l-Arap residues (Voragen et al., 1995). Pectins with type II arabinogalactans attached have been found, e.g. in apple, rapeseed, lemon, beet and grape (Voragen et al., 1995).

Manu/acture o/ industrial pectins Current raw materials

Raw materials presently employed in industrial practice are the dried press cake of apple juice manufacture (apple pomace, 10-15% extractable pectins) and the wet or dried peels and rags of citrus juice manufacture (citrus peel, 20-30% extractable pectins). Apple pomace and citrus peels are, in the wet state, very perishable commodities (May, 1990). The major part of the raw material is dried for shipment or warehousing, so that pectin can be produced outside the harvesting period and/or at another location. Washing in water is necessary prior to drying in order to minimise caramelisation (Rolin et al., 1998). Other sources have been considered for the extraction of commercial pectins, e.g. sugar beet, sunflower heads and the wastes from the processing of tropical fruits. These pectin-containing materials are potentially available in quantity and/or in logistically or economically favoured locations.

Pectin extraction

Commercially, pectin is extracted by treating the raw material with hot dilute mineral acid at ~pH 2. The hot pectin viscous extract is separated from the strongly swollen and partly disintegrated residue by a combination of centrifugation and filtration. The clarified extract is brought to pH 4 and concentrated under vacuum prior to alcoholic precipitation, pressing and drying. Throughout the process, unnecessary holding times at high temperatures are avoided in order to prevent pectin demethylation and depolymerisation. Pectins can be highly variable, depending mainly on the raw material quality, and it is usual to blend together a number of production batches and dilute them with sucrose or dextrose to a standard gelling performance. The process described above yields a pectin of around 75% methyl-esterification. To produce other types of pectins with diverse application properties, some saponification of the methyl groups is required. This is usually performed by the action of acid or alkali when pectins are in the alcoholic slurry. This process yields high-methoxy (HM) pectins (this term is used for commercial pectins of high DM (>50%)) with DM values in the range 55-75% or low-methoxy (LM) pectins (this term is used for commercial pectins of low DM (<50%)) with DM values in the range 2045%. Alkaline treatments at low temperature to avoid P-eliminations can also be used in the presence of ammonia. In these conditions, pectins are partly de-esterified and some of the methyl groups are replaced by amides, giving LM amidated pectins (the degree of amidation must be <25%). These amidated pectins need less calcium to gel and are less prone to precipitation at high calcium concentrations; furthermore they give gels that are more thermoreversible than the non-amidated LM pectins (Voragen et al., 1995).

Effluent has become a major consideration in the pectin industry; it is increasingly a major cost and sometimes a serious technical problem to the pectin manufacturer (May, 1990).

Pectin gelling properties and food applications

Pectin is first and foremost a gelling agent used to impart a gelled texture to foods, mainly fruit-based foods. The DM value is the key criterion that governs gelation conditions: HM pectins will gel with at least 55% of soluble solids and at pH < 3.5 while LM pectins will gel in the presence of calcium ions.

HM pectins. About 80% of the world production of HM pectin is used in the manufacture of jams and jellies, the pectin being added to supplement the gelling power of fruits. Jams and jellies are made with a soluble solids of 65%, a final pH of 2.9-3.2 and generally contain 0.3-0.5% HM pectin.

The analytical parameter that allows prediction of gelling behaviour (setting time, pH and temperature) is the DM. This has led to a subdivision of HM pectins into four commercial categories: ultra-rapid set (DM 74-77), rapid set (DM 71-74), medium set (DM 66-69) and slow set (DM 58-65). It is the art of the jam manufacturer to choose the correct pectin type for each jam or jelly type. For example, jams with whole fruits should gel shortly after filling in order to prevent fruit flotation. To keep the fruit evenly suspended, rapid- or ultra-rapid-set pectins that gel rapidly at high temperature (80 °C at pH 3.1; May, 1990) have to be used. At the other end of the scale, clear jellies need to gel slowly to avoid trapped air bubbles. Slow-set pectins that gel at low temperature (around 40 °C at pH 3.1; May, 1990) are the most obvious choice. Jam manufacturers also make a very wide range of jams, fillings and toppings for the confectionery industry. HM pectins are mainly used for making fruit jellies and jelly centres flavoured with natural fruit constituents and/or synthetic flavours. HM pectins find further applications in fruit drink concentrates, instant fruit drink powders, fruit juice-milk combinations and sour milk products (http://www.cpkelco. com/food/index.html).

LM pectins. LM pectin is used when the soluble solids content is between 20 and 55%, typically in low- or reduced-calorie fruit spreads. An addition of calcium salt is generally needed. The type of LM pectin (slow set, DM 50; medium set, DM 40; rapid set, DM 30) must be carefully selected according to the soluble solids/pH conditions in the application medium. The heat reversibility of LM pectin gels may be utilised in bakery jams and jellies for glazing purposes. Pectin manufacturers offer ready-made blends of LM pectins and sometimes other ingredients for products of this type. LM pectins are also often used in fruit preparations for yoghurt, fruit-milk desserts, gelled milk products and confectionery products. In the latter, LM pectins are used for jellies and centres in which the low pH range necessary for HM pectin gelation is not acceptable for flavour reasons (for example in cinnamon- or peppermint-flavoured jellies) (http://www.cpkelco.com/ food/index.html).

Pectin non-food applications

Sugar beet pectin suffers from several disadvantages as a competitor to apple or citrus pectin, especially its high content of acetyl groups which hinders 'traditional' gelation. However, in beet pectin, feruloyl groups naturally esterify neutral sugars side chains (see Section 16.5.2, Hydroxy cin-namic acids) and it is possible to take advantage of the presence of those feruloyl groups. Feruloylated pectins can indeed be oxidatively cross-linked by chemoenzymatical (peroxidase and hydrogen peroxide) or enzymatical (laccase) means (Rombouts and Thibault, 1986; Micard and Thibault, 1999). The gel formed is thermally stable and can be dehydrated and rehydrated (Rombouts and Thibault, 1986). It may thus lend itself to applications quite different from those of current commercial pectins, such as superadsorbant systems.

The resistance of pectin to degradation in the upper gastro-intestinal tract and its complete and rapid dissolution in the colon, make pectin an ideal ingredient for colon-specific drug delivery (Rolin et al., 1998). A colonic drug delivery system based on a pectin and galactomannan coating was proposed (Lee et al., 1999) and the site specificity of drug release was assessed in human subjects (Yang et al., 2002).

Pectins or some pectic-specific domains exhibit effects on the human immune system. The possible mechanisms of action include influence on the complement cascade, endocrinal functions, cytokine induction and the effect on chemotaxis of leucocytes (Wagner and Kraus, 2000; cited by Morra et al., 2004). Furthermore, some pectins, more particularly their RG-I domains, exhibit anti-ulcer and mitogenic activities (Yamada, 2000; cited by Morra et al., 2004). Finally, metastasis of some cancers depends on the cancer cells' specific recognition of galactoside epitopes, a recognition that can be inhibited by appropriately engineered pectins (Nangia-Makker et al., 2002). So far, limited studies have been performed with pectins or specific pectic domains, but this molecule has a clear potential for therapeutic significance and is undergoing clinical trials (Nangia-Makker et al., 2002).

Upgrading of isolated pectin domains: arabinans

Arabinans are present as pectin neutral sugar side chains in various plants (see Section 16.4.1, 'Hairy regions'). They can be extracted from isolated pectins or directly from plant by-products such as sugar beet pulp. Alkaline extraction at high temperature (70-98 °C) for 15-90 min followed by neutralisation and ultrafiltration yields a branched arabinan (molar mass of about 50 kDa) containing around 80% of l-Ara (McCleary et al., 1990) (Fig. 16.2). Branched arabinan exhibits surface active properties that make it suitable for use as an emulsifying agent. Additionally, flavour oil and fragrances may be encapsulated using arabinan (McCleary et al., 1990). However, the arabinan extraction and purification cost is a clear limitation for these uses.

Branched arabinan can be linearised using purified a-l-arabinofuranosi-dase to yield debranched arabinan (McCleary et al., 1990) (Fig. 16.2). The debranched arabinan forms an aqueous gel that has the properties of a fat substitute and may be used in foods (McCleary et al., 1990; Cooper et al., 1992). Linearised arabinans could also find applications as texture agents in cosmetic and pharmaceutical industries (Cooper et al., 1992).

16.4.2 Cellulose

Cellulose is the world's most abundant naturally occurring polymer, rivalled only by chitin. Commercial purification of cellulose is centred on

Pellet

Powdered lime (10 kg)

Juice

Decantation Filtration

Filtered juice

Ultrafiltration

Permeate

Retentate

Spray drying J Arabinofuranosidase

Branched arabinan -► Linearised arabinan

Fig. 16.2 Recovery of branched and debranched arabinan from sugar beet pulp.

wood pulp and cotton linters but fruit and vegetable wastes, which usually contain 25-30% of cellulose on a dry matter basis, could be interesting sources.

Chemical structure

Cellulose is a homopolymer of (1^4)-P-d-Glcp. The P-1,4 configuration results in a rigid and linear structure for cellulose. Cellulose chains exhibit a strong tendency to form intra- and intermolecular hydrogen bonds resulting in the formation of microfibrils whose length, width and crystallinity differ a lot depending on the cellulose origin. Cellulose arising from primary cell walls is particularly thin (2-3 nm width) and of low crystallinity.

Isolation and upgrading of cellulose from fruit and vegetable wastes Following the initial work of Weibel (1986, 1989) and Weibel and Myers (1990), Dinand et al. (1996, 1999) purified sugar beet cellulose and evaluated the application potential. A specific alkaline purification treatment led to a partial desincrustation of the cellulose microfibrils from the other cell wall components (pectins and hemicelluloses). Aqueous suspensions of cellulose microfibrils were then defibrillated using a high-pressure homog-eniser (Dinand et al., 1996, 1999) (Fig. 16.3(a)). This method was also applied in order to recover defibrillated cellulose fibres from lemon peels (Rondeau-Mouro et al., 2003). A similar method including an acid extraction was recently developed (Zykwinska et al., 2005) (Fig. 16.3(b)). In all cases 'desincrusted' parenchymal cell cellulose (PCC) - containing roughly 90% cellulose together with residual amounts of pectin, hemicellulose and inorganics - was obtained. After high-pressure homogenisation, defibril-lated PCC suspensions consisting of dispersions of cellulose microfibrils, either individual or still bundled together, were recovered (Fig. 16.3). One of the key properties of defibrillated PCC suspensions is that they do not sediment or flocculate and display liquid crystalline characteristics (Dinand et al., 1996, 1999). Homogenised PCC displays rheological properties (shear thinning properties together with pseudoplasticity) that are similar to those

Sugar beet pulp

Sugar beet pulp

Extract

Extract

Extensive washing with water

Residue

Sodium chlorite Parenchymal cell cellulose (PCC)

High-pressure homogeniser

Dispersions of cellulose microfibrils

Extract

HCl 0.1N, 3 x 30 min, 85 °C Extensive washing with water

Residue

Residue

Extract

NaOH 0.5N, 3 x 30 min, 80 °C Extensive washing with water

Parenchymal cell cellulose (PCC)

Extract

Parenchymal cell cellulose (PCC)

High-pressure homogeniser

High-pressure homogeniser

Dispersions of cellulose microfibrils

Dispersions of cellulose microfibrils

Fig. 16.3 Recovery of crude and defibrillated parenchymal cell cellulose (PCC) using the methods developed by (a) Dinand et al. (1996, 1999) and (b) Zykwinska et al. (2005).

observed for microfibrillated wood cellulose (commercialised under the trade name of Celish® by Daicel (http://www.daicel.co.jp/wsp/eZproduct/ product-c.html)) and for bacterial cellulose (Dinand et al., 1996, 1999). The interest in bacterial cellulose is reflected in a number of patents and publications devoted to this polymer (Bielecki et al., 2005). Bacterial cellulose has found a multitude of commercial applications in paper, textile and food industries, and as a biomaterial in cosmetics and medicine (Bielecki et al., 2005). The processing cost of bacterial cellulose is however still a pitfall to its competitive industrial production. Homogenised PCC could potentially replace bacterial cellulose in several applications: as a component of paper to reinforce paper mechanical properties (Dufresne et al., 1997), in surgical drapes and gowns, in various medical applications or in food applications.

Recently, cellulose microfibrils were isolated from swede roots and homogenised as described above (Bruce et al., 2005). Homogenised PCC suspensions were used to form composites with four matrix materials (poly-vinylacetate, acrylic polymer, epoxy and locust bean gum). The best of the composites had a stiffness and strength significantly greater than conventional flax-epoxy composites and as good as the best glass-fibre composites (Bruce et al., 2005).

Homogenised PCC suspensions were also successfully surface silylated (Gousse et al., 2004). The mildly silylated microfibrils retained their morphology, but could be dispersed in a non-flocculating manner into organic solvents. This highlights the potential for the use of these microfibrils in the reinforcing of non-polar polymers, such as polyolefins or other commodity polymers.

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