Oligosaccharides
The term oligosaccharide is derived from the Greek word olio-, which means few. They are composed of 3–10 monosaccharides joined together by glyco- sidic bonds. The most common oligosaccharides in the plant kingdom are a- galactosides (Kadlec, 2001). Oligosaccharides are not of great importance in foods except for a series of galactosylsucroses (often termed a-galactosides) that occur in legumes and some vegetables, and fructo-oligosaccharides found in cereals, onions and some other plants. The most ubiquitous group within the Table 6.1. Bioactive compounds and their presence in chickpea seeds.
Relative abundance, in comparison with
Class other legume seeds Biological activity
Protease inhibitors ++++ Impair protein digestion, antitumoral Oligosaccharides +++ Prebiotics, fl atulence
Phytates +++ Metal overloading, glycemic index lowering Saponins +++ Growth inhibitors, lower plasma cholesterol Polyphenols ++ Antioxidant
Isofl avones ± Phytoestrogens, metabolic control Lectins ± Severe growth depression, antitumoral ++++: very abundant; +++: abundant; ++: low; and ±: very low.
a-galactosides is the raffinose family of oligosaccharides. The family includes raffinose (a trisaccharide), stachyose (a tetrasaccharide) and verbascose (a pentasaccharide).
Raffinose is present in all parts of the chickpea plant but accumulates in the seeds and roots during development. The concentration of raffinose in the seeds increases to 0.09–3.0 g/100 g (desi) and 0.01–2.8 g/100 g (kabuli) as the seeds mature and dry (Table 6.2). Most of the drought-resistant leguminous crops contain higher raffinose contents and could contribute, in part, to their toler- ance (Arora, 1983). Both raffinose and stachyose occur in chickpea seeds and leaves, and have been shown to provide frost tolerance in plants (Castonguay et al., 1995). Stachyose contents range from 0.48 to 5.35 g/100 g for desi type and 0.35 to 6.48 g/100 g for kabuli types. Chickpea has only small amounts of verbascose, up to 0.41 g/100 g reported in desi (Saini and Knights, 1984) and is absent in most of kabuli and some desi varieties.
Ciceritol is also an a-galactoside, but does not belong to the raffinose family of oligosaccharides. It is an a-D-digalactoside of pinitol and was first discovered in chickpea (hence the name ciceritol) by Quemener and Brillouet (1983). It is thought that ciceritol was mistakenly identified as manninotriose in previous literature. Chickpea seeds contain 2.11–3.10 g /100 g and 1.24–2.79 g /100 g ciceritol in desi and kabuli types, respectively (Table 6.2).
The a-galactosides are well known as antinutritional factors for causing flatulence. The first information on the antinutritive effects of a-galactosides was reported back in 1917 by Kuriyama and Mendel (1917). Flatulence occurs because mammals (including humans) have no a-galactosidase present in their intestinal mucosa, which is required to hydrolyse these compounds. Hence, ingestion results in a-galactosides passing into the large intestine where anaero- bic fermentation of bacteria occurs producing gaseous by-products (e.g. hydro- gen, CO2 and traces of methane gas). Western populations have poor tolerance to flatulence, and are sometimes accompanied by diarrhoea and abdominal pain if consumed in large quantities. Raffinose and stachyose ingestion causes flatulence (Fleming, 1981), but not ciceritol, perhaps due to easier hydrolysis of this compound because to its different structure (Quemener and Brillouet, 1983). This may explain the reason of less flatulence of chickpea than lentil and beans (Sanchez-Mata et al., 1998).
The lack of digestion is also a constraint as far as intensive monogastric livestock producers are concerned, as oligosaccharides in the diet can reduce growth performance. In addition, a higher content of a-galactosides (higher than that found in normal diets) has been shown to reduce the absorption capacity of the small intestine by changing its osmotic pressure (Wiggins, 1984;
Zdunczyket al., 1998). In any case, intestinal digestion of a-galactosides can be improved by providing supplementary diets with exogenous a-galactosidase (Kozlowska et al., 2001). This is not an issue in the case of chickpea as only severely damaged seed would be economically competitive for animal feed compared to other grains.
Contrary to previous reports, research by Sandberg et al. (1993) recently showed that ~30% of dietary raffinose and stachyose was degraded and digested in the stomach and small intestine of humans.
147 Table 6.2. Oligosaccharide contents of desi and kabuli seeds.
Desi content Number of Kabuli content Number of
Oligosaccharides (g/100 g) cultivars References (g/100 g) cultivars References Raffi nose 0.09–3.0 29 Rao and Belavady (1978); 0.01–2.8 22 Rossi et al., (1984);
Rossi et al. (1984); Saini and Saini and Knights Knights (1984); Mulimani and (1984); Mulimani and Ramalingam (1997); Mansour Ramalingam (1997);
and Khalil (1998); Salgado Sanchez-Mata et al.
et al. (2001) (1998); Salgado et al.,
(2001)
Stachyose 0.48–5.35 19 Rossi et al. (1984); Saini and 0.35–6.48 21 Rossiet al., (1984); Saini Knights (1984); Mansour and and Knights (1984);
Khalil (1998); Salgado et al. Sanchez-Mata et al.,
(2001) (1998); Salgado et al.,
(2001)
Verbascose 0.01–0.41 8 Saini and Knights (1984); 0.00–0.37 2 Salgado et al., 2001
Salgado et al. (2001)
Ciceritol 2.11–3.10 10 Rossi et al. (1984) 1.24–2.79 12 Rossiet al., (1984);
Sanchez-Mata et al.,
(1998)
Althougha-galactosides have little food value (are partially digested, if at all), this does not imply the absence of health benefits. In fact, a-galactosides convey many benefits to both humans and monogastric animals. Since they pass mostly undigested into the lower gut, they are a constituent of dietary fibre and can act as a prebiotic. Dietary fibre also is highly beneficial (refer to Chapter 5, this volume). In addition, the prebiotic effect is derived from metab- olism of a-galactosides by gas-producing bacteria, which increase the colonic population of bifidobacteria. Bifidobacteria are advantageous to human health by suppressing intestinal putrefaction, reducing both constipation and diar- rhoea, stimulating the immune system and increasing resistance to infection (Mitsuoka, 1996).
Phytic acid
Phytic acid, myo-inositol-(1,2,3,4,5,6) hexakis-phosphate and its salts are the major sources of phosphorus in legume seeds (Urbano et al., 2000). Ravindran et al. (1994) found that the phytate phosphorus in chickpea was 51.2% of the total phosphorus content. The total phytic acid content in chickpea has been reported to vary from 0.3% to 1.8% (Duhan et al., 1989; Ravindran et al., 1994;
Burbano et al., 1995; Rincón et al., 1998; Martinez et al., 2002). Phytic acid content can vary with genotype, climate, type of soil and year.
Phytic acid has been considered an antinutrient as it binds with other nutri- ents and makes them indigestible. Excessive phytic acid in the diet can have a negative effect on mineral balance because of the insoluble complexes it forms with essential minerals (Cu2+, Zn2+, Fe3+ and Ca2+), which causes poor mineral bioavailability (Zhou and Erdman, 1995; Urbano et al., 2000). Phytic acid is able to make complex with proteins also, decreasing protein solubility. Therefore, phytates have negative impact on enzyme activity and there is evidence of its negative effects on key digestive enzymes like lipase, a-amylase, pepsin, trypsin and chymotripsin (Thompson, 1993; Greiner and Konietzny, 1996b; Urbano et al., 2000). The binding of phytic acid to these enzymes reduces nutrient digest- ibility. Phytic acid also binds with starch through phosphate linkages (Lajolo et al., 2004).
The ability of phytic acid to bind with minerals, proteins or starch, directly or indirectly, may alter solubility, functionality, digestibility and absorption of these nutrients. In addition, monogastric animals have a limited ability to hydrolyse phytates and release phosphate for absorption due to a lack of intesti- nal phytases (Zhou and Erdman, 1995; Greiner and Konietzny, 1996a; Urbano et al., 2000).
However, there are some beneficial effects of phytic acid, such as reduced bioavailability, and therefore toxicity, of heavy metals (e.g. cadmium and lead) present in the diet (Rimbach et al., 1996; Rimbach and Pallauf, 1997). Several studies have provided evidence of antioxidant properties of phytic acid in vitro (Lajolo et al., 2004). These effects are mainly mediated through its iron and copper chelating properties, although the molecular mechanisms are not fully understood. Contrary to this was research by Minihane and Rimbach (2002)
who demonstrated that under in vivo conditions phytic acid did not always have a significant effect on oxidant or antioxidant status.
Phytases are enzymes that are widely distributed throughout nature: in plants, certain animal tissues and microorganisms. They have been studied intensively in the last few years because of their ability to reduce the phytate content in monogastric and human foods (Greiner and Konietzny, 1996b). This is done by hydrolysing phytic acid to a series of lower phosphate esters of myo-inositol and phosphate. Two types of phytases are known: 3-phytases (EC 3.1.3.8) and 6-phy- tases (EC 3.1.3.26), indicating the susceptible phosphoester bond that is predomi- nantly attacked by the enzyme. The pathway of dephosphorylation of myo-inositol hexakis-phosphate by phytases purified from different legume seeds has been established by Maiti et al. (1974) and modified by Greiner et al. (2002).
Different structural isomers of myo-inositol phosphates can be generated during enzymatic degradation. They can have different physiological functions;
hence, their identification is of great importance to exploit the full potential of naturally occurring phytases (Greiner et al., 2002). The myo-inositol phosphates, IP6 and IP5, have the worst antinutritional effects, as the smaller molecules (IP4, IP3, IP2 and IP1) have a lower capacity to complex with inorganic cations. The major inositol phosphate in chickpea is IP6 (Burbano et al., 1995).
Some myo-inositol phosphates, including IP6 from soybean, have been sug- gested to have beneficial health effects, such as amelioration of heart disease by controlling hypercholesterolemia and atherosclerosis, prevention of kidney stone formation and a reduced risk of colon cancer (Greiner et al., 2002).
Tannins
The term ‘tannin’ originated from the ability of these compounds to tan animal skins to produce leather, and dates back to ancient times. Most, but not all, tannins have this ability and can also form insoluble complexes with carbohy- drates and protein. In fact, the precipitation of salivary proteins is responsible for the distinct astringency sensation of tannin-rich foods. Tannins are believed to play a role in plant protection (against infection, insect and animal damage) as these types of stresses cause increased plant tannin levels (Haslam, 1998).
Tannins are now defined as polyphenolic secondary metabolites of higher plants, which fall into four specific groups depending on their basic chemical struc- ture: (i) gallotannins; (ii) ellagitannins; (iii) complex tannins; and (iv) condensed tannins (also known as proanthocyanidins). Figure 6.1 shows this classification published by Khanbabaee and van Ree (2001). Currently, more than 1000 natu- rally occurring tannins are structurally identified (Quideau and Feldman, 1996).
Previously, tannins were divided into two groups based on their ability to fractionate hydrolytically (with acid, alkali, hot water or enzymatic action) as:
(i) hydrolysable tannins (including gallotannins and ellagitannins) or (ii) con- densed tannins. This definition was first elucidated by Freudenberg (1919). This simple categorization means that these definitions are still most commonly used in nutritional analyses today, despite ignoring tannins with molecular weights below 1000 Da (Khanbabaee and van Ree, 2001).
The tannin contents of chickpea from the literature are listed in Table 6.3.
Chickpea has been reported to contain approximately 0.36–0.72 g/100 g and 0.12–0.51 g /100 g total tannin (desi and kabuli types, respectively), of which 0–13% is condensed tannin. Chickpea contains low tannin contents, with con- centrations (both total and condensed) much lower than faba bean (Vicia faba), pigeon pea (Cajanus cajan) and lentil (Lens culinaris).
The hydrolysable tannins are not considered to have more antinutritional effects, but are responsible (with some other polyphenolic compounds) for the colour of chickpea seed coats. The condensed tannins (proanthocyanidins) are high-molecular-weight polymers (commonly ~5000 Da) and are known to bind dietary protein, thereby reducing protein digestion. This antinutritional effect can be significant in some other legumes, but only very small amounts of con- densed tannins are found in chickpea (<0.1% in desi and <0.04% in kabuli;
Table 6.3). Garcia-Lopez et al. (1990) found that tannins from chickpea caused no significant reduction in iron absorption, in contrast to tannins from tea, cof- fee and wine. In addition, tannins are located primarily in the seed coat; hence, the removal of the seed coat during or before food preparation will practically eliminate tannins from the diet.
Tannins
Gallotannins Ellagitannins Complex tannins Condensed
tannins
O C
1 2
OH
HO OH (G)
HO HO
HO OH
OH HO
HO HO O OR
OR RO RO
O
O OR
OR RO O
O C C O C
3 4
HO OH
OH
R = Galloyl moiety (G)
or other substituents (Catechin moiety)n (Catechin moiety)n
HO
HO HO
OH
OH O
O
HO
OR OH
HO OH
OR OR
RO O HO
O
O
OH (G) OH (G)
H (OH)
C CO C
OH OH OH
OH
Fig. 6.1. Tannin classifi cation. (From Khanbabaee and van Ree, 2001.)
Table 6.3. Tannin contents of desi and kabuli seeds.
Chickpea Content Number of
type Tannins (g/100 g) cultivars References
Desi Total tannin 0.36–0.72 53 Petterson et al. (1997); Salgado et al. (2001)
Condensed 0.01–0.09 47 Petterson et al. (1997); Salgado
tannin et al. (2001)
Kabuli Total tannin 0.12–0.51 39+ Zaki et al. (1996); Petterson et al.
(1997); Viveros et al. (2001)
Condensed 0.00–0.04 37 Petterson et al. (1997); Salgado
tannin et al. (2001)
Protein antinutrients
The most widely studied antinutrient proteins in legumes are the enzyme inhib- itors (pancreatic proteases and a-amylases) and the lectins (Lajolo et al., 2004;
Pusztaiet al., 2004).
Protease inhibitors
Legume seed protease inhibitors can have a major impact on seed nutritional value as they inhibit the function of digestive enzymes, such as trypsin and chymotrypsin, by competitive binding. These protease inhibitors contain no carbohydrates and belong to two different families: the Kunitz family and the Bowman-Birk family. Protease inhibitors from both families have been found in chickpea seeds (Domoney, 1999; Lajolo and Genovese, 2002; Lajolo et al., 2004; Srinivasan et al., 2005). Both families are capable of inhibiting trypsin and chymotrypsin. A large number of isoforms of the Bowman-Birk inhibitor (BBI) have been described in soybean, and have differing properties depending on their chemical structure. Saini et al. (1992) found 6–8 isoinhibitors in chick- pea capable of inhibiting trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1).
Smirnoffet al. (1976) found two active fragments of protease inhibitor in chick- pea seeds, designated as A and B. Fragment A inhibited trypsin but not chymo- trypsin, and fragment B inhibited chymotrypsin but not trypsin. The inhibition of human trypsin and chymotrypsin by chickpea seed extracts was reported by Gertler et al. (1982) and Belitz et al. (1982), and that of bovine enzymes by Borchers and Ackerson (1947), Abramova and Chernikov (1964) and Belitz et al. (1982). Although the porcine pancreatic proteinases have been subjected to inhibition studies using extracts from a range of leguminous species (Rascon et al., 1985), there is no report on similar inhibitory studies with chickpea.
The protease inhibitor contents of desi and kabuli seeds from the literature are reported in Table 6.4. Chickpea contains considerably higher amounts of tryp- sin inhibitor than the other commonly consumed Indian pulses, but contains much less than soybean (Sumathi and Patabhiraman, 1976). However, this is unlikely to be a problem as protease inhibitors are heat-labile and chickpea is usually cooked before consumption.
Antinutritional effects and health benefits have been ascribed to the pres- ence of certain amounts and types of protease inhibitors. The effect of trypsin inhibitors on animal growth is a consequence of inhibition of intestinal protein digestion, since the presence of inhibitors in diets consisting of free amino acids also led to decreased growth (Lajolo et al., 2004).
The Kunitz inhibitor and BBIs have been found to cause an enlargement of the pancreas (hypertrophy and hyperplasia) and hypersecretion of digestive enzymes (sulphur-rich proteins) in rodents and birds. This results in a loss of the sulphur-rich endogenous proteins that would cause growth depression, as legume seed proteins are generally deficient in sulphur amino acids (Lajolo and Genovese, 2002).
On the other hand, protease inhibitors have been linked, over the last two decades, to health-promoting properties (Champ, 2002) and are considered as
natural bioactive substances (Hill, 2004). Protease inhibitors may act as anticar- cinogenic agents (Clemente et al., 2004). BBIs have been shown to be effective in preventing or suppressing carcinogen-induced transformation in vitro and carcinogenesis in animal assays. The BBI achieved investigational new drug (IND) status from the Food and Drug Administration (FDA) in 1992 for this purpose (Kennedy, 1995), and studies with humans showed no toxic effects of BBI (Amstrong et al., 2000). Use of BBI inhibited the growth and survival of human prostate cancer cells (Kennedy and Wan, 2002). It also reduced the incidence and frequency of colon tumours in dimethylhydrazine-treated rats.
However, this effect was not observed with autoclaved BBI, suggesting that protease inhibitor activity was necessary for anticarcinogenic activity (Kennedy et al., 2002).
Dietary protease inhibitors have to survive digestive process in the gastro- intestinal tract in order to exert an effect, either local or systemic. There is not much information in the literature regarding the intestinal recovery of non- nutritional factors, probably due to methodological difficulties (Rubio et al., 2005). BBIs were effective in preventing or suppressing carcinogens when fed to animals (Pusztai et al., 2004). According to Clawson (1996), the effect of dietary protease inhibitors would be indirect. Clawson hypothesized that dietary protease inhibitors may act by inducing the synthesis and distribution of endogenous protease inhibitors, which would have widespread effects on cell growth and behaviour.
a-Amylase inhibitors
a-Amylases (a-1,4-glucan-4-glucanohydrolases) are endoamylases that catalyse the hydrolysis of a-D-(1,4) glycosidic linkages, which occur in starch and related compounds. They play a major role in the carbohydrate metabolism Table 6.4. Protease inhibitor contents of desi and kabuli seeds.
Chickpea Content Number of
type Inhibitor type (mg) cultivars References
Desi Trypsin inhibitor 1.16–15.7 129+ Singh and Jambunathan (1981);
Batterham (1989); Petterson et al. (1997); Saini (1997);
Salgado et al. (2001)
Chymotrypsin 2.40–13.19 77+ Singh and Jambunathan (1981);
inhibitor Batterham (1989); Petterson et al. (1997); Saini (1992) Kabuli Trypsin inhibitor 1.39–12.1 55+ Singh and Jambunathan (1981);
Batterham (1989); Zaki et al.
(1996); Petterson et al. (1997);
Salgado et al. (2001)
Chymotrypsin 3.0–10.74 25+ Singh and Jambunathan (1981);
inhibitor Batterham (1989); Petterson et al. (1997); Salgado et al. (2001)
of animals and humans by providing them glucose as an energy source and as a building block for synthesis of other sugars. Among a-amylase inhibitors (aAIs) found in plants, legume aAIs, especially aAIs from beans, have received considerable attention (Whitaker, 1988). Jaffe et al. (1973) screened 95 legume cultivars for aAI levels and found that lima beans (Phaseolus lunatus), mung beans (Phaseolus aureus) and horse gram (Dolichos biflorus L.) had the high- est levels of inhibitory activity. Although little is known about the screening ofaAIs in chickpea, Mulimani et al. (1994) determined the aAI activity of 28 varieties of chickpea and found variations ranging from 11.6 to 51.4 inhibi- tory units/g. Singh et al. (1982) observed that the amylase inhibitor activity on pancreatic amylase of chickpea cultivars ranged from 7.8 to 10.5 units/g (desi) and 5.6 to 10.0 units/g (kabuli), with considerable variations among these cul- tivars. A similar variation but of lower magnitude was observed using salivary amylase. A comparison under similar assay conditions indicated that the amy- lase inhibitor activity had a stronger influence on pancreatic amylase than sali- vary amylase for both desi and kabuli cultivars. Jaffe et al. (1973) reported that the partially purified kidney bean inhibited the salivary amylase more than the pancreatic amylase. This shows that amylase inhibitors from different legume seeds may exhibit unequal activity against different enzymes.
The most probable function of aAI in the plant is protection from predatory insects by inhibiting their digestive amylase. These aAIs have been shown to inhibit pancreatic amylases (human and porcine), human salivary amylase and insect amylase (Le Berre-Anton et al., 1997). aAIs are deleterious or antimeta- bolic for many predators of crop plants. The genes for both of these factors have been introduced into a number of plants and the resultant transgenic crops have a significantly increased resistance to predators (Gatehouse et al., 1994).
aAIs reduce amylase activity and starch digestion in the gut when given orally to humans (Singh et al., 1982). As a result, they lower postprandial increases in circulating glucose and insulin. These inhibitors may therefore prove to be useful in the treatments of obesity or diabetes mellitus.
The stability of aAI to proteolytic degradation in vivo does not seem to have been evaluated. However, the findings that orally administered aAI could greatly reduce intraluminal amylase levels and starch digestion in rats and humans suggests that nutritionally significant quantities of aAI survive passage through the small intestine in a fully functional form (Pusztai et al., 1995).
High dietary intakes of aAIs can however cause a number of potentially deleterious alterations in the body metabolism of experimental animals.
Significant enlargement of the small intestine and of the pancreas was evident in rats given aAI (Grant et al., 1998).
Lectins
Lectins (haemagglutinins) are glycoproteins, which are able to reversibly bind to specific sugars and glycoproteins on the surface of cells in the gut wall, thereby interfering with nutrient breakdown and absorption. This reaction is manifested, in vitro, by agglutination of red blood cells from various animal species. Lectins are extremely specific; different types having different interac- tions on toxicity, blood groups, mitogenesis, digestion and agglutination.