Monday, 29 August 2011

Anti-nutrients in Legumes and their Removal

Anti-nutrients in Legume Foods and their Removal

Anti-nutrients are the way nature protects the plants so that they can live long enough to effectively reproduce. They function as the immune system of the plant, offering protection from the radiation of the sun, foraging by animals and from invasion by bacteria, viruses, or fungi (Barbara 2009). According to Deshpande 2002, anti-nutrients may occur naturally in plants as secondary metabolites protecting plants from viral and fungal attack analogous to the immune system of animals or anti-nutrients may also produced in large amounts as a direct result of some adverse environment condition (Deshpande, 2002). The most important anti nutrient factors/ antiphysiological substances in legumes include protease inhibitors, phenolic substances, non-protein amino acids, lecithins, saponins, flatulence produces and non-starch polysaccharides (Vidivel & Janardhanen, 2001; Olguin et al., 2003).

Robin and Ross (1996) highlight that antinutrients are not something to be alarmed about since most foods typically have one or more antinutrients (figure 1 below) and that the issue is the concentration and type of antinutrient, and whether that specific antinutrient profile will adversely affect your health (Robin and Ross, 1996). All plants have some anti-nutrient properties, but the soybean plant is especially rich in these chemicals. If they are not removed by extensive preparation such as fermentation or soaking, soybeans are one of the worst foods a person can eat (Barbara 2009).
Groups most at risk of experiencing negative effects from the anti-nutrient properties of soy are infants taking soy baby formula, vegetarians eating a high soy diet, and mid-life women going heavy on the soy foods thinking they will help with symptoms of menopause (Barbara 2009). Figure 1 below shows the common anti-nutrients in foods.

Figure 1: Antinutrients in plant foods reported to reduce the bioavailability of iron, zinc, or both
Major dietary sources
Phytic acid or phytin
Whole legume seeds and cereal grains

Fibre (for example, cellulose, hemicellulose, lignin, cutin, suberin)
Whole cereal grain products (for example, wheat, rice, maize, oats, barley)

Tannins and polyphenols
Tea, coffee, beans, sorghum

Oxalic acid
Spinach leaves, rhubarb

Hemagglutinins (for example, lectins)
Most legumes and wheat

Heavy metals (for example, cadmium, mercury, lead, gold)
Plant foods obtained from crops grown on metal-polluted soils (for example, cdmium in rice)
Source: Robin and Ross, 1996.

Types of anti-nutrients in plant foods
Lectins are carbohydrate binding proteins present in most plants, especially in bean seeds (Pusztai A, 1991). Lectins are glycoproteins present in plant-based feed ingredients including soy bean meal, they bind to the intestinal epithelium and interfere with nutrient absorption (Lajolo and Genovese, 2002). According to Nancy and Bill (2006) Lectins are proteins in foods (both plant and animal) that bind themselves to carbohydrate molecules, especially the sugar part of the molecule.

Many lectins are toxic and/or inflammatory, resistant to cooking and digestive enzymes/stomach acids in the body (Pusztai A, 1991). They can damage gut wall allowing other proteins to cross undigested causing allergic reactions. When lectins are in the blood stream, they can bind to cell membranes in arteries and organs such as joints, kidney, pancreas and brain, causing antigen-antibody reactions leading to autoimmune disorders. Lectins can be blocked by simple sugars and oligosaccharides in the body (Pusztai A, 1991). Soybean lectins have been shown to depress nitrogen retention and increase nitrogen excretion via the urine, indicating interference with protein metabolism (Czerwinski et al., 2005). Lectins have been found in wheat, rye, barley, oats, corn and rice, but not in sorghum or millet. The lectins in grains appear to have somewhat similar biological activity (Nancy and Bill, 2006).

Lectins in soy beans and peanuts are not toxic but those from jack beans, winged beans, kidney beans, mung beans, lima beans and castor beans are all toxic when taken orally. The consumption of lectin containing foods may lead to endogenous loss of nitrogen and protein utilization (Shahidi, 1997). The carbohydrates and proteins that are undigested and unabsorbed in the small intestines reach the colon where they are fermented by the bacterial flora to short chain fatty acids and gases. The lectin induced disruption of the intestinal mucosa may allow entrance of the bacteria and their endotoxins to the blood stream and cause toxic response. Lectins may also be internalized directly and cause systematic effects such as increased protein catabolism and breakdown of stored fat and glycogen, and disturbance in mineral metabolism (Shahidi, 1997).
Fortunately, most lectins are easily destroyed by traditional methods of household cooking though quite resistant to inactivation by dry heat, practices such as the use of raw legume flours in baked goods should be viewed with caution (Deshpande, 2002). Lectins may be removed by soaking, sprouting, cooking, fermenting or heating (Pusztai A, 1991).

The terms phytic acid, phytate, and phytin refer to free acid, salt, and cacium/magnesium ion salts respectively  and in cereals and legumes phosphorus is present in significant amounts as phytin (myo-inositol hexaphosphate), (Deshpande, 2002). According to Robin and Ross (1996), phytic acid is a major metabolite in all mature seeds and grains and is the primary storage form of phosphorus in these plant components. Phytates are phosphorus compounds found mainly in cereal grains, legumes, nuts (Nancy and Bill, 2006). Phytic acid occurs naturally through out the plant kingdom and in particular soybean, rapeseed and cotton seed. Whole soybeans have been reported to contain 1-2% phytic acids and that the major part of the phosphorus contained within phytic acid are largely unavailable to animals due to the absence of the enzyme phytase within the digestive tract of monogastric animals,(Akande, Doma and Adamu, 2010). It rapidly accumulates in seeds during the ripening in the aleurone particles and accounting for up to 85% of the total phosphorus in many cereals and legumes (Deshpande, 2002).

Barbara (2009) notes that all legumes contain phytate (also known as phytic acid) to some extent, but the soybean is particularly rich in this anti-nutrient. Phytate works in the gastrointestinal tract to tightly bind minerals such as copper, iron, magnesium calcium and a particularly strong affinity for zinc, a mineral that supports wound healing, protein synthesis, reproductive health, nerve function, and brain development. It is believed that people living in developing countries are shorter than those in developed countries because of zinc deficiency caused by eating too many legumes. There is also evidence that mental development can be negatively impacted by a diet high in phytate (Barbara 2009). According to Deshpande, (2002) reduced bioavailability of minerals from phytate rich sources depends on several factors, which include the nutritional status of animals/humans, the concentration of minerals and phytate in foodstuffs, the ability of endogenous carriers in the intestinal mucosa to absorb essential minerals bound to phytate and other dietary substance.

Phytates bind proteins causing reduced protein solubility, inhibit enzymes including pepsin, trypsin, alpha-amylase and also reduce absorption of minerals such as zinc, calcium, magnesium, and iron, because they form insoluble complexes with these minerals (Pusztai A, 1991). Pytic acid also inhibits the action of gastrointestinal trysinase, trypsin, pepsin, lipase and amylase (Khare, 2000). Phytic acid, or its naturally occurring form, phytin ( a mixed magnesium and potassium salt of phytic acid), forms insoluble precipitates with several divalent metal cations (including Ca2+ , Fe3+, and Zn2+), (Robin and Ross (1996). Deshpande, (2002) highlights that phytic acid has six reactive phosphates and meets the criterion of chelating agent. Thus phytate is largely blamed for complexing dietary essential minerals especially zinc and copper in legumes and cereals and rendering them poorly available to monogastric animals.

Phytates are the main reservoir for phosphorus and other minerals in the seed that are mobilized by germination. Some recent studies have indicated that phytates enhance the immune system, may have anti-cancer and serum cholesterol-lowering effects. Antioxidant properties of Phytates help prevent free radical damage to DNA in the colon which can lead to cancer. (Pusztai A, 1991). Potential beneficial effects of Pa relate to its ability to lower blood glucose response to starchy foods by inactivating amylase enzyme; lowers plasma cholesterol and levels of triacylglycerols by binding to zinc and thus lower the ratio of plasma zinc to copper which is known to dispose humans to cardiovascular disease (Shahidi, 1997).

The mineral binding capacity of phytates is a mixed bag. On one hand, it may prevent absorption of minerals that you need while on the other hand, it may prevent absorption of minerals that you don’t need: for example, if you already have a high level of iron, the iron-binding capacity of phytates is a benefit because too much iron leads to serious free radical damage to your cells. Excessive free radical damage increases your risk of cancer and other degenerative disorders (Nancy and Bill, 2006). Phytates are generally found in foods high in fibre. Since fibre-rich foods protect against colon and breast cancers, it is now thought that phytates may be the protective agent in the fibre. Phytates, in combination with fibre, slow down the absorption of glucose from starch, thus helping you to balance blood sugar and insulin (Nancy and Bill, 2006).

Phytate is generally considered to be fairy heat-stable. Among the processing methods, germination and fermentation appear to be quite effective in decreasing the phytate concentrations, whereas soaking and cooking can remove more than 50% to 80% of the endogenous phytate in legumes and cereals (Deshpande, 2002). Baking and food processing will destroy phytates in grains and most legumes (Nancy and Bill, 2006). Fermented soy foods like miso and tempeh have the lowest levels of phytate and are the best choices for anyone wishing to eat soybean products. Tofu is also a good choice, as long as care is taken to replenish loss nutrients. Whole soybeans, soy milk, soy chips, soy protein isolates, soy flour and all the other myriad of products made from processed soybeans and advertised as health foods have much higher levels of phytate and are not worth eating (Nancy and Bill, 2006).

Soybeans have a high content of goitrogens, substances that can block the production of thyroid hormone as well as cause goiter formation. Low thyroid activity plagues women in America, particularly middle-aged women. Thyroid hormone stokes the cellular furnaces, known as mitochrondia. When thyroid production is low, energy levels as well as body heat are also low. Low thyroid level is what makes old people move so slowly and seem like every action is a huge chore. Low thyroid means the action of the heart is reduced, resulting in lack of oxygen to the cells, a prime condition for cancer (Barbara 2009).

Genistein, an isoflavone found in soybeans, can also block thyroid production. Phytate can accentuate these effects because it binds up zinc and copper, leaving little of these important minerals available to make thyroid hormone (Barbara 2009). A transport protein called GLUT1 is shut down by genistein. This protein sends glucose into the cells where it is used to generate energy. Slowing the transport of glucose means less energy production not only of thyroid hormone, but of every other action in the body (Barbara 2009). Another way in which soy isoflavones reduce energy in the body is by inhibiting tyrosine kinases, enzymes involved in the transfer of energy from one molecule to another. These enzymes drive cell division, memory consolidation, tissue repair, and blood vessel maintenance and regeneration.

It is this action of regulating cell division that made genistein a popular substance for fighting cancer. When research on this anti-cancer effect of genistein became know, the soy industry feverishly developed products that would appeal to Western women looking for genistein. In the middle of all this excitement, little attention was paid to how the energy reducing effects of genistein lowered cellular energy in normal cells (Barbara 2009).

The high cost of genistein
Women have been encouraged to use high genistein soy products to alleviate symptoms of menopause and as a guard against bone loss and breast cancer. But given the full range of effects of genistein in the body, high consumption could result in age-related memory loss. Commercial soybean products offer genistein levels as high as 20 to 60 mg per serving. Asians are presented as an example of the benefits of eating soybean products because their incidence of breast cancer and osteoporosis is low. However, the Asian diet of fermented soybean products such as miso and tempeh includes only around 5 mg of genistein a day (Barbara 2009). Genistein slows the growth of blood vessels to tumors, another action that makes it popular as a cancer fighter. However, it has the same effect on blood vessels serving normal cells. Eating a regular diet high in genistein could result in the starvation of healthy blood vessels, resulting in a reduced supply of oxygen to cells, setting up a cancer promoting situation (Barbara 2009).

Trypsin inhibitor
Inhibits the function of trypsin enzyme, causes pancreatic hypertrophy and dietary loss of cysteine. Lack of proper irrigation may increase the activity of trypsin inhibitor (Salunkhe and Kadam,1989). Trypsin inhibitors are proteins that interfere with nutrient absorption by reducing the activity of proteolytic enzymes trypsin and chymotrypsin (Norton 1991). The amount and activity of trypsin inhibitors in the diet has been shown to be inversely related to the availability of energy and protein (Krogdahl et al 1994). Proteases are enzymes (e.g trypsin and chymotrypsin) in human gastric juices that break down protein. Trypsin helps to regulate secretions from the pancreas. When trypsin is inhibited by protease inhibitors, the pancreas does not receive the signals it needs to slow down. Protease inhibitors are found in nearly all cereal grains and legumes. However, there is a lot of variability. For example, the amount of trypsin inhibitory activity in wheat is only 1.5% of the inhibitory activity found in soybeans (Nancy and Bill, 2006). 

The antinutrient activity of protease inhibitors is associated with growth inhibition and pancreatic hypertrophy. Trypsin inhibitors in soybean give rise to inactivation and loss of trypsin in the small intestine, thus trigger the release of cholecytokinin and induce pancreatic synthesis of excess trypsin and burden on sulphur containing amino acids in requirement of the body (Shahidi, 1997). The most characterised protein inhibi­tors of legume seeds are trypsin inhibitor of both, Bowman‑Birk type and Kunitz type, and α-amylase inhibitors. The presence of protease inhibitors in food decreases the appar­ent nutritional quality of proteins in the diet by affecting the ability of body digestive enzymes to degrade dietary protein, and thus limiting the intake of amino acids needed to con­struct new proteins. However, in certain situations the effects of inhibitors on protein digestion might be advantageous, e.g. by improving the intact absorption of some therapeutic pro­teins such as orally delivered insulin [Yamamoto et al., 1994]. Moreover, the control of proteases activity, considered to play a decisive role in a wide range of biological processes and misfunctioning related to cancer progression, may be consid­ered as anticarcinogenic mechanism (Clemente & Domon­ey, 2001).

Alpha-amylase inhibitor
Alpha amylase is a digestive enzyme that is present in your saliva and your pancreas. It helps you to begin the digestion of starches in your food. Alpha amylase inhibitors are compounds that inhibit alpha amylase from doing its job. Alpha amylase inhibitors are found in wheat, rye, barley, oats, rice and sorghum. They survive the baking process and are found in bread and baked products (Nancy and Bill, 2006).

The physiological role of alpha amylase inhibitors in plants is not well understood. They are not active against endogenous alpha or beta amylase of legumes or those in malt and barley, or microbial amylases. The bean inhibitors however inhibit insect larva alpha amylase and therefore may have a physiological role in protecting the seeds against insect attack (Deshpande, 2002). A general claim on the anti-diabetic role of α-amylase inhibitors has already been published [McCarty, 2005] and some patents concerning the use of food preparations containing suitable amounts of α-amylase inhibitors for the obesity control and the preven­tion and treatment of diabetes have appeared [Suzuki et al., 2003; Muri et al., 2004]. These finding confirmed the poten­tial of α-amylase inhibitors to be used as a nutraceutical com­pound. However, the positive or negative effect of all enzyme inhibitors depends on their level in different legumes and on the dose and frequency of consumption.

Proteases are enzymes in human gastric juices that break down protein. To examples of proteases are trypsin and chymotrypsin. Many plants, especially legumes, have protease inhibitors. Trypsin helps to regulate secretions from the pancreas. When trypsin is inhibited by protease inhibitors, the pancreas does not receive the signals it needs to slow down. Protease inhibitors are found in nearly all cereal grains and legumes. However, there is a lot of variability. For example, the amount of trypsin inhibitory activity in wheat is only 1.5% of the inhibitory activity found in soybeans (Nancy and Bill, 2006).

Raffinose Oligosaccharides
Oligosaccharides of the raffinose-series (namely raffinose, verbascose and stachyose) are major components in many food legumes1, and the antinutritional activity of grain legumes is frequently associated with the presence of these oligosaccharides. Raffinose-series oligosaccharides are not hydrolysed in the upper gut due to the absence of α-galactosidase. In the lower intestine they are metabolised by bacterial action, producing methane, hydrogen and carbon dioxide, which lead to flatulence and diarrhoea. Raffinose-series oligosaccharides are thus a factor limiting the use of grain legumes in monogastric diets (Megazyme International Ireland Limited, 2004). Flatulence is a complaint even among healthy individuals and is one of the common causes of abdominal discomfort. Its also associated with dyspepsia, constipation and diarrhoea (Madushini, 2002).

The pigmented varieties of certain cereals and legumes contain 2% to 4% condensed tannins, although amounts as high as 7% to 8% have been reported for red high-tannin sorgum varieties. Humans also consume a number of other foods containing considerable amounts of condensed tannins especially in beverages, such as cider, tea, cocoa and red wine(Deshpande, 2002).
Common beans contain polyphenolic compounds such as tannins which interfere with protein digestibility and protein quality. They are found primarily in the seed coat. They can bind to proteins preventing enzymatic susceptibility of the protein as well as inhibiting the proteolytic enzymes such as trypsin and Chymotrypsin (Pusztai A, 1991).

 Alkylresorcinols are compounds found in high amounts in rye and wheat, and to a lesser extent in oats, barley, millet and corn. They are found in the bran, or outer layer of the grain. They appear to act as both an appetite suppressant and as a toxin. As a toxin, they are implicated in many pathological processes, including death of red blood cells, as well as liver and kidney damage. They also stimulate a powerful inflammatory compound called thromboxane. On the other hand, low levels of alkylresorcinols may have anticancer and antioxidant properties (Nancy and Bill, 2006).

Saponins are a heterogeneous group of naturally occurring foam-producing triterpene or steroidal glycosides that occur in a wide range of plants, including pulses and oil seeds  such as kidney beans, chickpea, soybean, groundnut, lupin and sunflower (Akande, Doma and Adamu, 2010).

Other compounds: Phytoalexin are antimicrobial substances produced by a plant in response to infection by fungi or bacteria and that help to defend the plant by inhibiting the microbial growth. They are toxic interims of hemolytic effects and uncoupling of oxidative phophorylation. Phytoalexins can also be potent antioxidants (Free Radical Research, June 2002, pages 621–631; and Biochemical Pharmacology, January 2002, pages 99–104). Saponins are glycosidic and have foam forming ability. Arcelins are insecticidal storage proteins in beans (Pusztai A, 1991).

Anti-Nutrient Removal
Dietary quality is an important limiting factor to adequate nutrition in many resource-poor settings. One aspect of dietary quality with respect to adequacy of micronutrient intakes is bioavailability. Several traditional household food-processing and preparation methods can be used to enhance the bioavailability of micronutrients in plant-based diets. These include thermal processing, mechanical processing, soaking, fermentation, and germination/malting. These strategies aim to increase the physicochemical accessibility of micronutrients, decrease the content of antinutrients, such as phytate, or increase the content of compounds that improve bioavailability (Christine and Rosalind 2006).

Traditionally, several methods have been used to remove the toxicants and antinutrients present in plant foods in order to improve their nutritional quality and utilization. To accomplish this goal, several approaches may be considered. Breeding plant varieties containing low or no levels of toxicants is one such approach. Such an approach however requires long term efforts. Since the type and number of nutrients and toxicants present in plant foods are rather large and diverse with respect to their chemical and biochemical nature. Such efforts will also need to consider agronomic consequences of genetic manipulation, including crop yield, soil tolerance, light and water requirements and resistance to pests and diseases (Deshpande, 2002).

The physical and chemical means of removing undesirable antinutrients include several processing methods such as soaking, cooking, germination, fermentation, irradiation, selective etaction, membrane filtration and enzymatic treatments. In many instances, the use of only one method may not effectively remove all antinutrients present, and thus a combination of two or more methods may be required to accomplish the desired level of removal. The effectiveness of some of these methods in removing plant antinutrients is briefly described in the following discussion (Deshpande, 2002).

Biotechnology employs breeding techniques to eliminate the undesirable components from the beans. Such an approach requires long-term studies depending on the type and number of the undesirable components (Deshpande, 2002). Rigorous consideration reveals that this approach could lead to undesirable consequences for both crop growth and human health. This is especially true for certain antinutrients that are major metabolites in reproductive organs, such as phytic acid. 

Phytin, as the primary storage form of phosphorus in most mature seeds and grains, is an important compound required for early seed germination and seedling growth. Phytin is accumulated as part of globoid crystals in membrane-bound protein bodies of certain cell types within the developing seed, such as within protein bodies occurring in cells of the aleurone layer of cereal grains. Phytin deposits within globoid crystals of protein bodies also are associated with the accumulation of other minerals, including potassium, magnesium, iron, zinc, copper, manganese, and, in some seed types, calcium. As such, phytin plays an important role in determining mineral nutrient reserves of seeds and thus contributes to the viability and vigor of the seedling produced. Selecting for seeds and grain crops with substantially lower phytin content could have unacceptable effects on agricultural production, especially in regions of the world with soils of low phosphorus status, poor micronutrient fertility or both. Furthermore, recent reports have suggested that phytic acid may be an anticarcinogen for certain types of colon cancer (Robin and Ross, 1996).

Soaking beans and then discarding the soak medium can remove some of the unwanted components such as enzyme inhibitors and raffinose oligosaccharides. Soak temperature, medium type, bean type, length of soaking and solubility of the components are the factors affecting the extent of the removal. Salts and alkali in the solution increase the permeability of cell membrane increasing the amount of anti-nutrient leaching as well as some loss of desirable nutrients such as soluble vitamins and proteins (Pusztai A, 1991). Soaking usually forms an integral part of such processing methods as cooking, germination, fermentation and roasting. Soaking media frequently include water, salt solutions, and dilute aqueous alkali solutions. Salt and alkali help leach the soluble into the soaking medium by increasing the cell membrane permeability. However, such loss of antinutrients during soaking is also associated with loss of desirable nutrients, such as proteins, minerals and vitamins (Deshpande, 2002). Ramakrishna et al., (2006) and Shimelis and Rakshit, (2007) found that trypsin inhibitors activity was reduced in kidney bean to 9-18 %, by hydration. Khattab and Arntfield (2009) showed that soaking of cowpea, pea and kidney bean seeds significantly reduced their TIA by 10.22-19.85 %. Ibrahim et al. (2002) showed that long-time soaking (16 h) caused remarkable reduction in the anti-nutritional factors. Another study found that soaking cowpeas for 12 hours, dehulling of soaked seeds and germinating cowpeas contributed significantly to reducing phytic acid and tannin levels. Dehulling as well as germination increased the digestibility to of both the starches and the protein in the cowpea, (Preet and Punia, 2000). Chang et al. (1977) reported that phytic acid could be removed by simply soaking beans at 60 ˚C for 10 hrs. In her method, elimination of 90% of the phytic acid occurred. El-Hady and Habiba (2003) observed a 36% reduction in phytic acid in kidney beans after an overnight soaking in water at room temperature.

Soaking cereal and most legume flours (but not whole grains or seeds) in water can result in passive diffusion of water-soluble Na, K, or Mg phytate, which can then be removed by decanting the water (Perlas and Gibson 2002; Hotz and Gibson 2001). The extent of the phytate reduction depends on the species, pH, and length and conditions of soaking. Some polyphenols and oxalates that inhibit iron and calcium absorption, respectively, may also be lost by soaking (Erdman and Pneros, 1994).

Cooking inactivates heat sensitive trypsin and chymotrypsin inhibitors but not completely. Other factors such as saponins, flatulence factors and phytates may not be affected by cooking. If the soaking and cooking media are discarded, appreciable amounts of heat stable compounds can be removed (Pusztai A, 1991). When the soaking and cooking medium (such as water) is not discarded, a significant amount of heat-stable antinutrients and toxicants remain practically unchanged. On contrally if the medium is discarded, a significant amount of heat-stable antinutrients can be removed from plant foods. Excessive heat processing, however, should be prevented, since it affects the protein quality of foods (Deshpande, 2002).

Rasha, Gibriel, Nagwa, Ferial and Esmat (2011) proved that the ordinary boiling of soaked seeds for different time periods brought about a significant decrease in trypsin inhibitory activity as compared with raw seeds. The losses percent of trypsin inhibitory activity were 87.8, 96.8 and 86.5 % in soybean, mung bean and kidney bean, respectively after boiling for 30 min. Increasing the period of boiling caused greater losses in trypsin inhibitor activity. After 60 min. of boiling TIA was drastically decreased (4-10% residual activity) in tested seeds. Complete inactivation of trypsin inhibitor activity was observed for all tested seeds after boiling for 90 min.

Khattab and Arntfield (2009) stated that boiling, roasting, microwave cooking and autoclaving brought a total removal of trypsin inhibitor of cowpea, pea, and kidney bean. The loss of trypsin inhibitor activity during cooking may be due to destroying by high temperatures, due to their heat-sensitive nature to undetectable amounts when heating processes (cooking and autoclaving) were employed (Shimelis and Rakshit, 2007). The most effective methods for inactivation TIA were boiling (90 min) and autoclaving for 10 min (Rasha, Gibriel, Nagwa, Ferial and Esmat 2011).Pressure cooking of pigeon pea seeds completely destroyed the TIA while it was reduced to the extent of 86-88 per cent against the control in 48 h pigeon pea sprouts (Duhan et al., 2001). Alonso et al (2001) reported that extrusion reduced phytic acid content by 27 %, with a concomitant increase in the inositol tetrakisphosphate and inositol pentakisphosphate contents. El-Hady and Habiba (2003) also reported that phytic acid content decreased significantly for kidney beans processed at 180˚C and a bean feed moisture of 22%. In both of these studies, protein content was not affected indicating that reduction in phytic acid can be achieved without the destruction of protein.

Morris and Hill (1996), Chen (2004) reported a loss of phytic acid in cooked beans. They noted approximately 30, 27, 29, 30, 32 % reductions in phytic acid for cooked black, great northern, navy, pinto, and kidney beans, respectively. Nergiz and Gokgoz (2007) also reported 57-58% reduction in phytic acid after cooking beans that had been soaked 12 hours prior to cooking. Rehman and Salariya (2005) also noted cooking reduced phytic acid contents by 21 and 24 % in red and white kidney beans. Theses authors also observed greater reductions of phytic acids in kidney beans exposed to 121 ˚C (pressure cooking conditions). The reduction in phytic acid increased as the length of processing time increased. After 90 minutes at 121 ˚C, 48-50% of the phytic acid remained in the kidney beans.

Rehman and Salariya (2005) reported tannin reductions after ordinary cooking and during canning of the beans. Nergiz and Gokgoz (2007) reported a 71-77% reduction in total phenolic content compared to the raw bean. Xu and Chang (2008, 2009) observed similar reductions in phenolic compounds under boiling water conditions. Steam processing of the beans resulted in a lower phenolic loss (Xu and Chang 2008). Luthria and Pastor-Corrales (2006) reported that over 83% of the phenolic acids were retained beans during cooking. Their method of analysis was more complex and measured specifically the phenolic acids. These results are more indicative of the phenolic acid stability and may not be a comparable measure to the loss of total phenolic compounds, which is a more generic method for measuring total phenolic compounds in beans. Regardless of the phenolic reductions, cooked beans maintained good antioxidant activity (Boateng et al. 2008; Xu and Chang 2008, 2009).

El-Hady and Habiba (2003) reported that the extrusion process reduced total phenolics and tannins. The combination of soaking followed by extrusion had a greater impact on tannin reduction than extrusion processing of the raw kidney bean flour.  Korus et al. (2007) reported that extrusion caused a reduction in the total phenolic content for several bean varieties but did cause an increase in total phenolics in one variety tested. Overall, literature suggests that extrusion causes a slight reduction, usually less than 25%, in phenolic contents of beans.

Germination/malting increases the activity of endogenous phytase activity in cereals, legumes, and oil seeds through de novo synthesis, activation of intrinsic phytase, or both. Tropical cereals such as maize and sorghum have a lower endogenous phytase activity than do rye, wheat, triticale, buckwheat, and barley (Egli, Devidson, Juillerat, Barclay and Hurrell 2002). The rate of phytate hydrolysis varies with the species and variety as well as the stage of germination, pH, moisture content, temperature (optimal range 45–57_C), solubility of phytate, and the presence of certain inhibitors (Sandberg, Brune, Carlsson, Hallberg, Rossander and Skoglund; 1999). 

When the bean seeds are germinated, reserve nutrients are released. Available vitamin and mineral content increases while the phytate content and raffinose oligosaccharide contents decrease by 20-77% and over 70 %, respectively. The activities of trypsin, chymotrypsin and alpha-amylase are also reduced by germination depending on the bean type and germination conditions (Pusztai A, 1991). Germination moblises reserve nutrients required for the growth of plant seedlings and therefore, may help in the removal of at least some of the antinutrients, such as phytate and raffinose oligosaccharides which are thought to function as reserve nutrients. Significant reductions in phytate, lectin activity and raffinose sugars are reported on germination of various legumes. Beneficial effects of germination in terms of reduction in enzyme inhibitory activities, however, remain controversial (Deshpande, 2002). El-Hag et al., (1987) reported about 50% reduction in trypsin inhibitor activity in kidney bean (P. vulgaris) during 10-day germination. Ramakrishna et al., (2006) found that the raw dry Indian bean had a very high trypsin inhibitory activity which progressively decreased by 51% during the 12 h soaking period which decreased gradually to reach a level of 17% of the basal level of dry seeds at 32h germination. Sangronis and Machado, (2007) reported that the reduction in trypsin inhibitor was 52.5, 25.6 and 41.0 % for white beans, black beans and pigeon beans after germination for 5 days respectively. Germination is a mainly catabolic process as the reserved substances present in the cotyledon are used for the development and growth of the embryo.

Increase of total phenols after germination was reported by Khattak et al. (2007) for chickpea,
Duenas et al., (2009) for lupines and Tain et al., (2010) for oat. Lopez-Amoros et al., (2006) reported an increase in the antioxidant activity of beans and peas during germination an increase of antioxidant activity after germination was also observed in soybean, adzuki bean and mung bean The changes observed in these legumes were related to the increase of phenolics (Lin and Lai, 2006), and these may be attributed to the biochemical metabolism of seeds during germination. Duenas et al., (2009) reported that germination caused significant changes in the phenolic composition (increasing) due mainly to endogenous enzymes' activation and the complex biochemical metabolism of seeds during this process. Germination quantitatively reduces raffinose, stachyose and verbascose while sucrose is increased in all seeds except red lima beans and jack beans (Oboh et al., 2000).

Fermentation can induce phytate hydrolysis via the action of microbial phytase enzymes, which hydrolyze phytate to lower inositol phosphates. Such hydrolysis is important because myoinositol phosphates with ,5 phosphate groups (i.e., IP-1 to IP-4) do not have a negative effect on zinc absorption , and those with ,3 phosphate groups do not inhibit non-heme iron absorption (Hurrell, 2004; Sandberg, 1991). Microbial phytases originate either from the microflora on the surface of cereals and legumes or from a starter culture inoculate (sandberg, 1991). Low-molecular-weight organic acids (e.g., citric, malic, lactic acid) are also produced during fermentation and have the potential to enhance iron and zinc absorption via the formation of soluble ligands while simultaneously generating a low pH that optimizes the activity of endogenous phytase from cereal or legume flours (Teucher, Olivares and Cori; 2004).

Fermentation helps reduce raffinose oligosaccharides due to the alpha-galactosidase found in the bean seeds. Phytic acid content is also decreased due to the action of phytase present in the bean seeds. Microorganisms which are responsible for the fermentation also play a role (Pusztai A, 1991).  Similar to those in germination, most of the changes occurring during the fermentation of foods are of catabolic nature, and they help in the hydrolysis of such components as proteins and carbohydrates. Fermentation of foods can result in significant reduction in the quantity of certain antinutrients. The removal of raffinose oligosaccharides of legume during fermentation, for example, is primarily due to the alpha galactosidase activity present in legume seeds as well as in microorganisms involved in the process. Depending upon the type of legume as well as fermentation, phytic acid is also hydrolysed during fermentation to a variable degree (Deshpande, 2002). Fermentation of cereals reduces phytate content via action of phytases that catalyse conversion of phytate to inorganic orthophosphate and a series of myoinositols, lower phosphoric esters of phosphate. A 3-phytase appears to be characteristic of micro-organisms, while a 6-phatase is found in cereal grains and other plant food (Shahidi, 1997). The consumption of fermented food has been shown to improve the intestinal balance of beneficial lactic acid bacteria. Based on this and the phytase producing capability of the bacteria, the consumption of fermented foods may be another effective way to reduce the potential of dietary phytic acid impairing mineral absorption (Vin 2009).

Enzymatic Methods
Enzymatic removal of certain compounds such as raffinose oligosaccharides depends on the level of endogenous alpha-galactosidase which may be different in different beans. Enzymatic removal of phytate in white beans is successful up to 80 % reduction at 60 C when soaking and heating treatment are combined (Pusztai A, 1991). In addition to the traditional household processes for preparing plant foods for human consumption, enzymatic methods have been used to remove certain antinutrients of plant origin, including phytates and raffinose sugars. Endogenous enzymes, such as linamarinase, as well as externally added beta glycosidases are often used to remove the cynogenic glycosides of various legumes. The HCN thus produced is water soluble and volatile and can be easily removed by heating and or discarding the soaking water. In addition, processes such as ultrafiltration, irradiation of foods, addition of antibiotics or bacteriostats, extrusion cooking, and protein texturisation have proved useful in removing certain toxicants and anti-nutrients of plant foods (Deshpande, 2002).

Biological food-processing techniques that increase the activity of native enzymes of cereals and legumes are: soaking, germination, hydrothermal processing and fermentation. During germination, phytase enzymes are synthesized or activated. Lactic fermentation leads to lowering of pH as a consequence of bacterial production of organic acids, mainly lactic acid, which is favourable for cereal phytase activity (Sandberg, 2002). The microorganisms (e.g. fungi) of the starter culture used in fermentation, in some cases, exert phytase activity. However, in contrast to fungi and yeast, Lactobacillus sp. was not found to produce phytase (Fredrikson et al. 2002b). To optimize the food process to increase mineral availability by phytate degradation, it is essential to know the optimal conditions for the phytases, which are responsible for phytate degradation in the food process. There are differences in optimal conditions for phytate degradation between plant species (sandberg, 2002). For high-tannin cereals, it has been shown that treatment with polyphenol oxidase had a reducing effect on the phenolic content (Matuschek et al. 2001). Addition of 1500 U/g resulted in a 60% reduction of phenolic compounds and a significant improvement of Fe availability, estimated in vitro. A fungal tannase was used to decompose phenolic compounds in brown beans, but the influence on Fe availability was not determined (Gustafsson & Sandberg, 1995).

Irradiation and Ultra-filtration
Gamma irradiation results in the destruction of trypsin and chymotrypsin inhibitors in certain bean types at different extents. Ultra-filtration could help remove low molecular weight compounds if the beans are wet-processed (Pusztai A, 1991).

Removal of seed coat (dehulling) of soaked cowpeas reduced the polyphenols by 70 -71 per cent (Preet and Punia, 2000). Dehulling legumes substantially reduces the polyphenolic content in beans of P. vulgaris L. For example, Deshpande et al. (1982) reported that tannin content was reduced by 68.0 to 94.6% using a dehulling process.  Anton et al. (2008) reported that total phenolic content was substantially higher in the isolated seed coats of navy and pinto beans than in the whole bean. The high concentration of phenolic components in the bean hull was likely responsible for the increased antioxidant activity of the hull fraction observed by Anton et al. (2008). Household pounding is used to remove the bran and/or germ from cereals, which in turn may also reduce their phytate content when it is localized in the outer aleurone layer (e.g., rice, sorghum, and wheat) or in the germ (i.e., maize). Hence, bioavailability of iron, zinc, and calcium may be enhanced, although the content of minerals and some vitamins of these pounded cereals is simultaneously reduced (Christine and Rosalind 2006).

Combined Strategies
These effects emphasize that an integrated approach that combines a variety of the traditional food-processing and preparation practices discussed above, including the addition of even a small amount of animal-source foods, is probably the best strategy to improve the content and bioavailability of micronutrients in plant-based diets in resource-poor settings (Gibson, Yeudall, Drost, Mitimuni and Cullinan; 2003). Use of such a combination of strategies can almost completely remove phytate. This is important because phytic acid is a potent inhibitor of iron absorption, even at low concentrations (Hurrell, 2004).

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