Antinutritional Factors What is it?

? Simply defined, Nutrition or Nourishment is the provision of the materials (In the form of food) necessary to support life. An antinutritional factor would perform exactly the opposite function. It can be simply defined as a chemical substance, which, when present in human or animal food, impairs growth and normal functioning of the body. The effect of these anti nutrients in the body depends on the type and the concentration in which they are present in the food material. Antinutritional Factors What are the Sources (Few Examples)? 1. Whole Egg contains about 12-14% protein. Egg white contains a number of proteins frequently referred to as albumen. Conalbumin constitutes 13% of the proteins of the egg albumen. It is more easily heat coagulated. It easily binds metallic ions, such as Fe3+, Al3+, Cu2+ or Zn2+ forming heat stable complexes. Ovomucoid constitutes about 10% of the egg-white proteins. It exists in three forms, all of which are Trypsin Inhibitors. It is resistant to heat denaturation in acid media, but is rapidly denatured in alkaline solutions. Avidin is composed of three components A,B and C. It binds biotin (It is water soluble B vitamin called B7 and is required in various metabolic reactions including fat metabolism) and makes the vitamin unavailable. However, Avidin is easily denatured by heat and so its presence is not of concern when eggs are cooked. Ovoinhibitor is another protein capable of inhibiting trypsin and chymotrypsin. 2. Green Leafy Vegetables are a rich source of minerals but many of them also contain high amounts of Oxalic acid, which combines with minerals like Calcium, Manganese and Iron to form insoluble salts called Oxalates. The insoluble oxalates cannot be absorbed by the body. Nutrients Drumstick Spinach 0.72 15.1 46 Fenugreek 0.53 24.6 11.8 Radish Leaves 1.25 29.3 11.2

Total Iron 0.26 mg/g (Dry weight basis) Calcium mg/g 22.4 (Dry Weight Basis) Oxalic Acid 11.2 mg/g (Dry Weight Basis) Source: Plants in Human Health Gopalan

& Nutrition Policy by Artemis P. Simopoulos, C.

3. Phytate is a hexa phosphate of inositol. It is widely distributed in seeds. Unrefined cereals and millets are richest sources of phytates. They act as a source of bound phosphorous for the seeds during germination. These phytates bind iron, zinc, calcium and magnesium. In presence of Ca and Mg, it forms insoluble complexes with iron and thus makes iron unavailable.

Phytate Content in wheat flour Degree of Grinding 70% 85% 92% Phytate (mg/kg) 53 451 759

4. Tannins are condensed polyphenolic compounds which are widely distributed in plant kingdom. They are present in high amount in seed coat of most legumes, spices, tamarind, turmeric and in certain vegetables and fruits. Millets like bajra, ragi and sorghum also contain a fair amount of tannins. Tannin derivatives of Gallic acid are also present in Tea and Coffee. Tannins bind with iron irreversibly and interfere with iron absorption.

Antinutritional Factors Why are they relevant to us?

The World Food Programme, in India, works in collaboration with governments and non-governmental agencies to develop innovative hunger solutions. Through its various programmes, WFP aims to reduce food insecurity and malnutrition amongst vulnerable groups, especially women and children. For example, the Sahariya tribe in Madhya Pradesh is one of the poorest and the most-deprived communities in the region and suffers from high levels of anaemia and malnutrition. To enable the intake of irona vital micronutrientand curb the high prevalence of anaemia, WFP in collaboration with the State Government is providing access to fortified wheat flour to members of the Sahariya tribe living in the districts of Guna, Shivpuri and Sheopur. In this intervention, wheat flour milled locally at the

village flour mills is being fortified using a pre-blend comprising iron and folic acid as fortificant. In case of Sahariyas, the baseline study revealed that the population was consuming wheat as a staple cereal and that about 90% of the women were suffering from anaemia. The next step was to identify the chemical form of fortificant to be added in order to reduce the prevalence of anaemia. The wheat flour consumed by Sahariyas is being milled at local chakki mills where whole wheat flour is obtained from wheat grains. In wheat, phytic acid accounts for 60-85% of seed total phosphorous and its accumulation site in the grain is aleurone layer. Phytic acid is primarily present as a salt of mono and divalent cations (Ca2+, Mg2+, K+) and it is a form of storage of cations as well as phosphorous. The phytic acid content of cereals, pulses and legumes can be substantially reduced by several methods, some of which are particularly suitable for ensuring adequate iron absorption from cereal-based complementary foods or soy-based infant formulas. However, the molar ratio of phytic acid: iron needs to be decreased to at least 1 :1, or even to less than 0.5 :1, in order to achieve a meaningful increase in iron absorption. Milling removes about 90% of the phytic acid from cereal grains, but the remaining 10% is still strongly inhibitory. The action of phytases (enzymes) is usually necessary in order to achieve complete phytate degradation. Naturally occurring cereal phytases can be activated by traditional processes, such as soaking, germination and fermentation. At the industrial level, it is possible to completely degrade phytic acid in complementary food mixtures of cereals and legumes by adding exogenous phytases or by adding whole wheat or whole rye as a source of phytases, these being naturally high in phytases. Because of the risk of bacterial contamination, it is better to add the phytases under factory conditions, but as yet, this practice has not been adopted commercially. An assumption could be that since phytase (an enzyme) is present in significant concentrations in the wheat bran, the phytates would be gradually broken down post milling as the phytase enzyme from the bran would come into contact with the phytates from the aleurone layer. But naturally occurring wheat Phytase is functional at a particular temperature and pH, 45 to 50C and 5-6 pH respectively. (Germination could be used to increase the phytase activity (by about 6 folds) in wheat). But these conditions may or may not be prevailing in a fortification process/ programme. Therefore, it becomes critical to choose the fortificant in such way that the chemical form and dosage compensate for the adverse effect of phytates.

While selecting a fortificant, we have to consider its suitability with a particular food vehicle, dietary patterns of the target population, frequency of consumption of the selected food vehicle, bioavailability (Bioavailability of nutrients has been defined as the proportion in which ingested nutrients from a particular source is absorbed in a form that can be utilized in metabolism by animals/ humans) of the chemical form to be added, impact on organoleptic properties, cost etc. The fact that Sahariyas were consuming Whole wheat flour (high phytate content) without any further processing beyond milling, demanded the use of NaFeEDTA (Sodium Iron EDTA) as this water soluble compound displays 2-3 times better iron absorption than that from Ferrous sulphate in case of high phytate content foods. The other options could have been ferrous fumarate (x2 amount), encapsulated ferrous sulphate (x2 amount) or encapsulated ferrous fumarate (x2 amount). Or another method to enhance iron absorption would be to incorporate iron absorption enhancers in the preblend e.g. Ascorbic acid or Vitamin C. In the gastric juice, elemental iron is dissolved as ferrous ions. These ferrous ions tend to form insoluble precipitate with phytate, a strong binder of trace minerals such as iron and zinc. Therefore, iron absorption from high phytate diets decreases significantly. At low pH in the stomach, Sodium EDTA acts as a chelating agent and as such prevents iron from binding to phytic acid or Phenolic compounds, which would otherwise inhibit iron absorption.

The understanding of presence of antinutritional factors in the WFP food commodities or the food vehicle used for fortification programmes is critical in order to bring about a significant impact in the target population within a stipulated time frame.

Antinutritional Factors How can we reduce/ eliminate them? Trypsin Inhibitors in Soybeans Digestion is the preparation of ingested material for absorption. The major site of protein digestion is the small intestine. Proteins and polypeptides that exit from the stomach are digested to release free amino acids and small peptides that are absorbed. A major source of the intestinal proteolytic enzymes is the pancreas. The pancreas secrete three endopeptidases trypsinogen, chymotrypsinogen and proelastase, which are activated in the intestine to produce trypsin, chymotrypsin and elastase. Trypsin is specific for peptide bonds with basic amino acids while chymotrypsin is specific for bonds involving non-charged aromatic amino acids. Elastase is a protease with broad specificity, including the capacity to digest elastin which is the second major protein in the connective tissues (besides collagen). Many plants contain inhibitors of pancreatic proteolytic enzymes. The best known is raw soyabean. Soybeans contain large protein molecules that combine irreversibly with trypsin and chymotrypsin. Instead of being able to break the trypsin inhibitors apart, the trypsin is bound to the inhibitor and excreted. This reduces protein digestibility because of the tie up of trypsin and creates an amino acid deficiency from the increased excretion of trypsin. The pancreas gland enlarges in an attempt to produce more trypsin to overcome its inactivation in the gut. Trypsin inhibitors are inactivated by heat treatment.

Flatulence factors in Kidney Beans Starch accounts for the major proportion of carbohydrates in legumes (55-60% carbohydrates). The unavailable sugars in pulses include substantial levels of oligosaccharides of the raffinose family of sugars (raffinose, Stachyose and verbiscose), which are notoriously known for the flatulence production in man and animals. These sugars escape digestion, when they are ingested, due to lack of galactosidase activity in the mammalian mucosa. Consequently, the oligosaccharides are not absorbed into the blood and are digested by the microflora of the lower intestinal tract resulting in the production of large amounts of carbon dioxide and

hydrogen and a small amount of methane. Some of the methods used in processing pulses, such as germination, soaking, fermentation, cooking and autoclaving, reduce considerable amounts of oligosaccharides.

Processing Methods a) Decortication/ Dehulling - The seed coat (hull) of pulses is often indigestible and may have a bitter taste. Dehulling improves the palatability and taste of chickpea, pigeon pea, and lentil. Removal of hull facilitates a reduction of polyphenols or tannins, antinutritional factors mostly present in seed coat. It also reduces cooking time by removing the impermeable seed coat, which hinders water uptake during cooking. Dehulling is achieved by using Attrition type mills with carborundum coated stones (derived from silicon carbide) of various abrasive grades. The new improved attrition type mills are often called Under Runner Disc Shellers (URD). The orientation of the stones can be either horizontal or vertical, and the gap between the stones can be adjusted to the seed size to optimize dhal yields. The other modern mill type is a carborundum roller mill. It has a cylindrical carborundum stone that is tapered and rotates inside a perforated metal casing so that the gap between the roller and casing decreases from the inlet to the outlet. The stone or casing can sometimes be moved to adjust the gap depending on the seed size. These mills are often mounted horizontally or on a slight downward angle to facilitate passage of the seed.

Operation of an under runner Disc Sheller could be explained through the diagram below: (The same principle applies to Rice Dehusking)

Under Runner Disc Sheller

Carborundum Roller Mill

A number of other methods of Decortication include soaking the seeds for a short time in water; the husk/hull takes up more water than the seeds and may be easily separated by rubbing while still moist. In an alternative, the soaked grains may be dried and the husk/hull removed by pounding and winnowing. Roasting also renders the husk/hull easier to separate.

b) Soaking - usually forms an integral part of such processing methods as cooking, germination, fermentation and roasting. Discarding soak water before further processing can remove several water soluble antinutrients such as protease inhibitors, phytates, lathyrogens, goitrogens, cyanogenic glycosides

and polyphenols. The extent of their removal depends upon the soaking temperature, the type of soaking medium, the seed type, the length of soaking, and the solubility characteristics of the soluble antinutrients. Soaking media frequently include water, salt (or combination of salts) solutions, and dilute aqueous alkali solutions. Salt and alkali help leach the solubles 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, vitamins and minerals.

c) Germination & Fermentation - mobilizes 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 phytates 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. Similar to the changes in germination, most of the changes occurring during the fermentation of foods are of a 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 -galactosidase activity present in legume seeds as well as in the microorganisms involved in the process. Depending upon the type of legume as well as fermentation, phytic acid is also hydrolyzed during fermentation to a variable degree. 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 glycosidases are often used to remove the cyanogenic 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 texturization have proved useful in removing certain toxicants and antinutrients of plant foods.

d) Cooking - is probably the oldest known method of processing plant foods for human consumption. Cooking may be done at atmospheric pressure and temperature or at high pressure and high temperature (autoclaving). The primary purpose of cooking is to render the food palatable and to develop its aroma. The cooking water may or may not be discarded, depending on personal and cultural preferences. Cooking generally inactivates heat-sensitive factors, such as enzyme inhibitors, lectins, volatile compounds such as HCN and some of the off flavor components. The heat-stable factors, such as estrogens, saponins, phytates, polyphenols and allergens may not be affected to any significant extent by cooking. Even among the heat-labile antinutrients, complete inactivation may not always be possible. When the soaking and cooking medium (such as water) is not discarded, a significant amount of heatstable antinutrients and toxicants remain practically unchanged. On the contrary, 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 adversely affects the protein quality of foods.

Additional Information Development of Kunitz Trypsin Inhibitor Free Soyabean

Directorate of Soybean Research (Madhya Pradesh) has developed two new soybean genotypes free from Kunitz Inhibitor Polypeptide NRC 101 (INGR 10054) and NRC 102 (INGR 10055).

Major Pulse Crops

Common Name Bengal Gram or Chickpea (Chana) Red Gram or Tur (Arhar or Pigeon Pea) Black Gram or Urad Green Gram or Mung Moth or Tepary bean Lentil or Masur Horsegram or Kulthi Peas Khesari Dhal Black Beans, Kidney Beans, Pinto Beans Important Terms (Presentation)

Botanical Name Cicer arietinum Cajanus cajan Phaseolus mungo Phaseolus aureus Phaseolus aconitifolius Lens esculenta Dolichos biflorus Pisum sativum Lathyrus sativus Phaseolus vulgaris

a) Pancreatic Hypertrophy - The pancreas is a gland organ in the digestive and endocrine system of vertebrates. As a digestive organ, it secretes pancreatic juice containing digestive enzymes that assist in the digestion

and absorption of nutrients. These enzymes help to further break down the carbohydrates, proteins and lipids.

Hypertrophy in the increase in the volume of an organ or tissue due to enlargement of its component cells. It should be distinguished from hyperplasia, in which the cells remain approximately the same size but increase in number.

Raw Soybeans containing trypsin inhibitor may cause pancreatic hypertrophy and hyperplasia, an effect that is accompanied by an increase in the secretary activities of the pancreas. The enzyme-inhibitor complex is neither degraded nor reabsorbed. Since pancreatic proteases are particularly rich in Sulphur containing amino acids, this loss of essential amino acids from endogenous sources, in addition to the incomplete proteolysis of dietary proteins, contributes to the soy induced growth depression. This relationship is further supported by the fact that supplementing raw soy meal with Sulphur containing amino acids improves the growth rate when compared to heat treated meal.

b) Osteolathyrism Osteolathyrism is a collagen crosslinking deficiency brought on by dietary over-reliance on the seeds of Lathyrus sativus (Khesari Dhal). Symptoms of Osteolathyrism include weakness and fragility of connective tissue (i.e., skin, bones, and blood vessels (angiolathyrism) and the paralysis of the lower extremities associated with neurolathyrism. For these reasons, compounds containing lathyrogens should be avoided during pregnancy and growth of a child. c) Neurolathyrism - Lathyrism or Neurolathyrism is a neurological disease of humans and domestic animals, caused by eating certain legumes of the genus Lathyrus. This problem is mainly associated with Lathyrus sativus (also known as Grass pea, Kesari Dhal, Khesari Dhal or Almorta) and to a lesser degree with Lathyrus cicera, Lathyrus ochrus and Lathyrus clymenum containing the toxin ODAP (Oxalyldiaminopropionic acid). The consumption of large quantities of Lathyrus grain containing high concentrations of the glutamate analogue neurotoxin -oxalyl-L-,diaminopropionic acid (ODAP, also known as -N-oxalyl-amino-Lalanine, BOAA) causes paralysis, characterized by lack of strength in or inability to move the lower limbs. A unique symptom of lathyrism is the emaciation of gluteal muscles (buttocks). ODAP is a poison of mitochondria leading to excess cell death, especially in motor neurons. d) Goitrogens - Goitrogens are substances that suppress the function of the thyroid gland by interfering with iodine uptake, which can, as a result, cause an enlargement of the thyroid, i.e., a goitre. e) Cyanogenic glycosides They account for approximately 90% of the wider group of plant toxins known as cyanogens. The key characteristic of these toxins is cyanogenesis, the formation of free hydrogen cyanide. The major edible plants in which cyanogenic glycosides occur are almonds, sorghum, cassava, lima beans, stone fruits and bamboo shoots. Potential toxicity of cyanoglycosides arises from enzymatic degradation to produce hydrogen cyanide, resulting in acute cyanide poisoning. The enzyme responsible ( glucosidase) may arise from the plant material or from gut microflora. Clinical symptoms of acute cyanide poisoning include rapid respiration, drop in blood pressure, rapid pulse, headache, dizziness, vomiting, diarrhea, blue discoloration of the skin, twitching and convulsions (A convulsion is a medical condition where body muscles contract and relax rapidly and repeatedly, resulting in an uncontrolled shaking of the body) f) Saponins - Saponins are glycosides with a distinctive foaming characteristic. They are found in many plants, but get their name from the soapwort plant (Saponaria), the root of which was used historically as soap (Latin sapo ---> soap). Saponins have been reported in soyabean, sword

bean and jack bean. Toxic saponins cause nausea and vomiting. These toxins can be eliminated by soaking prior to cooking. g) Hemolysis Hemo means blood and lysis means setting free or releasing. It refers to the rupturing of erythrocytes or red blood cells. h) Proteolysis It refers to the breakdown of proteins by cellular enzymes called proteases. i) Estrogens They are the primary female sex hormones. j) Hemoglobinuria - In medicine, Hemoglobinuria or haemoglobinuria is a condition in which the oxygen transport protein hemoglobin is found in abnormally high concentrations in the urine. The condition is often associated with hemolytic anemia, in which red blood cells (RBCs) are destroyed, thereby increasing levels of free plasma hemoglobin. The excess hemoglobin is filtered by the kidneys, which release it into the urine, giving urine a red color. k) Rickets - Rickets is a softening of bones in children due to deficiency or impaired metabolism of vitamin D, magnesium , phosphorus or calcium, potentially leading to fractures and deformity. Rickets is among the most frequent childhood diseases in many developing countries. The predominant cause is a vitamin D deficiency, but lack of adequate calcium in the diet may also lead to rickets (cases of severe diarrhea and vomiting may be the cause of the deficiency). l) Turkey Poults Poults refers to a young fowl (turkey, chicken etc). m) Muscular dystrophy - Muscular dystrophy (abbreviated MD) refers to a group of hereditary and non-hereditary, muscle diseases that weaken the muscular skeletal system and hamper locomotion. Muscular dystrophies are characterized by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue. n) Hemagglutinins - Hemagglutinin or haemagglutinin refers to a substance that causes red blood cells to agglutinate. This process is called hemagglutination or haemagglutination. Agglutination is the clumping of particles. The word agglutination comes from the Latin agglutinare, meaning "to glue. This occurs in biology in three main examples (a) the clumping of cells such as bacteria or red blood cells in the presence of an antibody. The antibody or other molecule binds multiple particles and joins them, creating a large complex. (b) The coalescing of small particles that are suspended in a solution; these larger masses are then (usually) precipitated. (c) An allergic reaction type occurrence where cells become more compacted together to prevent

foreign materials entering them. This is usually the result of an antigen in the vicinity of the cells. o) Isoflavones - Isoflavones comprise a class of organic compounds, often naturally occurring, related to the isoflavonoids. Many act as phytoestrogens in mammals. Being phytochemicals, they are able to be termed antioxidants because of their ability to trap singlet oxygen. Some isoflavones, in particular soy isoflavones, when studied in populations eating soy protein, have indicated that there is a lower incidence of breast cancer and other common cancers because of its role in influencing sex hormone metabolism and biological activity through intracellular enzymes, protein synthesis, growth factor actions, malignant cell proliferations, differentiation and angiogenesis. Isoflavones are produced almost exclusively by the members of the Fabaceae (i.e., Leguminosae, or bean) family. p) Glycosides - In chemistry, a glycoside is a molecule in which a sugar is bound to a non-carbohydrate moiety, usually a small organic molecule.