CHICKPEA COMPOSITION

FIELD OF THE INVENTION

The present invention concerns compositions, infant formulae and nutraceuticals based on whole seeds of chickpeas. These formulae are free of known allergens, free of phytoestrogens and do not contain genetically modified organisms (GMOs). The invention further concerns a process for their production.

BACKGROUND OF THE INVENTION

Five to seven percent of infants consuming cow's milk formulae develop an allergy to cow's milk protein and are often switched to soy based formulae. Almost half of theses children are allergic to soy protein as well. About twenty five percent of children in the US are fed by soy based formulae. The soy based formulae contain phytoestrogens which have hazardous potential in infancy. Hence, the safety of soy for infant feeding is highly questionable.

Vegetarian soy-based infant formulae, initially developed for infants who are allergic to cow's milk-based formulae, now account for about 25% of the infant formulae sold. Formulations have changed over the years to improve digestibility, stability, availability of minerals, and protein quality. Even so many apprehensions have been raised regarding the phytoestrogenic isoflavone content of soy-based infant formulae. Although soy-based formulae support normal growth and nutritional status in healthy term infants in the first year of life, there are very limited data on sexual and reproductive development or outcomes such as immune function, visual acuity/cognitive development and thyroid function. Early exposure to soy and/or isoflavones might have long-term harmful effects. The daily intakes of isoflavone values for infants fed soy-based formulas were found to be from 1.6 to 6.6 mg/kg of body weight. The daily exposure of infants to isoflavones in soy infant formulas is 6-11 fold higher on a bodyweight basis than the dose that has hormonal effects in adults consuming soy foods. Circulating concentrations of isoflavones in infants fed soy-based formula were 13,000-22,000 times, higher than plasma oestradiol concentrations in early life, and may be sufficient to exert biological effects, whereas the contribution of isoflavones from breast-milk and cow-milk is negligible (Setchell KD and others. Isoflavone content of infant formulas and the metabolic fate of these early

phytoestrogens in early life. Am J Clin Nutr 1998 Dec;68(6 Suppl):1453S-1461S). The safety of soy for infant feeding is thus highly questionable; the need to produce a safe, nutritive, vegetarian infant formula free of possible anti-estrogen, anti-thyroid effects and other possible long term ill effects resulting from isoflavones (phytoestrogens) as are present in soy based infant formulae, is in dire need (e.g., Setchell KD, Zimmer-Nechemias L, CaiJ, HeubiJE. Exposure of infants to phyto-oestrogens -from soy-based infant formula. Lancet. 1997 JuI 5; 350(9070): 23-7; Setchell KD, Zimmer-Nechemias L, Cai J, Heubi JE. Isoflavone content of infant formulas and the metabolic fate of these phytoestrogens in early life Am J Clin Nutr. 1998 Dec;68(6 Suppl):1453S-1461S. Review; Irvine CH, Shand N, Fitzpatjϊck MG, Alexander SL. Daily intake and urinary excretion of genistein and daidzein by infants fed soy- or daiiγ-based infant formulas Am J Clin Nutr. 1998 Dec; 68(6 Suppl):1462S-1465S; Irvine CH, Fitzpatiick MG, Alexander SL. PhytoestiOgens in soy- based infant foods: concenfrations, daily intake, and possible biological effects. Proc Soc Exp Biol Med. 1998 Mar; 217(3):247-53; Knight DC, Eden JA ₁ Huang JL, Waring MA Isoflavone content of infant foods and formulas. J Paediatr Child Health. 1998 Apr;34(2): 135-8; Genovese ML, Lajolo FM. Isoflavones in soy-based foods consumed in Brazil: levels, distribution, and estimated intake. J Agric Food Chem. 2002 Oct 9; 50(21) :5987-93; Huggett AC, Pridmore S, Malnoe A, Haschke F, Offord EA Irvine et al, [Letter]. New Zealand Medical Journal 1955, 24 May:208-9; Fitzpatiick M. Soy formulas and the effects of isoflavones on the thyroid. New Zealand Medical Journal 2000 113:24-6).

Arenas et al. (1987), Arch. Latinoam Nut}'. 37(3): 551-559 determined in rats the chemical composition and protein quality of chickpea protein; Sotelo et al. (1987) Arch. Latinoam Nutr. 37(3): 468-47 evaluated the nutritive value of chickpeas in malnourished infants. Ulloa et al.(1993) Arch. Latinoam Nutr. 43(1): 50-54 evaluate the nutritive value of chickpea protein concentrate.

There is a need for a nutritive isoflavone-free, lactose-free and gluten-free composition which has similar protein value as soy infant formula and soy containing compositions and which is easily produced.

SUMMARY OF THE INVENTION

The subject invention thus provides a method for producing a composition comprising chickpeas, wherein the composition is free of isoflavones, gluten and lactose, the method comprising producing a dried chickpea composition and adding at least one additive to the dried composition.

The subject invention further provides a method for producing a composition comprising chickpeas, wherein the composition is free of isoflavones, gluten and lactose, the method comprising producing a dried chickpea composition, reducing the size of the chickpeas to obtain particles with an average particle size from about 1 to about 100 microns and with a particle size distribution of about 3-50 microns and adding at least one additive to the dried composition.

The subject invention also envisages a method for producing a composition comprising chickpeas, wherein the composition is free of isoflavones, gluten and lactose, the method comprising extruding a composition of chickpeas to obtain a paste or semi-dried chickpea composition.

The subject invention further provides a composition comprising chickpeas, wherein the composition is free of isoflavones, gluten and lactose obtainable by a method of the invention.

The invention further involves a dried formula comprising chickpeas, wherein the formula is free of isoflavones, gluten and lactose characterized by chickpea particles with an average particle size from about 1 to about 100 microns and with a particle size distribution of from about 3 to about 50 microns.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 displays a schematic flow chart for the method of preparation of a formula of the invention. Figure 2 shows the particle size distribution of regularly milled whole chickpeas after 3 hours of cooking.

Figure 3 shows the particle size distribution of superfine milled whole chickpeas after 1 hour of cooking.

Figure 4 shows the particle size distribution of superfine milled whole chickpeas after 3 hours of cooking.

DETAILED DESCRIPTION

Compositions based on legumes, such as whole chickpeas (which excel in nutritional values), alleviate most health and nutritional concerns, as well as furnish a product without phytoestrogens. In addition, such compositions offer a non GMO alternative for those that are concerned with possible risk associated with GMO soy.

Even though protein rich legumes in general contain isoflavones, the isoflavone estrogenic activity in leguminous varieties of chickpea is very low and practically negligible. While the soybean contains about 200 mg isoflavones per 100 g, chickpeas contain only 0.10 mg isoflavones per 100 g, (USDA-Iowa State University Database on the Isoflavone, ReL 1.3 - 2002).

Compositions and formulas of the subject invention can be administered to a subject. The subject can be, but is not limited to, a newborn, an infant, a toddler, a pre-school child, a child, an adolescent, an adult, an elderly, a geriatric subject, or subjects on special diets, such as, but not limited to vegetarian diets, diets as a result of allergies to cow milk protein and/or soy protein.

An infant as used herein can be any infant, such as, but not limited to a preterm and term infant, small premature infants, infants with very low birth weight (VLBW), extreme low birth weight (ELBW) ₅ infants suffering from gastrointestinal immaturity, diseases and disorders such as, but not limited to, vomiting, spitting up, dribbling, regurgitation, reflux (GERD), colic, diarrhea, gastroenteritis, constipation, and so forth.

A composition or formula as used herein can be, but is not limited to, a nutritional composition, a nutraceutical composition or a pharmaceutical composition.

A nutritional composition as used herein includes, but is not limited to, human milk fat substitute and infant formula. In a specific embodiment of the invention, the composition is an infant formula.

A imtraceutical composition as used herein can be any nutraceutical, which can be any substance that may be considered a food or part of a food and provides medical or health benefits, including the prevention and treatment of disease. Such nutraceutical compositions include, but are not limited to, a food additive, food supplement, dietary supplement, vegetables, herbal products, and processed foods such as cereals, soups and beverages and stimulant functional food and pharmafood.

For the sake of ease, we hereinafter refer to a composition of the invention as 'formula' or 'infant formula', but it should be understood that the subject invention can be any one of the compositions above and can be useful for any of the above age groups.

As used herein, "substantially free of isofiavones" means that amounts of Daidzin, Genistin, Glycitin, Daidzein, Genistein, Glycitein, Malonyl Daidzin, Malonyl Genistin, Malonyl Glycitin, Acetyl Daidzin, Acetyl Genistin, Acetyl Glycitin in the formula are negligible.

"Negligible amounts" as used herein are amounts below about 9 nanomoles per gram dry weight.

"Free of as used herein means at negligible amounts.

As used herein, the chickpeas can be any non-genetically modified variety of chickpeas obtained from any species of chickpeas such as, but not limited to, chickpeas varieties Desi and Kabuli, such as, but not limited to, Spanish-Kabuli, 3279 Kabuli, Desi 1408S ₅ Desi 8631, Hadas-Kabuli, and Desi 8575. In addition, the chickpea seeds may be processed, concentrated into a protein isolate/concentrate or into chickpea flour. Non- limiting examples of processed forms of chickpeas which can be used are heat treated and/or soaked, and/or fermented, and/or sprouted.

In one embodiment, heat treated chickpeas are employed. In another embodiment, soaked and heat treated chickpeas are to be employed. In a specific embodiment of the invention, the starting material is whole seed chickpeas.

"Hypo-allergenic" as used herein means the formula is a non-dairy fomiulation free of isoflavones, free of phytoestrogens, free of lactose and fee of gluten.

"Vegetarian" as used herein means that the formula does not contain animal-originated ingredients.

Isoflavone free formula of the invention can exist in many forms such as, but not limited to, liquid, freeze dried, spray dried, drum-dried, extruded or dry powder form. Dry powder can be further diluted with water and/or appropriate medium to form an edible formula.

Isoflavone free formula of the invention can also exist in a form ready for consumption, e.g., in a liquid form ready for consumption, in bottles ready for feeding, or in cans ready for use.

In one embodiment, isoflavone-free formula of the invention in powder form may be reconstituted with cold, warm or boiled water.

The methodology used to produce a formula of the invention is easy and cost effective, maintains the nutritional value of the formula and enables flexibility in the production of a final product

Any process for obtaining edible chickpea may be used in order to obtain chickpea in a suitable form to be incorporated into a formula of the invention, substantially free of isoflavones, gluten and lactose.

Isoflavone-free, gluten-free and lactose-free chickpea infant formulae can be made by various techniques by using simple home . processing means and by industrial manufacturing. Figure 1 demonstrates typical steps for obtaining the chickpea infant formulae. The steps disclosed in Figure 1 may be adapted for home or industrial processes.

In one embodiment, the processed chickpeas are converted into vegetarian infant formula by varying techniques based on heat-treated micromilled soaked chickpeas and/or hydrolyzed chickpea and/or chickpea protein concentrate and/or hydrolyzed chickpea proteins.

In general, the process for the production of the chickpea formula is carried out as follows: The chickpea whole seeds are soaked, the skin is removed, and then, if fiber and starch content needs to be reduced, or avoided otherwise, treated with GRAS (generally recognized as safe) enzymes. The chickpeas are then processed through a pressure cooker or an extruder to cook and to eliminate anti-nutritional factors and undesirable attributes, such as anti-nutritional factors, enzyme inhibitors and other harmful factors. The whole seeds (or the particles produced in the extruder) are (air-) dried where after their size is reduced by pulverization or grinding to achieve an average particle size from about 1 to about 100 microns and with a particle size distribution from about 3 to about 50 microns.

In one embodiment, the average particle size is from about 2 to about 90 microns; in another embodiment from about 3 to about 70 microns; In yet another embodiment, the average particle size is from about 4 to about 50 microns; in yet another embodiment from about 5-25; La yet another embodiment, the average particle size is from about 5 to about 20 microns; and in yet another embodiment from about 5 to about 15 microns;

It is further envisaged that the particle size distribution is from about 3 to about 40 microns.

Prior to the last stage, ash, nutritional quality, nutrients and mineral contents are analyzed. This step is applied at a known frequency to ensure the adequate data collection required for assessing the nutritional quality of the powder obtained.

In a specific embodiment, additives (nutrients) added to compositions of the invention are selected from the group consisting of vitamins, minerals, macro nutrients, amino acids, nucleotides, pro-biotics and anti-oxidants.

Non-limiting examples of such nutrients are vitamins (e.g., Bi, B ₂ , B ₃ , B ₆ , Bu, folic acid, vitamin C, biotin, pantothenic acid, K, A, D, E), minerals (e.g., Na ₅ K, Cl, Ca, P, Mg, Fe, I, Cu, Zn, Mn, Fl), macro nutrients (total fat, linoleic acid, inositol, phospolipids, long chain polyunsaturated fatty acids (LC-PUFA) e.g. DHA, AA, and ALA, carbohydrates, starch, protein, carnitine, taurine, choline, dietary fiber, crude fiber), amino acids (e.g., cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine). If required, additional nutrients (e.g., nucleotides, probiotics, and anti-oxidants) are added. Then the nutrients are mixed preferably as an add-mix or in other form such as sprayed liquid, encapsulated, agglomerated or other means used to add dry ingredients. The utilization of linear programirήng to establish the optimal weights and composition to meet all the necessary nutritional requirements specified by the health authorities, or equivalent calculations is performed and the derived nutrients quantities are then added and blended ensuring their uniform distribution in the final product.

An advantage, as opposed to adding such nutrients to the liquid, hydrated (in slurry or liquid) or reconstituted composition, is that different kinds of final products of infant formula (for example intended for different age groups) can be prepared from the same dried bulk (stock) of processed chickpeas by adding different nutrients/additives for each intended final product.

Another advantage as opposed to adding them to the liquid, hydrated (in slurry or liquid) or reconstituted composition, is that the heat exposure is reduced and damage to nutritional values is minimized.

Yet another advantage of admixing the nutrients after drying, as opposed to adding them to the liquid, hydrated (in slurry or liquid) or reconstituted composition is that different absorption patterns of the chickpeas may be achieved resulting in different products from the same dried bulk (stock) of processed chickpeas by adding different nutritional additives for each intended final product.

Home processing of chickpeas into vegetarian infant formulae (which can easily be processed by low-income populations and in developing nations) is carried out ^" by various ^"

techniques using simple operations e.g., the chickpeas are soaked overnight with water (that may include bi-carbonates) at room temperature, boiled for enough time to render the . chickpea softness. The boiling water is discarded and the soft and cooked chickpeas are wet-milled, well macerated with enough water and with available additives such as carbohydrates, oil/fat, vitamins, minerals, etc., in a. suitable wet mill, to render a liquid infant food that can provide the required amount of protein and calories. Vitamins and minerals may be added as liquid supplements.

The subject invention thus provides a method for producing a composition comprising chickpeas, wherein the composition is free of isoflavones, gluten and lactose, the method comprising producing a dried chickpea composition and adding at least one additive to the dried composition.

The subject invention further provides a method for producing a composition comprising chickpeas, wherein the composition is free of isoflavones, gluten and lactose, the method comprising producing a dried chickpea composition, reducing the size of the chickpeas to obtain particles with an average particle size from about 1 to about 100 microns and with a particle size distribution of about 3 to about 50 microns and adding at least one additive to the dried composition.

The subject invention also envisages a method for producing a composition comprising chickpeas, wherein the composition is free of isoflavones, gluten and lactose, the method comprising extruding a composition of chickpeas to obtain a paste or semi-dried chickpea composition. In one embodiment, the composition of chickpeas is enzymatically processed. In another embodiment, the particle size of the chickpeas is reduced to obtain particles with an average particle size from about 1 to about 100 microns and with a particle size distribution of about 3 to about 50 microns.

The subject invention also provides a method comprising extruding a composition of (processed) chickpeas, to obtain a paste or semi- dried chickpea composition, reducing the size of the chickpea composition to obtain chickpeas with an average particle size from about 1 to about 100 microns and with a particle size distribution of from about 3 to about 50 microns and adding to these chickpea particles at least one additive.

The subject invention further provides a method comprising enzymatically processing a chickpea composition, extruding the composition of (processed) chickpeas to obtain a paste or a semi-dried chickpea composition, reducing the size of the chickpea composition to obtain chickpeas with an average particle size from about 1 to about 100 microns and with a particle size distribution of from about 3 to about 50 microns and adding to these chickpea particles at least one additive.

In a specific embodiment, the average particle size is from about 2 to about 90 microns; in another embodiment from about 3 to about 70 microns; In yet another embodiment, the average particle size is from about 4 to about 50 microns; in yet another embodiment from about 5-25; In yet another embodiment, the average particle size is from about 5 to about 20 microns; and in yet another embodiment from about 5 to about

15 microns;

It is further envisaged that the particle size distribution is from about 3 to about 40 microns.

The subject invention further provides a composition comprising chickpeas, wherein the composition is free of isoflavones, gluten and lactose obtainable by a method of the invention. In one embodiment, the composition is an infant formula.

The invention further involves a dried formula comprising chickpeas, wherein the formula is free of isoflavones, gluten and lactose characterized by an average particle size from about 1 to about 100 microns and with a particle size distribution of from about 3 to about 50 microns. In one embodiment, the average particle size is from about 2 to about 90 microns; in another embodiment from about 3 to about 70 microns; In yet another embodiment, the average particle size is from about 4 to about 50 microns; in yet another embodiment from about 5-25; In yet another embodiment, the average particle size is from about 5 to about 20 microns; and in yet another embodiment from about 5 to about 15 microns. In a specific embodiment, the particle size distribution is from about 3 to about 40 microns. In one aspect of the invention, the dried formula is infant formula. ^{' '} -

EXAMPLES

The invention is further described in the following examples, which are not in any way intended to limit the scope of the invention as claimed.

EXAMPLE 1

The content of isofiavones in chickpea was tested in the following selected chickpea varieties Spanish-Kabuli, 3279 Kabuli, Desi 14088, Desi 8631, Hadas-Kabuli and Desi 8575 according to the analytical protocol of isofiavones analysis based on a method published by Pat Murphy of Iowa State University in J. Agric. Food Chem. 1994 Vol. 42, No. 8:1666-1673. A linear gradient varying from 85% to 65% of (A) 0.1% acetic acid in water to 15% to 35% (B) 0.1% acetic acid in acetonitrile over 50 minutes was used. UV detection at 254nm was utilized. Column was ODS-3 5um 4.6mm x 250mm.

The selected Chickpea varieties were analyzed for the presence of the following isofiavones: Daidzin, Genistin, Glycitin, Daidzein, Genistein, Glycitein, Malonyl Daidzin,

Malonyl Genistin, Malonyl Glycitin, Acetyl Daidzin, Acetyl Genistin, and Acetyl Glycitin.

The total amount of isofiavones in all the above noted selected varieties was negligible.

EXAMPLE 2 Chickpea varieties Spanish-Kabuli, Kabuli 3279, Desi 14088, Desi 8631, Hadas-Kabuli and Desi 8575 were analyzed for major nutritive components and were found to have ^* Energy 384-404 kcal/lOOg; Protein (Nx6.25) 20.4-24.9%; Fat 3.2-6.5%; Calcium 95-260 mg/lOOg; and Iron 5.3-7.6 mg/100 g.

Minerals were analyzed using ICP-MS, Kejldal methodology for energy, Lowry method for protein analysis and Soxhlet for fat analysis - all well established methods.

Vitamins were analyzed using HPLC. Macro-nutrients were analyzed using ICP-MS.

Amino acids were analyzed using spectrophotometry.

Table 1 shows the specific vitamin, mineral, macro-nutrient and amino acid content in 100 kcal of Kabuli chickpeas.

Table 1 also shows the recommended (FDA, EU) nutritive value of vitamin, mineral, macro-nutrient and amino acid content in 100 kcal required for infant formula.

The results indicated that all tested varieties of chickpea are suitable to be used as a component in infant formula although one may prefer to use a certain variety due to its nutritional value, ease of processing and/or color. Certain additives can be added to comply with the required amounts of vitamins, minerals, macro nutrients and amino acids.

Table 1

Comparison between chickpeas and target recommended nutritive value for infant formula (per 100 kcal).

CO

C co

CO

m

CO

I U) m I m c m

IO

* Target was calculated based on the average between MIN (minimum) and MAX (maximum).

EXAMPLE 3

The process for the production of the chickpea formula was carried out as follows:

Whole seeds of chickpeas were soaked in 1% salt solution (0.25 g/100 g NaCl + 0.75 g/10Og NaHCO _3.

The seeds were treated enzymatically using GRAS amylase and cellulase to reduce the dietary and crude fibers and to breakdown the starch into smaller carbohydrates in solid state fermentation (SSF).

The amylase used was termamyl 120L type L — α-amylase Novozymes (Novozymes A/S); 10 Kilo Novo Unit (KNU) were used per 100 g dry chickpeas. The cellulase used was Viscoflow β-glucanase 2.5 mg (dry)/100 g dry chickpeas Novozymes (Novozymes A/S)

KNU is defined as the amount of enzyme which hydrolyses 4870 mg (on a dry basis) of soluble Merck starch (Erg. B 6, lot number 6380528) per hour under standard conditions, pH 5.6, 37 ⁰ C ₅ and Ca ₂₊ concentration 0.0003 M (Novozymes A/S, 2001). Novozymes A/S Determination of Alpha- Amylase Activity (KNU and FAU). Novozymes Analytical Method EB-SM-0009.02/01 [online], February 2001 Novozymes A/S, Bagsvaerd, Denmark.

Alternatively, the seeds were cooked, the skin removed and then treated with enzymes. Or, the seeds were soaked in enzymes, the skin removed and cooked.

The chickpeas were then processed through a pressure cooker. Time, temperature and pressure in the cooker were optimized. The whole seeds were cooled, air dried or drum dried using a regular drier and pulverized using a commercial ultra-fine vortex pulverizing and classifying system of SUPER FINE Ltd. (P.O.Box: 532 Yokneam, ISRAEL ₅ 20692) to create a fine powder with an average particle size of about 5 to

about 15 microns with a narrow size distribution (i.e., 80% of the particle size population was between 3.0 and 40 microns).

Alternatively, the relatively high content of fibers in the chickpea skin ("coat") is used as pre-biotics in the new formed formulae. The starch and the fibers are treated enzymatically to control and enhance the prebiotic ingredient.

EXAMPLE 4

Clean chickpeas are soaked in water in room temperature overnight. The soaked chickpeas are passed through model X-20 extruder cooker and micromilled in an Ultra- Rotor to form a free flowing powder. This powder is then mixed with vitamins and minerals to obtain an infant formula and upon mixing with water a suitable dispersible and acceptable infant formula is obtained.

EXAMPLE 5

Whole chickpeas were divided into 3 groups.

Group I was soaked, pre-cooked, cooked for 3 hours, cooled, air-dried, and regularly milled. Figure 2 shows the particle size distribution of these chickpeas. Figure 2 shows that the average particle size of 10% of the chickpea population was below 7.1 microns, that the average particle size of 50% of the chickpea population was below 63.1 microns and that that the average particle size of 90% of the chickpea population was below

145.7 microns.

Group II was soaked, pre-cooked, cooked for 1 hour, cooled, air-dried, and milled superfine. Figure 3 shows the particle size distribution of. these chickpeas. Figure 3 shows that the average particle size of 10% of the chickpea population was below 3.1 microns, that the average particle size of 50% of the chickpea population was below 11.5 microns and that that the average particle size of 90% of the chickpea population was below 24.8 microns.

Group III was soaked, pre-cooked, cooked for 3 hours, cooled, air-dried, and milled superfine. Figure 4 shows the particle size distribution of these chickpeas. Figure 4

shows that the average particle size of 10% of the chickpea population was below 2.3 microns, that the average particle size of 50% of the chickpea population was below 13.9 microns and that that the average particle size of 90% of the chickpea population was below 37.5 microns.