Air Classified Pulse Protein Fractions Biology Essay

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Grain legumes, also known as pulses, are seeds of plants belonging to the family Leguminosae which are primarily harvested for their seeds. Pulses constitute a significant source of protein in food and feed (Duranti & Gius, 1997). They include beans peas, soybeans, peanuts and lentils. Pulses provide energy, dietary fibre, protein, minerals and vitamins required for human health. Research suggests that consumption of pulses may have potential health benefits which include reduced risk of cardiovascular diseases, cancer diabetes, osteoporosis, hypertension, gastrointestinal disorders, adrenal diseases and reduction of LDL cholesterol. Such studies have contributed to an increasing awareness of including pulses in the diet (Boye, Zare & Pletch, 2010).

Air classification is a process for separating components of dry materials according to their physical properties including size, shape, density and aerodynamic characteristics (Chrismon, 1978). It is a dry process used for the manufacture of starch-rich and protein-rich fractions from pulses pea in particular. Particle size is the primary basis for separation, which concentrates protein in the fine fraction and starch in the coarse fraction. The protein-rich resulting from air classification of pulses generally contain 50-60% protein (Youngs, 1975; Tyler, 1984; Sosulski, 1982; Wright, Bumstead, Coxon, Ellis, DuPont & Chan, 1984; Horvath, Ormi-Cserhalmi, & Czukor, 1989; Pokatong, 1994) and sometimes retain a strong flavor and suffer oxidative instability.

Protein concentrates can be prepared by using technologies such as isoelectric point washing, alkaline extraction and aqueous alcohol washing. It was demonstrated that with the exception of 90%- and 95%-ethanol-washed products, that could be classified as true protein concentrate could be prepared from air-classified pea protein- (Pokatong, 1994). The products were obtained in high yields and were essentially devoid of unwanted colour, flavour, lipid, oligosaccharides and trypsin inhibitor activity.

The focus of this research is

To optimize variables such as alcohol concentration, extraction temperature and extraction time which are important in the preparation of protein concentrates by aqueous- ethanol or aqueous- isopropanol) washing of air-classified pea protein.

To use the optimal washing conditions identified in the first objective for the preparation of protein concentrates from other air-classified pulse protein fractions such as fababean, lentil and to and compare the yields, composition and functionality of these products with those derived from air-classified pea protein.

To determine the composition, potential utilization, and value of the extracts obtained by aqueous-ethanol or aqueous-isopropanol washing of air-classified pulse protein fractions. To examine the effect of aqueous-ethanol washing of pea and chickpea flours on the protein separation efficiency achieved during air classification and to compare the composition of the air-classified starch and protein fractions.

To evaluate the composition and functionality of a by-product obtained in an Agriculture and Agri-Food Canada study where air-classified pea protein was extracted with 80% aqueous-ethanol.

1.2 Hypotheses

The following hypotheses will be tested.

Optimization of variables such as alcohol concentration, extraction temperature and extraction time which are important in the preparation of protein concentrates by aqueous-alcohol (ethanol or isopropanol) washing of air-classified pea protein will enable the preparation of protein concentrates with desirable composition and functionality and in high yield.

Protein concentrates containing 65-70% of protein can be prepared by aqueous-alcohol (ethanol or isopropanol) washing of air-classified protein fractions from pea, fababean, lentil and chickpea.

Aqueous-alcohol washing will substantially reduce the flavour and colour, and the lipid and oligosaccharide contents, of air-classified protein fractions.

The extract obtained by aqueous-alcohol washing of air- classified pulse protein fractions will exhibit interesting composition and/or functionality.

Aqueous-alcohol washing of pea and chickpea flours prior to air classification will increase the efficiency of starch and protein separation.

The by-product of an 80%-alcohol reflux extraction of air-classified pea protein would have potential for use as a protein-concentrate in food and feed applications.

1.3 Objectives

The following are the objectives of this research project:

To determine the optimum conditions (alcohol concentration, extraction temperature, and extraction time) for preparation of protein concentrates by aqueous-ethanol and aqueous-isopropanol washing of air-classified pea protein.

To compare the yield, composition and functionality of protein concentrates prepared by aqueous-ethanol or aqueous-isopropanol washing of air-classified protein fractions from pea, fababean, lentil and chickpea

To determine the composition, potential usefulness and value of the extract obtained from the aqueous-ethanol and aqueous-isopropanol washing of air-classified pulse protein fractions.

To study the effect of aqueous-ethanol washing of pea and chickpea flours prior to air classification on starch-protein separation efficiency and the composition of the air-classified fractions.

To determine the functionality of a product prepared by 80%-ethanol reflux extraction of air-classified pea protein.

2. LITERATURE SURVEY

2.1 Constituents of pulses

Grain legumes belong to the family Leguminosae and are subdivided into pulses and leguminous oilseeds (Michaels, 2004). Pulses serve as an important dietary protein source for a large segment of the world's population (Boye, Zare & Pletch, 2010). With the exception of peanut, chickpea, and soybean, grain legumes can be described as containing approximately 10% moisture, 21-25% protein, 0.8-1.5% lipid, 60-65% total carbohydrates and 2.2-4% ash (Dalgetty, Baik & Swangson, 2003). The chemical composition of bean, chickpea, fababean and field pea will be described in this section.

2.1.1 Chemical composition

2.1.1.1 Protein

Pulses during their development accumulate large amounts of proteins which is stored in membrane bound organelles, the storage vacuoles or protein bodies in the cotyledonary parenchyma cells (Duranti, 2006). The proteins in pulses are of two types enzymatic (metabolic) and structural. The majority of protein found within pulse seeds is in the form of storage protein, which is classified as albumins, globulins, and glutelins based on its solubility properties. Globulins soluble in dilute salt solution represent approximately 70% of the total protein in pulses. The globulin protein vicillin 7S and legumin 11S usually predominate. Albumins account for 10-20% of the total protein and are soluble in water. Glutelins soluble in dilute acid or base account for 10-20% of the total protein found in pulse seeds (Roy, Boye & Simpson, 2010). The non-storage proteins are enzymes, enzyme inhibitors, hormones, and transporting, structural and recognition proteins (Pokatong, 1994; Mosse & Pernollet, 1983).

The protein contents of pea varieties range from 23.1% to -30.9% (Nx5.6). Albumin and globulin represent 15-25% and 50-60% of the total protein respectively (Boye, Zare & Pletch, 2010; Guegen and Barbot, 1988). Chickpea varieties are reported to have protein contents ranging from 20.9- 25.27% with albumin, globulin, prolamin and glutelin contents ranging from 8.39-12.31%, 53.44-60.29%, 3.12-6.89% and 19.38-24.40% respectively. (Boye, Zare & Pletch, 2010; Fan and Sosulski, 1974) reported that the protein content of fababean was 32%. The storage globulins of Vicia-faba fall into two classes legumin and vicillin. Together these proteins contribute to approximately 20% of the mature seed dry weight (Ersland, Brown, Casey & Hall, 1983). Salt soluble globulins, including a major fraction of vicillin and minor fraction of legumin.are the predominant proteins in beans (Rui, Boye, Ribereau, Simpson & Prasher, 2011). The salt soluble protein fraction of an isolate from Great Northern Bean contains 62.2% of the total flour protein and consisted of 2S (35%), 7S (57%) and 11S (8%) proteins (Boye, Zare & Pletch, 2010).

2.1.1.1.1 Amino acid composition

The essential amino acid composition of pulse proteins exhibit wide variation. Pulses are mainly deficient in sulphur containing amino acids and tryptophan, but are rich in lysine (Salunkhe, Kadam & Chavan, 1985). The amino acid composition data for field pea seeds was summarized by Orr and Watt, (1957) Food and Agriculture Organization, (1970) Harvey, (1970). Holt & Sosulski (1979) stated that arginine, leucine, lysine, aspartic acid and glutamic acid occurred in highest amounts and accounted for 50% of the total amino acids, whereas histidine, methionine, threonine, tryptophan and cystine accounted for less than 11%. Chickpea and fababean have been found to contain high amounts of arginine, leucine, lysine, aspartic acid and glutamic acid but fababean is more deficient in methionine than chickpea and field pea. (Boye, Zare & Pletch, 2010; Kaldy & Kasting, 1974).The amino acid composition of pea, bean fababean and chickpea are shown in table 2.

Table : Essential amino acid composition (g/16g N) of various legumes

Amino Acid

Bean

Chickpea

Fababean

Field Pea

Arginine

6.9

10.3

10.5

9.5

Histidine

3.2

3.4

2.6

2.3

Isoleucine

5.3

4.1

4.3

7.4

Leucine

9.0

7.0

8.3

6.9

Lysine

7.7

7.7

6.6

7.2

Methionine

1.3

1.6

0.7

1.0

Phenylalanine

6.0

5.9

4.2

4.6

Threonine

4.9

3.6

3.3

3.8

Tryptophan

1.6

1.1

1.0

0.8

Valine

5.9

3.6

3.9

4.6

Source: Compiled from (Salunkhe, Kadam & Chavan, 1985; Adams, Coyne, Davis, Graham & Francis, 1985)

2.1.1.2 Carbohydrates

Carbohydrates, according to their role in plants, can be separated into three groups: the mono and disaccharides are sources of energy for growth, the oligosaccharides and starch are storage carbohydrates, and the non-cellulosic polysaccharides, pectins, hemicelluloses and cellulose comprise the structural components of the cell wall (Hedley, 2001). Starch is the most abundantly occurring carbohydrate in legumes. The endosperm of legume seeds is known to be a rich source of galactosides of sucrose and galactomannose (Arora, 1983). The total sugars represent only a small percentage of total carbohydrates in dry pulse seeds. The oligosaccharides of the raffinose family (raffinose, stachyose, verbacose and ajugose) predominate in most pulses and account for a significant percentage (31.1-76%) of the total sugars in several others. The predominant oligosaccharide depends on the type of pulse. Verbacose is the major oligosaccharide in fababean, whereas stachyose is the major oligosaccharide in Great Northern Beans and smooth and wrinkled peas (Reddy, Pierson, Sathe & Salunkhe, 1984). For most pulses the largest part of the carbohydrate fraction is starch accounting for 35-45% of the seed weight depending on the legume species (Hedley, 2001). In pulses amylose may constitute a significant portion of starch the range being from 10% to 66%. Starch solubility, lipid binding and other functional properties are influenced by amylose in starch. The solubility of starch granules is thought to be contributed by amylopectin (Reddy, Pierson, Sathe & Salunkhe, 1984). The anthrone method was used to determine the total available carbohydrate in chickpea and dry pea flour, and produced values that ranged from 625 to 657g/kg of dry matter (Berrios, Morales, Sanchez-Mata & Camara, 2010). They also reported that the total available carbohydrate in chickpea flour and dry pea flour consisted mainly of starch, based on the findings of Sosulski, Garrant & Slinkard (1976) and Swanson (1990) who determined the values to be 59.4% starch for chickpea flour and 53.6 % starch for dry pea flour.

Legumes contain a substantial amount of crude fibre commonly known as roughage. A heterogeneous group of cellulose and hemicellulose in which lignin, pectic and cutin substances are predominated by pentosans constitute crude fibre. Cellulose plays an important role in the utilization of nutrients. Increasing the level of cellulose in the diet decreases the utilization of ingested protein (Salunkhe, Kadam & Chavan, 1985)

2.1.1.3 Lipids

The lipid content of legumes is generally less than 7% the exceptions being the oilseed legumes peanut and soybean, which contain about 52% and 20% oil respectively. (Lam & Lumen, 2003). The total lipid in pulses consists of several classes of lipids such as neutral lipids, phospholipids and glycolipids. Their distribution in the seed varies with the species and variety. In most legume seeds neutral lipids are the predominant class; however, phospholipids and glycolipids are also present in appreciable amounts (Salunkhe, Sathe & Reddy, 1983). Neutral lipids consist of mono, di, and triacylglycerides and the remainder are metabolic polar lipids (phospholipids, glycolipids, sterols, sterol esters and lipoprotein) (Sosulski & Sosulski, 2006). The lipid of pea contains ten different neutral lipids including triacylglycerols, free sterols and sterol esters, which are major components, and monoglycerides, diglycerides, free fatty acids, waxes and certain pigments (Pokatong, 1994). The major fatty acids found in pea, bean and chickpea are oleic and linoleic acid.

2.1.1.4 Minerals and vitamins

Minerals and vitamins constitute the micronutrients of pulses. Pulses are good sources of thiamine, riboflavin and niacin. Carotene however is found only in small amounts (Salunkhe, Kadam & Chavan, 1985). Pulses are excellent sources of folate, which in addition to being an essential nutrient is thought to reduce the risk of neural tube defects. Beans are also a good source of thiamine and pantothenic acids. On average, 100g of pulses provide 23% of the nicotinic acid, 50% of the thiamine, 15% of the riboflavin, 20% of the vitamin B6, 19.5% of the folate and 30% of the pantothenic acid requirements of an adult. Pulses are poor sources of fat soluble vitamins and vitamin C (Lam & Lumen, 2003).

Pulses are good sources of minerals such as calcium, copper, zinc, potassium and magnesium. Potassium contributes 25-30% of the total mineral content of pulses (Salunkhe, Kadam & Chavan, 1985). Although pulses contain a good amount of phosphorous, it is mostly present as phytic acid which may affect the absorption and utilization of calcium, through the precipitation of insoluble salts in the stomach and duodenum. Pulses are also good sources of iron and other nutrients (Shukla, Dixit & Arora, 1983).

2.1.1.5 Antinutritional factors

Antinutritional compounds are molecules that disrupt the digestion process when raw seed or flour is consumed by monogastric species rendering the seed unpalatable. The antinutritional compounds found in pulse crops are classified into two categories: protein antinutritional compounds and non-protein antinutritional compounds (Roy, Boye & Simpson, 2010). The antinutritional factors include enzyme inhibitors, hemagglutinins (lectins), phytates, polyphenols, flatulence factors, cyanogenic compounds, lathyrogens, estrogens, goiterogens, saponins, antivitamins, and allergens (Salunkhe, Kadam & Chavan, 1985). Non-protein antinutritional compounds include alkaloids, phytic acid and phenolic compounds such as tannins and saponins. Protein antinutritional compounds commonly present in pulse crops include lectins or agglutinins, trypsin inhibitors, chymotrypsin inhibitors, anti-fungal peptides, and ribosome-inactivating proteins (Roy, Boye & Simpson, 2010).

2.2 Production of concentrated protein products

Pulses due to their significant protein content serve as good raw materials for the preparation of protein concentrates and isolates. Legumes due to the presence of antinutritional compounds require appropriate processing to make them palatable. Soy products have been widely used as nutritional and functional ingredients since 1960. Soy protein is categorised into three different groups based on protein content ranging from 40 to over 95% namely soy flours and grits, soy protein concentrates and soy protein isolates. Soy flours and grits are the least refined forms of soy protein made by grinding and screening soybean flakes either before the removal of oil or after. Defatted flour from which sugar and water and/or alcohol have been removed are called protein concentrates. The most refined soy protein products from which fat, sugars, cotyledonary fibers and water-soluble materials have been removed are called soy isolates (Endres, 2001). Many techniques used in the processing of soybeans have been adapted for other legumes. The available methods of protein concentrate and isolate production may be classified into two categories, namely, (1) separation of a protein-rich fraction using physical methods, and (2) solubilisation of proteins using a suitable solvent system followed by precipitation and/ or drying (Salunkhe, Kadam & Chavan, 1985).

2.2.1 Wet separation of starch and protein fractions from pulses

(Youngs, 1975) reported the production of protein concentrates from field pea using a wet process. Ripe, yellow peas, whole or dehulled, were ground to a fine flour in a pin mill and the flour was slurried with five parts of water. To increase the pH of the slurry to 9, lime was added to it. The slurry was centrifuged in order to yield a protein rich supernatant and starch solids. The high protein supernatant was spray or drum dried to yield a concentrated protein product (60% protein). The starch fraction, containing about 6% protein was reslurried with five parts of water and again centrifuged to produce starch solids containing about 2% protein. The next batch of flour was slurried using the wash water from the second extraction. A forced air oven at 60°C was used to dry the starch solids. A pale yellow, virtually bland protein concentrate was obtained. The extract was dried to avoid loss of solids as whey and to overcome effluent problems. High evaporation costs make wet processing expensive.

Lime

Water

Water

Starch

Drier

Drum Dryer

Starch

Flakes of Protein

Protein

Protein

Protein

Starch

Spray drier

Centrifuge

Slurry Tank

Centrifuge

Slurry Tank

Flour

Figure : Wet processing of pulses (Youngs, 1975).

2.2.2 Dry separation of pulse starch and protein fractions by air-classification

Protein concentrates can be produced from cereals and pulses by air classification (Emami, Tabil, Tyler & Crerar, 2002). The air classification process separates finely milled flour into starch and protein fractions. The composition of the seed plays a major role in determining the amount and composition of the processed products obtained from pulses and the distribution of fat, ash, fibre and non-starch carbohydrate between the fractions influences the purity of the separated fractions (Youngs, 1975). An air classifier is essentially an elutriator utilizing an air stream to separate a mixture of finer, particles from coarser ones (Tyler, 1984). Milling of pulses produces flours having particles of two discrete sizes and densities which aids the process of separation. Whole or de-hulled seed is ground into a very fine flour, followed by air classification in a spiral air stream to separate starch from protein (Boye, Zare & Pletch, 2010). The process can be repeated several times inorder to improve separation. Air classification of flours containing 21 % protein yielded 25% of fines with a protein content of 60% and a coarse fraction containing about 8% protein (Youngs, 1975). The protein content for fababean after the first air classification step ranged from 71-75%. Remilling the high starch fraction from fababean yielded a second high protein fraction containing 64-68% protein. Great Northern bean was reported to have 50 % protein in the fine fraction, and 41% protein in the fine fraction obtained after remilling.

Figure 2: The double pass pin milling and air classification process (Tyler, 1982).

2.2.3 Acid washing and alkaline extraction

Pokatong (1994) generated nitrogen solubility profiles with pH as the independent variable for soy flour and pea protein according to method 46-23 of the AACC. It was reported that pHs of 4.5 and 9 were appropriate for the production of protein concentrates by acid washing and alkaline extraction respectively. In alkaline extraction, a mixture of ground pulse flour and water in ratios ranging from 1:5 to 1:20 is made. The mixture is adjusted to a pH of 8-11 using dilute sodium hydroxide and allowed to stand for 30-180 min inorder to maximize the solubilization of proteins. The system may be subjected to elevated temperature (55-65°C) to further increase protein solubilization and extraction. Insoluble material is removed by filtration or centrifugation and the pH of the extract is adjusted to the isoelectric point (pH 4-5) to induce precipitation of protein. The extract is then centrifuged to recover proteins washed to facilitate removal of salts, neutralized and dried. Protein fractions extracted using alkaline extraction/isoelectric precipitation have been shown to have variable protein contents probably due to differences in processing conditions (Boye, Zare & Pletch, 2010). This method is extensively used in the preparation of protein isolates. Isolates are highly refined protein products.. Acid extraction works on principle similar to that of alkaline extraction with the exception that the initial extraction is conducted under acidic conditions. A low pH is used to solubilize proteins followed by precipitation at isoelectric point. The precipitated proteins recovered and dried.

2.2.4 Alcohol washing

Aqueous-alcohol washing can be used in the preparation of protein concentrates from air-classified pea flours. Aqueous-alcohol washing of soybean flour at a concentration of 60-70% is the commercially adapted method for the production of protein concentrates containing 65-70% protein. Pokatong (1994) prepared products having appropriate functionality from soybean and air-classified pea protein using an aqueous-alcohol washing process. Aqueous-alcohol can solubilize sugars and other partially water soluble compounds in oilseeds such as pigments and aflatoxins (Hron, 1997).

3. RESEARCH STUDIES

3.1 Optimization of aqueous-alcohol (ethanol or isopropanol) extraction of air-classified pea protein and comparative study of aqueous-ethanol and aqueous-isopropanol for preparation of protein concentrates from air-classified pea protein

3.1.1 Summary

Aqueous-alcohol (ethanol or isopropanol) washing of air-classified pea protein will be used in the preparation of protein concentrates. The optimal yield, protein concentration and functionality parameters will be identified.

3.1.2 Hypothesis

The following hypothesis will be tested as a part of this study.

Aqueous-alcohol-washed protein concentrates from air-classified pea protein containing 65-70% protein and having appropriate functionality can be prepared by optimizing extraction parameters such as alcohol concentration, temperature and extraction time.

3.1.3 Experimental approach

3.1.3.1 Materials

Air-classified pea protein will be supplied by Parrheim Foods, Saskatoon SK

3.1.3.2 Methods

Aqueous-alcohol (ethanol and isopropanol) washing of air-classified pea protein will be carried out using different concentrations of alcohol (50%, 60%, 70%). A flour to solvent ratio of 1:5 (200 g of product slurried in 1000 mL of solvent) will be employed. The mixture of aqueous-alcohol and pea protein will be homogenized for different time intervals and at varying temperatures and then centrifuged at 2000 x g for 10min at 4°C. The cake obtained after the extraction will be reslurried twice using aqueous-alcohol at the concentration used in the first extraction. The thrice extracted samples will be given a final wash with aqueous 95% aqueous-alcohol. The concentrate will be dispersion dried at ≤ 70°C.

Air classified pea protein

Aqueous-alcohol (50%, 60%, 70%) washing of pea protein 1:5 (w/v)

Centrifugation

Repeat

2X

Filtrate

Solids

Washing of solids with 95% aqueous-alcohol

Filtration

Drying

Grinding

Aqueous-alcohol-washed protein concentrate

Figure 3: Aqueous-alcohol washing of air-classified pea protein.

3.1.3.2.1 Chemical composition

The composition of the pea protein concentrates [protein (Nx6.25), starch, fat, ash and moisture] will be determined according to methods 46-13.01, 76-13.01, 30-25.01, 08-01.01 and 44-19.01 of the AACC (1999). Total lipid content will be determined by the method of Sahasrabudhe (1979). Oligosaccharide quantitation will be carried out by the method of Apostolos, Karoutis & Tyler (1992) or equivalent. Trypsin Inhibitor activity will be determined according to method 22-40.01 of the AACC (1999).

3.1.3.2.2 Functional analysis

Nitrogen solubility index (NSI) and water hydration capacity (WHC) will be determined according to methods 46-23.01 and 56-30.01 of the AACC (1999). Colour will be measured using a HunterLab spectrocolorimeter (Hunter Associates Laboratory, Inc., Reston, VA) and will be expressed in terms of L, a, and b values. For the determination of oil absorption capacity the modified method of Lin, Humbert & Sosulski, (1974) will be used. Emulsion capacity will be determined according to the method of Beuchat, (1977) as modified by (Sathe & Salunkhe (1981) and Han & Khan (1990b)). Emulsifying activity and emulsion stability will be determined according to the method of Yasumatsu et al., (1972) and foaming capacity and stability will be measured according to Bencini (1986) as modified by Han & Khan (1990b)

3.1.4 Discussion

A better understanding of the effect of physical parameters such as concentration of alcohol, extraction time agitation method and temperature would be crucial in maximizing the production of functional protein concentrates by aqueous-alcohol washing of air-classified pea or other pulse protein. The findings from this study would also provide a better understanding of the alcohol most suitable for the preparation of protein concentrates from air-classified pulse protein. A successful outcome of this study would encourage the commercial manufacture of aqueous-alcohol-washed protein concentrates.

3.2 Comparative study of the composition and functionality of protein concentrates prepared by aqueous- ethanol or aqueous-isopropanol) washing of air-classified pea, fababean and chickpea protein fractions

3.2.1 Summary

The objective of this study is to compare the composition and functionality of aqueous-alcohol-washed protein concentrates prepared from field pea, fababean, lentil and chickpea. For this study air-classified pulse protein concentrates will be prepared by aqueous-ethanol or aqueous-isopropanol washing at several alcohol concentrations (50-70%).The concentrates will be compared on the basis of yield, protein recovery, chemical composition (protein, fat, total lipid, oligosaccharides and trypsin inhibitor activity) and functionality (nitrogen solubility index, water hydration capacity, oil absorption, oil emulsification capacity, emulsifying activity, emulsion stability, foaming capacity, foam stability) as described in section 3.1.

3.2.2 Hypothesis

The following hypothesis will be tested as a part of this study.

Protein concentrates containing 65-70% protein and having appropriate functionality can be prepared by aqueous-alcohol washing of air-classified protein fractions from pea, fababean lentil and chickpea.

3.2.3 Experimental approach

3.2.3.1 Materials

Air-classified pulse protein fractions will be supplied by Parrheim Foods, Saskatoon SK.

3.2.3.2 Methods

The experimental protocols used in section 3.1 will be employed here.

3.2.4 Discussion

The knowledge gained through this study would encourage commercial production of protein concentrates by aqueous-alcohol washing of pulse protein fractions from pea, fababean, chickpea and other pulses

3.3 Composition of the extract obtained by aqueous-alcohol washing of air-classified pulse protein fractions

3.3.1 Summary

The main objective of this study is to determine the composition of the extract obtained by aqueous-ethanol or aqueous-isopropanol) washing of air-classified pulse protein fractions. The dried extract would be evaluated for the presence of ash, lipid, protein and sugars.

3.3.2 Hypothesis

Aqueous alcohol can extract a variety of lipid and protein components, sugars and pigments so it is conceivable that the extracts may exhibit interesting components.

3.3.3 Experimental approach

3.3.3.1 Materials

The extracts obtained by aqueous-alcohol washing of pulse flours ( section 3.1 and 3.2) will be used for this study.

3.3.3.2 Methods

The experimental protocols used in section 3.1 will be employed here.

3.3.4 Discussion

The findings from this study will lead to a better understanding of the extractability of the components from an air-classified pulse fraction by aqueous alcohol extraction, and of the potential value of the constituents of the extract.

3.4 Effect of aqueous- ethanol extraction of pea and chickpea flours prior to air-classification

3.4.1 Summary

Processing of pea by air classification yields high separation efficiency of starch and protein. Chickpea however due to its higher fat content exhibits poor separation efficiency). The overreaching aim of this study is to investigate the effect of extracting pea and chickpea flours with aqueous-ethanol prior to air classification. To accomplish the goal of this study, flours will be washed with aqueous-ethanol at various concentrations (50-70%). The extracted flour will be dried, milled and then air-classified. The protein fractions obtained after air classification by this method will be compared with those produced by the conventional method for yield, starch and protein separation efficiency and chemical composition (protein, fat, total lipid, oligosaccharides, and trypsin inhibitor activity).

3.4.2 Hypothesis

The following hypothesis will be tested during the course of this study

The separation efficiency of starch and protein during air classification will increase substantially due to the removal of fat from chickpea flour by aqueous-ethanol washing prior to air classification.

3.4.3 Experimental Approach

3.4.3.1 Materials

Pea and chickpea flour will be supplied by Parrheim Foods, Saskatoon, SK or obtained from commercial source.

3.4.3.2 Methods

Aqueous-ethanol washing of pea and chickpea flours will be carried out using different concentrations of ethanol (50%, 60%, and70%). A flour to solvent ratio of 1:5 (200 g of product slurried in 1000 mL of solvent) will be employed. The mixture of aqueous-ethanol and flour will be homogenized at room temperature and then centrifuged at 2000 x g for 10min at 4°C. The cake obtained after the extraction will be reslurried twice using aqueous-ethanol at the concentration used in the first extraction. The thrice extracted samples will be given a final wash with 95% aqueous-ethanol. The concentrate will be dispersion dried at ≤ 70°C re-milled as required and air-classified.

Flour

Aqueous-ethanol washing of flour 1:5 (w/v)

Centrifugation

Repeat

2X

Filtrate

Solids

Washing of solids with 95% aqueous-alcohol

Filtration

Drying

Air classification

Figure 4: Aqueous-alcohol washing of pulse flour prior to air classification.

Compositional analysis of starch and protein fractions according to methods described in section 3.1 will be undertaken.

3.4.4 Discussion

A successful outcome of this study would be the demonstration of effective air classification of aqueous-ethanol washed chickpea flour.

3.5 Evaluation of the by-product from extraction of air-classified pea protein with 80% aqueous ethanol

3.5.1 Summary

Scientists at Agriculture and Agri-Food Canada (AAFC) in Saskatoon and Winnipeg are currently engaged in research on isolation of insecticidal proteins from air-classified pea protein. The by-product from their process is a concentrated protein prepared by reflux extraction of air-classified pea protein with 80% (v/v) aqueous ethanol. The aim of this study is to evaluate the composition and functionality of the pea protein product prepared under these conditions.

3.5.2 Hypothesis

Aqueous-alcohol-washed protein produced by reflux extraction of air-classified pea protein with 80% aqueous ethanol will contain ≥ 65% protein and exhibit useful functionality.

3.5.3 Experimental approach

3.5.3.1 Materials

The by-product obtained from research at AAFC on isolation of insecticidal proteins from air-classified pea protein will be used for this study.

3.5.4.2 Methods

The experimental protocols adopted in section 3.1 for chemical composition and functionality will be employed here.

3.5.4 Discussion

Demonstration of acceptable protein concentration and useful functionality in pea protein product prepared by 80% alcohol reflux extraction of air-classified pea protein will aid in the identification of uses of the by-product from insecticidal protein extraction.

4. IMPORTANCE

A successful outcome from this research study will extend the prospects for the use of pulse proteins in food and feed. It will create interest in the commercial use of alcohol-washed pulse protein concentrates and also help overcome high effluent treatment costs and disposal problems.

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