**3. Technologies for extracting legume protein**

Several technologies exist for extraction of protein from legumes. Some have found success in commercial processing, while others have not mainly due to economics of the technologies. The methods include several wet extraction and dry processes. Extraction of protein from legume to an appropriate level of purity is necessary for its use in food formulations. Choice of extraction process depends on amount of protein required in the protein extract, legume type, functionality desired in the protein and nature of previous processing [35]. The processing methods can generally be classified into two categories, namely aqueous and dry processing. The various legumes differ in composition as some are lipid rich, while other are starch rich. Each is suitable for protein extraction using a particular method. Dry processing which involves fine milling and air classification is suitable for starch-rich legumes as pea and faba bean, but generally not used to process oil-rich legumes such as soybean, peanut and similar seeds [5, 39].

### **3.1 Aqueous extraction**

Aqueous processing involves using water or alkaline solution to solubilize protein from legume flour or flakes in the initial processing step. It uses solubility differences to separate protein from carbohydrates, fiber and other non-protein components. This is then followed by purification and drying. This method has been used for several decades in soy protein extraction. Extraction efficiency depends on pH, temperature, ionic environment and solvent to flour ratio. It has variations depending on raw material which is being used for protein extraction. It is desired to achieve highest protein recovery without the process being detrimental to protein functionality. The major extracted protein products in terms of application in food are protein concentrate and isolate.

#### *3.1.1 Protein concentrate*

Processing of grain legumes into protein concentrate follows the following general steps, namely cleaning, de-hulling, flaking, defatting, protein extraction, neutralization and drying. Production of protein concentrate from legumes is illustrated in **Figure 1**. De-fatted soy flakes or flour is the starting raw material. Protein extraction can be carried out using three methods. The methods are washing with (1) aqueous alcohol (60–90%), (2) acid leaching at pH 4.5 and (3) moist heat water leaching. If extraction was done using alcohol, solvent is removed in a process step referred to as desolventization. The protein may then be neutralized to pH 7 then dried. Drying options include drum drying and spray drying, with the latter being preferred and most commonly used. Protein concentrate products contain about 70% protein.

#### *3.1.2 Protein isolate*

Protein isolate is more refined than concentrate. Its processing generally involves alkaline extraction of protein followed by protein recovery at their isoelectric pH of about 4.0–5.0, depending on the legume. The general principles of this extraction approach can be applied, with appropriate modifications, to extract protein from different legumes. Process options exist for unit operations such as preparation, defatting, pH of carrying out protein recovery and drying. The general process and its possible modifications are described here. **Figure 2** illustrates processing steps involved in production of protein isolate from legumes.

*Legume Protein: Properties and Extraction for Food Applications DOI: http://dx.doi.org/10.5772/intechopen.100393*

#### **Figure 1.**

*Process flow diagram for production of protein concentrate.*

Processing starts with cleaning of legume seeds to remove impurities such as stones, mold infected seeds, discolored seeds, sand, soil and other foreign matter. Grains such as soybean, chickpea, and pigeon pea are du-hulled to remove the fiber-rich seed coat. The seed coat is also referred to as hull, husk, testa, or husk depending on grain legume. The grain is then prepared into flakes or flour. Then defatting is carried out. The most commonly used method to remove fat is by hexane extraction. It can be carried at either low or high temperature of around 60–80°C. The high temperature also inactivates lipoxygenase, which contributes to causing off-flavors in the resultant extracted protein. Defatting using hexane leaves residual oil of about 0.5–1% (w/w) of the extracted protein. The residual lipid is composed of phospholipids and polar lipids. They cannot be extracted with hexane. They are sources of off-flavors as they participate in auto-oxidation and lipoxygenase mediated reactions. Following defatting, the solvent is the recovered from the meal and oil. Alternative defatting processes include supercritical carbon dioxide extraction, enzymatically aided extraction, organic solvent extraction, and aqueous extraction [40]. They have various degrees of efficiency and cost implications. They have however not been used for commercial processing due to cost and efficiency.

The intermediate product obtained at the above step can be toasted and cooled to make defatted soy flour. Toasting is applied to inactivate trypsin inhibitors. The product obtained from removal of solvent is also called flakes, which can also be milled into flour, as starting materials for aqueous extraction into protein concentrate and isolate.

#### **Figure 2.**

*Process flow diagram for production of protein isolate.*

To extract proteins, the flakes is milled into flour and mixed with water at a ratio of 1:6. The pH is adjusted to 9 using lye so that protein goes into solution. Legume proteins are generally soluble in aqueous media. In solutions where pH is less or greater than isoelectric pH, a net positive or negative charge occurs resulting in repulsion and protein staying in solution [41]. Clarification is then carried out to remove non-protein insoluble materials such as carbohydrates and insoluble fiber. Clarification can be done using centrifugation, filtration or membrane process. Protein is then recovered from the solution. Recovery can be done using ultra-filtration or precipitation. Precipitation can be achieved using isoelectric precipitation, salting out, salting in, heating or organic solvents. Isoelectric precipitation is the most commonly used method during commercial scale processing. It is generally not detrimental to protein functionality. But it may cause protein to aggregate and also result in changes in solubility as a result of non-covalent interactions. Isoelectric precipitation is carried out by adjusting the acidity of the protein solution using dilute acid to pH 4.0–5.0. Separation is then done by centrifugation or decanting. The resultant protein is washed, then may be neutralized to pH 7. Final processing step involves drying by spray or drum drying. In the former method, a thin layer is applied to a heated drum to evaporate water. The preferred method is however spray drying because it gives protein that has less heat damage. Some aggregation may however occur. Protein isolate contain at least 90% protein.
