PROGRESS IN VEGETABLE PROTEINS ISOLATION TECHNIQUES: A REVIEW

Novel vegetable proteins, like those extracted from abundant raw materials (grass) or agri-food by-products and waste streams (oilseed meals), are expected to be used as replacers for animal-derived proteins, due to higher production efficiency, reduced life cycle environmental impact and possibility to meet consumers' dietary or cultural preferences. Although having a versatile functionality (emulsifying, foaming, gelling, texturizing agents), application of proteins is limited since their properties highly depend on their structure and composition, environmental factors (pH, ionic strength, presence of other microand macro-molecules in food matrices) and isolation method and conditions. The objective of this article is to review the current techniques used to isolate the proteins from vegetable raw materials and comment on the influence of extraction method and conditions (pH, ionic strength, extraction media temperature, extraction time, etc.) on protein properties (yield, purity, appearance, solubility, denaturation degree, emulsification efficiency, etc.). The utilization of novel technologies such as ultrasound assisted extraction, electro-activation technique and approaches (enzyme-assisted extraction) to improve protein extraction yield or functionality was also discussed.


INTRODUCTION
Although animal proteins have a competitive advantage over plant-based proteins in terms of their nutritional and functional properties, protein ingredient market is intensively seeking for alternative, underutilized sources of concentrated plant proteins in order to satisfy the demands of consumers with different ethnic, religious, dietary and moral preferences associated with consumption of animal-based products.There are numerous reasons for the increased global demand for novel, sustainable sources of proteins which are also of high nutritional value.According to Food and Agriculture Organization of the United Nations (FAO) in 2050 an increase in world human population up to 9 billion is expected.Moreover, a consumption of animal proteins has been continuously increasing which affects gas emission from cattle breeding and thus represents an ecological issue (Spiegel et al., 2013).On the other hand, in both underdeveloped and developing countries population is faced with proteinenergy malnutrition (PEM), which is quite often among small children as well as in the elderly population.Moreover, popula-rity of so-called "protein diets" has been also increasing as well as the demand for high protein food products.
Plant sources of proteins that are already widely consumed are the ones obtained from soy, wheat, peas and potatoes.Oilseed meals, by-products obtained after oil extraction, legume seeds and green plants represent excellent alternative protein sources (Karaca et al., 2011;Rodríguez-Ambriz et al., 2005).In order to exploit protein sources with low carbon footprint, higher production sustainability and lower production costs, the possibility to isolate proteins from canola, flax, hemp seed meal, rice bran, chickpea, sugar beet leaves, fababeans, lemna (water lentil), etc. was investigated (Papalamprou et al., 2009;Wanasundara and Shahidi, 1996;Xu and Diosady, 2000).Moreover, according to Stegeman et al. (2010) raw materials that are currently used for feed and biofuel products such are rapeseed, algae, grass, duckweed as well as some byproducts obtained from agricultural processing and other waste materials could be used as good sources of proteins.However, the major issue of these socalled "novel proteins" is their safety aspect concerning the occurrence of antinutritional factors, contaminants, allergens and other substances which are present or could be formed during the processing that might have negative effect on human health.Utilization of different plant protein isolates is mainly based on their versatility in the functional properties (solubility, viscosity, foam formation, emulsification, water and oil retention capacity etc.).Namely, their functional properties may originate from intrinsic factors such are protein composition and conformation, different environmental factors as well as the method of protein isolation (Fernández-Quintela et al., 1997).
Protein isolates are mostly obtained by solubilizing the protein rich source in an environment where the pH is far from the isoelectric point, followed by concentration with the aid of precipitation in an environment where the pH is close to the isoelectric point of the solubilized proteins.According to available literature data, iso-lation technique based on isoelectric protein precipitation in most of the cases results in coloured proteins due to coextracted chlorophylls and polyphenols which are very often of unpleasant and bitter taste that is undesirable from the technological and consumers point of view (Xu and Diosady, 2002).Another approach is to achieve protein solubilisation using saline solutions followed by protein precipitation due to salt removal through ultrafiltration and diafiltration membranes.The protein produced in this way has a micellar structure before being dried, with preserved native state (Arntfield et al., 1985).
The aim of this paper was to give an overview of protein isolation techniques and the effects of extraction conditions on the physicochemical and functional properties of the obtained protein isolates.The role of novel processing technologies and application of non-conventional approaches in protein isolation was also discussed.

PROTEIN ISOLATION TECHNIQUES
The most widely used procedures to prepare protein isolates from vegetable sources are presented in Figure 1.Alkaline extraction/isoelectric precipitation technique comprises alkaline solubilisation of the proteins, removal of the insoluble material by centrifugation, protein precipitation at pH which corresponds to isoelectric point and collection of precipitated protein by centrifugation.On the contrary, micellization involves protein extraction with salt solution, centrifugation to remove insoluble material, precipitation from a salt extract by ultrafiltration, diafiltration membranes or dilution in cold water, followed by protein recovery by centrifugation (Arntfield et al., 1985 1990), highly alkaline conditions during protein extrac-tion led to extensive protein denaturation.Therefore, a compromise has to be found between higher protein yields and extent of their denaturation.Similarly, rise in temperature and/or extraction time also contributes to better extractability of proteins.However, increase in temperature might also cause protein thermal denaturation and precipitation.Therefore, room or slightly higher temperatures are commonly advisable for protein extraction.According to different literature data extraction time is usually set between 10 and 60 min with constant stirring and at 5-15% (w/v) solid/solution ratio (Rodrigues et al., 2012).
Concerning micellization technique, increased ionic strength is often related to higher protein recoverability.Paredes-López and Ordorica-Falomir (1986) have increased safflower protein recoverability more than two times by increasing sodium chloride concentration from 0.1 M to 1.2 M. They have also shown that decrease in extraction pH from 7.0 to 5.8, at the same ionic strength, had slight influence on the increase in protein yield.
Depending on the extraction technique employed, the subsequent processes of protein concentration involve ultrafiltration, diafiltration membranes or simple precipitation at pH value close to isoelectric value of extracted solubilized proteins (Table 1).The concentrated protein solution is afterward bring to powder state using freeze drying, vacuum drying or spray drying techniques.
Electro-activated technique was also proposed as an alternative, non-invasive extraction method.According to this method, electric field was employed in order to produce alkaline water solutions which are claimed to have good extractive properties.According to Gerzhova et al.
(2015a), the use of electro-activation technique resulted in increased amount of extracted proteins in comparison to conventional alkaline extraction/isoelectric precipitation.The same group of authors also concluded that there were no significant differences in terms of solubility, surface hydrophobicity, water absorption as well as oil absorption capacity between the protein isolates obtained by conventional alkaline extraction method and electro-activated method (Gerzhova et al., 2015b).
Coustets and Teissié (2016) proposed pulsed-electric-field techniqueprocedure which involves electro-permeabilization of cell walls and/or membranes for protein extraction from Nanochloropsis and Chlorella.
Roselló-Soto et al. (2015) compared high voltage electrical discharges (HVED), pulsed electric field (PEF) and ultrasound (US) as pretreatments before extraction of protein and phenolic compounds from olive kernels.They found that HVED treatment was more effective than ultrasound and pulsed electric field in terms of energy input and effective treatment time.While PEF did not influence the increase in content of proteins, US and HVED treatments significantly improved amount of extracted proteins, which increase with the increase in input energy.

Table 1 .
Conditions/optimal conditions* used for protein isolation *In papers investigating extraction process optimization, only optimal parameters were reported ** Extraction solution was not reported if pH in water solution was simply adjusted using 0.5-2 mol/L NaOH or HCl

Table 2 .
Influence on novel technologies and approaches on protein recoverability