Functional Properties of Lupinus angustifolius Seed Protein Isolates

Protein isolates prepared by alkaline solubilization followed by isoelectric precipitation and freeze-drying from six varieties of Lupinus angustifolius (Haags Blaue, Sonate, Probor, Borlu, Boregine, and Boruta) grown in Mexico were evaluated for functional properties: nitrogen solubility, water-holding capacity (WHC), oil holding capacity (OHC), emulsion activity index (EAI), emulsion stability index (ESI), foaming capacity (FC), foam stability (FS), and gellingminimumconcentration (GMC).Thenitrogen solubility values, WHC, OHC, and FC did not show significant differences between the protein isolates. The solubility of the isolates was minimal at pH of 4.0 and 5.0 while the regions of maximum solubility were found at pH of 2.0 and 10.0. There were significant differences in EAI and ESI depending on the varieties used. The isolates of the Boregine and Borlu varieties showed the highest EAI with 29.3 and 28.3m g, respectively, while the lowest index was recorded in the isolate obtained from the Sonate variety (24.6m g). Like solubility, these indices also increased at both extremes of pH evaluated; both properties were minimal in the isoelectric pH range (4.0 to 5.0).


Introduction
Legume seeds represent an important and economical source of protein for human and animal feeding [1].Its amino acid composition is generally adequate; however, some species contain deficient amounts of some amino acids [2].Soybeans worldwide represent the main source of protein; nevertheless, the food industry is in the search for new sources of vegetable proteins of good nutritional quality and functional characteristics superior or equal to soybean proteins [1,2].Among the species most likely to compete with soybean proteins are legumes of the genus Lupinus [3,4].Historical antecedents indicate that different cultures of the Mediterranean and South America practiced for several years the cultivation of some species of the genus Lupinus to include them in their diet like Lupinus albus, L. luteus, L. angustifolius, and L. mutabilis.[5,6].Due to its high protein content and to the new varieties with low alkaloid content, the cultivation of these legumes has spread to other regions of the world for use in animal and human nutrition [7][8][9].In addition, Lupinus are more adaptable to regions with low fertility soils and mild or very cold winters compared to soybean cultivation [10].The seeds of these species represent a functional food ingredient because their proteins are related to the prevention of cardiovascular diseases, and it had been found that their proteins (conglutins) reduce blood glucose in hyperglycemic rats [11,12].In this way, obtaining protein isolates of Lupinus by isoelectric precipitation is an alternative for the commercial production of food ingredients.At present, the food industry has shown a growing interest in protein isolates, due to their functional properties such as water absorption, oil absorption, emulsification, foam formation, and gelation.These properties are essential to determine the possible uses of these as food ingredients.Functional properties provide information on how a particular ingredient would behave in a food matrix, play a prominent role during the preparation, processing, and storage of food, and, in turn, correlate with sensory properties.These properties could be considered as a result of different conformational changes or interactions between food components, such as interactions between proteins, proteins, and polysaccharides, lipids, phenolic compounds, or phytic acid [13].Furthermore, they constitute the functional base of diverse products mainly of low protein content and high fat content.On the other hand, legumes are source of beneficial compounds that have a protective effect on the development of various diseases [14].Therefore, the objective of the present research work was to determine the functional properties of protein isolates of six varieties obtained from Lupinus angustifolius cultivated in the state of Jalisco, Mexico.

Vegetable Materials.
Seeds of six varieties of L. angustifolius (Haags Blaue, Sonate, Probor, Borlu, Boregine, and Boruta) were used which were provided by the German company Saatzucht Steinach GmbH dedicated to genetic improvement.These seeds were sown and cultivated during the fall/winter period of 2014/2015 under irrigated conditions on agricultural soils of the University Center of Biological and Agricultural Sciences of the University of Guadalajara in Zapopan, Jalisco, Mexico.This site is located at the geographical coordinates 20 ∘ 44  47  N and 103 ∘ 30  43  W at an altitude of 1,523 m.It is characterized by a humid temperate climate with rains in summer, with precipitations ranging from 700 to 1400 mm per year and an annual average temperature of 12.0 to 18.0 ∘ C, with the presence of frost.After conventional land preparation with a moldboard plow and a disk harrow, seeding was performed manually under an experimental randomized block design, with four replicates.

Proximate Composition and Preparation of Protein Isolates.
After harvesting on the order of 50 kg of seeds, one batch of 1 kg each variety was separately ground with a knife mill (IKA5) until flour with a particle size of 2 mm was obtained.Every variety was subjected to triplicate a proximal chemical analysis according to the techniques described in AOAC (2000): moisture content (925.09),crude fat (920.30),crude protein (979.09),ash (923.03),crude fiber (962.09),and carbohydrate content [15].The flour was subjected to a degreasing process by Soxhlet extraction, using hexane as solvent [16].The defatted sample (100 g) was used to obtain the protein isolates.
The protein isolate was obtained using the isoelectric precipitation method [17].A dispersion of 100 g L −1 in Na 2 SO 3 (0.025%) was prepared and adjusted to pH of 9.0 using NaOH (1 N).Immediately it was stirred for one hour at room temperature (25 ∘ C) and centrifuged at 10,976 ×g.(HERMLE Labortechnik GmbH, Z 326, Germany) for 10 min.The supernatant was acidified to pH of 4.5 using HCl (1 N).This was subjected to centrifugation at 10,976 ×g for 10 min at 4 ∘ C, recovering the precipitate.A washing process with distilled water was applied to the precipitate and NaOH (1 N) was used to bring it to a pH of 7. The precipitate was lyophilized and stored at 4 ∘ C. The protein isolates obtained from the seeds of the six varieties were analyzed in triplicate for all functional properties.

Functional Properties
2.3.1.Solubility.The solubility of the protein isolates was evaluated according to the methodology proposed by Ogunwolu et al. [18] and Liu et al. [19] with slight modifications.Each isolate was prepared in a 0.1% w/v suspension in 10 mL of distilled water.The pH was adjusted in a range of 2.0-10.0 using NaOH (0.1 N) or HCl (0.1 N).The suspension was stirred for 30 min on an orbital shaker (Thermo Fisher Scientific, Multipurpose rotator, 2346, China).Subsequently, it was centrifuged for 10 min at 10,976 ×g (HERMLE Labortechnik GmbH, Z 326, Germany).The protein content in the supernatant was determined by the Bradford method.The standard curve was made from bovine serum albumin (BSA) and the absorbance of the samples was measured at 595 nm on a spectrophotometer (Thermo Fisher Scientific, G10S Uv-Vis, China).The solubility of the protein was calculated by the following equation: Solubility = protein in supernatant (g) protein in sample (g) × 100. (1)

Water-Holding Capacity (WHC).
The WHC was evaluated by the method of Chau et al. [20] with slight modifications.Samples of protein isolates (0.5 g) were dispersed in 5 mL of distilled water and stirred for 24 h.After this time, the centrifugation for 30 min at 1474 g (HERMLE Labortechnik GmbH, Z 326, Germany) was carried out.The volume of the supernatant was measured after centrifugation.The difference between the initial volume of water and the volume recovered after centrifugation determines the WHC expressed as milliliters of water by one gram of sample (mL g −1 ).
The WHC was determined using the following equation: where   is the initial volume of distilled water (mL);   is the volume of the supernatant (mL), and   is the weight of the sample (g).

Oil Holding Capacity (OHC).
The OHC was evaluated by the method of Chau et al. [20] with slight modifications.0.5 g of the protein isolates that were obtained was added 5 mL of canola oil contained in a 50 ml centrifuge tube, which was stirred for 30 min by magnetic stirring.Then, it is centrifuged at 1,474 g (HERMLE Labortechnik GmbH, Z 326, Germany) for 30 minutes.After centrifugation, the volume of the supernatant is measured.The difference between the initial volume of oil and the volume recovered corresponds to the OHC expressed in mL g −1 of protein isolates.The OHC was determined by the following equation: where   is the initial volume (mL);   is the volume of the supernatant (mL); and   is the weight of the sample (g).

Emulsion Activity Index (EAI) and Emulsion Stability Index (ESI)
. EAI and ESI were determined using the method described by Zheng et al. [21] with slight modifications, which consisted of mixing 10 mg of the protein isolates with 10 mL of distilled water together with 3.33 mL of canola oil.The pH of the dispersion was adjusted in a range of 2.0-10.0 by the addition of HCl (0.1 N) or NaOH (0.1 N) according to the case.Subsequently, the mixtures were homogenized on a vortex stirrer (IKA Mixing, Model MS1) for 1 min at 2,500 rpm.Then, a 5 mL aliquot of each emulsion formed was mixed with 5 mL of 0.1% sodium dodecylsulfate.The samples were analyzed on a spectrophotometer (Thermo Fisher Scientific, G10S Uv-Vis, China) and the absorbance was read at 500 nm.
The EAI was determined by the following equation: where  0 is the zero minute absorbance; DF is the dilution factor (100);  is the initial protein concentration (0.1 g 100 mL −1 ); 0 is optical path (0.01 m); and  is the fraction of oil (v/v) used to form the emulsion (0.25).After reading the absorbances in zero time, the emulsions were allowed to stand at room temperature (25 ∘ C) for 10 minutes ( 10 ).The absorbance of emulsions was obtained again, and the emulsion stability index (ESI) was calculated with the following equation: (5)

Foaming Capacity (FC) and Foam Stability (FS)
. FC and FS were determined following the method described by Cheung and Chau [22].0.5 g of protein isolates was mixed with 25 mL of distilled water adjusted to pH 7, by the addition of HCl (0.1 N) or NaOH (0.1 N).They were then shaken in a Oster (Sunbeam Mexicana, S.A de C.V, Model: BLSTEP7808W-013, Mexico) at the "lowest" speed (mode 1 of 10) for 5 min.The mixture was transferred to a graduated cylinder (50 mL).After 30 seconds, the initial foam volume was measured as well as after 5, 10, 15, 30, 60, and 120 minutes.
The foaming capacity was determined by the following equation: where VF30  is the volume of foam measured after 30 seconds of rest after the sample has been shaken; VF0 is the volume of foam measured after agitation.
The FS was determined using the following equation: where VF  is the foam volume after standing for 5, 10, 15, 30, 60, and 120 min.

Gelling Minimal Concentration (GMC).
The GMC of the protein isolates was determined according to the method described by Cheung and Chau [22].Suspensions of the protein isolates (50 mL) were prepared at a concentration of 4-20% (w/v) in distilled water, and the pH was adjusted to 7 by adding HCl (0.1 N) or NaOH (0.1 N).The suspensions were poured in 50 mL falcon tubes, which were placed in a vessel with water at 100 ∘ C for 1 h.Immediately thereafter, they were maintained at 4 ∘ C for 2 h.After the cooling time, the tubes were inverted to determine the degree of displacement of the suspensions through the walls of the tube.The absence of displacement at the different concentrations was considered as the GMC in the isolates.

Statistical Analysis.
The results obtained were subjected to analyses of variance (ANOVA) using the Statgraphics Plus software (v.4.1).Tukey's test was used to determine the significance of differences between treatments, with  < 0.05 taken to indicate a significant difference.

Chemical Composition of Lupine Seed Flour and Protein
Concentration of Isolates.Table 1 shows the results of the chemical analysis of the different flours of lupine seeds (Haags Blaue, Sonate, Probor, Borlu, Boregine, and Boruta).
The results showed that the main components of the seeds are carbohydrates and proteins (28.40-36.61%), of which the high content is similar to that of soybean seeds [23].
Significant differences (Table 1) were found between Probor and the other varieties, which showed the highest protein content (36.61%).In relation to the ash content, no significant differences ( > 0.05) were found.These values were lower than those reported for L. luteus and similar to that of L. angustifolius [24].The results of the crude fat determination showed lower values than those reported for soybean [25].
The varieties Haags Blaue (4.40), Boregine (3.88%), and Boruta (5.31%) showed significant difference ( < 0.05) among themselves.The results show that the fiber content varied from 8.05 to 15.12% in the flours of lupine seeds, which are similar to those of other legumes such as peas, beans, and lentils (13.6-28.9%)[26][27][28].Regarding the carbohydrates content, the values found ranged from 42.24 to 51.68%, similar to those reported to peas, beans, and lentils [26].The moisture values of the varieties of the present work were inferior to other legumes such as common bean (10%) [28] and higher to the Pardina lentil (4%) [29].
The final concentrations of proteins of Haags Blaue, Boregine, Borlu, Probor, Sonate, and Boruta obtained were 89.33%, 89.23%, 89.42%, 90.61%, 89.50%, and 88.40% respectively.It has been observed that both the purity and the amount of protein recovered are affected by the characteristics of the seeds, extraction time and temperature, and the relation between the flour with the solvent and the pH at which protein precipitation takes place [30].

Solubility of Protein.
The solubility of protein isolates provides important information about their possible technological applications [31].According to Horax et al. [32], the solubility of the protein at different pH values can serve as a useful indicator of the efficiency or performance of protein isolates in food and also of the degree of protein denaturation due to heat or chemical treatment.Figure 1 shows the variations of the solubility percentage of the protein in a range from pH 2.0 to 10.0, in which a similar behavior can be observed in all isolates.The results at pH 4 and 5 showed a sharp decrease in the solubility for all six isolates of Lupinus as reported Chau et al. [33].At pH 2 about 98-99% of the protein isolates were soluble, and about 98-99% were soluble at pH of 9 and 10, respectively, with no significant differences between them; meanwhile the minimum solubility percentages (1-2%) were recorded at pH of 4.0 and 5.0 as the isoelectric point of proteins.At this pH, the electrostatic intermolecular repulsion and its ionic hydration at this point are minimal, which causes precipitation of the proteins.The solubility pattern recorded in this study is similar to that reported in isolates obtained from L. albus and L. spp.where the minimum solubility value of the protein was less than 1% at pH 4.0 [34].However, El-Adawy et al. [35] reported a different behavior in Lupinus termis and Lupinus albus, since the minimum solubility value of the proteins (14 to 18%) was found in a range of pH of 4.0-5.0.On the other hand, Lqari et al. [36]  L. angustifolius isolate; the solubility of the protein was minimal (22.5%) at pH 4.3.In other legumes such as pea (Pisum sativum), bean (Vicia faba), and soybean (Glycine max), the lowest solubility percentage of the protein (1-10%) was found in pH values of 4.0-5.0[37].In a similar investigation of the present study but with different varieties of chickpea, Kaur and Singh [38] and Withana-Gamage et al. [39] found that protein solubility was minimal (2-6%) in a pH range of 4.0-4.5.The results showed good solubility in both the acidic and alkaline pH regions which was an important characteristic in food formulations.

Water-Holding Capacity (WHC) and Oil Holding Capacity (OHC).
Table 2 shows the WHC and OHC of protein isolates of Lupinus.The maximum values of water absorption were recorded in the isolates obtained from the Haags Blaue and Boruta varieties with 2.98 and 2.95 mL g −1 , respectively.Haags Blaue, Probor, Sonate, and Boruta did not show significant differences ( > 0.05).In general, the WHC values obtained in this study are considered high and are higher than those recorded in protein isolates of L. campestris, L. albus, L. splendens, L. termis, and L. angustifolius [34,36,40] and similar (Mean of 2.46 mL g −1 ) to those found in commercial soy protein isolates (Supro 670).This property is related to the ability of proteins to hydrate [18], and it is important in food systems due to their effects on the taste and texture of food [41].The water retention is a function of isolate obtention method, where alkaline conditions have an influence in the protein denaturation, its solubility, and the lower WHC [42].
The OHC did not show significant differences ( > 0.05) between the isolates (Table 2).The mean value of OHC in the six isolates was 2.63 mL g −1 , which is higher than that recorded in an isolate of L. campestris (1.7 mL g −1 ), but lower than that obtained in isolates of L. albus and L. splendens with 2.85 and 4.63 mL g −1 , respectively [34,40].Lqari et al. [36] in other leguminous isolates such as peas (Pisum sativum), bean (Vicia faba), and soybean (Glycine max) also reported OHC values lower (1.2, 1.6, and 1.1 mL g −1 protein) than those recorded in the isolates obtained and characterized in this study.
In general, as reported by Carvalho et al. [42], the OHC obtained in this study shows that Lupinus protein isolates possess well balanced proportions of externally oriented hydrophilic and hydrophobic groups; being able to reduce the interfacial tension in an emulsion system, they are very important in such as applications as in processing meat bases products.The values of WHC and OHC for these protein isolates that can be considered high in relation to others protein isolates used as ingredients in the preparation of cold meats, especially for sausages, are determining properties to develop a food of acceptable quality [18].

Emulsion Activity Index (EAI) and Emulsion Stability
Index (ESI).Significant differences ( < 0.05) were observed in both indexes according to the L. angustifolius varieties used (Table 2).The isolates obtained from the Boregine and Borlu varieties showed the highest EAI with 29.3 and 28.3 m 2 g −1 , respectively, while the lowest was recorded in the isolate obtained from the Sonate variety (26.4 m 2 g −1 ), while, in the ESI, the highest values were 14.9 and 14.7 min in the Probor and Haags Blaue isolates, respectively, and while the lower index was found in the protein isolate obtained from the Borlu variety (11.9 min) ( < 0.05).
The profiles of emulsifying activities (EAI and ESI) against pH are shown in Figures 2 and 3.The profiles of EAI and ESI are similar to solubility; these properties also increased at both pH extremes evaluated and were minimal in the isoelectric pH range (4.0 to 5.0).The profile of the emulsion properties as a function of pH for the different protein isolates studied was similar to that presented by the Indian and soybean protein isolates [18], as well as L. campestris isolates [40].The slight differences of the emulsion properties in the studied varieties can be due to the different level of solubilized proteins, which can influence the EAI and ESI by modifying the balance of Van der Waals attractive and repulsive electrostatic forces; a more soluble protein produces a rapid migration to the oil-water interface, thus favoring the formation and stability of emulsions [43].In general, the profiles of EAI (Figure 2) and ESI (Figure 3) presented variations similar to those reported in other isolates or concentrates, which are defined as a V shape pattern, indicating changes of the hydrophilic-lipophilic balance of the proteins in the pH gradient from 2 to 10.The result confirms the relationship between emulsifying and solubility properties of proteins.However, the results shown in this study and similar ones should be validated with other factors that may influence the emulsification properties, among them, droplet size, net charge, interfacial tension, viscosity, and protein conformation [42,44].Temperature is also a key factor affecting emulsifying properties of proteins.During the present study, the conditions used were 25 ∘ C, while other

Foaming Capacity (FC) and Foam Stability (FS).
The protein isolates showed similar FC values, between 116.3 and 116.8% (Table 2), without significant differences ( > 0.05).These results are similar to soybean (116%) [22], but higher than those reported in other beans [29,43,46,47].The high values of this property can be attributed to flexible protein molecules, which reduce surface tension and diffuse more quickly at the air-water interface, encapsulating air particles and thus favoring the formation of foam [48].With respect to FS, significant differences ( < 0.05) were recorded among the six isolates evaluated (Table 2), with the highest value in the Boruta variety (93.9%) and lowest value in Borlu variety (91.9%).The results for FS were similar to those reported for L. angustifolius protein isolates [36] and higher than those reported for soy protein isolates [39].When the foam stability values do not differ greatly from each other, it may be due to the ability to form a cohesive viscoelastic film via intermolecular interactions [49].According to the study of Adebowale et al. [49], the better stability of the foams might be attributed to formation of stable molecular layers in the acidic pH range.However, within food systems, foams are complex systems, including different phases such as a mixture of gases subdivided at solids, liquids, and multicomponent solutions of water, polymers, and surfactants which make necessary studies in a wide range of pH values [50].

Gelling Minimum Concentration (GMC).
The proteins are efficient gelling agents.A gel can be defined as an intermediate state between solid and liquid.Several factors influence gel formation, such as the protein size, molecular weight, amino acid composition, pH, and interaction with other components, since large molecules form extensive networks by cross-linking in three dimensions and also by the flexibility and ability of the proteins to undergo denaturation.The results obtained for GMC show that the protein isolates of the different lupine varieties need a minimum concentration of protein that ranged from 10% (Boregine) up to 12% (Probor) in order to form a stable gel (Table 2).The results are similar to those found in the literature for L. angustifolius protein isolates [36] in common varieties [29,47].The GMC of Boregine was slightly better than soybean protein isolate (10%).However, it is observed that lentil [29] and lima beans [47] show higher gelling capacity (7%), while other types of beans [43] have a less gelling capacity (15%).These variations between the different legumes seem to be related to the content of globulins which have high ease of forming gels [50,51].Hence, protein isolates of Lupinus are particularly useful in preparing comminuted meat emulsion as reported by Uvarova and Barrera-Arellano [44].

Conclusions
The chemical composition of L. angustifolius flour showed a significant difference among the different varieties evaluated, with protein contents of 28.40% in Boruta and 36.61% in Probor.Functional properties such as nitrogen solubility and the indexes of emulsion activity and emulsion stability were affected by the pH of the medium.In general, all these were minimal at values near the isoelectric point of the proteins (pH 4-6) and reached their maximum values at acidic (2) and alkaline pH (10).The maximum solubility of the protein isolates (99%) was observed at pH values of 2.0 and 10.0 without significant differences between varieties.Water-holding capacity showed significant differences between varieties, while oil holding capacity and foam formation were similar in all varieties.

Figure 1 :
Figure 1: Effect of pH on the solubility of six varieties of Lupinus angustifolius protein isolates.Different letters indicate significant differences among varieties at  < 0.05; ns indicates nonsignificance ( > 0.05).

1 )Figure 2 :
Figure 2: Effect of pH on the emulsion activity index (EAI) of six varieties of Lupinus angustifolius protein isolates.Different letters indicate significant differences among varieties at  < 0.05; ns indicates nonsignificance ( > 0.05).

Figure 3 :
Figure 3: Effect of pH on the emulsion stability index (ESI) of six varieties of Lupinus angustifolius protein isolates.Different letters indicate significant differences among varieties at  < 0.05.

Table 1 :
Chemical composition of seed flour of varieties of Lupinus angustifolius cultivated in Mexico (% dry basis).

Table 2 :
Functional properties of protein isolates obtained from seeds of varieties of Lupinus angustifolius cultivated in Mexico.Different letters in the same row indicate significant differences ( < 0.05).WHC: water holding capacity; OHC: oil holding capacity; EAI: emulsion activity index; ESI: emulsion stability index; FC: foaming capacity; FS: foam stability index; GMC: gelling minimum concentration.