3.1. Protein content, molecular weight distribution and surface hydrophobicity
In vegetable protein products protein concentration, molecular weight distribution and surface hydrophobicity determine their functional properties.
In this study, lupin protein isolate, with 87.9 ± 0,2% protein content, was produced from yellow lupin flour containing 39.3 ± 2.4% protein, by means of alkaline leaching and isoelectric precipitation. ElAdawy et al. [2] reported higher protein content in the isolate (about 91%); however, process parameters applied by the Authors were slightly different. In turn, Lampart-Szczapa and Mossor [23] received an isolate containing approx. 4% less of protein. The enzymatic hydrolysis had a limited effect on the content of protein substances. In the H-30, H-60 and H-120 hydrolysates they were found at the level of 89,4 ± 2,0, 90,1 ± 1,1, and 87,4 ± 0,3%, respectively. Also, Schlegel et al. [7] did not observe a significant change in protein content due to enzymatic proteolysis of lupin protein isolate.
The quantitative results of SDS-PAGE separation of proteins in LPI and hydrolysates are shown in Fig. 1 and Table 1.
Table 1. Relative content (%) of protein fractions in lupin protein preparations (results of SDS-PAGE).
Molecular weight
(kDa)
|
Protein content [%]
|
|
LPI
|
H-30
|
H-60
|
H-120
|
> 200
|
3.1
|
10.3
|
0.3
|
2.0
|
|
74–100
|
1.6
|
0.0
|
0.0
|
0.0
|
|
42–66
|
30.7
|
13.3
|
17.8
|
10.0
|
acidic subunits of α conglutin, HMW subunits of β conglutin
|
34–40
|
16.0
|
0.0
|
0.0
|
0.0
|
IMW subunits of β conglutin
|
26–34
|
8.8
|
0.3
|
1.1
|
0.0
|
IMW subunits of β conglutin, large subunits of γ-conglutin
|
20–22
|
16.3
|
0.5
|
6.8
|
4.7
|
basic subunits of α conglutin
|
18–20
|
6.4
|
1.3
|
0.0
|
0.0
|
LMW subunits of β conglutin
|
< 15
|
17.0
|
74.4
|
74.0
|
83.3
|
subunits of δ conglutin and products of hydrolysis
|
According to Capraro et al. [24] and Duranti et al. [25], 7S-β- and 11S-α-globulins are predominant in lupin flour proteins. This corresponds to the SDS-PAGE results, since the proportion of protein fractions of 42–66 kDa was the highest (30.7%) in the analysed non-hydrolyzed LPI. It is assumed that within this range there are subunits of one of the two main globulin fractions: legumin-like α conglutins (11S-α-globulins with above 330 kDa molecular weight) and vicilin-like βconglutins (7S-β-globulins with molecular weight of 143–260 kDa). Legumins are hexamers composed of heterogeneous acidic subunits (42–52 kDa) and basic subunits (20–22 kDa), while vicilins are trimers consisting of three subunits, which molecular weights range between 17 and 64 kDa (HMW: 53–64 kDa, IMW: 25–46 kDa and LMW: 17–20 kDa) [25]. Since these fractions contain polypeptides with similar molecular weights, it is not possible to distinguish these subunits clearly on the polyacrylamide gel used.
The next important protein fraction of the LPI (17%) were low-molecular-weight proteins below 15 kDa. These proteins are probably part of the δ conglutins (2S globulins) with molecular weight of about 13 kDa, composed of subunits: small (4 kDa) and large (9 kDa) [25]. In this work, the fraction of proteins in the molecular weight range of 34–40 kDa was represented by a fairly distinct band, representing 16% of all proteins in LPI. It is, most probably, a protein identified in the literature as an IMW subunit of β conglutins [24, 26]. A band with a MW in the range of 20–22 kDa had a similar relative share in LPI proteins representing basic subunits of legumins.
Analysis of the obtained electrophoretic data showed that some fractions were occurring only in individual lines and there were others, which repeated in all samples – namely in LPI and hydrolysates. The repetitive fractions visible on the gel scan (Fig. 1) were mainly proteins with molecular weights 42–66 kDa, partly 26–34 kDa, 20–22 kDa as well as low-molecular proteins (< 15 kDa).
In hydrolysates the main fractions (above 70%) were proteins with a molecular weight below 15 kDa. The relative content of these low-molecular-weight fractions in enzyme hydrolysates was 4–5 times higher than their level in the raw material (LPI). When comparing the LPI and products of its modification with Alcalase, a significant decrease in the content of the: high-molecular-weight (HMW) β conglutin (53–66 kDa) and acidic α conglutin (42–52 kDa) subunits as well as in other polypeptide chains of molecular weight above 15 kDa was observed. This is due to the breakdown of proteins into low molecular weight peptides and amino acids.
Analysis of protein band percentages showed differences between individual hydrolysates, which may result from differences in the examined functional properties. The product obtained as a result of two-hour proteolysis (H-120) had significantly higher content of low-molecular-weight protein fractions (below 15 kDa) than H-30 and H-60 hydrolysates.
In order to identify the products of proteolysis more accurately, the separation of peptides was performed according to the Schägger and von Jagow procedure [18], using a tricine cathode buffer. The results of the peptide separation are given in Fig. 2 and Table 2.
Table 2.Relative content (%) of protein fractions in lupin protein preparations (Schägger and von Jagow [18] procedure).
Molecular weight
(kDa)
|
Protein content [%]
|
LPI
|
H-30
|
H-60
|
H-120
|
> 100
|
2.5
|
|
|
|
74–100
|
2.8
|
|
|
|
43–65
|
26.9
|
31.2
|
27.1
|
28.8
|
34–40
|
8.0
|
14.7
|
6.0
|
0.0
|
24–33
|
20.3
|
9.2
|
27.7
|
25.5
|
20–23
|
4.9
|
8.9
|
11.8
|
11.1
|
18–20
|
7.7
|
0.6
|
0.0
|
0.0
|
12–16
|
16.8
|
9.0
|
5.2
|
3.1
|
< 10
|
10.1
|
26.4
|
22.2
|
31.5
|
The electrophoretic analysis by means of the Schägger and von Jagow procedure revealed that proteins with molecular weights in the range of 43–65 kDa (more than 26%) were predominant in LPI and this result confirmed the findings obtained by the standard SDS-PAGE electrophoresis. With regard to hydrolysates, however, the predominant protein fraction changed with a prolongation of the proteolysis length. It was found that in hydrolysates H-30, H-60 and H-120, the dominant compounds were those with molecular weight of 43–65 kDa, 24–33 kDa, and below 10 kDa, respectively.
The bands that represented the low molecular weight fractions in these preparations were wide and fuzzy, especially lines 6 and 7 in the gel area corresponding to the molecular weight below 6.2 kDa. The observed phenomenon is a result of polydispersity, i.e. an approximately continuous molecular weight distribution, with no dominant bands corresponding to a higher content of the molecular weight fraction.
The results obtained for the surface hydrophobicity of lupin protein preparations, presented as a plot and the simple equations fitted to them, are shown in Fig. 3. The highest value, expressed as a slope in a straight line equation representing changes in fluorescence intensity with increasing protein concentration, was observed for the LPI. In turn, the hydrophobicity of hydrolysates was distinctly lower and assumed similar values each other, showing no significant differences. This indicates that the initial hydrolysis of lupin proteins is already enough to reduce this parameter significantly; the prolongation of the process does not change it substantially.
Similar results of reducing surface hydrophobicity were obtained by Surówka et al. [27], who hydrolyzed soy protein concentrate and its extrudate using both Alcalase and Esperase.
3.2. Functional properties
Table 3 shows the results of the functional properties analysis of investigated preparations. The foam formed due to aeration of the LPI solution was quite difficult to obtain and was characterized by a coarse structure and very thin walls of the film. It was, however, relatively stable. The values of FC, which when higher reflect better foaming efficiency, increased noticeably as a result of hydrolysis. This index in all tested hydrolysates was about 10% higher than in the LPI. This means that the process of proteolysis significantly improves the foaming properties.
Table 3. Functional properties of LPI and its hydrolysates (H-30, H-60 i H-120).
|
LPI
|
H-30
|
H-60
|
H-120
|
FC (%)
|
62.5 ± 0.9a
|
72.7 ± 2.0b
|
70.6 ± 1.3b
|
74.1 ± 2.0b
|
FO (mL)
|
153.4 ± 6.8a
|
46.0 ± 5.0b
|
33.4 ± 3.9c
|
28.5 ± 2.4c
|
LD5 (%)
|
66.2 ± 7.2a
|
79.9 ± 2.9b
|
81.0 ± 4.7b
|
81.8 ± 1.3b
|
EAI (m2/g)
|
10.9 ± 0.6a
|
8.5 ± 0.3b
|
7.3 ± 0.3c
|
7.2 ± 0.6c
|
ESI (min)
|
59.1 ± 7.0a
|
13.3 ± 2.2b
|
9.4 ± 1.1b
|
11.6 ± 2.2b
|
The presented values are means ± standard deviations (n = 3). Values with different superscripts (a–c) in rows differ significantly at p ≤ 0.05.
|
In foams characterized by high FO values, gas bubbles are large and/or the foam walls are relatively thin. Thus, high FO values indicate a technologically unfavourable structure of the foam. As for hydrolysate preparations, the FO values decreased significantly compared to the FO values determined for the LPI. As a result, the foam obtained from the hydrolysate solutions had a desirable, more finely porous structure with strong walls. As in the case of FC, an improvement of this aeration property was noted due to enzymatic hydrolysis. However, according to the data in Table 3, the LD5 value increased, which means that foam stability decreased as compared to the LPI. Moreover, this decrease seems to be to some extent related to the length of the process. Therefore, in order to improve the foam stability of hydrolysate solutions, they should be used in preparations with stabilizing substances, e.g. polysaccharides increasing the viscosity.
According to Surówka et al. [27], who investigated soy protein hydrolysates, and Lqari et al. [5], who examined narrow-leafed lupin, partial enzymatic hydrolysis improves the foaming capacity compared to the raw material, from which the hydrolysate was prepared. However, the foams from these hydrolysates were not very stable. This agrees with our results obtained for the lupin hydrolysates, according to which peptides and δ conglutins, the LMW subunits and small subunits of β and γ conglutins had better adsorption capacity on the surface of air bubbles, providing good foaming properties to these products. Peptides present in hydrolysates can reach the interfacial surface much faster than proteins and create films that form the foam structure. The above authors concluded that degree of hydrolysis determines the foaming capacity of hydrolysates - with an increase in the degree of hydrolysis and a decrease in the average molecular weight, the ability to form foams increases. This is due to the fact that proteins with lower molecular weight have a greater ability to adsorb on the surface of air bubbles. Polar moieties located on the surface of the protein molecule more easily turn towards liquid and non-polar ones towards the air. As a result, a coherent, flexible film forms around the air bubbles.
Emulsion with the use of LPI is formed relatively efficiently as measured by EAI (Table 3). This index can be slightly improved by applying mild hydrolysis conditions, as was the case with limited enzymic hydrolysis of soy flour extrudates [8]. In this study, however, such improvement was not observed. The hydrolysate obtained after the half-hour process (H-30) produced emulsion with EAI lower by 22% compared to the isolate. Prolongation of the hydrolysis resulted in hydrolysates having even lower EAI values.
Alcalase hydrolysis had an even greater effect on ESI. While the emulsions obtained from LPI showed good stability, as a result of the hydrolysis, the ESI index decreased several times (Table 3).
It is believed that the surface hydrophobicity of proteins affects the emulsifying properties [28, 29]. When comparing the results of the analysis of emulsifying properties and surface hydrophobicity of LPI and its hydrolysates, it can be approximated that the results of hydrophobicity analysis and EAI and ESI are convergent, i.e. the hydrolysis leads to a deterioration of these indexes in line with the decrease of hydrophobicity.
In this study, as a result of α and β conglutins fragmentation, the number of peptides increased, but the number of both hydrophobic and hydrophilic moieties in their molecules was reduced. Even when the peptides are positioned on the surface of the dispersed lipids, interactions with the aqueous phase, which stabilize emulsions, will not be formed. In consequence, a loss is observed of emulsifying properties and decreased stabilization of the emulsion. Therefore, among the analysed preparations, LPI showed better emulsifying properties, than hydrolysates. Similar observations were reported in studies on rape protein hydrolyzed with Alcalase, where a decrease in emulsifying capacity as a result of hydrolysis was observed [30].