Effect of enzyme‐assisted hydrolysis on protein pattern, technofunctional, and sensory properties of lupin protein isolates using enzyme combinations

Abstract The modification of lupin protein isolates (LPI) by means of enzymatic hydrolysis (Lupinus angustifolius cultivar Boregine) was performed with four enzyme preparations (Alcalase 2.4 L, Papain, Corolase 7089, and Neutrase 0.8 L) in a one‐ and two‐step process to determine the efficacy for the destruction of major IgE‐reactive polypeptides and the evaluation of the technofunctional and sensory properties of lupin protein hydrolysates. Combinations of Alcalase 2.4 L and Papain were most effective in the degradation of polypeptides in L. angustifolius as measured by sodium dodecylsulfate–polyacrylamide gel electrophoresis. The enzymatic hydrolysis of the LPI increased their technofunctional properties such as protein solubility, foam activity, and emulsifying capacity almost independently of the enzyme preparation used. The sensory results showed a significant increase in bitterness from 1.9 for LPI to 5.7 for the combination of Alcalase 2.4 L and Papain in one‐step process. The aroma attributes of the hydrolysates were very similar to untreated LPI. The results of this study show the possibility of enzymatic hydrolysis of LPI to destroy the major IgE‐reactive polypeptides that increase the technofunctional properties of the isolates and thus their use in human nutrition as food ingredients.

Several attempts have been addressed to reduce the allergenic potential of food proteins to appeased allergic reactions in sensitive individuals (Chizoba Ekezie, Cheng, & Sun, 2018). One possible method is the inactivation of allergens by heat treatment, but this also has a considerable impact on food quality. Nonthermal technologies including pulsed light, high-pressure processing, gamma irradiation, cold plasma technology, ultrasonication, and pulsed electric fields were also described (Chizoba Ekezie et al., 2018;Meinlschmidt, Ueberham, et al., 2016), but most of these methods do not achieve the complete inactivation of allergens or have not been adequately studied.
However, protein hydrolysis can also affect the sensory properties of the products by producing a bitter taste which inhibits their use as a food ingredient (Spellman, O'Cuinn, & FitzGerald, 2004). Most of the studies described in literature and mentioned above focus on soy proteins. There is no literature data available that describe attempts to reduce the allergenic potential of lupin protein. In a previous study, Schlegel et al. (2019) investigated the impact of single protease treatments on technofunctional and sensory properties as well as on the molecular weight distribution to estimate the reduction of the immunoreactivity in lupin protein isolate (LPI) and hydrolysates.
The objective of the current study was to determine the effectiveness of different protease combinations for the degradation of major IgE-reactive polypeptides in L. angustifolius cultivar Boregine and the evaluation of the technofunctional characteristics of lupin hydrolysates. The influence of hydrolysis on the sensory attributes of LPIs was also investigated.

| Raw materials and chemicals
Lupin (L. angustifolius L. cultivar Boregine) seeds were purchased from Saatzucht Steinach GmbH & Co KG. The sources and properties of the used enzymes are listed in Table 1.

| Preparation of LPI
Lupin protein isolate was prepared from L. angustifolius L. cultivar Boregine. Seeds were dehulled and the hulls were separated by airsifting. Dehulled kernels were passed through a roller mill and the resulting flakes were de-oiled in n-hexane. Flakes were suspended in 0.5 M HCl at a 1:8 ratio. was separated with a decanter centrifuge (5,600 g, 4°C, 1 hr) and supernatant was discarded. The acid pre-extracted flakes were dispersed in 0.5 M NaOH (pH 8.0) at a 1:8 w/w ratio and stirred for 1 hr at room temperature. The suspension was separated (5,600 g, 4°C, 1 hr) and aliquots of 0.5 M HCl were added to the supernatant at room temperature to facilitate the protein precipitation at a pH of 4.5. The precipitated proteins were separated by centrifugation at 5,600 g for 130 min and then neutralized (0.5 M NaOH), pasteurized (70°C, 10 min) and spray dried.

| Enzymatic hydrolysis of LPI
For the enzymatic hydrolysis of LPI, four enzyme preparations were used (Table 1)  were chosen according to . Hydrolysis experiments were carried out with the enzyme combinations shown in Table 2 in a 4 L thermostatically controlled reaction vessel. Therefore, the protein isolate was dispersed with an Ultraturrax (IKA-Werke GmbH & Co. KG.) for 1 min at 5,000 rpm in deionized water at a protein concentration of 5% (w/w) and adjusted to 50°C and pH 8.0 with 3 M NaOH prior to enzyme addition.
Hydrolysis was performed either as one-step or two-step process leading to 12 combinations of two or three enzymes (Table 2) according to  with some modifications.
The one-step process was carried out with eight different enzyme combinations. The enzyme preparations were added simultaneously to the vessel and hydrolyzed for 4 hr. Aliquots were taken after 2 and 4 hr. For the two-step process, four different enzyme combinations were selected. The first enzyme preparation was incubated for 1 hr. Subsequently, the second enzyme preparation was added for another 4 hr. Aliquots were taken after 2 and 5 hr.
During hydrolysis, the suspension was continuously stirred at controlled pH and temperature. To avoid further hydrolysis, the reaction was stopped by heating the protein suspension to 90°C for 20 min,

| Chemical composition
The protein content was determined according to the Dumas combustion method AOAC 968.06 using a protein calculation factor of N × 5.8 according to Mosse, Huet, and Baudet (1987). The dry matter was analyzed according to AOAC methods 925.10 in a TGA 601 thermogravimetric system (Leco Corporation) at 105°C.

| Degree of hydrolysis
The degree of hydrolysis (DH) was quantified using the o-phthaldialdehyde (OPA) method with serine as the standard as previously described by Nielsen, Petersen, and Dambmann (2001). Abbreviation: E/S, Enzyme to solution ratio. a LPI was hydrolyzed in two-step process in the first stage for 1 hr with one enzyme (1) and after 1 hr the second enzyme (2) was added and hydrolysis was continued.

Number of enzymes
TA B L E 2 Combinations of protease preparations for LPI hydrolysis in one-step and two-step process

| Molecular weight distribution
The molecular weight distribution of LPI and its hydrolysates were determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) modified according to Laemmli (1970). SDS-

PAGE was performed in a vertical electrophoresis cell (Bio-Rad
Laboratories). LPI and LPI hydrolysates were applied at a protein equivalent of 10 µl sample per lane on a precast 4%-20% stain-free polyacrylamide gel (Bio-Rad Laboratories). Precision Plus Protein Unstained Standard with molecular weight of 10-250 kDa (Bio-Rad Laboratories) run alongside as size markers, and the protein subunits were visualized using a Gel Doc™ EZ Imager system (Bio-Rad Laboratories). The molecular weight distribution was determined using Image Lab software (Bio-Rad Laboratories).

| Protein solubility
Protein solubility (%) of LPI and its hydrolysates was determined in duplicate over the pH range of 4.0-9.0 following the method of Morr et al. (1985).

| Foaming properties
Foaming activity was determined in duplicate as recommended by Phillips, Haque, and Kinsella (1987). A 5% (w/w) protein solution (100 ml) at pH 7 and room temperature was whipped for 8 min in a Hobart 50-N device (Hobart GmbH). The increase in volume after whipping was used to calculate the foam activity. The foam density (g/L) was measured by weighing a selected amount of foam volume and was defined as a ratio of foam volume to foam weight. The percentage leftover of foam volume after 1 hr was described as foaming stability (%).

| Emulsifying capacity
Emulsifying capacity of 1% (w/w) sample solution was determined at pH 7.0 in duplicate according to the method described by Wang and

Johnson (2001) using a Titrino 702 SM titration system (Metrohm
GmbH & Co. KG) at a constant rate of 10 ml/min until a phase inversion. The volume of oil needed to achieve the phase inversion was used to calculate the emulsifying capacity (ml oil per g sample).

| Sensory analysis of protein hydrolysates
Sensory analysis was determined as previously described by Schlegel et al. (2019). Briefly, all samples were presented to the panel in plastic cups. Panelists (n = 10) were first required to record the retronasal aroma and taste attributes. The retronasal aroma attributes were rated on a scale from 0 (no perception) to 10 (strong perception) by each panelist. The taste attributes and trimeric astringent perception were also rated on a scale from 0 (no perception) to 10 (strong perception) with a nasal clamp by each panelist.

| Statistical analysis
Results are expressed as mean ± standard deviation. Data were analyzed using one-way analysis of variances (ANOVA) and means were generated and adjusted with Tukey's honestly significant difference post hoc test to determine the significance of differences between samples, with a threshold of p < .05. Statistical analysis was performed with Matlab R2018a for Windows (MathWorks). The protein content of LPI and its proteolytic hydrolysates was about 92% and the dry matter was about 90%.

| Degree of hydrolysis
The degree of hydrolysis (DH) was monitored to get-together with SDS-PAGE analysis-a first indication of the size reduction of the proteins in order to estimate the reduction of the allergenic potential of the lupin proteins. The results are shown in Table 3 (Chen, Scott, & Trepman, 1979).
The treatments of the LPI with the one-step process were able to achieve higher DH values after 4 hr than the two-step process after

| SDS-PAGE
Besides the determination of the DH, the molecular weight distribution of LPI and its hydrolysates was analyzed to get an indication Corolase 7089. The differences could be potentially-as described above-due to interactions between the cysteine residues released during hydrolysis with Papain (cysteine endopeptidase) and the OPA reaction components, which react to an weakly fluorescent product (Chen et al., 1979). TS 1 0.9 ± 0.1 a 9.5 ± 0.5 b,c 12.3 ± 0.2 c TS 2 0.9 ± 0.1 a 9.2 ± 1.1 b 11.9 ± 0.1 b TS 3 0.9 ± 0.1 a 9.4 ± 0.1 b,c 12.0 ± 0.1 b,c TS 4 0.9 ± 0.1 a 3.1 ± 0.2 a 5.3 ± 0.1 a Note: The data are expressed as mean ± standard deviation (n = 4). Values followed by different letter in a column indicate significant differences between groups (p < .05).

TA B L E 3
Degree of hydrolysis (DH) (%) of hydrolyzed LPI by different protease treatments

| Protein solubility
The solubility of each lyophilized hydrolysate and LPI was determined as a function of pH in the range of 4.0 and 9.0 as shown in Several studies described a correlation rate between solubility and the DH. This may be due to the fact that a higher DH showed a decrease in high-molecular-weight fractions, which exposed new ionizable groups and increased solubility. This study observed that the protein solubility of the hydrolysates with higher DH increased with a coefficient of correlation (R 2 ) of .88 (Figure 3).

| Foaming properties
The foaming properties (foam activity, stability, and density) of the hydrolysates are described in Note: The data are expressed as mean ± standard deviation (n = 4). Values followed by different letters in a column indicate significant differences between groups (p < .05). showed a significant decrease in foaming density compared to untreated LPI (Table 5). As expected, the hydrolysates OS 6 and TS 4 (Papain + Corolase 7089) showed a very low density of 29 and 33 g/L. This may be due to extensive hydrolysis and the resulting decrease in molecular weight, as shown by the SDS-PAGE profiles.

| Emulsifying capacity
As shown in Table 5, the emulsifying capacity of LPI (620 ml/g) was higher than most of the hydrolysates, with the exception of OS 6 and TS 4 (Papain + Corolase 7089) with 625 and 608 ml/g, respectively.
Both were combinations of Papain with Corolase 7089. A direct correlation between the emulsifying capacity of proteins and their solubility was described in literature (El-Adawy et al., 2001;Qi et al., 1997). Qi et al. (1997) described that more dissolved protein in an emulsion system will result in more protein in the interface between the oil phase and the continuous phase during emulsification. This correlation could not be observed in this study. Highly soluble hydrolysates, such as OS 4 (Alcalase 2.4 L + Neutrase 0.8 L) (87.6%) at pH 7.0 showed a decreased emulsifying capacity (370 ml/g) in comparison to the LPI (solubility 70.7% and emulsifying capacity 620 ml).

| Sensory analysis of the protein hydrolysates
The bitter taste of LPI and its hydrolysates was evaluated on a 10 cm continuous scale and the results are shown in Figure 4. Untreated LPI was judged with a bitterness intensity of 1.9. The bitter taste of the hydrolysates, with the exception of OS 5 (Alcalase 2.4 L + Papain) with a bitterness score of 5.7, was not significantly higher than that of untreated LPI. One of the most significant factors for bitterness is the hydrophobicity of peptides (Maehashi & Huang, 2009).
Besides the hydrophobicity, the molecular size of the proteins might also play an important role for bitter perception. Matoba and Hata (1972)   Note: The data are expressed as the median values scored on an unstructured 10 cm line between not noticeable at the left and very strong at the right, based on an evaluation by 10 panelists (n = 10). Values followed by different letters in a column indicate significant differences between groups (p < .05).

| CON CLUS ION
enzymatic hydrolysis with enzyme combinations to destroy the

ACK N OWLED G M ENT
This work was supported by the Fraunhofer-Zukunftsstiftung.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L R E V I E W
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.