3.2. Comparison of Microbial Growth on Lupin Protein Isolate Solutions
The growing parameters (CFU and pH) for all experiments after 0 h and 24 h of fermentation are shown in
Table 4 and
Table 5. The results showed that all microorganisms were able to grow in hydrolyzed LPI. The minimum increase in CFU/mL (ΔE
CFU/mL) was recorded for Pepsin hydrolysate S9 (
Lactobacillus helveticus) with 1.02 × 10
8 CFU/mL and the maximum for Pepsin hydrolysate S8 (
Lactobacillus amylolyticus) with 1.32 × 10
9 CFU/mL.
The lowest pH values after 24 h of fermentation were recorded for Papain hydrolysate S3 (
Lactobacillus helveticus) and Pepsin hydrolysate S9 (
Lactobacillus helveticus) with pH values of 3.3 and 3.7, respectively. Fermentation of hydrolysates obtained by Alcalase 2.4 treatment (S4–S6) tended to show higher pH values compared to hydrolysates obtained by other proteolytic enzyme preparations. Presumably, Alcalase 2.4 degradation leads to a higher buffer capacity of the respective samples. The lowest pH reduction (5.1) was achieved by Alcalase 2.4 hydrolysate S4 (
Lactobacillus sakei ssp.
carnosus). A former study described a rapid decrease in pH for
Lactobacillus helveticus and
Lactobacillus amylolyticus during the fermentation of LPI [
15]. In this work, the pH curve over 24 h of fermentation was also recorded (
Figure 1 shows exemplarily the pH curve for LPI samples (not hydrolyzed) fermented with
Lactobacillus helveticus. It was observed that
Lactobacillus helveticus showed an extended lag phase of 7 h in decreasing the pH value than
Lactobacillus helveticus did on Papain (S3) and Pepsin (S9) hydrolysate, respectively. However, after 24 h of fermentation,
Lactobacillus helveticus was able to decrease the pH in the range of 3.1 and 4.6 in all hydrolysates.
3.3. Molecular Weight Distribution (SDS-PAGE) and Immunoreactivity
The molecular weight distribution (SDS-PAGE) of LPI and its fermented hydrolysates was used to determine the protein integrity and is shown in
Figure 2. All treatments resulted in prominent changes in the SDS-PAGE profile with hydrolyzed polypeptides to smaller fragments with molecular weights below 30 kDa. Enzymatic hydrolysis seems to have the greatest influence on the degradation of polypeptides of LPI. Comparing the profiles of the molecular weight distribution of a previous study [
7] after hydrolysis of LPI, it is shown that the profiles did not change visibly compared to those after the combination of hydrolysis and fermentation. This observation is also supported by the results of a further study [
15], which show that fermentation has only minor influence on the profiles of molecular weight distribution. Furthermore, it is described that β-conglutin with a molecular weight of ~55–61 kDa is known as the major allergen of
L. angustifolius L. (Lup an 1) [
22]. The SDS-PAGE results of all fermented LPI hydrolysates showed a degradation of the described IgE-reactive polypeptide (
Figure 2) independent of the enzyme preparations used. These observations were confirmed by the results of the Bead-Assay (
Figure 3). A significant reduction in the signal intensity of Lup an 1 below 0.5% was observed for all fermented hydrolysates compared to unfermented LPI (100%) due to the combination of enzymatic hydrolysis and fermentation The highest decrease of immunological reactivity was achieved by treatment with Alcalase 2.4 L combined with fermentation S5 (
Lactobacillus amylolyticus) and S6 (
Lactobacillus. helveticus). No binding of Lup an 1 antibodies in sandwich format could be detected (read out not detectable). The assumption that enzymatic hydrolysis is a powerful approach to reduce allergenic potential is supported by further studies [
23,
24,
25,
26,
27].
3.5. Sensory Analysis
Fermentation enables changes in sensory profiling through the generation and degradation of flavor active compounds. Proteins, carbohydrates and fats from the raw materials provide the necessary precursors, e.g., for the formation of volatile aroma-active compounds. Most of the formation pathways of aroma-active and organic compounds are based on functional metabolic pathways of lactic acid bacteria.
Comparative retronasal aroma profile analysis (
Figure 4) shows the results of Papain hydrolysate S2 (
Lactobacillus amylolyticus) and Alcalase 2.4 L hydrolysate S4 (
Lactobacillus sakei ssp.
carnosus) compared to LPI across the panel and highlights the most impressive changes with significant differences (
p < 0.05) in aroma perception of cocoa-like and malty. For LPI, the major aroma perceptions were rated with a moderate perception with values of 3.5 for oatmeal-like, 3.0 for fatty and 3.0 for pea-like. All other aroma impressions were rated with an intensity of 2.6 and below. The Papain hydrolysate S2 (
Lactobacillus amylolyticus) and the Alcalase 2.4 L hydrolysate S4 (
Lactobacillus sakei ssp
. carnosus) were the only samples that showed significant differences (
p < 0.05) in the intensity of aroma perception (cocoa-like and malty) compared to untreated LPI. The cocoa-like impression increased with values of 2.5 for S2 and 6.3 for S4 compared to 0.8 for unmodified LPI. Furthermore, S4 showed a significant increase (
p < 0.05) in the intensity of the aroma perception of malty (3.7). All other samples showed no significant differences (
p < 0.05) of aroma perceptions compared to those of unmodified LPI, and are therefore not presented in the figure (see
Supplementary Materials).
The taste impressions bitter, salty and sour of LPI and fermented LPI hydrolysates were investigated by 10 panelists in the sensory evaluation. The bitter, salty and sour intensities of LPI were described with values of 3.0, 2.1 and 1.0, respectively. All treated samples did not show significant difference (p < 0.05) compared to untreated LPI, with the exception of the sample treated with Alcalase 2.4 L hydrolysate S5 (Lactobacillus amylolyticus), with a more bitter intensity of 5.6. Additionally, Pepsin hydrolysate S9 (Lactobacillus helveticus) showed significantly (p < 0.05) higher intensity of saltiness compared to untreated LPI. No significant differences could be observed between LPI and the treated samples regarding the intensity of sour.
Principal Component Analysis (PCA) was applied to identify the most relevant sensory attributes that affect sample characteristics.
Figure 5 shows the resulting biplots of the uncorrelated principal components (PCs) 1 and 2 based on the sensory data of the respective hydrolysates including untreated LPI, fermented LPI hydrolysates and the scaled loadings.
The first two components of PCA explained 39.92% and 26.12% of the observed variation (68.22% in total). The attribute with the strongest influence on PC1 was salty (−0.298) taste and cocoa-like (0.754) aroma impression. In contrast, PC2 was primarily described by the attributes bitter (−0.412) and salty (0.514). Unfermented LPI (−0.896/−1.108) was in close correlation with the pea-like aroma attribute and was found on the negative side of PC2, together with the Papain hydrolysates samples S1 (
Lactobacillus sakei ssp
. carnosus) and S3 (
Lactobacillus helveticus) and the Alcalase 2.4 L hydrolysates samples S5 (
Lactobacillus amylolyticus) and S6 (
Lactobacillus helveticus). The samples S2, S4 and the Pepsin hydrolysates S7 (
Lactobacillus sakei ssp
. carnosus), S8 (
Lactobacillus amylolyticus) and S9 (
Lactobacillus helveticus) were found on the positive side and were opposite of the unfermented LPI. The Pepsin hydrolysate fermented with
L. helveticus (S9) scored the highest in the PC2 (2.623) and was nearest with salty. S9 raised a low pH value of 3.7 after fermentation, which was neutralized (pH 7.0) with 1M NaOH. Due to the larger amount of NaOH, compared to other samples, the salty impression may have been increased. This assumption is further supported by the increased ash content for S9. In contrast, the Papain hydrolysate S1 (
Lactobacillus sakei ssp
. carnosus) and the Alcalase 2.4 L hydrolysates S5 (
Lactobacillus amylolyticus) and S6 (
Lactobacillus helveticus) scored the lowest in PC2 and were nearest ranged to the bitter taste attribute. It was observed that the Alcalase 2.4 L hydrolysate S5 (
Lactobacillus amylolyticus) showed a significant difference (
p < 0.05) of bitter intensity to untreated LPI. The Alcalase 2.4 L hydrolysates S4 (
Lactobacillus sakei ssp
. carnosus) and S6 (
Lactobacillus helveticus) did not show a significant difference (
p < 0.05), but a tendency of higher bitter intensity. Alcalase 2.4 L hydrolysates are known for bitter taste. Several studies have already shown that the hydrolysis of plant proteins such as lupin [
7,
10], soy [
6] and pea [
27] results in a bitter taste. However, S4 (
Lactobacillus sakei ssp
. carnosus) was not ranged close to the bitter attribute, rather in the positive range of PC2 (1.746), and was nearest ranged with cocoa-like and was scored the highest in the PC1 (4.483).