Comprehensive Identification of Short and Medium-Sized Peptides from Pixian Broad Bean Paste Protein Hydrolysates Using UPLC-Q-TOF-MS and UHPLC-Q Exactive HF-X.

Pixian broad bean paste (PBBP) is an indispensable food widely used in many East Asian countries, yet the knowledge about bioactive peptides released from parent proteins by enzymatic hydrolysis is limited. A total of 5867 low-molecular weight peptides were identified in the highly bioactive subfractions of the PBBP alcalase hydrolysates using traditional and peptidomics approaches. 19 short peptides (3-5 amino acids) were identified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry, including 5 tripeptides, 8 tetrapeptides, and 6 pentapeptides. 5848 medium-sized peptides (6-10 amino acids) were characterized using the peptidomics approach, including 1484 hexapeptides, 1217 heptapeptides, 1634 octapeptides, 927 nonapeptides, and 586 decapeptides. The comprehensive method can be used for the investigation of bioactive peptides in complex food matrices.

Fermentation characteristics of Pixian broad bean paste in closed system of gradient steady-state temperature field.

A closed system of gradient steady-state temperature field (GSTF) was constructed to ferment Pixian broad bean paste (PBP). The contents of physicochemical factors and organic acids in the fermentation under GSTF (FG) were closer to those in the traditional fermentation (TF). The taste intensities of 8 free amino acids in the FG were higher than those in the constant temperature fermentation (CTF), but 14 in the TF showed the highest among the processes of FG, CTF and TF. The FG product had the most volatiles with 87, and its flavor properties were more stable. The FG produced great effects on the microbe evolutions especially improved the fungal diversity. Bacillus were identified as the core microbes in the FG while the roles of Staphylococcus, Lactobacillus and Pantoea were strengthened. The results indicated that the fermentation characteristics in the FG had been further improved compared with the CTF.

Correlation between microbial communities and key flavors during post-fermentation of Pixian broad bean paste.

Pixian broad bean paste (PBP) is a traditional Chinese condiment, famous for its distinctive flavor. Microbial communities play a vital role in producing the unique flavor of PBP, and a significant accumulation of these volatile flavors occurs during the post-fermentation stage of its production. However, little is known about the relationship between the microbes and flavor compounds in PBP. In this study, high-throughput sequencing (HTS) analysis revealed that Leuconostoc (8.30%), Lactobacillus (7.05%), Weissella (5.80%) and Staphylococcus (4.03%) were the dominant bacterial genera, while the most prevalent yeast genera were Zygosaccharomyces (41.45%) and Pichia (5.83%). Gradual accumulations of free amino acids (glutamic acid and asparagine), organic acids (malic acid and tartaric acid), and unique volatiles (aldehydes, phenols and pyrazines) were evident throughout the post-fermentation process. Analysis of the Pearson's correlation coefficients between 66 key microbes and the key flavors was investigated. Nine core microbes were identified based on the linear discriminant analysis (LDA) scores ≥ 4 (or an average abundance >0.1%) and a high correlation with at least two flavor categories (P < 0.05, |ρ| > 0.8), namely Kosakonia, Kazachstania, Debaryomyces, Lactobacillus, Myroides, Stenotrophomonas, Ochrobactrum, Wohlfahrtiimonas, and Lactococcus genera. These results provide a clearer insight into microbial succession during PBP post-fermentation, thereby contributing to further quality improvement of PBP.

Improved protease activity of Pixian broad bean paste with cocultivation of Aspergillus oryzae QM-6 and Aspergillus niger QH-3

BACKGROUND: The preparation of broad bean koji is a key process in the production of Pixian broad bean paste (PBP). Protease is essential for the degradation of proteins during PBP fermentation. To obtain broad bean koji with high protease activity using the cocultivated strains of Aspergillus oryzae QM-6 (A. oryzae QM-6) and Aspergillus niger QH-3 (A. niger QH-3), the optimization of acid and neutral protease activities was carried out using Box­Behnken design with response surface methodology (RSM). RESULTS: The optimum conditions were found to be as follows: inoculation proportion (X1), 3:1 (A. oryzae QM-6: A. niger QH-3, w/w); culture temperature (X2), 33°C; inoculum size (X3), 0.5% (w/w); incubation time (X4), 5 d. The acid and neutral protease activities were 605.2 ± 12.4 U/g and 1582.9 ± 23.7 U/g, respectively, which were in good agreement with the predicted values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the broad bean koji extracellular proteins in the case of cocultivation were richer compared to those in the case of A. oryzae QM-6 or A. niger QH-3 strain only. In addition, the free amino acids (FAAs) in the fermentation product were 55% higher in the cocultivation process than in that involving only A. oryzae QM-6, further confirming the diversity of proteases in the fermentation products. CONCLUSIONS: The optimal conditions of koji-making in PBP were obtained using RSM. The cocultivation of A. oryzae and A. niger increases the overall enzyme activities in the culture medium and the FAAs content, which would thus have potential application in the PBP industry.

Flavor Compounds in Pixian Broad-Bean Paste: Non-Volatile Organic Acids and Amino Acids.

Non-volatile organic acids and amino acids are important flavor compounds in Pixian broad-bean paste, which is a traditional Chinese seasoning product. In this study, non-volatile organic acids, formed in the broad-bean paste due to the metabolism of large molecular compounds, are qualitatively and quantitatively determined by high-performance liquid chromatography (HPLC). Amino acids, mainly produced by hydrolysis of soybean proteins, were determined by the amino acid automatic analyzer. Results indicated that seven common organic acids and eighteen common amino acids were found in six Pixian broad-bean paste samples. The content of citric acid was found to be the highest in each sample, between 4.1 mg/g to 6.3 mg/g, and malic acid were between 2.1 mg/g to 3.6 mg/g ranked as the second. Moreover, fumaric acid was first detected in fermented bean pastes albeit with a low content. For amino acids, savory with lower sour taste including glutamine (Gln), glutamic acid (Glu), aspartic acid (Asp) and asparagines (Asn) were the most abundant, noted to be 6.5 mg/g, 4.0 mg/g, 6.4 mg/g, 4.9 mg/g, 6.2 mg/g and 10.2 mg/g, and bitter taste amino acids followed. More importantly, as important flavor materials in Pixian broad-bean paste, these two groups of substances are expected to be used to evaluate and represent the flavor quality of Pixian broad-bean paste. Moreover, the results revealed that citric acid, glutamic acid, methionine and proline were the most important flavor compounds. These findings are agreat contribution for evaluating the quality and further assessment of Pixian broad-bean paste.