Effects of Salinity on Physicochemical Properties, Flavor Compounds, and Bacterial Communities in Broad Bean Paste-Meju Fermentation.

Broad bean paste (BBP) is a traditional fermented soy food, and its high salt content not only prolongs the fermentation time but also threatens human health. In this study, three BBP-meju with different salt concentrations were prepared, and the effects of varying salinity on fermentation were comprehensively compared. The results showed that salt-reduced fermentation contributed to the accumulation of amino acid nitrogen, reducing sugars, free amino acids, and organic acids. Alcohols, esters, aldehydes, and acids were the main volatile flavor compounds in BBP-meju, and the highest total volatile flavor compounds were found in medium-salt meju. Bacillus, Staphylococcus, Aspergillus, and Mortierella were the dominant microbial communities during fermentation, and there were also three opportunistic pathogens, Enterobacter, Pantoea, and Brevundimonas, respectively. According to Spearman correlation analysis, Wickerhamomyces, Bacillus, Staphylococcus, and Mortierella all showed highly significant positive correlations with ≥3 key flavor compounds, which may be the core functional flora. Furthermore, the dominant microbial genera worked synergistically to promote the formation of high-quality flavor compounds and inhibit the production of off-flavors during salt-reduced fermentation. This study provides a theoretical reference for the quality and safety control of low-salt fermented soy foods.

Polymethoxylated flavones from the leaves of Vitex negundo have fungal-promoting and antibacterial activities during the production of broad bean koji.

Broad bean paste is a popular condiment in Asian countries. Leaves of Vitex negundo Linn. were used extensively in China during the koji-making of broad bean paste. Spreading V. negundo leaves on raw broad beans during fermentation was able to facilitate the rapid growth of fungi to form mature koji. We isolated two strains of fungi from mature koji, and four strains of bacteria from the rotten broad beans resulting from a failed attempt. According to microbial activity assays, two polymethoxylated flavones, 5-hydroxy-3,6,7,8,3',4'-hexamethoxy flavone (HJ-1) and 5,4'-dihydroxy-3,6,7,8,3'-pentamethoxy flavone (HJ-2) were isolated from V. negundo leaves, and the fungal growth promotion and inhibition of bacterial growth of these two compounds were found to improve the production of broad bean koji. This study reveals the compounds present in V. negundo leaves with bioactivity against important microbes in koji manufacture, and provides a theoretical basis for the application of V. negundo in broad bean paste production.

Investigation into the potential mechanism of Bacillus amyloliquefaciens in the fermentation of broad bean paste by metabolomics and transcriptomics.

Pixian broad bean paste is a renowned fermented seasoning. The fermentation of broad bean is the most important process of Pixian broad bean paste. To enhance the flavor of tank-fermented broad bean paste, salt-tolerant Bacillus amyloliquefaciens strain was inoculated, resulting in an increase in total amount of volatile compounds, potentially leading to different flavor characteristics. To investigate the fermentation mechanism, monoculture simulated fermentation systems were designed. Metabolomics and transcriptomics were used to explore Bacillus amyloliquefaciens' transcriptional response to salt stress and potential aroma production mechanisms. The results highlighted different metabolite profiles under salt stress, and the crucial roles of energy metabolism, amino acid metabolism, reaction system, transportation system in Bacillus amyloliquefaciens' hypersaline stress response. This study provides a scientific basis for the industrial application of Bacillus amyloliquefaciens and new insights into addressing the challenges of poor flavor quality in tank fermentation products.

Identification and molecular mechanisms of novel antioxidant peptides from fermented broad bean paste: A combined in silico and in vitro study.

This study aimed to investigate the antioxidative and cytoprotective activity of antioxidant peptides from fermented broad bean paste (FBBP) and explore their potential molecular mechanisms using a combined in silico and in vitro approach. Seven novel antioxidant peptides (VSRRFIYYL, SPAIPLP, PVPPPGG, KKDGYWWAKFK, LAWY, LGFMQF, and LPGCP) identified by integrated approaches of peptidomics and in silico bioinformatic analysis were synthesized, exhibiting strong antioxidant potential against in vitro radicals. Molecular docking results suggested that these peptides could form stable hydrogen bonds and solvent-accessible surface with key amino acid residues of Keap1, thus potentially regulating the Keap1-Nrf2 pathway by occupying the Nrf2-binding site on Keap1. Additionally, they exhibited strong cellular antioxidant activity and could protect HepG2 cells from AAPH-induced oxidative injury by reducing reactive oxygen species and MDA accumulation. This study firstly unraveled the molecular mechanisms of antioxidant peptides from FBBP, and provided a new theoretical basis for the high-value utilization of FBBP.

Taste Mechanism of Umami Molecules from Fermented Broad Bean Paste Based on In Silico Analysis and Peptidomics.

In this study, in silico analysis and peptidomics were performed to examine the generation mechanism of the umami taste of fermented broad bean paste (FBBP). Based on the information from peptidomics, a total of 470 free peptides were identified from FBBP, most of which were increased after fermentation. Additionally, the increase of the content of umami peptides, organic acids, and amino acids during fermentation contributed to the perception of umami taste in FBBP. Molecule docking results inferred that these umami molecules were easy to connect with Ser, Glu, His, and Gln in the T1R3 subunit through hydrogen bonds and electrostatic interaction force. The binding sites His145, Gln389, and Glu301 particularly contributed to the formation of the ligand-receptor complexes. The aromatic interaction, hydrogen bond, hydrophilicity, and solvent-accessible surface (SAS) played key roles in the receptor-peptide interaction. Sensory evaluation and electronic tongue results showed that EDEDE, DLSESV, SNGDDE, DETL, CDLSD, and TDEE screened from FBBP had umami characteristics and umami-enhancing effects (umami threshold values ranging from 0.131 to 0.394 mmol/L). This work provides new insight into the rapid and efficient screening of novel umami peptides and a deeper understanding of the taste mechanisms of umami molecules from FBBP.

Revealing the Succession of Spatial Heterogeneity of the Microbial Community during Broad Bean Paste Fermentation.

This study aimed to elaborate the assembly processes and metabolic regulation of the microbial community under the conditions of environmental factors and artificial intervention using broad bean paste (BBP) fermentation as a tractable research object. Spatial heterogenicity of amino acid nitrogen, titratable acidity, and volatile metabolites were observed between upper and lower layers after fermentation for 2 weeks. Amino nitrogen contents in the upper fermented mash reached 0.86, 0.93, and 1.06 g/100 g at 2, 4, and 6 weeks, respectively, which were significantly higher than those of mash located at the lower layer (0.61, 0.79, and 0.78 g/100 g). Moreover, higher concentrations of titratable acidity were accumulated in upper layers (2.05, 2.25 and 2.56 g/100g) than those in lower layers, and the differentiation of volatile metabolites was the greatest (R = 0.543) at 36 days, after which the BBP flavor profiles converged with the fermentation progress. The successive heterogenicity of the microbial community in the mid-late stage was also found during fermentation, and Zygosaccharomyces, Staphylococcus, and Bacillus had heterogeneous characteristics driven by sunlight, water activity, and microbial interactions. This study provided new insights into the mechanisms underlying the succession and assembly of the microbial community of BBP fermentation, which also laid new clues for researches of the microbial communities in complex ecosystems. IMPORTANCE Gaining insights into the community assembly processes is essential and valuable for the elaboration of underlying ecological patterns. However, current studies about microbial community succession in multispecies fermented food usually treat the research object as a whole, are focused exclusively on temporal dimensions, and have ignored the changes of community structure in spatial dimensions. Therefore, dissecting the community assembly process from the view of spatiotemporal dimensions will be a more comprehensive and detailed perspective. Here, we found the heterogenicity of the BBP microbial community under the traditional production technology from spatial and temporal scales, systematically analyzed the relationship between the spatiotemporal succession of community and the difference of BBP quality, and elucidated the roles of environmental factors and microbial interactions to drive the heterogeneous succession of the microbial community. Our findings provide a new insight into understanding the association between microbial community assembly and the quality of BBP.

The differences between broad bean koji fermented in laboratory and factory conditions by an efficient Aspergillus oryzae.

Broad bean paste-meju was fermented by a mixture of broad bean koji and saline; koji fermentation is an essential process for the production of broad bean paste-meju. Aspergillus oryzae was the most widely used in sauce fermentation. The purpose of this study was to research the factory adaptability of the highly efficient A. oryzae PNM003 and further evaluate the effect of fermentation conditions and fermentation strains on koji. A. oryzae PNM003 was compared with the widely used strain HN 3.042 not only in the laboratory but also in factory conditions (large scale). Results showed that the koji made with the same starter in the factory had a greater amount of fungi than that in the laboratory. Bacteria and yeast levels in HN_L koji were higher than in PN_L koji. As for fungi constitution, almost only Aspergillus survived in the end through the microorganism self-purification process during koji fermentation. As for the bacterial constitution, koji was grouped by fermentation conditions instead of fermentation starter. PN koji had higher protease activity and a higher content of total acids, amino acid nitrogen, amino acids, and organic acids in the laboratory conditions. Nevertheless, in factory conditions, PN koji and HN koji had similar indexes. As for volatile flavor compounds, koji made with the two starters in the same condition was grouped together. As for the same starter, there were more flavor compounds metabolized in the factory condition than in the laboratory condition, especially esters and alcohols. The results showed PN was a highly efficient strain to ferment koji, but the advantages were expressed more remarkably in laboratory conditions. In brief, the fermented condition had a greater influence than the fermentation starter for broad bean koji.

HS-SPME-GC × GC/MS combined with multivariate statistics analysis to investigate the flavor formation mechanism of tank-fermented broad bean paste.

With the advancement of industrialization, tank fermentation technology is promising for Pixian broad bean paste. This study identified and analyzed the general physicochemical factors and volatile metabolites of fermented broad beans in a thermostatic fermenter. Headspace solid-phase microextraction (HS-SPME)-two-dimensional gas chromatography-mass spectrometry (GC × GC-MS) was applied to detect the volatile compounds in fermented broad beans, while metabolomics was used to explore their physicochemical characteristics and analyze the possible metabolic mechanism. A total of 184 different metabolites were detected, including 36 alcohols, 29 aldehydes, 26 esters, 21 ketones, 14 acids, 14 aromatic compounds, ten heterocycles, nine phenols, nine organonitrogen compounds, seven hydrocarbons, two ethers, and seven other types, which were annotated to various branch metabolic pathways of carbohydrate and amino acid metabolism. This study provides references for subsequent functional microorganism mining to improve the quality of the tank-fermented broad beans and upgrade the Pixian broad bean paste industry.

Revealing the Secret of Umami Taste of Peptides Derived from Fermented Broad Bean Paste.

To understand the umami taste of fermented broad bean paste (FBBP) and explore the umami mechanism, eight peptides (PKALSAFK, NKHGSGK, SADETPR, EIKKAALDANEK, DALAHK, LDDGR, and GHENQR) were separated and identified via ultrafiltration, RP-HPLC, and UPLC-QTOF-MS/MS methods. Sensory experiments suggested that eight novel peptides showed umami/umami-enhancing and salt-enhancing functions. Significantly, the threshold of EIKKAALDANEK in aqueous solution exceeded that of most umami peptides reported in the past 5 years. The omission test further confirmed that umami peptides contributed to the umami taste of FBBP. Molecular docking results inferred that all peptides easily bind with Ser, Glu, His, and Asp residues in T1R3 through hydrogen bonds and electrostatic interactions. The aromatic interaction, hydrogen bond, hydrophilicity, and solvent-accessible surface (SAS) were the main interaction forces. This work may contribute to revealing the secret of the umami taste of FBBP and lay the groundwork for the efficient screening of umami peptides.

Safety evaluation and application of lactic acid bacteria and yeast strains isolated from Sichuan broad bean paste.

Broad bean paste is one of the most popular characteristic traditional fermented bean products in China, which is prepared by mixed fermentation of a variety of microorganisms, among which lactic acid bacteria and yeast played an important role in the improvement of the fermented broad bean paste quality. However, the traditional open-air fermentation of broad bean paste brought some risks of harmful microorganisms. In this study, the safety and fermentation ability of lactic acid bacteria and yeast strains isolated from traditional broad bean paste was evaluated. The results showed that the protease activity of the strain Lactobacillus plantarum DPUL-J5 (366.73 ± 9.00 U/L) and Pichia kudriavzevii DPUY-J5 (237.18 ± 10.93 U/L) were the highest. Both strains produced little biogenic amines, and did not exhibit α-hemolytic activity or antibiotic resistance for some of the antibiotics most used in human medicine. Furthermore, the broad bean paste fermentation involving DPUL-J5 and DPUY-J5 was beneficial for accumulating higher total acid (1.69 ± 0.01 g/100 g), amino-acid nitrogen (0.85 ± 0.03 g/100 g), and more volatile flavor compounds, meanwhile, reducing the levels of biogenic amines and aflatoxin B1. Therefore, this study provided a new strategy to improve the safety and quality of traditional broad bean paste.

Patterns of biogenic amine during broad bean paste fermentation: microbial diversity and functionality via Bacillus bioaugmentation.

BACKGROUND: Broad bean paste is a high nitrogen and high salt traditional Chinese condiment, which triggers biosynthesis of nitrogen hazards like biogenic amines (BAs). Mechanisms of association and applied research of functional safety and community assembly within multiple-microbial fermentation are currently lacking. Here, bioaugmentation was performed based on the profiles of BAs accumulation and microbial succession to evaluate the functional variation within broad bean paste fermentation. RESULTS: Putrescine, spermine, and spermidine were the main BAs during traditional broad bean paste fermentation. Staphylococcus, Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, and Bacillus were the predominant bacteria, whereas Aspergillus and Zygosaccharomyces dominated in fungal species, and community structure shifted upon salt exposure. PICRUSt software uncovered that Bacillus contributed significantly (>1%) to the amine oxidase gene family. Bacillus amyloliquefaciens 1-G6 and Bacillus licheniformis 2-B3 were screened to perform the bioaugmentation of broad bean paste, which achieved a 29% and 16% BA decrease respectively. Interaction network analysis showed that Cronobacter and Lactobacillus were significantly negatively correlated with Bacillus (ρ = -0.829 and ρ = -0.714, respectively, P < 0.05) in the B. amyloliquefaciens 1-G6 group, and Staphylococcus and Buttiauxella were inhibited by Bacillus (ρ = -0.657 and ρ = -0.543, respectively, P < 0.05) in the B. licheniformis 2-B3 group. CONCLUSION: The synergism of amine oxidase activity and microbial interactions led to the decline of BAs. Thus, this study improves our understanding of the underlying mechanisms of microbial succession and functional variation to further facilitate the optimization of the fermented food industry.

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.

Roles of sunlight exposure on chemosensory characteristic of broad bean paste by untargeted profiling of volatile flavors and multivariate statistical analysis.

This study aimed to elaborate the role of sunlight on chemosensory characteristics of broad bean paste (BBP). Two groups of BBP fermentation with sunlight treated (ST) or darkness treated (DT) were prepared and the volatile compounds were revealed and compared via GC-MS and GC-IMS and multivariate statistical analysis. A total of 125 compounds were identified and 24 markers with potential contribution to the BBP flavor characteristics were screened. Sunlight exposure was proposed to favor the accumulation of volatile compounds providing fruity, floral, buttery and roasted aroma during fermentation while volatile flavor compounds with unfavorable aroma were enriched in the darkness-treated group. Through KEGG-enrichment analysis, phenylalanine metabolism was found to play critical roles in regulating volatile flavor compound formation and the predicted synthesis network for 24 marker volatile flavor compounds was constructed. This study may unravel the scientific meaning of the role of sunlight on BBP chemosensory quality.

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.

Development of a defined autochthonous starter through dissecting the seasonal microbiome of broad bean paste.

Bean-based fermentation foods are usually ripened in open environment, which would lead to inconsistencies in flavor and quality between batches. The physicochemical metabolism and microbial community of seasonal broad bean paste (BBP) were compared to distinguish discriminant metabolites and unique taxa, as well as their specific reasons for different flavor and quality in this study. Here, we found that environmental variables led to the seasonal distribution of microbiota, and differential microorganisms further contributed to the inconsistency of flavor quality, in which Lactobacillales was responsible for the higher titratable acid and amino acid nitrogen concentration in winter pei, while Saccharomycetales benefited the formation of volatile flavor substances in autumn pei. Additionally, we compared the effect of different combinations of Lactobacillales with Zygosaccharomyces rouxii on the quality of BBP, and found that W. confusa was more suitable for BBP fermentation rather than T. halophilus in terms of sensory characteristics and physicochemical metabolites.

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.

A Bottom-Up Approach To Develop a Synthetic Microbial Community Model: Application for Efficient Reduced-Salt Broad Bean Paste Fermentation.

Humans have used high salinity for the production of bean-based fermented foods over thousands of years. Although high salinity can inhibit the growth of harmful microbes and select functional microbiota in an open environment, it also affects fermentation efficiency of bean-based fermented foods and has a negative impact on people's health. Therefore, it is imperative to develop novel defined starter cultures for reduced-salt fermentation in a sterile environment. Here, we explored the microbial assembly and function in the fermentation of traditional Chinese broad bean paste with 12% salinity. The results revealed that the salinity and microbial interactions together drove the dynamic of community and pointed out that five dominant genera (Staphylococcus, Bacillus, Weissella, Aspergillus, and Zygosaccharomyces) may play different key roles in different fermentation stages. Then, core species were isolated from broad bean paste, and their salinity tolerance, interactions, and metabolic characteristics were evaluated. The results provided an opportunity to validate in situ predictions through in vitro dissection of microbial assembly and function. Last, we reconstructed the synthetic microbial community with five strains (Aspergillus oryzae, Bacillus subtilis, Staphylococcus gallinarum, Weissella confusa, and Zygosaccharomyces rouxii) under different salinities and realized efficient fermentation of broad bean paste for 6 weeks in a sterile environment with 6% salinity. In general, this work provided a bottom-up approach for the development of a simplified microbial community model with desired functions to improve the fermentation efficiency of bean-based fermented foods by deconstructing and reconstructing the microbial structure and function.IMPORTANCE Humans have mastered high-salinity fermentation techniques for bean-based fermented product preparation over thousands of years. High salinity was used to select the functional microbiota and conducted food fermentation production with unique flavor. Although a high-salinity environment is beneficial for suppressing harmful microbes in the open fermentation environment, the fermentation efficiency of functional microbes is partially inhibited. Therefore, application of defined starter cultures for reduced-salt fermentation in a sterile environment is an alternative approach to improve the fermentation efficiency of bean-based fermented foods and guide the transformation of traditional industry. However, the assembly and function of self-organized microbiota in an open fermentation environment are still unclear. This study provides a comprehensive understanding of microbial function and the mechanism of community succession in a high-salinity environment during the fermentation of broad bean paste so as to reconstruct the microbial community and realize efficient fermentation of broad bean paste in a sterile environment.

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.

Adaptive evolution of Aspergillus oryzae 3.042 strain and process optimization to reduce the formation of tyrosine crystals in broad bean paste.

Tyrosine crystals occasionally appeared on the broad bean paste surface, which will cause economic losses. This study aimed to eliminate the tyrosine crystals in broad bean paste through decreasing the activities of proteolytic enzymes produced by Aspergillus oryzae and process optimization. Broad bean pastes containing no more than 6.16 mg/g dry material tyrosine showed low possibility to form tyrosine crystals. Using tyrosine as substrate, the A. oryzae 3.042 was adaptively evolved and the tyrosine content in the broad bean paste fermented by the evolved A. oryzae was reduced from 6.49 mg/g dry material to 6.14 mg/g dry material (p < 0.05). When the production process was optimized, the tyrosine content in broad bean paste was further reduced to 5.67 mg/g dry material (p < 0.05). In this condition, no tyrosine crystals were formed in broad bean paste after the 12-month storage while the product quality was not influenced. PRACTICAL APPLICATIONS: Tyrosine crystals were one of the most important factors which negatively influence the quality of traditionally fermented food, including broad bean paste, soybean paste, and sausages. The appearance of tyrosine crystals in these foods will cause economic losses to manufacturers. This study tried to eliminate the appearance of tyrosine crystals through decreasing the activities of proteolytic enzymes produced by Aspergillus oryzae 3.042 and fermentation process optimization. The adoption of modified A. oryzae and optimized fermentation process successfully guaranteed the elimination of tyrosine crystals formation in the production and storage period of broad bean paste. This will not only benefit the broad bean paste manufacturers but also provide guidance for other fermented food producers to deal with tyrosine crystals problem.

Effect of extrusion pretreatment on the physical and chemical properties of broad bean and its relationship to koji preparation.

The physicochemical properties of broad bean paste koji made from extruded and traditional steam-cooked raw materials were investigated by scanning electron micrographs (SEM), confocal laser scanning micrographs (CLSM), circular dichroism (CD) and Molecular weight (Mw) analysis. The results showed that the protease activity of koji made from the extruded materials was higher than that of the traditional cooking method. A network porous structure in the extruded samples was observed by SEM. Furthermore, the CLSM results showed that the starch of the extrudate was embedded well in its protein network. CD spectroscopy implied that the α-helix decreased while the ß-turn and random coil increased after both treatments. Mw analysis determined that the starch molecules were degraded during extrusion, the proportion of micromolecules was more in the extruded samples than in the steam-cooked samples. However, the chain length distribution of starch exhibited no significant difference between the extruded and steam-cooked broad beans.