Dynamics and correlation of microbial community and flavor in Pixian Douban fermented with closed process of constant temperature.

BACKGROUND: As a Chinese traditional flavor condiment, Pixian Douban (PXDB) is produced using a traditional open fermentation process. In this study, an experimental fermentation of PXDB was conducted at 40 °C for 90 days in a closed system, which has not been applied to PXDB production. The flavor, microbial community and correlations of the samples in the closed system were compared with those in the traditional fermentation. RESULTS: The content of organic acids and free amino acids in the closed fermentation of constant temperature (CFCT) achieved the standards of product quality, although they were lower than those in the traditional fermentation. Of the 140 detected aroma components, 98 were shared in the two fermentation processes. Enterobacter, Bacteroides and Megamonas were the core microbial genera related to 26 flavor components in the traditional fermentation, while Pantoea was the core microbial genus related to 18 flavor components in CFCT. The CFCT has its own unique advantages over traditional fermentation in forming aromas. It produced a greater impact on the succession of fungi than those of bacteria after changing traditional fermentation to CFCT. The influence of microorganisms on the formation of flavor components was relatively more balanced in CFCT, while the changed fermentation process impacted greatly on the functions of Zygosaccharomomyces and Pichia but little on those of Sphingomonas, Megamonas and Parabacteroides. CONCLUSION: The study indicated that it was feasible to ferment PXDB in the closed system, and provides a basis to realize controllable PXDB production. © 2020 Society of Chemical Industry.

An auto-inducible expression and high cell density fermentation of Beefy Meaty Peptide with Bacillus subtilis.

Currently, some cases about the expression of flavor peptides with microorganisms were reported owing to the obvious advantages of biological expression over traditional methods. However, beefy meaty peptide (BMP), the focus of umami peptides, has neither been concerned in its safe expression nor its overproduction in fermenter. In this study, multi-copy BMP (8BMP) was successfully auto-inducibly expressed and efficiently produced in Bacillus subtilis 168. First, 8BMP was successfully auto-inducibly expressed with srfA promoter in B. subtilis 168. Further, the efficient production of 8BMP was researched in a 5-L fermenter: the fermentation optimized by Pontryagin's maximum principle obtained the highest 8BMP yield (3.16 g/L), which was 1.2 times and 1.8 times than that of two-stage feeding cultivation (2.67 g/L) and constant-rate feeding cultivation (1.75 g/L), respectively. Overall, the auto-inducible expression of 8BMP in B. subtilis and fermentation with Pontryagin's maximum principle are conductive for overproduction of BMP and other peptides.

Successful expression of the Bordetella petrii nitrile hydratase activator P14K and the unnecessary role of Ser115.

BACKGROUND: The activator P14K is necessary for the activation of nitrile hydratase (NHase). However, it is hard to be expressed heterogeneously. Although an N-terminal strep tagged P14K could be successfully expressed from Pseudomonas putida, various strategies for the over-expression of P14K are needed to facilitate further application of NHase. RESULTS: P14K was successfully expressed through fusing a his tag (his-P14K), and was over-expressed through fusing a gst tag (gst-P14K) at its N-terminus in the NHase of Bordetella petrii DSM 12804. The stability of gst-P14K was demonstrated to be higher than that of the his-P14K. In addition, the Ser115 in the characteristic motif CXLC-Ser115-C of the active center of NHase was found to be unnecessary for NHase maturation. CONCLUSIONS: Our results are not only useful for the NHase activator expression and the understanding of the role of Ser115 during NHase activation, but also helpful for other proteins with difficulty in heterologous expression.

Oxidation of Chlorpromazine by Lipoxygenase Immobilized on Magnetic Fe3O4 Nanoparticles.

In this work, soybean lipoxygenase enzyme was immobilized on the magnetic Fe3O4 nanoparticles to oxidate chlorpromazine (CPZ). The physicochemical properties of the nanoparticles were characterized with transmission electronic microscopy (TEM), fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermal gravimetric analysis (TGA), and the magnetic properties were detected by a vibrating sample magnetometer (VSM). The TEM images indicated the surface and the size of the immobilized enzyme were much rougher and thicker than those of the naked particles. The analyses of XRD patterns showed that the structure of the magnetic nanoparticles had no significant change while the nanoparticles also exhibited higher superparamagnetism at room temperature. Compared to free enzyme or the enzyme immobilized with other methods, the optimal pH, temperature and H2O2 concentration for the activity of immobilized enzyme did not have great changes, but the immobilized enzyme was more stable and less sensitive to the change of the influence factors than free counterpart. The immobilized enzyme could be recovered easily by magnetic separation and could be reused for many times in the process of CPZ oxidation. The results obtained by simulating the model of Michaelis-Menten indicated that the reaction of CPZ oxidation followed Michaelis-Menten kinetics and the enzyme still retained its affinity for CPZ while the enzyme was immobilized.

Estimates of minor ocean tide loading displacement and its impact on continuous GPS coordinate time series.

The site displacement due to ocean tidal loading is regarded as one of the largest uncertainties in precise geodetic positioning measurements, among which the effect of minor ocean tides (MOT), except for the 11 main tidal constituents, are sometimes neglected in routine precise global positioning system (GPS) data processing. We find that MOT can cause large vertical loading displacements with peak-to-peak variations reaching more than 8 mm at coastal/island stations. The impact of MOT on the 24-hour GPS solution is slightly larger than the magnitude of MOT loading itself, with peak-to-peak displacement variation at about 10 mm for the horizontal and 30 mm for the vertical components. We also find that the vertical velocity of all the selected stations in the Southwest Pacific was reduced by more than 10% after considering the MOT effect, while stations with weighted root mean square reduced data account for 62%, 59%, and 36% for the up, east, and north components respectively, in particular for most coastal/island stations. Furthermore, MOT correction could significantly reduce the annual signal of the global stacked east component, the near fortnightly and the long-term periodic signals in the up component. The power of some anomalous harmonics of 1.04 cycle per year is also decreased to some extent. These results further proved the benefits of MOT correction in precise GPS data processing.

Revealing the formation mechanisms of key flavors in fermented broad bean paste.

Pixian Douban (PXDB) is a popular Chinese condiment for its distinctive flavor. Broad bean fermentation (Meju) is the most important process in the formation of flavor substances. Key flavors were analyzed qualitatively and quantitatively, and metagenomic technology was applied to study the microbial diversity during broad bean fermentation. In addition, the main metabolic pathways of key flavors were explored. Results indicated that Staphylococcus_gallinarum was the main microorganism in the microbial community, accounting for 39.13%, followed by Lactobacillus_agilis, accounting for 13.76%. Aspergillus_flavus was the fungus with the highest species abundance, accounting for 3.02%. The KEGG Pathway enrichment analysis showed that carbohydrate metabolism and amino acid metabolism were the main metabolic pathways. Glycoside hydrolase and glycosyltransferase genes were the most abundant, accounting for more than 70% of the total number of active enzyme genes. A total of 113 enzymes related to key flavors and 39 microorganisms corresponding to enzymes were annotated. And Staphylococcus_gallinarum, Lactobacillus_agilis, Weissella_confusa, Pediococcus_acidilactici, Staphylococcus_kloosii, Aspergillus_oryzae, and Aspergillus_flavus played a key role in the metabolic pathway. This study reveals the formation mechanism of key flavors in fermented broad bean, it is important for guiding the industrial production of PXDB and improving product quality.

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.

Improving superamphiphobicity by mimicking tree-branch topography.

when a droplet impacts on a superhydrophobic structured surface below a certain impact velocity, the droplet can bounce off completely from the surface. However, above such velocity a fraction of the droplet will pin on the surface. Surfaces capable of repelling water droplets are ubiquitous in nature or have been artificially fabricated. However, as the surface tension of the liquid is reduced, the capability of the surface to remain non-wetting gets hindered. Despite progress in previous research, the understanding and development of superamphiphobic surface to impacting low surface tension droplets remains elusive. It is proposed that multi-layer re-entrant like roughness can further enhance the anti-wetting properties also for low surface tension fluids. In this work, we produce patterned conical micro-structures with lateral nano-sized roughness. Furthermore, the droplet impact experiments are conducted on various surfaces with variable surface tensions (27 mN/m - 72 mN/m) by using droplets with different Weber numbers (2-170). We show that conical microstructures with lateral roughness mimicking tree-branches provides a surface topology capable of absorbing the force exerted by the droplet during the impact which prevents the droplet from pinning on the surface at higher impact velocity even for low surface tension droplets. Our study has significance for understanding the liquid interaction mechanism with the surface during the impact process and for the associated surface design considerations.

Sensory and Volatile Compounds Characteristics of the Sauce in Bean Paste Fish Treated with Ultra-High-Pressure and Representative Thermal Sterilization.

This study investigated the differences between three sterilized samples to reveal the unique aroma characteristics of the sauce in bean paste fish by multiple analysis methodologies. Samples were subjected to pasteurized (PS), high-temperature sterilization (HTS), and ultra-high-pressure treatment (UHP) tests. The UHP had a higher sensory evaluation and could better maintain the original flavor of the sample. A total of 92, 83, 85, and 76 volatile compounds were detected via comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) techniques in the control (CT), PS, HTS, and UHP groups, respectively. According to the analysis of gas chromatograph-olfactometry and odor activity value, 7 compounds were considered to have an aromatic influence on the sauces, in which four compounds (1,8-Cineole, Linalool, Hexanal, and Dimethyl trisulfide) exhibited a positive contribution to the aroma of the sauces. PLS-DA results showed that the UHP group positively correlated with volatiles (Isoamylol and 1-Octen-3-ol), color, and gloss. In general, the UHP treatment could retain the original state and flavor of the sauce, showing a high similarity to the control group. The HTS significantly altered the flavor and status of the samples.

Insight into the protein degradation during the broad bean fermentation process.

Broad bean fermentation is of vital importance in PixianDouban (PXDB) production, as well as a key process for microorganisms to degrade protein, which lays the foundation for the formation of PXDB flavor. In this study, two fungi and bacteria were screened, and their morphology, molecular biology, growth, and enzyme production characteristics were analyzed, and then they were applied to the broad bean fermentation simulation system. The protein, peptide, amino acid, amino nitrogen, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system were evaluated. The results showed that the four microorganisms were Aspergillus oryzae, Aspergillus jensenii, Staphylococcus gallinarum, and Enterobacter hormaeche. Aspergillus oryzae had the highest protease activity at pH 7.0, while the other three strains had better enzyme activity stability under neutral acidic conditions. And the total protein (F1 and F2 were 18.32 g/100 g, 19.15 g/100 g, respectively), peptides (11.79 ± 0.04 mg/g and 12.06 ± 0.04 mg/g), and amino acids (55.12 ± 2.78 mg/g and 54.11 ± 1.97 mg/g) of the fungus experimental groups (F) were higher than the bacterial experimental groups (B). In addition, the enzyme system produced by fungi exhibited a stronger ability for albumin (20 kDa) and glutenin (<30 kDa) deterioration in neutral conditions, while the bacterial enzyme system was more efficient in degrading albumin (<30 kDa) and glutenin (20-30 kDa) in acidic conditions, as indicated by SDS-PAGE. These findings showed that both bacteria and fungi played an important role in the degradation of protein in different fermentation stages of broad bean fermentation. Practical applications: There is a lack of comprehensive understanding of the protein composition and protein degradation mechanism of broad beans in the fermentation stage of PXDB. This research work explored the differences in the degradation of PXDB fermented protein by different microorganisms, and provided a theoretical basis for optimizing the production of PXDB and improving the quality of PXDB.

Isolation, identification and characterization of taste peptides from fermented broad bean paste.

Pixian broad-bean paste (PBBP) is a famous fermented condiment in China, which may produce abundant flavor peptides during the fermentation process. Herein, the flavor peptides from fermented broad-bean (FB) were separated and identified via ethanol precipitation, macroporous resin, and ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) methods. The results showed that there were fifteen novel flavor peptides in FB. Among them, six peptides (i.e. ALDELGT, AELTPEP, SAALQAG, SFEAVEAAPT, EQDNDGNNIFSGFKR, QTFNTEEDTAK, and DAPASGN) were perceived umami with threshold values ranging from 0.76 to 1.84 mmol L-1. And they had both salty-enhancing and umami-enhancing abilities. Meanwhile, novel peptides (i.e. GGLRIINPEGQQ, SIITPPERQ, GGLSIITPPERQA, GGLRIINPEG, SIITPPER, RIINPEGQQ, DALNVNRHIV, and LPKILLLQLV) were perceived bitter with threshold values ranging from 0.56 to 2.89 mmol L-1. And the monosodium glutamate (MSG) solution had a certain masking effect on bitter peptides. GFSSEFLA showed a strong sweet taste. From peptide sequence analysis, it was found that Aspergillus oryzae and broad-bean were important taste-active sources of the flavor peptides in FB. Besides, the synthetic peptides showed a much better sensory taste than the mixtures of the corresponding amino acids. It indicated that peptides might play an important role in deciding the taste of FB. Significantly, it is the first time that the isolated flavor peptides were identified from FB.

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.

Characterization of volatile compounds of Pixian Douban fermented in closed system of gradient steady-state temperature field.

As an essential flavor condiment in Sichuan cuisine, Pixian Douban (PXDB) is usually produced by open fermentation process in strip pools or ceramic vats. In this study, an experiment of PXDB fermentation was conducted for 90 days in a closed system of gradient steady-state temperature field (GSTF). To investigate the characterization of volatile compounds of PXDB in the closed system, the volatiles in three kinds of samples including samples of GSTF (SGT), samples of constant temperature (SCT), and samples of traditional fermentation (STF) were analyzed. The results showed that 75, 67, and 68 volatile compounds were detected in SGT, SCT, and STF, respectively. Compared with the traditional fermentation, the process in the closed system of GSTF was conducive to produce more kinds of esters and alcohols. A total of 22 major aroma active compounds were identified in three samples by combination analyses of gas chromatography-olfactometry (GC-O) and odor activity value (OAV). The appearance, smell, texture, and taste of the three different samples had shown different changes, but the sensory characteristics of the SGT were more similar to those of the STF by quantitative descriptive analysis (QDA) and principal component analysis (PCA). This study indicated that the closed system of GSTF could be applied in PXDB fermentation to obtain higher quality products, which brought a bright prospect of replacing the traditional fermentation process to realize the controllable industrialized production of PXDB.