Separation, biochemical characterization and salt-tolerant mechanisms of alkaline protease from Aspergillus oryzae.

BACKGROUND: The salt tolerance of proteases secreted by Aspergillus oryzae 3.042 closely relates to the utilization of raw materials and the quality of soy sauce. However, little is known about the salt-tolerant proteases and their salt-tolerant mechanisms. RESULTS: In this study, we isolated and identified a salt-tolerant alkaline protease (AP, approximately 29 kDa) produced by A. oryzae 3.042. It was considered as a metal-ion-independent serine protease. The optimum and stable pH values were both pH 9.0 and the optimum temperature was 40 °C. Over 20% relative activity of AP remained in the presence of 3.0 mol L-1 NaCl after 7 days, but its Km and Vmax were only mildly influenced by the presence of 3.0 mol L-1 NaCl, indicating its outstanding salt tolerance. Furthermore, AP was more stable than non-salt-tolerant protease at high salinity. The salt-tolerant mechanisms of AP could be due to more salt bridges, higher proportion of ordered secondary structures and stronger hydrophobic amino acid residues in the interior. CONCLUSIONS: The above results are vital for maintaining, activating and/or modulating the activity of AP in high-salt environments. They would also provide theoretical guidance for the modification of AP and the engineering of A. oryzae 3.042 so as to secrete more AP. © 2018 Society of Chemical Industry.

A novel method for beef potentiator preparation and identification of its characteristic aroma compounds.

BACKGROUND: Beef potentiator (BP) is the most popular savoury flavour and regarded as the soul of the modern food industry. In this work, BP was prepared by a novel method with Aspergillus oryzae and Aspergillus niger (BPSF). Three other BPs prepared using commercial enzymes (Protamex, Flavourzyme and papain; BPCEs) were used as controls to investigate its aroma characteristics and related compounds. RESULTS: Sensory evaluation showed that BPSF possessed more favourable and distinctive sauce-like, meat-like, roast and alcoholic attributes when compared with BPCEs. Significantly higher contents (peak areas) and proportions of pyrazines, pyrroles, sulfurous compounds and alcohols in BPSF were responsible for its sensory characteristics, and most of these aroma compounds were derived from microbial metabolism during beef koji preparation and the Maillard reaction. CONCLUSION: BP prepared by synergistic fermentation with A. oryzae and A. niger is a potential alternative for BP preparation.

Natural pigments in the food industry: Enhancing stability, nutritional benefits, and gut microbiome health.

Natural pigments are increasingly favored in the food industry for their vibrant colors, fewer side effects and potential health benefits compared to synthetic pigments. However, their application in food industry is hindered by their instability under harsh environmental conditions. This review evaluates current strategies aimed at enhancing the stability and bioactivity of natural pigments. Advanced physicochemical methods have shown promise in enhancing the stability of natural pigments, enabling their incorporation into food products to enhance sensory attributes, texture, and bioactive properties. Moreover, recent studies demonstrated that most natural pigments offer health benefits. Importantly, they have been found to positively influence gut microbiota, in particular their regulation of the beneficial and harmful flora of the gut microbiome, the reduction of ecological dysbiosis through changes in the composition of the gut microbiome, and the alleviation of systemic inflammation caused by a high-fat diet in mice, suggesting a beneficial role in dietary interventions.

Quercetin inhibits caspase-1-dependent macrophage pyroptosis in experimental folic acid nephropathy.

BACKGROUND: The role of pyroptosis in kidney disease is limited and incomplete. Quercetin, a flavonoid compound present in a variety of fruits, vegetables, and plants, has shown antioxidant and anti-inflammatory properties. This study was designed to validate the importance of pyroptosis in an experimental model of folic acid nephropathy and to explore the effect of quercetin in protecting against pyroptosis. METHODS: Gene set enrichment analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) were used to establish the correlation between pyroptosis and folic acid nephropathy. Immune cell infiltration, network pharmacology and single-cell RNA sequencing analysis were utilized to ascertain the specific target of quercetin in relation to pyroptosis. Finally, quercetin's role was verified in vivo and in vitro. RESULTS: The GSEA analysis revealed a significant correlation between pyroptosis and folic acid nephropathy (NES = 1.764, P = 0.004). The hub genes identified through WGCNA were closely associated with inflammation. Molecular docking demonstrated a strong binding affinity between quercetin and caspase-1, a protein known to be involved in macrophage function, as confirmed by immune cell infiltration and single-cell analysis. Quercetin demonstrated a significant amelioration of kidney injury and reduction in macrophage infiltration in the animal model. Furthermore, quercetin exhibited a significant inhibition of caspase-1 expression, subsequently leading to the inhibition of pro-inflammatory cytokines expression, such as IL-1ß, IL-18, TNF-α, and IL-6. The inhibitory effect of quercetin on macrophage pyroptosis was also confirmed in RAW264.7 cells. CONCLUSION: This study contributes substantial evidence to support the significant role of pyroptosis in the development of folic acid nephropathy, and highlights the ability of quercetin to downregulate caspase-1 in macrophages as a protective mechanism against pyroptosis.

Ultrasound-Assisted Enzymatic Protein Hydrolysis in Food Processing: Mechanism and Parameters.

Ultrasound has been widely used as a green and efficient non-thermal processing technique to assist with enzymatic hydrolysis. Compared with traditional enzymatic hydrolysis, ultrasonic-pretreatment-assisted enzymatic hydrolysis can significantly improve the efficiency of enzymatic hydrolysis and enhance the biological activity of substrates. At present, this technology is mainly used for the extraction of bioactive substances and the degradation of biological macromolecules. This review is focused on the mechanism of enzymatic hydrolysis assisted by ultrasonic pretreatment, including the effects of ultrasonic pretreatment on the enzyme structure, substrate structure, enzymatic hydrolysis kinetics, and thermodynamics and the effects of the ultrasonic conditions on the enzymatic hydrolysis results. The development status of ultrasonic devices and the application of ultrasonic-assisted enzymatic hydrolysis in the food industry are briefly described in this study. In the future, more attention should be paid to research on ultrasound-assisted enzymatic hydrolysis devices to promote the expansion of production and improve production efficiency.

Improving the Flavour of Enzymatically Hydrolysed Beef Liquid by Sonication.

Beef potentiator is an important flavour enhancer in the food industry, while it is prone to generating insufficient compounds with umami and sweet tastes and compounds with a fishy odour during enzymatic hydrolysis of beef, resulting in poor flavour of beef potentiator. It has been extensively reported that sonication is capable of improving food flavour. However, the effect of sonication on the flavour of enzymatically hydrolysed beef liquid (EHBL) was scarcely reported. Herein, we investigated the effect of sonication on the flavour of EHBL using quantitative descriptive analysis (QDA), physicochemical analysis and SPME-GC-olfactometry/MS. QDA showed that sonication had a significant effect on taste improvement and off-odour removal of EHBL. Compared with the control, sonication (40 kHz, 80 W/L) increased the contents of total nitrogen, formaldehyde nitrogen, total sugars, reducing sugars, free amino acids (FAAs) and hydrolysis degree of EHBL by 19.25%, 19.80%, 11.83%, 9.52%, 14.37% and 20.45%. Notably, sonication markedly enhanced the contents of sweet FAAs, umami FAAs and bitter FAAs of EHBL by 19.66%, 14.04% and 9.18%, respectively, which contributed to the taste improvement of EHBL. SPME-GC-olfactometry/MS analysis showed that aldehydes and alcohols were the main contributors to aroma compounds of EHBL, and sonication significantly increased the contents of key aroma compounds and alcohols (115.88%) in EHBL. Notably, sonication decreased the contents of fishy odorants, hexanoic acid and nonanal markedly by 35.29% and 26.03%, which was responsible for the aroma improvement of EHBL. Therefore, sonication could become a new potential tool to improve the flavour of EHBL.

Efficient degradation of soybean protein B3 subunit in soy sauce by ultrasound-assisted prolyl endopeptidase and its primary mechanism.

Prolyl endopeptidase can partially degrade soybean protein B3 subunit and alleviate soy sauce secondary precipitate. In this study, the influences of ultrasound-assisted prolyl endopeptidase on the degradation of soybean protein B3 subunit of soy sauce and primary mechanism were investigated using SDS-PAGE, MALDI-TOF-MS, circular dichromatic spectrometer, fluorescence spectra, etc. Results showed that ultrasound-assisted prolyl endopeptidase enhanced 72% degradation rate of B3 subunit and reduced soy sauce secondary precipitate remarkably, meanwhile significantly increased content of organic taste compounds of soy sauce compared with control (p < 0.05). Sonication markedly reduced percentage of α-helix and increased percentage of random coil, made hydrophobic amino acids inside prolyl endopeptidase exposed to its surface and enhanced its flexibility, which facilitated the binding of prolyl endopeptidase active center with B3 subunit and finally enhanced the latter's degradation rate and appearance quality of soy sauce. This work laid a foundation for solving soy sauce secondary precipitate.

Research Advances in Preparation, Stability, Application, and Possible Risks of Nanoselenium: Focus on Food and Food-Related Fields.

Nanoselenium is a promising selenium supplement as a result of its low toxicity and high bioavailability. However, the understanding on the preparation, stability, bioavailability, possible risks, and related underlying mechanisms of nanoselenium is not in-depth. Thus, the above aspects were reviewed on the basis of the latest literature. The reducing capacity and stability of the reducing agent and binding force between nanoselenium and the template decide the nanoselenium stability. Although research on nanoselenium application in food, agriculture, livestock, and aquaculture has been widely carried out, it is not widely applied in the fields. Se-containing amino acids are synthesized using nanoselenium adsorbed by organisms, and they constitute Se-containing proteins with other amino acids, which improves the health of organisms via scavenging excessive radicals. Notably, excessive nanoselenium intake generates redundant Se-containing amino acids, leading to dysfunction of key proteins in organisms, and its toxic doses vary with organisms. Furthermore, some issues related to nanoselenium still need to be solved urgently.

The latest advances on soy sauce research in the past decade: Emphasis on the advances in China.

As an indispensable soybean-fermented condiment, soy sauce is extensively utilized in catering, daily cooking and food industry in East Asia and Southeast Asia and is becoming popular in the whole world. In the past decade, researchers began to pay great importance to the scientific research of soy sauce, which remarkably promoted the advances on fermentation strains, quality, safety, function and other aspects of soy sauce. Of them, the screening and reconstruction of Aspergillus oryzae with high-yield of salt and acid-tolerant proteases, mechanism of soy sauce flavor formation, improvement of soy sauce quality through the combination of novel physical processing technique and microbial/enzyme, separation and identification of soy sauce functional components are attracting more attention of researchers, and related achievements have been reported continually. Meanwhile, we pointed out the drawbacks of the above research and the future research directions based on published literature and our knowledge. We believe that this review can provide an insightful reference for international related researchers to understand the advances on soy sauce research.

Improving Soy Sauce Aroma Using High Hydrostatic Pressure and the Preliminary Mechanism.

Using high hydrostatic pressure (HHP) to treat liquid foods can improve their aroma; however, no information about the effects of HHP on soy sauce aroma has yet been reported. The effects of HHP on the aroma of soy sauce fermented for 30 d were investigated using quantitative descriptive analysis (QDA), SPME-GC-olfactometry/MS, hierarchical cluster analysis (HCA) and principal component analysis (PCA). Results showed that the pressure used during HHP treatment had a greater influence on soy sauce aroma than the duration of HHP. Compared to the control, soy sauce that was treated with HHP at 400 MPa for 30 min (HHP400-30) obtained the highest sensory score (33% higher) by increasing its sour (7%), malty (9%), floral (27%) and caramel-like (47%) aromas, while decreasing its alcoholic (6%), fruity (6%) and smoky (12%) aromas; moreover, the aroma of HHP400-30 soy sauce was comparable with that of soy sauce fermented for 180 d. Further investigation demonstrated that HHP (400 MPa/30 min) enhanced the OAVs of compounds with sour (19%), malty (37%), floral (37%), caramel-like (49%) and other aromas (118%), and lowered the OAVs of compounds with alcoholic (5%), fruity (12%) and smoky (17%) aromas. These results were consistent with the results of the QDA. HHP treatment positively regulated the Maillard, oxidation and hydrolysis reactions in raw soy sauce, which resulted in the improvement and accelerated formation of raw soy sauce aroma. HHP was capable of simultaneously improving raw soy sauce aroma while accelerating its aroma formation, and this could treatment become a new alternative process involved in the production of high-quality soy sauce.

Evaluation of dual-frequency multi-angle ultrasound on physicochemical properties of tofu gel and its finished product by TOPSIS-entropy weight method.

The effects of dual-frequency (40 + 20 kHz) and multi-angle ultrasound (0°, 30°, 45°) on the coagulation state, network structure, flavor and protein conformation of tofu gel were studied. The results showed that the gel flavor of 40 + 20 kHz 0° group was the best and fluorescence intensity was low. The gel flavor in the 40 + 20 kHz 30° group was better than the group without ultrasound, and hydrophobic interaction and disulfide bond content was the largest. Meanwhile, the degree of protein cross-link was increased. The gel in 40 + 20 kHz 45° group had tightly gel state, high thermal stability, but poor flavor. Combined with The Order Preference by Similarity to Ideal Solution (TOPSIS)-entropy weight method, the 40 + 20 kHz 30° group, was the best ultrasonic treatment of gel. It can change the interaction between proteins, promote protein cross-link, and form a uniform and dense gel network. Finally, the hardness and moisture content of finished tofu were increased significantly, and the quality was improved.

Ultrasound-Assisted Synthesis of Potentially Food-Grade Nano-Zinc Oxide in Ionic Liquids: A Safe, Green, Efficient Approach and Its Acoustics Mechanism.

In food application, nano-zinc oxide (nano-ZnO) is a very important nano metal material; thus, it is necessary to prepare potentially food-grade nano-ZnO. Nano-ZnO synthesized by the ultrasound-assisted method can reach a safe level because of its import physical processing characteristics. Firstly, the micromorphology and microstructure of nano-ZnO synthesized by the ultrasonic method were compared with that by the mechanical stirring method through atomic force microscopy, X-ray diffraction, and Fourier-transform infrared. Secondly, the on-line monitoring of different ultrasonic fields in real-time was studied during the whole synthesis process of nano-ZnO by polyvinylidene fluoride sensor, and two control groups (water medium) were set. The results showed that nano-ZnO obtained by the ultrasonic method were smaller in size and had less surface roughness compared with the mechanical stirring method. The nucleation and crystallization process of nano-ZnO was controlled by the ultrasonic method with sharp diffraction peaks of higher intensities. Moreover, for the ultrasonic mechanism, it was found that the oscillation behavior of bubbles varied from liquid to liquid, and variation was also found in the same liquid under different restraint of interfaces. Based on voltage waveforms monitored in the three liquid media, differences in the life cycle of cavitation bubble oscillation, cycle of collapse stage, maximum voltage amplitude, and acoustic intensity were observed. The physical mechanism of ultrasound-assisted synthesis of nano-ZnO was revealed through voltage fluctuations of the acoustics signal, which can lay a theoretical foundation for the controllability of food ultrasonic physical processing.

Degradation Mechanism of Soybean Protein B3 Subunit Catalyzed by Prolyl Endopeptidase from Aspergillus niger during Soy Sauce Fermentation.

Soy sauce secondary precipitate formed due to the B3 subunit seriously affects soy sauce's appearance quality. In this study, a prolyl endopeptidase (APE) from Aspergillus niger, which could degrade approximately 50% of the B3 subunit and increase proline content by 24% in soy sauce, was isolated and identified. The results showed that APE was an acidic salt-tolerant serine protease (62 kDa), which was optimally active at 40 °C and pH 4.0, and retained more than 69% activity in 3 M NaCl solution over 10 days. As a potential substrate of APE, the B3 subunit contains 10 proline residues. High salinity could not damage the hydrogen bonds, salt bridges, and interior hydrophobicity of APE; thus, the spatial structures and activity of APE in 3 M NaCl solution were stable within 3 days and decreased thereafter. High salinity made the B3 subunit more rigid and lowered the catalytic activity of APE on the B3 subunit, hindering complete hydrolysis of the B3 subunit. This was the first report about the APE capable of degrading the B3 subunit and reducing the secondary precipitate of soy sauce, providing a new possibility to solve the secondary precipitate of soy sauce.

The Physicochemical Properties and Antioxidant Activity of Spirulina (Artrhospira platensis) Chlorophylls Microencapsulated in Different Ratios of Gum Arabic and Whey Protein Isolate.

Spirulina (Artrhospira platensis) is rich in chlorophylls (CH) and is used as a potential natural additive in the food industry. In this study, the CH content was extracted from spirulina powder after ultrasound treatment. Microcapsules were then prepared at different ratios of gum Arabic (GA) and whey protein isolate (WPI) through freeze-drying to improve the chemical stability of CH. As a result, a* and C* values of the microcapsules prepared from GA:WPI ratios (3:7) were -8.94 ± 0.05 and 15.44 ± 0.08, respectively. The GA fraction increased from 1 to 9, and encapsulation efficiency (EE) of microcapsules also increased by 9.62%. Moreover, the absorption peaks of CH at 2927 and 1626 cm-1 in microcapsules emerged as a redshift detected by FT-IR. From SEM images, the morphology of microcapsules changed from broken glassy to irregular porous flake-like structures when the GA ratio increased. In addition, the coated microcapsules (GA:WPI = 3:7) showed the highest DPPH free radical scavenging activity (SADPPH) (56.38 ± 0.19) due to low moisture content and better chemical stability through thermogravimetric analysis (TGA). Conclusively, GA and WPI coacervates as the wall material may improve the stability of CH extracted from spirulina.

Improvement of betalains stability extracted from red dragon fruit peel by ultrasound-assisted microencapsulation with maltodextrin.

Natural betalains can be potential food additives because of their antioxidant activities, but they have poor thermal stability. In this study, betalains were extracted from red dragon fruit peel, and then encapsulated with maltodextrin by ultrasound method to increase the physicochemical properties of betalains microcapsules. The encapsulation efficiency of the betalains was above 79%, and the particle size and Zeta potential values were 275.46 nm and -29.01 mV, respectively. Compared to the control sample, onset temperature and DPPH free radical scavenging of betalains microcapsules under the modest ultrasound treatment (200 W, 5 min) was increased by 1.6 °C and 12.24%, respectively. This increase could be due to the ability of ultrasonification to create interactions between maltodextrin and betalains (as evidenced by FT-IR). Therefore, modest ultrasound treatment can be used for microcapsulation to improve the stability of betalains, and then expand the application of betalains in heat processed food field.

Enhancing organic selenium content and antioxidant activities of soy sauce using nano-selenium during soybean soaking.

Nano-selenium has a greater potential than inorganic selenium in preventing selenium-deficiency diseases due to its higher safety. In this study, spherical nano-selenium particles (53.8 nm) were prepared using sodium selenite, ascorbic acid and chitosan. Selenium-enriched soy sauces were prepared by soaking soybean in nano-selenium and sodium selenite solutions (2-10 mg/L), respectively. Total selenium and organic selenium contents of soy sauces prepared by nano-selenium and sodium selenite were increased by 32-191-fold and 29-173-fold compared to the control (without selenium), and organic selenium accounted for over 90% of total selenium. Soy sauce prepared using 6 mg/L nano-selenium had the strongest antioxidant activities, which were 9.25-28.02% higher than the control. Nano-selenium (6 mg/L) markedly enhanced the koji's enzyme activities (9.76-33.59%), then the latter promoted the release of total phenolics (27.54%), total flavonoids (27.27%) and the formation of free amino acids (16.19%), Maillard reaction products (24.50%), finally the antioxidant activities of selenium-enriched soy sauce were enhanced.

Influence of Low-Intensity Ultrasound on ε-Polylysine Production: Intracellular ATP and Key Biosynthesis Enzymes during Streptomyces albulus Fermentation.

The effect of low-intensity sonication treatment on cell growth, ε-polylysine (ε-PL) yield and its biological mechanism were investigated, using a 3-L-jar fermenter coupled with an in situ ultrasonic slot with a Streptomyces albulus strain SAR 14-116. Under ultrasonic conditions (28 kHz, 0.37 W cm-2, 60 min), a high biomass of SAR 14-116 and concentration of ε-PL were realized (i.e., they increased by 14.92% and 28.45%, respectively) when compared with a control. Besides this, ultrasonication increased the mycelia viability and intracellular ATP as well as activities of key enzymes involved in the ε-PL biosynthesis pathway, resulting in an improvement in the production of ε-PL. Data on qRT-PCR revealed that ultrasonication also affected the gene expression of key enzymes in the ε-PL biosynthesis pathway, including ε-PL synthetase (PLS). These outcomes provided the basis for understanding the effects of ultrasound-assisted fermentation on the stimulation of metabolite production and fermentation procedure in a fermenter.

Ougan juice debittering using ultrasound-aided enzymatic hydrolysis: Impacts on aroma and taste.

The optimal sonication conditions (40 kHz, 80 W/L and 60 min) during Ougan juice debittering by Aspergillus niger koji extract were established. Enzymatic hydrolysis degrees of naringin and limonin were enhanced to 89.90% and 36.16%, and enzymatic hydrolysis time was shortened by 33%. Sonication significantly enhanced activities of α-l-rhamnosidases, ß-glucosidases and limoninases from A. niger koji extract and facilitated break of CO bonds in naringin (p < 0.05). These accounted for the enhanced enzymatic hydrolysis degrees and velocities of bitter compounds. Meanwhile, sonication lowered 40%, 7% and 21%, 13%, 11%, 25% of bitter, sour tastes and green, citrus-like, floral, woody notes, but enhanced 18% and 15% of fruity and sweet notes, resulting in 38% and 33% increases in over-all taste and aroma scores. Lowered levels of bitter compounds, organic acids, green, citrus-like, floral, woody aroma compounds and enhanced levels of fruity, sweet aroma compounds caused by sonication accounted for the flavor improvements.

Hawthorn pectin: Extraction, function and utilization.

Pectin has been widely used as emulsifiers, gelling agents, glazing agents, stabilizers, and thickeners in food products. Hawthorn pectin has a higher viscosity than other foods-derived pectin such as lemon and apple pectin. It is also reported as a multifunctional fruit substance, which reduces the risk of hyperlipidemia and dyslipidemia. Therefore, hawthorn pectin is a potential resource for the development of new drugs, functional foods, and health-care products. This review symmetrically summarized the extraction methods, physiological characteristics, functional properties, and processing technologies of hawthorn pectin. It laid a foundation for the further research of hawthorn pectin and promoted the diversified utilization of hawthorn.

Characterization of the aroma-active compounds in black soybean sauce, a distinctive soy sauce.

Black soybean sauce's (BSS) aroma was scarcely investigated, which seriously affected BSS's quality and consumers' preference. Thus the aroma compounds in BSS were characterized using gas chromatography-mass spectrometry/gas chromatography-olfactometry coupling with recombination and omission experiments. Sensory evaluation showed the fruity odor was increased by 63% and the malty, alcoholic, floral, smoky, caramel-like and sour odors were decreased by 24-35% when compared to the control soy sauce (SS, p < 0.05). Totally, 126 volatile compounds, 44 aroma-active compounds and 22 vital aroma-active compounds were identified in BSS. Compared to SS, BSS exhibited a distinctive aroma characteristics which was caused by significantly higher odor activity values (OAVs) of 3-methylbutyl acetate (357%), ethyl propanoate (144%), ethyl 3-methylbutanoate (70%), ethyl 2-methylbutanoate (102%) and lower OAVs of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (52%), 4-hydroxy-2-ethyl-5-methyl-3(2H)-furan-3-one (50%), ethanol (48%), 4-vinylguaiacol (41%), 3-methylthiopropanal (37%), 3-methylbutanol (33%), 4-ethylguaiacol (28%). The results would contribute to BSS's quality control and aroma improvement.