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A novel aspartic-type endopeptidase was previously obtained from Moringa oleifera seeds; however, its specific milk-clotting properties have remained unclear. Here, we used various biophysical and molecular simulation approaches for characterizing the structure and function of the aspartic-type endopeptidase. The endopeptidase was preferentially active toward κ-casein (CN) and hydrolyzed it more than calf rennet; however, its ability to hydrolyze α-CN and β-CN was weaker than that of calf rennet. The endopeptidase cleaved κ-CN at Gln135-Asp136 and generated a 15 588.18 Da peptide with 135 amino acids. We further simulated the docking complex of the endopeptidase and κ-CN and found out that they possibly combined with each other via hydrogen bonds. The flocculation reaction between the endopeptidase and κ-CN indicated that milk coagulation occurred within 60 min. Overall, our observations suggest that the aspartic-type endopeptidase can be a potential rennet alternative for cheese making.

Thanks to low cost, high safety, and large energy density, aqueous zinc-ion batteries have attracted tremendous interest worldwide. However, it remains a challenge to develop high-performance cathode materials with an appropriate method that is easy to realize massive production. Herein, we use a molten salt method to synthesize nanostructured manganese oxides. The crystalline phases of the manganese oxides can be tuned by changing the amount of reduced graphene oxide added to the reactant mixture. It is found that the α-MnO2/Mn2O3 nanocomposite with the largest mass ratio of Mn2O3 delivers the best electrochemical performances among all the products. And its rate capability and cyclability can be significantly improved by modifying the Zn anode with carbon black coating and nanocellulose binder. In this situation, the nanocomposite can deliver high discharging capacities of 322.1 and 213.6 mAh g–1 at 0.2 and 3 A g–1, respectively. After 1000 cycles, it can retain 86.2% of the capacity at the 2nd cycle. Thus, this nanocomposite holds great promise for practical applications.

Due to their low cost, high safety, environmental friendliness, and impressive electrochemical performances, aqueous zinc-ion batteries are considered promising alternative technologies to lithium-ion batteries for use in large-scale applications. However, existing aqueous zinc-ion batteries usually suffer from poor cyclability and cannot operate at subzero temperatures. Herein, to solve these problems, the electrolyte in aqueous zinc-ion batterie is optimized by adding the appropriate amounts of diethyl ether and ethylene glycol. Results show that the addition of 1% diethyl ether contributes to the best cyclability at 25 °C. Furthermore, the addition of 30% ethylene glycol results in the best electrochemical performances at 0 and − 10 °C. This significant performance improvement at low temperatures is ascribed to the high ionic conductivity of the modified electrolyte and the low charge transfer impedance of the battery with the modified electrolyte at 0 and −10 °C. It is also shown that the modified electrolyte can decrease the nucleation overpotential of zinc plating, enhance the interfacial stability between the zinc metal and electrolyte, suppress the zinc dendritic growth and side reactions, and decrease the self-corrosion rate of the zinc anode. This work offers a facile strategy to realize aqueous zinc-ion batteries with excellent cyclability and antifreezing ability and may inspire research on other aqueous energy storage systems.

It is urgent to develop high-performance cathode materials for the emerging aqueous zinc-ion batteries with a facile strategy and optimize the related components. Herein, a Ca0.23V2O5·0.95H2O nanobelt cathode material with a rather large interlayer spacing of 13.0 A is prepared via a one-step hydrothermal approach. The battery with this cathode material and 3 M Zn(CF3SO3)2 electrolyte displays high specific capacity (355.2 mAh g−1 at 0.2 A g−1), great rate capability (240.8 mAh g−1 at 5 A g−1), and excellent cyclability (97.7% capacity retention over 2000 cycles). Such superior performances are ascribed to fast electrochemical kinetics, outstanding electrode/electrolyte interface stability, and nearly dendrite-free characteristic. Instead, when ZnSO4 or Zn(ClO4)2 is used to replace Zn(CF3SO3)2, the electrochemical performances become much inferior, due to the slow electrochemical kinetics, inhomogeneous Zn stripping/plating process, and the formation of large dendrites and byproducts. This work not only discloses a high-performance cathode material for aqueous zinc-ion batteries but also offers a reference for the choice of electrolyte salt.

Aqueous zinc-ion batteries have received significant attention due to their low cost and high safety. However, the unsatisfactory cycling performances caused by the dendritic growth on the Zn anode limit their practical applications. Herein, we propose to modify the conventional Zn foil anode by using carbon black coating and nanofibrillated cellulose binder. The carbon black can form an electrically conductive network, thus greatly enlarging the electroactive surface area, while the nanofibrillated cellulose can act as an electrolyte reservoir to facilitate charge transports. Thanks to that, the modified anode can significantly eliminate the dendritic growth and side reactions, therefore ensuring excellent interface stability with the electrolyte even at a commercial-level areal capacity of 5 mAh g-1. With the modified anode, the Zn-MnO2 battery gives a high capacity retention of 87.4% after 1000 cycles, much higher than that with the unmodified Zn foil (42.6%). This study discloses a facile, scalable, and cost-effective strategy to achieve dendrite-free metal electrodes towards great cyclability.

This study aimed to investigate the effect of coatings on the quality of ripened dry-cured hams during long-term storage, especially the profile of volatile compounds. The coatings were made up of 33% palm oil, 16.5% water, 39.7% cassava starch, 6.8% corn starch, 1.6% mono- and diglycerides of fatty acids, 0.6% tert-butylhydroquinone (TBHQ), and 1.8% sodium carbonate. The results showed that the moisture content of the coated ham (48.93–49.59%) was higher than that of the noncoated ham (44.37%). The average peroxide value (POV) and b* value were lower in the coated hams than in the noncoated hams (5.52 and 8.99 meq/kg, respectively), and the sensory attributes of the coated hams had better overall acceptability scores. The changes in the contents of 39 volatile flavor compounds were evaluated through a multivariate statistical analysis, revealing that 20 identified compounds could be related to the decrease in fat pungent aroma, and most belonged to the long-chain benzene and carboxylic acid family. Meanwhile, 2-nonanone, nonanal, amyl alcohol, and 2-heptanone indicated that they could be used as markers to distinguish between the coated and noncoated groups. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-020-04579-z) contains supplementary material, which is available to authorized users.

Aims Staphylococcus aureus has emerged as a serious threat to food safety owing to biofilm formation. The study aimed to examine the antibiofilm mechanism of a novel milk-derived antimicrobial peptide BCp12 against it. Methods and results Antibiofilm activity of BCp12 was studied by crystal violet staining, MTT assay, motility, SEM and CLSM. TMT proteome, real-time PCR and molecular docking in silico were conducted to evaluate the mechanism of BCp12 against S. aureus biofilm. The results showed that BCp12 had significant antibiofilm activity at 1 × MIC and sub-MIC. BCp12 induced the dispersion of structure of S. aureus biofilm BCp12 inhibited the movement of S. aureus. A total of 703 proteins were downregulated and 334 proteins were upregulated after BCp12 treatment. The proteins (agrA, agrB, agrC and psmβ) of the QS systems were downregulated. Additionally, the expression of the agr-related genes, agrA, agrB, agrC and psmβ, was downregulated. BCp12 was bound to the receptor proteins agrA and agrC through hydrogen bonds and π–π bonds. Conclusions The results showed the antibiofilm activity of BCp12 and it inhibits the biofilm formation by interfering agr QS system. Significance and Impact of Study BCp12 has the potential to be a novel antibiofilm agent against S. aureus biofilm and used in the food industry.

This study aimed to characterize a novel antimicrobial peptide (AMP) obtained from Moringa oleifera seed protein hydrolysates. Cell membrane chromatography and live bacteria adsorption were combined into a single step to efficiently isolate the active fraction of the AMP. Five peptides were identified by LC-MS/MS, among which the MCNDCGA peptide (termed MOp3) showed the greatest inhibitory effect against Staphylococcus aureus [minimum inhibitory concentration (MIC): 2 mg/mL]. MOp3 was identified as a hydrophobic anionic AMP rich in β-sheet structures with negligible hemolytic activity at 2.0 × MIC. MOp3 had good tolerance to salt solutions at 5 % and pH range 6.0-8.0, but was sensitive to high temperatures (100 °C) and acid protease. Microscopic observation further revealed that MOp3 induced irreversible damage onto the cell membrane of S. aureus and interacted with dihydrofolate reductase and DNA gyrase by hydrogen bonding and hydrophobic interaction. These findings highlight the potential application of a new antimicrobial agent against S. aureus in the food industry.

The prevalence of staphylococcal infection and the emergence of multidrug resistance of Staphylococcus aureus (S. aureus) are major concerns in food safety and public health. This study aimed to investigate the prevalence of S. aureus isolated from traditional Chinese Rubing and Rushan cheese, antimicrobial resistance profiles, genomic characteristics, and predict antimicrobial resistance genes (ARGs). From 124 samples, 18 of 62 (29.03%) of Rubing and 5 of 62 (8.06%) of Rushan cheese were confirmed to be S. aureus positive by standard culture-based methods. Twenty-three coagulase-positive staphylococci isolates were grouped into 16 clusters by pulsed-field gel electrophoresis and subjected to routine susceptibility testing to 12 antibiotics. Those isolates exhibited high resistance to penicillin (100%), erythromycin, trimethoprim-sulphamethoxazole (34.78%), oxacillin, clindamycin, and cefoxitin (21.74%). Multidrug-resistant (MDR) S. aureus was found in 34.78% (8 of 23) of isolates. Further, S. aureus strain DC.RB_015 isolated from Rubing cheese, recognized as the most resistant to six antibiotics, was selected for whole-genome sequencing (WGS), continued with in silico approaches. S. aureus DC.RB_015 had a single chromosome size of 2,794,578 bp and a plasmid size of 22,961 bp. The strain harbored 18 predicted ARGs, including eight efflux pump genes (mepA, tet(K), arlR, arlS, norA, mgrA, tet(38), LmrS), one peptidoglycan biosynthesis gene (bacA), two β-lactams resistance genes (mecA, blaZ), and seven genes conferring other antimicrobial resistance (APH(3′)-IIIa, aad(6), ErmB, SAT-4, mecR1, GlpT, murA). The results of this study expand the knowledge of S. aureus strain DC.RB_015, increase food safety awareness, and will be helpful in establishing therapeutic therapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03072-4.

A cysteine peptidase was previously identified from germinated Moringa oleifera seeds, but its milk-clotting properties on bovine caseins was still unclear. In this study, this novel cysteine peptidase (MoCP) showed preferential activity on κ-casein (κ-CN), with greater hydrolytic activity compared with calf rennet, whereas weak hydrolysis of α-casein and β-casein made MoCP suitable for application in cheesemaking and may yield various functional peptides. All 3 evaluated caseins were hydrolyzed to form relatively stable peptide bands within 3 h of proteolysis with MoCP. Cleavage sites were determined by gel electrophoresis, liquid chromatography mass spectrometry/mass spectrometry, and peptide sequencing, which revealed that cleavage of κ-CN by MoCP occurred at residue Ile129-Pro130 and generated a 14,895.37-Da peptide. The flocculation reaction between MoCP and κ-CN determined by 3-dimensional microscopy with super-depth of field revealed that the initial 30 min of reaction were key for milk coagulation, which may affect curd yield. Overall, the findings presented herein suggest that the cysteine peptidase from germinated M. oleifera seeds can be considered a promising plant-derived rennet alternative for use in cheese manufacture.

In this study, label-free quantitative proteomics was used to investigate the biological functions of M. oleifera seed proteins, which resulted in the identification of milk-clotting proteases. In total, 921 proteins were identified, and proteins within the molecular weight range of 30–50 kDa were abundant. The identified proteins were mainly involved in catalytic activity and metabolic processes associated with carbohydrate and protein metabolism, among which, proteases in the observed molecular weight range could possibly be responsible for the previously reported milk-clotting activity. An aspartic-type endopeptidase with molecular mass of 45,517 Da was purified from M. oleifera seeds using ammonium sulfate precipitation, ultrafiltration, and preparative high performance liquid chromatography, and was characterized using liquid chromatography-mass spectrometry (LC-MS)/MS. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the purified protease exhibited hydrolase activity and was involved in several metabolic pathways, which further confirmed that proteomic analysis can assist in the purification of the milk-clotting protease. The optimal temperature and pH required for protease activity were 60 °C and 5.0, respectively. The high thermal stability and good pH stability of the protease indicated that it can be used in the dairy industry.

Flexible aqueous zinc-ion batteries (AZIBs) are promising to satisfy the emerging wearable electronics. However, conventional hydrogel electrolytes are unable to work at subzero temperatures because they inevitably freeze. In this work, a borax-crosslinked polyvinyl alcohol (PVA)/glycerol gel electrolyte is developed, in which glycerol can strongly interact with PVA chains, thus effectively prohibiting the formation of ice crystals within the whole gel network. Thanks to this, the freezing point of this gel electrolyte is below −60 °C, which allows it to work in extremely cold environments. Even at −35 °C, it still exhibits a high ionic conductivity of 10.1 mS cm−1 and great mechanical properties. On the basis of this anti-freezing gel electrolyte, a flexible quasi-solid-state aqueous Zn–MnO2 battery is assembled and realizes an impressive energy density of 46.8 mW h cm−3 (1330 μW h cm−2) at a power density of 96 mW cm−3 (2.7 mW cm−2) at 25 °C, outperforming nearly all the reported AZIBs. More importantly, when the temperature is reduced to −35 °C, a rather high energy density (25.8 mW h cm−3, 732 μW h cm−2) can still be achieved, and 53.3% of that value can be retained when the power density is increased to about 10-fold. This battery also shows excellent cycling durability (around 90% capacity retention over 2000 cycles) and great tolerance to various extreme conditions even when the temperature is down to −35 °C. These findings provide valuable insights into designing aqueous batteries/supercapacitors that can work in cold climates and high-altitude areas.

Aqueous zinc-ion batteries (AZIBs) are promising due to their intrinsic safety and low cost. However, the unsatisfactory cathode materials with low mass loading (

As a new food source, Moringa oleifera Lam. leaves containing large amounts of proteins attract increasing attention. This study detected and characterized hydrolytic and milk-clotting enzymes in these leaves. The proteins were examined by proteomics. A total of 3378 proteins were identified, mostly comprising enzymes of carbohydrate and protein metabolism, including 676 hydrolases, 548 oxidoreductases, glycoside hydrolases and proteinases. A serine/threonine-endopeptidase with a molecular mass of 56.146 kDa and an isoelectric point of 5.27 with milk-clotting activity was obtained from M. oleifera leaves by multistage ultrafiltration and anion exchange chromatography, and characterized by ESI mass spectrometry. The milk-clotting activity was inhibited over 95% by chymostatin, which confirmed its chymotrypsin-like serine protease nature. The protease had a milk-clotting activity/proteolytic activity (MCA/PA) activity ratio of 126.76, an optimal pH of 8.0, and an optimal temperature of 65 °C, indicating its potential use for cheese production and in food industry.

The protein extract of Moringa oleifera flowers is reported to have milk-clotting activity (MCA), but information regarding its protease is unclear. In this study, two milk-clotting proteases (MoFP 12 and MoFP 13) with molecular weights of 42.304 kDa and 31.741 kDa, respectively, were isolated and identified from M. oleifera flowers using liquid chromatography-mass spectrometry (LC-MS/MS). Bioinformatics analysis showed that these two milk-clotting proteases were primarily involved in hydrolase activity and catabolic process, and exhibited hydrophilicity. The secondary structure of MoFP 12 consisted of 43.65% helix, 13.23% strand, and 43.12% coil, and MoFP 13 consisted of 26.51% helix, 20.14% strand, and 53.35% coil. The proteases were stable in the pH range of 5.0 to 8.0 and showed their maximum MCA at 70℃. Additionally, by investigating the effect of proteases on caseins, κ-casein (CN) was observed to preferentially be hydrolyzed by the two proteases, followed by α-CN, and to a lesser extent β-CN. These findings revealed the main milk-clotting proteases in M. oleifera flowers and its milk-clotting properties, indicating its potential for application in the dairy and food sectors, especially in the cheese-making industry.

A novel aspartic-type endopeptidase was previously obtained from

Rubing cheese is a traditional Chinese Protected Designation of Origin (PDO) cheese consumed for more than six hundred years, but to date, the digestion properties and peptide profiling during simulated gastrointestinal digestion are still uncertain. This study aimed to investigate the effects of traditional direct acidification technology (TRB) and fermentation acidification technology on digestion properties and peptide profiling of rubing cheese (FRB) proteins after simulated gastrointestinal digestion by protein digestomics, coupled with bioinformatic in silico analyses to identify potential bioactive peptides. The results demonstrated that FRB could significantly improve the in vitro digestibility, protein degradation, and polypeptide content than TRB (P ＜ 0.05). Furthermore, a total of 369 and 332 peptides were identified in FRB- and TRB-pancreatic digests, respectively, using LC-MS/MS. FRB could release more low molecular weight peptides of 400-1200 Da from α-casein and β-casein after digestion. These low peptides included 16 reported potential ACEIPs (angiotensin I-converting enzyme inhibitory peptides), 11 dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides, and 6 antioxidant peptides, while TRB contained more than the reported potential antimicrobial peptides (10). In vitro activity determination showed that FRB had significantly higher ACEI, α-glucosidase inhibitory, and antioxidant activities than TRB during the entire digestion time (P ＜ 0.05), which was correlated to the reported potential bioactive peptides released during the digestion of FRB. Our study is the most comprehensive protein digestomic analysis of Chinese rubing cheese to date and provides a new positive outlook on rubing cheese consumption.

With the booming development of wearable electronics, flexible zinc-based batteries are attracting significant attention due to their high safety, low cost, environmental benignity, and relatively large energy/power densities. However, in a conventional segregated configuration, the electrodes could be easily detached from the separator when the battery is subjected to bending strain, which would dramatically depress electrochemical performances. Moreover, severe zinc dendrite growth and parasitic side reactions at the anode are extremely detrimental to the durability and the reliability of zinc-based batteries. Herein, a flexible self-standing composite film anode consisting of zinc microspheres, carbon nanotubes, and nanocellulose is constructed to replace the conventional Zn foil. It is found that the use of this anode can effectively inhibit the dendrites and side reactions, thereby substantially improving the cyclability. In addition, a layer-by-layer vacuum filtration method is used to integrate the composite film anode with a cellulose separator and a MnO2-based composite film cathode into a single matrix. The unique integrated battery realizes great rate capability and cycling stability, and more importantly, superior affordability to bending deformations. Besides, the commonly used thick, heavy, and expensive current collectors are no longer required in the integrated configuration, therefore enabling the battery to be smarter and cheaper. This study not only opens a new option for building dendrite-free zinc anodes but also discloses a facile strategy to achieve integrated configuration for energy storage devices.

The present study aimed to investigate the biological functions of germinated M. oleifera seed proteins and to identify the identity of milk-clotting proteases. A total of 963 proteins were identified, and those with molecular weights between 10 and 30 kDa were most abundant. The identified proteins were mainly involved in energy-associated catalytic activity and metabolic processes, and carbohydrate and protein metabolisms. The numbers of proteins associated with the hydrolytic and catalytic activities were higher than the matured dry M. oleifera seeds reported previously. Of the identified proteins, proteases were mainly involved in the milk-clotting activity. Especially, a cysteine peptidase with a molecular mass of 17.727 kDa exhibiting hydrolase and peptidase activities was purified and identified. The identified cysteine peptidase was hydrophilic, and its secondary structure consisted of 27.60% alpha helix, 9.20% beta fold, and 63.20% irregular curl; its tertiary structure was also constructed using M. oleifera seed 2S protein as the protein template. The optimal pH and temperature of the purified protease were pH 4.0 and 60 °C, respectively. The protease had high acidic stability and good thermostability, thus could potentially be applied in the dairy industry.

Background Protein lysine malonylation, a novel post-translational modification (PTM), has been recently linked with energy metabolism in bacteria. Staphylococcus aureus is the third most important foodborne pathogen worldwide. Nonetheless, substrates and biological roles of malonylation are still poorly understood in this pathogen. Results Using anti-malonyl-lysine antibody enrichment and high-resolution LC-MS/MS analysis, 440 lysine-malonylated sites were identified in 281 proteins of S. aureus strain. The frequency of valine in position − 1 and alanine at + 2 and + 4 positions was high. KEGG pathway analysis showed that six categories were highly enriched, including ribosome, glycolysis/gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), valine, leucine, isoleucine degradation, and aminoacyl-tRNA biosynthesis. In total, 31 malonylated sites in S. aureus shared homology with lysine-malonylated sites previously identified in E. coli, indicating malonylated proteins are highly conserved among bacteria. Key rate-limiting enzymes in central carbon metabolic pathways were also found to be malonylated in S. aureus, namely pyruvate kinase (PYK), 6-phosphofructokinase, phosphoglycerate kinase, dihydrolipoyl dehydrogenase, and F1F0-ATP synthase. Notably, malonylation sites were found at or near protein active sites, including KH domain protein, thioredoxin, alanine dehydrogenase (ALD), dihydrolipoyl dehydrogenase (LpdA), pyruvate oxidase CidC, and catabolite control protein A (CcpA), thus suggesting that lysine malonylation may affect the activity of such enzymes. Conclusions Data presented herein expand the current knowledge on lysine malonylation in prokaryotes and indicate the potential roles of protein malonylation in bacterial physiology and metabolism.

This study aims to isolate the antimicrobial peptide (AMP) from buffalo casein hydrolyzed by Dregea sinensis Hemsl. protease. The AMP was isolated from hydrolysate by live bacteria adsorption, then analyzed using reversed-phase high-performance liquid chromatography, and the fraction with highest antimicrobial activity was identified by liquid chromatography-tandem MS. Further, we characterized the peptide in terms of its peptide sequence, structure, and antimicrobial activity. The results identified the AA sequence of the peptide as YLGYLEQLLRLK, which corresponds to residues 106 to 117 of bovine αS1-casein, and we named it BCp12. BCp12 displays α-helical structure, with high hydrophobic moments and net positive charge. BCp12 can inhibit the growth of indicator bacteria, with minimum inhibitory concentration values ranging from 0.8 to 1.6 mg/mL, and can induce low toxicity in mammalian cells. Antimicrobial activity of the BCp12 peptide remained stable under different salt concentrations but was sensitive to trypsin and high temperatures (121°C and above). The results support further research in the application of our newly generated AMP as an antimicrobial agent in the food industry and in food processing facilities.