Insights into the impact of complex phosphates on acid-induced milk fan gel properties: Texture, rheological, microstructure, and molecular forces.

Milk fan cheese, a type of stretched -cheese, presents challenges in its stretch-forming. This study investigated the impacts of complex phosphates (sodium tripolyphosphate and sodium dihydrogen phosphate, STPP-DSP) on the gelling properties of acid-induced milk fan gel and the mechanisms contributing to its stretch-forming. The treatment of milk fan gel with STPP-DSP resulted in improved functional and textural properties compared with the control group. In particular, drawing length increased significantly from 69.67 nm to 80.33 nm, and adhesiveness increased from 1737.89 g/mm to 1969.79 g/mm. The addition of STPP-DSP also led to increased viscosity, elastic modulus (G'), and viscous modulus (G"). Microstructural analysis revealed the formation of a fibrous structure within the gel after STPP-DSP treatment, facilitating uniform embedding of fat globules and emulsification. Structural analysis showed that the addition of STPP-DSP increased ß-fold and decreased random coiling of the gel, facilitating the unfolding of protein structures. Additionally, UV absorption spectroscopy and excitation-emission matrix spectroscopy results indicated the formation of a chelate between STPP-DSP and milk fan gel, increasing protein-protein molecular interactions. Evidence from differential scanning calorimetry and x-ray diffraction demonstrated the formation of sodium caseinate chelate. Fourier transform infrared spectroscopy and zeta potential analysis revealed that the sodium caseinate chelate formed through hydrophobicity, hydrogen bonding, and electrostatic forces. These findings provided theoretical insights into how phosphates can improve the stretch-forming of milk fan gel, facilitating the application of phosphate additives in stretched -cheese processing.

Identification and molecular mechanism of novel bifunctional peptides from Duroc × (Landrace × Yorkshire) pig dry-cured ham: A peptidomics and in silico analysis.

Duroc × (Landrace × Yorkshire) pigs are popular in the Chinese market because of their rapid growth, leanness, and economic value. Despite their widespread use in dry-cured ham processing, there is a lack of research on the bioactive peptides of Duroc × (Landrace × Yorkshire) pig ham (DLYH). This study aimed to investigate the presence of peptides with antioxidant and α-glucosidase inhibitory activities in DLYH using peptidomics and in silico analysis. A total of 453 peptides were identified from DLYH, originating mainly from myosin, actin, and the EF-hand domain-containing protein. Notably, two peptides, YDEAGPSIVH (YH10) and FAGDDAPRAVF (FF11), emerged as novel bioactive peptides with antioxidant and α-glucosidase inhibitory activities. Among these peptides, YH10 exhibited a high DPPH radical scavenging activity (IC50 = 1.93 mM), ABTS radical scavenging activity (IC50 = 0.10 mM), α-glucosidase inhibitory activity (IC50 = 2.13 mM), and good gastrointestinal tolerance. Molecular docking analysis showed that YH10 was bound to the ABTS and DPPH radicals and the active site of α-glucosidase (3A4A) primarily through hydrogen bonding and hydrophobic interactions. Furthermore, molecular dynamics (MD) simulation indicated that the YH10-3A4A complexes maintained stable and compact conformations. In conclusion, our findings indicated that peptide YH10 derived from DLYH possesses bifunctional properties of α-glucosidase inhibition and antioxidant activity, which could be beneficial for maintaining ham quality and promoting human health.

Identification, structural characterization, and molecular dynamic simulation of ACE inhibitory peptides in whey hydrolysates from Chinese Rushan cheese by-product.

To realize the high-value utilization of Rushan cheese by-product, Rushan cheese whey was used as a raw material to prepare angiotensin-Ⅰ-converting enzyme inhibitory peptides (ACEIPs). After enzymatic hydrolysisn and ultrafiltration, the sequences of peptides were identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Two novel ACE inhibitory peptides Phe-Asp-Arg-Pro-Phe-Leu (FDRPFL) and Lys-Trp-Glu-Lys-Pro-Phe (KWEKPF) were identified. Additionally, both of the peptides exhibited good water-solubility and no toxicity according to in-silico prediction. Fourier transform infrared spectroscopy results show that both FDRPFL and KWEKPF were enriched in ß-turn and ß-sheet structures. Lineweaver-Burk plots revealed that FDRPFL and KWEKPF exhibited non-competitive and mixed inhibition patterns, respectively. Molecular docking and MD simulation showed that hydrogen bonds and ionic bonds forces allowed FDRPFL and KWEKPF to form stable and compact complexes with ACE. In conclusion, enzymatic hydrolysis of Rushan cheese by-products yields bioactive peptides, increases the added value of whey and reduces environmental pollution.

Plant-derived rennet: research progress, novel strategies for their isolation, identification, mechanism, bioactive peptide generation, and application in cheese manufacturing.

Rennet, an aspartate protease found in the stomach of unweaned calves, effectively cuts the peptide bond between Phe105-Met106 in κ-casein, hydrolyzing the casein micelles to coagulate the milk and is a crucial additive in cheese production. Rennet is one of the most used enzymes of animal origin in cheese making. However, using rennet al.one is insufficient to meet the increasing demand for cheese production worldwide. Numerous studies have shown that plant rennet can be an alternative to bovine rennet and exhibit a good renneting effect. Therefore, it is crucial and urgent to find a reliable plant rennet. Based on our team's research on rennet enzymes of plant origin, such as from Dregea sinensis Hemsl. and Moringa oleifer Lam., for more than ten years, this paper reviews the relevant literature on rennet sources, isolation, identification, rennet mechanism, functional active peptide screening, and application in cheese production. In addition, it proposes the various techniques for targeted isolation and identification of rennet and efficient screening of functionally active peptides, which show excellent prospects for development.

Integrated metabolomics and peptidomics to delineate characteristic metabolites in milk fermented with novel Lactiplantibacillus plantarum L3.

A novel wild-type Lactiplantibacillus plantarum (L. plantarum) L3 with good fermentation characteristics and protein degradation capacity was isolated from raw milk samples. In this study, the metabolites in milk fermented with L. plantarum L3 were investigated by metabolomic and peptidomics analyses. The metabolomics results revealed that the metabolites in milk fermented with L. plantarum L3 were Thr-Pro, Val-Lys, l-creatine, pyridoxine, and muramic acid, which improved the taste and nutritional qualities of the milk. Moreover, the water-soluble peptides derived from L3 fermented milk exhibited high antioxidant properties and angiotensin I-converting enzyme inhibitory (ACEI) activities. Additionally, 152 peptides were found using liquid chromatography-mass spectrometry (LC-MS/MS). Furthermore, endogenous enzymes secreted by L. plantarum L3 cleaved ß- and α-casein to release six ACEI peptides (ACEIPs), nineteen antioxidant peptides (AOPs), and five antimicrobial peptides (AMPS). Overall, these findings could be valuable in improving the quality of fermented milk.

Proteomic approach-based comparison of metabolic pathways and functional activities of whey proteins derived from Guishan and Saanen goat milk.

Guishan goats, a unique goat breed in Yunnan Province, have a long history and representation, but their whey protein and function remain unclear. In this study, we carried out a quantitative analysis of the Guishan and Saanen goat whey proteome using a label-free proteomic approach. A total of 500 proteins were quantified from the 2 kinds of goat whey proteins, including 463 common proteins, 37 uniquely expressed whey proteins (UEWP), and 12 differentially expressed whey proteins (DEWP). Bioinformatics analysis indicated that UEWP and DEWP were mainly involved in cellular and immune system processes, membrane, and binding. In addition, UEWP and DEWP in Guishan goats participated primarily in metabolism and immune-related pathways, whereas Saanen goat whey proteins were associated mostly with environmental information processing-related pathways. Guishan goat whey promoted the growth of RAW264.7 macrophages more than Saanen goat whey, and significantly reduced the production of nitric oxide in lipopolysaccharide-stimulated RAW264.7 cells. This study provides a reference for further understanding these 2 goat whey proteins and finding functional active substances from them.

Discovery of specific antioxidant peptide from Chinese Dahe black pig and hybrid pig dry-cured hams based on peptidomics strategy.

The quality of hams obtained from different pig breeds can vary depending on endogenous antioxidant peptides in the hams. The aims of this study were (i) to investigate the specific peptides in Chinese Dahe black pig ham (DWH) and hybrid pig ham (Yorkshire × Landrace × Dahe black ham, YLDWH) and their antioxidant activity, and (ii) to elucidate the relationship between ham quality and antioxidant peptides. iTRAQ quantitative peptidomic method was used to discover specific peptides of DWH and YLDWH. In addition, in vitro assays were performed to evaluate their antioxidant activity. A total of 73 specific peptides were identified from DWH and YLDWH by LC-MS/MS. Forty-four specific peptides in DWH were primarily hydrolysed from myosin and myoglobin by endopeptidases, while 29 specific peptides in YLDWH were primarily hydrolysed from myosin and troponin-T. Six specific peptides that were statistically significantly different based on their fold changes and P-values were selected for the identification of DWH and YLDWH. DWH-derived specific peptide AGAPDERGPGPAAR (AR14), which had high stability and was non-toxic, had the highest DPPH• and ABTS•+ scavenging activity (IC50 = 1.657 mg/mL and 0.173 mg/mL, respectively) and cellular antioxidant capacity. Molecular docking showed that AR14 interacted with Val369, and Val420 of Keap1 via hydrogen bonds. Furthermore, AR14 bound to DPPH and ABTS through hydrogen bonding and hydrophobic interactions. Together, our results demonstrate that the DWH-derived antioxidant peptide AR14 exhibits the free radical scavenging and cellular antioxidant activity and can be used to preserve ham quality and promote human health.

Insights into in vitro digestion properties and peptide profiling of Chinese rubing PDO cheese prepared using different acidification technology.

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 ß-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.

iTRAQ-Based Quantitative Proteomic Analysis of Antibacterial Mechanism of Milk-Derived Peptide BCp12 against Escherichia coli.

BCp12 is a novel casein-derived antibacterial peptide with a broad-spectrum antibacterial effect. However, its action mechanism against E. coli is unknown. In this study, the growth curve showed that BCp12 had excellent antibacterial activity against E. coli. Red (propidium iodide staining) and green (fluorescein isothiocyanate staining) fluorescence signals were detected at the edges of the E. coli cells treated with BCp12. scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images showed that E. coli cells became rough and shrunken, and part of the cell contents leaked to form a cavity. Furthermore, the iTRAQ proteome analysis showed that 193 and 174 proteins were significantly up-regulated and down-regulated, respectively, after BCp12 treatment. Four enzymes involved in fatty acid degradation of E. coli were down-regulated, disrupting the synthesis of cell membranes. Molecular docking and gel retardation assays showed that BCp12 could bind to genes encoding four key enzymes involved in the fatty acid degradation pathway through hydrogen bonding and hydrophobic interactions, thus significantly inhibiting their activities. Overall, the results indicate that BCp12 inhibits the growth of E. coli, causing metabolic disorders, thus destroying the structure of cell membranes.

Antimicrobial Peptide BCp12 Inhibits Staphylococcus aureus Growth by Altering Lysine Malonylation Levels in the Arginine Synthesis Pathway.

To adapt to external stimuli, bacteria fine-tune important protein activities using post-translational modifications. The present study provides novel insights into the molecular mechanism of the antimicrobial peptide BCp12. We demonstrate that BCp12 significantly suppressed bacterial growth, induced cell apoptosis, and modulated overall malonylation levels in Staphylococcus aureus cells. Malonylateomic analysis was performed to identify the proteins malonylated by the BCp12 treatment of S. aureus. In total, 53 malonylated proteins (17 up-regulated, 36 down-regulated) were identified as differentially expressed malonylated proteins (DMPs; > 1.5-fold or <0.67-fold, P < 0.05). This result was confirmed via the identification of 21 differential metabolites (DMs; VIP > 1, P < 0.05) in the arginine and proline metabolome. Bioinformatic analysis revealed that the DMPs and DMs were especially enriched in the arginine synthesis pathway. By integrating our lysine malonylational and metabolomic data, we provide new insights into the mechanism by which BCp12 inhibits S. aureus.

Proteomics analysis of the bio-functions of Dregea sinensis stems provides insights regarding milk-clotting enzyme.

Dregea sinensis (D. sinensis) stems have traditionally been used as milk coagulant in Dali of Yunnan Province, China. In this study, proteomics was used to investigate the bio-functions of D. sinensis stem proteins, leading to the purification and identification of the milk-clotting enzyme. A total of 205 proteins mainly involved in the catalytic and metabolic processes were identified, of which 28 proteins exhibited hydrolase activity. Among the 28 proteins, we focused on two enzymes (M9QMC9 and B7VF65). Based on proteomics, a cysteine protease (M9QMC9) with a molecular weight of 25.8 kDa and milk-clotting activity was purified from D. sinensis stems using double ammonium sulfate precipitation and was confirmed using liquid chromatography-mass spectrometry (LC-MS/MS). The milk-clotting temperature using the purified enzyme was around 80 °C (specific activity at 314.38 U/mg), and it was found to be stable in the pH range of 6-9 in NaCl concentration of <0.8 mol/L. These findings indicated that the enzyme isolated from D. sinensis stems has potential in the dairy and food sectors, especially in the cheese-making industry.

Simulated in vitro gastrointestinal digestion of traditional Chinese Rushan and Naizha cheese: Peptidome profiles and bioactivity elucidation.

Chinese Rushan and Naizha, the traditional acid coagulated cheese types produced from cow and yak milk, respectively, have been consumed for more than thousands of years. In this study, we aimed to characterise peptides of Rushan and Naizha in simulated in vitro gastrointestinal digestion using label-free based peptidomic. The identified peptide sequences were subjected to BIOPEP database driven bioactivity search. In total, 309 and 225 peptides were identified from Rushan and Naizha cheese, respectively, corresponding to 20 protein annotations. Analysis of label-free quantification found different protein digestibility, where casein was the primary source of peptides in Rushan, among which 62% represented ß-casein by peptide count. The release of peptides was concentrated in specific residues 145-155 of ß-casein in Rushan. In contrast, κ-casein and 7 minor milk proteins were dominant in digestion of Naizha cheese (p < 0.05). In particular, there were 11 peptides from digestion that were exact matches in databases to sequences with immunomodulatory, antibacterial, ACE-inhibition, DPP IV inhibition and antioxidant activities. Four novel angiotensin I-converting enzyme inhibitory (ACEI) activities peptides (YPFPGPIH, LKNWGEGW, RELEEIR, and HPHPHLS) were explored using molecular docking, chemically synthesized, and in vitro ACEI activity. The peptides had lower estimated free energy values (-5.34 to -7.66 kcal/mol), and exhibited the lowest IC50 value of 109.5, 77.7, 196.6, and 64.30 µM, respectively. Our study is the most comprehensive peptidomic analysis of Chinese Rushan and Naizha cheese to date.