Rapid Revealing of Quorum Sensing (QS)-Regulated PLA, Biofilm and Lysine Targets of Lactiplantibacillus plantarum L3.

Quorum sensing (QS) can regulate the production of multiple functional factors in bacteria, but the process of identifying its regulatory targets is very complex and labor-intensive. In this study, an efficient and rapid method to find QS targets through prediction was used. The genome of Lactiplantibacillus plantarum (L. plantarum) L3 was sequenced and characterized, and then linked the L. plantarum L3 genome to the STRING database for QS system regulatory target prediction. A total of 3,167,484 base pairs (bps) were examined from the genome of L. plantarum L3, and 30 QS-related genes were discovered (including luxS). The STRING database prediction indicated that the 30 QS-related genes are mainly involved in the regulation of nine metabolic pathways. Furthermore, metE, metK, aroB, cysE, and birA1 were predicted to be regulatory targets of the LuxS/AI-2 QS system, and these five targets were validated based on quantitative real-time PCR and content determination. Successful elucidation of the LuxS/AI-2 QS system's key targets and regulation mechanism in L. plantarum L3 demonstrated the effectiveness of the new approach for predicting QS targets and provides a scientific basis for future work on improving regulation of functional factor production.

Revealing the mechanism of flavor improvement of fermented goat milk based on lipid changes.

The mechanism of goat milk (GM) flavor improvement based on lipid changes requires understanding. According to sensory evaluation results, the texture, taste, appearance, aroma, and overall acceptability score of Guishan fermented goat milk (GMF) were higher than those of GM. In total, 779 lipid molecules and 121 volatile compounds were formed from the metabolite-lipid level in the GM and GMF, as determined through lipidomics and gas chromatography-mass spectrometry. The key volatile flavor compounds in the GMF were (E,E)-2,4-decadienal, ethyl acetate, acetoin, 2,3-pentanedione, acetic acid, and 2,3-butanedione. Of them, 60 lipids significantly contributed to the flavor profiles of the GMF, based on the correlation analysis. The triacylglycerides (TAGs) 12:0_14:0_16:0 and 13:0_13:0_18:2 contributed to aroma retention, while TAG and phosphatidylethanolamine were identified as key substrates for flavor compound formation during fermentation. Lipids associated with glycerophospholipid and linoleic acid metabolism pathways significantly affected volatile compound formation in the GMF. This study provides an in-depth understanding of the lipids and flavors of the GMF, and this information will be useful for the development of specific GMF products.

Isolation and identification of milk-clotting proteases from Prinsepia utilis Royle and its application in cheese processing.

The aim of this study was to isolate and identify the main milk-clotting proteases from Prinsepia utilis Royle. Protein isolates obtained using precipitation with 20 %-50 % ammonium sulfate (AS) showed higher milk-clotting activity (MCA) at 154.34 + 0.35 SU. Two milk-clotting proteases, namely P191 and P1831, with molecular weight of 49.665 kDa and 68.737 kDa, respectively, were isolated and identified using liquid chromatography-mass spectrometry (LC-MS/MS). Bioinformatic analysis showed that the two identified milk-clotting proteases were primarily involved in hydrolase activity and catabolic processes. Moreover, secondary structure analysis showed that P191 structurally consisted of 40.85 % of alpha-helices, 15.96 % of beta-strands, and 43.19 % of coiled coil motifs, whereas P1831 consisted of 70 % of alpha-helices, 7.5 % of beta-strands, and 22.5 % of coiled coil motifs. P191 and P1831 were shown to belong to the aspartic protease and metalloproteinase types, and exhibited stability within the pH range of 4-6 and good thermal stability at 30-80 °C. The addition of CaCl2 (<200 mg/L) increased the MCA of P191 and P1831, while the addition of NaCl (>3 mg/mL) inhibited their MCA. Moreover, P191 and P1831 preferably hydrolyzed kappa-casein, followed by alpha-casein, and to a lesser extent beta-casein. Additionally, cheese processed with the simultaneous use of the two proteases isolated in the present study exhibited good sensory properties, higher protein content, and denser microstructure compared with cheese processed using papaya rennet or calf rennet. These findings unveil the characteristics of two proteases isolated from P. utilis, their milk-clotting properties, and potential application in the cheese-making industry.

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.

Novel insights into whey protein among Yak, Yellow Cattle, and Cattle-Yak milk.

This study identified characteristic whey proteins from Zhongdian Yak (ZY), Diqing Yellow Cattle (DYC), and Cattle Yak (CY), revealing insights into their potential functions and released peptides. A total of 118 whey proteins were quantified in milk obtained from the three breeds of cattle, including seven characteristic proteins (IGL@ protein, 40S ribosomal protein S9, calreticulin, etc.) in CY milk and two characteristic proteins (RNA helicase and uncharacterized protein (A0A3Q1LFQ2)) in ZY milk. These characteristic proteins are involved in the phagosome and Fc gamma R-mediated phagocytosis pathways, exhibiting immunoprotective activities, verified through molecular docking. Furthermore, the molecular docking results showed five whey proteins (IGL@ protein, rho GDP-dissociation inhibitor 1, small monomeric GTPase, action-like protein 3, and adenylyl cyclase-associated protein) interacted with TLR4 through multiple hydrogen and hydrophobic bonds. Therefore, these proteins may exert immunomodulatory functions by inhibiting TLR4. Meanwhile, whey proteins produced bioactive peptides, such as antioxidant peptides and ACE inhibitory peptides after simulated gastrointestinal digestion (SGID). The whey proteins and bioactive peptides from CY exhibited more types and activities than the ZY and DYC whey proteins. This study provides a theoretical basis for promoting formula milk powder production.

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.

Preparation and characterization of Pickering emulsion with directionally embedded antimicrobial peptide MOp2 and its preservation effect on grass carp.

The peptide MOp2 obtained from Moringa oleifera seeds showed good antimicrobial activity. However, the stability of its activity has not yet been studied. In the present study, MOp2-loaded thiolated chitosan-stabilized (CMOp2) Pickering emulsion was prepared and applied to prolong the shelf life of grass carp. The encapsulation rate of MOp2 was 57.7% in CMOp2. In addition, the effects of different concentrations of CMOp2 solid particles and pH on droplet size, zeta optional and storage stability of Pickering emulsions were evaluated; the best condition for preparing Pickering emulsion through experiment was 1.75% CMOp2 solid particles at pH 9.5. Moreover, morphological observations and rheological analysis indicated that Pickering emulsions were considered a water-in-oil emulsion with typical non-Newtonian fluid characteristics. Furthermore, the prepared Pickering emulsion could significantly inhibit the growth of Escherichia coli and Staphylococcus aureus. Besides, Pickering emulsion effectively prevented spoilage of grass carp, and the Pickering emulsion-treated group reduced its pH, TVB-N and color values, inhibited microbial growth, and extended shelf life to 9 day at the storage of 4 °C. Overall, the present findings provide a reference for the application of MOp2-loaded Pickering emulsions in food preservation.

BCp12/PLA combination: A novel antibacterial agent targeting Mur family, DNA gyrase and DHFR.

The combination of natural antimicrobial peptide BCp12/phenyllatic acid (BCp12/PLA) presents a more efficient antibacterial effect, but its antibacterial mechanism remains unclear. This study studied the synergistic antibacterial mechanism of BCp12 and PLA against S. aureus. The results demonstrated that the BCp12/PLA combination presented a synergistic antibacterial effect against S. aureus, with a fractional inhibitory concentration of 0.05. Furthermore, flow cytometry and scanning electron microscope analysis revealed that BCp12 and PLA synergistically promoted cell membrane disruption compared with the group treated only with one compound, inducing structural cell damage and cytoplasmic leakage. In addition, fluorescence spectroscopy analysis suggested that BCp12 and PLA synergistically influenced genomic DNA. BCp12 and PLA targeted enzymes related to peptidoglycan and DNA synthesis and interacted by hydrogen bonding and hydrophobic interactions with mur enzymes (murC, murD, murE, murF, and murG), dihydrofolate reductase, and DNA gyrase. Additionally, the combined treatment successfully inhibited microbial reproduction in the storage of pasteurized milk, indicating that the combination of BCp12 and PLA can be used as a new preservative strategy in food systems. Overall, this study could provide potential strategies for preventing and controlling foodborne pathogens.

Identification and Characterization of Novel Umami Peptides from Protein Hydrolysates of Morchella esculenta and Their Interaction with T1R1/T1R3 Receptor.

The study aimed to explore umami peptides derived from protein hydrolysates of Morchella esculenta. According to the electronic tongue and sensory evaluation, the ultrafiltration fractions (<3 kDa) of the protein hydrolysates exhibited the strongest umami taste. The overall flavor of the screened fractions was significantly improved after the Maillard reaction, based on the electronic nose and electronic tongue analyses, and the content of total free amino acid increased from 387.35 to 589.30 µg/mL. A total of 37 peptides with high confidence were identified from the fractions using LC-MS/MS. Additionally, two novel umami peptides were screened through bioinformatics and molecular docking, and their recognition threshold was 0.43 (EYPPLGRFA) and 0.52 mmol/L (TVIDAPGHRDFI), respectively. In addition, molecular docking analysis revealed that the key binding sites, such as Ser148, Leu51, Arg327, and Leu468 in T1R1/T1R3 contributed to docking, and hydrogen bonding and hydrophobic interactions were the dominant interaction forces between the two umami peptides and T1R1/T1R3 receptor. This study contributes to the development and utilization of Morchella esculenta in flavored foods.

Anti-inflammatory effect of two novel peptides derived from Binglangjiang buffalo whey protein in lipopolysaccharide-stimulated RAW264.7 macrophages.

Whey protein hydrolysate from Binglangjiang buffalo, a unique genetic resource, has anti-inflammatory activity, but its anti-inflammatory composition and effects are unknown. The aim of this study was to investigate the anti-inflammatory peptides from Binglangjiang buffalo whey protein hydrolysate. A total of 1483 peptides were identified using LC-MS/MS, and 12 peptides were chosen for chemical synthesis using peptidomics, and then two novel anti-inflammatory peptides (DQPFFHYN (DN8) and YSPFSSFPR (YR9)) were screened out using LPS-stimulated RAW264.7 cells. The molecular weights of DN8 and YR9 with ß-turn conformations were 1067.458 Da and 1087.52 Da, respectively, and showed a high in-vitro safety profile and thermal stability, but were intolerant to pepsin. Furthermore, ELISA and Western blot analysis indicated that peptides DN8 and YR9 significantly suppressed the secretion of pro-inflammatory cytokines NO, TNF-α, and IL-6 and the expression of mediators iNOS, TNF-α, and IL-6 in LPS-stimulated RAW264.7 cells. The study provides insights into the development of novel food-based anti-inflammatory nutritional supplements.

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.

Dissecting LuxS/AI-2 quorum sensing system-mediated phenyllactic acid production mechanisms of Lactiplantibacillus plantarum L3.

The phenyllactic acid (PLA) produced by lactic acid bacteria (LAB) inhibits fungi and facilitates the quality control of fermented milk. A strain of Lactiplantibacillus plantarum L3 (L. plantarum L3) with high PLA production was screened in the pre-laboratory, but the mechanism of its PLA formation is unclear. The amount of autoinducer-2 (AI-2) increased with increasing culture time, as did cell density and PLA. The results in this study suggest that PLA production in L. plantarum L3 may be regulated by the LuxS/AI-2 Quorum Sensing (QS) system. Tandem mass tag (TMT) quantitative proteomics analysis showed that a total of 1291 differentially expressed proteins (DEPs) were quantified in the incubated for 24 h compared with the incubated for 2 h, of which 516 DEPs were up-regulated and 775 DEPs were down-regulated. Among them, S-ribosomal homocysteine lyase (luxS), aminotransferase (araT), and lactate dehydrogenase (ldh) are the key proteins for PLA formation. The DEPs were mainly involved in the QS pathway and the core pathway of PLA synthesis. Furanone effectively inhibited the production of L. plantarum L3 PLA. In addition, Western blot analysis demonstrated that luxS, araT, and ldh were the key proteins regulating PLA production. This study reveals the regulatory mechanism of PLA based on the LuxS/AI-2 QS system, which provides a theoretical basis for the efficient and large-scale production of PLA in industries in the future.

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.

Identification, characterization and in vitro activity of hypoglycemic peptides in whey hydrolysates from rubing cheese by-product.

The by-product of Chinese rubing cheese is rich in whey protein. Whey hydrolysates exhibit good hypoglycemic activity, but which specific peptide components are responsible for this effect have not yet been investigated. Herein, the α-glucosidase inhibitory activity of the ultrafiltered fraction (<3 kDa) of rubing cheese whey hydrolysates was evaluated with the inhibition rate of 37.89 %. In addition, peptide identification was conducted using LC-MS/MS, and three peptides YPVEPF, VPYPQ, and LPYPY were identified. Among these, YPVEPF had higher α-glucosidase inhibitory activity (IC50 = 3.52 mg/mL) and interacted with α-glucosidase via hydrogen bonding and hydrophobic forces. YPVEPF was characterized as an amphipathic peptide rich in antiparallel (50.50 %) and random coil (35.20 %) structures, as well as showed good tolerance to gastrointestinal digestion and incubation under the temperature range of 20-80 °C. Notably, YPVEPF activity increased in the presence of Al3+ and Fe3+, as well as within the pH range of 2.0-6.0. Furthermore, YPVEPF had negligible hemolytic activity at a concentration of 1.0 mg/mL, no toxicity at concentrations below 0.5 mg/mL, and significantly promoted glucose consumption in HepG2 cells (p < 0.0001). Collectively, these findings indicate the potential of YPVEPF to be used as a novel hypoglycemic peptide in functional foods.

Isolation, identification and characterization of a novel antimicrobial peptide from Moringa oleifera seeds based on affinity adsorption.

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 ß-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.

Identification, structure, and caseinolytic properties of milk-clotting proteases from Moringa oleifera flowers.

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 ß-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.

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.