Use of proteomics to elucidate characteristics of Cronobacter sakazakii under mild heat stress.

Cronobacter sakazakii is an opportunistic pathogen known for causing severe diseases. Mild heat treatment is commonly used in food processing, however, the pathogenic characteristics and underlying mechanisms of Cronobacter sakazakii strains remain poorly understood. In this study, we found that mild heat stress (MHS) at 52 °C can induce several deleterious effects in Cronobacter sakazakii, including damage to the cell wall, genomic DNA breakage, and misfolding of cytoplasmic proteins. These conditions lead to a decreased survival ability under acid, desiccation, and osmotic stress; a reduction in biofilm formation; and diminished motility. Notably, surviving C. sakazakii cells retain their pathogenicity, causing significant intestinal damage in newborn mice. This damage is characterized by epithelial sloughing and disruption of the intestinal structure. Tandem mass tag (TMT)-based proteomics identified 736 proteins with differential abundance across C. sakazakii strains subjected to mild heat stress, highlighting adaptations in biofilm formation, motility, and stress tolerance. Key regulatory changes were observed in phospholipid metabolism and protein synthesis, which underpin this complex stress response. This data illustrates a sophisticated balance between environmental adaptability and pathogenic potential. The metabolic and pathogenic responses of C. sakazakii to mild heat stress are closely linked to its phospholipid metabolism and the production of secretory proteins, both crucial for its virulence and reliant on membrane transport. This complex interplay emphasizes the need to understand these mechanisms to develop effective control strategies.

Preparation, characterization, and mechanism of DPP-IV inhibitory peptides derived from Bactrian camel milk.

In this study, double enzyme hydrolysis significantly enhanced the DPP-IV inhibition rate compared to single enzyme. The α + K enzymes exhibited the highest inhibition rate. Ultrasonic pretreatment for 30 min improved the hydrolysis efficiency and DPP-IV inhibition rate, potentially due to the structural changes in hydrolysates, such as the increased surface hydrophobicity, and reduced particle size, α-helix and ß-turn. Six peptides were screened and verified in vitro. QPY, WPEYL, and YPPQVM displayed competitive inhibition, while LPAAP and IPAPSFPRL displayed mixed competitive/non-competitive inhibition. The interactions between these six peptides and DPP-IV primarily occurred through hydrogen bonds, electrostatic and hydrophobic interactions. Network pharmacological analysis indicated that LPAAP might inhibit DPP-IV activity trough interactions with diabetes-related targets such as CASP3, HSP90AA1, MMP9, and MMP9. These results uncover the potential mechanism of regulating blood glucose by camel milk hydrolysates, establishing camel milk peptide as a source of DPP-IV inhibitory peptide.

The casein in sheep milk processed by cold plasma technology: Phosphorylation degree, functional properties, oxidation characteristics, and structure.

Cold plasma is a nonthermal process used for modification of proteins. The objective of this study was to investigate the effect of cold plasma technology on the phosphorylation degree, functional and oxidation properties, and structure of casein in sheep milk. Cold plasma treatment for 3-4 min significantly increased the phosphorylation degree and enhanced functional properties, including water-holding capacity, solubility, foaming capacity and stability. Besides, plasma treatment time profoundly influenced protein oxidation, and treatment for 2 and 3 min could be the preferred conditions to minimize protein change. The protein conformation became unstable with the extension of treatment time. Particle size, polymer dispersity index, and microscopy images confirmed alterations in the protein structure following 3 min of processing. Consequently, using cold plasma treatment at 10 kHz 20 kV for 3 min could be suggested for milk protein modification, providing a basis for the application of high-quality caseins in food processing.

Ratiometric fluorescence platform for the ultrasensitive detection of kanamycin based on split aptamer co-recognition triggers Mg2+-DNAzyme-driven DNA walker systems.

In this work, a novel 3D-DNA walker signal amplification strategy was designed to construct a fluorescent aptasensor for the detection of kanamycin (KAN). The aptasensor utilizes split aptamers for the synergistic recognition of KAN. The presence of KAN induces the split aptamers recombination to form the Mg2+-DNAzyme structure, which is activated by Mg2+ to drive the 3D-DNA walker process for cascading signal amplification. Employing gold nanoflowers (AuNFs) as walking substrate material increases the local DNA concentration to enhance the walker efficiency. The prepared fluorescent aptasensor achieved efficient and sensitive detection of KAN with satisfactory results in the concentration range of 1 × 10-8 - 1 × 10-3 µg/kg and the detection limit of 5.63 fg/kg. Meanwhile, the designed fluorescent aptasensor exhibited favorable specificity, anti-interference, storage stability and reproducibility, and verified the feasibility of its application in milk samples. The present work provides an effective tool for the regulation of KAN contamination in animal-derived foods with promising prospects.

Colorimetric aptasensor utilizing MOF-235 with exceptional peroxidase-like activity for the detection of oxytetracycline residues in raw milk.

In this work, a simple, convenient and cost-effective colorimetric aptasensor was successfully constructed for the detection of antibiotic residues in raw milk based on the property that aptamer (Apt) synergistically enhances the catalase-like activity of MOF-235. Under optimised conditions, the proposed colorimetric aptasensor exhibited a wide detection range (15-1500 nM) with a low detection limit (6.92 nM). Furthermore, the proposed aptasensor demonstrated high selectivity, good resistance to interference and storage stability. The proposed aptasensor was validated by spiking recovery in camel milk, cow milk and goat milk with satisfactory recoveries, which demonstrated the great potential of the aptasensor for further application in real food samples, and also suggested that MOF-235 can be used as a potential universal platform to build a sensitive detection platform for other targets.

Oligosaccharide profiles as potential biomarkers for detecting adulteration of caprine dairy products with bovine dairy products.

Adulteration of caprine dairy products raises concerns among consumers. This study aimed to identify the differences in oligosaccharide profiles of caprine dairy products, including raw milk, colostrum powder, and lactose powder, and their corresponding bovine dairy products, and provide new insights for detecting adulteration of bovine dairy products in caprine dairy products. Twenty-seven oligosaccharides were detected in caprine and bovine dairy products. The principal component analysis plot of the oligosaccharide profiles clearly differentiated among the six types of dairy products. Specific oligosaccharides that were most distinctive for caprine and bovine dairy products were identified. Lacto-N-triose (LNTri) could be used as a potential biomarker for distinguishing caprine milk from bovine milk, caprine colostrum powder from bovine colostrum powder, and caprine lactose powder from bovine lactose powder. The results demonstrated that oligosaccharides could be used as biomarkers for detecting bovine dairy products in caprine dairy products, especially caprine lactose powder.

Conjugation of dual-natural milk-derived proteins with fucoidan to prepare controllable glycosylation products via dielectric barrier discharge cold plasma.

This study reported that fibrillar bridges (whey protein isolate nanofibrils, WPNs) were used to associate the casein (CA) nanoparticles through the pH-driven method to obtain the self-assembled WPN-CA complexes. Then, a novel technology involving cold plasma (CP) was innovatively proposed to enhance the protective properties of complexes. The confirmation of structural transitions and interactions resulting from the adjustment of WPN-to-CA ratios (WtCs) led to the identification of the complexes named WPCA (WtC1.0:1). Next, the results showed a rapid conjugation between WPCA and fucoidan (FD) with a degree of grafting of 16.03 % after 10 min CP treatment. The coupling of WPCA with FD to form conjugates was confirmed by SDS-PAGE analysis, indicating covalent bonds' formation. FTIR spectroscopy revealed an augmentation in the intensity of the OH stretching vibration of the WPCA-FD conjugate, concomitant with a decrease in ß-turns and an elevation in ß-sheets content. Furthermore, the application of glycosylation treatment to WPCA-FD resulted in a noteworthy enhancement of both the thermal stability and antioxidant activity characteristics of WPCA. Our findings move a step forward, as CP-assisted Maillard reaction has shown potential as an efficient and energy-saving method to enhance the functional properties of milk-derived proteins in the food industry.

Galactooligosaccharide Mediates NF-κB Pathway to Improve Intestinal Barrier Function and Intestinal Microbiota.

The use of antibiotics to treat diarrhea and other diseases early in life can lead to intestinal disorders in infants, which can cause a range of immune-related diseases. Intestinal microbiota diversity is closely related to dietary intake, with many oligosaccharides impacting intestinal microorganism structures and communities. Thus, oligosaccharide type and quantity are important for intestinal microbiota construction. Galactooligosaccharides (GOS) are functional oligosaccharides that can be supplemented with infant formula. Currently, information on GOS and its impact on intestinal microbiota diversity and disorders is lacking. Similarly, GOS is rarely reported within the context of intestinal barrier function. In this study, 16S rRNA sequencing, gas chromatography, and immunohistochemistry were used to investigate the effects of GOS on the intestinal microbiota and barrier pathways in antibiotic-treated mouse models. The results found that GOS promoted Bifidobacterium and Akkermansia proliferation, increased short-chain fatty acid levels, increased tight junction protein expression (occludin and ZO-1), increased secretory immunoglobulin A (SIgA) and albumin levels, significantly downregulated NF-κB expression, and reduced lipopolysaccharide (LPS), interleukin-IL-1ß (IL-1ß), and IL-6 levels. Also, a high GOS dose in ampicillin-supplemented animals provided resistance to intestinal damage.

Camel milk peptides alleviate hyperglycemia by regulating gut microbiota and metabolites in type 2 diabetic mice.

This study aimed to investigate the hypoglycemic effect of Camel milk peptides (CMPs) on Type 2 diabetes mellitus (T2DM) mice and reveal its related mechanism from the aspect of gut microbiota and metabolites. The administering CMPs significantly alleviated the weight loss, polydipsia and polyphagia, reduced fasting blood glucose (FBG), improved insulin resistance and sensitivity, and restored the level of serum hormones, lipopolysaccharide (LPS), lipid metabolic and tissue damage. Furthermore, CMPs intervention remarkably reversed gut microbiota dysbiosis in T2DM mice by reducing the relative abundance of Proteobacteria, Allobaculum, Clostridium, Shigella and the Firmicutes/Bacteroidetes ratio, while increasing the relative abundance of Bacteroidetes and Blautia. Metabolomic analysis identified 84 different metabolites between T2DM and CMPs-treated groups, participating in three pathways of Pantothenate and CoA biosynthesis, Phenylalanine metabolism and Linoleic acid metabolism. Ureidopropionic acid, pantothenic acid, hippuric acid, hydrocinnamic acid and linoleic acid were identified as key acidic metabolites closely related to hypoglycemic effect. Correlation analysis indicated that CMPs might have a hypoglycemic effect through their impact on gut microbiota, leading to variations in short-chain fatty acids (SCFAs), acidic metabolites and metabolic pathways. These findings suggest that CMPs could be a beneficial nutritional supplement for intervention T2DM.

Comprehensive investigation of milk oligosaccharides in different mammalian species and the effect of breed and lactation period on sheep milk oligosaccharides.

Milk oligosaccharides (MOs) have unique health benefits for newborns, and MOs are important components in mammalian milk. The present study was conducted to provide a comprehensive analysis of MOs in important domestic animals, including goats, cows, camels and sheep. The comparison with human MOs was conducted simultaneously. Furthermore, analysis of the relative abundance of sheep MOs among different breeds (Hu sheep, East Friesen sheep, East Friesen-Hu crossbred sheep) and lactation periods (colostrum, mature milk) was performed. In general, 35, 24 19, 26, and 16 MOs were identified in human, goat, bovine, camel and sheep milk, respectively. The type of sheep MOs was not greatly influenced by the breeds and lactation period. Hu sheep colostrum had the highest abundance of MOs among six sheep milks, followed by East Friesen sheep colostrum, while East Friesen-Hu crossbred sheep mature milk had the lowest abundance of MOs. These findings provide evidence for the potential value of MOs from domestic animal milk for the commercial applications.

A new insight into the polar lipid composition in mature breast milk and ewe milk with comparative lipidomics analysis.

Polar lipids play essential biological functions in energy storage, both as structural components of cell membranes and as signaling molecules. In this study, a comprehensive UHPLC-QTRAP-MS-based lipidomic analysis of mature breast milk (BM) and ewe milk (EM) was conducted. Through the analysis, a total of 362 polar lipid species from 14 subclasses were characterized, including 60 phosphatidylethanolamines (PEs), 59 phosphatidylcholines (PCs), 38 phosphatidylinositols (PIs), 35 sphingomyelins (SMs), and 34 ceramides (Cers). Of these, 139 lipid molecules were screened as significantly differentially expressed polar lipids (SDPLs) between the two kinds of milk based on the following criteria: a variable importance in projection (VIP) value > 1.0, a false discovery rate-adjusted P-value < 0.0001, and a fold change (FC) of either > 2.0 or < 0.5; these included 111 upregulated and 28 downregulated SDPLs in EM compared to BM. Among these SDPLs, the content of PE (16:1_18:0) was found to be significantly higher in EM compared to BM (FC = 69.5853, P < 0.0001). Moreover, sphingolipid metabolism and glycerophospholipid metabolism were determined to be vital metabolic pathways. This was derived from the finding that PE, PC, SM, and PI were key lipid metabolites in the two kinds of milk that were related to these two metabolic pathways. This study provides new insights into the characterization of SDPLs in mammalian milk, and also provides a theoretical basis for optimizing infant formula.

Comparison of properties and application of starch nanoparticles optimized prepared from different crystalline starches.

Starch nanoparticles (SNPs) were produced by nanoprecipitation combined with ultrasonication with the use of different starches (corn, potato and sago starch) and used to stabilize Pickering emulsions. The orthogonal experiment was used to optimize preparation conditions of gelatinization pretreatment duration of 30 min, ultrasonic power of 600 W, and ultrasonic time of 40 min. Compared with native starch, the SNPs were spherical in shape and displayed a V-type crystalline structure with low relative crystallinity and higher degree of double-helix. Compared with native starch-Pickering emulsion, the SNP-Pickering emulsion had a smaller droplet size, more uniform distribution, clearer oil/water interface, and higher static stability of droplets. The sago SNP-Pickering emulsion had the great gelatinous structure and emulsion stability. In addition, the SNP-Pickering emulsion had the better loading efficiency and controlled release performance of curcumin. Meanwhile, the bioavailability of curcumin in sago SNP-Pickering emulsion was highest.

Characterization and comparison of lipids from human and ewe colostrum based on lipidomics analysis.

Colostrum is essential for immune system development and has a protective role for infants in early life. However, the lipid compositions of human and ewe colostra have not been characterized. We hypothesized that lipidomics can be used to compare lipids in two mammalian colostra. Herein, 1004 lipids assigned to 26 subclasses were identified in both human and ewe colostra using a quantitative lipidomics approach. In total, 173 significantly different lipids (SDLs) were investigated (variable importance in projection > 1.1, fold change (FC) ≥ 2 or ≤0.5, and P < 0.0001). Four potential lipid biomarkers, namely, DG (19:0/18:0), TG (10:0/15:0/16:0), FFA (22:0), and TG (18:1/24:1/18:2), were selected from the 173 SDLs based on FC values. These different lipids were involved in 44 metabolic pathways, of which sphingolipid metabolism and glycerophospholipid metabolism were the major pathways. Our results improve the understanding of the differences between human and ewe colostra lipids.

Mechanisms of Mitochondrial Apoptosis-Mediated Meat Tenderization Based on Quantitative Phosphoproteomic Analysis.

This study investigates the mechanism of phosphorylation in the regulation of apoptosis-mediated meat tenderization during postmortem aging. The results found that the pork muscle exhibited apoptotic potential at early postmortem (48 h) and showed more tenderness at late postmortem, as evidenced by the increase in mitochondrial membrane permeability (MMP), Ca2+ level, reactive oxygen species (ROS) content, and caspases activity at 0 h to 48 h, and decreases in ATP level at 0 h to 24 h and shear force at 12 h to 120 h (p < 0.05). Phosphoproteomic analysis revealed that phosphorylation regulated apoptosis by modulating ATP and calcium bindings as well as apoptotic signaling, which occurred within early 12 h and mainly occurred at 12 h to 48 h postmortem. Moreover, differential expression of phosphoproteins demonstrated that phosphorylation regulated oxidative stress-induced apoptosis and rigor mortis, thereby promoting the development of meat tenderness. Our results provide insights into the roles of phosphorylation in various physiological processes that affect meat tenderness.

Variations in Oligosaccharides and N/O-Glycans in Human Milk through the Eight-Month Lactation Period.

Oligosaccharides and N/O-glycans are abundant in human milk and have numerous biological functions (for instance sialylated glycans provide sialic acid for the growth of infant brains), but their variation trends during lactation need further exploration. Qualitative and quantitative analyses of oligosaccharides and N/O-glycans in human milk at different lactation stages (from 7 days to 8 months) were performed using UHPLC-ESI-MS/MS. Thirty-four oligosaccharides, twenty-three N-glycans, and six O-glycans were identified. Oligosaccharides showed the highest abundance in human colostrum and decreased with the progression of lactation, and the abundance of N/O-glycans fluctuated as lactation progressed, while a high abundance of sialylated oligosaccharides and sialylated N/O-glycans was observed in human colostrum. These findings provide evidence for breastfeeding support and contribute to the development of infant formula supplemented with human milk glycans.

Changes of bacterial microbiota and volatile flavor compounds in ewe milk during dielectric barrier discharge cold plasma processing.

Dielectric barrier discharge cold plasma (DBD-CP) processing can be considered as a potential alternative to conventional pasteurization in ewe milk, which has previously been shown. However, residual microorganisms in ewe milk during DBD-CP processing have not yet been evaluated. This study investigated the microbiota present in ewe milk that was cold plasma-treated. Pasteurized ewe milk was used as a positive control. The bacterial community analysis demonstrated that 8 phyla and 16 genera were identified as dominant microbiota, and Proteobacteria and Pseudomonas were the predominant phyla and genera in cold plasma-treated milk samples. In addition, 23, 22, and 16 volatile flavor compounds (VFCs) were detected in raw milk, pasteurized milk, and cold plasma-treated milk samples, respectively. Fourteen different VFCs were produced according to the variable importance in the projection (VIP ≥ 1.0). The application of DBD-CP might lead to the absence of mutton odor produced by some carboxylic acids, further improving the characteristic flavor of ewe milk by facilitating lipid oxidation. Six bacterial genera, including Porphyromonas, Streptococcus, Fusobacterium, Prevotella, Treponema_2 and Leptotrichia, were highly negatively correlated (|ρ| > 0.5 and P < 0.05) with the flavor substances, according to Spearman correlation coefficient analysis. Overall, the systematic method employed in this study could identify core microbiota present and important VFCs in cold plasma-treated milk ewe milk. The study also provided a better understanding of the potential relationship between bacterial profiles and flavor compounds, which might be useful for further enhancing the quality of ewe milk products.

Comparative Lipidomics Analysis of Human and Ruminant Milk Reveals Variation in Composition and Structural Characteristics.

In the present study, the different lipidomes between human milk and ruminant milk were compared. The 471, 376, 467, and 87 differential lipids were identified in human versus cow, goat, sheep, and camel groups, respectively. According to multivariate statistical analysis, lipids in human and camel milk were closer but differed from other milk. The distributions of long-chain and polyunsaturated fatty acids of triglycerides (TGs), the proportions of functional TGs (OPO, OPL, and PPO), and many kinds of phospholipids (PLs) (PS, PI, GD, GM3, and Cer) in human milk were similar to those in camel milk. The similar structure of TGs and proportion of PLs in human milk to camel milk might contribute to their similar digestion and bioactivity properties. Camel milk could be considered as a new resource of lipid base for infant formula. Minor PLs should also be considered for designing formula. Our results provide a new sight for humanized lipids in infant formula.

The probiotic Companilactobacillus crustorum MN047 alleviates colitis-associated tumorigenesis via modulating the intestinal microenvironment.

Supplementation of probiotics is a promising method to alleviate colorectal cancer (CRC) via modulating the intestinal microenvironment. This study aimed to assess the potential anti-CRC effect of Companilactobacillus crustorum MN047 on an azoxymethane and dextran sulfate sodium-induced colitis-associated (CA)-CRC mouse model. Mice were gavaged with C. crustorum MN047 once daily (∼1 × 109 CFU per mouse). The CA-CRC ameliorating effect of this strain was investigated based on the gut microbiota, inflammation and intestinal barrier integrity. Results showed that C. crustorum MN047 could significantly attenuate tumorigenesis and inflammation via suppressing the TLR4/NF-κB pathway. Moreover, this probiotic could improve the intestinal barrier integrity by increasing the mRNA level of some tight junction-related proteins and reducing goblet cell loss. In addition, C. crustorum MN047 administration led to an increase in beneficial bacteria and a decrease in harmful bacteria, thereby increasing SCFAs and reducing LPS levels. These results suggested that C. crustorum MN047 could partially ameliorate the formation of CA-CRC by modulating the gut microbiota, attenuating inflammation and enhancing the intestinal barrier integrity. Therefore, C. crustorum MN047 was a promising probiotic supplement for attenuating CA-CRC.

Structural evaluation of cytochrome c by Raman spectroscopy and its relationship with apoptosis and protein degradation in postmortem bovine muscle.

Structural changes of cytochrome c and its relationship with apoptosis and protein degradation of bovine muscle during postmortem aging were investigated. Results from amide I and amide II ~ VI showed that the π* orbital d electron decreased, the π electron density increased, and the frequency of the C-N stretching vibration increased. The distance between heme Fe and N atoms of the porphyrin decreased, the bond length decreased, and the heme core size decreased. Besides, Fe ligand vibration related Raman bands of cytochrome c had red (right) shift gradually with the extension of aging. The apoptotic rate and the degradation products of desmin and troponin-T were increased (P < 0.05). Correlation analysis results suggested that Fe ligand vibration, not amide I ~ VI related Raman bands were correlated with cytochrome c mediated apoptosis and degradation of myofibrillar protein of bovine muscle during aging.

Microbiome-metabolomic analyses of the impacts of dietary stachyose on fecal microbiota and metabolites in infants intestinal microbiota-associated mice.

BACKGROUND: The intestinal microbiota and metabolites play an important role in human health and immunity. However, few studies have investigated the long-term effects of stachyose on the human intestinal microbiota and metabolism. Therefore, in this study, the feces of infants were transplanted into germ-free mice, and the effect of long-term stachyose intake on intestinal metabolism was examined by comparing the results of microbiome and metabolome analyses. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to study the effects of stachyose intake on the metabolites and metabolic pathways of the transplanted human intestinal microbiota. RESULTS: We observed that stachyose significantly altered the composition of the intestinal microbiota and metabolites, up-regulated production of the metabolite taurocholic acid, down-regulated amino acid metabolism, and significantly regulated the metabolism of taurine and hydroxytaurine, pantothenate and coenzyme A (CoA) biosynthesis, and other signaling pathways. CONCLUSION: These findings may provide a basis for elucidating the mechanism by which stachyose promotes host health. © 2020 Society of Chemical Industry.