Enhancing immune regulation in vitro: the synergistic impact of 3'-sialyllactose and osteopontin in a nutrient blend following influenza virus infection.

Natural components of breast milk, human milk oligosaccharides (HMOs) and osteopontin (OPN) have been shown to have a variety of functional activities and are widely used in infant formulas. However, the preventive and therapeutic effects of both on influenza viruses are not known. In this study, antiviral assays using a human laryngeal carcinoma cell line (HEP-2) showed that 3'-sialyllactose (3'-SL) and OPN had the best antiviral ability with IC50 values of 33.46 µM and 1.65 µM, respectively. 3'-SL (10 µM) and OPN (4 µM) were used in combination to achieve 75% inhibition. Further studies found that the combination of 200 µg/mL of 3'-SL with 500 µg/mL of OPN exerted the best antiviral ability. The reason for this was related to reduced levels of the cytokines TNF-α, IL-6, and iNOS in relation to mRNA expression. Plaque assay and TCID50 assay found the same results and verified synergistic effects. Our research indicates that a combination of 3'-SL and OPN can effectively reduce inflammatory storms and exhibit anti-influenza virus effects through synergistic action.

Characteristics of casein phosphopeptides in Chinese human milk and its correlation with infant growth: A cross-sectional study.

This research aimed to investigate the characteristics of casein phosphopeptides in Chinese human milk, and their potential relationship to infant growth. Using the liquid chromatography-Orbitrap-mass spectrometry technique, a total of 15 casein phosphopeptides were identified from 200 human milk samples. Also, our results indicate that casein phosphopeptides were phosphorylated with only one phosphate. The relative concentrations of casein phosphopeptides at 6 months postpartum were increased compared with milk at 2 months (FDR < 0.05). Significantly positive correlations were observed between casein phosphopeptides and infant growth, as shown by four casein phosphopeptides were positively correlated with the infants' weight-for-age Z-scores (rs range from 0.20 to 0.29), and three casein phosphopeptides were positively correlated with the infants' length-for-age Z-scores (rs range from 0.19 to 0.27). This study is the first to reveal the phosphorylated level and composition of casein phosphopeptides in Chinese human milk, and their potential relationship with infant growth.

Effect of different salts on the foaming properties of model protein systems for infant formula.

This multiscale study aimed to evaluate the effects of different salts (NaCl, KCl, MgCl2, and CaCl2) on the foaming capacity (FC) and foam stability (FS) of model protein systems (MPS) for infant formula via changes in surface and structural properties. Our results showed that the FC and FS of MPS were increased with the addition of NaCl, KCl, and MgCl2, whereas CaCl2 significantly decreased FC (79.5 ± 10.6%) and increased FS (93.2 ± 2.2%). The surface hydrophobicity was increased and the net charge and surface tension were reduced after the addition of salts. Structural analysis revealed the reduction of intensity of intrinsic fluorescence spectroscopy and UV absorption, and the conversion of α-helix into ß-strand, which was attributed to protein agglomeration. Additionally, MgCl2 and CaCl2 exhibited larger size and lower net charge compared with NaCl and KCl, indicating a greater ability to bind to charged amino acids and form larger aggregates. Correlation analysis indicated that FC was positively related to adsorbed protein and ß-turn and negatively correlated with particle size. In addition, FS showed a positive correlation with ß-strand, apparent viscosity, and zeta potential. However, it exhibited a negative correlation with ß-turn, α-helix, and sulfhydryl content. These results provide a theoretical reference for further understanding of the effect of salts on the foaming properties of MPS.

pH-induced physiochemical and structural changes of milk proteins mixtures and its effect on foaming behavior.

Milk proteins are well known to produce aerated food due to the amphiphilicity. However, milk proteins are commonly added in blends for the desirable properties in food industry. In this study, the foaming properties of milk protein mixtures (MPM), a mixtures of whey protein isolated (WPI) and milk protein concentrate (MPC), was studied through foaming capacity (FC), foam stability (FS), and foam morphology at pH 3.0-9.0. Physiochemical, structural, surface properties, and Pearson correlation analysis were measured to gain insight into foaming behavior. Results indicated that MPM showed excellent FC (113.0-114.3 %) and FS (90.7-93.0 %) at pH 6.0-9.0, and foam displayed a smaller size and uniform distribution. MPM solutions showed smaller particles, higher solubility, and lower apparent viscosity at pH 6.0-9.0, which resulted in an increase in surface pressure and adsorption rate (Kdiff), facilitating more protein absorbed to interface. To further investigate structural changes, various spectral methods were used, in which the structure of MPM was changed with pH. Correlation analysis further suggests that Kdiff and solubility positively affect the formation of foam, while free sulfhydryl and ß-sheet contributed to stabilizing foams. These findings provide valuable information on MPM as ingredients for aerated foods under acidic, neutral, and alkaline conditions.

Effects of Transglutaminase Concentration and Drying Method on Encapsulation of Lactobacillus plantarum in Gelatin-Based Hydrogel.

Lactobacillus plantarum is a kind of probiotic that benefits the host by regulating the gut microbiota, but it is easily damaged when passing through the gastrointestinal tract, hindering its ability to reach the destination and reducing its utilization value. Encapsulation is a promising strategy for solving this problem. In this study, transglutaminase (TGase)-crosslinked gelatin (GE)/sodium hexametaphosphate (SHMP) hydrogels were used to encapsulate L. plantarum. The effects of TGase concentration and drying method on the physiochemical properties of the hydrogels were determined. The results showed that at a TGase concentration of 9 U/gGE, the hardness, chewiness, energy storage modulus, and apparent viscosity of the hydrogel encapsulation system were maximized. This concentration produced more high-energy isopeptide bonds, strengthening the interactions between molecules, forming a more stable three-dimensional network structure. The survival rate under the simulated gastrointestinal conditions and storage stability of L. plantarum were improved at this concentration. The thermal stability of the encapsulation system dried via microwave vacuum freeze drying (MFD) was slightly higher than that when dried via freeze drying (FD). The gel structure was more stable, and the activity of L. plantarum decreased more slowly during the storage period when dried using MFD. This research provides a theoretical basis for the development of encapsulation technology of probiotics.

Structure Characterization and Functional Properties of Flaxseed Protein-Chlorogenic Acid Complex.

This study aimed to investigate the effects of the covalent binding of flaxseed protein (FP) and chlorogenic acid (CA) on the structure and functional properties of FP-CA complexes fabricated using the alkali method. The results suggested that the encapsulation efficiency of CA encapsulated by FP ranged from 66.11% to 72.20% and the loading capacity of CA increased with an increasing addition ratio of CA with a dose-dependent relationship, which increased from 2.34% to 10.19%. The particle size, turbidity, zeta potential and PDI of FP and the FP-CA complexes had no significant discrepancy. UV-Vis and fluorescence spectra showed the existence of the interaction between FP and CA. SEM images showed that the surface of the FP-0.35%CA complex had more wrinkles compared to FP. Differential scanning calorimetry analysis indicated the decomposition temperature of FP at 198 °C was higher than that (197 °C) of the FP-0.35%CA complex, implying that the stability of the FP-CA complexes was lower than FP. The functional properties suggested that the FP-CA complex with 1.40% CA had a higher water holding capacity (500.81%), lower oil holding capacity (273.495%) and lower surface hydrophobicity. Moreover, the FP-CA complexes had better antioxidant activities than that of FP. Therefore, this study provides more insights for the potential application of FP-CA covalent complexes in functional food processing.

Noncovalent Interaction of Lactoferrin with Epicatechin and Epigallocatechin: Focus on Fluorescence Quenching and Antioxidant Properties.

Lactoferrin (LF) from bovine milk possesses antioxidant activity, immune regulatory and other biological activities. However, the effects of epicatechin (EC) and epigallocatechin (EGC) interacting with LF on the antioxidant activity of LF have not been investigated. Therefore, this study aimed to explore their interaction mechanism and the antioxidant activity of LF. UV spectra revealed that EGC (100 µM) induced a higher blue shift of LF at the maximum absorption wavelength than that of EC (100 µM). Fluorescence spectra results suggested that LF fluorescence was quenched by EC and EGC in the static type, which changed the polarity of the microenvironment around LF. The quenching constants Ksv (5.91 × 103-9.20 × 103) of EC-LF complexes at different temperatures were all higher than that (1.35 × 103-1.75 × 103) of the EGC-LF complex. EC could bind to LF via hydrophobic interactions while hydrogen bonding and van der Waals forces drove the binding of EGC to LF. Both the EC-LF complex and EGC-LF complex could bind to LF with one site. EGC formed more hydrogen bonds with LF than that of EC. The antioxidant activity of LF was increased by the high addition level of EC and EGC. These findings would provide more references for developing LF-catechin complexes as functional antioxidants.

Supplementing maternal diet with milk oligosaccharides and probiotics helps develop the immune system and intestinal flora of offsprings.

Intestinal flora is very important for improving the development of the immune system in newborns. Maternal diet during pregnancy and lactation is one of the key factors affecting the growth and development of offspring. The objective of the present study was to examine whether supplementation of maternal diet with milk oligosaccharides and Bifidobacterium could influence the development of the intestinal flora and immune system of neonatal mice. In total, 30 pregnant Institute of Cancer Research (ICR) mice were randomly divided into six groups: a control group (basal diet) and five intervention groups (basal diet supplemented with different doses of 2'-fucosyllactose [2'-FL] and Bifidobacterium Bb12) during the pregnancy period. All female mice were monitored for physical health during gavage. After delivery, the number of mice in each litter, any deformity, and the development of the offspring were recorded. The spleen, blood, and fecal samples of six groups of 10-12 day-old offspring were collected. The results demonstrated that maternal milk oligosaccharides and probiotics conferred protective effects against lipopolysaccharide (LPS)-induced immunosuppression in mice offspring by significantly enhancing the immune organ indexes, splenocyte proliferation, immunoglobulin (immunoglobulin G, A, M) production as well as improving the macrophage phagocytosis (p < .05). The abundance of Lactobacilli and Bifidobacteria in the feces of offspring mice in the intervention groups was significantly higher than that of the offspring mice in the control group (p < .05). These findings suggest that the combination of 2'-FL and Bifidobacterium Bb12 displayed synergistic interactions between the two components that could promote the development of the immune system of the offsprings and improve their microbiota through maternal ingestion.

Characterization of the odor compounds in human milk by DHS/GC × GC-O-MS: A feasible and efficient method.

Odor analysis of human milk (HM) is often challenging. Here, a new strategy for the analysis of odorants in HM using dynamic headspace sampling combined with comprehensive two-dimensional gas chromatography-olfactometry-mass spectrometry (DHS/GC × GC-O-MS) was established based on the comparison of different extraction methods and instrument modes. Overall, DHS/GC × GC-O-MS was proved to be able to provide higher extraction efficiency and better analytical results of the odor-active compounds (OACs) in HM, meanwhile, the salt addition during the extraction further promoted the release of the odorants. Twenty key OACs in HM were identified by flavor dilution analysis and odor activity calculation, of which 1-octen-3-one, 2,3-butanedione, (E)-2-nonenal, and nonanal contributed significantly to the odor of HM. In addition, 2,3-pentanedione was detected as a key OAC in HM for the first time. This study provided a powerful analytical strategy for the comprehensive odor analysis of HM.

Analytical Ultracentrifugation-Calibrated Anion-Exchange Chromatography for Sensitive and Intact Determination of Osteopontin in Infant Formula and Dairy Products.

Osteopontin is a crucial protein ingredient that has been applied in fortified dairy products and infant formula. It has great significance to infant gut health and brain development. However, current techniques including enzyme-linked immunosorbent assay and liquid chromatography coupled with mass spectrometry are still facing the bottleneck of low sensitivity and indirect quantification. Moreover, the unavailable certified commercial OPN standard hinders its accurate quantification. Herein, a novel method of anion-exchange chromatography was established to determine OPN concentration in several dairy matrices. The polarity-reversed capillary isoelectric focusing was utilized to measure the exact isoelectric point (pI) to support method development for OPN separation. Analytical ultracentrifugation was used to calibrate the purity of intact OPN to develop an in-house reference standard. The method showed the merits of limits of detection to 0.04 mg/100 g, relative standard deviation of reproducibility <5% for 13 out of 14 tested matrices, and an average recovery rate of 101.3%. This method has shown the potential to be adopted as an international standard method for the quantification of intact OPN in infant formula and dairy products.

Determination of oxylipins and their precursors in breast milk by solid phase extraction-ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry.

Oxylipins and their precursors (long-chain polyunsaturated fatty acids, LCPUFAs) are key intercellular signaling molecules influencing the inflammatory response. Each oxylipin has pro- and/or anti-inflammatory effects, and the relative abundance of different oxylipins can alter the inflammatory balance, making it important to clarify the oxylipin profile of breast milk for optimal infant health. The extraction, identification, and simultaneous quantification of oxylipins in breast milk are challenging due to the structural similarity, limited stability, and the low endogenous concentration of oxylipins and the complex matrix of breast milk. This study aimed to develop a solid phase extraction-ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry (SPE-UPLC-MS/MS) method for the comprehensive and specific quantification of oxylipins and their precursors in breast milk. The LC conditions (including column, mobile phase, and gradient conditions) and SPE procedure (including SPE cartridges, elution solvent, and elution volume) were optimized to achieve accurate quantification and better analyte recovery. A single 18-minute chromatographic run allows for the quantification of 20 oxylipins and 5 PUFAs. The results showed good linearity (R2 > 0.99) over the concentration range of 2 to 100 ng/mL, with the instrument detection limits ranging from 0.01 to 0.90 ng/mL for oxylipins and 0.02 to 0.59 ng/mL for PUFAs. The method is rapid, sensitive, and reproducible (RSD ≤ 10%) and is suitable for the quantitative analysis of oxylipins and their precursors in infant formula samples.

Antibiofilm effects of punicalagin against Staphylococcus aureus in vitro.

Staphylococcus aureus is a common foodborne pathogen which can form biofilms to help them resist to antimicrobials. It brings great harm to human health. Punicalagin has good antimicrobial activities against S. aureus, but its effect on biofilm formation has not been clearly illustrated. The aim of this study was to explore the antibiofilm effects of punicalagin against S. aureus. Results showed that punicalagin did not significantly interfere with the growth of S. aureus at the concentrations of 1/64 MIC to 1/16 MIC. The biomass and metabolic activity of biofilms were significantly reduced when exposed to sub-inhibitory concentrations of punicalagin. The number of viable cells in the biofilms was also decreased after punicalagin treatment. Scanning electron microscopy and confocal laser scanning microscopy images confirmed that punicalagin damaged the structure of biofilms. The antibiofilm mechanism was partly due to the modification of the cell surface which led to the reduction of cell surface hydrophobicity. These findings suggest that punicalagin has the potential to be developed as an alternative to control S. aureus biofilms.

Bifidobacterium bifidum E3 Combined with Bifidobacterium longum subsp. infantis E4 Improves LPS-Induced Intestinal Injury by Inhibiting the TLR4/NF-κB and MAPK Signaling Pathways In Vivo.

Changes in the functions of the intestinal barrier occur in parallel with the development of sepsis. The protection by Bifidobacterium strains (BB, BL, BB12, and BLBB) was evaluated in mice injected with lipopolysaccharide (LPS). The results revealed an increase in the ratio of ileal villus length to crypt depth in the BLBB group compared with that in the LPS group, as were the number of IgA+ plasma, CD4+/CD8+ T, and dendritic cells. The levels of diamine oxidase (DAO) and d-lactic acid in the serum were lessened in the BLBB group after LPS injection compared with that in the LPS group. In addition, the BLBB group exhibited an increased expression level of tight junction proteins (zonula occludens-1, occludin, and claudin-1), mucin (MUC2) mRNA, reduced NFκB/MAPK signaling pathways, and decreased expression levels of inflammatory cytokines (IL-1ß, IL-6, and TNF-α). The BLBB group enriched the relative abundance of Muribaculaceae, Lachnospiraceae_NK4A136_group, Clostridia_Ucg-014, and Alistipes, resulting in an increase in strains producing short-chain fatty acids. Furthermore, the BLBB group leads to higher levels of deoxycholic acid and biosynthesized linoleate. This study suggested that the BLBB group could enhance the capacity of the intestinal barrier and intestinal mucosal immunity, reduce intestinal inflammation, and improve the composition of gut microbiota. Bifidobacterium bifidum E3 combined with Bifidobacterium longum subsp. infantis E4 may thus serve as a probiotic against the intestinal injury caused by LPS.

Elucidating gut microbiota and metabolite patterns shaped by goat milk-based infant formula feeding in mice colonized by healthy infant feces.

The gut microbiota plays an evolutionarily conserved role in host metabolism, which is influenced by diet. Here, we investigated differences in shaping the gut microbiota and regulating metabolism in cow milk-based infant formula, goat milk-based infant formula, and mix milk-based infant formula compared with pasteurized human milk. 16S rRNA results showed that goat milk-based infant formula selectively increased the relative abundance of Blautia, Roseburia, Alistites and Muribaculum in the gut compared to other infant formulas. Metabolomics identification indicated that goat milk-based infant formula mainly emphasized bile acid biosynthesis, arachidonic acid metabolism and steroid biosynthesis metabolic pathways. Metabolites associated with these metabolic pathways were positively associated with increased microorganisms in goat milk-based infant formula, particularly Alistipes. Furthermore, we found a deficiency of Akkermansia abundance in three infant formula-fed compared to pasteurizedhuman milk-fed. This study presents new insights into the improvement and application of goat milk-based infant formulas in terms of intestinal microecology.

Antibiofilm Effects of Oleuropein against Staphylococcus aureus: An In Vitro Study.

Staphylococcus aureus has posed a huge threat to human health and the economy. Oleuropein has antibacterial activities against various microorganisms but research on its effect on the S. aureus biofilm is limited. This research aimed to estimate the antibiofilm activities of oleuropein against S. aureus. The results suggest that the minimum inhibitory concentration of oleuropein against S. aureus ATCC 25923 was 3 mg/mL. The biomass of biofilms formed on the microplates and coverslips and the viability of bacteria were significantly reduced after the treatment with oleuropein. The scanning electron microscopy observation results indicated that the stacking thickness and density of the biofilm decreased when S. aureus was exposed to oleuropein. It had a bactericidal effect on biofilm bacteria and removed polysaccharides and proteins from mature biofilms. The effects of oleuropein on the biofilm could be explained by a reduction in bacterial secretion of extracellular polymeric substances and a change in bacterial surface hydrophobicity. Based on the above findings, oleuropein has the potential to be used against food pollution caused by S. aureus biofilms.

Flavor Wheel Construction and Sensory Profile Description of Human Milk.

To explore the flavor characteristics of human milk, we constructed a three-tiered human milk flavor wheel based on 53 sensory descriptors belonging to different sensory categories. Fifteen sensory descriptors were selected using M-value and multivariate statistical methods, and the corresponding references were set up to realize qualitative and quantitative sensory evaluation of the human milk samples. To ensure the accuracy and reliability of the sensory evaluation, the performance of the sensory panelists was also tested. The sensory profile analysis indicated that the established sensory descriptors could properly reflect the general sensory properties of the human milk and could also be used to distinguish different samples. Further investigation exposed that the fat content might be an important factor that influence the sensory properties of human milk. To the best of our knowledge, this is the first report on the flavor wheel of human milk.

Foaming and Physicochemical Properties of Commercial Protein Ingredients Used for Infant Formula Formulation.

Protein, as one of the main ingredients for infant formula, may be closely related to the undesirable foam formed during the reconstitution of infant formula. Demineralized whey powder (D70 and D90), whey protein concentrate (WPC), and skimmed milk powder (SMP) are the four protein ingredients commonly used in infant formula formulation. The foaming and physicochemical properties of these four protein ingredients from different manufacturers were analyzed in the present study. Significant differences (p < 0.05) in foaming properties were found between the samples from different manufacturers. SMP showed a highest foaming capacity (FC) and foam stability (FS), followed by D70, D90, and WPC. Although the protein composition was similar based on reducing SDS-PAGE, the aggregates varied based on non-reducing SDS-PAGE, probably resulting in the different foaming properties. Particle size, zeta potential, and solubility of the protein ingredients were assessed. The protein structure was evaluated by circular dichroism, surface hydrophobicity, and free sulfhydryl. Pearson's correlation analysis demonstrated that FC and FS were positively correlated with random coil (0.55 and 0.74), ß-turn (0.53 and 0.73), and zeta potential (0.55 and 0.51) but negatively correlated with ß-strand (−0.56 and −0.71), free sulfhydryl (−0.56 and −0.63), particle size (−0.45 and −0.53), and fat content (−0.50 and −0.49). The results of this study could provide a theoretical guidance for reducing formation of foam of infant formula products during reconstitution.

Promotion effect of the blend containing 2'-FL, OPN and DHA on oligodendrocyte progenitor cells myelination in vitro.

During early neurodevelopment of infant, myelination plays an essential role in brain connectivity and emergence of behavioral and cognitive function. Early life nutrition is an important factor to shape myelination and consequently cognitive appearance. To analyze the effects of additive nutrients, including 2'-fucosyllactose (2'-FL), osteopontin (OPN), docosahexaenoic acid (DHA), on neurocognitive function and brain structure, the current study evaluated the effects of different composition of breast milk nutrients on oligodendrocyte progenitor cells (OPCs) myelination with a neural primary cell model in vitro. The study showed that the three nutrients promoted the proliferation, maturation and differentiation of OPCs into mature oligodendrocytes (OLs) in each phage of the cell growth, and the effect of the nutrients blend is obviously stronger than that of the nutrient treatment alone, showing a synergistic effect in promotion of OPCs. The results of this experiment clarified the effects of 2'-FL OPN and DHA to promote myelination development of neural cells, and laid an experimental basis for further optimization of infant formula.

2'-Fucosyllactose Promotes the Production of Short-Chain Fatty Acids and Improves Immune Function in Human-Microbiota-Associated Mice by Regulating Gut Microbiota.

As a natural prebiotic in human milk, 2'-fucosyllactose (2'-FL) is actively used in infant formula (IF). However, the 2'-FL influence on the improvement of gut microbiota and the regulation of the immune function remains unknown. In this study, human microbiota-associated (HMA) mice were used to demonstrate that feeding 2'-FL-containing IF was comparable to human milk at levels of immune cytokines (IL-2, IL-9, IL-10, and sIgA) and short-chain fatty acids (SCFAs, i.e., acetate and propionate). In addition, 2'-FL increased the abundance of Blautia and Olsenella and improved the anti-inflammatory cytokine IL-10 levels. The abundance of Blautia and Olsenella positively correlated with the IL-10 levels. 2'-FL also decreased the abundance of Enterorhabdus and Lachnospiraceae_UCG-006 and elevated SCFA levels, showing a negative correlation between these genera and SCFAs. Our findings revealed that feeding 2'-FL-containing IF drives the levels of cytokines and SCFAs toward human milk levels by shaping the beneficial gut microbiota profile.

Co-fermented cow milk protein by Lactobacillus helveticus KLDS 1.8701 and Lactobacillus plantarum KLDS 1.0386 attenuates its allergic immune response in Balb/c mice.

Milk protein is one of the major food allergens. As an effective processing method, fermentation may reduce the potential allergenicity of allergens. This study aimed to evaluate the therapeutic potential of co-fermented milk protein using Lactobacillus helveticus KLDS 1.8701 and Lactobacillus plantarum KLDS 1.0386 in cow milk protein allergy (CMPA) management. This study determined the secondary and tertiary structures of the fermented versus unfermented proteins by Fourier-transform infrared spectroscopy and surface hydrophobicity to evaluate its conformational changes. Our results showed that different fermentation methods have significantly altered the conformational structures of the cow milk protein, especially the tertiary structure. Further, the potential allergenicity of the fermented cow milk protein was assessed in Balb/c mice, and mice treated with the unfermented milk and phosphate-buffered saline were used as a control. We observed a significant reduction in allergenicity via the results of the spleen index, serum total IgE, specific IgE, histamine, and mouse mast cell protease 1 in the mice treated with the co-fermented milk protein. In addition, we analyzed the cytokines and transcription factors expression levels of spleen and jejunum and confirmed that co-fermentation could effectively reduce the sensitization of cow milk protein by regulating the imbalance of T helper (Th1/Th2 and Treg/Th17). This study suggested that changes of conformational structure could reduce the potential sensitization of cow milk protein; thus, fermentation may be a promising strategy for developing a method of hypoallergenic dairy products.