Advances in the Study of Probiotics for Immunomodulation and Intervention in Food Allergy.

Food allergies are a serious food safety and public health issue. Soybean, dairy, aquatic, poultry, and nut products are common allergens inducing allergic reactions and adverse symptoms such as atopic dermatitis, allergic eczema, allergic asthma, and allergic rhinitis. Probiotics are assumed as an essential ingredient in maintaining intestinal microorganisms' composition. They have unique physiological roles and therapeutic effects in maintaining the mucosal barrier, immune function, and gastrointestinal tract, inhibiting the invasion of pathogenic bacteria, and preventing diarrhea and food allergies. Multiple pieces of evidence reveal a significant disruptive effect of probiotics on food allergy pathology and progression mechanisms. Thus, this review describes the allergenic proteins as an entry point and briefly describes the application of probiotics in allergenic foods. Then, the role of probiotics in preventing and curing allergic diseases by regulating human immunity through intestinal flora and intestinal barrier, modulating host immune active cells, and improving host amino acid metabolism are described in detail. The anti-allergic role of probiotics in the function and metabolism of the gastrointestinal tract has been comprehensively explored to furnish insights for relieving food allergy symptoms and preventing food allergy.

Cinnamaldehyde inhibit Escherichia coli associated with membrane disruption and oxidative damage.

In this study, the antimicrobial mechanism of cinnamaldehyde (CIN) against Gram-negative Escherichia coli ATCC 25922 (E. coli) based on membrane and gene regulation was investigated. Treatment with low concentration (0, 1/8, 1/4, 3/8 MIC) of CIN can effectively suppress the growth of E. coli by prolonging its lag phase and Raman spectroscopy showed obvious distinction of the E. coli after being treated with these concentration of CIN. The determination of relative conductivity indicated that CIN at relatively high concentration (0, 1, 2, 4 MIC) can increase the cell membrane permeability, causing the leakage of cellular content. Besides, the content of malondialdehyde (MDA) and the activity of total superoxide dismutase (SOD) of E. coli increased with increasing treatment concentration of CIN, implying that CIN can cause oxidative damage on E. coli cell membrane and induce the increase of total SOD activity to resist this oxidative harm. Moreover, quantitative real-time RT-PCR (qRT-PCR) analysis revealed the relationship between expression of antioxidant genes (SODa, SODb, SODc) and treatment CIN concentration, suggesting that SOD, especially SODc, played a significant role in resistance of E. coli to CIN. The underlying inactivation processing of CIN on E. coli was explored to support CIN as a potential and natural antimicrobial agent in food industry.

Modification of membrane properties and fatty acids biosynthesis-related genes in Escherichia coli and Staphylococcus aureus: Implications for the antibacterial mechanism of naringenin.

In this work, modifications of cell membrane fluidity, fatty acid composition and fatty acid biosynthesis-associated genes of Escherichia coli ATCC 25922 (E. coli) and Staphylococcus aureus ATCC 6538 (S. aureus), during growth in the presence of naringenin (NAR), one of the natural antibacterial components in citrus plants, was investigated. Compared to E. coli, the growth of S. aureus was significantly inhibited by NAR in low concentrations. Combination of gas chromatography-mass spectrometry with fluorescence polarization analysis revealed that E. coli and S. aureus cells increased membrane fluidity by altering the composition of membrane fatty acids after exposure to NAR. For example, E. coli cells produced more unsaturated fatty acids (from 18.5% to 43.3%) at the expense of both cyclopropane and saturated fatty acids after growth in the concentrations of NAR from 0 to 2.20mM. For S. aureus grown with NAR at 0 to 1.47mM, the relative proportions of anteiso-branched chain fatty acids increased from 37.2% to 54.4%, whereas iso-branched and straight chain fatty acids decreased from 30.0% and 33.1% to 21.6% and 23.7%, respectively. Real time q-PCR analysis showed that NAR at higher concentrations induced a significant down-regulation of fatty acid biosynthesis-associated genes in the bacteria, with the exception of an increased expression of fabA gene. The minimum inhibitory concentration (MIC) of NAR against these two bacteria was determined, and both of bacteria underwent morphological changes after exposure to 1.0 and 2.0 MIC.

Temperature-mediated variations in cellular membrane fatty acid composition of Staphylococcus aureus in resistance to pulsed electric fields.

Effects of growth temperature on cell membrane fatty acid composition, fluidity and lethal and sublethal injury by pulsed electric fields (PEF) in Staphylococcus aureus ATCC 43300 (S. aureus) in the stationary phase were investigated. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) revealed that branched chain fatty acids (iso C14:0, iso C15:0, anteiso C15:0 and anteiso C17:0) and straight chain fatty acids (C12:0, C14:0, C16:0, C17:0 and C18:0) were primary constituents in the membrane. The S. aureus changed its membrane fatty acid composition and its overall fluidity when exposed to different temperatures. The PEF lethal and sublethal effects were assessed, and results suggested that the degree of inactivation depended on the cell membrane structure, electric field strength and treatment time. The PEF inactivation kinetics including lethal and sublethal injury fractions were fitted with non-linear Weibull distribution, suggesting that inactivation of the first log cycle of S. aureus population was significantly affected by growth temperature, and the membrane of cells became more fluid, and easier to induce electroportion in low temperatures. Moreover, the morphology of S. aureus cells were investigated by electron microscopy, showing that various temperature-modified cells were distorted to differing extents and some even collapsed due to deep irreversible electroporation after PEF treatment.

An in vitro investigation of the inhibitory mechanism of ß-galactosidase by cinnamaldehyde alone and in combination with carvacrol and thymol.

BACKGROUND: Some antibacterial agents exert their antimicrobial action by targeting the cytoplasmic macromolecules, such as proteins or nucleic acids, to disturb the properties of macromolecules that may deeply influence their biological activities and functions. Cinnamaldehyde (CIN) is a natural antibacterial ingredient found in the bark and leaves of cinnamon trees. METHODS: The inhibitory mechanism of a typical enzyme, ß-galactosidase by CIN was investigated by UV-visible, fluorescence, 3-D spectroscopy, circular dichroism, atomic force microscopy and molecular modeling studies. RESULTS: CIN decreased the activity of ß-galactosidase by competitive inhibition through a multiphase kinetic process. 3-D spectroscopy and circular dichroism showed that the binding of CIN to ß-galactosidase resulted in changes in micro-environment of tryptophan and tyrosine residues, and conformation of ß-galactosidase. The molecular recognition was also analyzed through modeling which indicated that CIN was inserted into the active site pocket of ß-galactosidase and interacted with amino acid residues, such as Met502, Trp568, Phe601 and Trp999. Atomic force microscopy showed that a serious destabilization of the native conformation of ß-galactosidase occurred after binding with CIN, e.g., morphological changes and increased dimensions of the ß-galactosidase molecule. Moreover, it was found that the combinations of CIN, carvacrol and thymol exposure displayed synergistic effects on the inhibition of ß-galactosidase. GENERAL SIGNIFICANCE: This study exhibits a comprehensively understanding about the action mechanism of CIN that affects the conformation and activity of ß-galactosidase in biochemical processes and provides some new insights into the possible intracellular targeting behaviors of CIN at a molecular level.

Combination of microbiological, spectroscopic and molecular docking techniques to study the antibacterial mechanism of thymol against Staphylococcus aureus: membrane damage and genomic DNA binding.

Thymol (2-isopropyl-5-methylphenol) is a natural ingredient used as flavor or preservative agent in food products. The antibacterial mechanism of thymol against Gram-positive, Staphylococcus aureus was investigated in this work. A total of 15 membrane fatty acids were identified in S. aureus cells by gas chromatography-mass spectrometry. Exposure to thymol at low concentrations induced obvious alterations in membrane fatty acid composition, such as decreasing the proportion of branched 12-methyltetradecanoic acid and 14-methylhexadecanoic acid (from 22.4 and 17.3% to 7.9 and 10.3%, respectively). Membrane permeability assay and morphological image showed that thymol at higher concentrations disrupted S. aureus cell membrane integrity, which may decrease cell viability. Moreover, the interaction of thymol with genomic DNA was also investigated using multi-spectroscopic techniques, docking and atomic force microscopy. The results indicated that thymol bound to the minor groove of DNA with binding constant (K a) value of (1.22 ± 0.14) × 104 M-1, and this binding interaction induced a mild destabilization in the DNA secondary structure, and made DNA molecules to be aggregated. Graphical Abstract Thymol exerts its antibacterial effect throught destruction of bacterial cell membrane and binding directly to genomic DNA.

Characterization and application in yogurt of genipin-crosslinked chitosan microcapsules encapsulating with Lactiplantibacillus plantarum DMDL 9010.

Microcapsules could improve the protection of probiotics in the lyophilization and gastrointestinal digestion process. The purpose of this study was to prepare Lactiplantibacillus plantarum DMDL 9010 (LP9010) microcapsules by cross-linking chitosan with genipin and to determine the encapsulation efficiency, morphological characterization, storage stability and the application of the microcapsules in fermentation. The results showed that the LP9010 microcapsules embedded in 1.00 wt% genipin cross-linked chitosan were in a uniform spherical shape with a smooth surface and satisfying agglomeration. The LP9010 microcapsules demonstrated the reasonable thermal stability and persistence of biological activity in the range of -20 °C to 25 °C. Additionally, yogurt obtained from the ST + LB + ELP9010 strain formulation with the addition of microencapsulated LP9010 had smaller particles, better taste, and better stability compared with the yogurt obtained from other strain formulations. As detected by GC-MS, the yogurt formulated with ST + LB + ELP9010 as a strain retained more flavor substances and the content of flavor substances was greater than that of the yogurt obtained from other strain formulations. Therefore, genipin cross-link chitosan could be a suitable microencapsulated material for producing yogurt fermentation strains.

Pulsed electric field-assisted esterification improves the freeze-thaw stability of corn starch gel by changing its molecular structure.

The influence of pulsed electric field (PEF) combined with octenyl succinic anhydride (OSA) on the freeze-thaw stability of corn starch gel was investigated. After five freeze-thaw cycles, the syneresis value of OSA starch treated with PEF-assisted esterification for 15 min was lower by 29.5 %, while that of OSA starch without PEF treatment was lower by 10.17 %, compared to that of native starch. Low-field nuclear magnetic resonance data showed that the introduction of OSA groups greatly increased the water-holding capacity of starch. Results from differential scanning calorimetry (DSC) and X-ray diffraction (XRD) showed that the PEF-assisted esterification markedly hindered the re-formation of the helical structure of starch during freeze-thaw cycles. Moreover, PEF-assisted esterification improved the viscoelastic properties of the starch gel. It is found that the freeze-thaw stability of the PEF-modified starch depends not only on the degree of substitution but also on the starch molecular fine structure. PEF-assisted OSA starch with a high degree of substitution, a low content of amylose, and a high content of short amylopectin chains were found to have high freeze-thaw stability. This study shows that PEF-assisted esterification is a promising technique that should be used for preserving the quality of frozen foods.

Enhanced encapsulation of lutein using soy protein isolate nanoparticles prepared by pulsed electric field and pH shifting treatment.

In this study, soy protein isolate (SPI) was modified by a pulsed electric field (PEF) combined with pH shifting treatment (10 kV/cm, pH 11) to prepare SPI nanoparticles (PSPI11) for efficient loading of lutein. The results showed that when the mass ratio of SPI to lutein was 25:1, the encapsulation efficiency of lutein in PSPI11 increased from 54% to 77%, and the loading capacity increased by 41% compared to the original SPI. The formed SPI-lutein composite nanoparticles (PSPI11-LUTNPs) had smaller, more homogeneous sizes and larger negative charges than SPI7-LUTNPs. The combined treatment favored the unfolding of the SPI structure and could expose its interior hydrophobic groups to bind with lutein. Nanocomplexation with SPIs significantly improved the solubility and stability of lutein, with PSPI11 showing the greatest improvement. As a result, PEF combined with pH shifting pretreatment is an effective method for developing SPI nanoparticles loaded and protected with lutein.

Investigation on the impact of quality characteristics and storage stability of foxtail millet induced by air cold plasma.

The aim of this work was to investigate the effects of dielectric barrier discharge-air cold plasma (DBD-ACP, 15-35 kV, 2-12 min) on the quality of foxtail millets. The L and b* values were evaluated by a digital colorimeter representing that the color of millets was significantly changed at 25 kV for 4-12 min or at 35 kV for 2-12 min. The results were consistent with the change of total yellow pigment in millets, indicating that DBD-ACP damaged the carotenoids if the treatment condition was too high. The activity of lipoxygenase and lipase, involving the oxidation and hydrolysis of lipids of millet, decreased significantly induced by DBD-ACP. For example, the lipoxygenase and lipase activity of Mizhi millet was decreased from 44.0 to 18.7 U g-1min-1, 56.0-15.1 U/(mg pro) (p<0.05) after being exposed to 25 kV for 2-12 min, respectively. Changes of color, lipoxygenase and lipase activity, and malondialdehyde content of millets were determined during accelerated storage (40 ± 2°C and 75% Relative Humidity) for 15 days after being treated by DBD-ACP under 15 and 25 kV for 4 min. Results showed that millets treated by DBD-ACP at 15 kV kept a better color with lower malondialdehyde content, and lower lipoxygenase and lipase activity compared to control. This work implied that DBD-ACP is an underlying approach for the storage of foxtail millets.

Inactivation efficacy and mechanisms of atmospheric cold plasma on Alicyclobacillus acidoterrestris: Insight into the influence of growth temperature on survival.

The bactericidal effect of dielectric barrier discharge-atmospheric cold plasma (DBD-ACP, 20, and 30 kV) against Alicyclobacillus acidoterrestris on the saline solution and apple juice was investigated. Results show that DBD-ACP is effective for the inactivation of A. acidoterrestris by causing significant changes in cell membrane permeability and bacterial morphology. The effect of culture temperatures on the resistance of A. acidoterrestris to DBD-ACP was also studied. A. acidoterrestris cells grown at 25°C had the lowest resistance but it was gradually increased as the culture temperature was increased (25-45°C) (p < 0.05). Moreover, results from Fourier transform infrared spectroscopy (FT-IR) and Gas Chromatography-Mass Spectrometer (GC-MS) analysis showed that the increase in the culture temperature can gradually cause the decreased level of cyclohexaneundecanoic acid in the cell membrane of A. acidoterrestris (p < 0.05). In contrast, cyclopentaneundecanoic acid, palmitic acid, and stearic acid showed an increasing trend in which the fluidity of the bacterial cell membrane decreased. This study shows a specific correlation between the resistance of A. acidoterrestris and the fatty acid composition of the cell membrane to DBD-ACP.

Determination of membrane disruption and genomic DNA binding of cinnamaldehyde to Escherichia coli by use of microbiological and spectroscopic techniques.

This work was aimed to investigate the antibacterial action of cinnamaldehyde (CIN) against Escherichia coli ATCC 8735 (E. coli) based on membrane fatty acid composition analysis, alterations of permeability and cell morphology as well as interaction with genomic DNA. Analysis of membrane fatty acids using gas chromatography-mass spectrometry (GC-MS) revealed that the proportion of unsaturated fatty acids (UFA) and saturated fatty acids (SFA) were the major fatty acids in plasmic membrane, and their levels were significantly changed after exposure of E. coli to CIN at low concentrations. For example, the proportion of UFA decreased from 39.97% to 20.98%, while the relative content of SFA increased from 50.14% to 67.80% as E. coli was grown in increasing concentrations of CIN (from 0 to 0.88mM). Scanning electron microscopy (SEM) showed that the morphology of E. coli cells to be wrinkled, distorted and even lysed after exposure to CIN, which therefore decreased the cell viability. The binding of CIN to genomic DNA was probed using fluorescence, UV-Visible absorption spectra, circular dichroism, molecular modeling and atomic force microscopy (AFM). Results indicated that CIN likely bound to the minor groove of genomic DNA, and changed the secondary structure and morphology of this biomacromolecule. Therefore, CIN can be deem as a kind of natural antimicrobial agents, which influence both cell membrane and genomic DNA.

Hydroxyl-related differences for three dietary flavonoids as inhibitors of human purine nucleoside phosphorylase.

In this work, the hydroxyl-related differences of binding properties and inhibitory activities of dietary flavonoids, namely chrysin, baicalein and apigenin against purine nucleoside phosphorylase (PNP) were investigated. It was found that the hydroxylation on position C4' of chrysin (→apigenin) mildly decreased the binding affinities for PNP, whereas on the position C6 of chrysin (→baicalein) significantly increased binding affinities. Comparatively, the hydroxylation on position C4' and C6 greatly improved their PNP inhibitory effects. The IC50 values of apigenin and baicalein were 6.09 × 10-5 M and 8.94 × 10-5 M, respectively, which is significantly lower than that of chrysin (2.13 × 10-4 M). Results from molecular modeling revealed that there are two binding sites, i.e. active site (major) and tryptophan site (minor) on PNP, and the binding of these flavonoids might induce a serious conformational destabilization of PNP as a result of altering the micro-environment and morphology by flavonoids.

Membrane and genomic DNA dual-targeting of citrus flavonoid naringenin against Staphylococcus aureus.

The antimicrobial mechanism of naringenin, one of the citrus antibacterial flavonoids against food-borne Staphylococcus aureus ATCC 6538, was investigated in this study. Analysis of gas chromatography-mass spectrometry (GC-MS) and fluorescence showed that relatively low concentrations of naringenin caused perturbations in the membrane fatty acid composition and the conformation of membrane proteins through changing the microenvironment of phenylalanine, tyrosine, and tryptophan residues. Exposure of naringenin at higher levels significantly increased membrane permeability and changed the morphology of S. aureus cells. The genomic DNA-binding of naringenin was also quantitatively monitored using UV-vis spectra in combination with multivariate curve resolution-alternating least squares (MCR-ALS) analysis, and the concentration and pure spectra profiles for the three reaction species (DNA, naringenin, and DNA-naringenin) were obtained. Moreover, the thermal behavior of DNA and docking studies revealed that naringenin preferentially bound to the A-T base pair regions of genomic DNA via groove binding, and atomic force microscopy and circular dichroism showed that naringenin induced mild secondary structure and obvious morphological variations of this biomacromolecule. These results suggested that naringenin exerting its antibacterial effects might be connected with disruption of the cytoplasmic membrane and DNA targeting effects in Staphylococcus aureus.

Membrane Destruction and DNA Binding of Staphylococcus aureus Cells Induced by Carvacrol and Its Combined Effect with a Pulsed Electric Field.

Carvacrol (5-isopropyl-2-methylphenol, CAR) is an antibacterial ingredient that occurs naturally in the leaves of the plant Origanum vulgare. The antimicrobial mechanism of CAR against Staphylococcus aureus ATCC 43300 was investigated in the study. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) showed that exposure to CAR at low concentrations induced a marked increase in the level of unbranched fatty acids (from 34.90 ± 1.77% to 62.37 ± 4.26%). Moreover, CAR at higher levels severely damaged the integrity and morphologies of the S. aureus cell membrane. The DNA-binding properties of CAR were also investigated using fluorescence, circular dichroism, molecular modeling, and atomic-force microscopy. The results showed that CAR bound to DNA via the minor-groove mode, mildly perturbed the DNA secondary structure, and induced DNA molecules to be aggregated. Furthermore, a combination of CAR with a pulsed-electric field was found to exhibit strong synergistic effects on S. aureus.