Incorporation of probiotics into 3D printed Pickering emulsion gel stabilized by tea protein/xanthan gum.

Different xanthan gum (XG) concentrations on the rheological/texture properties of Pickering emulsion (PE) gel stabilized by tea protein/xanthan gum (TP/XG) were studied to achieve an ink feasible for 3D printing. Afterwards, the effects of 3D printing and digestion process on the viability of probiotics were studied when encapsulated in the PE gel. Results indicated that gel strength, stability, storage modulus (G') and loss modulus (G″) increased as XG concentration increased. Nozzle diameter and printing temperature of 45 and 55℃ had no significant effect on probiotic's viability, but printing temperature of 65℃ reduced viable probiotics from 8.07 to 6.59 log CFU/g. No significant change of probiotics viability in 3D printed samples was observed during 11-day storage at 4℃. PE gel encapsulated probiotic's viability was significantly improved under heat treatment and simulated gastrointestinal environment. This study gives insights on the production of 3D printed foods using PE gel incorporating probiotics.

Ferrous sulfate efficiently kills Vibrio parahaemolyticus and protects salmon sashimi from its contamination.

The primary seafood-borne pathogen Vibrio parahaemolyticus seriously threats the health of consumers preferring raw-fish products, becoming a global concern in food safety. In the present study, we found ferrous sulfate (FeSO4), a nutritional iron supplement, could efficiently induce the death of V. parahaemolyticus. Further, the bactericidal mechanisms of FeSO4 were explored. With a fluorescent probe of Fe2+, a significant influx of Fe2+ was determined in V. parahaemolyticus exposed to FeSO4, and the addition of an intracellular Fe2+ chelator was able to block the cell death. This suggested that cell death in V. parahaemolyticus induced by FeSO4 was dependent on the influx of Fe2+. It was intriguing that we did not observe the eruption of reactive oxygen species (ROS) and lipid hydroperoxides by Fe2+, but the application of liproxstatin-1 (a ferroptosis inhibitor) significantly modified the occurrence of cell death in V. parahaemolyticus. These results suggested FeSO4-induced cell death in V. parahaemolyticus be a ferroptosis differing from that in mammalian cells. Through transcriptome analysis, it was discovered that the exposure of FeSO4 disturbed considerable amounts of gene expression in V. parahaemolyticus including those involved in protein metabolism, amide biosynthesis, two-component system, amino acid degradation, carbon metabolism, citrate cycle, pyruvate metabolism, oxidative phosphorylation, and so on. These data suggested that FeSO4 was a pleiotropic antimicrobial agent against V. parahaemolyticus. Notably, FeSO4 was able to eliminate V. parahaemolyticus in salmon sashimi as well, without affecting the color, texture, shearing force, and sensory characteristics of salmon sashimi. Taken together, our results deciphered a unique ferroptosis in V. parahaemolyticus by FeSO4, and highlighted its potential in raw-fish products to control V. parahaemolyticus.

Exogenous Iron Induces NADPH Oxidases-Dependent Ferroptosis in the Conidia of Aspergillus flavus.

Aspergillus flavus is saprophytic soil fungus that contaminates seed crops with the carcinogenic secondary metabolite aflatoxin, posing a significant threat to humans and animals. Ferrous sulfate is a common iron supplement that is used to the treatment of iron-deficiency anemia. Here, we identified an unexpected inhibitory role of ferrous sulfate on A. flavus. With specific fluorescent dyes, we detected several conidial ferroptosis hallmarks in conidia under the treatment of 1 mM Fe2+, including nonapoptosis necrosis, iron-dependent, lipid peroxide accumulation, and ROS burst. However, unlike traditional ferroptosis in mammals, Fe2+ triggered conidial ferroptosis in A. flavus was regulated by NADPH oxidase (NOXs) activation instead of Fenton reaction. Transcriptomic and some other bioinformatics analyses showed that NoxA in A. flavus might be a potential target of Fe2+, and thus led to the occurrence of conidial ferroptosis. Furthermore, noxA deletion mutant was constructed, and both ROS generation and conidial ferroptosis in ΔnoxA was reduced when exposed to Fe2+. Taken together, our study revealed an exogenous Fe2+-triggered conidial ferroptosis pathway mediated by NoxA of A. flavus, which greatly contributes to the development of an alternative strategy to control this pathogen.

Transcriptome Analysis and Weighted Gene Co-expression Network Reveal Multitarget-Directed Antibacterial Mechanisms of Benzyl Isothiocyanate against Staphylococcus aureus.

Staphylococcus aureus can cause many diseases and has a strong tendency to develop resistance to multiple antibiotics. In this study, benzyl isothiocyanate (BITC) was shown to have an excellent inhibitory effect on S. aureus ATCC25923 and methicillin-resistant S. aureus strains, with a minimum inhibitory concentration of 10 µg/mL. Under a scanning electron microscope, shrinkage and lysis of the cellular envelope were observed when exposed to BITC, and a bactericidal mode of BITC against S. aureus was further confirmed through flow cytometry. Additionally, the RNA profiles of S. aureus cells exposed to BITC indicated a violent transcriptional response to BITC. Through Kyoto Encyclopedia of Genes and Genomes analysis, it was found that many pathways involving bacterial survival were significantly affected, such as RNA degradation, oxidative phosphorylation, arginine biosynthesis, and so forth. A gene co-expression network was constructed using weighted gene co-expression network analysis, and six biologically meaningful co-expression modules and 125 hub genes were identified from the network. Among them, EfeB, GroES, SmpB, and Lsp were possibly targeted by BITC, leading to the death of S. aureus. Our results indicated a great potential of BITC to be applied in food safety and pharmaceuticals, highlighting its multitarget-directed bactericidal effects on S. aureus.

Fabrication of food-grade Pickering high internal phase emulsions stabilized by the mixture of ß-cyclodextrin and sugar beet pectin.

Food-grade Pickering high internal phase emulsions (HIPEs) stabilized by a mixture of ß-cyclodextrin (ß-CD) and sugar beet pectin (SBP) were fabricated for the first time. The factors affecting the microstructures, mechanical properties, and stabilities of the Pickering HIPEs were systematically investigated. The corresponding hybrid particles were also separated and characterized to reveal the formation mechanism. The results indicated that the mixture could induce the formation of HIPEs with an oil phase volume fraction (φ) of 75% using a one-step high-speed shearing process at room temperature. The composition (the mass ratio of ß-CD to SBP, Rc/s) and concentration (W) of the mixture had significant effects on the formation and properties of HIPEs. When W ≥ 1.0% and Rc/s = 2:2 or 3:1, HIPEs had smaller oil droplets, higher gel strengths, better centrifugation stabilities and lutein protection effects. The spectral analysis suggested that SBP could adhere to the surface of ß-CD particles to form hybrid particles during the homogenization. Compared with native ß-CD particles, these hybrid particles had higher ζ-potential absolute values, and the SBP could also increase the viscosity of the aqueous phase, which contributed to the formation and properties of these HIPEs.

Fabrication of chondroitin sulfate calcium complex and its chondrocyte proliferation in vitro.

Chondroitin sulfate (CS)-calcium complex (CSCa) was fabricated, and the structural characteristics of CSCa and its proliferative bioactivity to the chondrocyte were investigated in vitro. Results suggested calcium ions could bind CS chains forming polysaccharide-metal complex, and the maximum calcium holding capacity of CSCa reached 4.23 %. Characterization of CSCa was performed by EDS, AFM, FTIR, UV, XRD and 1H-NMR. It was found that calcium ions were integrated with CS by binding the sulfate or carboxyl groups. The thermal properties analysis indicated CSCa had a good thermal stability by TGA and DSC. CSCa could interact the calcium-sensing receptor increasing the intracellular calcium ions and influence the cell cycle. The TGF-ß1 secretion induced by CSCa could activate the TGF-ß/Smads pathway and change the genes associated proliferation expression ultimately leading to the chondrocyte proliferation. This research probably has an important implication for understanding the effect of CSCa on bone care as food supplements.

ROS Involves the Fungicidal Actions of Thymol against Spores of Aspergillus flavus via the Induction of Nitric Oxide.

Aspergillus flavus is a well-known pathogenic fungus for both crops and human beings. The acquisition of resistance to azoles by A. flavus is leading to more failures occurring in the prevention of infection by A. flavus. In this study, we found that thymol, one of the major chemical constituents of the essential oil of Monarda punctate, had efficient fungicidal activity against A. flavus and led to sporular lysis. Further studies indicated that thymol treatment induced the generation of both ROS and NO in spores, whereas NO accumulation was far later than ROS accumulation in response to thymol. By blocking ROS production with the inhibitors of NADPH oxidase, NO generation was also significantly inhibited in the presence of thymol, which indicated that ROS induced NO generation in A. flavus in response to thymol treatment. Moreover, the removal of either ROS or NO attenuated lysis and death of spores exposed to thymol. The addition of SNP (exogenous NO donor) eliminated the protective effects of the inhibitors of NADPH oxidase on thymol-induced lysis and death of spores. Taken together, it could be concluded that ROS is involved in spore death induced by thymol via the induction of NO.

Characterization of the Supermolecular Structure of Polydatin/6-O-α-Maltosyl-ß-cyclodextrin Inclusion Complex.

Polydatin is the main bioactive ingredient in many medicinal plants, such as Hu-zhang (Polygonum cuspidatum), with many bioactivities. However, its poor aqueous solubility restricts its application in functional food. In this work, 6-O-α-Maltosyl-ß-cyclodextrin (Malt-ß-CD), a new kind of ß-CD derivative was used to enhance the aqueous solubility and stability of polydatin by forming the inclusion complex. The phase solubility study showed that polydatin and Malt-ß-CD could form the complex with the stoichiometric ratio of 1:1. The supermolecular structure of the polydatin/Malt-ß-CD complex was characterized by ultraviolet-visible spectroscopy (UV), Fourier transform infrared spectroscopy (FT-IR), X-ray diffractometry (XRD), thermogravimetric/differential scanning calorimetry (TG/DSC), and proton nuclear magnetic resonance ((1) H-NMR) spectroscopy. The changes of the characteristic spectral and thermal properties of polydatin suggested that polydatin could entrap inside the cavity of Malt-ß-CD. Furthermore, to reasonably understand the complexation mode, the supermolecular structure of polydatin/Malt-ß-CD inclusion complex was postulated by a molecular docking method based on Autodock 4.2.3. It was clearly observed that the ring B of polydatin oriented toward the narrow rim of Malt-ß-CD with ring A and glucosyl group practically exposed to the wide rim by hydrogen bonding, which was in a good agreement with the spectral data.

Tissue distribution and developmental changes of ghrelin and GOAT expression in broiler chickens during embryogenesis.

Ghrelin plays important roles, such as regulating growth hormone release and energy metabolism, but little is known about its developmental changes in the proventriculi of chicken embryos. This study was designed to elucidate the distributions and developmental changes of ghrelin and ghrelin-O-acyltransferase (GOAT) expression in broiler embryos using qRT-PCR and immunohistochemistry. Our results demonstrated the following: (1) on E18, ghrelin and GOAT are ubiquitously expressed in every tissue examined. The expression level of ghrelin mRNA was the highest in the proventriculus, reaching a level that was 50-fold higher than that in the hypothalamus, while GOAT mRNA expression was low in the proventriculus and it was only 67.6% as high as that of hypothalamus; (2) ghrelin and GOAT mRNA expression were detected in the proventriculus on E9, but only at 1.9% and 1.7% of the level expressed on E18, respectively, and their expression levels increased rapidly from E18 to E21. There was similar developmental pattern in the ghrelin and GOAT mRNA expression; and (3) ghrelin-immunopositive cells were first detected in the proventriculus on E15, were located only in the compound tubular glands of the proventriculus, and were of the closed-cell type. The density of ghrelin-immunopositive cells increased significantly from E15 to E21. These results suggest that ghrelin may be an important regulating factor that plays a vital role during the development of chicken embryos.

Comparative evaluation of tannic acid inhibiting α-glucosidase and trypsin.

In this work, the inhibitory effects of tannic acid on the α-glucosidase and trypsin were systematically evaluated by comparing with the clinical diabetes healer acarbose and the soybean-derived trypsin inhibitor using fluorescence spectroscopy and enzymatic kinetics methods. We showed that the anti-α-glucosidase activity of tannic acid (IC50=0.44µg/mL) was higher than that of acarbose (IC50>0.60µg/mL), while its anti-trypsin activity (IC50=0.79mg/mL) was significantly lower than that of the trypsin inhibitor from soybean (IC50<0.20mg/mL). Enzymatic kinetics measurements confirmed that the inhibitory pattern of tannic acid toward two tested enzymes was a mixed competitive and noncompetitive inhibition. Tannic acid could bind the enzymes to form new complexes, presenting a strong static fluorescence quenching. The presence of tannic acid led to the hypsochromic shift of the maximum fluorescence in trypsin, but not in α-glucosidase. The thermodynamic parameters indicated that the main driving force between tannic acid and both the enzymes was the hydrophobic interaction followed by the electrostatic interaction. Our work suggests that tannic acid is a strong anti-α-glucosidase natural inhibitor with a low inhibitory activity for trypsin, thus its roles in functional food and medicinal plants should be re-recognized.

The endogenous nitric oxide mediates selenium-induced phytotoxicity by promoting ROS generation in Brassica rapa.

Selenium (Se) is suggested as an emerging pollutant in agricultural environment because of the increasing anthropogenic release of Se, which in turn results in phytotoxicity. The most common consequence of Se-induced toxicity in plants is oxidative injury, but how Se induces reactive oxygen species (ROS) burst remains unclear. In this work, histofluorescent staining was applied to monitor the dynamics of ROS and nitric oxide (NO) in the root of Brassica rapa under Se(IV) stress. Se(IV)-induced faster accumulation of NO than ROS. Both NO and ROS accumulation were positively correlated with Se(IV)-induced inhibition of root growth. The NO accumulation was nitrate reductase (NR)- and nitric oxide synthase (NOS)-dependent while ROS accumulation was NADPH oxidase-dependent. The removal of NO by NR inhibitor, NOS inhibitor, and NO scavenger could alleviate Se(IV)-induced expression of Br_Rbohs coding for NADPH oxidase and the following ROS accumulation in roots, which further resulted in the amelioration of Se(IV)-induced oxidative injury and growth inhibition. Thus, we proposed that the endogenous NO played a toxic role in B. rapa under Se(IV) stress by triggering ROS burst. Such findings can be used to evaluate the toxic effects of Se contamination on crop plants.

Selenium inhibits root elongation by repressing the generation of endogenous hydrogen sulfide in Brassica rapa.

Selenium (Se) has been becoming an emerging pollutant causing severe phytotoxicity, which the biochemical mechanism is rarely known. Although hydrogen sulfide (H2S) has been suggested as an important exogenous regulator modulating plant physiological adaptions in response to heavy metal stress, whether and how the endogenous H2S regulates Se-induce phytotoxicity remains unclear. In this work, a self-developed specific fluorescent probe (WSP-1) was applied to track endogenous H2S in situ in the roots of Brassica rapa under Se(IV) stress. Se(IV)-induced root growth stunt was closely correlated with the inhibition of endogenous H2S generation in root tips. Se(IV) stress dampened the expression of most LCD and DCD homologues in the roots of B. rapa. By using various specific fluorescent probes for bio-imaging root tips in situ, we found that the increase in endogenous H2S by the application of H2S donor NaHS could significantly alleviate Se(IV)-induced reactive oxygen species (ROS) over-accumulation, oxidative impairment, and cell death in root tips, which further resulted in the recovery of root growth under Se(IV) stress. However, dampening the endogenous H2S could block the alleviated effect of NaHS on Se(IV)-induced phytotoxicity. Finally, the increase in endogenous H2S resulted in the enhancement of glutathione (GSH) in Se(IV)-treated roots, which may share the similar molecular mechanism for the dominant role of H2S in removing ROS by activating GSH biosynthesis in mammals. Altogether, these data provide the first direct evidences confirming the pivotal role of endogenous H2S in modulating Se(IV)-induced phytotoxicity in roots.

Neuroprotective effect of tea polyphenols on oxyhemoglobin induced subarachnoid hemorrhage in mice.

Tea polyphenols are of great benefit to the treatment of several neurodegenerative diseases. In order to explore the neuroprotective effects of tea polyphenols and their potential mechanisms, an established in vivo subarachnoid hemorrhage (SAH) model was used and alterations of mitochondrial function, ATP content, and cytochrome c (cyt c) in cerebral cortex were detected. This study showed that the alteration of mitochondrial membrane potential was an early event in SAH progression. The trend of ATP production was similar to that of mitochondrial membrane potential, indicating that the lower the mitochondrial membrane potential, lesser the ATP produced. Due to mitochondrial dysfunction, more cyt c was released in the SAH group. Interestingly, the preadministration of tea polyphenols significantly rescued the mitochondrial membrane potential to basal level, as well as the ATP content and the cyt c level in the brain cortex 12 h after SAH. After pretreatment with tea polyphenols, the neurological outcome was also improved. The results provide strong evidence that tea polyphenols enhance neuroprotective effects by inhibiting polarization of mitochondrial membrane potential, increasing ATP content, and blocking cyt c release.