Physicochemical properties and cell proliferation and adhesive bioactivity of collagen-hyaluronate composite gradient membrane.

Membrane materials were widely used in guided tissue regeneration (GTR) to prevent fibroblast invasion and form a confined area for preferentially growing of osteoblast. A novel collagen-hyaluronate composite gradient membrane was prepared by Tilapia (Oreochromis mossambicus) skin collagen and sodium hyaluronate for potential GTR applications and their bioactivities were investigated by cellular viability. SEM results indicated the membrane showed a dense outer and a porous inner surface for effectively guiding the growth of bone tissue. Physicochemical and biosafety experiments showed the tensile strength of membrane was 466.57 ± 44.31 KPa and contact angle was 74.11°, and the membrane showed perfect biocompatibility and cytocompatibility as well, which met the requirements of GTR material. Cell morphology revealed that the membrane could facilitate the adherence and proliferation of fibroblast and osteoblast. The results of qRT-PCR and ELISA demonstrated that the membrane could effectively activate TGF-ß/Smad pathway in fibroblast, and promote the expressions of TGF-ß1, FN1 and VEGF. Remarkably, RUNX2 was stimulated in BMP2 pathway by the membrane to regulate osteoblast differentiation. In summary, the collagen-hyaluronate composite gradient membrane not only fulfills the prerequisites for use as a GTR material but also demonstrates substantial potential for practical applications in the field.

The α-Helical Cap Domain of a Novel Esterase from Gut Alistipes shahii Shaping the Substrate-Binding Pocket.

The human gut microbiota regulates nutritional metabolism, especially by encoding specific ferulic acid esterases (FAEs) to release functional ferulic acid (FA) from dietary fiber. In our previous study, we observed seven upregulated FAE genes during in vitro fecal slurry fermentation using wheat bran. Here, a 29 kDa FAE (AsFAE) from Alistipes shahii of Bacteroides was characterized and identified as the type-A FAE. The X-ray structure of AsFAE has been determined, revealing a unique α-helical domain comprising five α-helices, which was first characterized in FAEs from the gut microbiota. Further molecular docking analysis and biochemical studies revealed that Tyr100, Thr122, Tyr219, and Ile220 are essential for substrate binding and catalytic efficiency. Additionally, Glu129 and Lys130 in the cap domain shaped the substrate-binding pocket and affected the substrate preference. This is the first report on A. shahii FAE, providing a theoretical basis for the dietary metabolism in the human gut.

Four Citrus Flavanones Exert Atherosclerosis Alleviation Effects in ApoE-/- Mice via Different Metabolic and Signaling Pathways.

Citrus flavanones have the potential to alleviate atherosclerosis. The metabolism and anti-atherosclerosis signaling pathways of four citrus flavanones (naringin, naringenin, hesperidin, and hesperetin) were compared in ApoE-/- mice. Naringin had the most potent anti-atherogenic effect, followed by hesperidin, naringenin, and hesperetin with reductions of 55.92, 34.98, 42.87, and 24.70% in the atherosclerotic plaque rate compared with the control, respectively. Oral naringin mainly existed in the intestine due to the high water solubility of 7-O-nohesperidoside and alleviated atherosclerosis mainly by enhancing bile acid synthesis in the gut microbiota-FXR/FGF15-CYP7A1 pathway. The other three flavanones mainly alleviated atherosclerosis in the liver after absorption from the intestine. Hesperidin upregulates ABCA1 by 1.8-fold to enhance cholesterol reverse transport, while the aglycones naringenin and hesperetin inhibited cholesterol synthesis via downregulating HMGCR by 2.4- and 2.3-fold, respectively. Hesperetin was more resistant to absorption than naringenin due to the existence of a 4'-methoxyl group and had relatively weak effects on atherosclerosis. The alleviation of atherosclerosis by the four citrus flavanones was tightly related to differences in their in vivo metabolism and signaling pathways. This provides new insights into the anti-atherosclerotic mechanisms of food functional flavanones and guidance for the design of novel, efficient strategies for preventing atherosclerosis based on citrus flavanones.

Directed evolution of feruloyl esterase from Lactobacillus acidophilus and its application for ferulic acid production.

Producing ferulic acid (FA) from the natural substrate with feruloyl esterase is promising in industries, screening and engineering new enzymes with high efficiency to increase the FA yield is of great concern. Here, the feruloyl esterase of Lactobacillus acidophilus (FAELac) was heterologous expressed and the FAELac with different oligomerization states was separated. Interestingly, the activity of dimer was 37-fold higher than high-polymer. To further enhance the efficiency of FAELac, eight mutants were generated based on the simulated structure, of which Q198A, Q134T enhanced the catalytic efficiency by 5.4- and 4.3-fold in comparison with the wild type. Moreover, higher yields of FA (2.21, 6.60, and 1.67 mg/g substrate, respectively) were released by the mutants from de-starched wheat bran, insoluble wheat arabinoxylan, and steam-exploded corn stover. These results indicated that improving the purification process, engineering new FAELac and substrates bias studies hold great potential for increasing FA production yield.

Upper digestion fate of citrus pectin-stabilized emulsion: An interfacial behavior perspective.

Citrus pectin can serve as a naturally digestion-resistant emulsifier, although how it achieves this effect is still unknown. In this study, the upper digestion fate of an emulsion stabilized by different concentrations of citrus pectin, and changes in its interfacial properties during digestion, were investigated. Emulsions stabilized by high-concentration citrus pectin (3 %) were relatively stable during digestion and had a lower free fatty acid (FFA) release rate than emulsions stabilized by low-concentration citrus pectin (1 %). At the low concentration, the citrus pectin interface had a thin absorbing layer and was largely replaced by bile salts, while at high concentration the citrus pectin interface possessed a uniform and thick adsorbing layer that resisted the replacement of bile salts and enabled lipase adsorption. This study has improved our understanding of the digestion of emulsion from the interface and will be useful for designing emulsion-based functional foods that can achieve targeted release.

Simultaneous determination of 14 bioactive citrus flavonoids using thin-layer chromatography combined with surface enhanced Raman spectroscopy.

Citrus flavonoids consist of diverse analogs and possess various health-promoting effects dramatically depending on their chemical structures. Since different flavonoids usually co-exist in real samples, it's necessary to develop rapid and efficient methods for simultaneous determination of multiple flavonoids. Thin layer chromatography combined with surface enhanced Raman spectroscopy (TLC-SERS) was established to simultaneously separate and detect 14 citrus flavonoids for the first time. These target compounds could be characterized and discriminated when paired with SERS at 6-500 times greater the sensitivity than TLC alone. TLC-SERS exhibited high recovery rates (91.5-121.7%) with relative standard deviation lower than 20.8%. Moreover, the established TLC-SERS method was successfully used to simultaneously detect multiple flavonoids in real samples, which exhibited comparable accuracy to high performance liquid chromatography with shorter analytical time (10 vs 45 min). All the results demonstrated that this could be a promising method for simultaneous, rapid, sensitive and accurate detection of flavonoids.

Naringin Alleviates Atherosclerosis in ApoE-/- Mice by Regulating Cholesterol Metabolism Involved in Gut Microbiota Remodeling.

Naringin, a major flavonoid in citrus, has potential for preventing atherosclerosis. The presence in the colon of a large amount of naringin after oral intake might affect the gut microbiota. We investigated the role of gut microbiota remodeling in the alleviation of atherosclerosis by naringin. Naringin significantly alleviated atherosclerosis and lowered the serum and liver cholesterol levels by 24.04 and 28.37% in ApoE-/- mice fed with a high-fat diet. Nontarget metabolomics showed that naringin modulated the hepatic levels of cholesterol derivatives and bile acids. Naringin increased the excretion of bile acids and neutral sterols by 1.6- and 4.3-fold, respectively. The main potential pathway by which naringin alleviated atherosclerosis was the gut microbiota-liver-cholesterol axis. Naringin modulated the abundances of bile salt hydrolase- and 7α-dehydroxylase-producing bacteria, promoting bile acid synthesis from cholesterol by upregulating CYP7A1 via suppression of the FXR/FGF15 pathway. In addition, naringin facilitated reverse cholesterol transport by downregulating PCSK9/IDOL. The results provide insight into the atherosclerosis-alleviation mechanisms of citrus flavonoids and a scientific basis for the development of functional foods containing citrus flavonoids.

Effect of mesoscopic structure of citrus pectin on its emulsifying properties: Compactness is more important than size.

We previously explored citrus oil emulsion stabilized by citrus pectin. In this report, we characterized key parameters of the citrus pectin mesoscopic structure and their effect on emulsifying capacity, and explored the underlying mechanism by determining the interfacial properties, emulsifying ability, and micromorphology. To generate different mesoscopic structure, citrus pectins were hydrolyzed or regulated by pH and NaCl. Hydrolysis decreased the size of citrus pectin mesoscopic structure with constant compactness, leading to superior interfacial properties but inferior emulsifying ability. In contrast, pH and NaCl regulation decreased the mesoscopic structure size and increased the compactness, and pH- and NaCl-regulated citrus pectin formed a compact absorbed layer at the interface to resist droplet coalescence/flocculation during homogenization. Our results support the importance of compactness of the citrus pectin mesoscopic structure on emulsifying capacity. This study increased our understanding on the relationship between the mesoscopic structures of polysaccharide emulsifier and emulsifying ability.

Gold Nanobones Enhanced Ultrasensitive Surface-Enhanced Raman Scattering Aptasensor for Detecting Escherichia coli O157:H7.

Sensitive, robust, and highly specific detection of Escherichia coli O157:H7, one of the most hazardous foodborne pathogens and the cause of numerous diseases, is needed to ensure public health. Herein, a one-pot step method is reported for the preparation of multifunctional gold nanobones (NBs) (GNRApt-1+RhB) from gold nanorods (GNRs) comediated by an aptamer (Apt-1) and the signal molecule rhodamine B (RhB) for surface-enhanced Raman scattering detection of E. coli O157:H7. The characterized result showed that Apt-1 and RhB were embedded in the gold NBs, and then, this combination exhibited good recognition, excellent stability, and significant Raman signal intensity enhancement. The Raman enhancement derived from a strong electromagnetic field distribution with the locations at the apex of both ends of the GNRApt-1+RhB and the signal stability was because of the firm embedment of Apt-1 (poly A20 + E. coli O157:H7 aptamers) and RhB on the surface of the GNRApt-1+RhB. Optimization experiments established that surface-enhanced Raman-scattered RhB absorption at 1350 cm-1 had a strong linear relationship (y = 180.30x - 61.49; R2 = 0.9982) with E. coli O157:H7 concentration over the range of 10-10,000 cfu/mL with a limit of detection of 3 cfu/mL. This novel aptasensor sensitively detects E. coli O157:H7 and has great promise for food pathogenic bacteria detection.

The structure-property relationships of acid- and alkali-extracted grapefruit peel pectins.

Structure and properties of pectin can be affected by extraction methods. In this study, grapefruit peel pectins extracted by HCl (at pH 1 [P1], 2 [P2], and 3 [P3]) and NaOH (at pH 9 [P9], 10 [P10], and 11 [P11]) were prepared and characterized. Atomic force microscopy (AFM) provided direct evidence of complex nano-structural patterns of pectins and revealed cross-linked networks of P10 and P11. Small-angle X-ray scattering (SAXS) demonstrated that P1, P2, and P3 possessed a relatively extended conformation, whereas P9, P10, and P11 displayed a three-dimensional structure and folded conformation. The compact and extended conformations of P3 contributed to its high viscosity in solution and the stability of the formed emulsion (75%). Porous surface and larger three-dimensional nanostructure (Dmax: 23 nm) of P10 facilitated its ion-binding capacity. Our results provide valuable insight into relationship between extraction methods and structure-properties of pectin, facilitating design of functional pectins.

Characterization of polymethoxyflavone demethylation during drying processes of citrus peels.

Polymethoxyflavones (PMFs) are found almost exclusively in citrus peel and have attracted much attention due to their potential health benefits. Dried citrus peel is an important ingredient for applications in food and traditional Chinese medicine. However, the structural changes of PMFs during drying processes of citrus peel remain unknown. In this study, for the first time we discovered that four major permethoxylated PMFs, i.e. sinensetin, nobiletin, heptamethoxyflavone and tangeretin, underwent demethylation at the 5-position on the A ring of their flavonoid structures to yield corresponding 5-demethylated PMFs during the drying process of citrus peel. Our results further demonstrated that the aforementioned PMF demethylation was through two mechanisms: acid hydrolysis and enzyme-mediated catalysis. PMF demethylation in citrus peels was systematically characterized during hot-air drying (HAD), vacuum-freeze drying (VFD) and sun drying (SD). The highest PMF demethylation was obtained in SD followed by HAD and VFD. This study provided a solid scientific basis for rational control of PMF demethylation in citrus peels, which could facilitate the production of high-quality citrus peel and related products.

The stability of three different citrus oil-in-water emulsions fabricated by spontaneous emulsification.

In this study, emulsions were prepared through spontaneous emulsification, using three different citrus oils as the oil phase and Tween 80 as the surfactant. Utilizing 4% Tween 80, three types of citrus oil emulsions were prepared with small particle size, monomodal distribution and high transmission. After 24 h, each emulsion exhibited different degrees of gravitational separation. Mandarin oil emulsions were the most unstable, showing coalescence of small droplets with an obvious cream layer formed at 9 h. Bergamot oil emulsions possessed small droplets with the best stability over 24 h, due to their relatively polar components (e.g. linalyl acetate) and water-insoluble constituents (e.g. γ-terpinene). These results suggest that the emulsifying properties and instability mechanism of citrus oil emulsions are strongly dependent on the inherent properties and composition of citrus oils. This study is significant for the development of an effective strategy to improve the stability of citrus oil-based colloidal systems.

Characterization of physical properties and electronic sensory analyses of citrus oil-based nanoemulsions.

Citrus oils and their emulsions have been widely used in food and beverage products due to their flavor, various beneficial health functions and relative high solubility for lipophilic bioactive components. However, the non-digestibility and instability has limited the application of emulsions made from a single type of citrus oil. In this study, common triacylglycerol oils (i.e. corn oil and MCT oil) and citrus oils (i.e. bergamot oil and sweet orange oil) were used in combination with different mixing ratios (triacylglycerol oil:citrus oil = 1:0, 9:1, 5:1, 3:1, 1:1 and 0:1) to produce various nanoemulsions (10% oil phase), and their physical and electronic sensory properties were systematically characterized. The results demonstrated that the mixed oil nanoemulsions were much more stable than pure citrus oil emulsions. Electronic nose, electronic eye and electronic tongue were shown to be able to provide informative evaluation of the electronic sensory of the emulsions. Data-fitting of these electronic sensory devices significantly improved the effective discrimination and accuracy of sensory evaluation of the emulsions. These results provided basis for using triacylglycerol oils and citrus oils in combination to produce nanoemulsions with superior physical and electronic sensory properties. Moreover, the electronic sensory evaluation method utilized in this study provided a useful approach for evaluation of emulsion-based food and beverage products.

Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.

Verticillium dahliae transcription factor VdFTF1 regulates the expression of multiple secreted virulence factors and is required for full virulence in cotton.

Fungal transcription factors (TFs) implicated in the regulation of virulence gene expression have been identified in a number of plant pathogens. In Verticillium dahliae, despite its agricultural importance, few regulators of transcription have been characterized. In this study, a T-DNA insertion mutant with significantly reduced virulence towards cotton was identified. The T-DNA was traced to VdFTF1, a gene encoding a TF containing a Fungal_trans domain. Transient expression in onion epidermal cells indicated that VdFTF1 is localized to the nucleus. The VdFTF1-deletion strains displayed normal vegetative growth, mycelial pigmentation and conidial morphology, but exhibited significantly reduced virulence on cotton, suggesting that VdFTF1 is required exclusively for pathogenesis. Comparisons of global transcription patterns of wild-type and VdFTF1-deletion strains indicated that VdFTF1 affected the expression of 802 genes, 233 of which were associated with catalytic processes. These genes encoded 69 potentially secreted proteins, 43 of which contained a carbohydrate enzyme domain known to participate in pathogenesis during infection of cotton. Targeted gene deletion of one VdFTF1-regulated gene resulted in significantly impaired vascular colonization, as measured by quantitative polymerase chain reaction, as well as aggressiveness and symptom severity in cotton. In conclusion, VdFTF1, which encodes a TF containing a Fungal_trans domain, regulates the gene expression of plant cell wall degradation enzymes in V. dahliae, which are required for full virulence on cotton.

Effects of Preheating and Storage Temperatures on Aroma Profile and Physical Properties of Citrus-Oil Emulsions.

Citrus oils are used as good carrier oil for emulsion fabrication due to their special flavor and various health-promoting functions. In this study, the effects of preheating temperature (30, 40, 50, 60, and 70 °C) and storage temperature (4, 25, and 37 °C) on aroma profiles and physical properties of three citrus-oil (i.e., mandarin, sweet orange, and bergamot oils) emulsions were systematically investigated for the first time. The results demonstrated the significant impact of temperature on aroma profile and physical properties. The abundance of d-limonene was found to be the main factor determining the aroma of the three citrus-oil emulsions at different preheating and storage temperatures, while ß-linalool and linalyl acetate were important for the aroma of bergamot oil emulsion. Preheating temperature showed a profound impact on the aroma of citrus-oil emulsions, and the aroma of different citrus oil emulsions showed different sensitivity to preheating temperature. Storage temperature was also able to alter the properties of citrus oil emulsions. The higher was the storage temperature, the more alteration of aroma and more instability of the emulsions there was, which could be attributed to the alteration of the oil components and the properties of emulsions. Among all three emulsions, bergamot-oil emulsion was the most stable and exhibited the most potent ability to preserve the aroma against high temperature. Our results would facilitate the application of citrus-oil emulsions in functional foods and beverages.

Genome-wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.).

Verticillium wilt (VW), caused by infection by Verticillium dahliae, is considered one of the most yield-limiting diseases in cotton. To examine the genetic architecture of cotton VW resistance, we performed a genome-wide association study (GWAS) using a panel of 299 accessions and 85 630 single nucleotide polymorphisms (SNPs) detected using the specific-locus amplified fragment sequencing (SLAF-seq) approach. Trait-SNP association analysis detected a total of 17 significant SNPs at P < 1.17 × 10-5 (P = 1/85 630, -log10 P = 4.93); the peaks of SNPs associated with VW resistance on A10 were continuous and common in three environments (RDIG2015, RDIF2015 and RDIF2016). Haplotype block structure analysis predicted 22 candidate genes for VW resistance based on A10_99672586 with a minimum P-value (-log10 P = 6.21). One of these genes (CG02) was near the significant SNP A10_99672586 (0.26 Mb), located in a 372-kb haplotype block, and its Arabidopsis AT3G25510 homologues contain TIR-NBS-LRR domains that may be involved in disease resistance response. Real-time quantitative PCR and virus-induced gene silencing (VIGS) analysis showed that CG02 was specific to up-regulation in the resistant (R) genotype Zhongzhimian2 (ZZM2) and that silenced plants were more susceptible to V. dahliae. These results indicate that CG02 is likely the candidate gene for resistance against V. dahliae in cotton. The identified locus or gene may serve as a promising target for genetic engineering and selection for improving resistance to VW in cotton.

Verticillium dahliae manipulates plant immunity by glycoside hydrolase 12 proteins in conjunction with carbohydrate-binding module 1.

Glycoside hydrolase 12 (GH12) proteins act as virulence factors and pathogen-associated molecular patterns (PAMPs) in oomycetes. However, the pathogenic mechanisms of fungal GH12 proteins have not been characterized. In this study, we demonstrated that two of the six GH12 proteins produced by the fungus Verticillium dahliae Vd991, VdEG1 and VdEG3 acted as PAMPs to trigger cell death and PAMP-triggered immunity (PTI) independent of their enzymatic activity in Nicotiana benthamiana. A 63-amino-acid peptide of VdEG3 was sufficient for cell death-inducing activity, but this was not the case for the corresponding peptide of VdEG1. Further study indicated that VdEG1 and VdEG3 trigger PTI in different ways: BAK1 is required for VdEG1- and VdEG3-triggered immunity, while SOBIR1 is specifically required for VdEG1-triggered immunity in N. benthamiana. Unlike oomycetes, which employ RXLR effectors to suppress host immunity, a carbohydrate-binding module family 1 (CBM1) protein domain suppressed GH12 protein-induced cell death. Furthermore, during infection of N. benthamiana and cotton, VdEG1 and VdEG3 acted as PAMPs and virulence factors, respectively indicative of host-dependent molecular functions. These results suggest that VdEG1 and VdEG3 associate differently with BAK1 and SOBIR1 receptor-like kinases to trigger immunity in N. benthamiana, and together with CBM1-containing proteins manipulate plant immunity.

Characterization of the Verticillium dahliae Exoproteome Involves in Pathogenicity from Cotton-Containing Medium.

Verticillium wilt, caused by the Verticillium dahliae phytopathogen, is a devastating disease affecting many economically important crops. Previous studies have shown that the exoproteome of V. dahliae plays a significant role in this pathogenic process, but the components and mechanisms that underlie this remain unclear. In this study, the exoproteome of V. dahliae was induced in a cotton-containing C'zapek-Dox (CCD) medium and quantified using the high-throughput isobaric tag technique for relative and absolute quantification (iTRAQ). Results showed that the abundance of 271 secreted proteins was affected by the CCD medium, of which 172 contain typical signal peptides generally produced by the Golgi/endoplasmic reticulum (ER). These enhanced abundance proteins were predominantly enriched in carbohydrate hydrolases; 126 were classified as carbohydrate-active (CAZymes) and almost all were significantly up-regulated in the CCD medium. Results showed that CAZymes proteins 30 and 22 participate in pectin and cellulose degradation pathways, corresponding with the transcription levels of several genes encoded plant cell wall degradation enzyme activated significantly during cotton infection. In addition, targeted deletion of two pectin lyase genes (VdPL3.1 and VdPL3.3) impaired wilt virulence to cotton. This study demonstrates that the V. dahliae exoproteome plays a crucial role in the development of symptoms of wilting and necrosis, predominantly via the pathogenic mechanisms of plant cell wall degradation as part of host plant infection.

The fungicide triadimefon affects beer flavor and composition by influencing Saccharomyces cerevisiae metabolism.

Despite the fact that beer is produced on a large scale, the effects of pesticide residues on beer have been rarely investigated. In this study, we used micro-brewing settings to determine the effect of triadimefon on the growth of Saccharomyces cerevisiae and beer flavor. The yeast growth in medium was significantly inhibited (45%) at concentrations higher than 5 mg L(-1), reaching 80% and 100% inhibition at 10 mg L(-1) and 50 mg L(-1), respectively. There were significant differences in sensory quality between beer samples fermented with and without triadimefon based on data obtained with an electronic tongue and nose. Such an effect was most likely underlain by changes in yeast fermentation activity, including decreased utilization of maltotriose and most amino acids, reduced production of isobutyl and isoamyl alcohols, and increased ethyl acetate content in the fungicide treated samples. Furthermore, yeast metabolic profiling by phenotype microarray and UPLC/TOF-MS showed that triadimefon caused significant changes in the metabolism of glutathione, phenylalanine and sphingolipids, and in sterol biosynthesis. Thus, triadimefon negatively affects beer sensory qualities by influencing the metabolic activity of S. cerevisiae during fermentation, emphasizing the necessity of stricter control over fungicide residues in brewing by the food industry.