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The effect of casein phosphopeptides (CPP) and ferrous bisglycinate (FebisGly) at different ratios (1:20, 1:10, and 1:5 w/w) on iron supplementation was investigated. The in vitro bioaccessibility, structural changes, antioxidant activity, and the effect of absorption inhibitors were also explored. The results demonstrated that CPP enhanced the bioaccessibility of FebisGly by 68.72 % ± 0.18 % and increased the ß-sheet content from 21.60 % ± 0.23 % to 67.92 % ± 0.12 %, forming a stable secondary structure. The particle size distribution (PSD) and rheological analyses indicated that CPP significantly contributed to the formation of chelated irons, resulting in a uniform PSD and enhanced viscoelasticity. Moreover, it prolonged the gastric emptying time, reducing gastric irritation further. The carboxyl and amino groups in the CPP molecules participated in chelation reaction, improved the antioxidant activity, and competed with phytic acid, tannic acid, and cellulose for iron. Overall, these results laid a foundation for developing novel iron supplementation strategies.

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Sodium nitrite (SN), a prevalent food preservative, is known to precipitate hepatotoxicity upon exposure. This study elucidates the hepatoprotective effects of corn oligopeptide (COP) and vitamin E (VE) against SN-induced hepatic injury in canine hepatocytes. Canine liver cells were subjected to SN to induce hepatotoxicity, followed by treatment with COP and VE. Evaluations included assays for cell viability, oxidative stress markers, apoptosis, and inflammatory cytokines. Additionally, transcriptomic and metabolomic analyses were performed to delineate the underlying molecular mechanisms. The findings demonstrated that COP and VE significantly ameliorated SN-induced cytotoxicity, oxidative stress, and apoptosis. It was evidenced by restored cell viability, enhanced antioxidant enzyme activity, reduced cytoplasmic enzyme leakage, and decreased levels of malondialdehyde and inflammatory cytokines, with COP showing superior efficacy. The RNA sequencing revealed that COP treatment suppressed the SN-activated aminoacyl-tRNA biosynthesis pathway and TGF-ß/NF-κB signaling pathways, thereby mitigating amino acid depletion, apoptosis, and inflammation. Moreover, COP treatment upregulated genes associated with protein folding, bile acid synthesis, and DNA repair. Metabolomic analysis corroborated these results, showing that COP restored amino acid levels and enhanced bile acid metabolism, alleviating SN-induced metabolic disruptions. These findings offered significant insights into the protective mechanisms of COP underscoring its prospective application in treating liver injuries.

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AIMS: This study evaluates the antibacterial characteristics and mechanisms of combined tea polyphenols (TPs), Nisin, and ε-polylysine (PL) against Streptococcus canis, Streptococcus minor, Streptococcus mutans, and Actinomyces oris, common zoonotic pathogens in companion animals. METHODS AND RESULTS: Pathogenic strains were isolated from feline oral cavities and assessed using minimum inhibitory concentration (MIC) tests, inhibition zone assays, growth kinetics, and biofilm inhibition studies. Among single agents, PL exhibited the lowest MIC values against all four pathogens. TP showed significant resistance against S. minor, and Nisin against S. mutans. The combination treatment (Comb) of TP, Nisin, and PL in a ratio of 13:5:1 demonstrated broad-spectrum antibacterial activity, maintaining low MIC values, forming large inhibition zones, prolonging the bacterial lag phase, reducing growth rates, and inhibiting biofilm formation. RNA sequencing and metabolomic analysis indicated that TP, Nisin, and PL inhibited various membrane-bound carbohydrate-specific transferases through the phosphoenolpyruvate-dependent phosphotransferase system in S. canis, disrupting carbohydrate uptake. They also downregulated glycolysis and the citric acid cycle, inhibiting cellular energy metabolism. Additionally, they modulated the activities of peptidoglycan glycosyltransferases and d-alanyl-d-alanine carboxypeptidase, interfering with peptidoglycan cross-linking and bacterial cell wall stability. CONCLUSIONS: The Comb therapy significantly enhances antibacterial efficacy by targeting multiple bacterial pathways, offering potential applications in food and pharmaceutical antimicrobials.

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CD19 is a surface antigen on B cells that regulates B cell activation and proliferation, participating in B cell signaling. It is expressed in all B cell lineage tumor diseases, making CD19 a significant marker for detecting B cell tumor diseases and an important target for related immunotherapies. In recent years, with the deepening research on canine and feline diseases and the establishment of animal models, the demand for cat CD19 monoclonal antibodies (mAbs) has been steadily increasing. We successfully prepared cat CD19-specific monoclonal antibodies using a KLH-conjugated cat CD19 peptide as an antigen and optimized the antibody production method. The obtained monoclonal antibodies' molecular and cellular affinities were identified using CD19 peptides, eukaryotic overexpressed proteins, and peripheral blood mononuclear cells (PBMCs). The results indicate that the CD19-3H9 and CD19-8A7 monoclonal antibodies prepared in this study specifically bind to the CD19 molecule, demonstrating their suitability for use in ELISA, Western blot, and cell assays. This study successfully produced cat CD19 monoclonal antibodies with specificity and optimized the antibody preparation method, laying the foundation for the diagnosis and targeted drug combination therapy of B cell tumor diseases in both humans and pets.

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Omega-3 polyunsaturated fatty acids (n-3 PUFAs), mainly including α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), possess antioxidant properties and play a crucial role in growth and development. However, the combined effects of ALA, EPA, and DHA at different concentrations have rarely been reported. This work explored the effects of EPA, ALA, and DHA on the viability and antioxidant capacity of mouse hepatocytes, with the objective of enhancing the antioxidant capacity. Within the appropriate concentration range, cell viability and the activity of glutathione S-transferase, superoxide dismutase, and catalase were increased, while the oxidation products of malondialdehyde and the level of intracellular reactive oxygen species were obviously reduced. Thus, oxidative stress was relieved, and cellular antioxidant levels were improved. Finally, response surface optimization was carried out for EPA, ALA, and DHA, and the model was established. The antioxidant capacity of the cells was highest at EPA, ALA, and DHA concentrations of 145.46, 405.05, and 551.52 µM, respectively. These findings lay the foundation for further exploration of the interactive mechanisms of n-3 PUFAs in the body, as well as their applications in nutraceutical food.

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In this study, the utilization mechanism of oligosaccharides by Bifidobacterium was investigated through the transcriptome sequencing and non-targeted metabolomics technology of Bifidobacterium animalis cultured with fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS). The results showed that FOS affected the synthesis of adenosine triphosphate binding transporters (ABC transporters) by increasing the expression levels of msmE, msmG, and gluA. Similarly, GOS improved aminoacyl-tRNA synthases by upregulating the expression of tRNA-Ala, tRNA-Pro, and tRNA-Met. Bifidobacterium animalis cultured with FOS and GOS produced different metabolites, such as histamine, tartaric acid, and norepinephrine, with the functions of inhibiting inflammation, alleviating depression and diseases related to brain and nervous system and maintaining body health. Furthermore, the transcriptome and metabolome analysis results revealed that FOS and GOS promoted the growth and metabolism of Bifidobacterium animalis by regulating the related pathways of carbohydrate, energy, and amino acid metabolism. Overall, the experimental results provided significant insights into the prebiotic effects of FOS and GOS.

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AIMS: To construct the microemulsion delivery system (ME) loading ATSO and NA and study their physicochemical characteristics to enhance their stability and water solubility. METHODS: By plotting ternary phase diagrams, the composition and proportions of the MEs were determined. The physicochemical characteristics and stability of MEs were evaluated by mean diameter, polydispersity index (PDI), pH, electrical conductivity, transmission electron microscopy (TEM), rheological behaviour measurement, and phase inversion temperature (PIT). RESULTS: The MEs was composed with EL-40 as a surfactant and specifically with the addition of ethanol as a cosurfactant in NA-loaded ME. The mean diameters of ATSO-loaded ME and NA-loaded ME were 39.65 ± 0.24 nm and 32.90 ± 2.65 nm, and PDI were 0.49 ± 0.01 and 0.28 ± 0.14, respectively. The TEM confirmed the spherical and smooth morphology of MEs. The rheological results indicated that MEs are dilatant fluids with the advantages of low viscosity, high fluidity, and tolerance to temperature fluctuations. The mean diameter and PDI of MEs showed no significant change after storage at 25 °C for 28 days and centrifugation. CONCLUSION: The prepared microemulsions could expand the application prospects of ATSO and NA products in cosmetics, medicine, foods and other fields.

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Cassava protein (CP), barley protein (BP) and yellow pea protein (YPP) are important nutrient and integral constituent of staple in pet foods. It is known that the digestion of proteins directly influences their absorption and utilisation. In the present work, we performed in vitro simulated gastrointestinal digestion of three plant proteins as a staple for dog and cat food. The digestion rate of CP, BP and YPP in dog food was 56.33 ± 0.90%, 48.53 ± 0.91%, and 66.96 ± 0.37%, respectively, whereas the digestion rate of CP, BP, and YPP in cat food was 66.25 ± 0.72%, 43.42 ± 0.83%, and 58.05 ± 0.85%, respectively. Using SDS-polyacrylamide gel electrophoresis to determine the molecular weight (MW) of each protein and the products of their digestion, it was revealed that MW of digestion samples decreased, and MW during the small intestine phase was lower than that during the gastric phase. Peptide sequences of digested products were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and it was found that the total number of peptides in the small intestine digestion samples was higher than that in the gastric phase samples. The MW of peptides obtained from CP was within the range of 1000-1500 Da, while MW of peptides derived from BP and YPP was within the range of 400-2000 Da. In addition, free amino acids were mainly produced in the small intestine phase. Furthermore, the percentage of essential amino acids in the small intestine phase (63 ~ 82%) was higher than that in the gastric phase (37 ~ 63%). Taken together, these findings contribute to the current understanding of the utilisation of plant proteins in dog and cat foods and provide important insights into the selection and application of plant proteins as a staple in dog and cat foods.

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Identification of the phosphatidylserine (PS) discrepancies occurring on the cellular membrane during apoptotic processes is of the utmost importance. However, monitoring the quantity of PS molecules in real-time at a single-cell level currently remains a challenging task. Here, we demonstrate this objective by leveraging the specific binding and reversible interaction exhibited by the zinc(II) dipyridinamine complex (ZnDPA) with PS. Lipoic acid-functionalized ZnDPA (LP-ZnDPA) was subsequently immobilized onto the surface of an atomic force microscopy cantilever to form a force probe, ALP-ZnDPA, enabling a PS-specific dynamic imaging and detection mode. By utilizing this technique, we can not only create a heat map of the expression level of PS with submicron resolution but also quantify the number of molecules present on a single cell's surface with a detection limit of 1.86 × 104 molecules. The feasibility of the proposed method is demonstrated through the analysis of PS expression levels in different cancer cell lines and at various stages of paclitaxel-induced apoptosis. This study represents the first application of a force probe to quantify PS molecules on the surface of individual cells, providing insight into dynamic changes in PS content during apoptosis at the molecular level and introducing a novel dimension to current detection methodologies.

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BACKGROUND: Calcium is important in the formation of bones and teeth, cell metabolism, and other physiological activities. In this work, casein phosphopeptide-calcium chelate (CPP-Ca) was synthesized and the optimal process parameters for the chelation reaction were obtained. The bioavailability of calcium in CPP-Ca was investigated by in vitro gastrointestinal simulated digestion. The existence of phytic acid and oxalic acid in the digestion system was evaluated to clarify the calcium holding ability of casein phosphopeptide (CPP). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify oligopeptides from CPP-Ca. RESULTS: The optimal process parameters for the chelation reaction were: peptide concentration 7.76 mgmL-1 , pH 8.54, and reaction temperature 43.3 °C. The digestion in vitro results indicated that the calcium release rate of CPP-Ca in the stomach for 2 h reached 85%, and about 50% of the ionized calcium was re-chelated with CPP in the intestine. Phytic acid and oxalic acid could lead to a sharp decrease in soluble calcium but around 50% of the calcium was still retained in the form of chelates in the presence of CPP. The LC-MS/MS identified 19 casein-derived oligopeptides after digestion, and calcium modifications were found on eight peptides derived from ß-casein and αs2 -casein. CONCLUSIONS: This study clarified the excellent calcium holding capacity of CPP in the presence of phytic acid and oxalic acid. Liquid chromatography-tandem mass spectrometry also revealed peptide changes, and identified peptides that chelate with calcium. These findings provided significant insights that could be relevant to the further utilization and product development of peptide-calcium chelate in the food industry. © 2023 Society of Chemical Industry.

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Changes in diet and environment can lead to acute diarrhea in companion animals, but the composition and interactions of the gut microbiome during acute diarrhea remain unclear. In this multicenter case-control study, we investigated the relationship between intestinal flora and acute diarrhea in two breeds of cats. Acutely diarrheic American Shorthair (MD, n = 12) and British Shorthair (BD, n = 12) and healthy American Shorthair (MH, n = 12) and British Shorthair (BH, n = 12) cats were recruited. Gut microbial 16S rRNA sequencing, metagenomic sequencing, and untargeted metabolomic analysis were performed. We observed significant differences in beta-diversity (Adonis, P < 0.05) across breeds and disease state cohorts. Profound differences in gut microbial structure and function were found between the two cat breeds. In comparison to healthy British Shorthair cats, Prevotella, Providencia, and Sutterella were enriched while Blautia, Peptoclostridium, and Tyzzerella were reduced in American Shorthair cats. In the case-control cohort, cats with acute diarrhea exhibited an increased abundance of Bacteroidota, Prevotella, and Prevotella copri and a decreased abundance of Bacilli, Erysipelotrichales, and Erysipelatoclostridiaceae (both MD and BD cats, P < 0.05). Metabolomic analysis identified significant changes in the BD intestine, affecting 45 metabolic pathways. Moreover, using a random forest classifier, we successfully predicted the occurrence of acute diarrhea with an area under the curve of 0.95. Our findings indicate a distinct gut microbiome profile that is associated with the presence of acute diarrhea in cats. However, further investigations using larger cohorts of cats with diverse conditions are required to validate and extend these findings. IMPORTANCE Acute diarrhea is common in cats, and our understanding of the gut microbiome variations across breeds and disease states remains unclear. We investigated the gut microbiome of two cat breeds (British Shorthair and American Shorthair) with acute diarrhea. Our study revealed significant effects of breeds and disease states on the structure and function of the gut microbiota in cats. These findings emphasize the need to consider breed-related factors in animal nutrition and research models. Additionally, we observed an altered gut metabolome in cats with acute diarrhea, closely linked to changes in bacterial genera. We identified a panel of microbial biomarkers with high diagnostic accuracy for feline acute diarrhea. These findings provide novel insights into the diagnosis, classification, and treatment of feline gastrointestinal diseases.

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For different breeds of dogs with acute diarrhea, the gut microbiota and metabolome profiles are unclear. This prospective observational study analyzed the gut microbiomes of poodles with acute diarrhea and Labrador retrievers with acute diarrhea based on 16S amplicon sequencing, with respective healthy dogs as controls. Fecal non-target metabolomics and metagenomics were performed on poodles with acute diarrhea. This study found that the diversity and structure of the microbial community differed significantly between the two breeds in cohorts of healthy dogs. Two breeds of dogs with acute diarrhea demonstrated different changes in microbial communities and metabolic functions. The metabolism of starch and sucrose was significantly decreased in dogs with acute diarrhea, which may be attributed to the reduced activity of dextran dextrinase. Non-targeted metabolomics identified 21 abnormal metabolic pathways exhibited by dogs with acute diarrhea, including starch, amino acid, bile acid metabolism, etc., and were closely related to specific intestinal flora. This study provided new insights into breed specificity and the development of dietary treatment strategy in canine gastrointestinal disease.

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In this work, CPP-Ca chelate was synthesized by chelating casein phosphopeptide (CPP) and calcium and characterized by Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) Energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). The antioxidant activity and calcium holding capacity of CPP-Ca were evaluated and its secondary structure transition was monitored during gastrointestinal digestion by in situ Raman spectroscopy. The results demonstrated that calcium chelating rate reached 40 % and calcium ion was bound to CPP mainly through the interaction of carboxyl and amino groups. The result of calcium holding capacity confirmed the formation of calcium phosphate precipitates could be delayed by 10-15 min with increasing CPP concentration. In vitro simulated digestion revealed CPP-Ca exhibited excellent calcium solubility and its secondary structural changes occurred, especially α-helix and ß-sheet content. These findings provided significant insights into enhancing bioavailability of calcium supplements and developing of calcium functional foods for human and animals.

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Exposure to environmental particulate matter (PM) causes lung damage in humans, but it is not sufficiently studied in companion animals. In this work, we found that organic extracts (OE) of PM induced oxidative stress and were more cytotoxic than water-soluble extracts (WE) of PM in canine (Canis familiaris) pulmonary alveolar type II epithelial (PAE) cells. Subsequently, we evaluated the alleviating effects of polyunsaturated fatty acid mixtures (eicosapentaenoic and docosahexaenoic acids), vitamin E, and lycopene on OE-exposed PAE cells. The results indicated that the three nutrients attenuated OE-induced oxidative stress. Compared with OE-exposed PAE cells, cells pretreated with the three nutrients exhibited a 1.7 ~ 2.2-fold reduction in reactive oxygen species, 15.58% ~ 19.96% increase in cell viability, 26.19% ~ 29.32% reduction in lactate dehydrogenase release and 33.87% ~ 40.10% reduction in intracellular malondialdehyde. Meanwhile, the activities of superoxide dismutase, catalase, and glutathione peroxidase increased by 35.22% ~ 47.70%, 45.36% ~ 64.13%, and 48.56% ~ 68.18%. Besides, microscopic observation revealed that nutrients improved cell morphology, as evidenced by reduced cell shrinkage and increased apposition. Finally, the mechanisms of OE toxicity and antioxidant nutrients were explored respectively. The results revealed that OE triggered cytotoxicity by directly disrupting antioxidant enzyme activity through activation of ROS, while nutrients inhibited OE-induced cellular oxidative stress via upregulation of the Nrf2/HO-1 signaling pathway. The present study may provide significant insights into the prevention of PM-induced lung diseases by antioxidant supplementation in animals.

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Acer truncatum seed oil (ATSO) contains abundant unsaturated fatty acids, with significant quantities of nervonic acid (NA, > 5%), which was authenticated as a new food resource in China. For the sake of minimizing animal consumption and the importance of NA for human health, extraction of NA from plants has become a research hotspot. In the present study, three extraction factors were determined to significantly influence the saponification reaction based on single-factor experiments: NaOH dosage, reaction time, and reaction temperature. These three factors were used to further optimize the saponification process through the response surface methodology, and the highest yield of mixed fatty acids was 83.12%. Moreover, the activation energy (40.8228 kJ/mol), the pre-exponential factor [2.568 × 106 m3 /(kmol·min)], and the kinetic equation [rA = kcA cB = 2.568 × 106 ·exp(- 4970 . 1 T ) $\frac{{{\rm{4970}}{\rm{.1}}}}{{\rm{T}}})$ cA cB ] of the ATSO saponification reaction were determined by combining the chemical reaction rate equation of the elementary reaction, the Arrhenius equation, and the NaOH concentration in the substrate. Finally, the mixed fatty acids of ATSO were crystallized by triple-stage low-temperature crystallization, and we achieved 25.05% purity for NA. This study provides a technological basis and strategy for specific fatty acid production from ASTO, as well as other vegetable oils important in the field of food and health supplement products. PRACTICAL APPLICATION: Nervonic acid (NA) is an essential component of neural cells and neural tissue, and it is vital for maintaining the normal work of nerve tissues in organisms and promotes neurodevelopment. NA has traditionally been mainly obtained from shark hunting, which is now restricted due to an international ban on shark fishing. The alternative way to produce NA cheaply and in large quantities is from plant sources. The techniques utilized in this study provide an effective method of NA separation from Acer truncatum seed oil for industrial production.

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Ambient particulate matter (APM) is extremely harmful to life's health. In this study, we investigated cellular injury in cat (Felix catus) lung cells (FCA-L2) exposed to organic and water-soluble extracts from APM. As well, the protective effect of vitamin E (VE), lycopene and a mixture of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (molar concentration ratio of 2:1) against this damage was evaluated. Organic and water-soluble extracts induced oxidative stress in FCA-L2 cells, as evidenced by excess reactive oxygen species production and mitochondrial damage, while treatment with VE, lycopene and EPA: DHA remarkably alleviated these indices. It was further found that treatment with EPA: DHA decreased lactate dehydrogenase and malondialdehyde, as well as increased activities of superoxide dismutase, glutathione peroxidase and catalase. Our study confirmed that nutrients mediates APM-induced oxidative stress via antioxidant proteins. Also, these findings could provide new insights into reducing APM-induced cytotoxicity by nutritional supplementation based on antioxidant compounds for animals.

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BACKGROUND: Melt crystallization is typically recognized as a highly efficient and green method for oil fractionation. This work concentrated on novel layer melt crystallization for preparing desirable olein and stearin products from palm oil and the evaluation of fraction quality. Layer melt crystallization was performed at various temperatures and the effects on fractions were evaluated using iodine value (IV), solid fat content (SFC) and melting point. The lipid composition, thermal and crystallization properties, and phase behaviors of the final optimized fractions were determined using gas chromatography, high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry and differential scanning calorimetry. RESULTS: Increasing crystallization tube temperatures under the same jacket temperature increased the melting point and SFC, while decreasing the IV of the olein product. Opposite results were observed for the stearin product. Major fatty acids in fractions were determined as palmitic acid and oleic acid. 1,2-Dioleoyl-3-palmitoylglycerol and 1,3-dipalmitoyl-2-oleoylglycerol were identified as the main triacylglycerols in olein and stearin fractions, respectively. A critical effect of crystallization temperature on co-crystallization of oleins and stearins was revealed. A transition from plate-like crystal growth to spherulitic growth with spontaneous nucleation was indicated in palm oil and stearin fractions with increasing crystallization temperature. As for olein fractions, a temperature increase resulted in heterogeneous nucleation from instantaneous nucleation. CONCLUSIONS: Novel layer melt crystallization was successfully applied and optimized for fractionating palm oil. The composition and property changes of obtained fractions were analyzed and explained at both macroscopic and microscopic levels. © 2021 Society of Chemical Industry.

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BACKGROUND: Ziziphus jujuba Miller cv. Dongzao is extremely susceptible to reddening, browning, nutritional loss, and perishability after harvest. In this study, we evaluated the mechanisms of calcium chloride and chitosan/nano-silica composite film treatments on the quality, especially in reddening, by physiological and metabolomic assays. RESULTS: The treatment delayed the decline of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and chalcone isomerase (CHI) activities. Meanwhile, the treated groups retarded the increases in anthocyanin and quercetin contents by inhibiting the gene expressions of flavonol synthase (ZjFLS), dihydroflavonol 4-reductase (ZjDFR), and anthocyanidin synthase (ZjANS), while promoting leucoanthocyanidin reductase (ZjLAR) expression, which leads to retardation of fruit reddening. Anthocyanins were found to be responsible for post-harvest winter jujube reddening through principal component analysis. Results from the technique for order preference by similarity to an ideal solution indicated that the treated group delayed the decline of the quality of 'Dongzao' and extended its shelf life. CONCLUSION: The treatment induced the heightening of flavonoids metabolism. They enhanced the nutritional value and the ability to resist stress by delaying the decline of PAL, CHS, and CHI activities. Meanwhile, the treated groups retarded the increase in anthocyanin and quercetin contents by inhibiting the gene expressions of ZjFLS, ZjDFR, and ZjANS and promoting ZjLAR expression, which leads to retardation of fruit reddening. Anthocyanins are responsible for post-harvest winter jujube reddening. Coating treatment effectively delayed the decline of winter jujube quality. © 2020 Society of Chemical Industry.

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In this work, a microemulsion emitting fluorescence was fabricated as a potential oral delivery system for bioactive compounds. In simulated oral administration, the microemulsion was characterized for its microstructure by 1hydrogen-nuclear magnetic resonance (1H-NMR). Results showed that microemulsions not only have good resistance to oral and gastric phases, but also lay a solid foundation for the release of bioactive compounds in the intestine. Fluorescence stability tests showed that microemulsions exhibit a remarkable fluorescence intensity in the digestive environment, indicating feasibility as a label-free delivery carrier. Moreover, in vitro release tests of bioactive compounds confirmed that an α-linolenic acid (ALA)-loaded microemulsion mainly released in the intestine, thereby achieving the aim of controlling the release of bioactive compounds. These results suggest that the synthesized fluorescent microemulsion, combining the favorable features of nontoxicity, antidigestive stability, remarkable fluorescence intensity, and controllable release, can be regarded as a promising label-free delivery carrier for oral administration.

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In this work, the effects of α-linolenic acid (ALA) loaded in oil-in-water (O/W) and water-in-oil-in-water (W/O/W) microemulsions on cell viability, lactic dehydrogenase (LDH) viability, and reactive oxygen species (ROS) levels were examined using Cell Counting Kit-8 (CCK-8), an LDH assay kit, and a fluorescence microscope, respectively. The CCK-8 assay demonstrated that ALA inhibited MDA-MB-231 human breast cancer cell proliferation in a dose-dependent manner. Further, the results of LDH activity and ROS levels revealed that ALA-induced cancer cell damage was closely related to oxidative stress. Under the irradiation of ultraviolet light, the microemulsion without any added fluorescent dye would emit bright blue fluorescence, and the fluorescent images of the cells treated with ALA-loaded O/W and W/O/W microemulsions at different incubation times were taken, which exhibited long-term photostability and biocompatibility. In addition, the fluorescence mechanism of the microemulsion was explained by immobilizing surfactant molecules with aggregation-induced emission (AIE) properties at the water-oil interface through the microemulsion with a self-assembled structure. These findings showed the potential application of O/W and W/O/W microemulsions as the label-free delivery carriers in long-term imaging of living cells and real-time release monitoring of nutrients.

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