Effect of brewing conditions on the chemical and sensory profiles of milk tea.

The brewing conditions of beverage milk tea determine the taste of milk tea. This study investigated the changes in sensory characteristics and small molecule compounds in milk tea made from large-leaf yellow tea under different brewing conditions by sensory analysis, colorimeter, and LC-MS. The results show that the tea to milk ratio is the most important process affecting the taste, and the color values of b* (+yellow, - blue) can be used to evaluate the taste of milk tea made from large leaf yellow tea. The composition of small molecular compounds is affected by tea to milk ratio, which can change the taste of milk tea. l-cysteine and 8-methylsulfinyloctyl glucosinolate are significantly positively correlated with taste by metabolomics analysis. l-cysteine was used to verify the analysis results by LC-MS. The total acceptance of milk tea is improved by adding l-cysteine at a low level (0.025-0.035 mM).

Ascorbic acid regulates mouse spermatogonial stem cell proliferation in a Wnt/ß-catenin/ROS signaling dependent manner.

Spermatogonial stem cells (SSCs) provide a foundation for spermatogenesis, but the mechanism of SSC proliferation is still poorly understood. To investigate whether and how ascorbic acid (AA) regulates the growth of mouse SSCs in vitro, the SSCs were cultured in different concentration AA medium for 14 days. The proliferation, apoptosis and the reactive oxygen species (ROS) levels of SSCs in different AA groups were respectively detected. Moreover, the SSC activity in 40 µg/mL AA group and the control was tested by a transplantation assay. To explore the mechanism of AA regulating mouse SSCs proliferation, the dishevelled homolog 2 (DVL2) and nucleoredoxin (NRX) protein levels, the expression of axis inhibition protein 2 (Axin2), leucine-rich G-protein coupled receptor 5 (Lgr5), B-cell lymphoma-2 (Bcl-2), BCL2-Associated X (Bax), c-myc and cyclin D1 genes in Wnt/ß-catenin pathway were respectively confirmed. The results showed that the adding concentration of AA did not affect the main shape of SSCs. A 40 µg/mL AA in culture medium promoted the proliferation, and decreased the ROS production and apoptosis rate of SSCs. Moreover, colonization efficiency in the seminiferous tubules of the recipient testis in 40 µg/mL AA group was higher compared with the control group by a transplantation assay. Finally, the appropriate ROS in the 40 µg/mL AA group further adjust the levels of DVL2 and NRX protein in the Wnt/ß-catenin pathway to maintain the nuclear intensity of ß-catenin, in turn, the expression of apoptosis gene Bax decreased, while the expression of Bcl2, Axin2, Lgr5, c-myc and cyclin D1 genes increased. The study confirmed that AA adjusts the endogenous ROS level to impact on SSC proliferation in a dose-dependent manner by Wnt/ß-catenin signaling pathway.

l-Theanine improves emulsification stability and antioxidant capacity of diacylglycerol by hydrophobic binding ß-lactoglobulin as emulsion surface stabilizer.

Diacylglycerol (DAG) is commonly used as fat substitute in food manufacture due to its functional properties, but DAG has poor emulsification and oxidation stability, which limits its wide application in food industry. In this work, fluorescence quenching data and thermodynamic parameters were analyzed to investigate the interaction mechanism between l-theanine (L-Th) and ß-lactoglobulin (ß-LG). DAG emulsion was prepared by using ß-lactoglobulin-theanine (ß-LG-Th) as surface stabilizer, and its emulsification and oxidation stability were evaluated. The results showed that the hydrophobic interaction played an important role on the conjugate of ß-LG and L-Th due to the negative values for ΔG, positive values for ΔH and ΔS at pH 4.0, pH 6.0 and pH 8.0. The DAG has been better embedded by using ß-LG-Th as surface stabilizer, and the droplet size was about 0.2 µm to 1.5 µm when the pH was 6.0, the ratio of L-Th to ß-LG was 1:1. ß-LG-Th as surface stabilizer for DAG can increase the ζ-potential and emulsion index, make the emulsion droplet size distribution more uniform. The l-theanine was better to be used to improve the emulsification stability and antioxidant capacity of DAG by binding ß-LG as surface stabilizer.

Aptamer-Based Fluorescence Quenching Approach for Detection of Aflatoxin M1 in Milk.

Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, is a feed and food contaminant of global concern. In this study, we developed a fast and simple method for detection of AFM1 based on a structure-switching signaling aptamer. This aptasensor is based on the change in fluorescence signal due to formation of an AFM1/aptamer complex. To generate the aptasensor, the specific aptamer was modified with FAM (carboxyfluorescein), and their complementary DNAs (cDNA) were modified with a carboxytetramethylrhodamine (TAMRA) quenching group. In the absence of AFM1, the aptamers were hybridized with cDNA, resulting in quenching of the aptamer fluorescence due to the proximity of the aptamer's fluorophore to the quenching group on the cDNA. On the other hand, in the presence of AFM1, a structural switch in the aptamer was induced by formation of an AFM1/aptamer complex. Changes in the structure of the aptamer led to the release of the cDNA, causing the generation of a fluorescence signal. Thus, AFM1 concentrations could be quantitatively monitored based on the changes in fluorescences. Under optimized conditions, this assay exhibited a linear response to AFM1 in the range of 1-100 ng/mL and a limit of detection of 0.5 ng/mL was calculated. This proposed aptasensor was applied to milk samples spiked with a dilution series of AFM1, yielding satisfactory recoveries from 93.4 to 101.3%. These results demonstrated that this detection technique could be useful for high-throughput and quantitative determination of mycotoxin levels in milk and dairy products.

Moderate hypoxia modulates ABCG2 to promote the proliferation of mouse spermatogonial stem cells by maintaining mild ROS levels.

The aim of this study was to investigate the effects of different oxygen (O2) concentrations on the growth of mouse spermatogonial stem cells (SSCs) and the possible mechanisms of cell proliferation in vitro. The SSCs from testicular cells were cultured in various O2 concentrations (1%, 2.5%, 5%, and 20% O2) for 7 days. Colonies of SSCs were identified morphologically and by immunofluorescence. The number of mouse SSC colonies and the area covered by them were measured. Cell cycle progression of the SSCs was analyzed to identify the state of cell proliferation. The effects of O2 concentrations on the levels of intracellular reactive oxygen species (ROS) and expression of ATP binding cassette subfamily G member 2 (ABCG2) were also analyzed in the SSCs. Following culturing for 7 days, the SSCs were treated with Ko143 (a specific inhibitor of ABCG2) for 1 h, and the ROS level and expression of bcl-2, bax, and p53 were analyzed. The results showed that mouse SSCs formed compact colonies and had unclear borders in different O2 concentrations for 7 days, and there were no major morphologic differences between the O2 treatment groups. The expression of the SSC marker, GFR α1 was studied in each O2 treatment group. The number and area of SSC colonies, and the number of GFR α1 positive cells were the highest in the 2.5% O2 treatment group. Compared with other O2 concentrations, the number of cells in G0 cycle was significantly higher, while the level of intracellular ROS was lower at 1% O2. Moreover, the intracellular ROS levels gradually increased with increasing O2 concentration from 1% to 20%. The expression of ABCG2 in the SSCs cultured at 2.5% O2 was higher than in the other O2 groups. Inhibition of ABCG2 increased intracellular ROS generation, and the expression of the pro-apoptotic genes bax and p53, and decreased the expression of the anti-apoptotic gene bcl-2. In conclusion, moderate to low O2 tension increases ABCG2 expression to maintain mild ROS levels, triggers the expression of the anti-apoptotic genes, suppresses the proapoptotic gene pathway, and further promotes the proliferation of mouse SSCs in vitro.

Effects of duration of thermal stress on growth performance, serum oxidative stress indices, the expression and localization of ABCG2 and mitochondria ROS production of skeletal muscle, small intestine and immune organs in broilers.

The purpose of the current study was to investigate that effect of duration of thermal stress on growth performance, oxidative stress indices in serum, the expression and localization of ABCG2, and mitochondria ROS production in skeletal muscle, small intestine and immune organs, and then to further reveal correlations between indicators. At 28 days of age, sixty broilers were randomly divided into the control group (25 ±â€¯2 °C; 24 h/day) and the heat stress group (36 ±â€¯2 °C; 8 h/day lasted for 1 week or 2 weeks). Fifteen broilers per group were respectively euthanized, and some samples were respectively collected from the control and the heat stress groups at the end of the 1st week or the 2nd week of heat stress. A typical heat stress response has been observed at this temperature. Compared with the control group, the birds subjected to heat stress at the end of the 1st week reduced (P ＜ 0.05) body weight (BW), average daily feed intake (ADFI), average daily gain (ADG), the activity of serum antioxidant enzyme and content of glutathione (GSH), while increased (P ＜ 0.05) feed conversion ratio (FCR), serum corticosterone and malondialdehyde (MDA) levels. However, when the heat stress lasted for the end of the 2nd week, there was no significant difference (P ＞ 0.05) in ADFI, ADG, FCR and serum contents of corticosterone, MDA and GSH. Regardless of duration of thermal stress, the localization of ABCG2 protein had no change. Moreover, heat stress also did not affect (P ＞ 0.05) the IOD of the ABCG2 positive portion and the expression of the ABCG2 mRNA in the pectorales, crureus, duodenum, jejunum, ileum and spleen, while significantly increased (P ＜ 0.05) the corresponding tissues ROS production at the end of the 1st week of heat stress. In contrast, at the end of the 2nd week of heat stress, IOD of the ABCG2 positive portion and the expression of the ABCG2 mRNA in heat stress group significantly increased (P ＜ 0.05), while the corresponding tissues ROS production had no difference (P ＞ 0.05) compared to the control group. Collectively, duration of thermal stress affects growth performance, serum oxidative stress indices, and the expression of ABCG2 and the ROS production of broiler tissues in a time-dependent manner. There is a negative correlation between the expression of ABCG2 and the ROS production in the corresponding tissues under heat stress.

[Cloning and Ion Transportation of Cryptosporidium andersoni ATP-binding Cassette 1 Gene].

Objective: To investigate the transportation of intracellular and extracellular K(+), Ca(2+), Na(+) and Mg(2+) under the function of Cryptosporidium andersoni ATP-binding cassette (CaABC) 1 gene. Methods: CaABC1 gene was amplified by PCR using specifically designed primers. The eukaryotic expression plasmid pEGFP-C1-CaABC1 was constructed, and transfected into mouse intestinal epithelial cells via liposome transfection. The blank (with no transfection) and control groups (transfected with empty plasmid pEGFP-C1) were also set. Changes in intracellular and extracellular K(+), Ca(2+), Na(+) and Mg(2+) concentrations were examined by the ion concentration assay kit. Results: PCR amplification resulted in a 544 bp product. The recombinant plasmid pEGFP-C1-CaABC1 was successfully constructed. Green fluorescence was seen in the control and transfected groups, but not in the blank group. The concentrations of K(+), Ca(2+), Na(+) and Mg(2+) in intracellular fluid were （5.51 ± 0.51）, （1.98 ± 0.06）, （108.33 ± 1.33） and （0.93 ± 0.03） mmol/L in the blank group; （6.25 ± 0.70）, （1.90 ± 0.13）, （107.73 ± 1.79） and （0.87 ± 0.05） mmol/L in the control group; and （14.84 ± 0.90）, （3.40 ± 0.14）, （127.64 ± 1.49） and （1.72 ± 0.20） mmol/L in the transfected group. The concentrations of K+, Ca2+, Na+ and Mg2+ in extracellular fluid were （12.72 ± 0.83）, （3.72 ± 0.03）, （116.83 ± 1.04） and (2.02 ± 0.18) mmol/L in the blank group; （10.11 ± 0.90）, （3.58 ± 0.06）, （115.89 ± 1.86） and (1.71 ± 0.41) mmol/L in the control group; and （5.77 ± 0.21）, （1.29 ± 0.18）, （96.21 ± 1.19） and (0.64 ± 0.02) mmol/L in the transfected group. There were significant differences in K+, Ca2+ and Mg2+ concentrations between the transfected group and the control group. Conclusion: CaABC1 participates in the transportation of K+, Ca2+ and Mg2+.

Cloning and Iron Transportation of Nucleotide Binding Domain of Cryptosporidium andersoni ATP-Binding Cassette (CaABC) Gene.

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of Ca(2+), Mg(2+), K(+), and HCO3 (-) in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.

Establishment of recipient model for spermatogonial stem cells transplantation in Kunming mice.

The objective of this study was to establish a recipient model for spermatogonial stem cells (SSCs) transplantation in the Kunming mice after different doses busulfan treatment. The results showed that the most optimal dose of busulfan was 20mg/kg and the most appropriate time for transplantation was 5-7 wk after busulfan treatment. Then, the cloned fragments existed in the testis of recipient mice after 20mg/kg busulfan treatment and the offspring with enhanced green fluorescent protein (EGFP) were produced by the transplanting SSCs. Hence, we established the effective recipient model for donor-derived SSCs transplantation in Kunming mice.

Effect of vitamin C on growth of caprine spermatogonial stem cells in vitro.

The genetic manipulation of spermatogonial stem cells (SSCs) can be used for the production of transgenic animals in a wide range of species. However, this technology is limited by the absence of an ideal culture system in which SSCs can be maintained and proliferated, especially in domestic animals like the goat. The aim of this study therefore was to investigate whether the addition of vitamin C (Vc) in cell culture influences the growth of caprine SSCs. Various concentrations of Vc (0, 5, 10, 25, 40, and 50 µg/mL(-1)) were added to SSC culture media, and their effect on morphology and alkaline phosphatase activity was studied. The number of caprine SSC colonies and area covered by them were measured at 10 days of culture. The expression of various germ cell and somatic cell markers such as VASA, integrins, Oct-4, GATA-4, α-SMA, vimentin, and Thy-1 was studied to identify the proliferated cells using immunostaining analyses. Further, the intracellular reactive oxygen species (ROS) level was measured at the 3rd, 6th, and 9th day after culture, and expression of Bax, Bcl-2, and P53, factors involved in the regulation of apoptosis, were analyzed on the 7th day after culture using reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. The results showed that the SSCs formed compact colonies and had unclear borders in the different Vc-supplemented groups at 10 days, and there were no major morphologic differences between the groups. The number and area of colonies were both the highest in the 40 µg/mL(-1) Vc group. Differential expression of markers for germ cells, undifferentiated spermatogonia, and testis somatic cells was observed. Cultured germ cell clumps were found to have alkaline phosphatase activity regardless of the Vc dose. The number of Thy-1- and Oct-4-positive cells was the most in the 40 µg/mL(-1) Vc group. Moreover, the level of ROS was dependent on the Vc dose and culture time. The Vc dose 40 µg/mL(-1) was found to be optimum with regard to decreasing ROS generation, and increasing the expression of the antiapoptotic gene Bcl-2 and decreasing the expression of the proapoptotic genes Bax and P53. In conclusion, the addition of 40 µg/mL(-1) Vc can maintain a certain physiological level of ROS, trigger the expression of the antiapoptosis gene Bcl-2, suppress the proapoptotic gene P53 and Bax pathway, and further promote the proliferation of caprine SSCs in vitro.

Recognition and enrichment specificity of Fe3O4 magnetic nanoparticles surface modified by chitosan and Staphylococcus aureus enterotoxins A antiserum.

Fe(3)O(4) modified by chitosan (CTS) and Staphylococcus aureus enterotoxin A (SEA) antiserum immunomagnetic beads (Fe(3)O(4)-CTS-SEA-IB) that targeted enrich SEA were prepared in aqueous solution and using CTS and SEA antiserum as surface modification stabilizer. Recognition experiments of the prepared Fe(3)O(4)-CTS-SEA-IB on SEA were conducted to determine adsorption capacity and recognition specificity. The results showed that the SEA antiserum were prepared and successfully conjugated onto Fe(3)O(4)-modificated CTS magnetic beads. The Fe(3)O(4)-CTS-SEA immunomagnetic beads displayed a high adsorption capacity and recognition specificity for SEA, and the adsorption quantity could reach 6.48 × 10(-3) µmol/g. The specificity evaluation results showed that the Fe(3)O(4)-CTS-SEA immunomagnetic beads had high enrichment and affinity property for SEA compared to SEB (Staphylococcus aureus enterotoxin B) and SPA (Staphylococcus aureus protein A).

[Isolation and identification of cow-origin Cryptosporidium isolates in Hefei].

OBJECTIVE: To isolate cow-origin Cryptosporidium in Hefei, and identify its species. METHODS: 285 dairy cattle fecal samples collected from a farm in Hefei were examined by using floating saturated solution of sucrose and modified acid-fast staining. Cryptosporidium oocysts were isolated and purified from positive fecal samples. Genetic DNA was extracted to be the template. According to the sequence of 18S rRNA gene and HSP70 gene from Cryptosporidium sp., the primers were designed and synthesized. The PCR products were amplified by PCR and nested-PCR. The nested PCR products were cloned and sequenced. Homology searches and phylogenic tree construction were done by DNAStar software. RESULTS: Five fecal samples were positive by morphological methods with an infection rate of 1.8% (5/285). Oocysts from the 5 positive fecal samples were elliptical or ovoid detected by using floating saturated solution of sucrose and modified acid-fast staining with the size of 7.37 microm x 6.13 microm and 7.58 microm x 6.20 microm, and a shape index of 1.20 and 1.22, respectively. Nested-PCR resulted in a 18S rRNA and HSP70 gene fragments with approximately 250 bp and 325 bp, respectively. The five isolates showed a high level of nucleic acid identity with sequence data of the 18S rRNA gene of Cryptosporidium andersoni (DQ989573), and they were clustered in the same clade. The highest HSP70 gene sequence identity was found among the five isolates and other reported C. andersoni isolates (AY954892 and DQ989576), and they were placed into the same clade. CONCLUSION: The cow-origin Cryptosporidium isolates derived from Hefei is Cryptosporidium andersoni.

[Research progress on nutrient transport and metabolism of Cryptosporidium].

Cryptosporidiosis is a worldwide zoonotic disease, and Cryptosporidium is coccidia-like parasite that develops in epithelial cells in digestive and respiratory tracts of human and animals. This review summarizes the specific function structure of Cryptosporidium, nutrient uptake, transport, metabolism, and the impact of Cryptosporidium on host nutrient absorption.

[Determination of enrofloxacin residue in chicken muscle using molecular imprinted solid phase extraction-high performance capillary electrophoresis].

Molecular imprinted polymer targeted for enrofloxacin (ENR) was synthesized using ENR as the template molecules, alpha-methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross-linking agent. With the ENR molecular imprinted polymer, a molecular imprinted solid phase extraction-high performance capillary electrophoresis (MISPE-HPCE) method for the determination of ENR in chicken muscle was developed. The results showed that this method exhibited high selectivity and sensitivity for the determination of ENR in chicken muscle. Under the optimized conditions, the limit of detection and limit of quantification were 92.02 microg/kg and 336.04 microg/kg, respectively. The recoveries of the ENR spiked in chicken muscle at different levels were in the range of 77.84%-86.52%, and the relative standard deviations (RSDs) were in the range of 2.18%-3.76%. This MISPE-HPCE method is feasible for the analysis of ENR residue in chicken muscle.

Fluorescence detection of total count of Escherichia coli and Staphylococcus aureus on water-soluble CdSe quantum dots coupled with bacteria.

The aim of this paper was to demonstrate a fluorescence measurement method for rapid detection of two bacterial count by using water-soluble quantum dots (QDs) as a fluorescence marker, and spectrofluorometer acted as detection apparatus, while Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were as detection target bacteria. Highly luminescent water-soluble CdSe QDs were first prepared by using thioglycolic acid (TGA) as a ligand, and were then covalently coupled with target bacteria. The bacterial cell images were obtained using fluorescence microscopy. Our results showed that CdSe QDs prepared in water phase were highly luminescent, stable, and successfully conjugated with E. coli and S. aureus. The fluorescence method could detect 10(2)-10(7)CFU/mL total count of E. coli and S. aureus in 1-2h and the low detection limit is 10(2)CFU/mL. A linear relationship of the fluorescence peak intensity and log total count of E. coli and S. aureus have been established using the equation Y=118.68X-141.75 (r=0.9907).