Chen Niu

Kinetics of adsorption of bovine serum albumin on magnetic carboxymethyl chitosan nanoparticles

The magnetic carboxymethyl chitosan nanoparticles (MNPs-CMC) were developed as effective magnetic affinity adsorbent for Bovine serum albumin and the adsorption reactions were investigated. The obtained experimental data were compared with the adsorption kinetics models and equilibrium isotherms. The experimental kinetic data were modeled using Pseudo-first order, Pseudo-second order, Banghams equation, Intra-particle diffusion model and Elovich equations. It was found that the adsorption reactions followed the Pseudo-second order kinetics equation. The experimental isotherm data were analyzed using Langmuir, Freundlich, Dubinin-Radushkevich and Temkin equations. By comparing the correlation coefficients determined for each linear transformation of isotherm analysis, it was found that the Langmuir equation was the best fit equilibrium model for the adsorption of BSA. Error functions have been used to determine the alternative single component parameters by nonlinear regression due to the inherent bias in using the correlation coefficient resulting from linearization. It showed that the Langmuir equation resulted in the lowest values for the error function and thus fitted the data better than the other isotherm. Various thermodynamic parameters such as enthalpy (ΔH°), entropy (ΔS°) and Gibbs free energy (ΔG°) were evaluated. MNPs-CMC nanoaprticles were shown to be a promising material for adsorption of BSA from aqueous solutions.

An immunomagnetic separation-real-time PCR system for the detection of Alicyclobacillus acidoterrestris in fruit products

Alicyclobacillus acidoterrestris is the most important spoilage species within the Alicyclobacillus genus and has become a major issue in the pasteurized fruit juice industry. The aim of this study was to develop a method combining immunomagnetic separation (IMS) with real-time PCR system (IMS-PCR) for rapid and specific detection of A. acidoterrestris in fruit products. A real-time PCR with the TaqMan system was designed to target the 16S rDNA genes with specific primer and probe set. The specificity of the assay was confirmed using 9 A. acidoterrestris strains and 21 non-A. acidoterrestris strains. The results indicated that no combination of the designed primers and probe was found in any Alicyclobacillus genus except A. acidoterrestris. The detection limit of the established IMS-PCR was less than 10CFU/mL and the testing process was accomplished in 2-3h. For the three types of samples (sterile water, apple juice and kiwi juice), the correlation coefficient of standard curves was greater than 0.991, and the calculated PCR efficiencies were from 108% to 109%. As compared with the standard culture method performed concurrently on the same set of samples, the sensitivity, specificity and accuracy of IMS-PCR for 196 naturally contaminated fruit products were 90.0%, 98.3% and 97.5%, respectively. The results exhibited that the proposed IMS-PCR method was effective for the rapid detection of A. acidoterrestris in fruit products.

Development and evaluation of an immunomagnetic separation-ELISA for the detection of Alicyclobacillus spp. in apple juice.

The immunomagnetic separation (IMS) technique was used in combination with an enzyme-linked immunosorbent assay (ELISA) procedure to shorten the total analysis time and improve the sensitivity for the detection of Alicyclobacillus spp. in apple juice samples. The specificity of IMS-ELISA for twenty strains of Alicyclobacillus spp. and eighteen strains of non-Alicyclobacillus spp. was determined and there was little cross-reaction with non-Alicyclobacillus strains. Artificially contaminated apple juice with different concentrations of Alicyclobacillus acidoterrestris was detected by IMS-ELISA, and the detection limit of the assay in apple juice was 10(3)CFU/mL. Furthermore, the sample inoculated with 1CFU/mL of A. acidoterrestris could be detected as positive after incubation for 24h. The IMS-ELISA described, allows for the identification of suspect positive samples within 3h of testing versus 3-5days required by standard culture methods while significantly reducing the materials and labor required for the detection of Alicyclobacillus spp. in apple juice samples. As compared with the standard culture method performed concurrently on the same set of samples, the sensitivity, specificity and accuracy of IMS-ELISA for 102 naturally contaminated apple juice samples were 91.3%, 96.02% and 95.09%, respectively. These results demonstrated that the newly proposed IMS-ELISA procedure can be a potentially useful analytical method for the detection of Alicyclobacillus spp. in apple juice.

Immunomagnetic separation combined with polymerase chain reaction for the detection of Alicyclobacillus acidoterrestris in apple juice.

A combination of immunomagnetic separation (IMS) and polymerase chain reaction (PCR) was used to detect Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice. The optimum technological parameters of the IMS system were investigated. The results indicated that the immunocapture reactions could be finished in 60 min and the quantity of IMPs used for IMS was 2.5 mg/mL. Then the combined IMS-PCR procedure was assessed by detecting A. acidoterrestris in apple juice samples. The agarose gel electrophoresis results of 20 different strains showed that the IMS-PCR procedure presented high specificity to the A. acidoterrestris. The sensitivity of the IMS-PCR was 2×101 CFU/mL and the total detection time was 3 to 4 h. Of the 78 naturally contaminated apple juice samples examined, the sensitivity, specificity and accuracy of IMS-PCR compared with the standardized pour plate method were 90.9%, 97.0% and 96.2%, respectively. The results exhibited that the developed IMS-PCR method will be a valuable tool for detecting A. acidoterrestris and improving food quality in juice samples.

Development of Polyclonal Antibody-Based Indirect Enzyme-Linked Immunosorbent Assay for the Detection of Alicyclobacillus Strains in Apple Juice

A sort of specific polyclonal anti-Alicyclobacillus antibody was generated by immunizing New Zealand white rabbits, and a sensitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for Alicyclobacillus detection in apple juice. A set of experimental parameters such as concentration of antigen, dilutions of the antibody and goat anti-rabbit IgG-horseradish peroxidase conjugate, selection of the blocking reagent, incubation time, and temperature was optimized. The cross-reactivity of the antibody was evaluated by ELISA and the result was consistent with Western blot analysis. The detection limit of the ELISA was about 10(5) colony forming units (CFU)/mL in apple juice samples. Samples were detected by ELISA and conventional culture method, and the ELISA results gave a good agreement with the results obtained by plating on Alicyclobacillus acidoterrestris medium agar. ELISA takes a total detection time of 6 to 7 h, which is less than the time of conventional techniques requiring more than 24 to 48 h. These results indicated that the established ELISA was a potential useful analytical method for detection of Alicyclobacillus in apple juice.

Characterization of Osmotolerant Yeasts and Yeast-Like Molds from Apple Orchards and Apple Juice Processing Plants in China and Investigation of Their Spoilage Potential

Yeasts and yeast-like fungal isolates were recovered from apple orchards and apple juice processing plants located in the Shaanxi province of China. The strains were evaluated for osmotolerance by growing them in 50% (w/v) glucose. Of the strains tested, 66 were positive for osmotolerance and were subsequently identified by 26S or 5.8S-ITS ribosomal RNA (rRNA) gene sequencing. Physiological tests and RAPD-PCR analysis were performed to reveal the polymorphism of isolates belonging to the same species. Further, the spoilage potential of the 66 isolates was determining by evaluating their growth in 50% to 70% (w/v) glucose and measuring gas generation in 50% (w/v) glucose. Thirteen osmotolerant isolates representing 9 species were obtained from 10 apple orchards and 53 target isolates representing 19 species were recovered from 2 apple juice processing plants. In total, members of 14 genera and 23 species of osmotolerant isolates including yeast-like molds were recovered from all sources. The commonly recovered osmotolerant isolates belonged to Kluyveromyces marxianus, Hanseniaspora uvarum, Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Candida tropicalis, and Pichia kudriavzevii. The polymorphism of isolates belonging to the same species was limited to 1 to 3 biotypes. The majority of species were capable of growing within a range of glucose concentration, similar to sugar concentrations found in apple juice products with a lag phase from 96 to 192 h. Overall, Z. rouxii was particularly the most tolerant to high glucose concentration with the shortest lag phase of 48 h in 70% (w/v) glucose and the fastest gas generation rate in 50% (w/v) glucose.

A novel method to quantify the activity of alcohol acetyltransferase Using a SnO2-based sensor of electronic nose

Alcohol acetyltransferase (AATFase) extensively catalyzes the reactions of alcohols to acetic esters in microorganisms and plants. In this work, a novel method has been proposed to quantify the activity of AATFase using a SnO2-based sensor of electronic nose, which was determined on the basis of its higher sensitivity to the reducing alcohol than the oxidizing ester. The maximum value of the first-derivative of the signals from the SnO2-based sensor was therein found to be an eigenvalue of isoamyl alcohol concentration. Quadratic polynomial regression perfectly fitted the correlation between the eigenvalue and the isoamyl alcohol concentration. The method was used to determine the AATFase activity in this type of reaction by calculating the conversion rate of isoamyl alcohol. The proposed method has been successfully applied to determine the AATFase activity of a cider yeast strain. Compared with GC-MS, the method shows promises with ideal recovery and low cost.

Preparation and Characterization of Carboxymethyl Chitosan Modified Magnetic Nanoparticles for Bovine Serum Albumin Adsorption

Carboxymethyl chitosan (CMC) were prepared from the carboxymethylation reaction of chitosan under mild conditions, and magnetic carboxymethyl chitosan nanoparticles (MNPs-CMC) were developed by binding CMC on the surface of magnetic Fe3O4 nanoparticles (MNPs) via EDC/NHS activation. The characterization results indicated that MNPs were successfully modified by CMC and the modified nanoparticles had a good magnetic property. By investigating the adsorption of BSA, it was found that the maximum BSA immobilization capacity was up to 159.51 mg·g−1. The adsorption characteristics of BSA can fit the pseudo-second-order model and Langmuir equation well. Furthermore, a high desorption efficiency of 81.47% was achieved under an alkaline condition (pH 9.6).

Accessing spoilage features of osmotolerant yeasts identified from kiwifruit plantation and processing environment in Shaanxi, China.

Osmotolerant yeasts originating from kiwifruit industrial chain can result in spoilage incidences, while little information is available about their species and spoilage features. This work identified possible spoilage osmotolerant yeasts from orchards and a manufacturer (quick-freeze kiwifruit manufacturer) in main producing areas in Shaanxi, China and further characterized their spoilage features. A total of 86 osmotolerant isolates dispersing over 29 species were identified through 26S rDNA sequencing at the D1/D2 domain, among which Hanseniaspora uvarum occurred most frequently and have intimate relationships with kiwifruit. RAPD analysis indicated a high variability of this species from sampling regions. The correlation of genotypes with origins was established except for isolates from Zhouzhi orchards, and the mobility of H. uvarum from orchard to the manufacturer can be speculated and contributed to spoilage sourcing. The manufacturing environment favored the inhabitance of osmotolerant yeasts more than the orchard by giving high positive sample ratio or osmotolerant yeast ratio. The growth curves under various glucose levels were fitted by Grofit R package and the obtained growth parameters indicated phenotypic diversity in the H. uvarum and the rest species. Wickerhamomyces anomalus (OM14) and Candida glabrata (OZ17) were the most glucose tolerant species and availability of high glucose would assist them to produce more gas. The test osmotolerant species were odor altering in kiwifruit concentrate juice. 3-Methyl-1-butanol, phenylethyl alcohol, phenylethyl acetate, 5-hydroxymethylfurfural (5-HMF) and ethyl acetate were the most altered compound identified by GC/MS in the juice. Particularly, W. anomalus produced 4-vinylguaiacol and M. guilliermondii produced 4-ethylguaiacol that would imperil product acceptance. The study determines the target spoilers as well as offering a detailed spoilage features, which will be instructive in implementing preventative measures to increase production safety of kiwifruit.

Protein abundance changes of Zygosaccharomyces rouxii in different sugar concentrations

Zygosaccharomyces rouxii is a yeast which can cause spoilage in the concentrated juice industries. It exhibits resistance to high sugar concentrations but genome- and proteome-wide studies on Z. rouxii in response to high sugar concentrations have been poorly investigated. Herein, by using a 2-D electrophoresis based workflow, the proteome of a wild strain of Z. rouxii under different sugar concentrations has been analyzed. Proteins were extracted, quantified, and subjected to 2-DE analysis in the pH range 4-7. Differences in growth (lag phase), protein content (13.97-19.23mg/g cell dry weight) and number of resolved spots (196-296) were found between sugar concentrations. ANOVA test showed that 168 spots were different, and 47 spots, corresponding to 40 unique gene products have been identified. These protein species are involved in carbohydrate and energy metabolism, amino acid metabolism, response to stimulus, protein transport and vesicle organization, cell morphogenesis regulation, transcription and translation, nucleotide metabolism, amino-sugar nucleotide-sugar pathways, oxidoreductases balancing, and ribosome biogenesis. The present study provides important information about how Z. rouxii acts to cope with high sugar concentration at molecular levels, which might enhance our global understanding of Z. rouxiis high sugar-tolerance trait.