CD-MOF-1 Growth on Polysaccharide Gels through Only C2-OH/C3-OH or C5-O/C6-OH Group Formed Four-Coordinated K+ Ions for Developing Porous Biogels.

The γ-cyclodextrin (γ-CD) metal-organic frameworks (CD-MOF-1) consist of γ-CD and potassium (K+) ions through coordinating an eight-coordinated K+ ion with two C5-linked oxygen and C6-linked hydroxyl (C5-O/C6-OH) groups in the primary faces of adjacent γ-CD units and two C2- and C3-linked hydroxyl (C2-OH/C3-OH) groups in the secondary faces. Herein, we found polysaccharide gels with only C2-OH/C3-OH or C5-O/C6-OH groups in pyranoid rings can form four-coordinated K+ ions and then coordinate γ-CD in a KOH solution for CD-MOF-1 growth. Exposure of C2-OH/C3-OH or C5-O/C6-OH groups in polysaccharide gels is important to form active four-coordinated K+ ions. Mechanism supporting this work is that four-coordinated K+ ion sites are first formed after coordinating C2-OH/C3-OH groups in pectin and then coordinating C5-O/C6-OH groups in the primary faces of γ-CD units. Alternatively, four-coordinated K+ ions with C5-O/C6-OH groups in chitosan can coordinate the C2-OH/C3-OH groups in the secondary faces of γ-CD units. Mechanism of CD-MOF-1 growing on pectin and chitosan gels through the proposed four-coordinated K+ ions is also universally applicable to other polysaccharide gels with similar C2-OH/C3-OH or C5-O/C6-OH groups such as alginate gel. Based on this mechanism, we developed pectin and chitosan gel-based CD-MOF-1 composites and exemplified applications of them in antibacterial and organic dye removal. To help future research and applications of this mechanism, we share our theoretical assumption for further investigations that any matrices with an ortho-hydroxyl carbon chain or ortho-hydroxyl ether structures may form four-coordinated K+ ions for CD-MOF-1 growth. The proposed mechanism will broaden the development of novel CD-MOF-1 composites in various fields.

Discovery of Antibacterial Dietary Spices That Target Antibiotic-Resistant Bacteria.

Although spice extracts are well known to exhibit antibacterial properties, there is lack of a comprehensive evaluation of the antibacterial effect of spices against antibiotic-resistant bacteria. In the present study, ethanolic extracts from a total of 67 spices were comprehensively investigated for their in vitro antibacterial activities by agar well diffusion against two common food-borne bacteria, Staphylococcus aureus and Salmonella enteritidis, with multi-drug resistance. Results showed that S. aureus was generally more sensitive to spice extracts than S. enteritidis. Of the 67 spice extracts, 38 exhibited antibacterial activity against drug-resistant S. aureus, while only four samples were effective on drug-resistant S. enteritidis. In addition, 11 spice extracts with inhibition zones greater than 15 mm were further verified for their broad-spectrum antibacterial properties using another 10 drug-resistant S. aureus strains. It was found that five spice extracts, including galangal, fructus galangae, cinnamon, yellow mustard seed, and rosemary, exhibited the highest antibacterial capacity. Further cytotoxicity of these 11 spices was determined and LC50 values were found to be more than 100 µg/mL except for galangal, rosemary, and sage, whose LC50 values were 9.32 ± 0.83, 19.77 ± 2.17, and 50.54 ± 2.57, respectively. Moreover, the antioxidant activities (ferric-reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC) values) and total phenolic content (TPC) of spice extracts were determined to establish possible correlations with the antibacterial activity. Although the antibacterial effect was positively correlated with the antioxidant activities and TPC, the correlation was weak (r < 0.5), indicating that the antibacterial activity could also be attributed to other components besides antioxidant polyphenols in the tested spice extracts. In conclusion, dietary spices are good natural sources of antibacterial agents to fight against antibiotic-resistant bacteria, with potential applications as natural food preservatives and natural alternatives to antibiotics in animal feeding.

Separation, Identification, and Bioactivities of the Main Gallotannins of Red Sword Bean (Canavalia gladiata) Coats.

The red sword bean (Canavalia gladiata) is an underutilized edible bean cultivated in China. It was previously found to have the highest content of antioxidant polyphenols among 42 edible beans, mainly gallic acid, and gallotannins in its red bean coat, an apparently unique characteristic among edible beans. In this study, the main phenolic compounds in red sword bean coats were further separated by Sephadex LH-20 column chromatography, and identified by LC-MS/MS. Furthermore, the FRAP and ABTS antioxidant activities and antibacterial activity (diameter of inhibition zone, DIZ) of main gallotannin-rich fractions were tested. Our results showed that gallotannins of red sword bean coats were mainly comprised of monogalloyl to hexagalloyl hexosides. Interestingly, tetragalloyl, pentagalloyl, and hexagalloyl hexosides were identified as the possible candidates responsible for the red color of the coats. On the other hand, gallotannin-rich fractions exhibited diverse antioxidant and antibacterial activities, and tetragalloyl hexoside overall had the highest free radical scavenging and antibacterial activities. The degree of galloylation did not completely explain the structure-function relationship of gallotannins isolated from red sword bean coats, as there should exist other factors affecting their bioactivities. In conclusion, red sword bean coats are excellent natural sources of gallotannins, and their gallotannin-rich extracts can be utilized as natural antioxidant and antibacterial agents with potential health benefits as well as application in food industry.

[Assessment and application of a molecular diagnostic method on the detection of four types of diarrheagenic Escherichia coli].

OBJECTIVE: To establish and evaluate a molecular diagnostic method for routine monitoring of four types of diarrheagenic Escherichia (E.) coli (DEC)and to study the distribution of four types of DEC isolated from diarrheal patients in Shanghai. METHODS: DEC-PCR standard operation procedure(SOP)had been developed for DEC detection and isolation, using the Statens Serum Institute (SSI) DEC PCR kits with multiplex PCR technique after verification tests on reference strains. Diarrhea specimens from 3 clinical hospitals in Shanghai were tested from June to September, 2012. RESULTS: Specificity of the PCR kit was 100% by verification on the 26 DEC reference strains. A total number of 218 DEC isolates, including 181 fermented lactose and 37 unfermented lactose were identified from the 1887 stool specimens of diarrhea patients, with positive rate as 11.6%. The most common pathogen(54.1%, 118/218)was enteropathogenic E. coli(EPEC), followed by enterotoxigenic E. coli(ETEC, 41.3%, 90/218), enteroinvasive E. coli (EIEC, 4.1%, 9/ 218) and Shigatoxin-producing E. coli(STEC, 0.5%, 1/218)in addition to 18 Shigella isolates. ETEC dominated in diarrhea patients with foreign residency, as well as 1/3 were perinatal stage of neonatal ETEC of all diarrhea cases under the age of 5, while EPEC dominated in the Chinese diarrhea patients especially among young kids under the age of 2. CONCLUSION: Data was reliable after assessment on this molecular diagnostics and seperation procedures used for the routine monitoring on four types of DEC, while the diagnosis and reference ability of DEC regarding the laboratories net-working on food-borne pathogens need to be built up and improved.

Mutations in gltB and gltC reduce oxidative stress tolerance and biofilm formation in Listeria monocytogenes 4b G.

The foodborne pathogen Listeria monocytogenes has the capability to persist on surfaces in food-processing environments, and the organism is resistant to environmental stresses. In this study, a Tn917 insertion mutant of L. monocytogenes 4b G showing reduced biofilm formation and sensitivity to oxidative stress was identified and characterized. The transposon insertion site within the gltB gene was identified by inverse PCR. The gltC gene is located upstream and is reported to be transcribed divergently from gltB. Mutants with deletions in gltB and gltC were constructed and both showed reduced biofilm formation and increased sensitivity to H2O2 compared to the wild-type. In the wild-type strain, gltB and gltC expressions were induced approximately 8-fold and 14-fold by quantitative RT-PCR, respectively, with exposure to H2O2, providing further evidence that their gene products may be involved in the response to oxidative stress. In addition, after the induction by H2O2 and compared with the wild-type, the gltB expression in ΔgltC and the gltC expression in ΔgltB were down-regulated about 4-fold (p<0.05) and 3-fold (p<0.05) respectively. These data demonstrate a possible mutual regulation between gltB and gltC expressions under oxidative stress conditions, partly explaining the similar oxidative stress responses of ΔgltB and ΔgltC. Furthermore, ΔgltB and ΔgltC exhibited decreased adherence to a glass surface compared to the wild-type, while the cell motility of wild-type and mutant strains was similar. It is hypothesized that some cell surface characteristics unrelated with cell motility may be introduced into the mutants by the inactivation of gltB or gltC, which might lead to the reduction in biofilm formation. We conclude that both gltB and gltC are involved in the biofilm formation as well as the oxidative stress tolerance in L. monocytogenes 4b G, by pathways that remain yet unclear.

Diversity of Salmonella isolates using serotyping and multilocus sequence typing.

One hundred and twenty-one Salmonella isolates were obtained from food, feed, and live chicken samples derived from 13 countries or regions. In this study, their subtypes were evaluated by serotyping and multilocus sequence typing (MLST), and their genetic profiles were also characterized. It was demonstrated by serotyping on these isolates that 36 various serovars were obtained in this study, of which three serotypes S. Babelsberg, S. Fresno, and S. II were first found in mainland China. Based on Simpson's index of diversity, the serotyping method had a 0.943 discriminatory power. Meanwhile, there were a total of 42 unique sequence types (STs) characterized by MLST, and the discriminatory power of MLST (D = 0.947) was close to that of the serotyping method. In MLST, hisD revealed the highest levels of nucleotide diversity. In addition, ST-92 was the most common ST represented by 16 Salmonella isolates, followed by ST-367 which was represented by 14 isolates. Seven new alleles were identified, which were associated with other alleles and resulted in the assignment of nine new STs. It was concluded from the results that MLST was generally associated with serotype, but not associated with the epidemiological source of the samples, and antimicrobial resistance patterns.

Serotype, genotype, and antimicrobial susceptibility profiles of Salmonella from chicken farms in Shanghai.

Five hundred fifty samples were collected from five chicken farms in Shanghai during March 2005 to October 2006. Twenty-five samples tested positive for Salmonella from a total of 550 samples, of which 500 were obtained from feces of healthy chickens and 50 were obtained from diseased chicks. The 25 presumptive Salmonella isolates were confirmed by the API 20E identification kit. Serotyping of these isolates by agglutination tests with antiserum displayed seven serovars; genotyping of these isolates with multilocus sequence typing demonstrated six sequence type (ST) patterns (i.e., ST-11, ST-19, ST-92, ST-96, ST-290, and ST-367). The multilocus sequence typing data revealed that some of these strains, isolated from different farms, might have the same ST and might come from the same source. The susceptibilities of these strains to 14 antimicrobials were determined; most of the isolates (13 of 25) were resistant to doxycycline and tetracycline, and two isolates were resistant to cefotaxime and ceftazidime, but none was resistant to gentamicin or kanamycin.

Canna edulis Ker by-product: chemical composition and characteristics of the dietary fiber.

Canna edulis Ker by-product was recycled and utilized after starch extraction. The chemical composition, physical properties and antioxidant activity of the by-product were investigated. The by-product was mainly composed of dietary fiber (54.84% measured by AOAC method), and the insoluble dietary fiber constituted the major fraction. Then, the chemical composition of dietary fiber was tested using modified AOAC and Englyst methods. The results showed that dietary fiber was comprised of cellulose, hemicelluloses (including xyloglucans, arabinoxylans and glucuronoxylans), pectin and lignin. Moreover, the by-product contained relatively high content of phenolic compounds and exhibited a moderate antioxidant activity. In addition, the by-product showed both high water-holding capacity (12.5 mL/g) and oil-holding capacity (14 mL/g), and its suspension exhibited controllable viscosity. Therefore, the by-product from C. edulis is not only a source of dietary fiber but also a functional ingredient for food industry.

Development of a quantitative polymerase chain reaction method using a live bacterium as internal control for the detection of Listeria monocytogenes.

A novel real-time quantitative polymerase chain reaction (Q-PCR) assay was developed for rapid and accurate detection of Listeria monocytogenes. In this Q-PCR assay, a computational DNA random shuffling method was used to design an internal amplification control (IAC) sequence, which was the same in length and G + C content to the hly amplicon. This IAC sequence was inserted into the genome of L. monocytogenes to create a mutant strain named L. monocytogenes-IAC. The LM-IAC was used as an internal control during the PCR assay and produced accurate quantification of L. monocytogenes due to similar DNA extraction and amplification efficiencies between LM-IAC strain and wild-type L. monocytogenes. Quantification by this method was over a 5-log linearity range of initial L. monocytogenes with an R(2) value of 0.9997. This PCR method will provide accurate quantification of L. monocytogenes and can be used in the clinic and food assays for diagnostic purposes.

Phosphate assimilation by Chlorella and adjustment of phosphate concentration in basal medium for its cultivation.

Assimilation of phosphate by Chlorella pyrenoidosa was 0.81-8.1 mg PO(4)-P/g dry weight for heterotrophic cultures and 0.81-16.1 mg/g for mixotrophic cultures. Optimal carbon:phosphorous (C/P) ratios were 206:1-2060:1 and 103:1-2060:1 for heterotrophic and mixotrophic cultivations, respectively. These requirements for phosphate for growth of C. pyrenoidosa under either heterotrophic or mixotrophic conditions are much less (6.25-62.5 or 3.12-62.5-fold at 10 g glucose/l) than its concentration in basal medium.

Species-specific diagnostic marker for rapid identification of Staphylococcus aureus.

Staphylococcus aureus is a common bacterial pathogen that has emerged as an increasingly important health concern. Following the recent publication of the genome sequences of 9 S. aureus strains (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi), a gene of S. aureus that relates signal transduction as a target for rapid detection and identification of the pathogen has been investigated. By sequence analysis of S. aureus signal transduction genes from the complete genome of S. aureus ATCC N315 and their comparison with other DNA sequences using GenBank BLAST searches, we identified a unique gene, vicK. Polymerase chain reaction primers (vicK1 and vicK2) derived from this gene allowed amplification of a 289-bp DNA fragment only from S. aureus and not from other Staphylococcus species and other common bacteria tested. Besides offering an additional target for specific confirmation of S. aureus, further analysis of the signal transduction gene vicK and its related protein product may lead to new insights into the molecular mechanisms of S. aureus maintenance and pathogenicity.

[Study on the application and evaluation of methods for gene and antigen detection in plague surveillance program].

OBJECTIVE: To apply and evaluate new methods regarding specific gene and antigen detection in plague surveillance program. METHODS: 1798 samples from natural foci of plague were tested, using internal quality control multiple-polymerase chain reaction, F1 antigen marked by immuno chromatographic assay and enzyme linked immunosorbent assay. Culture of Yersinia pestis and reverse indirect hemagglutination assay were used as reference diagnostic methods. RESULTS: The overall positive rate of culture on Yersinia pestis together with gene and antigen detection was 7.34%, showing an 16.81% increase when comparing to 6.28% using Yersinia pestis culture method alone. The rate of coincidence was 97.13%. CONCLUSION: The new standard being used for specific gene and antigen detection could increase the positive rate of diagnosis on plague.

[Mutagenesis on biofilm formation of Listeria monocytogenes by Tn917 transposon insertion].

Surface-attached populations of bacteria comprising either single or multiple species were referred to as biofilm. The bacteria in the biofilm had more resistant ability against antibiotics and disinfectors, which could cause the constituent clinical affection or food contamination. In order to isolate and characterize the genes involved in biofilm formation of Listeria monocytogenes, the plasmid pTV1-OK containing the transposon Tn917 was transformed into the protoplasm of Listeria monocytogenes. After cultivating for 4 days, twenty positive transformants per microgram DNA were generated. Some transformants were selected for inducing transposition in water bath overnight at 42 degrees C, and the transposition efficiency of Tn917 was 10(-7). The LM-49 strain with higher ability on biofilm formation than the wild strain was obtained by microtiter plate assaying, and the purple ring formed by the LM-49 was thicker than that by the wild after a 4-day incubation. The PCR result using specific primers of Tn917 proved that Tn917 was inserted into the genome of L. monocytogenes. So the LM-49 strain can be regarded as the mutant of biofilm formation, and the higher ability of biofilm formation is caused by the insertion of Tn917.

[Molecular biological characteristics and genetic significance of Yersinia pestis in China].

OBJECTIVE: To understand the molecular biological characteristics in order to analyse the genetic background of Yersinia pestis in China. METHODS: Primary datum on ribotyping, pulsed field gene electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and insertion sequence (IS) of Yersinia pestis were used and under cluster analysis. Genetic interval and various methods of recognized molecular feature between different strains were evaluated. RESULTS: Ribotypes the PFGE types seemed to be corresponding. Stains from Microtus fuscus and area in Tibet Zhongba belonged to 7 copy rRNA gene and the genetic interval were the far more with 6 copy rRNA gene stains, and not definite with RAPD, but with many exceptions. The genetic interval between strains were showed by resemble value. CONCLUSION: Yersinia pestis in China had its own manifold, particular molecular biological characteristics due to natural barriers, geographical complex, circumstances in Tianshan Mountains and Gandise Mountains areas. Yersinia pestis were limited to separateness, evoluted only in certain areas to form a great many gene types.

[Genetic analysis of Yersinia pestis strains isolated in China].

OBJECTIVE: The strains of Yersinia pestis isolated in different period and different natural foci in China were analyzed. METHODS: Traditional and molecular biological methods were used. Rhamnose fermentation, rRNA gene copy number, nitrite reduction, and the glycerol fermentation were important characters for typing, and pulse field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) profile could reflect the genetic distance between the strains. RESULTS: The strains could be divided into 15 genetic types by those 6 characters with each of them covered an isolated geographical territories. CONCLUSION: The characters of strains were described; the genetic relationship of different types, their evolution, and the forming and shift of plague natural foci were analyzed.

High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture.

The green microalga Chlorella protothecoides was grown heterotrophically in batch mode in a 3.7-L fermenter containing 40 g/L glucose and 3.6 g/L urea. In the late exponential phase, concentrated nutrients containing glucose and urea were fed into the culture, in which the nitrogen source was sufficient compared to carbon source. As a result, a maximum cell dry weight concentration of 48 g/L was achieved. This cell dry weight concentration was 28.4 g/L higher than that obtained in batch culture under the same growth conditions. In another cultivation run, the culture was provided with the same initial concentrations of glucose (40 g/L) and urea (3.6 g/L) as in the batch mode, followed by a relatively reduced supply of nitrogen source in the fed-batch mode to establish a nitrogen-limited culture. Such a modification resulted in an enhanced lutein production without significantly lowering biomass production. The cellular lutein content was 0.27 mg/g higher than that obtained in the N-sufficient culture. The improvements were also reflected by higher maximum lutein yield, lutein productivity, and lutein yield coefficient on glucose. This N-limited fed-batch culture was successfully scaled up from 3.7 L to 30 L, and a three-step cultivation process was developed for the high-yield production of lutein. The maximum cell dry weight concentration (45.8 g/L) achieved in the large fermenter (30 L) was comparable to that in the small one (3.7 L). The maintenance of the culture at a higher temperature (i.e., 32 degrees C) for 84 h resulted in a 19.9% increase in lutein content but a 13.6% decrease in cell dry weight concentration as compared to the fed-batch culture (30 L) without such a treatment. The enhancement of lutein production resulted from the combination of nitrogen limitation and high-temperature stress.