Nondestructive characterization gender of chicken eggs by odor using SPME/GC-MS coupled with chemometrics.

It's a difficult task for researchers to identify the gender of chicken eggs by nondestructive approach in the early of incubation, which not only could reduce the cost of incubation, but also could improve the welfare of chicks. Therefore, SPME/GC-MS has been applied to investigate its potential as a nondestructive tool for characterizing the differences of odor between male and female chicken eggs during early of incubation and even before hatch. The results showed that more volatiles were found in female White leghorn eggs during early of incubation and 6,10-dimethyl-5,9-undecadien-2-one, 6-methyl-5-hepten-2-one, nonanal, decanal, octanal, 2-nonen-1-ol, etc. were important for the distinction of male and female White leghorn eggs during E1-E9 of incubation. 2-ethyl-1-hexanol; octanal, nonanal, 2,2,4-trimethyl-3-carboxyisopropyl pentanoic acid isobutyl ester; 2-nonen-1-ol, cyclopropanecarboxamide, heptadecane were correlated with gender of unhatched White leghorn, Hy-line brown and Jing fen eggs, respectively. Moreover, sex-related volatiles have been strongly influenced by incubation process and egg breed, and to be related to steroid hormone biosynthesis. What's more, this study enables us to develop a new visual for ovo sexing of chicken eggs and advances our understanding of the biological significance behind volatiles emitted from chicken eggs.

Separation and Identification of Highly Efficient Antioxidant Peptides from Eggshell Membrane.

The enzymatic hydrolysates (EHs) of the eggshell membrane (ESM) were obtained after incubating eggshell membrane in solutions prepared with Na2SO3 and alkaline protease combinations. The effects of enzyme species, enzyme dosage, Na2SO3 concentration, and hydrolysis time on the antioxidant activity of the ESM-EH were determined. Also, the correlation between the degree of hydrolysis (DH) and the antioxidant activity of ESM-EH was analyzed. The DH of ESM-EH showed a highly positive correlation with the reducing power (R2 = 0.857) and total antioxidant activity (TAA) (R2 = 0.876) and performed negative correlation with the Fe2+-chelating ability (R2 = -0.529). The molecular weight distribution of the ESM-EH was determined by MALDI-TOF/MS. Cation exchange chromatography (Sephadex C-25) was used to isolate the ESM-EH and then the enzymatic hydrolysis fragment (EHF) was obtained. Among the five isolated fragments (F1~F5), fragment 3 (F3), which was composed of 28 polypeptides, showed the highest ability to quench ABTS• (2,2-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid) (90.44%) and also displayed stronger TBARS (thiobarbituric acid- reactive substances) (58.17%) and TAA (303.82 µg /mL) than the ESM-EH. Further analysis of the 28 peptides in F3 identified using LC-MS/MS indicated that five peptides (ESYHLPR, NVIDPPIYAR, MFAEWQPR, LLFAMTKPK, MLKMLPFK) showed high water-solubility, biological activities, and antioxidant characteristics. Finally, the TAA of the synthetic peptide was verified, the synthetic peptides ESYHLPR and MFAEWQPR performed the best activity and have high potentials to be used as antioxidant agents in functional foods, pharmaceuticals, or cosmetics.

Differentiation of Bacillus thuringiensis From Bacillus cereus Group Using a Unique Marker Based on Real-Time PCR.

The efficiency of a novel biomarker (the transcriptional regulator, XRE) was tested and evaluated in differentiating Bacillus thuringiensis from Bacillus cereus group species in environmental and spiked samples based on PCR and real-time PCR. Totally 120 strains, representing two bacterial groups, B. cereus group and non-Bacillus sp., were used to evaluate the performance of XRE and crystal protein (cry2, an existing biomarker). Further, three diverse samples (kimbap, lettuce, and spinach) were inoculated with B. thuringiensis and prominent biomarkers XRE and cry2 were used as targets. Direct analysis of the detection results for the pure cultures of B. cereus group wild-types, references and type strains revealed an accuracy rate of 97.5% targeting XRE, and 83.3% targeting cry2. The real-time PCR was constructed with a R 2-value of 0.993. For the artificially contaminated samples, a concentration of 103 CFU/g of B. thuringiensis in spiked food samples could be detected using real-time PCR targeting XRE. A good performance was obtained with XRE in discriminating B. thuringiensis from B. cereus groups, as well as detecting B. thuringiensis in spiked food samples with PCR or real-time PCR. Therefore, this real-time PCR targeting XRE can be used as a dependable and promising tool to identify B. thuringiensis in foods.

Characterization and classification of volatiles from different breeds of eggs by SPME-GC-MS and chemometrics.

Volatiles of shell eggs were identified by SPME-GC-MS to characterize and discriminate white Leghorn (W), Hy-line brown (H) and Jing fen (J) hatching eggs with comparison, principal components (PC), partial least squares (PLS), random forest classification (RFC) and canonical discriminant (CD) analyses. DVB/CAR/PDMS fiber and extraction 60 min were suited to analyze the volatiles emitted from eggs. A total of 17 or 18 volatile compounds were identified in raw shell hatching eggs, namely, nonanal, decanal and 6-methly-5-hepten-2-one were the main volatile components with contributions that over 70%. The composition and/or profile of volatile compounds from W and H eggs were much more similar than J eggs. Hexanal, decanal, 6-methly-5-hepten-2-one, heptanal, etc. have greatly contributed to the distinction of W, H and J eggs in sparse (S)-PLS and orthogonal (O)-PLS models. The accuracy of RFC and CD model were 100%, 100% (initial) and 83.3% (cross-validation), respectively. Heptanal, 6-methly-5-hepten-2-one, octanal, etc. were contributed positively to the classification of W, H, J eggs in RFC, especially for heptanal.

Whole genome sequence of Bacillus thuringiensis ATCC 10792 and improved discrimination of Bacillus thuringiensis from Bacillus cereus group based on novel biomarkers.

BACKGROUND: Among the Bacillus cereus group, B. thuringiensis, is one of the most extensively used biological control agent. The present study reports the complete genome and four novel plasmid analysis of the type strain B. thuringiensis ATCC 10792. METHODS: Complete genome sequencing of Bacillus thuringiensis ATCC 10792, assembled using de-novo (v.3.2.0, assembly name MIRA3), Pac-Bio sequencers and Hierarchical Genome Assembly Process software (version 4.1) and real-time polymerase chain reaction (qPCR) is a consistent technique for quantifying gene expression based on specific biomarkers, in addition the efficiency of the primers were analysed based on artificially spiked food samples on lettuce, kimbab and spinach with B. thuringiensis ATCC 10792. RESULTS: Complete genome annotation was performed, and a total of 6269 proteins with 5427594 bps were identified and four novel plasmid (poh2, poh3, poh4, poh5) a total of 134, 131, 96, 21 proteins with 113294; 92,949; 86488; 11332 bps were identified. Six selective genes (lipoprotein-lipo, methyltransferase-MT, S-layer homology domain protein-BC, flagellar motor protein-motB, transcriptional regulator-XRE, crystal protein-cry2) and associated four novel plasmids were investigated along with the characteristics and expression profiles of two housekeeping genes (chaperonin protein-GroEL and topoisomerase enzyme-gyrB). Although from the assessment of 120 strains, both GroEL and gyrB showed 100% specificity towards detection of both B. thuringiensis in artificially spiked vegetable samples. All the eight genes revealed no specificity towards any of the 9 non- Bacillus strains. CONCLUSION: In our study based on the complete genome and plasmid sequence of B. thuringiensis ATCC 10792, among the six discriminating genes, specifically GroEL, gyrB and XRE showed promising results in identifying B. thuringiensis ATCC 10792, and there detection limit was 3.0-9.6 log CFU/g in the food samples respectfully. The key role in control of the predatory biological agent.

New perspectives on Mega plasmid sequence (poh1) in Bacillus thuringiensis ATCC 10792 harbouring antimicrobial, insecticidal and antibiotic resistance genes.

Bacillus thuringiensis promotes the growth of numerous economically important crops. The present study presents the complete genome sequence for a mega plasmid present in the type strain of B. thuringiensis ATCC 10792, a typical spore-forming Gram-positive bacterium with insecticidal activity, and investigates its genetic characteristics. The genome was sequenced and assembled de novo using Pac-Bio sequencers and the Hierarchical Genome Assembly Process, respectively. Further genome annotation was performed, and a total of 489 proteins and a novel mega-plasmid (poh1) with 584,623 bps were identified. The organization of poh1 revealed the genes involved in the insecticidal toxin pathway. The genes responsible for antimicrobial, insecticidal and antibiotic activities were well conserved in poh1, indicating an intimate association with plant hosts. The poh1 plasmid contains the gene encoding a novel crystal protein kinase responsible for production of zeta toxin, which poisons insects and other Gram-negative bacteria through the global inhibition of peptidoglycan synthesis. Lantibiotics are a group of bacteriocins that include the biologically active antimicrobial peptide Paenibacillin. Further, poh1 also contains the genes that encode the gramicidin S prototypical antibiotic peptide and tetracycline resistance protein. In conclusion, the strain-specific genes of B. thuringiensis strain ATCC 10792 were identified through complete genome sequencing and bioinformatics data based on major pathogenic factors that contribute to further studies of the pathogenic mechanism and phenotype analyses.

Modeling the effect of pH, water activity, and ethanol concentration on biofilm formation of Staphylococcus aureus.

In this work, the effect of environmental factors on Staphylococcus aureus (ATCC 13150) biofilm formation in tryptic soy broth was investigated under different ranges of pH (3.0-9.5), ethanol concentration (EtOH 0.0-20.0%), and aw (NaCl, 0.866-0.992). Biofilm formation was quantified using the crystal violet staining method and optical density (OD: 590 nm) measurements. Biofilm formation was significantly stronger at pH and aw close to S. aureus optimal growth conditions, while it was high at EtOH around 2.5-3.5%. Data sets from the difference between the OD measurements of the test and control (ΔOD) were fitted to the cardinal parameter model (CPM) and cardinal parameter model with inflection (CPMI) to describe the effect of the environmental factors. The models showed good quality of fit for the experimental data in terms of calculated RMSE, with the latter ranging from 0.276 to 0.455. CPM gave a good quality of fit compared to CPMI for the environmental factors tested. Optimal pH was close to neutral (6.76-6.81) and biofilm formation was possible till pH = 3.81-3.78 for CPM and CPMI, respectively. Optimum EtOH and aw conditions for biofilm formation were in the range of 1.99-2.75 and 0.98-0.97, respectively. Predicted OD values observed using strain 13150 were very closely correlated to the OD values predicted with strain 12600 with R2 of 0.978, 0.991, and 0.947 for pH, EtOH, and aw, respectively. The cultivable bacterial cells within the biofilm were enumerated using standard plate counting and a linear model was applied to correlate the attached biofilm cells to ΔOD of biofilm formation. It was found that the biofilm formation correlated with S. aureus population growth. At 2.5-3.5% of EtOH the maximum population density was lower than that observed at 0.0% of EtOH. As 2.5-3.5% of EtOH initiated a stronger biofilm formation, biofilm formation seems to be induced by ethanol stress. The development of cardinal parameter models to describe the effect environmental factors of importance to biofilm formation, offers a promising predictive microbiology approach to decrypting the S. aureus population growth and survival ability on food processing surfaces.

A New Secondary Model Developed for the Growth Rate of Escherichia coli O157:H7 in Broth.

This study was attempted to develop a new exponential sum model to describe the effect of temperature on growth rate (GR) of Escherichia coli O157:H7 in broth. The growth rates of E. coli O157:H7 at different storage temperatures (4, 10, 15, 20, 25, 30, and 35°C) estimated by fitting with the modified Gompertz model were used to develop secondary models such as square root model, Ratkowsky model and exponential sum model. Measures of coefficient of determination (R(2)), root mean square error (RMSE) and the sum of squares due to error (SSE) were employed to compare the performances of these three secondary models. Based on these criteria, the developed exponential sum model showed the better goodness-of-fit and performance.

Combination treatment of alkaline electrolyzed water and citric acid with mild heat to ensure microbial safety, shelf-life and sensory quality of shredded carrots.

The objective of this study was to determine the synergistic effect of alkaline electrolyzed water and citric acid with mild heat against background and pathogenic microorganisms on carrots. Shredded carrots were inoculated with approximately 6-7 log CFU/g of Escherichia coli O157:H7 (932, and 933) and Listeria monocytogenes (ATCC 19116, and 19111) and then dip treated with alkaline electrolyzed water (AlEW), acidic electrolyzed water (AcEW), 100 ppm sodium hypochlorite (NaOCl), deionized water (DaIW), or 1% citric acid (CA) alone or with combinations of AlEW and 1% CA (AlEW + CA). The populations of spoilage bacteria on the carrots were investigated after various exposure times (1, 3, and 5 min) and treatment at different dipping temperatures (1, 20, 40, and 50 °C) and then optimal condition (3 min at 50 °C) was applied against foodborne pathogens on the carrots. When compared to the untreated control, treatment AcEW most effectively reduced the numbers of total bacteria, yeast and fungi, followed by AlEW and 100 ppm NaOCl. Exposure to all treatments for 3 min significantly reduced the numbers of total bacteria, yeast and fungi on the carrots. As the dipping temperature increased from 1 °C to 50 °C, the reductions of total bacteria, yeast and fungi increased significantly from 0.22 to 2.67 log CFU/g during the wash treatment (p ≤ 0.05). The combined 1% citric acid and AlEW treatment at 50 °C showed a reduction of the total bacterial count and the yeast and fungi of around 3.7 log CFU/g, as well as effective reduction of L. monocytogenes (3.97 log CFU/g), and E. Coli O157:H7 (4 log CFU/g). Combinations of alkaline electrolyzed water and citric acid better maintained the sensory and microbial quality of the fresh-cut carrots and enhanced the overall shelf-life of the produce.

Combined effects of alkaline electrolyzed water and citric acid with mild heat to control microorganisms on cabbage.

Effects of alkaline electrolyzed water (AlEW), acidic electrolyzed water (AcEW), 100 ppm sodium hypochlorite (NaClO), deionized water (DIW), 1% citric acid (CA) alone, and combinations of AlEW with 1% CA (AlEW + CA), in reducing the populations of spoilage bacteria and foodborne pathogens on cabbage were investigated at various dipping times (3, 5, and 10 min) with different dipping temperatures (1, 20, 40, and 50 degrees C). Inhibitory effect of the selected optimal treatment against Listeria monocytogenes and Escherichia coli O157 : H7 on cabbage were also evaluated. Compared to the untreated control, AlEW treatment most effectively reduced the numbers of total bacteria, yeast, and mold, followed by AcEW and 100-ppm NaClO treatments. All treatments dip washed for 5 min significantly reduced the numbers of total bacteria, yeast, and mold on cabbage. With increasing dipping temperature from 1 to 50 degrees C, the reductions of total bacteria, yeast, and mold were significantly increased from 0.19 to 1.12 log CFU/g in the DIW wash treatment (P < 0.05). Combined 1% CA with AlEW treatment at 50 degrees C showed the reduction of around 3.98 and 3.45 log CFU/g on the total count, and yeast and mold, effective reduction of L. monocytogenes (3.99 log CFU/g), and E. coli O157 : H7 (4.19 log CFU/g) on cabbage. The results suggest that combining AlEW with CA could be a possible method to control foodborne pathogens and spoilage bacteria effectively on produce.

[Effect of erythromycin on apoptosis of epithelial cell in nasal polyps].

OBJECTIVE: To study the effect of erythromycin on apoptosis and expression of Bax and Bcl-2 of epithelial cell in nasal polyps. METHODS: Epithelial cells from thirty nasal polyps and fifteen inferior turbinates were cultured in Dulbecco Eagle and Ham F12 (1:1) and divided into two groups (one group were treated with Erythromycin). Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling and expression of Bcl-2 and Bax detected by immunohistochemistry at 1, 3, 5 days after culture. RESULTS: The apoptosis indexes (AI) of epithelial cell in nasal polyps after cultured for 1, 3, 5 days with erythromycin were (33.23 +/- 6.50)%, (38.21 +/- 7.22)% and (52.63 +/- 7.86)% respectively. The AI of epithelial cell in inferior turbinates were (31.02 +/- 5.60)%, (32.13 +/- 7.15)% and (39.64 +/- 7.48)% respectively. There was significant difference between two groups at 5 day after culture (P < 0.05). Expression of Bax and Bcl-2 of epithelial cell was significantly higher in nasal polyps than that in inferior turbinates (P < 0.01). Expression of Bax of epithelial cell cultured with erythromycin was significantly higher than that in the controls (P < 0.05). Apoptosis was clearly promoted after 5 day culture with Erythromycin (P < 0.05). CONCLUSIONS: Erythromycin promoted expression of Bax and apoptosis of epithelial cell in nasal polyps.