Yongtao Wang

New Insights into the Formation of Viable but Nonculturable Escherichia coli O157:H7 Induced by High-Pressure CO2

ABSTRACT The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l-serine and l-threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation. IMPORTANCE Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment. Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment.

Effects of high pressure processing on activity and structure of soluble acid invertase in mango pulp, crude extract, purified form and model systems

The effects of high pressure processing (HPP) on the activity of soluble acid invertase (SAI) in mango pulp, crude extract, purified SAI and purified SAI in model systems (pectin, bovine serum albumin (BSA), sugars and pH 3-7) were investigated. The activity of SAI in mango pulp was increased after HPP, and that in crude extract stayed unchanged. The activity of purified SAI was decreased after HPP at 45 and 50°C. Pectin exhibited a concentration-dependent protection for purified SAI against HPP at 50°C/600MPa for 30min. Pectin that had an esterification degree (DE) of 85% exhibited a greater protection than pectin that had a DE of 20-34%. BSA, acidic pH (3-6) and sucrose also exhibited protection for purified SAI against HPP. HPP at 50°C/600MPa for 30min disrupted the secondary structure and tertiary structure of purified SAI, but no aggregation of purified SAI was observed after HPP.

Role of peach proteins in juice precipitation induced by high pressure CO2.

To better understand the role of peach proteins in juice precipitation induced by high pressure CO2 (HPCD), proteins extracted from peach juice were subjected to HPCD and heat, and changes in particle size distribution (PSD) and structure were investigated. PSD analysis showed aggregations of proteins were both induced by HPCD and heat, but HPCD induced a stronger aggregation. The endotherm of HPCD- and heat-treated proteins moved to lower temperature, indicating that higher-order structures were altered after treatments. Furthermore, proteins related to HPCD- and heat-induced precipitation were analyzed by proteomics and bioinformatics. It was found that proteins with low content of α-helix and hydrogen bonds were more inclined to precipitate under HPCD, and HPCD precipitated proteins with more compact structures than heat, which might cause the stronger aggregation of proteins by HPCD. In conclusion, HPCD could induce the aggregation of peach proteins by destroying higher-order structures, which contributes to juice precipitation.

Investigating the Inactivation Mechanism of Bacillus subtilis Spores by High Pressure CO2

The objective of this study was to investigate the inactivation mechanism of Bacillus subtilis spores by high pressure CO2 (HPCD) processing. The spores of B. subtilis were subjected to heat at 0.1 MPa or HPCD at 6.5-20 MPa, and 64-86°C for 0-120 min. The germination, the permeability of inner membrane (IM) and cortex, the release of pyridine-2, 6-dicarboxylic acid (DPA), and changes in the morphological and internal structures of spores were investigated. The HPCD-treated spores did not lose heat resistance and their DPA release was lower than the inactivation, suggesting that spores did not germinate during HPCD. The flow cytometry analysis suggested that the permeability of the IM and cortex of HPCD-treated spores was increased. Furthermore, the DPA of the HPCD-treated spores were released in parallel with their inactivation and the fluorescence photomicrographs showed that these treated spores were stained by propidium iodide, ensuring that the permeability of IM of spores was increased by HPCD. The scanning electron microscopy photomicrographs showed that spores were crushed into debris or exhibited a hollowness on the surface, and the transmission electron microscopy photomicrographs exhibited an enlarged core, ruptured and indistinguishable IM and a loss of core materials in the HPCD-treated spores, indicating that HPCD damaged the structures of the spores. These findings suggested that HPCD inactivated B. subtilis spores by directly damaging the structure of the spores, rather than inducing germination of the spores.

The Synergistic Effect of High Pressure CO2 and Nisin on Inactivation of Bacillus subtilis Spores in Aqueous Solutions.

The inactivation effects of high pressure CO2 + nisin (simultaneous treatment of HPCD and nisin, HPCD + nisin), HPCD→nisin (HPCD was followed by nisin), and nisin→HPCD (nisin was followed by HPCD) treatments on Bacillus subtilis spores in aqueous solutions were compared. The spores were treated by HPCD at 6.5 or 20 MPa, 84–86°C and 0–30 min, and the concentration of nisin was 0.02%. Treated spores were examined for the viability, the permeability of inner membrane (IM) using flow cytometry method and pyridine-2, 6-dicarboxylic acid (DPA) release, and structural damage by transmission electron microscopy. A synergistic effect of HPCD + nisin treatment on inactivation of the spores was found, and the inactivation efficiency of the spores was HPCD + nisin > HPCD→nisin or nisin→HPCD. Moreover, HPCD + nisin caused higher IM permeability and DPA release of the spores than HPCD. A possible action mode of nisin-enhanced inactivation of the spores was suggested as that HPCD firstly damaged the coat and cortex of spores, and nisin penetrated into and acted on the IM of spores, which increased the damage to the IM of spores, and resulted in higher inactivation of the spores.

Characterization of physico-chemical and bio-chemical compositions of selected four strawberry cultivars

The physico-chemical and bio-chemical compositions of Hongyan, Tiangxiang, Tongzi Ι and Zhangji strawberries inChinawere analyzed. Their values were pH 3.42~3.73, titration acidity 0.63~0.79%, total soluble sugars 5.26~8.95 g/100 gfresh weight (FW), ascorbic acid 21.38~42.89 mg/100 gFW, total phenolics 235.12~444.73 mg/100 gFW, pectin 82.84~96.13 mg/100 gFW, total organic acids 874.30~1216.27 mg/100 gFW, Individual phenolic compounds other than anthocyanins 7.60~12.18 mg/100 gFW, free amino acids 13.35~32.66 mg/100 gFW, monomeric anthocyanins 4.47~47.19 mg/100gFW, antioxidant capacity of ·DPPH 14.14~18.87 and FRAP 7.97~10.54 equal to mg/100 gVc, polyphenol oxidase (PPO) activity 0~0.42 Abs/min, peroxidase (POD) activity 0.17~0.34 Abs/min and pectin methyl esterase (PME) activity 0.012~0.018 mL/min. Tongzi Ι was most suitable for food processing due to the highest titration acidity, total phenolics, pectin, total organic acids, monomeric anthocyanins, antioxidant capacity of ·DPPH and FRAP with lower PPO, POD and PME activity.

Inactivation kinetics, structural, and morphological modification of mango soluble acid invertase by high pressure processing combined with mild temperatures

The activity, structure and morphology of mango soluble acid invertase (SAI) were investigated after high pressure processing (HPP) combined with mild temperature at 50-600MPa and 40-50°C. The activity of mango SAI was efficiently reduced by HPP at 50MPa/45 and 50°C, or 600MPa/40, 45 and 50°C, while it was increased by 10-30% after HPP at 50-200MPa/40°C. Significant antagonistic effect of pressure and temperature on the activity of SAI was observed at 50-400MPa/50°C. The secondary structure of SAI was not influenced by HPP. However, its tertiary structure was modified by HPP, and severer modification occurred with higher pressure, higher temperature, and longer treatment time. Results of atomic force microscope suggested that HPP at 400MPa/50°C for 2.5min induced dissociation of SAI, and HPP at 600MPa/50°C for 30min resulted aggregation of SAI.

iTRAQ-Based Proteomic Analysis of Sublethally Injured Escherichia coli O157:H7 Cells Induced by High Pressure Carbon Dioxide

High pressure carbon dioxide (HPCD) could cause sublethally injured cells (SICs), which may cause food poisoning and spoilage during food storage and limit its application. Therefore, the formation of SICs of Escherichia coli O157:H7 was investigated by isobaric tag for relative and absolute quantification (iTRAQ) proteomic methods in this study for better controlling the SICs induced by HPCD. A total of 2,446 proteins was identified by iTRAQ, of which 93 and 29 were significantly differentially expressed in the SICs compared with live control cells (CKL) and dead control cells (CKD), respectively. Among the 93 differentially expressed proteins (DEP) in the SICs compared with CKL, 65 proteins showed down-regulation and 28 showed up-regulation. According to the comprehensive proteome coverage analysis, the SICs survived under HPCD by reducing carbohydrate decomposing, lipid transport and metabolism, amino acid transport and metabolism, transcription and translation, DNA replication and repair. Besides, the SICs showed stress response, DNA damage response and an increased carbohydrate transport, peptidoglycan synthesis and disulfide bond formation to HPCD. Among the 29 DEP in the SICs compared with CKD, 12 proteins showed down-regulation and 17 showed up-regulation. According to the comprehensive proteome coverage analysis, the SICs survived under HPCD by accumulation of cell protective agents like carbohydrates and amino acids, and decreasing transcription and translation activities. Results showed that the formation of the SICs with low metabolic activity and high survival ability was a survival strategy for E. coli O157:H7 against HPCD.