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Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, ChinaState Key Laboratory of Applied Microbiology, South China (the Ministry–Province Joint Development), Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, 510070, China
Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China
Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China
Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China
Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China
Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, ChinaState Key Laboratory of Applied Microbiology, South China (the Ministry–Province Joint Development), Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, 510070, China
State Key Laboratory of Applied Microbiology, South China (the Ministry–Province Joint Development), Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, 510070, China
Powdered infant formula is considered as the main transmission vehicle for Cronobacter sakazakii infections including meningitis, septicemia, and necrotizing enterocolitis. The effects of high-pressure processing treatment on inactivation of C. sakazakii ranging from 100 to 400 MPa for 3.0, 5.0, and 7.0 min in whole milk and skim milk were studied. Significant differences in inactivation of C. sakazakii were observed in milk samples under different pressures for 3 to 7 min compared with untreated samples, and C. sakazakii was not detected after 400 MPa for 3 min. The lethality rates of C. sakazakii cells in whole and skim milk with an initial level of 104 cfu/mL after 100 and 200 MPa treatments were not significantly different, but relatively higher lethality rates were found in whole milk after 300 MPa treatment than in skim milk. Finally, the scanning electron micrographs indicated that cellular envelope and intracellular damage of C. sakazakii cells were apparent after 300 and 400 MPa for 5.0 min compared with the untreated cells, and a progressive increase of injured cells with increased pressure treatment was found. It was concluded that C. sakazakii was sensitive to high-pressure processing treatment and that high-pressure processing treatment with 400 MPa for 3.0 min can be used to control C. sakazakii contamination in milk samples.
The genus Cronobacter is a group of foodborne pathogens, including C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. condimenti, C. universalis, and C. dublinensis, that cause meningitis, septicemia, and necrotizing enterocolitis in neonates (
Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomo-species 1, and of three subspecies, Cronobacter dublinensis ssp. dublinensis ssp. nov., Cronobacter dublinensis ssp. lausannensis ssp. nov. and Cronobacter dublinensis ssp. lactaridi ssp. nov.
Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg infection, water and food ingredients.
FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization) Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up formulae. Microbiological Risk Assessment Series No. 15..
Food and Agriculture Organization of the United Nations/World Health Organization,
Rome, Italy2008
reported that C. sakazakii was the most frequent species within the genus of Cronobacter from powdered milk samples. Under various environmental stresses, several studies have shown that Cronobacter species have unusual abilities to survive (
). Consequently, the control of C. sakazakii is of importance for ensuring microbial safety of milk samples.
High-pressure processing (HPP) is a promising alternative method to conventional thermal pasteurization for improving food safety due to the inactivation of foodborne pathogens (
). High-pressure processing has been approved to be applied in food processing procedures by the US Food and Drug Administration and the USDA. In recent years, HPP has been used to inactivate the important foodborne pathogens such as Staphylococcus aureus in milk or meat products (
) in various food samples. However, little research has focused on the inactivation of HPP on C. sakazakii in milk, which is considered the main transmission vehicle.
The current study was to determine the effects of HPP on inactivation of C. sakazakii with different inoculum levels into whole milk and skim milk samples. In addition, the morphological changes in HPP-treated C. sakazakii cells was also evaluated using a scanning electron microscope.
The type stain ATCC 29544 was incubated into tryptic soy broth for 16 h at 37°C and different concentrations (3.2 × 105 and 1.7 × 103 cfu/mL) were obtained using the 10-fold method. Then, 1.0 mL of C. sakazakii suspension (105 and 103 cfu/mL) was incubated with 9 mL of whole milk and skim milk, respectively. Thus, the whole milk and skim milk samples containing 104 or 102 cfu/mL were prepared. The whole milk and skim milk samples incubated with C. sakazakii were treated with HPP using 100, 200, 300, and 400 MPa for 0 min (control samples), 3.0, 5.0, and 7.0 min. Each experiment was performed in triplicate.
After HPP treatment, the survival of C. sakazakii was counted using the 10-fold dilution method. The lethality rate was determined as the decreasing percentage between colony counts of the control (untreated) and treated milk samples. Each experiment was replicated 3 times.
Under 100 to 400 MPa pressure treatment for 3.0, 5.0, and 7.0 min, a significant difference (P < 0.05) of survival of C. sakazakii under the holding times was observed (Table 1). In addition, different inactivation under 200 to 400 MPa treatments was extremely significant (P < 0.01) compared with untreated whole milk samples. The inactivation effect of HPP was significantly enhanced by increased pressure intensity (P < 0.05), and cell counts were not detected at 400 MPa for 3.0 min. Similar results of survival of C. sakazakii in skim milk samples after HPP treatment are shown in Table 1.
reported that S. aureus in sterile saline and minced meat samples can survive after 450 MPa for 15 min, but no S. aureus can be detected after 550 MPa for 3.0 min. Less than 0.5 log cfu/mL of S. aureus survived after 500 MPa for 5.0 min in buffered peptone water with an initial inoculum of 104 cfu/mL (
reported that reduction of Listeria monocytogenes in nonselective medium was about 3 log cfu/mL after 400 MPa for 3.0 min. Gram-negative bacteria might be more sensitive to HPP treatment due to the structure and composition of the outer membrane. For example, the high hydrostatic pressure (300 MPa for 10 min) could extensively inactivate Vibrio parahaemolyticus from 109 cfu/mL to no viable cells (
). In this study, lethality rates of C. sakazakii in whole milk and skim milk were also analyzed. In Table 2, the lethality rates of C. sakazakii cell in whole and skim milk with an initial level of 104 cfu/mL after the 100 and 200 MPa treatment were not significantly different (P > 0.05). In addition, the total tendency of lethality rates in whole milk was lower than in skim milk after 100 and 200 MPa treatment for 3.0 and 5.0 min, indicating that whole milk provided a certain protective effect for inactivation of C. sakazakii under relatively lower pressure. Interestingly, significant differences of lethality rates (P < 0.05) were observed in whole milk and skim milk after 300 MPa for 3.0, 5.0, and 7.0 min, and higher lethality rates were found in whole milk than in skim milk. These contradictory findings might be related to the interaction between structure or compositions of outer membrane and fat after HPP, but a detailed mechanism remains to be revealed. Previous studies have indicated that the food matrix has a protective effect on the microorganism against high pressure (
Modelling the effect of high pressure on the inactivation kinetics of a pressure-resistant strain of Pediococcus damnosus in phosphate buffer and gilt-head seabream (Sparus aurata).
). A comparison between the survival curves in phosphate buffer and gilt-head seabream showed a clear protective effect of the food matrix on the resistance of Pediococcus damnosus, especially at 500 and 550 MPa (
Modelling the effect of high pressure on the inactivation kinetics of a pressure-resistant strain of Pediococcus damnosus in phosphate buffer and gilt-head seabream (Sparus aurata).
). After high hydrostatic pressure of 350 and 450 MPa for 3.0, 6.0, 9.0, 12, and 15 min, the inactivation of Staphylococcus aureus in sterile saline was higher than in minced meat (
). However, no significant differences in inactivation of E. coli were observed in cooked ham homogenized with water (3:1) and in phosphate buffer after 500 MPa for 10 min (
Furthermore, C. sakazakii cells were prepared from pressure-treated and untreated cell suspensions via centrifugation at 5,000 × g for 10 min at room temperature after washing the pellet twice in 0.1 M phosphate buffer (pH 7.4). The subsequent steps were described by
. Finally, the morphological changes of C. sakazakii after HPP were analyzed by performing scanning electron microscopy (Hitachi S4700, Hitachi, Tokyo, Japan) at 15 kV voltage, and photomicrographs were obtained.
From Figure 1, scanning electron microscopy indicated that morphological changes of cell structure such as cytomorphosis and cellular crimple were observed after 300 and 400 MPa for 5.0 min, whereas untreated cells exhibited intact cell structure and clear cell wall of C. sakazakii. In addition, a progressive increase of injured cells occurred with increased pressure treatment, which was consistent with the survival of C. sakazakii under 300 and 400 MPa. After 300 MPa treatment, the structure of some cells was intact, whereas C. sakazakii cells were completely injured under 400 MPa. In V. parahaemolyticus, some cell disruptions were caused by 300 MPa treatment. Similar results were observed in E. coli and S. aureus by transmission electron microscopy as described by
). In addition, some bud scars were caused and the membrane integrity of cells was lost after HPP treatment in this study, suggesting that the cellular wall and outer membrane might be the main target of HPP.
) suggested that protein denaturation might be the main mechanism for inactivation by HPP treatment. Moreover, metabolic activity and membrane potential of foodborne pathogens decreases after HPP treatment (
). We inferred that inactivation of C. sakazakii after HPP treatment was associated with cell disruption, and simultaneously the release of cytoplasmic content caused the loss of viability of cells and denaturation of functional components of cells such as enzymes and structural proteins.
Figure 1Scanning electron micrographs of Cronobacter sakazakii cells. (A) Untreated cells, (B) cells treated at 300 MPa for 5 min, and (C) cells treated at 400 MPa for 5 min.
We gratefully acknowledge the financial support of the Anhui Provincial Grand Project Special of Science and Technology (15czz03109; China) and the Science and Technology Planning Project of Guangdong Province, China (2013B021500012).
References
Alpas H.
Alma L.
Bozoglu F.
Inactivation of Alicyclobacillus acidoterrestris vegetative cells in model system, apple, orange and tomato juices by high hydrostatic pressure.
Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomo-species 1, and of three subspecies, Cronobacter dublinensis ssp. dublinensis ssp. nov., Cronobacter dublinensis ssp. lausannensis ssp. nov. and Cronobacter dublinensis ssp. lactaridi ssp. nov.
Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg infection, water and food ingredients.
Modelling the effect of high pressure on the inactivation kinetics of a pressure-resistant strain of Pediococcus damnosus in phosphate buffer and gilt-head seabream (Sparus aurata).
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