• KOREAN JOURNAL OF PACKAGING SCIENCE & TECHNOLOGY
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• v.26 no.1
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• pp.19-23
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• 2020

Al-laminated packaging film incorporating ascorbic acid or tocopherol at inner food contact layer was tested in the potential to improve antioxidative preservation of powdered infant formula under CO₂-enriched atmosphere. Product of 200 g was packaged with the packaging film containing 0.3% antioxidant in sealant layer of low density polyethylene and stored at 30℃ for 286 days with periodic measurement of package atmosphere and product's quality attributes. The CO₂-flushed package resulted in shrinkage of tight contact between the product and the film not allowing gas sampling of package atmosphere after 140 days. Package of tocopherol-incorporated film allowed some ingress of oxygen after 112 days presumably due to its weakening of heat-seal area. The increased oxygen concentration in the tocopherol-added film package led to the concomitant increase of peroxide value, an index of lipid oxidation. On the other hand, packaging of ascorbic acid-added film pouch could suppress lipid oxidation marginally in consistent manner compared to control package without any antioxidant.

• Food Science of Animal Resources
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• v.31 no.6
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• pp.944-951
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• 2011

An improved cholesterol analysis method was developed for powdered infant formula by gas chromatographic separation after liquid-liquid extraction and partition. In the official Korea Food Standard method for cholesterol analysis, the water phase and solvent phase were not well separated in the case of emulsified foods such as powdered infant formulas and baby foods. For the rapid and simple sample preparation method, an optimized direct saponification condition was established for heating temperature, heating time, and KOH concentration. From the results, the optimum conditions were as follows: heating temperature $90^{\circ}C$, heating time 60 min, and 16 M KOH 10 mL for a 2 g infant formula sample; improved separation condition for gas chromatography was as follows: the initial oven condition was $250^{\circ}C$ for 25 min, the oven temperature was increased to $290^{\circ}C$ by $10^{\circ}C$/min ratio, and finally the oven temperature remained at $290^{\circ}C$for 9 min. The developed method could be implemented for the study of cholesterol, providing the advantages of reduced inspection time and cost in emulsified foods such as infant formula.

• Food Science of Animal Resources
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• v.28 no.5
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• pp.555-561
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• 2008

Ninety-nine samples of powdered infant formula in a market were collected from the local market and their contaminations for total aerobic bacteria, coliform, FAO/WHO Category A, B, and C pathogens were analyzed. Total aerobic bacteria were detected in 92 of 99 samples (93%) at levels of $1.83{\pm}0.68\;Log\;MPN/g$. These levels were below legal levels specified for infant formulas except for one sample detected by 4.5 Log CFU/g. Coliform was detected in 12 of 99 samples (12%) at levels of $1.26{\pm}1.03\;Log\;MPN/g$ whereas non-detection was required according to the specification of coliform in infant formulas. Escherichia coli was detected in 1 of 99 samples by 0.48 Log MPN/g. Salmonella and Enterobacter sakazakii among Category A weren't detected in all the samples. Enterobacteriaceae, Category B group, were detected in 25 samples of total 99 samples (25%) by $0.83{\pm}1.37\;Log\;MPN/g$. Enterobacteriaceae identified by API 20E were Escherichia vulneris, Es. hermannii, Pantoea spp., Citrobacter koseri, Klebsiella pneumoniae, En. cloaceae. Bacillus cereus among Category C was highly detected in 29 of 99 samples (29%) at levels of $0.69{\pm}0.32\;Log\;MPN/g$ with the most probable number count method, which were below legal levels for the specification of B. cereus in infant formulas. Clostridium perfringens, E. coli O157, Staphyloccus aureus, Listeria monocytogenes, Yersinia enterocolitica, and Campylobacter jejuni/coli were not detected. Contamination level of major pathogens was low and falls within the range of specification of infant formulas. However, Enterobacteriaceae and B.cereus showed the high prevalence and some Enterobacteriaceae causing disease were detected. Therefore, it is necessary to monitor the potential pathogens continually and reduce them to improve the microbial quality of non-sterilized powdered infant formulas.

• Journal of Food Hygiene and Safety
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• v.31 no.6
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• pp.439-444
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• 2016

In the present study, the performance of culture methods using two selective agars and real-time PCR were compared for selective isolation of Cronobacter in powdered infant formula and dried pumpkin. Two food samples were spiked with the pathogen and then preenriched in distilled water. A small portion of preenrichment (10 mL) was incubated in Enterobacteriaceae enrichment both, followed by inoculation onto Druggan-Forsythe-Iversen agar (DFI agar) and Cronobacter sakazakii chromogenic plating agar (R&F agar). The preenrichment and enrichment (1 mL each) was used in real-time PCR assay. In powdered infant formula (PIF), no statistical difference was observed between both culture methods and real-time PCR with preenrichemt (p > 0.05). However, the number of positives obtained by R&F agar and real-time PCR was much higher than that of culture method using DFI agar in dried pumpkin (p < 0.05). In particular, R&F agar yielded a significantly greater selectivity than DFI agar in dried pumpkin (p < 0.05). Real-time PCR and R&F agar, which are currently recommended by US FDA, could be used as an alternative detection tools for the isolation of Cronobacter in PIF and ingredient of child foods such as dried pumpkin that has high number of competing natural microflora.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.87-91
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• 2011

The aim of this study was to compare the performance of conventional culture and real-time PCR for detection of Cronobacter spp. in powdered foods. Infant formula, baby food and Misugaru inoculated with Cronobacter were enriched in distilled water as first enrichment step, followed by incubating in Enterobacteriaceae enrichment (EE) broth as second enrichment step. A loopful of enriched sample was streaked onto Druggan-Forsythe-Iversen agar, followed by incubating at $37^{\circ}C$ for 24 h. One milliliter of the enriched distilled water and EE broth were used in real-time PCR assay. No statistical differences were observed in the number of positive samples between culture method and real-time PCR (p>0.05) in all types of food samples. The number of positives of real-time PCR was higher in the first enrichment media (distilled water) than the second enrichment media (EE broth), though there was no significant difference (p>0.05). It appears that some components of the second enrichment broth, EE broth, inhibit the reaction of real-time PCR. These results show that real-time PCR using a single enrichment with distilled water could be useful as an effective screening method for detection of Cronobacter while saving much time and labor compared to conventional culture method.

• Food Science of Animal Resources
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• v.30 no.3
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• pp.479-486
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• 2010

Tolerance against heat, sanitizers, and antibiotics of 112 Cronobacter isolates classified by desiccation was determined to permit effective biocontrol in powdered foods. The isolates were classified into three groups: dry-tolerant (n=37), dry-sensitive (n=7), and dry-intermediate (n=68). The strains that were highly tolerant to drying also showed high heat tolerance that they seemed to have high tolerance to heat after dry stress in powdered foods like infant formula. Sodium hypochlorite and benzalkonium chloride concentrations necessary to achieve a 5-log reduction in viable counts (CFU/mL) were 15-25 ppm and 5-15 ppm, respectively. However, there was little difference of the efficacy of these sanitizers between dry-sensitive and -tolerant strains for planktonic cells suspended in 3% albumin. The minimal inhibition concentration (MIC) of $\beta$-lactam ampicillin was 64-128 ppm for 90% of the strains. The isolates were consistently sensitive to kanamycin and naldixic acid (MIC=4 ppm). Dry-tolerant strains displayed more antibiotic resistance than dry-sensitive strains. The results indicate that dry-tolerant Cronobacter isolates often possess heat and antibiotic resistance, indicated the need for potent sterilization treatments of powdered foods.

• Food Science of Animal Resources
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• v.36 no.5
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• pp.635-640
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• 2016

Lactobacillus acidophilus n.v. Er2 317/402 strain Narine is known as a health beneficial functional probiotic culture and supplementary source of nutrition for newborns. In this study, in vitro antimicrobial activities of Narine-lyophilized (Narine-L), Narine-heat treated (Narine-HT), and Narine crude cell-free extract (Narine-CCFE) were evaluated against pathogen Cronobacter sakazakii (C. sakazakii) in agar as well as in a reconstituted powdered infant formula (RPIF) model. Inhibition zones of 30 mg Narine-L and Narine-HT were both 150 U, whereas inhibition zone of 30 mg Narine-CCFE was 200 U. Narine-L (1 g) and Narine-HT (1 g) were added to 10 mL of artificially contaminated RPIF, respectively, containing 100 μL of C. sakazakii (1.62×10⁸ colony forming unit (CFU)/mL). After treatment with Narine-L and Narine-HT for 3 h and 6 h at 37℃, less than ≤107 CFU/mL of C. sakazakii was detected in RPIF. Without Narine-L and Narine-HT treatment, the population of C. sakazakii increased up to 5.36×10⁹ CFU/mL after 6 h. Examination by transmission electron microscopy confirmed C. sakazakii cells were damaged by Narine-CCFE. Thus, employing Narine culture as a natural and safe bio-preservative may protect infants from C. sakazakii.

• Journal of Food Hygiene and Safety
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• v.23 no.2
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• pp.157-162
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• 2008

Enterobacter sakazakii was initially referred to as yellow-pigmented Enterobacter cloacae and reclassified in 1980. E. sakazakii infection cause life-threatening meningitis, septicemia, and necrotizing enterocolitis in infants. Powdered infant formula (PIF) and baby foods may be the important vehicle of E. sakazakii infection. It has been reported that E. sakazakii was isolated from PIF and sunsik ingredients produced in Korea. Some infants have been fed sunsik as a weaning diet. Therefore, it is necessary that this organism should be inactivated on preparing PIF and sunsik at homes and in hospitals. The cocktail of three Korean E. sakazakii strains (human, sunsik and soil isolates) were used to investigate the inactivation of this organism with hot water at 50, 60, 65, 70 and $80^{\circ}C$ and microwave heating for 60, 75, 90, 105 and 120 sec. Reconstituted PIF and sunsikwere inoculated with cocktailed vegetative cells of E. sakazakii at 6 log CFU/mL. Thermal inactivation of vegetative cells of E. sakazakii were achieved by reconstituted PIF and sunsik with hot water at $60^{\circ}C$ or greater and with microwave heating at 2,450 MHz for 75 sec or longer. Considering that biofilm formation of E. sakazakii was adapted to survive the dry environment that is PIF and sunsik and thermal resistance increased, it is suggested that inactivation of E. sakazakii was used by hot water at $70^{\circ}C$ or greater and microwave heating for 90 sec or longer. Reconstituted PIF and sunsik were inoculated with cocktailed vegetative cells of E. sakazakii at 2 to 3 log CFU/mL to investigate the growth curve of this organism and stored at 5, 10, 15, 20, 25, 30 and $35^{\circ}C$. Viable counts slightly changed at 5, $10^{\circ}C$ during 48 h but grew at $15^{\circ}C$ or greater. Considering that E. sakazakii is able to grow in infant formula milk at refrigerator temperature, reconstituted PIF and sunsik that are not immediately consumed should be discarded or stored at refrigeration temperatures within 24 h.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1430-1437
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• 2021

Cronobacter sakazakii is an opportunistic pathogenic bacterium found in powdered infant formula and is fatal to neonates. Antibiotic resistance has emerged owing to overuse of antibiotics. Therefore, demand for high-yield bacteriophages as an alternative to antibiotics has increased. Accordingly, we developed a modified mass-production method for bacteriophages by introducing a two-stage self-cycling (TSSC) process, which yielded high-concentration bacteriophage solutions by replenishing the nutritional medium at the beginning of each process, without additional challenge. pH of the culture medium was monitored in real-time during C. sakazakii growth and bacteriophage CS01 propagation, and the changes in various parameters were assessed. The pH of the culture medium dropped to 5.8 when the host bacteria reached the early log phase (OD₅₄₀ = 0.3). After challenge, it decreased to 4.65 and then recovered to 4.94; therefore, we set the optimum pH to challenge the phage at 5.8 and that to harvest the phage at 4.94. We then compared phage production during the TSSC process in jar-type bioreactors and the batch culture process in shaker flasks. In the same volume of LB medium, the concentration of the phage titer solution obtained with the TSSC process was 24 times higher than that obtained with the batch culture process. Moreover, we stably obtained high concentrations of bacteriophage solutions for three cycles with the TSSC process. Overall, this modified TSSC process could simplify large-scale production of bacteriophage CS01 and reduce the unit cost of phage titer solution. These results could contribute to curing infants infected with antibiotic-resistant C. sakazakii.

• Journal of Dairy Science and Biotechnology
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• v.41 no.3
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• pp.103-112
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• 2023

This study aimed to assess the effectiveness of 10 different pre-enrichment methods using Real-Time polymerase chain reaction (PCR) in support of the FDA method. When the initial Cronobacter spp. (Enterobacter sakazakii) inoculation was 7.2 CFU/g, the Ct values were observed in the following order: 21.37 (Enterobacteriaceae enrichment [EE] broth), 21.95 (brain heart infusion [BHI]), 22.72 (tryptic soy broth [TSB]), 23.02 (violet red bile lactose [VRBL]), 22.31 (TSB-0.1% sodium pyruvate [SP]), 23.43 (distilled water [DW]), 24.34 (phosphate buffered saline [PBS]), 24.95 (nutrient broth [NB]), 25.82 (TSB-0.6% yeast extract [YE]), and 28.27 (violet red bile glucose [VRBG]). For an inoculation of 1.82% CFU/g of Cronobacter spp. (E. sakazakii), the Ct values were recorded in this sequence: 20.34 (EE broth), 22.16 (TSB-0.6% YE), 22.37 (BHI), 22.71 (VRBL), 22.88 (TSB), 23.01 (DW), 23.19 (NB), 23.79 (TSB-0.1% SP), 24.66 (VRBG), and 24.70 (PBS). Finally, when the inoculum of Cronobacter spp. (E. sakazakii) was 0.182 CFU/g, the Ct values followed this order: 21.93 (VRBL), 23.07 (TSB-0.6% YE), 23.31 (DW), 23.47 (PBS), 23.70 (BHI), 24.14 (TSB-0.1% SP), 25.14 (TSB), 29.00 (VRBG), 31.55 (EE broth), and were undetected in the case of NB. Consequently, these results indicate that there were no significant differences among the 10 different pre-enrichment broths. Future studies should focus on exploring pre-enrichment broths that can improve the limit of detection at very low Cronobacter spp. (E. sakazakii) concentrations and enhance the selective recovery of Cronobacter spp. (E. sakazakii) under acid, antibiotic, cold, and heat damage conditions.