Minimum Inhibitory Concentration (MIC) of Propionic Acid, Sorbic Acid, and Benzoic Acid against Food Spoilage Microorganisms in Animal Products to Use MIC as Threshold for Natural Preservative Production.

Some preservatives are naturally contained in raw food materials, while in some cases may have been introduced in food by careless handling or fermentation. However, it is difficult to distinguish between intentionally added preservatives and the preservatives naturally produced in food. The objective of this study was to evaluate the minimum inhibitory concentration (MIC) of propionic acid, sorbic acid, and benzoic acid for inhibiting food spoilage microorganisms in animal products, which can be useful in determining if the preservatives are natural or not. The broth microdilution method was used to determine the MIC of preservatives for 57 microorganisms. Five bacteria that were the most sensitive to propionic acid, benzoic acid, and sorbic acid were inoculated in unprocessed and processed animal products. A hundred microliters of the preservatives were then spiked in samples. After storage, the cells were counted to determine the MIC of the preservatives. The MIC of the preservatives in animal products ranged from 100 to 1,500 ppm for propionic acid, from 100 to >1,500 ppm for benzoic acid, and from 100 to >1,200 ppm for sorbic acid. Thus, if the concentrations of preservatives are below the MIC, the preservatives may not be added intentionally. Therefore, the MIC result will be useful in determining if preservatives are added intentionally in food.

Effect of Starter Cultures on Quality of Fermented Sausages.

The expansion and advancement of the meat product market have increased the demand for fermented sausages. A typical method for manufacturing high-quality fermented sausages is using a starter culture, which improves the taste, aroma, and texture. Currently, the starter culture for manufacturing fermented sausages is mainly composed of microorganisms such as lactic acid bacteria, yeast, and fungi, which generate volatile compounds by the oxidation of fatty acids. In addition, protein decomposition and changes in pH occur during the fermentation period. It can positively change the texture of the fermented sausage. In this review, we discuss the requirements (improving food safety, the safety of starter culture, enzyme activity, and color) of microorganisms used in starter cultures and the generation of flavor compounds (heptanal, octanal, nonanal, hexanal, 2-pentylfuran, 1-penten-3-ol, and 2-pentanone) from lipids. Furthermore, quality improvement (hardness and chewiness) due to texture changes after starter culture application during the manufacturing process are discussed.

High Prevalence of Listeria monocytogenes in Smoked Duck: Antibiotic and Heat Resistance, Virulence, and Genetics of the Isolates.

This study aimed at determining the genetic and virulence characteristics of the Listeria monocytogenes from smoked ducks. L. monocytogenes was isolated by plating, and the isolated colonies were identified by PCR. All the obtained seven L. monocytogenes isolates possessed the virulence genes (inlA, inlB, plcB, and hlyA) and a 385 bp actA amplicon. The L. monocytogenes isolates (SMFM2018 SD 1-1, SMFM 2018 SD 4-1, SMFM 2018 SD 4-2, SMFM 2018 SD 5-2, SMFM 2018 SD 5-3, SMFM 2018 SD 6-2, and SMFM 2018 SD 7-1) were inoculated in tryptic soy broth (TSB) containing 0.6% yeast extract at 60°C, followed by cell counting on tryptic soy agar (TSA) containing 0.6% yeast extract at 0, 2, 5, 8, and 10 min. We identified five heat resistant isolates compared to the standard strain (L. monocytogenes ATCC13932), among which three exhibited the serotype 1/2b and D-values of 5.41, 6.48, and 6.71, respectively at 60°C. The optical densities of the cultures were regulated to a 0.5 McFarland standard to assess resistance against nine antibiotics after an incubation at 30°C for 24 h. All isolates were penicillin G resistant, possessing the virulence genes (inlA, inlB, plcB, and hlyA) and the 385-bp actA amplicon, moreover, three isolates showed clindamycin resistance. In conclusion, this study allowed us to characterize L. monocytogenes isolates from smoked ducks, exhibiting clindamycin and penicillin G resistance, along with the 385-bp actA amplicon, representing higher invasion efficiency than the 268-bp actA, and the higher heat resistance serotype 1/2b.

Development of a Selective Agar for Improving Campylobacter jejuni Detection in Food.

BACKGROUND: Campylobacter jejuni is a major gastroenteritis-causing foodborne pathogen. However, it is difficult to isolate when competing bacteria or cold-damaged cells are present. OBJECTIVE: Herein, a medium (Campylobacter selective agar, CSA) was developed and supplemented with catalase, L-serine, L-cysteine, and quercetin for the selective detection of C. jejuni in food. METHOD: The C. jejuni-detection efficiency in media broth and chicken tenders was evaluated. The pathogen was enumerated on modified charcoal-cefoperazone-deoxycholate agar (mCCDA), CSA supplemented with 4 µM catalase (CSA-C4), 8 µM catalase (CSA-C8), 20 mM L-serine (CSA-S20) or 50 mM L-serine (CSA-S50), and mCCDA supplemented with 0.5 mM L-cysteine (mCCDA-LC0.5), 1 mM L-cysteine (mCCDA-LC1), 40 µM quercetin (mCCDA-Q40) or 320 µM quercetin (mCCDA-Q320). The detection efficiency was then evaluated by counting colonies on the selective agar media. Quantitative assessment was also performed using chicken and duck carcasses. RESULTS: The C. jejuni detection efficiencies were higher (P < 0.05) in the groups CSA-C4 or CSA-C8, and CSA-S20 or CSA-S50, than mCCDA, and the detection efficiencies were maintained even in the presence of Acinetobacter baumannii, a competing bacterium. In the quantitative test, CSA-C8 and CSA-S50 demonstrated higher C. jejuni-detection efficiencies than mCCDA (control). CONCLUSIONS: Therefore, CSA-C8 and CSA-S50 improved the detection efficiency of C. jejuni in poultry products by promoting the recovery of cold-damaged cells. HIGHLIGHTS: When using CSA-C8 or CSA-S50 developed in this study for detection of C. jejuni in food, detection efficiency was higher than mCCDA.

Lactobacillus fermentum SMFM2017-NK4 Isolated from Kimchi Can Prevent Obesity by Inhibiting Fat Accumulation.

This study evaluated the anti-obesity effects of lactic acid bacteria. Thirty-one lactic acid bacteria were examined in vitro for their ability to inhibit α-glucosidase activity, lipase activity, and 3T3-L1 cell differentiation. Four selected lactic acid bacteria were administered to obese C57BL/6J mice models for 8 weeks. The degree of improvement in obesity was determined by weight gain and serum biochemical analysis. The expression levels of genes (Fas and Cpt-2) related to obesity in the liver were analyzed by quantitative reverse transcription (qRT)-PCR. In addition, antioxidant protein levels (SOD-2, CAT, and GPx-1) in the liver were evaluated. The lactic acid bacteria-treated groups (PPGK1, LFNK3, LPNK2, and LFNK4) showed lower weight increase rate than the control group. The total cholesterol (T-chol), triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in the blood serum of the LFNK4 group were the lowest among other groups, compared to the control group. The expression levels of lipid metabolism-related genes (Fas and Cpt-2) in the liver of the LFNK4 group were lower in Fas and higher in Cpt-2 than in the control group. The antioxidant protein expression levels (SOD-2, CAT, and GPx-1) in the liver tissue were also higher in the LFNK4 group. These results indicate that L. fermentum SMFM2017-NK4 has anti-obesity effects.

Contamination of Clostridium perfringens in soy sauce, and quantitative microbial risk assessment for C. perfringens through soy sauce consumption.

The objective of this study was to conduct QMRA (quantitative microbial risk assessment) of Clostridium perfringens through soy sauce consumption. Four hundred and ninety soy sauce samples from markets were analyzed to detect C. perfringens. Temperature and time were also measured during transportation and display of soy sauce. A primary model was developed by fitting the Weibull model to the C. perfringens cell counts in soy sauce at 7-35°C, and δ (the time needed to decrease 1 log CFU/ml) and ρ (curve shape) were calculated. The parameters were analyzed, using the Exponential model (secondary model) as a function of temperature. The consumption amount and percentage of soy sauce were surveyed, and a dose-response model was searched. Using all collected data, a simulation model was prepared in the @RISK program to estimate the probability of C. perfringens foodborne illness by soy sauce consumption. C. perfringens were negative in 490 samples. Thus, the initial contamination level was estimated to be -2.9 log CFU/ml. The developed predictive models showed that C. perfringens cell counts decreased during transportation and display. The average consumption amounts, and the percentage of soy sauce were 7.81 ml and 81.2%, respectively. The simulation showed that the probability of C. perfringens foodborne illness by consumption of soy sauce was 1.7 × 10-16 per person per day. Therefore, the risk of C. perfringens by consumption of soy sauce is low in Korea.

Improvement of the detection efficiency of 3M™ molecular detection system for Campylobacter in poultry using nitrogen-doped carbon nanodots.

This study was performed to examine whether the use of nitrogen-doped carbon nanodots (N-CNDs) can improve the detection sensitivity of the 3 M™ molecular detection system (MDS) for Campylobacter. N-CNDs were added to a Campylobacter enrichment broth (CEB) at concentrations of 5 and 10 mg/mL (NCEB-5 and NCEB-10, respectively). Campylobacter coli, C. jejuni, and C. lari were inoculated into the broths. The broth cultures were then irradiated with light-emitting diode (LED) at 425 nm for 1 h and incubated at 42 °C for 6 h, and then grown on modified charcoal cefoperazone deoxycholate agar (mCCDA). The detection rates of the MDS and a conventional method (plating an enriched sample on mCCDA and analyzing a colony on mCCDA with PCR) for Campylobacter in chicken and duck carcasses were compared. The detection rates from the MDS were compared after enrichment in CEB and NCEB-5 at 3, 5, 6, 7, 9, 12, and 24 h. When 5 mg/mL of N-CNDs was added to the CEB followed by irradiation at 425 nm, growth of the Campylobacter was accelerated. In addition, the qualitative test was more sensitive in the MDS than in the conventional method, and the detection time was shortened in CEB enriched with N-CNDs. These results indicate that adding N-CNDs to CEB can improve the detection efficiency of MDS.

Identification of Pathogenic Variations in Seafood Vibrio parahaemolyticus Isolates by Comparing Genome Sequences.

ABSTRACT: An investigation of the pathogenic characteristics of isolates of Vibrio parahaemolyticus was conducted by identifying the pathogenic tdh gene and then performing adherence and cytotoxicity assays. Genome sequences of the seafood isolates were analyzed using the Illumina HiSeq 2500 platform. The isolated strains were then mapped by comparing the genomes to the reference genome, and variations in the nucleotide sequences and amino acids were identified with the CLC Genomics Workbench program. The tdh gene was identified in four isolates of V. parahaemolyticus, of which three-SMFM201809-CPC7-3, SMFM201809-CF8-2, and SMFM201809-CF8-3-showed high cytotoxicity and differences in cell adhesion. These isolates were selected to identify virulence factors and genomic variations. All three isolates had the same virulence factors, such as adherence, secretion system, and toxin. In addition, amino acid variants were identified in the regions of type IV pilus, T3SS1 and T3SS1 secreted effectors, and thermolabile hemolysin. These results indicate that variations in amino acids found in regions related to adherence and cytotoxicity result in differences in adhesion efficiency and cytotoxicity; therefore, the isolates with these variations may cause more serious foodborne illness compared with other strains.

Synthesis of nitrogen-doped carbon nanodots to destroy bacteria competing with Campylobacter jejuni in enrichment medium, and development of a monoclonal antibody to detect C. jejuni after enrichment.

The objective of this study was to develop a method with improved sensitivity for Campylobacter jejuni detection in foods. Nitrogen-doped carbon nanodots (N-CNDs) were synthesized and added to an enrichment medium (Bolton broth) at a concentration of 10 mg/mL. A light-emitting diode (LED) at a wavelength of 425 nm was used to irradiate the N-CNDs-supplemented enrichment medium to induce an exothermic reaction for 1 h. Additionally, a monoclonal antibody specific to C. jejuni NCTC11168 was developed using hybridoma cells to aid detection. The C. jejuni detection capabilities of N-CNDs-supplemented enrichment medium and the conventional Bolton broth enrichment, were compared using duck samples. C. jejuni in the enrichment was detected with the monoclonal antibody based-indirect enzyme-linked immunosorbent assay (ID-ELISA). The N-CNDs-supplemented enrichment medium showed a better C. jejuni detection capability than the conventional Bolton broth enrichment. Additionally, data from ID-ELISA showed excellent detection efficiency and a shortened detection time in the N-CNDs-supplemented enrichment medium after LED irradiation at 425 nm. These results indicate that 1-h LED irradiation at 425 nm to Bolton broth supplemented with the N-CNDs increased the detection efficiency and shortened the detection time with the monoclonal antibody for C. jejuni in food.

Influence of milk microbiota on Listeria monocytogenes survival during cheese ripening.

This study aimed to compare the three strains of Listeria monocytogenes survival in raw milk cheese and pasteurized milk cheese and to suggest the effect of milk microbiota on survival. L. monocytogenes cell counts decreased in all cheese as ripening time increased, and the survival rate was different for the strains of L. monocytogenes. Furthermore, L. monocytogenes survived longer in raw milk cheese than in pasteurized milk cheese. The difference of bacterial survival in each cheese was independent of Aw or the Lactobacillus spp. populations in cheeses; there was no difference in Aw or Lactobacillus spp. populations in all cheeses. The richness of microbiota in raw milk was little higher than in pasteurized milk, and five phyla (Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Lentisphaerae, and Verrucomicrobia) were present only in raw milk. Also, organic acid-producing bacteria were presented more in pasteurized milk compared with raw milk; thus, the growth of L. monocytogenes was slower in pasteurized milk. In conclusion, differences in the microbial community of milk can affect the growth of L. monocytogenes. Making cheese using raw milk is a risk of L. monocytogenes infection; thus, efforts to prevent growth of L. monocytogenes such as the use of appropriate food additives are required.

Quantitative microbial risk assessment for Clostridium perfringens foodborne illness following consumption of kimchi in South Korea.

The purpose of this study was to conduct a risk assessment for Clostridium perfringens foodborne illness via kimchi consumption in South Korea. Prevalence of C. perfringens in kimchi, kimchi consumption amount and frequency, and distribution conditions (time and temperature) from manufacture to the home were determined. C. perfringens initial contamination level was estimated using Beta distribution [Beta (6, 79)]. Potential C. perfringens cell counts during distribution were predicted using the Weibull model (primary models, R 2 = 0.923-0.953) and a polynomial model [(δ = 1/(0.2385 + (- 0.0307 × Temp) + (0.0011 × Temp2)), R 2 = 0.719]. Average daily consumption data was assessed using Gamma distribution [1.0444, 91.767, RiskShift (0.16895), RiskTruncate (0, 1078)]. The mean risk of C. perfringens-associated foodborne illness following kimchi consumption was found to be 1.21 × 10-17. These results suggest that the risk of C. perfringens foodborne illness from kimchi consumption, under current conditions, can be considered to be very low in S. Korea.

Anti-Inflammatory Effect of a Peptide Derived from the Synbiotics, Fermented Cudrania tricuspidata with Lactobacillus gasseri, on Inflammatory Bowel Disease.

The objective of this study was to evaluate the effects of peptides derived from synbiotics on improving inflammatory bowel disease (IBD). Five-week-old male C57BL/6 mice were administered with dextran sulfate sodium (DSS) via drinking water for seven days to induce IBD (IBD group). The mice in the IBD group were orally administered with PBS (IBD-PBS-positive control), Lactobacillus gasseri 505 (IBD-Pro), fermented powder of CT extract with L. gasseri 505 (IBD-Syn), ß-casein: LSQSKVLPVPQKAVPYPQRDMP (IBD-Pep 1), or α s2-casein: VYQHQKAMKPWIQPKTKVIPYVRYL (IBD-Pep 2) (both peptides are present in the synbiotics) for four more days while inducing IBD. To confirm IBD induction, the weights of the animals and the disease activity index (DAI) scores were evaluated once every two days. Following treatment of probiotics, synbiotics, or peptides for 11 days, the mice were sacrificed. The length of the small and large intestines was measured. The expression of the proinflammatory cytokines IL-1ß, IL-6, TNF-α, and COX-2 in the large intestine was measured. Large intestine tissue was fixed in 10% formalin and stained with hematoxylin and eosin for histopathological analysis. The body weights decreased and DAI scores increased in the IBD group, but the DAI scores were lower in the IBD-Pep 2 group than those in the IBD group treated with PBS, Pro, Syn, or Pep 1. The lengths of the small and large intestines were shorter in the IBD group than in the group without IBD, and the expression levels of the proinflammatory cytokines were lower (p < 0.05) in the IBD-Pep 2 group than those in the IBD-PBS-positive control group. In addition, histopathological analysis showed that IBD was ameliorated in the Pep 2-treated group. These results indicate that Pep 2 derived from α s2-casein was effective in alleviating IBD-associated inflammation. Thus, we showed that these peptides can alleviate inflammation in IBD.

Akkermansia muciniphila Prevents Fatty Liver Disease, Decreases Serum Triglycerides, and Maintains Gut Homeostasis.

The objective of this study was to elucidate the effect of intestinal Akkermansia muciniphila bacteria on fatty liver disease. Five-week-old C57BL/6N mice were administered either phosphate-buffered saline (PBS; control) or A. muciniphila at 108 to 109 CFU/ml, and were fed either a 45% fat diet (high-fat diet [HFD]) or a 10% fat diet (normal diet [ND]) for 10 weeks. After 10 weeks, the mice were euthanized, and blood and tissue samples, including adipose tissue, cecum, liver, and brain, were immediately collected. Biochemical and histological analyses were conducted, and the expression levels of related factors were compared to determine the antiobesity effects of Akkermansia muciniphila The gut microbiome was analyzed in fecal samples. Oral administration of A. muciniphila significantly (P < 0.05) lowered serum triglyceride (TG) and alanine aminotransferase (ALT) levels in obese mice. Compared to the non-A. muciniphila-treated group, the expression of SREBP (regulator of TG synthesis in liver tissue) was decreased in the A. muciniphila-treated group. The expression of IL-6 in the liver of obese mice was decreased following the administration of A. muciniphila Furthermore, alterations in the ratio of Firmicutes to Bacteroidetes and the decrease in bacterial diversity caused by the HFD were restored upon the administration of A. muciniphila These results indicate that A. muciniphila prevents fatty liver disease in obese mice by regulating TG synthesis in the liver and maintaining gut homeostasis.IMPORTANCE This study investigated the effect of Akkermansia muciniphila on fatty liver disease. Although some research about the effects of A. muciniphila on host health has been published, study of the relationship between A. muciniphila administration and fatty liver, as well as changes in the gut microbiota, has not been conducted. In this study, we demonstrated that A. muciniphila prevented fatty liver disease by regulation of the expression of genes that regulate fat synthesis and inflammation in the liver.

Combined Enrichment and Quantitative Polymerase Chain Reaction to Improve Sensitivity and Reduce Time of Detection of Listeria monocytogenes in Mushrooms.

This study evaluated a combined method for the detection of Listeria monocytogenes in mushrooms, involving enrichment and quantitative real-time polymerase chain reaction (qPCR), to improve sensitivity and reduce detection time. The growth of L. monocytogenes was evaluated in Listeria enrichment broth (LEB) with modified carbon and nitrogen sources, increasing sodium concentrations, and added micronutrients. Primers targeting the L. monocytogenes iap (iap1 and iap2), hlyA (hlyA1-hlyA6), and prfA (prfA1-prfA4) genes were developed and their sensitivity and specificity were evaluated. The greatest increase in L. monocytogenes cell count was observed after 6-h incubation at 30°C in LEB+2 × FAC (LEB plus 20 mL/L ferric ammonium citrate), where cell count increased by 1.4 log CFU (colony-forming unit)/mL, compared with 0.9 log CFU/mL in LEB (p < 0.05). iap2 primers targeting the iap gene showed high specificity and were the most sensitive among those tested, with a detection limit of 2 log CFU/mL in LEB medium, 3.1 log CFU/g in golden needle mushroom, and 3.5 log CFU/g in large oyster mushroom. When applied to detection in golden needle mushrooms, a combination of 3-h incubation in LEB+2 × FAC medium and qPCR analysis with iap2 primers permitted detection of L. monocytogenes, even at an inoculum of 1 log CFU/g. Similarly, in large oyster mushrooms, 10-h enrichment in LEB+2 × FAC medium resulted in a cell count of 3.7 log CFU/g. These results indicate that a combined detection method, using LEB+2 × FAC medium for enrichment followed by qPCR with iap2 primer pair, can reduce enrichment time and improve the sensitivity and specificity of L. monocytogenes detection in mushrooms.

Mathematical Models to Describe the Kinetic Behavior of Staphylococcus aureus in Jerky.

The objective of this study was to develop mathematical models for describing the kinetic behavior of Staphylococcus aureus (S. aureus) in seasoned beef jerky. Seasoned beef jerky was cut into 10-g pieces. Next, 0.1 mL of S. aureus ATCC13565 was inoculated into the samples to obtain 3 Log CFU/g, and the samples were stored aerobically at 10°C, 20°C, 25°C, 30°C, and 35°C for 600 h. S. aureus cell counts were enumerated on Baird Parker agar during storage. To develop a primary model, the Weibull model was fitted to the cell count data to calculate Delta (required time for the first decimal reduction) and ρ (shape of curves). For secondary modeling, a polynomial model was fitted to the Delta values as a function of storage temperature. To evaluate the accuracy of the model prediction, the root mean square error (RMSE) was calculated by comparing the predicted data with the observed data. The surviving S. aureus cell counts were decreased at all storage temperatures. The Delta values were longer at 10°C, 20°C, and 25°C than at 30°C and 35°C. The secondary model well-described the temperature effect on Delta with an R2 value of 0.920. In validation analysis, RMSE values of 0.325 suggested that the model performance was appropriate. S. aureus in beef jerky survives for a long period at low storage temperatures and that the model developed in this study is useful for describing the kinetic behavior of S. aureus in seasoned beef jerky.

Development of Hydrogels to Improve the Safety of Yukhoe (Korean Beef Tartare) by Reducing Psychrotrophic Listeria monocytogenes Cell Counts on Raw Beef Surface.

This study developed an antimicrobial hydrogel to control Listeria monocytogenes in Yukhoe (Korean beef tartare). Four hydrogels (hydrogel 1: 5% alginate+1% chitosan+0.2% CaCl2, hydrogel 2: 1% κ-carrageenan+1% chitosan, hydrogel 3: 2% κ-carrageenan+1% CaCl2, and hydrogel 4: 2% κ-carrageenan+3% CaCl2) were prepared. The hydrogels then absorbed 0.1% grapefruit seed extract (GSE) and 0.1% citrus extract (CE) for 30, 60, 120, and 240 min to be antimicrobial hydrogels. To select the most effective antimicrobial hydrogel, their swelling ratio (SR) and antilisterial activities were determined. The selected hydrogel (2×2 cm) was then placed on surface of beef (round; 3×3 cm), where L. monocytogenes (ca. 106 CFU/g) were inoculated, and the cell counts were enumerated on PALCAM agar. Among the hydrogels, the SR of hydrogel 1 increased with absorbing time, but other hydrogels showed no significant changes. Antimicrobial hydrogel 1 showed higher (p<0.05) antilisterial activity than other antimicrobial hydrogels, especially for the one absorbed the antimicrobial for 120 min. Thus, the antimicrobial hydrogel 1 absorbed antimicrobials for 120 min was applied on raw beef at 4℃, and reduced (p<0.05) more than 90% of L. monocytogenes on raw beef. These results indicate that antimicrobial hydrogel 1 formulated with 0.1% GSE or 0.1% CE is appropriate to improve the safety of Yukhoe by reducing psychrotrophic L. monocytogenes cell counts on raw beef.