Minimal effects of high-pressure treatment on Salmonella enterica serovar Typhimurium inoculated into peanut butter and peanut products

About 1.2 billion pounds of peanut butter are consumed annually in the United States. In 2008 to 2009, an outbreak involving Salmonella Typhimurium in peanut butter led to a recall of over 3900 products by over 200 companies. More than 700 people became sick, 100 were hospitalized, and 9 people died from this outbreak. This study examines the efficacy of high-pressure processing (HPP) to decrease S. Typhimurium American Type Culture Collection (ATCC) 53647 inoculated into peanut butter and model systems. The viability of S. Typhimurium in peanut butter stored at room temperature was investigated. A culture of S. Typhimurium (6.88 log CFU/g) was inoculated into peanut butter. Following 28 d at 20 °C there was a 1.23-log reduction. Approximately 10(6) to 10(7) CFU/g S. Typhimurium were inoculated into 4 brands of peanut butter, 3 natural peanut butters and peanut flour slurries at 2, 5, and 10% peanut flour protein in peanut oil and in distilled water. All were treated at 600 MPa for 5 min at 45 °C. While significant differences were found between natural peanut butter and peanut protein mixtures, the reduction was <1.0 log. The peanut flour/oil mixtures had a 1.7, 1.6, and 1.0-log reduction from HPP (2, 5, and 10% protein, respectively) whereas peanut flour/water mixtures had a 6.7-log reduction for all protein levels. Oil had a protective effect indicating HPP may not help the microbial safety of water-in-oil food emulsions including peanut butter. Practical Application: There have been multiple outbreaks of foodborne illness involving peanut butter products. This study looks at the potential use of high-pressure processing to reduce the bacteria that may be in peanut butter.     

Evaluation of protein quality of peanut (Arachis hypogaea) cultivars using Tetrahymena pyriformis

Protein quality of eight peanut (Arachis hypogaea L) cultivars was evaluated using chemical, biological and microbiological techniques. The chemical score for S-amino acids and lysine in peanut cultivars ranged from 37 to 50 and 47 to 55%, respectively. Protein efficiency ratio and relative nutritive value of peanut proteins varied significantly from 1.45 to 1.76 and 45.6 to 54.2%, respectively. Relative nutritive value was significantly associated with chemical score (r = 0.98**) and protein efficiency ratio (r = 0.88**), and therefore could be used as a quick method to measure protein quality of peanut cultivars.     

Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars

Salmonella enterica serovar Typhimurium is a major foodborne pathogen throughout the world. Until now, the specific target genes for the detection and identification of serovar Typhimurium have not been developed. To determine the specific probes for serovar Typhimurium, the genes of serovar Typhimurium LT2 that were expected to be unique were selected with the BLAST (Basic Local Alignment Search Tool) program within GenBank. The selected genes were compared with 11 genomic sequences of various Salmonella serovars by BLAST. Of these selected genes, 10 were expected to be specific to serovar Typhimurium and were not related to virulence factor genes of Salmonella pathogenicity island or to genes of the O and H antigens of Salmonella. Primers for the 10 selected genes were constructed, and PCRs were evaluated with various genomic DNAs of Salmonella and non-Salmonella strains for the specific identification of Salmonella serovar Typhimurium. Among all the primer sets for the 10 genes, STM4497 showed the highest degree of specificity to serovar Typhimurium. In this study, a specific primer set for Salmonella serovar Typhimurium was developed on the basis of the comparison of genomic sequences between Salmonella serovars and was validated with PCR. This method of comparative genomics to select target genes or sequences can be applied to the specific detection of microorganisms.     

花生种子中凝集素的凝集活性及性质研究

利用血凝实验及糖抑制实验测定花生种子中凝集素的凝血活性及糖抑制专一性,同时进行热稳定性及pH影响实验。结果显示：花生种子的凝集素粗提液不能凝集人的A、B、O型血细胞,但是可以凝聚唾液酸酶处理后的A、B、O型血细胞。凝集素对红细胞的凝集可被乳糖、蜜二糖、棉籽糖和D-半乳糖四种糖所抑制,在pH为5.0～11.0范围内较稳定,经55℃保温10min后,活性完全消失。在检测的6个不同品种的花生中,河北高油的种子中凝集素活性最强。在花生的生长初期,这种凝集素的存在部位是子叶和根。     

Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs

Despite washing and decontamination, outbreaks linked to consumption of fresh or minimally-processed leafy greens have been increasingly reported in recent years. In order to assure the safety of produce it is necessary to gain knowledge regarding the exact routes of contamination. Leaf internalization through stomata was previously reported as a potential route of contamination, which renders food-borne pathogens protected from washing and disinfection by sanitizers. In the present study we have examined the incidence (percentage of microscopic fields harboring ≥ 1 GFP-tagged bacteria) of Salmonella Typhimurium on the surface and underneath the epidermis in detached leaves of seven vegetables and fresh herbs. The incidence of internalized Salmonella varied considerably among the different plants. The highest incidence was observed in iceberg lettuce (81 ± 16%) and arugula leaves (88 ± 16%), while romaine (16 ± 16%) and red-lettuce (20 ± 15%), showed significantly lower incidence (P < 0.05). Internalization incidence in fresh basil was 46 ± 12%, while parsley and tomato leaves demonstrated only marginal internalization (1.9 ± 3.3% and 0.56 ± 1.36%, respectively). Internalization of Salmonella in iceberg lettuce largely varied (0-100%) through a 2 year survey, with a higher incidence occurring mainly in the summer. These results imply that Salmonella internalization occurs in several leafy vegetables and fresh herbs, other than iceberg lettuce, yet the level of internalization largely varies among plants and within the same crop. Since internalized bacteria may evade disinfection, it is of great interest to identify plants which are more susceptible to bacterial internalization, as well as plant and environmental factors that affect internalization.     

Heat tolerance of Salmonella enterica serovars Agona, Enteritidis, and Typhimurium in peanut butter

Recent large foodborne outbreaks caused by Salmonella enterica serovars have been associated with consumption of foods with high fat content and reduced water activity, even though their ingredients usually undergo pasteurization. The present study was focused on the heat tolerance of Salmonella enterica serovars Agona, Enteritidis, and Typhimurium in peanut butter. The Salmonella serovars in the peanut butter were resistant to heat, and even at a temperature as high as 90 degrees C only 3.2-log reduction in CFU was observed. The obtained thermal inactivation curves were upwardly concave, indicating rapid death at the beginning (10 min) followed by lower death rates and an asymptotic tail. The curves fitted the nonlinear Weibull model with beta parameters < 1, indicating that the remaining cells have a lower probability of dying. beta at 70 degrees C (0.40 +/- 0.04) was significantly lower than beta at 80 degrees C (0.73 +/- 0.19) and 90 degrees C (0.69 +/- 0.17). Very little decrease in the viable population (less than 2-log decrease) was noted in cultures that were exposed to a second thermal treatment. Peanut butter is a highly concentrated colloidal suspension of lipid and water in a peanut meal phase. We hypothesized that differences in the local environments of the bacteria, with respect to fat content or water activity, explained the observed distribution and high portion of surviving cells (0.1%, independent of the initial cell number). These results demonstrate that thermal treatments are inadequate to consistently destroy Salmonella in highly contaminated peanut butter and that the pasteurization process cannot be improved significantly by longer treatment or higher temperatures.     

Antimicrobial resistance of Salmonella enterica serovar Typhimurium isolated from cattle in Japan

The antimicrobial susceptibility of 144 Salmonella enterica serovar Typhimurium isolates collected from all over Japan between 1973 and 1998 were investigated. All the isolates exhibited resistance to four or more antimicrobials and 22 resistance patterns were observed. Isolates showing resistance patterns to ampicillin (A), chloramphenicol (C), streptomycin (S), sulfonamides (Su) and tetracycline (T), which are typical resistance patterns for S. Typhimurium DT104 (DT104), were predominant. Thirty-six of the 68 isolates that exhibited resistance to five or more antimicrobials (ACSSuT+) were identified as DT104 by phage typing. Another 103 S. Typhimurium strains gathered from cattle between 1977 and 1999 in a limited area of Japan were analyzed for molecular epidemiological studies. Results using fluorescent amplified-fragment length polymorphism and pulsed-field gel electrophoresis suggest that clonal exchange of S. Typhimurium among cattle in Japan has occurred since 1992, and that contemporary strains show a remarkable degree of homogeneity with DT104 at a molecular level. The clonal replacement by DT104 affected the antimicrobial resistance pattern of S. Typhimurium from cattle in Japan.     

Inactivation kinetics of Listeria monocytogenes,Salmonella enterica serovar Typhimurium,and Campylobacter jejuni in ready-to-eat sliced ham using UV-C irradiation

To investigate the applicability of UV-C irradiation on the inactivation of foodborne pathogenic bacteria in ready-to-eat sliced ham, UV-C treatment was evaluated. Irradiation dose required for 90% reduction of the populations of Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Campylobacter jejuni were determined to be 2.48, 2.39, and 2.18 J/m². Ready-to-eat sliced hams were inoculated with the pathogens and irradiated with UV-C light of 1000, 2000, 4000, 6000, and 8000 J/m². Microbiological data indicated that foodborne pathogen populations significantly (p < 0.05) decreased with increasing UV-C irradiation. In particular, UV-C irradiation at 8000 J/m² reduced the populations of L. monocytogenes, S. Typhimurium, and C. jejuni in the ham by 2.74, 2.02, and 1.72 log CFU/g. The results indicate that UV-C irradiation can be used as a microbial inactivation method for ready-to-eat sliced ham, and inactivation kinetics of the foodborne pathogens fit the Weibull model better than the first-order kinetics model.     

花生发根培养条件的优化

利用单因素实验和正交设计研究了培养基中激素、碳源、氮源、磷酸盐、初始pH等对鲁花11号花生发根生长的影响。结果表明,花生发根的最适基本液体培养基为无激素的MS培养基;培养基中加入NAA、6-BA、IBA等三种激素,在不同程度上抑制发根的生长;花生发根培养最适条件为:碳源为1.5%蔗糖、氮源含量与标准MS培养基相同、磷酸盐为3.75mmol/L、初始pH为6.1。100μmol/L水杨酸（SA）可以提高花生发根白藜芦醇苷合成分泌以及白藜芦醇的合成。      

Quantification of low and high levels of Salmonella enterica serovar Typhimurium on leaves

Precise and rapid quantification of low levels of pathogens associated with fresh produce may be particularly challenging and yet evermore necessary to guarantee microbial safety of the produce and to carry out research on the subject of persistence of the pathogens. Here, microbiological and molecular based methods were examined for their ability to precisely quantify different amounts of Salmonella enterica serovar Typhimurium artificially inoculated on parsley leaves. Recovery of S. Typhimurium from parsley by mechanical detachment using stomacher, mortar and pestle, vortex, sonicator or homogenizer followed by plating resulted in underestimation with less than 1% recovery when leaves were inoculated with 3.5–6.5 log CFU/g. Lower levels were undetectable by most assayed methods, and only recovery with mortar and pestle or adding of enrichment step resulted in partial detection of 300 CFU/g. Implementation of PCR based methods with/without pre extraction of the DNA from the contaminated leaves resulted in more accurate values of the pathogen (about 20% of the initial inocula) and as low as 300 CFU/g were detected even without an enrichment step. These methods can be applied to study transfer of Salmonlla from contaminated water or soil to plants using low and more reasonable levels of contamination.     

Microfluidic rapid quantification of Salmonella enterica serovar Typhimurium collected from chicken meat using immunomagnetic separation after formaldehyde treatment

Foodborne pathogens such as Salmonella enterica serovar Typhimurium, which cause self-limiting gastroenteritis, are important in food safety and public health. Detection of Salmonella in complex food matrices is required for rapid and effective monitoring of contaminated food products. In this study, we collected Salmonella Typhimurium in chicken using immunomagnetic separation after formaldehyde treatment. Collected Salmonella Typhimurium cells were then quantified microfluidic device or fluorescence microscopy. The recovery ratio obtained with immunomagnetic separation after formaldehyde treatment was 77 (±5)% for the microscope count and 63 (±9)% for the microfluidic count. According to the fluorescence microscope images, the food matrix was effectively removed from the collected cell suspension. The counts of Salmonella Typhimurium cells obtained by the microfluidic device and fluorescence microscope were closely correlated (R² = 0.993) with a range of 3.3 (±0.5) × 10³ to 1.2 (±0.5) × 10⁶ cells g⁻¹. Our method provides quantitative detection of Salmonella Typhimurium within 3–4 h and could be used to efficiently evaluate microbial risk of quality control of foodstuffs and prevention of foodborne disease.     

Virulence-resistance plasmids (pUO-StVR2-like) in meat isolates of Salmonella enterica serovar Typhimurium

During a screening of Salmonella enterica in foods of animal origin four isolates of serovar Typhimurium carrying hybrid virulence-resistance plasmids were detected. Three of them, one from pork and two from chicken meat, contained pUO-StVR2, a derivative of the pSLT virulence plasmid with the bla_OXA-1, catA1, aadA1, sulI and tet(B) genes, which confer resistance to ampicillin, chloramphenicol, streptomycin-spectinomycin, sulfonamides and tetracycline, respectively. The fourth isolate, from pork, harbored a pUO-StVR2 variant (termed ν8) with an additional dfrA10 gene responsible for resistance to trimethoprim. This gene is part of the orf513-dfrA10-qacEΔ1-sul1 element characteristically found in complex class 1 integrons. Pulsed-field gel electrophoresis identified two XbaI-BlnI combined profiles: X2-B2 generated from the three pUO-StVR2 isolates, and X12-B17 shown by the pUO-StVR2-ν8 isolate. The same profiles have also been found in clinical and ill pig isolates, supporting chicken and pork meat as vehicles for transmission of Typhimurium carrying pUO-StVR2-like plasmids.     

Recovery of Salmonella enterica serovars Typhimurium and Tennessee in peanut butter after electron beam exposure

The effect of electron beam (e-beam) radiation on the recovery of Salmonella serotypes Tennessee (ATCC 10722) and Typhimurium (ATCC 14028) in creamy peanut butter over a 14-d storage period at 22 °C was studied. Each Salmonella type was independently inoculated into peanut butter and subjected to e-beam doses that ranged from 0 to 3.1 kGy, confirmed by film dosimetry. After 2-, 4-, 6-, 8-, and 14-d of storage, microbial analyses were conducted. Survivors were recovered on growth and selective media using standard spread-plating methods. Microbial counts (CFU/g) were log-converted and differences were determined by ANOVA and Tukey's Honestly Significant Differences test. When samples were not e-beam-treated, there were no significant changes (P > 0.05) in microbial numbers over time. In e-beamed samples, microbial numbers decreased over time; however, reductions were not always significant. Initial recovery rates (R-rates) 2 d after e-beam treatment were significantly different for the 2 strains of Salmonella and between recovery media (P < 0.05); however, these differences did not persist for the remainder of the storage period (P > 0.05) indicating that injured cells were not able to survive in the high-fat, low-water activity peanut butter environment. R-rates for both strains of Salmonella were maintained until day 14 when there were significant reductions in Salmonella Typhimurium (P < 0.05). These results indicate that Salmonella Tennessee and Salmonella Typhimurium will survive in peanut butter when exposed to nonlethal doses of e-beam irradiation. PRACTICAL APPLICATION: Electron beam (e-beam) irradiation is an alternative to thermal processing; this technique inactivates microorganisms and insects that might be present in a food by generating radiation by accelerated electrons that inactivate organisms directly because of interaction with cell components and indirectly by producing free radicals that disrupt integrity of the cell membrane. E-beam radiation will reduce the number of probable microbiological hazards that could be present while the food remains generally unaffected in texture, taste, and nutritional value. A recent study showed e-beam irradiation to be effective at reducing both Salmonella Tennessee and Typhimurium in peanut butter by one log after exposure to less than 1 kGy, highlighting the need to explore this process further.     

Effects of tomato variety, temperature differential, and post-stem removal time on internalization of Salmonella enterica serovar Thompson in tomatoes

Tomatoes have been implicated in salmonellosis outbreaks due to possible contamination through bacterial internalization during postharvest handling. This study was conducted to determine the effects of tomato variety, temperature differential between tomato pulp and bacterial suspension, and the time delay between stem removal and immersion in bacterial suspension on internalization of Salmonella enterica serovar Thompson in tomato fruit. Mature green tomatoes at 32.2°C were immersed in water containing approximately 10(6) CFU/ml S. enterica bacteria. Different tomato varieties (Mountain Spring, Applause, and BHN961), temperature differentials (-10, 0, and 10°F, or -5.6, 0, and 5.6°C, respectively), and post-stem removal times (0, 2, and 16 h) were evaluated for their effects on S. enterica internalization. The incidence and density of internalized cells were determined by culture enrichment and most-probable-number methods, respectively. Overall, variety and post-stem removal time by variety interaction significantly affected the incidence of S. enterica internalization (P < 0.0001), while temperature differential had no significant effect (P = 0.36). Mountain Spring tomatoes were less susceptible to S. enterica internalization than were Applause and BHN961. Increasing the time interval between stem removal and immersion greatly reduced pathogen internalization in BHN961 and Applause, while it had no effect in Mountain Spring tomatoes. The variety and interactions between varieties and post-stem removal times (P = 0.0363) and between temperature differentials and post-stem removal times (P = 0.0257) had significant effects on the populations of internalized S. enterica. Furthermore, all internalized S. enterica cells were found within the core tissue segments immediately underneath the stem scars.     

Characterization of antimicrobial resistance in Salmonella enterica serovar Typhimurium isolates from food animals in the U.S.

Salmonella enterica subspecies enteric serovar Typhimurium is the second most common serovar implicated in human diseases in the United States. In this study, 120 S. Typhimurium isolates from animal sources were characterized by antimicrobial susceptibility testing, antimicrobial resistance gene detection and plasmid analysis. Overall, 94 (78%) of the isolates were resistant to one or more antimicrobials and 63 (53%) were resistant to at least five antimicrobials. Resistance was most commonly observed to streptomycin (62%), sulfisoxazole (62%), or tetracycline (61%). When resistance was detected, a corresponding resistance gene was detected in 89% of cases. Class 1 integrons were detected in 51 isolates, all which contained the aadA2 gene. A plasmid Inc group was detected in 68 (57%) isolates. Thirty nine (57%) of these isolates were resistant to 5 or more antimicrobials.     

Comparison of sublethal injury induced in Salmonella enterica serovar Typhimurium by heat and by different nonthermal treatments

We have studied sublethal injury in Salmonella enterica serovar Typhimurium caused by mild heat and by different emerging nonthermal food preservation treatments, i.e., high-pressure homogenization, high hydrostatic pressure, pulsed white light, and pulsed electric field. Sublethal injury was determined by plating on different selective media, i.e., tryptic soy agar (TSA) plus 3% NaCl, TSA adjusted to pH 5.5, and violet red bile glucose agar. For each inactivation technique, at least five treatments using different doses were applied in order to cover an inactivation range of 0 to 5 log units. For all of the treatments performed with a technique, the logarithm of the viability reductions measured on each of the selective plating media was plotted against the logarithm of the viability reduction on TSA as a nonselective medium, and these points were fined by a straight line. Sublethal injury between different techniques was then compared by the slope and the y intercept of these regression lines. The highest levels of sublethal injury were observed for the heat and high hydrostatic pressure treatments. Sublethal injury after those treatments was observed on all selective plating media. For the heat treatment, but not for the high-pressure treatment, sublethal injury occurred at low doses, which were not yet lethal. The other nonthermal techniques resulted in sublethal injury on only some of the selective plating media, and the levels of injury were much lower. The different manifestations of sublethal injury were attributed to different inactivation mechanisms by each of the techniques, and a mechanistic model is proposed to explain these differences.     

Prevention of pre-harvest aflatoxin production and the effect of different harvest times on peanut (Arachis hypogaea L.) fatty acids

The aim of this study was to investigate the relationship between aflatoxin and fatty acids and to determine the optimum harvest time to avoid pre-harvest aflatoxin formation. It was established that harvest time had statistically significant effects on the levels of saturated fatty acids: myristic acid (C14:0), palmitic acid (C16:0), heptadecanoic acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0), behenic acid (C22:0), lignoceric acid (C24:0), monounsaturated fatty acids; palmitoleic acid (C16:1), heptadecenoic acid (C17:1), oleic acid (C18:1) and gadoleic acid (C20:1); and on polyunsaturated fatty acids: linoleic acid (C18:2) and linolenic acid (C18:3). By delaying the harvest time, the ratio of saturated fatty acids decreased and unsaturated fatty acids increased. It was shown that the longer harvesting was delayed, the greater the quantity of oleic acid that was produced. Before harvest time, if the soil moisture was 5% or higher, aflatoxin was produced by fungi. It was found that the weather conditions of the region were suitable for aflatoxin production. Soil moisture appears to be more important than soil temperature for aflatoxin formation. The production of aflatoxin was not observed in the first and second harvests, both of which are at early harvest times. It was found that aflatoxin B1 during harvest time was the most significant of the four toxins. The third harvest time, which is the most widely used, was observed to have significant problems due to aflatoxin formation. Therefore, it is suggested as a result of this study that the harvest of peanuts must be done considering seed yield before the middle of September to avoid aflatoxin formation at harvest time.