Detection of Salmonella in food over 30 h using enrichment and polymerase chain reaction

A 30-h method for the detection of Salmonella spp. in food was developed. The method involved preenrichment in buffered peptone water for 6–8 h, immunomagnetic separation (IMS) using Dynabeads^®anti-Salmonella , selective enrichment in Rappaport-Vassiliadis broth for 16–18 h, lysis of bacterial cells in sodium dodecylsulfate and NaOH solution at 95°C, and the polymerase chain reaction (PCR) using primers ST11 and ST15. The detection limit of the method was 10⁰cfu 25g⁻¹, as determined by the analysis of food samples artificially contaminated with S. enteritidis. When the reference material containing on average 5 cfu of S. panama was used for the artificial contamination, 3 out of 22 food samples were found to be false-negative. When the method was evaluated in comparison with the standard ISO 6579 method on 42 possibly naturally-contaminated food samples, one sample was found positive by the 30-h method, one sample was found positive by the ISO-method, and two samples were found positive by both methods. The developed method proved rapid, but produced a non-zero level of false-negative results.     

The potential application of plant essential oils as natural food preservatives in soft cheese

Investigations were carried out to assess the efficiency of four plant essential oils; bay, clove, cinnamon and thyme as natural food preservatives. The effect of the plant essential oils at concentrations of 0·1, 0·5 and 1% was studied in low-fat and full-fat soft cheese against Listeria monocytogenes and Salmonella enteritidis at 4° and 10°C respectively, over a 14-day period. The composition of the cheese was shown to be an important factor in determining the effectiveness of the plant essential oils. In the low-fat cheese, all four oils at 1% reduced L. monocytogenes to ≤1·0 log₁₀cfu ml⁻¹. In contrast, in the full-fat cheese, oil of clove was the only oil to achieve this reduction. Oil of thyme proved ineffective against S. enteritidis in the full-fat cheese, yet was equally as effective as the other three oils in the low-fat cheese, reducing S. enteritidis to ≤1·0 log₁₀cfu ml⁻¹from day 4 onwards. It is concluded that selected plant essential oils can act as potent inhibitors of L. monocytogenes and S. enteritidis in a food product.     

Salmonella Detection in Meat and Fish by Membrane Hybridization with Chromogenic/Phosphatase/Biotin DNA Probe

The hybridization specificity of a biotin labeled 1.8 kb HindIII DNA fragment was confirmed by colony hybridization with Salmonella and non-Salmonella isolates. Culture conditions were then tested for the enrichment of salmonellae in foods with large populations of competitive flora. Different conditions of preenrichment and selective enrichment could be used for detecting low populations of salmonellae in foods. Enrichment using lactose combined tetrathionate (CTET) broth and Salmonella-Shigella (SS) agar followed by spotting the suspected isolates on a nitrocellulose membrane [CETE → SS (S)] was better. As low as 1.6 × 10⁰ salmonellae/g food could be detected.     

The detection of Salmonella serovars from animal feed and raw chicken using a combined immunomagnetic separation and ELISA method

The immunomagnetic separation technique was used in conjunction with an ELISA-based detection system to recover Salmonella enteritidis, Salmonella typhimurium and Salmonella virchow (2–2×10³cfu 25 g⁻¹) from raw chicken and animal feed. The detection time was less than 27 h and included the conventional pre-enrichment (18 h) step in buffered peptone water and an enrichment step (6 h) in glucose nutrient broth. In contrast, the conventional method using Rappaport-Vassiliades enrichment broth and selective plating on XLD agar required 72–96 h.     

Enrichment of injuredSalmonellain poultry products for detection by polymyxin–cloth enzyme immunoassay

Because injuredSalmonellacells have the potential to cause salmonellosis, their detection in foods is important to food safety testing programs. In pure cultures, a small number of heat-injuredSalmonella enteritidiscells were detected by the polymyxin–cloth enzyme immunoassay after 2-h static pre-enrichment in brain–heart infusion (BHI) medium supplemented with 0.05% yeast extract and 0.05% cholate (BYC), followed by overnight static enrichment in the presence of 0.3% selenite. However, in chicken carcass rinse, heat-injuredS. enteritidiscells (400 cfu ml^–1) could not be detected even after 6-h pre-enrichment in BYC, while uninjured cells were detectable. Prolonged (20 h) pre-enrichment in BYC, followed by 20-h incubation of a 10-fold dilution with BYC with addition of 0.03% selenite, permitted the detection of heat-injuredS. enteritidis(less than 7 cfu ml^–1) in the chicken carcass rinse.     

Recovery of Salmonella from refrigerated preenrichment and refrigerated enrichment media

The productivity of the standard cultural procedure for the isolation of Salmonella using preenrichment in buffered peptone water (BPW) followed by inoculation into Rappaport-Vassiliadis (RV), tetrathionate (TBG) (Difco) and selenite-cystine (SC) (Difco) enrichment broths, was compared with that using preenrichment and enrichment cultures which had been held under refrigeration (72 h at 5–10°C). Seventy-seven of 251 samples of food products were found to contain Salmonella. Refrigerated preenrichment cultures inoculated into RV medium (43°C), TBG broth (43°C) and SC broth (37°C) yielded salmonellae from 93.5, 85.7 and 54.5% contaminated samples, respectively. Refrigerated cultures in RV, TBG and SC broths, inoculated onto three selective plating media, identified 100, 87.0 and 41.6% of the contaminated samples, respectively.The selective plating medium brilliant green deoxycholate agar was at least as productive as brilliant green sulpha agar and bismuth sulphite agar, when streaked from RV and TBG broths, but was less effective when streaked from SC broth.     

Nonstarter lactic acid bacteria biofilms and calcium lactate crystals in Cheddar cheese

A sanitized cheese plant was swabbed for the presence of nonstarter lactic acid bacteria (NSLAB) biofilms. Swabs were analyzed to determine the sources and microorganisms responsible for contamination. In pilot plant experiments, cheese vats filled with standard cheese milk (lactose:protein = 1.47) and ultrafiltered cheese milk (lactose:protein = 1.23) were inoculated with Lactococcus lactis ssp. cremoris starter culture (8 log cfu/mL) with or without Lactobacillus curvatus or Pediococci acidilactici as adjunct cultures (2 log cfu/mL). Cheddar cheeses were aged at 7.2 or 10°C for 168 d. The raw milk silo, ultrafiltration unit, cheddaring belt, and cheese tower had NSLAB biofilms ranging from 2 to 4 log cfu/100 cm². The population of Lb. curvatus reached 8 log cfu/g, whereas P. acidilactici reached 7 log cfu/g of experimental Cheddar cheese in 14 d. Higher NSLAB counts were observed in the first 14 d of aging in cheese stored at 10°C compared with that stored at 7.2°C. However, microbial counts decreased more quickly in Cheddar cheeses aged at 10°C compared with 7.2°C after 28 d. In cheeses without specific adjunct cultures (Lb. curvatus or P. acidilactici), calcium lactate crystals were not observed within 168 d. However, crystals were observed after only 56 d in cheeses containing Lb. curvatus, which also had increased concentration of d(−)-lactic acid compared with control cheeses. Our research shows that low levels of contamination with certain NSLAB can result in calcium lactate crystals, regardless of lactose:protein ratio.     

The isolation and identification of yeasts obtained during the manufacture and ripening of Cheddar cheese

Sources of yeast, which may contaminate the curd during the manufacture of Cheddar cheese, were examined in a single cheese factory. A total of 77 yeast species present in the factory environment, manufacturing and ripening of Cheddar cheese were identified according to cellular long-chain fatty acid analysis and verified with conventional identification techniques. Product line samples were taken at critical control points in the manufacturing process and analysed after incubation at 25°C for 96 h. The progression of yeast species during cheese-making and ripening was monitored after renneting and at subsequent 48-h intervals. Dominant species wereDebaryomyces hanseniiandCryptococcus albidus, whileYarrowia lipolytica, Rhodotorula minuta, Torulaspora delbrueckii, Rhodotorula glutinisandKluyveromyces marxianuswere present at low numbers. The results obtained showed that yeasts were present in all cheese samples examined, at quantities ranging from 9×10²to 1·4×10⁷cfu g⁻¹.     

Rapid and simple method for detectingSalmonellain chicken feces using polymyxin-cloth enzyme immunoassay

The presence of three strains ofSalmonellacells in chicken feces was detected by polymyxin-cloth enzyme immunoassay. A cotton swab-full ofSalmonella-free chicken feces (containing an aerobic count of about 3×10⁷cfu) was mixed with a small number ofSalmonella typhimurium(6 or 12 cfu),Salmonella enteritidis(6 or 15 cfu) orSalmonella hadar(5 or 10 cfu) in 5 ml of brain–heart infusion medium supplemented with 0.5% yeast extract, 0.3% sodium hydrogen selenite, and 0.5% sodium cholate. After 20 h static incubation at 37°C, heat-extracted lipopolysaccharide (LPS) antigens were captured by polymixin B adsorbed onto macroporous polyester cloth. The LPS antigens were then detected via sequential reactions with a mixture of mouse anti-SalmonellaLPS antibody and rabbitSalmonellaO antiserum followed by a mixture of horseradish peroxidase conjugates of antibody to mouse and rabbit immunoglobulin G. The total procedure took about 22 h. This method was also used to detect endogeneousSalmonellain feces of chicken inoculated withSalmonellaand to measure the level of fecalSalmonella. This simple, rapid and sensitive method forSalmonelladetection in feces should be useful not only for routine screening ofSalmonellashedding in poultry, but also forSalmonellatesting of stool from human patients and food handlers.     

Response of two Salmonella enterica strains inoculated in model cheese treated with high hydrostatic pressure

The aim of this work was to determine the response to high hydrostatic pressure and the ability for survival, recovery, and growth of 2 strains of Salmonella enterica (Salmonella enteritidis and Salmonella typhimurium) inoculated in a washed-curd model cheese produced with and without starter culture. Inoculated samples were treated at 300 and 400 MPa for 10 min at room temperature and analyzed after treatment and after 1, 7, and 15 d of storage at 12° C to study the behavior of the Salmonella population. Cheese samples produced with starter culture and treated at 300 and 400 MPa showed maximum lethality; no significant differences in the baroresistant behavior of both strains were detected. Nevertheless, when starter culture was not present, the maximum lethality was only observed in cheese samples treated at 400 MPa, in the case of S. enteritidis. Ability to repair and grow was not observed in model cheese produced with starter culture and cell counts of treated samples decreased after 15 d of storage at 12° C. In cheese produced without starter culture, Salmonella cells showed the ability to repair and grow during the storage period, reaching counts over 3 log₁₀ (cfu/mL) in both applied treatments and serotypes. These results suggest that high hydrostatic pressure treatments are effective to reduce Salmonella population in this type of cheese, but the presence of the starter culture affects the ability of this microorganism to repair and grow during the storage period.     

Optimization of a rapid dot-blot immunoassay for detection of Salmonella enterica serovar Enteritidis in poultry products and environmental samples

An immunoassay was developed for the detection of Salmonella serovar Enteritidis in poultry and environmental samples. This assay consisted of a two-step procedure that involved an enrichment step using whole egg homogenate (EH) as the enrichment medium and detection by a monoclonal antibody (MAb)-based dot-blot assay. Egg homogenate enriched Salmonella Enteritidis was heated to 100°C for 10 min in the presence of cholic acid, a detergent, to liberate the lipopolysaccharide (LPS) antigen in gelled egg matrix. This was subsequently transferred onto a nitrocellulose membrane for detection with MAb 2F11. Several commercially available media were compared with egg homogenate for their relative ability to resuscitate and propagate Salmonella Enteritidis to detectable levels. Incubation in EH, trypticase soy broth (TSB), and lactose broth (LB) resulted in comparable levels of Salmonella Enteritidis as demonstrated by viable plate counts. Salmonella Enteritidis grown in TSB exhibited the greatest visual intensity showing a positive test when tested by the dot-blot assay. Incubation time necessary to detect one cfu of Salmonella Enteritidis was reduced from 20 to 10 h using TSB as the enrichment broth. Addition of ferrous sulphate or ferrioxamine E or cholic acid in the enrichment broth had negligible negative effects on the growth of Salmonella. Salmonella Enteritidis when incubated with a mixture of naturally contaminated or artificially inoculated competitive micro-organisms in environmental samples at a ratio of 1:10², was able to reproduce to detectable numbers for the immunoassay. This method was able to detect all phage types (PT 1, 6, 7, 8, 13, 13a, 14b, 21 and 28) with unique ribopatterns. The results demonstrated that Salmonella Enteritidis, when preenriched in a medium containing ferrous sulphate or cholic acid, could be readily detected in the presence of 100-fold higher competition of other micro-organisms.     

Interactions between Lactococcus lactis and Streptococcus thermophilus strains in Cheddar cheese processing conditions

The growth of pure and mixed cultures of Lactococcus lactis and Streptococcus thermophilus under simulated Cheddar cheese manufacture was examined. Cell-free wheys (CFW) of the cultures were prepared for analysis by automated spectrophotometry (AS). The maximal growth rate of the lactococci in S. thermophilus R0083 CFW was 13% higher than that noted in their own CFW and three lactococci also gave higher biomass levels (OD_max). During simulated Cheddar cheese fermentations with four paired cultures, one L. lactis strain grew 20% less when paired with S. thermophilus R0083, and an increase in colony forming units (cfu) was found with one other lactococcal strain. Viable counts of S. thermophilus in mixed cultures varied by less than 0.1 log cfu mL^?1. The AS data on OD_max in CFW were useful in predicting the evolution of cfu in the fermented mixed cultures. As a function of strain, the presence of S. thermophilus in a Cheddar fermentation process can enable extended growth of the lactococci.     

Effect of galactose metabolising and non-metabolising strains of Streptococcus thermophilus as a starter culture adjunct on the properties of Cheddar cheese made with low or high pH at whey drainage

Cheddar cheese was made using control culture (Lactococcus lactis subsp. lactis), or with control culture plus a galactose-metabolising (Gal⁺) or galactose-non-metabolising (Gal⁻) Streptococcus thermophilus adjunct; for each culture type, the pH at whey drainage was either low (pH 6.15) or high (pH 6.45). S. thermophilus affected the levels of residual lactose and galactose, and the volatile compound profile and sensory properties of the mature cheese (270 d) to an extent dependent on the drain pH and phenotype (Gal⁺ or Gal⁻). For all culture systems, reducing drain pH resulted in lower levels of moisture and lactic acid, a higher concentration of free amino acids, and higher firmness. The results indicate that S. thermophilus may be used to diversify the sensory properties of Cheddar cheese, for example from a fruity buttery odour and creamy flavour to a more acid taste, rancid odour, and a sweaty cheese flavour at high drain pH.     

MODELLING of TIME-TEMPERATURE EFFECTS ON BACTERIA POPULATIONS DURING COOLING of CHEDDAR CHEESE BLOCKS1

Differences in cooling rate of Cheddar cheese from pressing (35C) to aging temperature (3.5–12C) has been reported to be responsible for flavor variation within a production lot. During aging, starter and nonstarter bacteria contribute extensively to flavor quality. Temperature effects on these bacteria were quantified using cheese from a local processor. At day 1, starter counts were 8 × 10⁷ cfu/g but as aging continued, starter counts decreased and non-starters became dominant. At 35C, starter counts reached 3 × 10⁶ cfu/g by day 3 and were below 10⁶ cfu/g by day 5. At 25, 20, 15 and 12C, starter bacteria were below 10⁶ cfu/g by day 10, 20, 24 and 40, respectively. Nonstarter counts, initially at 10⁴ cfu/g, reached = 10⁸ cfu/g at increasingly shorter times with higher temperatures. Kinetic analysis of growth in cheese and in a liquid medium suggested the possibility of diffusion growth limitations in cheese. Computer simulations for the growth of nonstarters suggests the individual cooling of small blocks (18 kg) would reduce the contribution of nonstarter counts to Cheddar cheese aging.     

An improved method for the recovery of Salmonella serovars from orange juice using universal preenrichment broth

The relative effectiveness of three methods for the recovery of Salmonella serovars from orange juice was determined. One method, a modified Bacteriological Analytical Manual (BAM) procedure consisted of preenrichment in lactose broth at 35 degrees C for 24 h, selective enrichment, and selective plating. Another method, a National Centers for Disease Control and Prevention (CDC 1) procedure, consisted of direct enrichment in tetrathionate broth at 35 degrees C for 24 and 48 h, followed by selective plating. The third method (also from CDC and designated CDC 2) consisted of preenrichment in Universal Preenrichment (UP) broth at 35 degrees C for 24 h, selective enrichment, and selective plating. In 10 experiments encompassing five different Salmonella serovars and 200 test portions per broth, the CDC 1 method recovered 141 Salmonella-positive test portions, the BAM method recovered 151, and the CDC 2 method recovered 171. In 2 of the 10 experiments, with two different Salmonella serovars, the BAM recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did the CDC 2 method. On the basis of the above results, the second phase of this study focused on a comparison of the effectiveness of the BAM-recommended lactose broth and the CDC 2-recommended UP broth as preenrichment media for the recovery of Salmonella serovars from pasteurized and unpasteurized orange juice. Subsequent culture treatment of the two preenrichments was identical so that the effect of other variables (e.g., different selective enrichment media, various incubation temperatures, and different selective plating agars) on the relative performance of these two preenrichment media was excluded. In one of nine experiments, with pasteurized orange juice, lactose broth recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did UP broth. For the combined results of the nine pasteurized orange juice experiments (180 test portions per broth), lactose broth recovered 99 Salmonella-positive test portions, and UP broth recovered 116. In three of seven experiments, with unpasteurized orange juice, lactose broth recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did UP broth. For the combined results of the seven unpasteurized orange juice experiments (140 test portions per broth), lactose broth recovered 73 Salmonella-positive test portions, and UP broth recovered 117. For both pasteurized and unpasteurized orange juice, the total number of Salmonella-positive test portions recovered with UP broth was significantly greater than the number recovered with lactose broth. These results indicate that UP broth is a more effective enrichment broth for the recovery of Salmonella from orange juice than is lactose broth.     

Detection of Salmonella enteritidis and Salmonella typhimurium in foods using a rapid,multiplex real-time recombinase polymerase amplification assay

Salmonella has been recognized as a major foodborne pathogen for humans and animals. In this study, a multiplex real-time recombinase polymerase amplification (RPA) was developed for simultaneous detection of Salmonella enterica serovars, Salmonella enteritidis and Salmonella typhimurium, from chicken, eggs, lettuce, and papaya. The reaction was performed for 20 min at 35°C, and the detection limit of the assay was 10² CFU/ml for pure culture. In food application, the limit of detection (LOD) of S. enteritidis and S. typhimurium using multiplex real-time RPA without enrichment procedure was 10² CFU/25 g, respectively. After enrichment, the LOD of S. enteritidis and S. typhimurium was 10 CFU/25 g. Moreover, the result for Salmonella spp. was not significantly different from those obtained using a culture-based method. Additionally, the assay has a lower cross-reactivity with other pathogenic microorganisms and a good stability performance. Thus, the developed multiplex RPA assay could be used as a rapid tool for the detection of S. enteritidis and S. typhimurium in food.     

Effect of sample preparation and bacterial concentration on Salmonella enterica detection in poultry meat using culture methods and PCR assaying of preenrichment broths

We evaluated the sensitivity of a PCR assay in the detection of Salmonella enterica at the broth preenrichment step of poultry meat. A total of 162 retail poultry meat samples, which were prepared by manual massaging, stomacher or no homogenization were compared for Salmonella recovery. Using these homogenization methods, the PCR assay at the broth preenrichment step detected Salmonella in, respectively, 48.9%, 62.2% and 50.0% of meat and giblet samples detected as Salmonella-positive using the culture method. In ground chicken, however, Salmonella was detected in 21.7% of samples treated by stomacher homogenization, compared to 40.7% and 48% of untreated and hand-massaged samples, respectively. These results suggest that stomaching of ground chicken causes excessive effusion of food constituents, which affects PCR results. Using the most probable number (MPN) technique, Salmonella was detected at under 1.0 CFU/g in 12 ground chicken samples and under 10³ CFU/ml of broth in seven of the 12 broth-enriched samples, which considered the minimum concentration detectable by PCR assay. These results show that Salmonella detection using routine PCR assays is difficult in poultry meat, and in particular ground chicken, due to low amounts of Salmonella and the presence of inhibitors.     

Enhanced nutty flavor formation in cheddar cheese made with a malty Lactococcus lactis adjunct culture

Nutty flavor in Cheddar cheese is desirable, and recent research demonstrated that 2- and 3-methyl butanal and 2-methyl propanal were primary sources of nutty flavors in Cheddar. Because malty strains of Lac-tococcus lactis (formerly Streptococcus lactis var. malti-genes) are characterized by the efficient production of these and other Strecker aldehydes during growth, this study investigated the influence of a malty L. lactis adjunct culture on nutty flavor development in Cheddar cheese. Cheeses made with different adjunct levels (0, 10⁴ cfu/mL, and 10⁵ cfu/mL) were ripened at 5 or 13°C and analyzed after 1 wk, 4 mo, and 8 mo by a combination of instrumental and sensory methods to characterize nutty flavor development. Cheeses ripened at 13°C developed aged flavors (brothy, sulfur, and nutty fla-vors) more rapidly than cheeses held at 5°C. Additionally, cheeses made with the adjunct culture showed more rapid and more intense nutty flavor development than control cheeses. Cheeses that had higher intensities of nutty flavors also had a higher concentration of 2/3-methyl butanal and 2-methyl propanal compared with control cheeses, which again confirmed that these compounds are a source of nutty flavor in Cheddar cheese. Results from this study provide a simple methodology for cheese manufacturers to obtain consistent nutty flavor in Cheddar cheese.     

Galactose-positive adjunct cultures prevent gas formation by Paucilactobacillus wasatchensis WDC04 in a model gas production test

Gas production by obligatory heterofermentative lactic acid bacteria such as Paucilactobacillus wasatchensis is a sporadic problem in Cheddar cheese and results in undesired slits and cracks in the cheese. Growth of Pa. wasatchensis is not rapid, which makes investigations of gas production difficult to consistently execute. A primary objective of this study was to develop a model gas production test that could be used to investigate the effect of galactose and ribose utilization on gas production by Pa. wasatchensis and determine whether galactose-fermenting adjunct cultures could prevent gas formation. Paucilactobacillus wasatchensis WDC04 was inoculated at 10¹ to 10⁶ cfu/mL into carbohydrate-restricted MRS broth containing different ribose and galactose levels and incubated for up to 21 d at 23°C. Gas production in the broth was detected using a Durham tube inverted on a 6-cm-long capillary tube; cells were enumerated at 4, 8, and 12 d; and residual galactose was also measured. Gas production was sporadic except for when 10⁵ cfu/mL of Pa. wasatchensis WDC04 was inoculated into broth containing 0.3% ribose and 0.7% galactose. In those tubes, gas production was consistently observed after 8-d incubation, by which time galactose levels had decreased to 0.15%. Co-inoculation of Pa. wasatchensis WDC04 with as few as 10³ cfu/mL of a lactose-negative galactose-positive adjunct culture (Pediococcus acidilactici 23F, Lacticaseibacillus paracasei UW4, or Lactobacillus helveticus 7995) resulted in galactose depletion by d 4 and no observable gas production by d 12. With less galactose available to the slower-growing Pa. wasatchensis WDC04, its growth was limited to 10⁸ cfu/mL when any of the adjunct cultures was co-inoculated, compared with 10⁹ cfu/mL when grown on its own. We concluded that galactose-fermenting adjunct cultures have potential for preventing unwanted gas production in cheese by competition for resources and especially by removing the 6-carbon galactose before it can be utilized for energy by an obligatory heterofermentative lactobacilli such as Pa. wasatchensis and produce carbon dioxide.     

Growth kinetics of Salmonella in mixed cultures incubated in Rappaport–Vassiliadis medium

Reproducible numbers ofSalmonella enteritidis,Klebsiella pneumoniaeandStaphylococcus aureuswere mixed in various proportions and their growth in Rappaport–Vassiliadis enrichment broth quantified at 4 h intervals over a 24-h period. No limit in the ability of the RV medium to detectSalmonellain the presence of low or high numbers of competing organisms could be observed. After incubation salmonellae were found in all mixtures regardless of the composition of the mixtures.