Leora A. Shelef

Enhanced antimicrobial effects of combination of lactate and diacetate on Listeria monocytogenes and Salmonella spp. in beef bologna

The antimicrobial activities of salts of organic acids such as lactate and acetate are well documented, but there is limited information on their effect when used in combination. We previously reported enhanced inhibition of Listeria monocvtogenes and Salmonella enterica serovar Enteritidis in sterile comminuted beef at 5 and 10 degrees C by combinations of sodium lactate (SL) (2.5%) and sodium diacetate (SDA) (0.2%). The present study was undertaken to evaluate the inhibitory effect of these salts, alone and in combination, in ready-to-eat (RTE) meat. Single strains and six-strain mixtures of each of the pathogens ( approximately 3 log CFU/g) were tested in beef bologna during aerobic storage at 5 and 10 degrees C for up to 60 days. The growth rate of the six-strain mixture of Listeria was faster than that of the single strain (Scott A) in the lactate/diacetate-free product. While each of the salts delayed growth of the listeriae at 5 degrees C, the effect of their combination was listericidal for the single strain and listeriostatic for the six-strain mixture. Enhanced inhibition by the salt combination was also observed at 10 degrees C. Salmonella numbers declined to undetectable levels in the untreated meat product and in each of the treatments after 20-30 days. However, the decline was more rapid in meat with the combination of the salts during storage at both 5 and 10 degrees C. Each of the salts further delayed the growth of the background microflora during storage at 5 degrees C, with their combinations showing the most effect.

Enhanced inhibition of Listeria monocytogenes and salmonella enteritidis in meat by combinations of sodium lactate and diacetate.

The antimicrobial activities of sodium lactate (SL) and sodium acetate (SA) are well documented, but there is limited information on the effect of their combination or of the combination of SL and sodium diacetate (SDA) on survival and growth of Listeria monocytogenes and salmonellae in meat. Effects of SL (1.8 and 2.5%), SDA (0.1 and 0.2%), or SA (0.2%) and their combinations on the behavior of L monocytogenes and Salmonella enterica serovar Enteritidis were investigated in sterile comminuted beef (pH 6.3, 79% moisture) during storage at 5 and 10 degrees C. Although L. monocytogenes grew faster than Salmonella Enteritidis in control samples at 10 degrees C, numbers of both pathogens increased from 3.5 to approximately 8.0 log CFU/g after 20 days. SL (1.8%) decreased the growth rate of both L. monocytogenes and Salmonella Enteritidis. SDA (0.2%) was more effective than SL in decreasing the growth rate of L monocytogenes, and it caused a more than 1 log CFU/g decline in initial numbers of Salmonella Enteritidis during storage for 25 days at 10 degrees C. Synergy was observed by combinations of SL and SDA. Combinations of 2.5% SL and 0.2% SDA were bacteriostatic to L. monocytogenes and bactericidal to Salmonella Enteritidis after 20 days at 10 degrees C. At 5 degrees C, a listeriostatic effect was produced by 1.8% SL + 0.1% SDA, whereas numbers of Salmonella Enteritidis were less than 10 cells/g after refrigeration for 30 days. Although SA was consistently and significantly less inhibitory than SDA, its mixtures with SL also demonstrated synergistic activity against both pathogens. Combinations of 2.5% SL and 0.2% SDA can be expected to greatly enhance the safety of refrigerated and temperature-abused ready-to-eat meats.

Sensitivity of six strains of Listeria monocytogenes to nisin

The sensitivity of six strains of Listeria monocytogenes (serotype 1, 1a, 2, 4a, 4b, and a nonhemolytic strain) to nisin was compared at pH 5.0, 6.0, and 7.0. The effects of 240 and 600 IU/ml of nisin in broth on 103, 104, 106, and 108 CFU/ml of each of the strains were tested, and cell numbers were determined after incubation for 30 min, 60 min, and 24 h at 35°C. Although populations decreased after a contact time of 30 min, survivors of 240 IU of nisin per ml reached high numbers after 24 h. Similar results were obtained for 600 IU of nisin per ml but there were no survivors after 24 h of incubation when inocula of less than 104 CFU/ml were tested irrespective of the strain. The sensitivity of L. monocytogenes to nisin at each pH was strain dependent, and it was enhanced at the low pH.

Behaviour of foodborne pathogens in cooked liver sausage containing lactates

Previous reports from our laboratory showed sensitivity of Listeria monocytogenes to lactates in cooked pork liver sausage. Further studies were undertaken to explain the antilisterial effects and to examine the sensitivity of Escherichia coli O157:H7 and Salmonella typhimurium strains to lactates. To determine the effect of heat treatment on the antilisterial activity during storage at 5 and 20°C, 3% sodium or calcium lactate (NaL or CaL) were added to sausage emulsion, followed by (1) sterilization (121°C, 15 min); (2) processing in a water bath to internal temperature of 70°C; or (3) added to ready-to-eat sausage (no additional heat treatment). Inhibition of L. monocytogenes strain Scott A was highest after heat sterilization and storage at 5°C. Growth of S. typhimurium and E. coli O157:H7 was abated in sterilized sausage by 3% NaL and CaL. Changes in log cfu g −1 after 4 days at 20°C were (1) L. monocytogenes : control, 5·96; 3% NaL, −0·17; 3% CaL, −0·77; (2) E. coli : control, 4·01; NaL, 0·34; CaL, −0·06; (3) S. typhimurium : control, 3·92; NaL, 1·14; CaL, 0·92. While CaL was consistently more effective than NaL, examination of other calcium salts (carbonate, chloride, citrate and phosphate, dibasic) confirmed that lactate is the principal antibacterial factor.

Automated simultaneous detection of low levels of listeriae and salmonellae in foods.

Salmonella spp. and Listeria monocytogenes continue to be major pathogens of concern to food processors. However, routine screening of food samples to detect these pathogens is generally labor intensive and costly. Automated optical procedures for the detection of salmonellae and listeriae in foods were developed in our laboratory. In the present study we report their adaptation to a simultaneous recovery and detection procedure. Milk, shell eggs, fresh and ready-to-eat (RTE) meats or raw chicken contaminated with a combination of sub-lethally injured salmonellae and listeriae (10-50 cells each) were incubated for 6 h at 35 degrees C in modified universal pre-enrichment broth (MUPB). Volumes (4 ml) were then transferred to vials containing selective liquid media for these pathogens (4 ml), and incubated overnight at 35 degrees C in a BioSys instrument. The presence of the pathogens was identified by a black coloration of the media and a sharp drop in light transmittance caused by hydrogen sulfide production (Salmonella organisms), or esculin hydrolysis (Listeria organisms). There was no difference in the detection time of salmonellae when incubated alone or with listeriae, but listeriae grew at a slower rate in the presence of salmonellae, resulting in a delay of < or = 1 h in their detection. Overall, the detection of 10-50 salmonellae and 10-50 listeriae in 25 g of the tested foods required a total of 24 h. Confirmation of the pathogens by PCR-based assay (6 h) was completed the following day directly from positive vials, requiring a total of < or = 30 h for detection and confirmation. Negative samples required no confirmation. The testing system was confirmed in 70 naturally contaminated foods.

Rapid Optical Measurements of Microbial Contamination in Raw Ground Beef and Effects of Citrate and Lactate

The levels of microbial contamination in purchased pre-wrapped fresh ground beef samples were estimated by using the BioSys™ automated optical system for rapid detection of the presence of microorganisms. Additionally, the response of the system to the activity of antimicrobial agents in meat was evaluated after adding (separately) the sodium salts of lactic and citric acid to meat samples. Meat homogenates in peptone water were incubated at 30°C in broth containing the indicators bromcresol purple (pH) or resazurin (redox potential). Curves (light transmittance versus time) generated by the instrument showed significant and rapid changes in the indicator color, and times of detection of the initial changes were inversely proportional to the standard plate counts in the meat samples. This relationship indicated that the detection time can provide a good estimate of the microbial quality of fresh meat. Treatment of meat with 2% citrate or 2% lactate extended the shelf life by about 2 and 4 days. respectively. Detection time values confirmed the ability of the salts to delay the microbial spoilage of meat.

A new rapid automated method for the detection of Listeria from environmental swabs and sponges.

Many food and meat processors test environmental swabs and sponges to confirm the absence of Listeria spp. Spectral pattern changes in a liquid growth medium, resulting from esculin hydrolysis by Listeria in contaminated swabs and sponges, were automatically monitored by the BioSys instrument in a semifluid layer (SFL). The blackening of SFL in modified MOX broth resulted in sharply declining curves, which were easily detected by the instrument. The instrument detected all nine strains of Listeria monocytogenes tested. None of the gram negative organisms (Proteus, Escherichia coli, Pseudomonas, Citrobacter and Yersinia) were detected by the system, nor were most gram positive organisms, including Bacillus, Streptococcus, and Lactobacillus strains, Staphylococcus aureus, Enterococcus faecium and E. faecalis, which hydrolyze esculin, produced black colonies on PALCAM and Oxford media and were also detected in the system. A total of 122 sponges and swabs collected at food processing plants were evaluated by this method. Of these, 99 were negative, and 11 were positive. L. innocua was the dominant Listeria species in these environmental samples. Good correlation was obtained between numbers of Listeria and detection times of esculin hydrolysis: 1000 CFU/swab were detected in 10-13 h, whereas 1-10 CFU/swab were detected in less than 22 h. The total assay time was 26 h.

Automated detection of Salmonella spp. in foods

An automated method to detect salmonellae in foods was developed and tested in food samples intentionally contaminated with the test organisms. Liquid eggs, shell eggs, dry eggs, skim milk and chicken were spiked with Salmonella enteritidis, S. typhimurium or S. newport to yield 2 to 25 CFU per 25 g or ml of sample. Following pre-enrichment in universal pre-enrichment broth at 42 degrees C for 6 h (eggs and milk) or 16 h (chicken), Salmonella cells were captured by immunomagnetic beads coated with Salmonella antibody (Vicam, Watertown, MA). The beads were transferred to selective liquid media containing carbohydrate (dulcitol or xylose), amino acid (lysine or ornithine), and H2S indicator, and incubated at 42 degrees C in the BioSys instrument (MicroSys, Ann Arbor, MI). Salmonella positive samples were identified by black discoloration of the media during incubation, while negative samples remained colorless. These color changes were recorded by the instrument. All the artificially contaminated samples tested positive within 15-18 h, while control samples remained negative during 24 h incubation. The results agreed with standard identification procedures. A total of 24 h was required to detect 2 to 25 CFU of the pathogen in 25 g or ml of eggs and milk, and up to 36 h in chicken, compared to 72 h in the standard methods.

Rapid detection of low levels of Listeria in foods and next-day confirmation of L. monocytogenes

Outbreaks of foodborne listeriosis caused by Listeria monocytogenes in recent years, and the high mortality rate associated with listeriosis, have raised the need for reliable and rapid detection of the pathogen. A simple, automated method was developed for the detection of Listeria organisms in foods. It consists of a 6-h pre-enrichment step followed by overnight incubation in selective broth at 35 degrees C. Changes in light transmittance in the selective broth are registered continuously by an optical sensor of the BioSys instrument (MicroSys, Ann Arbor, MI), and recorded in the computer. Esculin hydrolysis by listeriae results in black coloration of the media that causes a sharp drop in light transmittance, whereas negative samples remain colorless. Confirmation of L. monocytogenes is carried out only on esculin-positive samples and is completed within 6 h. Detection of 10-50 cells of Listeria inoculated into 25 g of food was confirmed in shell eggs, milk and ground beef. Naturally contaminated raw and ready-to-eat foods were further screened to validate the procedure.

Novel selective and non‐selective optical detection of micro‐organisms

A new instrument, capable of detecting metabolic changes due to microbiological activity, is described. Optical changes in growth media are monitored in a semi‐fluid zone that separates the liquid medium containing the sample. Data demonstrate that common media can be utilized in conjunction with this rapid automated technology. Nutrient broth with the pH dye indicator bromocresol purple was suitable for total counts. Selective media containing dyes were utilized to assess the presence or absence of specific groups of organisms. Biochemical reactions, such as lysine decarboxylase activity, were identified by the unique generated patterns, and specific enzymatic cleavage reactions with chromogenic substrates, such as 5‐bromo‐4 chloro‐3 indolyl‐β‐D‐glucuronic acid (X‐GLUC), were monitored.