Growth and adherence on stainless steel by Enterococcus faecium cells.

Enterococcus faecium isolated from Brazilian raw milk was used in this study. For growth studies, E. faecium was inoculated into 10% RSM (reconstituted skim milk) and MRS both, incubated at 6.5 and 9 degrees C for 10 days and at 30, 42, and 45 degrees C for 48 h. Cells were enumerated after spread-plating onto MRS agar and incubating at 30 degrees C for 48 h. The ability of E. faecium cells to adhere to stainless-steel chips (6 by 6 by 1 mm, AISI 304, finish #4) was investigated. MRS broth containing stainless steel chips was inoculated to an initial concentration of 10(3) or 10(6) CFU/ml of E. faecium. Adherent cells were stained with acridine orange and enumerated by epifluorescence microscopy. E. faecium grew between 6.5 and 42 degrees C in MRS and between 9 and 40 degrees C in RSM. In MRS broth with 10(6) or 10(3) CFU/ml, the g (generation time) values were 0.62 and 0.42 h and R (growth rate) values were 1.6 and 2.4 h-1. Values of R = 2.3 h-1 and g = 0.43 h were determined for E. faecium growing in RSM with 10(3) CFU/ml. In MRS broth, for samples with a starting concentration of 10(6) cells per ml, adherence to stainless-steel chips was first observed at 2 h. However, adherence was first observed at 4 h in samples with an initial concentration of 10(3) cells per ml. After 10 h of exposure the number of adherent cells was similar for all samples regardless of initial inoculum. These results indicate that E. faecium readily adheres to stainless steel. It also underscores the need to control E. faecium by using appropriate low storage temperatures and adequate sanitizing practices in the dairy industry.

Bacteriocidal activity of sanitizers against Enterococcus faecium attached to stainless steel as determined by plate count and impedance methods.

Enterococcus faecium attached to stainless steel chips (100 mm2) was treated with the following sanitizers: sodium hypochlorite, peracetic acid (PA), peracetic acid plus an organic acid (PAS), quaternary ammonium, organic acid, and anionic acid. The effectiveness of sanitizer solutions on planktonic cells (not attached) was evaluated by the Association of Official Analytical Chemists (AOAC) suspension test. The number of attached cells was determined by impedance measurement and plate count method after vortexing. The decimal reduction (DR) in numbers of the E. faecium population was determined for the three methods and was analyzed by analysis of variance (P < 0.05) using Statview software. The adhered cells were more resistant (P < 0.05) than nonadherent cells. The DR averages for all of the sanitizers for 30 s of exposure were 6.4, 2.2, and 2.5 for the AOAC suspension test, plate count method after vortexing, and impedance measurement, respectively. Plate count and impedance methods showed a difference (P < 0.05) after 30 s of sanitizer exposure but not after 2 min. The impedance measurement was the best method to measure adherent cells. Impedance measurement required the development of a quadratic regression. The equation developed from 82 samples is as follows: log CFU/chip = 0.2385T2-0.96T + 9.35, r2 = 0.92, P < 0.05, T = impedance detection time in hours. This method showed that the sanitizers PAS and PA were more effective against E. faecium than the other sanitizers. At 30 s, the impedance method recovered about 25 times more cells than the plate count method after vortexing. These data suggest that impedance measurement is the method of choice when evaluating the number of bacterial cells adhered to a surface.

Adherence to stainless steel by foodborne microorganisms during growth in model food systems.

Biofilm formation on stainless steel by Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7, Pseudomonas fragi and Pseudomonas fluorescens during growth in model food systems was studied. Test growth media included tryptic soy broth (TSB), diluted TSB (dTSB), 1% reconstituted skim milk (RSM) and diluted meat juice (DMJ). Adherent cells were stained with acridine orange and enumerated using epifluorescent microscopy and computerized image analysis. Cells were observed on the stainless steel surface after 1 h in all of the media. However, the increases in the number of adherent cells over time was seen only with S. typhimurium in DMJ, E. coli O157:H7 in TSB, dTSB and DMJ, P. fragi in RSM and P. fluorescens in RSM. The medium which produced the highest observed level of adherent cells was different for each microorganism.

Isolation and identification of antimicrobial furocoumarins from parsley.

Photoactive furocoumarins extracted from four varieties of fresh and freeze-dried parsley leaves inhibited a DNA repair-deficient Escherichia coli in a photobiological assay. Using media-modified assays, the human pathogens E. coli O157:H7 and Listeria monocytogenes, the spoilage microorganism Erwinia carotovora, and Listeria innocua were also inhibited. Pseudomonas fragi was not inhibited. Minimum concentrations of Forest Green parsley powder in agar which showed inhibition ranged from 0.12% to 8.0% depending on the microorganism. Ultraviolet light (UV) at 365 nm for 60 min used to photoactivate the furocoumarins in the bioassay had little effect on L. monocytogenes and L. innocua. A slight UV inhibitory effect was detected with E. carotovora. Furocoumarins, psoralen, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), oxypeucedanin and isopimpinellin were identified using gas chromatography-mass spectrometry. Psoralen, 8-MOP, and 5-MOP were quantified. A difference in relative furocoumarin concentration (weight of furocoumarin per weight of dry parsley leaves) for all varieties of parsley was revealed. The concentration of 5-MOP was significantly greater than 8-MOP (P < 0.05), but not significantly greater than psoralen. Psoralen and 8-MOP were not significantly different in concentration.

Recovery of thermally-stressed Escherichia coli O157:H7 by media supplemented with pyruvate.

Unheated and heat-stressed (57 degrees C, 50 min and 60 min) cells of Escherichia coli O157:H7, were enumerated using three media supplemented with 1% sodium pyruvate (NaPyr): plate count agar (PCA), tryptic soy agar (TSA) and phenol red sorbitol agar (PhRSA) using the spread plate method. The medium recovering the greatest numbers of severely heated E. coli O157:H7 was PCA with 1% NaPyr. Recovery of heat stressed E. coli O157:H7 on this medium was significantly higher (P < 0.05) than the two other media with pyruvate: 16.3% (50 min heating) and 0.55% (60 min heating) of the total population was recovered with TSA + 1% NaPyr when compared to those numbers found on PCA + 1% NaPyr. The ability of PhRSA + 1% NaPyr to recover heat-stressed E. coli O157:H7 was similar to that of TSA + 1% NaPyr. Using PhRSA + 1% NaPyr media. 12.9% (50 min heating) and 0.61% (60 min heating) of the total population were recovered when compared with the cells enumerated on PCA + 1% NaPyr. Recovery of the heat-stressed cells using the spread plate method was greater than using pour plate method. Recovery was significantly higher (P < 0.05) on the spread plates for highly stressed E. coli O157:H7(1.2 log) heated for 60 min than on the pour plates. Overall, the populations on the TSA spread and pour plates were low compared with the same heat-stressed cells recovered on media containing pyruvate. The

Use of Nisin in Ice Cream and Effect on the Survival of Listeria monocytogenes †.

The survival characteristics of Listeria monocytogenes V7 were investigated in a full fat (10% fat) and reduced fat (3%) ice cream. The effect of nisin on the survival of Listeria monocytogenes in the ice creams was also evaluated. Ice cream mixes varying in composition were manufactured and inoculated with L. monocytogenes , passed through a "soft serve" freezer and then frozen at -18°C for up to 3 months. Samples were removed from storage throughout the three months, thawed, and then plated on Listeria -selective agar. In the samples that did not contain nisin, no reduction in the cell population was observed throughout manufacture and frozen storage. When nisin was present in the ice cream, a significant reduction in the cell population (P < .05) was observed. At the end of 3 months of frozen storage, no Listeria cells were detected in the 3% fat ice cream containing nisin. The effect of nisin on Listeria cells was decreased somewhat in the higher fat ice cream but this decrease was not significant over the 3 month storage. The stability of nisin in the ice cream remained constant throughout storage at -18°C.

Utilization of cheddar cheese containing nisin as an antimicrobial agent in other foods.

Cheddar cheese made with nisin-producing lactococci contained between 400 and 1200 IU of nisin per gram of cheese. Cultures used were Lactococcus lactis ssp. cremoris JS102, a nisin-producing transconjugant developed in the laboratories of Dr. L.L. McKay and Lactococcus lactis ssp. lactis NCDO 1404 obtained from the National Collection of Food Bacteria, Reading, England. Pasteurized process cheese spreads with 53% and 60% moisture and 0, 301 and 387 IU nisin/g were manufactured and inoculated with 2000 spores of Clostridium sporogenes PA 3679 during manufacture. The heat process did not reduce nisin activity in the cheese spreads. The spreads were incubated at 22 degrees and 37 degrees C for 90 days. Spoilage was detected by the presence of gas and/or odor in the packages. The shelf-life of the nisin-containing cheese spreads was significantly greater than that of the control cheese spreads at the lower temperature at both moisture levels, whereas the keeping quality of the higher moisture cheeses at the higher temperature was not significantly different. Club cheese or cold pack cheese spreads with moisture levels of 44% and 60% and 0, 100 and 300 IU nisin/g were made. These cold processed cheese spreads were inoculated with 1000 cfu per g of Listeria monocytogenes V7, Staphylococcus aureus 196E and spores of C. sporogenes PA 3679. Heat shocked spores of PA 3769 at the same number were added to separate lots of the cheese spread. The cold pack cheese spreads were incubated at 23 degrees and 37 degrees C for up to 8 weeks. Samples were taken weekly and analyzed for surviving organisms. Significant reductions in numbers of the non-sporeforming test microbes were noted at both temperatures, at both moisture levels and both levels of nisin. Heat shocking the spores was needed to show reduction in numbers during the storage of the cold pack cheese spreads. The data obtained in this study suggest that the use of nisin-containing cheese as an ingredient in pasteurized process cheese or cold pack cheese spreads could be an effective method of controlling the growth of undesirable microorganisms in these processed foods.

Microbial biofilms in the food processing industry--should they be a concern?

Biofilm formation will occur on solid surfaces in contact with a liquid. Organic and inorganic material in the liquid sediment onto the solid material. Subsequently, biologically active microorganisms will be attracted to this conditioned surface and adhere to it. The microbial cells will initiate growth, form an attachment matrix and develop into a complex community forming a microbial biofilm. Such microbial biofilms are common on solid surfaces in contact with many different kinds of liquids, fresh water, sea water, oil, milk and so on. These biofilms may be of benefit or be detrimental to the environment where they form. The goal of this review has been to summarize the literature on the development of microbial biofilms in these different environments with particular emphasis on what occurs in the environment of a food processing plant. Methods to control adherent microorganisms and subsequent biofilms in the food processing plant are discussed. It is apparent from the data that has been reviewed that the potential for the development of microbial biofilms in the environment of the food processing plant exists. However, the cleaning and sanitizing practices carried out in the food industry have been shown to control biofilm formation on food contact surfaces. Microbial attachment has been shown to occur on non-food contact surfaces and these attached microbes, if left undisturbed, will form biofilms. The potential for contamination of food with undesirable spoilage and pathogenic bacteria from attached microbes and biofilms exists in these food processing systems. Biofilm formation on non-food contact surfaces needs to be studied further and methods developed to prevent and control these biofilms.

Shelf-life of pasteurized process cheese spreads made from cheddar cheese manufactured with a nisin-producing starter culture.

Cheddar cheese made with a nisin-producing starter culture and Cheddar cheese made with a commercially available starter culture were used to manufacture pasteurized process cheese spreads at low and high moisture percentages (53 and 60%, respectively). Composition did not differ between spreads of similar moisture content with and without nisin. The nisin contents of cheese spreads were 301 and 387 IU/g at the high and low moisture percentages, respectively. Nisin was not inactivated by the thermal process used during cheese spread manufacture. Shelf-life of pasteurized process cheese spreads was determined during storage at 22 and 37 degrees C. Low moisture cheese spreads with nisin had a longer shelf-life than corresponding cheese spreads without nisin when cheeses were incubated at either temperature. High moisture cheese spreads with nisin had a longer shelf-life than control spreads when cheeses were incubated at 22 degrees C. However, shelf-life did not differ between high moisture spread with nisin and cheese spreads without nisin when cheeses were incubated at 37 degrees C.

Bacterial Survival on Cornstarch-Containing Polyethylene Film Held Under Food Storage Conditions.

Plastics in which cornstarch is incorporated into the polymer network have been developed. The effect of cornstarch in plastic film on the survival of spoilage and pathogenic bacteria was evaluated. Cornstarch-containing polyethylene film (CSPE) and control polyethylene film (PE) were inoculated with Salmonella typhimurium , Aeromonas hydrophila , Bacillus cereus , and Pseudomonas fragi and held under various combinations of temperature and relative humidity to mimic food storage conditions. Bacterial recovery from film samples indicated that, in general, survival was not enhanced by the presence of cornstarch. Enhanced growth of S. typhimurium , A. hydrophila , and P. fragi was observed under saturated relative humidity at some storage temperatures when a CSPE-supplemented minimal salts medium was used as compared to PE-supplemented medium. Enhanced growth was not apparent when a nutritionally complex growth medium supplemented with CSPE or PE was used. These results indicated that, from a microbiological viewpoint, cornstarch-containing polyethylene film could be successfully used to package foods.

Stability of Cornstarch-containing Polyethylene Films to Starch-degrading Enzymes.

Processes have been developed to incorporate cornstarch into plastics with the intent of increasing the rate of plastic biodegradation. The effect of starch-degrading enzymes on food-grade polyethylene film that contained 6% cornstarch (CSPE) was examined. Control polyethylene film with no added starch, CSPE and laboratory grade soluble starch were treated with α-amylase, ß-amylase, or amyloglucosidase. Samples were removed periodically and were subjected to the Nelson-Somogyi method for the determination of reducing sugar content. Treatment with α-amylase and ß-amylase released over 30% of the soluble starch as glucose, while less than 1 % of the starch in CSPE was released. Amyloglucosidase activity released up to 50% of the soluble starch as reducing sugar. However, less than 4% of the CSPE starch was liberated. Image analysis of iodine-stained films showed that enzymatic treatment did not remove surface granules. These results indicated that breakdown of CSPE by starch degrading enzymes was limited.

Bacterial Survival on Lean Beef and Bologna Wrapped With Cornstarch-Containing Polyethylene Film.

Cornstarch-containing plastic films could be used to package foods if the presence of cornstarch had no adverse effect on food safety. The survival of pathogenic bacteria on meat samples that had been wrapped with cornstarch-containing plastic film was evaluated. Cornstarch-containing polyethylene film and control polyethylene film were used to cover lean beef and bologna that had been inoculated with Salmonella typhimurium , Staphylococcus aureus , and Bacillus cereus . Additional samples were prepared in which inoculum was applied to the outer surface of plastic-covered meat. Samples were stored at 4 and 21°C. Bacterial recovery from meat samples indicated that survival was not enhanced by the presence of cornstarch. No migration through polyethylene film or cornstarch-containing polyethylene film into lean meat or bologna was observed. These results indicated that, from a microbiological viewpoint, cornstarch-containing polyethylene film could be successfully used to package foods.

The behavior of selected microorganisms during the manufacture of high moisture Jack cheeses from ultrafiltered milk.

Whole milk was pasteurized and concentrated two times by ultrafiltration. Starter cultures, Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis, were propagated in either reconstituted skim milk, two times UF retentate, or UF permeate, or a direct vat system was used for the starter culture. The cheese milk was simultaneously inoculated with starter culture and Pseudomonas fragi 4973, Staphylococcus aureus 196E, and Salmonella typhimurium var. Hillfarm. Control whole milk, UF control milk, inoculated whole milk, and inoculated UF milk were made into Monterey Jack cheese using traditional procedures. The process of cheese manufacture was followed by determination of pH, titratable acidity, and microbial population levels. The cheeses were stored for 6 mo and analyzed every month for percentage solids and microbial population levels. Generally, numbers of contaminant microbes increased at a similar rate during manufacture in all cheeses. During the 6-mo ripening period, bacterial starter culture population levels remained high, psychrotrophs declined slowly, Staphylococcus levels remained stable, and Salmonella populations decreased. No Staphylococcus enterotoxin was detected by reverse passive latex agglutination assay.

Relationship between bile tolerance and the presence of a ruthenium red staining layer on strains of Lactobacillus acidophilus.

Seventeen strains of Lactobacillus acidophilus were evaluated to determine the relationship between bile tolerance and the presence of an outer polysaccharide layer exterior to the cell wall when viewed by transmission electron microscopy. Bile tolerance is necessary for survival of lactobacilli in the intestinal tract, and the polysaccharide layer may be responsible for adherence to human intestinal tissue. These two factors may be the basis for use of L. acidophilus as a dietary adjunct. Ten strains exhibited a ruthenium red-stained outer polysaccharide layer. Three of the 10 strains had extremely dense layers, which may indicate stronger adherence properties. Seven strains did not contain a ruthenium red-stained outer layer; however, six strains that did not have the stained layer were resistant to 1.0% bile concentration. Fourteen strains were tolerant to 1% bile, one strain was tolerant to 6% bile, and two strains were sensitive to bile. No relationship between bile tolerance and the presence of the ruthenium red-stained outer polysaccharide layer was apparent.

Comparison of Methods for Isolation of Listeria from Raw Milk.

Methods used to isolate Listeria spp. from raw milk were compared during a 13-month period (April 1989-April 1990). Raw milk was obtained bimonthly from 12 dairy farms during this time period. Three enrichments were compared: Listeria enrichment broth (LEB); University of Vermont medium (UVM), followed by a secondary enrichment with Fraser broth; and LPALCAMY. Four selective plating media, including Listeria selective agar-Oxford formulation; Modified McBride Listeria agar (MMLA); Lithium chloride-phenylethanol-moxalactam (LPM) agar; and L-PALCAM agar, were compared. L-PALCAMY enrichment broth enhanced isolation of L. monocytogenes and other Listeria spp. The greatest numbers of positive Listeria samples were obtained on Oxford and PALCAM agars, while the poorest isolation was obtained using the combination of LEB with MMLA. L. monocytogenes was found to be present in 3.0% (9/300) of the samples analyzed. The incidence of Listeria spp. was 28.0%; Listeria innocua , 26.7%; and Listeria welshimeri , 1.7%. Listeria ivanovii was not isolated. The incidence of Listeria was highest during the warmer months of April-July, while the lowest incidences occurred in December and February. Somatic cell counts (SCC) and standard plate counts (SPC) were determined by the cooperative that supplied the raw milk. In general, a producer's milk with high SPC corresponded to a high incidence of Listeria spp., while low SPC corresponded to a low incidence. A particular producer's milk with the highest incidence of Listeria spp. also had the highest SCC.

Growth and Attachment of Listeria monocytogenes to Cast Iron.

The growth and potential attachment of Listeria monocytogenes to cast iron in drains was investigated in this study. L. monocytogenes was grown in rich and dilute nutritive media in free-standing cast iron drains. The pH in the drain was adjusted over the growth period to pH 4.5, 7.0, and 9.0. L. monocytogenes was found to survive pH adjustments in drains for 28 d. Scanning electron microscopy (SEM) was used to investigate the attachment of L. monocytogenes to cast iron chips. SEM observation showed L. monocytogenes cells apparently attached to the iron surface of the chip. Listeria does not appear to attach as readily to cast iron as to stainless steel surfaces.

The Survival of Listeria monocytogenes in Aerosols.

Aerosols of Listeria monocytogenes were generated in 0.1 % reconstituted nonfat dried milk, 0.1% peptone water, and distilled water. Suspensions of L. monocytogenes Scott A and L. monocytogenes V37CE were prepared in three population ranges: high, between 108-109 CFU/ml; intermediate, between 105-106 CFU/ml; and low, between 103-104 CFU/ml. Aerosols were generated into an enclosed hood by pressurized Freon. Prepoured petri plates containing tryptic soy agar with 0.6% yeast extract (Difco, Detroit, MI) were spread out in the bottom of the hood. Fallout was collected by removing the lids of randomly selected plates and leaving the lids off for selected time periods. The time periods used for collecting fallout varied and were based on the initial populations of the solutions that were aerolized. Fallout was reported as CFU/cm2 at each time interval. Results showed that high populations of L. monocytogenes Scott A and L. monocytogenes V37CE survived in aerosol suspensions generated under these conditions for 210 min.

Characterization and Behavior of Salmonella javiana During Manufacture of Mozzarella-type Cheese 1.

A patient isolate of Salmonella javiana implicated in an outbreak of salmonellosis in Minnesota was characterized and used to examine its response to Mozzarella manufacturing conditions. The strain possessed biochemical-metabolic activities typical of Salmonella species. Growth was observed in 6.5% NaCl Trypticase Soy Broth (TSB) but not in 12% NaCl TSB. This S. javiana strain was resistant to two antibiotics, penicillin G and erythromycin. Pasteurization trials indicated the strain did not survive pasteurization and that pasteurization affected a log reduction of greater than 9 cycles. Mozzarella-type cheese was manufactured from milk inoculated with S. javiana at levels of 1 × 104 and 1 × 106 per ml milk. Manufacturing process was monitored by following pH, titratable acidity, and temperature. Survival of S. javiana was monitored using traditional enrichment procedures and direct plating procedures. S. javiana survived and grew through the acid-ripening phase, but temperatures attained in cheese mass during stretching and molding (60°C, 140°F) killed all Salmonella present. No subsequent process steps were found positive for Salmonella .

Preliminary characterization of a food-borne multiple-antibiotic-resistant Salmonella typhimurium strain.

Plasmid characterization studies were conducted on a Salmonella typhimurium strain isolated from pasteurized milk and from a symptomatic patient during the 1985 Illinois salmonellosis outbreak. This strain (Hf) was reported to possess an unusual plasmid profile which distinguished it from all Salmonella strains isolated in the United States prior to 1984. Antibiotic susceptibility testing revealed that the strain was resistant to tetracycline, erythromycin, clindamycin, sulfisoxazole, sulfadiazene, triple sulfa, cefoperazone, streptomycin, mezlocillin, piperacillin, carbenicillin, penicillin, ampicillin, and kanamycin. Plasmid analysis revealed that the strain possessed four plasmids with sizes of approximately 158, 98, 10.2, and 6.0 kilobase pairs (kb). Successive transfer at 43 degrees C led to increased antibiotic sensitivity in 75.5% of the isolates screened. Electroporation and calcium chloride treatment were each used to transform plasmid-free Escherichia coli strains with the plasmid pool from S. typhimurium Hf. Plasmids introduced by transformation ranged in size from 4.4 to 23.2 kb and correlated with resistance to penicillin G, ampicillin, carbenicillin, cephalothin, cefoperazone, cefamandole, mezlocillin, piperacillin, and in some cases, tetracycline and kanamycin. DNA-DNA hybridization experiments localized these resistance genes to a highly duplicated 6.3-kb fragment of the total EcoRI restriction digest of the S. typhimurium Hf plasmid pool.

The Survival of Salmonella typhimurium in Propylene Glycol/Water Mixtures.

The effect of propylene glycol, a coolant used in dairy processing plants, on the growth and survival of Salmonella typhimurium was studied. Cultures were inoculated into 0.1% reconstituted nonfat dry milk with glycol concentrations ranging from 0 to 50% (Aw 0.99-0.83) and were incubated at 37°C or -1°C. Serial dilutions were plated on tryptic soy agar (Difco), incubated at 37°C for 24 h and enumerated. At 37°C, 5% or more glycol inhibited the growth of S. typhimurium . At concentrations >20% glycol, Aw<0.96, no recovery was possible after 4 h at 37°C. At -1°C, a slow decline in population was seen regardless of glycol concentration. The higher the concentration, the faster the rate of decline in population. Aeration of the system resulted in a more rapid decrease in the number of S. typhimurium than the static system.