Psychrotrophic Growth Research Articles

EFFECTIVENESS OF CARNOBACTERIUM PISCICOLA LK5 FOR CONTROLLING THE GROWTH OF LISTERIA MONOCYTOGENES SCOTT A IN REFRIGERATED FOODS1

ABSTRACT The potential for controlling the growth of Listeria monocytogenes in refrigerated foods using Carnobacterium piscicola LK5, a bacteriocin‐producing strain originally isolated from raw ground beef, was studied using co‐culture techniques. Eight foods, including UHT milk, canned “all‐beef”dog food (cooked meat), raw ground beef, irradiation‐sterilized raw ground beef, chicken roll, pasteurized crabmeat, canned creamed corn, and frankfurters, were inoculated with 103 cfu/g L. monocytogenes Scott A, with and without 104cfu/g LK5, and incubated at 5 and 19C. Samples were removed periodically and assayed for total aerobic plate count using Brain Heart Infusion Agar and L. monocytogenes using Vogel‐Johnson Agar or Modified Vogel Johnson Agar. The growth of L. monocytogenes was suppressed in milk, dog food, crabmeat, creamed corn, and frankfurters stored at 5C. The microorganism was less inhibitory at 19C. In sterile raw ground beef, LK5 inactivated the pathogen at 5C and prevented its growth at 19C. No activity attributable to LK5 was observed in refrigerated nonsterile ground beef or chicken roll; however, these products did not support the psychrotrophic growth of the pathogen even in the absence of LK5. LK5 was most effective in products where the background microflora was reduced by either thermal processing or irradiation treatment. The results indicate that C. piscicola LK5 has potential as a means for preventing the growth of L. monocytogenes in a variety of refrigerated food products.

Some insight into the mechanism of inhibition of psychrotrophic bacterial growth in raw milk by lactic acid bacteria

Addition of lactic acid bacteria to refrigerated raw milk inhibited growth of Gram‐negative psychrotrophic bacteria. In order to obtain complete inhibition, population levels of lactic acid bacteria approaching 1× 108 cfu/ml were required. At this inoculum level a substantial pH change was observed in the milk during storage. The degree of inhibition was related to the final pH of the milk. Inhibition of the psychrotrophs was brought about by adjustment of pH with L(+) lactic acid but not by HCI. Inhibition by the lactic acid bacteria was observed in heat‐treated milks seeded with a small volume of raw milk, but no inhibition of Gram‐negative psychrotrophic growth was observed when cell‐free preparations of lactic acid bacteria were used. A cell‐free ultrafiltrate of one lactic acid culture did inhibit a strain of Bacillus circulans.

Growth characteristics of meat starter cultures

The Australian Code of Practice for manufacture of dry and semi-dry sausage (salami) states that fermentation temperatures must not exceed 25°C and that a pH of 5·2 must be achieved in the product within 48 h. In order to select the most appropriate starter cultures for fermentation, Lactobacillus plantarum, Pediococcus pentosaceus and Staphylococcus carnosus were characterised with respect to growth and acid production determined at constant pH (4·7, 5·5 and 6·3). L. plantarum and P. pentosaceus showed similar characteristics over the pH range studied while S. carnosus was sensitive to lower pH. Also, P. pentosaceus showed greater psychrotrophic growth without pH control than L. plantarum or S. carnosus. Salami made with P. pentosaceus maintained higher viable numbers in the product over 8 days than did L. plantarum. Growth of S. carnosus in salami could not be detected in the presence of the more pH-tolerant organisms. The growth of L. plantarum and P. pentosaceus did not prevent the development of high levels of non-starter flora, a factor that can be important in determining salami quality. However, the more psychrotrophic P. pentosaceus ensured a greater dominance of starter over non-starter flora.

Growth of Octopine-Catabolizing Pseudomonas spp. under Octopine Limitation in Chemostats and Their Potential To Compete with Agrobacterium tumefaciens

The growth characteristics of five octopine-catabolizing pseudomonads have been determined in batch and continuous cultures. All five strains belonged to rRNA homology group I and showed a more psychrotrophic growth pattern than did Agrobacterium tumefaciens B6 and ATCC 15955. In chemostats limited by octopine, either as the source of carbon and nitrogen or the sole source of nitrogen, maximum specific growth rates and substrate affinities were lower than those in chemostats limited by glutamate. These growth dynamics were similar to those observed for Agrobacterium strains B6 and ATCC 15955 even though the catabolic genes and pathways are believed to be different in the two genera. An analysis of the yields in octopine-limited chemostats indicated that the use of octopine as the sole source of carbon and nitrogen was grossly inefficient. Octopine and presumably lysopine and octopinic acid provided a better source of nitrogen than of carbon. One of the Pseudomonas fluorescens strains, E175D, was able to produce its highest yield on octopine as a nitrogen source. Competition models formulated on pure culture parameters indicated that two of the Pseudomonas spp. would dominate A. tumefaciens B6 and ATCC 15955 when in simple competition for octopine as a limiting substrate.

Survival and Growth of Listeria monocytogenes on Lettuce as Influenced by Shredding, Chlorine Treatment, Modified Atmosphere Packaging and Temperature

ABSTRACTThe effects of shredding, chlorine treatment and modified atmosphere packaging on survival and growth of Listeria monocytogenes, mesophilic aerobes, psychrotrophs and yeasts and molds on lettuce stored at 5°C and 10°C were determined. With the exception of shredded lettuce which had not been chlorine treated, no significant changes in populations of L. monocytogenes were detected during the first 8 days of incubation at 5°C; significant increases occurred between 8 and 15 days. Significant increases occurred within 3 days when lettuce was stored at 10°C; after 10 days, populations reached 108‐109 CFU/g. Chlorine treatment, modified atmosphere (3% O2, 97% N2) and shredding did not influence growth of L. monocytogenes. It was concluded that L. monocytogenes is capable of growing on lettuce subjected to commonly used packaging and distribution procedures used in the food industry.

Inhibition of the Psychrotrophic Bacteria of Raw Milk by Addition of Lactic Acid Bacteria

Two commercial cultures of lactic acid bacteria (LAB) were added to raw milk in order to inhibit the development of psychrotrophic bacteria. The effects of inoculation level, pH development, media for culture preparation, and milk heat treatment on the inhibitory activity of LAB were studied. Inoculation levels of 2.5 × 106/mL and less of LAB did not significantly reduce psychrotrophic growth. Maximum inoculation level of LAB studied was 25 × 106/mL and this reduced psychrotrophic development by a factor of 10; however, milk pH was reduced to 6.54 over 48 h of incubation at 7°C with this inoculation level. This acidification was not responsible for the inhibitory activity of LAB on psychrotrophs. The LAB were more effective when psychrotroph populations had high multiplication rates. Initial populations of Pseudomonas putida in pasteurized milk did not influence the activity of the LAB. Pseudomonas putida grew faster in sterile milk than in pasteurized milk, but average inhibition rates of LAB were similar in both media.

Effectiveness of Cold-serving Units Using Two Cold-holding Methods in Foodservice Operations

Effectiveness of two cold-holding methods commonly used to maintain temperatures of products held on cold-serving units (CSU) was determined by time-temperature and bacterial growth patterns of three products. Products used were bulk (2.27 kg) and portioned (100 g) cottage cheese, portioned (100 g) tuna salad, and deviled eggs halves (100 ± 10 g). All products were held on a cold-serving unit using the mechanical/ice cold-holding method (mechanical cooling used in combination with 3 to 10 cm ice) for 24 h (control; laboratory setting), as well as on three separate cold-serving units using the mechanical cold-holding method (at three university residence hall field sites under actual operating conditions) for 4 h (maximum length of service). Temperatures of all bulk and portioned products held on CSUs using the mechanical/ice cold-holding method (initial temperatures of food were 4 to 8.2°C) were >7.2°C after 2 h with a 50% load factor. When the mechanical cold-holding method was used, all portioned products (initial temperatures were 8.2 to 11°C) were <7.2°C after 2 h with a 75% load factor. Temperature differences between the mechanical and mechanical/ice cold-holding methods were attributed to ice on the cold-serving unit. The ice insulated the products from the mechanically cooled basin and allowed internal temperatures of the products to increase. Statistical significance for bacterial growth patterns was reported only for products held on cold-serving units using the mechanical/ice cold-holding method: mesophilic growth in deviled eggs (p<0.05) and psychrotrophic growth in tuna salad (p<0.001). As expected, bulk cottage cheese had a significantly higher temperature over time (p<0.05) than did portioned cottage cheese for both methods of cold-holding. Based on results of this study, portioned foods on cold-serving units should be held less than 2 h when the mechanical/ice cold-holding method is used, or up to 4 h when the mechanical cold-holding method is available.

Growth of Listeria monocytogenes in soymilk

Pasteurized or sterilized commercial soymilk was inoculated with two levels of Listeria moncytogenes strain Scott A, Brie-1 or DA-3, and stored at 22°C or 5°C. Similar growth patterns were noted for all three strains in either product. At 22°C, cell numbers increased rapidly, reaching 10 8-10 9 ml −1 within 72 h in samples inoculated with 10 2 or 10 4 CFU ml −1. Numbers of 5–8 × 10 8 cells ml −1. were reached after 30 days at 5°C. Doubling times were 1·33 h at 22°C and 1·57 days at 5°C. These data indicate the potential rapid psychrotrophic growth of Listeria in soymilk contaminated by these organisms.

Shelf‐life of Algin/Calcium Restructured Turkey Products Held under Aerobic and Anaerobic Conditions

ABSTRACTThe shelf‐life of restructured products made with ground turkey or turkey breast pieces and formulated with combinations of 0.5–1.0% sodium alginate, 0.1–0.2% calcium carbonate and 0.15–0.30% lactate (ACL) was compared to product containing a combination of 1.4% NaCl and 0.32% sodium tripolyphosphate and to no‐additive controls stored at 4°C under aerobic and anaerobic conditions. No significant (p &gt; 0.05) differences were observed between ACL and salt/phosphate restructured turkey products in the rate and extent of growth of psychrotrophs or lactic acid bacteria. The salt/phosphate combination, however, repressed (p&lt;0.05) the growth of pseudomonads. Overall, inclusion of ACL did not influence spoilage of restructured turkey meat products held under aerobic or anaerobic conditions.

Effect of Gas Atmosphere Packaging on Psychrotrophic Growth and Succession on Steak Surfaces

ABSTRACTThe influence of gas atmosphere packaging (1) 15% CO2, 40% 02, 45% N2; (2) 60% CO2 40% 02 (3) 10% CO2, 5% 02, 85% N2; (4) film overwrap (control) on the microbial growth and succession on steaks was studied during storage. Treatment 3 was most effective in reducing psychrotrophic growth on steaks. Pseudomonas species dominated the microflora for all packaging treatments during early storage. In all gas atmospheres. Serratia liquefaciens increased with storage time and the 10% CO2 atmosphere showed the greatest increase. Enterobacter aerogenes appeared late in the storage period in all atmospheres and Yersinia enterocolitica was isolated after 12 days of storage from steaks in atmosphere 1.

CORRELATION OF TIME-TEMPERATURE INDICATOR RESPONSE WITH MICROBIAL GROWTH IN PASTEURIZED MILK

Commercially obtained pasteurized whole milk was stored at three constant temperatures (0°C, 5°C, and 10°C), and one variable temperature condition (cyclic exposure of 0°C for 14 days and 10°C for 2 days). Daily analyses were conducted to enumerate the growth of total bacteria, coliforms, psychrotrophs, and spore forming organisms in samples from each storage treatment. Microbial growth was correlated with the response of the I-POINT and LifeLine full-history time-temperature indicators. Response of the I-POINT model 2140 was strongly related to germination of the psychrotrophic bacteria, and significant correlations (r > 0.95) were found between total count enumeration and the LifeLine model 57 indicator.

Growth of Aerobic Psychrotrophs and Color Changes of Precooked Beef Slices as Affected by Packaging Procedure

Precooked beef slices from top round roasts were used in replicate trials to determine the effects of packaging treatment upon shelf-life characteristics (microbial growth and color attributes) during retail storage. Roasts were dry-roasted to an internal temperature of 60°C, cooled for 1 h, then sliced (3–4 mm). Slices were packaged in: (a) vacuum; (b) 15% CO2, 40% O2, 45% N2; (c) 15% CO2, 20% O2, 65% N2; or (d) 15% CO2, 10% O2, 75% N2 and held at 4°C for up to 18 d. Enumeration of microorganisms and determination of color attributes were done at 0, 3, 6, 9, 12, 15, and 18 d of storage. Psychrotrophic counts were higher (P<.05) on slices stored 9–18 d in atmospheres containing ambient (20%) oxygen level as compared to the other packaging treatments. Vacuum packaging exhibited a greater lag phase for psychrotrophs. Vacuum packaged slices maintained a superior cooked beef color for 18 d and had greater (P<.05) chroma readings, indicating a greater color intensity.

The relation between temperature and growth of bacteria in dairy products

The effect of temperature on the growth of psychrotrophs in dairy products has been studied using the ‘square root’ and Arrhenius models. The square root model has been shown to accurately predict temperature effects on the growth of several psychrotrophs in a number of dairy products. The formula can be applied for individual bacteria regardless of growth medium. This was not true for the Arrhenius equation. In this case, the apparent activation energy for growth (μ) was dependent on source of isolate and growth medium. For isolates from pasteurized cream, μ values were lower during growth in double cream than those obtained during growth in full-fat milk. This was due to shorter generation times at lower temperatures when these isolates were grown in double cream. Temperature-growth parameters were similar for bacteria grown in pasteurized as well as UHT products.

Psychrotrophic Growth and Thermal Inactivation of Listeria monocytogenes as a Function of Milk Composition

Listeria monocytogenes strains 19111, 19113, 19115, F5027 and F5069 were grown in 11% nonfat milk solids, skim milk and whole milk at 4, 10, 22, and 37°C to determine the influence of temperature and milk composition on growth and thermal resistance. Milk composition affected cellular growth. The psychrotrophic growth of L. monocytogenes serotype 4b strains was enhanced in whole milk when compared to skim milk or 11% NFMS. This enhancement of psychrotrophic growth was not observed for serotype 1 or 3 strains. The stimulatory effect of whole milk on serotype 4b L. monocytogenes strains was most dramatic at 10°C where cells increased from 7.9 × 10° to 5.8 × 106 CFU/ml within 48 h. Milk composition did not affect the thermal resistance of L. monocytogenes. All strains used in this study had a D62.7°C value of 1.0 min or less, therefore, pasteurization as defined by current FDA guidelines should eliminate this organism from raw milk with a large margin of safety. Post-pasteurization contamination of dairy products with L. monocytogenes must be eliminated since the psychrotrophic nature of this organism ensures survival and proliferation during refrigerated storage.

Shelf-Life Extension of Vacuum Packaged Frankfurters Made from Beef Inoculated with Streptococcus lactis

Fresh beef, containing .5% dextrose, was inoculated with Streptococcus lactis, incubated for 3, 5 and 7 d at 7±1°C, and then used to manufacture frankfurters. The frankfurters were vacuum-packaged and stored for 1, 2, 4 and 6 weeks at 3±1°C. Microbial quality was evaluated by examining frankfurters for aerobic and psychrotrophic counts at the end of each storage period. The level of nisin, an antibiotic substance produced by this strain of S. lactis, was also monitored in fresh meat and processed products. During the incubation period, no appreciable reduction in pH occurred in fresh meat. Growth of psychrotrophs was slower in treated than untreated fresh meat, but whether this was due to presence of nisin or competition resulting from the heavy inoculation with S. lactis is unclear. Before and after processing, nisin levels in the 5- and 7-d incubation-treated groups were higher than in the 3-d group, and all treated groups had higher nisin levels than untreated groups. Little difference in aerobic and psychrotrophic growth was noted with storage time between treated and untreated groups for the 3-d incubation period, but well defined differences were noted in the 5- and 7-d incubation groups, with aerobic growth in treated groups being significantly slowed through the 6-week storage period, and psychrotrophs being 1 to 1.5 log cycles lower at the 6-week evaluation. The growth patterns suggest that nisin played a role in the reduced growth rate of bacteria in treated groups.

Growth of Indicator, Pathogenic and Psychrotrophic Bacteria in Mechanically Separated Beef, Lean Ground Beef and Beef Bone Marrow

Growth of Escherichia coli, Salmonella anatum, Staphylococcus aureus, Clostridium perfringens and naturally occurring psychrotrophs in mechanically separated beef (MSB), lean ground beef (LGB) and beef bone marrow (BBM) was studied. Six good grade steers were slaughtered and samples of MSB, LGB and BBM were prepared under proper sanitary care. Six hundred grams of each sample were collected; 100 g were used for chemical analysis. The remaining 500 g were divided into 10-g portions, each mixed with 10 ml of water and either frozen to −20°C or used immediately for bacteriological analysis. For growth studies, samples were inoculated with E. coli or one of the pathogens, incubated at 37°C up to 24 h and enumerated for colony forming units (CFU) on specific selective agar plates. During the first 8 h of incubation, E. coli and S. anatum multiplied rapidly in MSB and LGB but rather slowly in BBM. By 24 h, both species had multiplied to the same population level. Initial growth of S. aureus was rapid in MSB and LGB, but by 24 h its number was higher in LGB than in MSB or BBM. C. perfringens grew faster in LGB and slower in BBM during the 24-h period. Growth of psychrotrophs was determined by incubating the materials at 10, 7 and 3°C up to 10 d. The psychrotrophs grew fastest in MSB and slowest in LGB at all three temperatures, especially at 3°C. Rapid growth of various bacteria in MSB should be considered in its production, storage and subsequent use.

Influence of Combinations of Lactobacillus lactis and Potassium Sorbate on Growth of Psychrotrophs in Raw Milk

Cells of Lactobacillus lactis from frozen concentrated cultures were added (1×108/ml) to refrigerated raw milk to determine their effect on growth of psychrotrophs during storage of the milk at 5 or 7°C. Some strains of Lactobacillus lactis were significantly inhibitory toward the psychrotrophs in refrigerated raw milk at both temperatures. Two selected strains of Lactobacillus lactis were added along with potassium sorbate (.1 and .2%) to refrigerated raw milk stored at 5°C to determine their combined effect on growth of the psychrotrophs. The combinations of cells of lactobacilli and sorbate were more inhibitory than either lactobacilli or sorbate alone. The measurement of hydrogen peroxide produced by strains of Lactobacillus lactis indicated that the strains most antagonistic toward psychrotrophs also were the more active producers of hydrogen peroxide. Addition of sorbate had no apparent effect on hydrogen peroxide production by the cultures during refrigerated storage.

Growth of Bacteria in Soy-Extended Ground Beef Stored at Three Temperatures

The objective of this study was to determine the influence of five separate levels of textured soy protein (TSP) on growth of psychrotrophs, mesophiles, coliforms, Staphylococcus aureus, and fecal streptococci in soy-extended ground beef stored at −16°, 0° and 6°C. Highly significant increases in psychrotroph and mesophile counts accompanied increased levels of soy at 0° and 6°C, but not at −16°C. Soy-extended beef samples containing 20 and 40% TSP spoiled one day faster at 6°C and four days sooner at 0°C than non-extended ground beef. No significant differences in coliform, fecal streptococci or S. aureus counts could be attributed to increasing levels of TSP in extended ground beef at −16°, 0° or 6°C. Protein content did not vary significantly with TSP concentration; however, fat decreased as soy level increased. Moisture and carbohydrate-ash content increased significantly as soy level increased, as did pH which reached a maximum of 6.5.

COMPARISON OF ANTIMICROBIAL ACTIONS OF MONOLAURIN AND SORBIC ACID

The antimicrobial effects of monolaurin and sorbic acid were compared in laboratory media, cottage cheese and a pork homogenate. Sorbic acid (250–1000 ppm) was more effective than monolaurin (250–1000 ppm) in inhibiting the growth of Staphylococcus aureus S‐6 and Salmonella enteritidis 13076 in pH 5.2 trypticase soy broth. Monolaurin was more effective than sorbic acid against the growth of S. typhimurium 13311 under similar conditions. No synergistic effects were observed when combinations of monolaurin and sorbic acid were tested. In the cottage cheese study, 800 ppm sorbic acid effectively inhibited the growth of coliforms and yeasts and molds, but only slightly inhibited the growth of psychrotrophs. Monolaurin at 800 ppm was ineffective in controlling the growth of any of the microorganisms tested. The use of 3000 ppm monolaurin in a pork homogenate had no effect on the outgrowth and gas formation by Clostridium sporogenes PA3679, whereas 3000 ppm sorbic acid delayed outgrowth and gas formation. Combinations of monolalurin and sorbic acid indicated that sorbic acid was the active anticlostridial agent.

Role of Psychrotrophic Bacteria in the Degradation of Waste Activated Sludge

Abstract A series of batch and semi-continuous laboratory scale aerobic reactors was evaluated, with emphasis on the microbiological aspects. Batch treatment of waste activated sludge (COD 10,000 mg/l) was carried out over a period of eight weeks in three separate reactors at 5, 10 and 20°C. A semi-continuous unit operating at 5°C was investigated at a loading rate of 0.64 kg VSS/m3/day. Analyses performed included pH, O2 uptake rate, total solids, suspended solids, volatile suspended solids and COD determinations. The microbiological tests included total plate counts (20°C) and psychrotrophic counts (l°C). Results indicated that long (impractical) detention times were required to aerobically treat waste activated sludge at low temperatures. At temperatures supposedly favourable to psychrotrophic growth, it was found that the proportion of psychrotrophic bacteria could not develop to a significant level and remained at less that 1% of the total viable bacterial population, regardless of the mode of operation or the detention time. This is in contrast to the finding that approximately 54% of the viable bacteria in the aeration tank of a large conventional activated sludge plant, operating at a mixed liquor temperature of 13°C, were psychrotrophic. It is apparent that psychrotrophic bacteria played an insignificant role in the degradation of waste activated sludge at low temperatures, (5 and 10°C) and that biological oxidation was performed primarily be mesophilic bacteria. The study indicated that under the experimental conditions employed, the amount of available substrate present was one of the major regulating factors in the development or selection of psychrotrophic bacteria.