Genetic Diversity and Spoilage Potentials among Pseudomonas spp. Isolated from Fluid Milk Products and Dairy Processing Plants

Bacterial strains and culture conditions. Pseudomonas spp. isolates from raw and pasteurized milk samples were obtained as part of the shelf life testing program of the Cornell University Milk Quality Improvement Program (2). Raw and processed milk samples and environmental samples from the four processing plants involved in this program were collected over a 15-month period. Each processing plant was visited at least three times. Processed milk samples were collected during the first and second visits, raw milk was collected during the second visit, and environmental samples were collected during the third visit.

The numbers of viable bacteria in pasteurized and raw milk samples were determined by standard plate counts (SPC) and psychrotrophic plate counts (PPC) (19). Pasteurized milk samples were plated at days 0, 7, and 14 after receipt. For each sample, 10 to 15 Pseudomonas-like colonies were picked randomly from representative SPC plates and streaked to purification on plate count agar (PCA; Difco, Detroit, Mich.). Environmental samples were collected from the processing environment (floor, drains, pipes, filling bowl interior, pasteurized-milk storage tanks, and valves) by using sterile sponges moistened with neutralizing buffer (Hardy Diagnostics, Santa Maria, Calif.). Environmental samples were transported to the laboratory in sterile stomacher bags, stored at 4°C, and analyzed within 24 h. A volume of 25 to 50 ml of phosphate buffer was added to the bags, and 10-fold serial dilutions were plated onto cetrimide-fucidin-cephaloridine (Oxoid, Basingstoke, United Kingdom)-supplemented Pseudomonas agar base (Oxoid) plates. Ten to fifteen colonies were chosen randomly from each sample. All single colonies were initially characterized according to Gram staining results and oxidase and catalase activities. The colonies that were confirmed as putative Pseudomonas spp. (gram negative, oxidase positive, and catalase positive) were further characterized as described below.

Phenotypic characterization.All isolates were characterized by using API 20 NE strips (BioMerioux Vitek, Inc., Hazelwood, Mo.) according to the manufacturer's instructions. Species identifications were obtained by using the API database. Oxidase reactions of the isolates were determined using BBLDrySlide oxidase slides (Becton Dickinson Company, Sparks, Md.) according to the manufacturer's instructions. For catalase activity, 1.5% H₂O₂ was dropped onto a bacterial smear that had been placed on a glass slide. Gas production indicated a positive reaction.

Extracellular-enzyme production.To determine the production of extracellular proteases, lipases, and lecithinases, Pseudomonas colonies were streaked to isolation on PCA and then single colonies were transferred to agar plates containing the appropriate substrates. Production of extracellular proteolytic enzymes was determined on PCA (Difco) containing 1% skim milk powder (skim milk agar) (31). After incubation at 30°C for 72 h, the plates were flooded with 1 N HCl for observation of clearance zones formed by protease-positive strains. For each isolate, clearance zones around two to five colonies were measured and the ratio of the clearance zone diameter to the colony diameter was calculated. The assay was repeated at least three times, and the mean ratios and standard deviations are reported. Production of extracellular lipase was assessed using single-layer agar (31) consisting of 5% (wt/vol) clarified butterfat (16) and Victoria blue B blended into tryptic soy agar (Difco) (1:7,500 dilution). After being incubated at 30°C for up to 5 days, the plates were observed for the presence of colonies surrounded by dark blue zones. Lipase activity was assessed by noting the color intensity surrounding the colonies. Following visual inspection, the color intensity was categorized on a scale of 1 to 4, where 1 represents light blue and 4 represents very dark blue. Production of extracellular lecithinase was determined on PCA containing 10% egg yolk emulsion (Sigma, St. Louis, Mo.). After incubation at 30°C for up to 5 days, the diameters of the opaque rings surrounding lecithinase-positive colonies were measured for two to three representative colonies for each isolate. The ratio of the size of the opaque zone to that of the colony was calculated. The mean ratios and standard deviations from three assays are reported.

In addition to the 338 isolates from this study, a total of 27 Pseudomonas isolates were selected from a previous study for the enzyme assays (32).

Ribotyping.Isolates with different API 20 NE codes were selected for ribotyping. Colonies obtained from different samples but with identical API codes were treated as independent isolates.

Bacteria were grown on brain heart infusion agar (Becton Dickinson) overnight at 30°C and ribotyped by using the restriction enzyme EcoRI in a RiboPrinter (Qualicon Inc., Wilmington, Del.) as described previously (5).

Sensory analyses.Sensory analyses of the pasteurized milk samples were performed by 10 trained members of a sensory panel as follows. Milk samples were mixed by inversion, and then, in dim light, 60 ml of each sample was poured into 148-ml plastic cups, which were capped with plastic lids and presented at 15°C to panel members. Panelists evaluated the milk according to the procedures of Chapman et al. (7). Milk flavor was scored on a scale from 1 to 10, where any rating below 6 was considered poor, 6 to 7 was fair, and 8 to 10 was good. The overall scores were assigned with a predominant sensory criticism. Unsalted crackers and spring water were provided to cleanse the palate. Panelists were provided monetary incentives to encourage participation through the completion of the experiments. The Compusense five (Compusense Inc., Guelph, Canada) computerized data collection system was used to determine the order of presentation of the samples and to collect the data.

Ecology of Pseudomonas in dairy processing plants.The genetic diversity of Pseudomonas spp. isolated from four dairy processing plants was studied. A total of 338 putative Pseudomonas isolates were obtained from raw milk, processed milk, and environmental samples (Table 1). All 338 isolates were characterized using API 20 NE strips. Based on the API 20 NE results, 51% of the isolates were identified as P. fluorescens, 14% were identified as P. putida, and 25% were identified as either P. fluorescens or P. putida. The API 20 NE system was unable to identify 10% of the isolates to the species level.

Ribotyping was completed for 81 isolates with different API codes. The resulting ribotypes are shown in Table 2. Initially, ribotype patterns with similarity coefficients of >0.93, as calculated by the Riboprinter's software by use of band positions and intensities, were considered identical and were grouped together as one ribogroup with the same designation. Further refinement of these groupings was performed by visual evaluation of closely related ribotype patterns. A total of 42 different ribotypes were found among the 81 isolates tested (Table 1).

Eight of 42 (19%) ribotypes were found in more than one dairy, while the remaining 81% were found only in one dairy. None of the raw milk isolates was of the same ribotype as that of pasteurized milk isolates in any of the four dairies. Except for one ribotype, 72-S-3, the ribotype patterns of the isolates from milk collected in the first plant visit did not resemble the patterns from isolates collected in the second plant visit. In dairy plant D, ribotype 112-S-2 was found in both processed milk samples and in environmental samples, including those collected from the valve, floor, and drain. However, in the other three dairies, no connection was established between isolates from processed milk samples and those from environmental samples. In two of the dairies, the same ribotypes (82-S-4 and 57-S-8) were isolated from raw milk samples and environmental samples.

From dairy plant A, seven different ribotypes were isolated from raw milk, processed milk, and environmental samples (Table 1). Pseudomonas spp. could not be isolated from processed milk samples obtained during the first plant visit (the major contaminating organisms in these milk samples were Acinetobacter spp.). Pseudomonas spp. were isolated from raw milk and processed milk samples obtained during the second plant visit. None of the raw milk or environmental ribotypes matched the ribotypes from processed milk. Only one ribotype was isolated from the processed milk samples. As we were unable to isolate Pseudomonas from samples collected during the first visit, samples were also obtained during three additional plant visits following the second visit. No Pseudomonas was isolated from the additional samples. The SPC from milk obtained during those three visits were also very low, averaging 500 CFU/ml at 14 days postprocessing. Only four Pseudomonas colonies were isolated from environmental samples.

In dairy plant B, 14 different ribotypes were isolated from raw and processed milk and environmental samples (Table 1). Ribotype 82-S-4 was isolated from both raw milk and environmental (drain) samples. A total of 15 colonies were isolated from the milk samples collected during the first sampling period that had been plated at 7 days postprocessing. All of these were identified as P. fluorescens by API 20 NE (Table 2). The three strains selected bore ribotype 51-S-5. Another 15 colonies were isolated from the samples plated on day 14, and all of these were identified as P. putida by API 20 NE (Table 2). Of these, one isolate bore ribotype 384-S-2.

In dairy plant C, 13 different ribotypes were isolated from raw and processed milk and environmental samples (Table 1). Ribotype 72-S-3 was isolated from processed milk samples obtained during both the first and second plant visits.

Seventeen different ribotypes were isolated from raw and processed milk and environmental samples collected from dairy plant D (Fig. 1). Ribotype 112-S-2 was observed among isolates from processed milk, floor, valve, and drain samples. Ribotype 57-S-8 was isolated from both raw milk and environmental (drain) samples.

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FIG. 1.

Riboprints of Pseudomonas isolates obtained from dairy processing plant D.

Spoilage potentials of Pseudomonas and relationships between ribotypes and enzyme activities.All Pseudomonas isolates were evaluated for production of extracellular protease, lipase, and lecithinase. Of the 338 Pseudomonas isolates, 51% were protease positive, 47% were lecithinase positive, and 67% were lipase positive. Lipolytic and proteolytic activities varied among the Pseudomonas strains. Most of the P. fluorescens strains were positive for all enzyme activities (69%), while most P. putida strains (87.5%) were negative for all enzyme activities. Isolates that were characterized as either P. fluorescens or P. putida by API 20 NE were mostly protease negative (93%); however, the majority of these isolates (75%) had lipase activity. No clear differences emerged among the enzyme activities between the milk isolates and the environmental isolates (Table 2).

Isolates bearing the same ribotype generally had similar extracellular enzymatic profiles (Table 3). The extracellular enzymatic activities and ribotypes were also closely related. Specifically, ribotypes 50-S-8 and 72-S-3 were composed of isolates with the highest extracellular lipase activities while the highest extracellular protease activity was seen in isolates grouped in ribotype 55-S-6 (Fig. 2).

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FIG. 2.

Relationship between ribotypes and enzyme activities of Pseudomonas spp. Mean activities and standard deviations are reported as follows: mean ratio reflecting protease activity = clearance zone diameter/colony size; mean ratio reflecting lecithinase activity = opaque zone diameter/colony size; lipase activity = mean color intensity; n = the number of isolates within a ribotype.

In general, the presence or absence of both extracellular lecithinase and lipase activities appeared to be correlated in this collection of isolates. Forty-five percent of the isolates had both lecithinase and lipase activities, while 31% had neither activity. On the other hand, 22% of the isolates had only lipase activity and just 2% of the isolates had lecithinase activity but no lipase activity.

Effect of Pseudomonas on the flavor of milk samples.For sensory analysis, milk was scored on a scale from 1 to 10, where any rating below 6 was considered poor, 6 to 7 was fair, and 8 to 10 was good. The overall scores were assigned with a predominant criticism. Table 4 presents the SPC, mean flavor scores of processed milk samples, and percentages of enzymatically active Pseudomonas isolates from the samples.

In general, sensory scores were considered “good” at 7 days postprocessing, with an average score of 7.5. Two products also retained good flavor scores (7.6 and 7.5) at 14 days postprocessing. In general, good flavor scores were associated with low numbers of bacteria. All rancid flavors were associated with the presence of lipase-producing Pseudomonas. We conclude that, with the exception of two samples examined in this study, the flavor defects of processed milk were associated with both high numbers of bacteria and the presence of degradative enzymatic activities among the contaminating Pseudomonas strains. One of the two exceptions was the plant B milk sample from the first visit which coagulated at 14 days postprocessing even though the Pseudomonas isolates obtained from the processed milk samples had none of the degradative enzymatic activities measured in this study. Despite the apparent absence of degradative Pseudomonas spp., the overall numbers of bacteria were high in this product (1.3 × 10⁸ CFU/ml). Milk samples from dairy plant D (first visit) were positive for all enzyme activities tested and the SPC was high (1.9 × 10⁸ CFU/ml), but the sensory panelists reported no serious flavor defects.

Spoilage potentials of the Pseudomonas strains.SPC, PPC, and coliform counts are methods that are frequently used to monitor milk quality (3). However, poor correlations exist among the SPC, the PPC, the coliform count, and product shelf life (14). With the exception of the determination of the presence of very high initial bacterial counts, simple measurement of bacterial numbers present in freshly pasteurized products does not provide useful predictive insight into product shelf life. Craven and Macauley (11) showed that the presence of large populations of Pseudomonas spp. generally resulted in shorter shelf lives for pasteurized milk than if products were contaminated with other types of organisms. However, they also reported that some samples predominantly containing Pseudomonas spp. had shelf lives that were equal to or greater than those of samples predominantly containing other types of microorganisms. The growth rates and metabolic activities of contaminating organisms at refrigeration temperatures are the two most important factors that influence product shelf life (14).

Our results showed that 69% of the P. fluorescens strains were positive for all enzyme activities, while most P. putida strains (87.5%) were negative for all enzyme activities. These results correspond with those of Wiedmann et al. (32) in that the majority of the P. fluorescens strains have extracellular protease, lipase, and lecithinase activity while P. putida strains are usually negative for all enzyme activities. We conclude that P. fluorescens is an important spoilage organism in processed milk products. Our results show that the lecithinase and lipase activities of pseudomonads are usually associated. Seventy-six percent of the isolates either had both lecithinase and lipase activity or neither lecithinase nor lipase activity.

Ribotyping is very useful for the characterization of spoilage microflora. In this study, isolates that had the same ribotype, even if obtained from different sources, usually had the same extracellular-enzyme profiles (Table 3). We found considerable variation in the lipolytic and proteolytic activities of the Pseudomonas strains; however, isolates with the same ribotype usually had similar extracellular-enzyme activities (Fig. 2).

Effect of Pseudomonas on the flavor of milk samples.Growth of psychrotrophic bacteria after pasteurization is an important factor in milk lipolysis and proteolysis. Flavor defects generally develop in processed milk when the bacterial population of the milk rises to ≥10⁷ CFU/ml (24). Off-flavors generally develop in three stages. First, milk loses its freshness. Then, it is perceived as stale. Finally, its products develop rancid, fruity, and bitter flavors. Bitter flavors usually accompany protein degradation. Soapy and rancid flavors usually are a result of lipid breakdown (8). Our results indicate that sensory characteristics depend on both the number of bacteria and their enzymatic activities. For example, although all Pseudomonas isolates obtained from plant A were positive for all three enzyme activities, no major flavor defects were reported, probably because of the low SPC of the samples. All rancid flavors were associated with lipase production capability among the Pseudomonas isolates. Although Pseudomonas isolates obtained from the first visit to plant B had no enzyme activity, milk coagulated by day 14 postprocessing. This defect was probably the consequence of protease production by other, non-Pseudomonas bacteria. Although Pseudomonas spp. are the main concern with regard to proteolytic degradation of milk, other proteolytic organisms such as Achromobacter, Aeromonas, Flavobacterium, and Xanthomonas spp. can be found in milk and can cause proteolytic degradation and milk coagulation (11, 26). Pseudomonas spp. isolates from milk samples obtained during the first visit to plant D were positive for all enzyme activities, but pronounced flavor defects were not detected despite the high SPC. The most likely explanation for this phenomenon is that the presence of the Pseudomonas strains able to cause spoilage was low relative to the presence of other organisms with lower spoilage potentials.

Conclusions.We found that ribotyping is a discriminatory and useful typing method for differentiating Pseudomonas spp. capable of decreasing the shelf lives of fluid milk products. Ribotyping revealed a large genomic diversity among Pseudomonas spp. isolated from these dairy plants. Our results indicate that there is a relationship between bacterial ribotypes and their spoilage potentials. Ribotyping is a convenient and reliable method for identifying strains with food spoilage potential.