Acid/heat resistance of Salmonella serotypes in powdered egg albumin

In Argentina, the egg/egg product industry keeps growing at the rate of 3% per year, while exports are increasing at annual rates of up to 20%. Entre Ríos contributes 21% egg production in the country, and participates with 27% of the eggs that are further processed in Argentina (Irigoyen, 2010). On the other hand, the epidemiological history of Salmonella Enteritidis shows that this particular bacterium has found a biological niche to grow in hen eggs. Various research reports have shown the association between human food borne disease and egg products (Gantois et al., 2009). Salmonella infections are a major cause of morbidity in Argentina, and these bacteria are among the most frequently isolated pathogens from food poison disease (Sandoval & Terzolo, 1989; Caffer & Pichel, 2006).

It has been seen that some salmonellae that have been previously adapted to acidic conditions (i.e., that have been previously in contact with an acidic medium then inoculated to different substrates including culture media and foods) were also more resistant to stress factors, including heat. This inducible mechanism is known as the "acid tolerance response". There are several ways by which organisms can adapt to acidic conditions, either in a gradual fashion or by the use of an acid shock, in which case adaptation occurs suddenly. In the acid shock adaptation the organisms are inoculated and grown for a given time period in a given culture medium, at a nearly neutral end pH. Afterwards, these organisms are suddenly subjected to an abrupt pH level decrease by the addition of an acidifying agent (final acidic pH). On the other hand, for gradual adaptation, the organisms are inoculated and cultured for a given period of time in a culture medium containing excess glucose levels. As a result of glucose utilization, medium pH gradually decreases thus the organism gets adapted slowly. These induced acid adaptation mechanisms could also provide the bacterium with resistance to other stressors such as heat (Malheiros et al., 2009).

The purpose of this research was to study the heat resistance of Salmonella serotypes isolated from eggs, in powdered egg albumin, at the temperatures used in the pasteurization process using both acid-adapted and non-acid-adapted Salmonella spp. isolates.

Salmonella isolates were studied in 120 5-15 hen egg batches selected on purpose (dirty or cracked) eggs or eggshells. Sample collection lasted for 2 years, during April and November. Samples were collected in commercial farms, households, and egg shops in the city of Gualeguaychú, Entre Ríos, Argentina. The eggs were kept at ambient temperature until analyzed. For isolation purposes, the ISO 6579-2002 technique was used. Somatic and flagellar serotyping was performed as per provisions in the Manual for Salmonella Procedures Part I: Isolation, Identification and Serotyping, INEI-ANLIS, Dr. Carlos G. Malbrán, jointly with the Biologic Production Institute, INEI-ANLIS (Manual de Procedimientos Salmonella Parte I: Aislamiento, Identificación y Serotipificación del INEI-ANLIS "Dr. Carlos G. Malbrán; Instituto de Producción de Biológicos). All strains were stored at -30 ºC in 50% (v/v) glycerol. For study purposes, the strains were stored at 4ºC in tryptein-soy agar, or subcultured in brain-heart infusion (BHI) broth at 37ºC for 24h, prior to being used in the assays.

Parallel cultures of the strains were prepared in nutrient broth (NB) as well as in 10 g/L glucose-supplemented nutrient broth (GNB). The cultures were incubated at 37ºC for 18 hours (h) under static conditions. Culture pH was measured then seeded in trypticase soy agar (TSA) for livability assessment (Malheiros et al., 2009). For the acid shock adaptation studies, then isolates were incubated in acetic acid-supplemented NB (AANB) at the following pH levels: 3.5, 4.0, and 4.5, for 24-72h. For the survival studies, cultures were seeded on the surface of 0.6% yeast extract-enriched TSA plates, and then incubated for 6h at 37°C, in order to enhance the recovery of injured cells.

For the heat inactivation assays of non-acid-adapted strains, cultures were started in BHI medium, while for the acid-adapted strains cultures were started in GNB medium, Cultures were centrifuged, the supernatant was discarded, and the pellet was re-suspended in sterile 0.1% peptone water up to an optical density (OD) of 0.4-0.5 at 600nm. Industrially-pasteurized powdered albumin using the "hot room" process at 82ºC for 14h was used. This albumin was subjected to microbiological analysis then spray-inoculated with ~1mL/g and dried in the incubator at 50ºC. In both homogenized substrates, the bacterial population density in each inoculum was determined by surface seeding on TSA plates at 37ºC for 24h. Sterile capillary tubes were then loaded with each of the test substrates, sealed, and fully submerged in a controlled-temperature water bath (Oteiza et al., 2003). After different treatment times, two capillary tubes were removed from the water bath then cooled down on ice in order to halt the heating process. The capillary tubes were cleaned with alcohol and one of their ends was aseptically broken., Serial dilutions (1:10) were prepared in 0.1% peptone water, and seeded in duplicate on the surface of 0.6% yeast extract-added TSA plates. Plates were incubated at 37ºC for 24h. Counts were expressed as colony-forming units (CFU)/ml or grams (g), depending on the substrate. For each temperature, both the D and z temperature inactivation values were determined by linear regression (D = the necessary time to reduce 90% of bacterial populations; z = the temperature increase necessary to reduce 90% of the D value).

In total, 14 Salmonella sp. isolates were obtained then typed. The following results were obtained: 2 S. Enteritidis isolates and 1 S. Oranienburg isolate, from 30 whole egg batches. Also, 1 S. Enteritidis, 3 S. Typhimurium, 4 S. Montevideo, 2 S. Hadar, and 1 S. Infantis isolates were obtained from 90 eggshell batches. Similar serotypes were isolated by Gantois et al. (2009) from hen eggs.

The bacteria present in the powdered, pasteurized albumin were studied, and the following results were obtained: Total heterophils, 6-9x10² CFU/g; total coliforms: 10-100 CFU/g; molds and yeast, 10-70 CFU/g; Bacilluscereus, > 10 CFU/g; Salmonella sp: absent in 25g.

One isolate of each serotype was subjected to this study. Prior to inoculation, the NB pH was 7.37±0.12. End pH values varied from 4.28 to 4.42±0.02 for S. Enteritidis; and 4.10-4.38±0.03 for S. Typhimurium. Initial and final bacterial counts were 8.34 log CFU/ml^-1-7.02 log CFU ml^-1 for S. Enteritidis, and 8.22 log CFU/ml^-1-7.30 log CFU/ml^-1 for S. Typhimurium. These cultures were inoculated into NB, pH 3.5, 4.0, and 4.5 in AA. D values for S. Enteritidis were: 26.33± 4.35min at pH 3.5; 44.23 ± 5.7 min at pH 4.0 and 157.88± 48.7 min at pH 4.5. D values for S. Typhimurium were 20.35± 1.75 min at pH 3.5; 36.83±18.26 min at pH 4.0 and 85.98±22.7 min at pH 4.5. The analysis of variance showed significant differences in the low pH-induced decimal reduction times for both serotypes, particularly for pH 4.5 (p<0.05). S. Enteritidis showed higher resistance at the various pH levels tested.

We were only able to determine the D values for 0.1% peptone water in the acid-adapted cultures, whilst for the albumin studies, both heat-adapted and non-heat-adapted cultures were used. All assays were performed in duplicate. Only as an example, Figure 1 shows the survival graphs for S. Enteritidis in powdered albumin at all 3 temperatures studied.

Figure 1. Survival curves of acid-adapted cultures (A) and non-acid-adapted cultures (B) of S. Enteritidis in powdered egg albumin throughout the treatment at 72, 77, and 82ºC

                                                                       Heating times (min)

 

 

Both serotypes showed to be protected against the lethal effects of heat thanks to acid adaptation

D and z values are shown in Table 1.

Table 1. Salmonella heat resistance

Significant differences existed among the D values for each temperature and for each serotype studied, in peptone water. Acid-adapted (AA) S. Enteritidis showed higher D values tan AA S. Typhimurium (AA). The D value was increased 1,270 fold for S. Enteritidis and 1,400 fold for S. Typhimurium, in the powdered albumin, when compared in terms of heat resistance in peptone water, which can be attributed to the low water activity of albumin (0.366±0.05) together with other substrate components potentially exerting a protective effect on the bacteria.

In the powdered albumin, the previous adaptation to the acid increased, in average, 3 times the D value for S. Enteritidis and 2,3 times the D value for S. Typhimurium as compared to the non-acid-adapted cultures.

The analysis of variance showed no significant differences in the decimal reduction times for each temperature in the serotypes studied. Also, acid adaptation has a significant effect on the D values for these bacteria (p< 0.05).

These results are important in the food technology/food safety arena. Careful attention should be paid to pasteurization times and temperatures for powdered egg whites in order to assure the destruction of both acid adapted- and non-acid-adapted bacterial strains.

S. Enteritidis and S. Typhimurium showed increased resistance to the effects of pH 4.5. In peptone water, the heat-killing studies showed that the acid-adapted S. Enteritidis had higher D values than acid-adapted S. Typhimurium. At temperatures between 72 and 82ºC the acid-adapted Salmonella strains have increased heat resistance in powdered egg albumin.

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