of Freeze-dried Salmonella typhimurium1

APPLIED MicRoBioLoGY, Jan., 1967, p. 22-3 Copyright 1967 American Society for Microbiology Vol. 15, No. 1 Printed in US.A. Influence of Platen Temperatures and Relative Humidity During Storage on the Survival of Freeze-dried Salmonella typhimurium1 T. J. SINSKEY, G. J. SILVERMAN, AND S. A. GOLDBLITH Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts Received for publication 16 June 1966 ABsTACr Salmonella typhimurium survived freeze-drying at a platen temperature of 12 F (48.9 C) and also, though to a much lesser degree, at 16 F (82.6 C). The extent of the survival at these temperatures was dependent on the composition of the model system employed. The incidence of damage immediately after freeze-drying was greater for cells dried at the higher platen temperature and was influenced by the composition of the menstruum in which the cells were dried. In model systems having protein-dominant isotherms, survival during subsequent storage depended greatly on relative humidity, with recovery highest at relative humidities below those corresponding to moisture contents at which a monomolecular layer is formed. In menstrua having a higher sugar content, survival was best at low relative humidities corresponding to a very low equilibrium moisture content in the model system used. Damage during storage tended to be a function of the composition of the gels in which the organisms were freeze-dried, and also depended greatly on the presence of air and on the relative humidity. The maximal percentage of damage usually occurred at the low relative humidities as storage time increased. The technique of lyophilization of microorganisms avoids exposing the cells to elevated temperatures during the vacuum-drying stage. In contrast, freeze-dried foods are dried at elevated temperatures to accelerate the removal of water. Microbial contaminants on foods may, therefore, be more severely affected by commercial lyophilization procedures than are cells lyophilized for preservation. Scott (15, 16) studied the effects of relative humidity on the survival of dried microorganisms, but his work did not consider effects of high platen temperatures during freeze-drying on the survival of microorganisms stored at various relative humidities under air or nitrogen. In addition to being rendered nonviable, a population of freeze-dried microorganisms can also sustain injury. Sinskey et al. (17) detected metabolic damage, as defined by Straka and Stokes (19), to Salmonella typhimurium immediately after it was freeze-dried in foods. The pres- 'Contribution no. 723 from the Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge. ent study was designed to evaluate more fully the extent of such damage as influenced by various platen temperatures and by the composition of the model system used during storage at 2 C at various relative humidities in air and in nitrogen. The components of foods, such as proteins, amino acids, peptides, and sugars, have all been shown to be important in the preservation of cultures by lyophilization (4, 6). S. typhimurium, the species of Salmonella responsible for the highest incidence of food-poisoning cases in most countries and the species which occurs most frequently in foods (2, 2), was chosen as the test organism. Salmonellae are commonly present in certain dried foods such as eggs (1) and in dried animal feeds (7). McIntosh et al. (Food Technol., in press) compared the survival of S. typhimurium in beef and egg melange with that of S. derby and S. senftenberg after freeze-drying and during storage at a low moisture content. They found that S. typhimurium had the highest resistance to processing and exhibited a high level of survival during storage at 2 C. 22

VOL. 1 5,1967 SURVIVAL OF FREEZE-DRIED S. TYPHIMURIUM 23 MATERLMLS AND METHODS Organism. We grew S. typhimurium (ATCC 7823) for 24 hr in Nutrient Broth on a rotary shaker at 37 C. The cells were centrifuged and washed three times with cold diluent (.1% Trypticase in.3 M K2HPO4, ph 7). We added 2 ml of a suspension of the washed organisms containing 18 organisms per milliliter to 1 liter of a given model system. The model system was sterilized and cooled to room temperature before the organisms were added. This procedure resulted in an initial concentration of about 16 organisms per milliliter in each model system. Model systems. We selected 2% gelatin (Difco) as the material common to all of the model systems and added 5% dextrose, with or without.5% Nutrient Broth (Difco), to the gelatin solution. The ph for all of these formulations varied between 6.8 and 7. and was not readjusted. Processing. Amounts of 1 ml of the inoculated gelatin solutions were pipetted into sterile, prechilled, plastic ice-cube trays and frozen at -4 F (-4 C) in an air blast freezer for 12 hr. The cubes were then quickly removed from the ice-cube trays and placed in the precooled (-3 C) freeze-dryer on perforated metal trays. The samples were freeze-dried at a platen temperature of either 12 or 16 F (48.9 or 82.2 C) in a modified VirTis freeze-dryer at an initial chamber pressure of 1,u of Hg. In addition to the modifications previously described (17), the dryer was further refined for more precise platen temperature control by introducing a proportional temperature-control unit (Dynapac, Lab-Line Instruments, Inc., Melrose Park, Ill.) so that the temperature between platens did not vary more than 2 F (.94 C), at the desired platen temperature, throughout the drying cycle. At the termination of the drying cycle, the vacuum was broken with nitrogen. Equilibration during storage at various relative humidities. After freeze-drying, the samples to be stored in air were equilibrated in desiccator jars over selected saturated salt solutions at 2 C. For samples stored under nitrogen, tubes (18 by 15 mm) were half-filled with the saturated salt solutions and frozen to prevent any flashing-off during subsequent evacuation steps. After the samples were freezedried, an appropriate tube of a frozen salt solution was placed in a no. 2 can together with two freezedried samples. The can was rapidly evacuated, flushed with nitrogen, evacuated again, and sealed under nitrogen. The residual oxygen content in the sealed cans was found to be less than 2% in all cases and usually less than 1% as determined by gas chromatographic analysis of the headspace (9). All samples were stored at 2 C. Water-vapor equilibrium between the samples and the saturated salt solutions was achieved within 1 week for samples stored in nitrogen or air. Viability and damage. By replicate plate counts, the number of viable cells was determined initially, after freezing, after freeze-drying, and at intervals during storage on Trypticase Soy Agar (BBL) supplemented with.5% yeast extract (Difco). Samples of 1 ml of the frozen material were homogenized in a blender in 9 ml of diluent, giving a 1:1 dilution. Freeze-dried samples examined prior to storage were rehydrated and blended in sufficient diluent to give a 1:1 dilution and were homogenized in a blender. Sufficient diluent was added to stored samples to compensate both for the water removed during freeze-drying and for the water absorbed during storage. This procedure allowed direct calculation of S. typhimurium survival for each cube. Survival is expressed as the fraction of frozen cells surviving freeze-drying. Damage to the test organism was enumerated by the difference in recovery on Trypticase Soy-yeast extract-agar and on the minimal salt medium of Davis (3). It had been found (17) that, initially and after freezing, recovery on the minimal medium was essentially equal to that on the complete medium (Trypticase Soy-yeast extract-agar). Statistical analyses were made of survival data, and distinctions between experimental conditions were at the 5% level of significance. RESULTS Survival immediately after freeze-drying. Table 1 gives the percentage of survival after freezedrying. At platen temperatures of 12 F (48.9 C), the 2%-gelatin samples exhibited the lowest survival. Addition of either Nutrient Broth or dextrose improved survival; however, Nutrient Broth and dextrose added together appeared to act synergistically, since survival with both after freeze-drying was considerably higher than with either alone. At 16 F (82.2 C), survival in 2% gelatin was extremely low. The addition of Nutrient Broth resulted in a 5-fold improvement in survival which was equivalent to survival obtained by the addition of dextrose. The synergistic action resulting from the combination of Nutrient Broth and dextrose noted at 12 F was not observed at 16 F. The incidence of metabolic damage after freeze-drying varied with the platen temperature and the composition of the drying menstruum. No damage was detected for the 2 %-gelatin samples dried at 16 F. This is due to the fact that the number of organisms surviving 16 F in 2% gelatin was too small to apply the technique of measuring damage with any degree of confidence. Gelatin samples dried at 16 F which contained dextrose alone or in combination with Nutrient Broth exhibited a very high percentage of damage: 65 and 71 %, respectively, as compared with 24% damage noted for the sample containing only Nutrient Broth. Survival during storage for samples freeze-dried at 12 F. Survival was lowest for S. typhimurium dried in gelatin alone; after 1 week viable cells could be enumerated only in those samples stored at relative humidities of 23% or less. No viable organisms were recovered after 25 days

24 SINSKEY, SILVERMAN, AND GOLDBLITH APPL. MICROBIOL. TABLE 1. Percentage of survival and damage immediately after freeze-drying Platen temp Drying time Moisture Moisture freeze-drying after Substrate Per survival cent metabolic ~~~~~~damage F hr after a Per cent ~~~~~~~Per cent 12 2 1. 2% Gel 2.4 31 2 1.2 2% Gel + NB 18.5 31 18 1.1 2% Gel + 5% Dex 24 28 18 1. 2% Gel + NB + 5% Dex 53 15 16 15 1.3 2% Gel.9 b 15 1.7 2% Gel + NB.5 24 15 1.8 2% Gel + 5% Dex.6 65 14 1.2 2% Gel + NB + 5% Dex.6 71 a Substrate refers to initial composition; Gel = 2 g of gelatin per liter; NB = 8 g of Nutrient Broth per liter; Dex = 5 g of dextrose per liter. b No damage detected because number of surviving organisms was too small to measure for damage. z &1=- 1-2 > D 4 io-4 1-5L 1 2 3 4 5 6 7 1 2 3 4 5 6 STORAGE TIME - (DAYS) FIG. 1. Survival of Salmonella typhimurium freeze-dried at 12 F and stored under air or nitrogen at various relative humidities in gelatin. (Fig. 1). The addition of glucose or Nutrient Broth to gelatin significantly increased survival at all relative humidities and, with the exception of 75% relative humidity, the survival curves tended to become asymptotic with the storage period (Fig. 2 and 3). Dextrose was more effective than Nutrient Broth in maintaining viability, but, generally, the most protection was afforded by the combination of dextrose and Nutrientl Broth (Fig. 4). This was most evident for samples stored at the higher relative humidities. The differences between samples stored in air or in nitrogen was minor for samples equilibrated at relative humidities of and 11%, but became more significant as the relative humidity was increased. Samples stored in nitrogen at relative

VoL. 15, 1967 SURVIVAL OF FREEZE-DRIED S. TYPHIMURIUM 25 12F-AIR 12F-NITROGEN '-1 1 1zio2 11% % 11 % z -J ~~~~33% 75% 23% 12 3 4 5671 23 4 56 7 STORAGE TIME -(DAYS) FIG. 2. Survival of Salmonella typhimnuriun freeze-dried at 12 F and stored under air or nitrogen at various relative humidities in gelatin + Nutrient Broth. lo-, 23% z -J ~~33% % % 11% 11% 75% > ~~~~~12F- AIR 12F-NITROGEN ii: D U)'o- IU 1Q 2 3 4 5 6 7 1 2 3 4 5 6 7 STORAGE TIME -(DAYS) Survival of Salmonella typhimurium freeze-dried at 12 F and stored under air or nitrogen at various FIG. 3. relative humidities in gelatin + dextrose.

26 SINSKEY, SILVERMAN, AND GOLDBLITH APPL. MICROBIOL. I zi -J a: c, io-5 L- 1 2 3 4 5 6 7 1 2 3 4 STORAGE TIM E-( DAYS) FIG. 4. Survival of Salmonella typhimurium freeze-dried at 12 F and stored under air or nitrogen at various relative humidities in gelatin + dextrose + Nutrient Broth. humidities of 23 % and higher were protected to a greater extent than were samples stored in air; the largest effect was noted for those samples with glucose. Survival during storage for samples freeze-dried at 16 F. Freeze-drying at a platen temperature of 16 F not only resulted, immediately after the freeze-drying, in a survival fraction lower than that of the freeze-drying at a platen temperature of 12 F, but also in a higher death rate during storage at 2 C. In the presence of either Nutrient Broth or dextrose, the survival of S. typhimurium during storage is equivalent and appreciably higher than in samples containing only gelatin (Fig. 5 and 6). There was no survival in samples containing gelatin alone at 7 days. The highest survival for any sample was noted in the combination of Nutrient Broth and dextrose stored under nitrogen (Fig. 7). In contrast to most of the samples dried at 12 F, storage in air was highly lethal to test organisms dried at 16 F. Metabolic damage during storage. Figures 8 to 11 are representative of distinctions between protein - dominated and carbohydrate model containing - systems. The data for gelatin and gelatin plus dextrose are not presented since gelatin did not show survival for the 16 F samples, and the damage noted for the gelatin and dextrose samples was equivalent to that of samples containing gelatin plus Nutrient Broth and dextrose. There appeared to be an optimal relative humidity for damage as storage progressed, especially for those samples processed at 16 F; the optimal point occurred at low relative humidities during the later stages of storage. With one exception, storage in nitrogen, compared to storage in air, yielded a higher percentage of damaged cells in samples with a higher survival of S. typhimurium. This difference was more pronounced at the high relative humidities early in the storage period and decreased with increasing storage time. The one exception was gelatin plus Nutrient Broth at 14 days of storage, where air showed a higher evidence of damage at the lower relative humidities and none at the high. Figure 2 indicates that, for gelatin and Nutrient Broth, survival in air was essentially equivalent to survival in nitrogen. This was the only model system in this study in which this occurred. Damage appeared to be highest with Nutrient Broth and dextrose stored in air at 6% relative humidity. Whether or not there was an optimal

VOL. 15, 1967 SURVIVAL OF FREEZE-DRIED S. TYPHIMURIUM 27 z io-2 I c-j Cn - lo : \^V33% 1 I I I I I _I I I 1I I I -_ O 1 2 3 4 5 6 7 1 2 3 4 5 6 7 STORAGE TIME- (DAYS) Fio. 5. Survival of Salmonella typhimurium freeze-dried at 16 F and stored under air or nitrogen at various relative humidities in gelatin + Nutrient Broth. _o51 I I I I I I L i I I I I I 1 2 3 4 5 6 7 1 2 3 4 5 6 7 STORAGE TIME- (DAYS) STORAGE TIME- (DAYS) FIG. 6. Survival of Salmonella typhimurium freeze-dried at 16 F and stored under air or nitrogen at various relative humidities in gelatin + dextrose.

28 SINSKEY, SILVERMAN, AND GOLDBLITH APPL. MICROBIOL. ZIY 1M X F: D %% 23% ~~~75% > ~~33% IU io3 1 23 4 5 6 1 3 5 7 75% 1 2 3 4 5 6 7 1 2 3 4 5 6 7 FIG. 7. Survival of Salmonella typhimurium freeze-dried at 16 F and stored under air or nitrogen,at various relative humidities in gelatin + dextrose + Nutrient Broth. relative humidity for damage at 6% relative humidity under nitrogen storage could not be determined, since sulfuric acid could not be frozen to prevent flashing during the canning operation. In general, drying at 16 F and storing in nitrogen yielded a higher percentage of damaged organisms than did processing at 12 F although survival was less at the higher temperature. This difference was less apparent with organisms stored in air. DISCUSSION It has already been shown that various protective agents are needed to increase microbial survival after freeze-drying (4, 6). Under the conditions imposed on the model systems in this study, the relative magnitude of protection afforded by dextrose or Nutrient Broth, or both, was dependent on the temperature employed during freeze-drying. At the lower drying temperature of 12 F, dextrose alone or in combination with Nutrient Broth was highly effective in maintaining stability during storage, especially at lower relative humidities. Scott (15) investigated the possibility of there being an optimal relative humidity for survival of 16 F 9 _ 16 DAYS NITROGEN 8 -- / 8-1 (! 6 c 5 -- 14 DAYS AIR,e 4 3-2 1 I 2 II 4 16 F 45 DAYS AIR _< \ 6 2 % RH (2C) 51 DAYS NITROGEN 4 6 FIG. 8. Metabolic damage during storage of Salmonella typhimurium freeze-dried at 16 F and stored at various relative humnidities at 2 C in gelatin + Nutrient Broth. lyophilized microorganisms during storage. For Staphylococcus aureus and Pseudomonas fluorescens, dried in papain digest and stored in vacuo, survival was somewhat better at 7, 11, and 16% relative humidities than at or at 22% or higher. When Salmonella newport was dried in the same medium, the optimal relative humidity was less

VOL. 15, 1967 SURVIVAL OF FREEZE-DRIED S. TYPHIMURIUM 29 16 F 6 F so~~~~~~~ 8 / 7,- -D 14 DAYS NITROGEN 52 DAYS NITROGEN u6k - 4-14 DAYS AIR 3 2 AIR 2 4 6 8 2 4 6 8 %RH (2 C) FIG. 9. Metabolic damage during storage of Salmonella typhimurium freeze-dried at 16 F and stored at various relative humidities at 2 C in gelatin + dextrose + Nutrient Broth. % R H (2 C) FIG. 1. Metabolic damage during storage ofsalmonella typhimurium freeze-dried at 12 F and stored at various relative humidities at 2 C in gelatin + Nutrient Broth., II- O_\t38DAYS AIR 2 4 6 8 2 4 6 8 %RH (2 C) FIG. 11. Metabolic damage during storage ofsalmonella typhimurium freeze-dried at 12 F and stored at various relative humidities at 2 C in gelatin + dextrose + Nutrient Broth. clearly defined in vacuo than in air in which it was close to 2%. For S. newport dried in a buffered-salts medium or in dialyzed horse serum and stored in vacuo, optimal survival was at 22% relative humidity; in whole serum or in the dialyzable serum fraction, optimal survival in air was at 43 % relative humidity. In the presence of sucrose, the effect of variations in relative humidity was much less. When glucose or arabinose was present, survival decreased with increasing relative humidities, and when all three sugars were present, the differences between results in vacuo and in air were small. In the present study, no optimal relative humidity was noted for S. typhimurium. Survival was highest at the lower relative humidities of and 11%, and lowest at the highest relative humidity of 75 %. At the intermediate humidities of 22 and 33%, survival tended to reflect the composition of the model system, the presence or absence of air, and the platen temperature. The presence of dextrose or Nutrient Broth, or both, was necessary for prolonging the storage life of the test organism processed at both platen temperatures, and air was much more lethal to cells processed at the higher platen temperature. From the standpoint of the quality of freezedried foods, the optimal storage conditions for foods with sigmoidal isotherms, characteristic of high protein and low carbohydrate contents, indicate a relative humidity corresponding to the monomolecular-layer water content (13). However, the optimal point for survival of S. typhimurium in the model systems used in this study did not usually occur at 23%, the relative humidity corresponding to the monomolecular-layer water content for gelatin and gelatin plus Nutrient Broth. In the model systems having a nonsigmoidal isotherm (those containing dextrose), the optimal relative humidity for survival was generally at the low humidities. For foods high in sugars, Salwin (13) pointed out that optimal relative humidities for the maintenance of high quality during storage are the lowest attainable, and this was also true for the survival of S. tryphimurium freeze-dried and stored under the conditions used in these experiments. Metabolic damage in frozen menstrua was not a stable characteristic of a bacterial population exposed to sublethal stresses. Straka and Stokes (19) believe that during frozen storage damaged organisms can either undergo repair or increase in damage and that organisms which were not initially damaged will, during storage, incur damage. The method employed by Straka and Stokes (19) measures only net recoverability of damaged cells and cannot detect the behavior of individual

3 SINSKEY, SILVERMAN, AND GOLDBLITH APPL. MICROBIOL. cells during storage. A possible explanation for the increase in the damage occurring in the presence of dextrose may be found in the work of Scott (16). He postulated that carbonyl-amino reactions may be sufficient to counteract the protection normally afforded by dextrose. The addition of Nutrient Broth would, if anything, tend to favor this reaction and, in these experiments, would cause a percentage of damage equivalent to that of samples containing dextrose only. Since the nature of bacterial damage is not yet well defined, difficulties in recovery of freeze-dried cells may be anticipated and would depend upon the techniques employed (17). Other investigators (5, 1, 11, 18) have found that the quantitation of organisms exposed to stress by lethal agents can be modified by recovery techniques, and that damage appears to be a profound physiological phenomenon involving fundamental cellrecovery mechanisms (8, 12). Our current investigations, which will be reported at a later date, indicate that this is also true for freeze-dried cells. ACKNOWLEDGMENT This investigation was supported by Public Health Service research grant EF-314 from the Division of Environmental Engineering and Food Protection. LITERATURE CITED 1. ANONYMOUS. 1964. An evaluation of public health hazards from microbiological contamination of foods. Natl. Acad. Sci. Natl. Res. Council Publ. 1195. 2. BOWMER, E. J. 1965. Salmonellae in food-a review. J. Milk Food Technol. 28:74-86. 3. DAvIs, B. D. 195. Studies on nutritional deficient bacterial mutants isolated by means of penicillin. Experientia 6:41-5. 4. GREAvES, R. I. N. 196. Some factors which influence the stability of freeze-dried cultures, p. 23. In A. S. Parkes and A. U. Smith [ed.], Recent research in freezing and drying. Blackwell Scientific Publications, Oxford. 5. HARRIS, N. D. 1963. The influence of the recovery medium and the incubation temperature on the survival of damaged bacteria. J. Appl. Bacteriol. 26:387-397. 6. HELLER, G. 1941. A quantitative study of environmental factors involved in survival and death of bacteria in the desiccated state. J. Bacteriol. 41:19-126. 7. HOBBS, B. C. 1962. Salmonellae, p. 224-227. In J. C. Ayres, A. A. Kraft, H. E. Snyder, and H. W. Walker [ed.], Chemical and biological hazards in food. Iowa State Univ. Press, Ames. 8. IANDOLO, J. J., AND Z. J. ORDAL. 1966. Repair of thermal injury of Staphylococcus aureus. J. Bacteriol. 91:134-142. 9. KAREL, M., P. ISSENBERG, L. RONSIVALLI, AND V. JURIN. 1963. Application of gas chromatography to the measurement of gas of packaging material. Food Technol. 17:91-94. 1. NELSON, F. E. 1943. Factors which influence the growth of heat-treated bacteria. I. A comparison of four agar media. J. Bacteriol. 45:395-44. 11. NELSON, F. E. 1944. Factors which influence the growth of heat-treated bacteria. II. Further studies on media. J. Bacteriol. 48:473-477. 12. RECORD, B. R., R. TAYLOR, AND D. S. MILLER. 1962. The survival of Escherichia coli on drying and rehydration. J. Gen. Microbiol. 28:585-598. 13. SALWIN, H. 1962. The role of moisture in deteriorative reactions of dehydrated foods, p. 58-73. In Freeze-drying of foods. Natl. Acad. Sci. Natl. Res. Council Publ., Washington, D.C. 14. SCOTT, W. J. 1957. Water relations of food spoilage microorganisms. Advan. Food Res. 7:83-127. 15. Scorr, W. J. 1958. The effect of residual water on the survival of dried bacteria during storage. J. Gen. Microbiol. 19:624-633. 16. Scorr, W. J. 196. A mechanism causing death during storage of dried microorganisms in recent research in freezing and drying, p. 188-22. In A. S. Parkes and A. U. Smith [ed.], Recent research in freezing and drying. Blackwell Scientific Publications, Oxford. 17. SINSKEY, T. J., A. H. MCINTOSH, I. S. PABLO, G. J. SILVERMAN, AND S. A. GOLDBLITH. 1964. Considerations in the recovery of microorganisms from freeze-dried foods. Health Lab. Sci. 1:297-36. 18. STAPLETON, G. E., D. BILLEN, AND A. HOL- LAENDER. 1953. Recovery of X-irradiated bacteria at sub-optimal incubation temperatures. J. Cellular Comp. Physiol. 41:345-357. 19. STRAKA, R. P., AND J. L. STOKES. 1959. Metabolic injury to bacteria at low temperatures. J. Bacteriol. 78:181-185. 2. WILSON, E., R. S. PAFFENBORGER, J. J. FOTER, AND K. H. LEwIS. 1961. Prevalence of salmonellae in meat and poultry products. J. Infect. Diseases 19:166-171.