Effects of Ingredients Used in Condensed and Frozen Dairy Products on Thermal Resistance of Potentially Pathogenic Staphylococci'

Transcription

1 Effects of Ingredients Used in Condensed and Frozen Dairy Products on Thermal Resistance of Potentially Pathogenic Staphylococci' RANJIT S. KADAN, W. H. MARTIN, AND Ross MIICKELSEN Kansas Agricultural Experimnent Station, and Department of Dairy Science, Kansas State MIanhattan, Kansas Received for publication 3 August 1962 ABSTRACT KADAN, RANJIT (Kansas State University, Manhattan), W. H. MARTIN, AND Ross AIICKELSEN. Effects of ingredients used in condensed and frozen dairy products on thermal resistance of potentially pathogenic staphylococci. Appl. M\icrobiol. 11: A cell suspension of Staphylococcus aureus (196E) was injected into raw skim milk which contained different concentrations of sugar, serum solids, fat, stabilizers, and emulsifiers. The ingredient samples were exposed for the desired length of time in a constant-temperature water bath (60 C). Standard plate counts were made, and the number of surviving organisms was determined. Regression coefficients for each ingredient concentration were calculated and plotted against the per cent ingredient concentration to give an indication of protective action. Analyses of variance were conducted on bacterial counts to test the protective action of each ingredient. A comparison of the number of survivors in different sugar concentrations showed that with up to 14 % sugar all the organisms were killed within 30 min. In sugar concentrations above 14 %, the number of survivors increased regularly with each increase in sugar concentration up to 57 %, which was the maximum used. In concentrations of serum solids above 9 %, some organisms survived 35 min of heat treatment. Butter fat, stabilizer, and emulsifier did not offer any protective action in the concentrations observed. Several workers (Anzulovic, 1932; Fay, 1934; Robertson, 1927) have studied effects of various ingredients on thermal resistance of microorganisms. Those related to Staphylococcus aureus are somewhat limited. Generally, the investigators have studied the effects of the ingredients on thermal resistance by varying the concentration in media other than milk. Very few investigators have reported that protective action afforded on the thermal resistance of S. aureus by the concentration of the ingredients 1 Contribution no. 309, Department of Dairy Science, Kansas Agricultural Experiment Station, Manhattan. Material in this manuscript was presented by the senior author to partially fulfill requirements for the degree of Master of Science at Kansas State University. University, in amounts used in industry. Recenit reports (Anderson and Stone, 1955; Armijo et al., 1957; Hendricks, Belknap, and Hausler, 1959) incriminiating dairy products in outbreaks of staphylococcal food poisoninlg have called for research on the problem. This investigatioin was undertaken to measure the protection that various inigredienits used in the manufacture of condensed and frozeni dairy products have against a potentially pathogenic strain of staphylococcus. Anzulovic (1932) reported that gelatin, serum solids, and fat gave some protection to bacteria, in the order mentioned, during pasteurization. Robertson (1927) heated Streptococcus thermiiopailus, Sarcina lutea, Escherichia coli, and Mlicr ococcus aureus (Staphylococcus aur eus) in increasing percentages of sugar, and found that the concentration of sugar and of surviving bacteria increased together. However, he did not determine the maximal concentration at which the number of cells began to decrease. Fay (1934) observed a definite protective action with a hypertonic solution of sugar and dextrose that caused thermal destruction of microorganisms, including a strain of staphylococcus. In a study on thermal destruction of Escherichia coli, Read, Schwartz, and Litsky (1961) observed a lower z value (degrees F necessary to reduce thermal death time tenfold) in 40 % cream than in milk, chocolate milk, or ice cream mix. Daoust, Read, and Litsky (1961), while studying heat resistance of Corynebacteriuin diphtheriae 296 in ice cream mix, chocolate milk, and cream, found protective action in the order mentioned. MIATERIALS AND MIETHODS A known culture of a potentially pathogenic S. aureus strain (196E) was obtained from G. 1\I. Dack, Food Research Institute, University of Chicago. The culture was maintained on slants of proteose peptone by transferal every 14 days, incubation at 37 C for 24 hr, and storage at 3 to 5 C. These served as stock cultures from which the test inocula were prepared during the 2nd to 14th day of storage. The cell suspension of organism was prepared by washing 24-hr proteose-peptone plates with 15 ml of 45

2 46 6KADAN, MARTIN, AND MICKELSEN APPL. MICROBIOL. sterile physiological saline. The concentrated suspension was shaken in a bacteriological tube with a plastic cap to break the clumps. It was then filtered through sterile Whatman no. 12 filter paper to remove any suspended portions of the media. The filtrate was standardized with physiological saline to 59 % transmittance, as measured by a Bausch and Lomb Spectronic 20 colorimeter at a wavelength of 500 m,u. It was previously determined that 0.05 ml of this suspension, when injected into 2 ml of sterile milk, gave a staphylococcal population of about one million per nil of milk. Raw skim milk was used as a base. The different concentrations of sugar, serum solids, fat, stabilizers, and emulsifiers were prepared by adding the required amount of material to the milk on a weight basis. For example, 6 % sugar solution was prepared by placing 94 g of skim milk in an Erlenmeyer flask and adding 6 g of sugar. The sugar was dissolved by gentle stirring and, in the high concentrations, by warming. The percentages of sugar tested were 6, 10, 14, 25, 30, 45, and 57. The various fat emulsions were prepared by adding the required amount of prestandardized 40 %7 raw cream to the skim milk. The suspension was mixed by pouring it several times from one flask to another. A sample of the resultant suspensions was again checked for fat percentage, by the Babcock test. Three percentages of fat (6, 10, and 14) were tested. Skim milk was standardized to 20 and 30 % serum-solids concentrations by adding high-quality instant nonfat dry milk. The mixture was blended for about 10 min in a sterilized Waring Blendor. The resultant products were stored overnight in the refrigerator at 3 to 5 C to permit all incorporated air to escape. The percentages of serum solids tested were 9, 20, and 30. The stabilizers and emulsifiers were dissolved in skim milk to a concentration of 0.5%. All ingredient samples, except 20 and 30 % serumsolids concentrations, were autoclaved at 121 C and 15 psi for 15 min. The autoclaved ingredient samples were held overnight at room temperature and used the next day. Sterilized-ingredient samples (2 ml) were transferred with a continuous pipetter to bacteriological test tubes (13 by 100 mm); 40 tubes from each ingredient sample were prepared. Each tube was fitted with a sleeveless rubber diaphragm. A 22-gauge 1.5-in. needle was inserted through the diaphragm for subsequent inoculation. The needles were kept covered with steel caps until the bacterial suspension was injected. A constant-temperature, electrically heated and controlled water bath was used to heat the ingredient samples. A Cenco no Dekhotinsky mercury thermometer, in conjunction with a Cenco no relay, controlled the temperature within +0.1 C. A high-speed turbine stirrer provided constant agitation and maintained a uniform temperature in the water bath. A metal plate with several holes (14 mm diam) was installed in the upper part of the water bath. Rubber diaphragm-stoppers with diameters larger than the holes held the tubes containing the ingredient samples and, at the same time, facilitated the agitation with constant movement of water. The water level was maintained at the level of the plate. The temperature of the water bath was kept constant at 60 C throughout each trial. The rate of heating and cooling of the ingredient samples in the bacteriological tubes was determined at the beginning of the study. It was found that no ingredient sample took more than 1.5 min to reach the desired temperature. For further certainty, a tempering period of 5 min was followed throughout the study. After the desired exposure, the tubes were transferred immediately to ice water, which cooled the heated ingredient samples to about 25 C within 30 sec and to about 8 C in 90 sec. The technique for inoculation and timing described by Grosche, Lucas, and Speck (1952) was used, with some modifications. To provide more comparative data, three successive samples were run in one day. Five tubes from each concentration were transferred and tempered for 5 min. Then, by means of a tuberculin syringe, a 0.05-ml suspension of the test organism was inoculated through the 22-gauge needle. Simultaneously, a stop watch was actuated. Immediately after the inoculation, the needle TABLE 1. Effects of different concentrations of sugar in skim milk on the survival of Staphylococcus aureus (196E) at 60 C Sugar concn Initial count/ml Survivors/ml at (min) Skim milk... 1,134,000 18,590 2, Sugar (6%)... 1,351,000 13,750 2, Sugar (10%)... 1,351,000 16,060 2, Sugar (14%)... 1,351,000 28,550 2, Sugar (25%)... 1,562,000 87,550 15,310 2, Sugar (30%)... 1,620, ,100 98,000 2, Sugar (45%)... 1,082, , ,000 51,400 38,000 19,800 18,280 4,600 Sugar (57%)... 1,082, , , , ,000 91,400 77,000 69,000 Sugar (6%) + corn sugar (10%)... 1,002,000 4, Sugar (10%) + corn sugar (6%)... 1,005,000 4, Sugar (12%) + corn sugar (4%)... 1,214,000 3, Plated on Standard Plate Count agar. Results expressed as an average of 10 individual counts at each concentration.

3 THERMAL RESISTANCE OF STAPHYLOCOCCI IN DAIRY PRODUCTS VOL. 11)X was drawn from the stopper to avoid drainage of the residual inoculum into the ingredient sample. As the needle was withdrawn, the tubes were gently agitated to aid in the distribution of the inoculum. The inoculum in the second tube of the series of five was made after 30 sec. Remaining inoculations were made similarly, and ingredient samples exposed in the water bath for from 5 to 35 min. During the process of inoculation of the heating tubes, two samples of 0.05 ml of inoculum were taken for each time interval in two tubes having 2 ml of cooled ingredient sample. These tubes were kept in ice water until the time of plating. Before being plated, the contents of each tube containing 2 ml of ingredient sample and 0.05 ml of bacterial suspension were thoroughly mixed; 1 ml from each tube was serially diluted and plated. Standard Plate Count (SPC; BBL) agar was used as a plating medium except in the case of serum-solids concentration, where Staphylococcus Medium No. 110 (S-10; BBL) was used. The plates were incubated for 48 i 4 hr at 37 C. At the end of incubation, the colonies were counted and the number was recorded. Counts of the surviving organisms were averaged and are reported in Tables 1, 3, and 5. The logs of the counts of survivals were plotted on the vertical scale (Y axis) against the respective exposure time on the horizontal scale (X axis), on ordinary graph paper. The regression coefficients for each ingredient concentration were calculated, using the method of least square (Snedecor, 1955). D values (time in min required for the destruction of one log cycle or 90 % of the organisms at a given temperature) were obtained on the slope of these lines, using the method of Collins and Dunkley (1956). In cases where organisms survived 35 mim of exposure, all values from 5 to 35 inin of exposure were used in calculations. When organisms died earlier than 35 min, the values up to death of all organisms were used. Regression coefficients were plotted TABLE 2. Effect of sugar concentration on regression coefficients of log of bacterial count on time and D values Sugar concn bt SEt No. ofsob- Skim milk alone Sugar (6%) Sugar (10%) Sugar (14%) Sugar (25%) Sugar (30%) Sugar (45%) Sugar (57%) Sugar (6%) + corn sugar (10%) Sugar (10%) + corn sugar (6%) Sugar (12%) + corn sugar (4%) Compiled from Table 1. t Regression coefficient of log of bacterial count on time. I Standard error of the trend line. D against per cent ingredient concentration, to give an indication of protective action. Analyses of variance were conducted on the bacterial counts to test the protective action of each ingredient. RESULTS Effect of sugar. In Table 1 are presented the initial count and the number of organisms which survived the various heat treatments for each sugar concentration. A comparison of the number of survivors in different sugar concentrations showed that with up to 14 % sugar, all the organisms were killed within 30 min. With increases in sugar concentration in skim milk above 14 %,o the number of survivors also increased regularly and reached a maximum at 57% sugar concentration (the highest concentration used). The protective action at 57 % sugar was so marked that more than half the number of initial population survived the 35 min of exposure. It may also be noticed that with the addition of even a small portion of corn sugar, the number of survivors was greatly reduced. In a solution containing 12 % sucrose plus 4 % corn sugar, the organisms were eliminated within 20 min, whereas it took 30 min for complete destruction in 10 % sucrose solution. The regression coefficients and D values obtained from these data are presented in Table 2. -o ~~~~~~~0 0o \ O \ \ z Z Slope Sugar APs _n l wa Fa_ +0_0erums-u.U3 J Fat A PERCENT INGREDIENT CONCENTRATION FIG. 1. Effect of ingredient concentrations on regression coefficients of log of survival rate of Staphylococcus aureus (1962E) at 60 C.

4 48 KADAN, MARTIN, AND MICKELSEN APPL. MICROBIOL. TABLE 3. Effect of different concentrations of fat or serum solids in sktm milk on the-survival of Staphylococcus aureus (196E) at 60 C (Avg of 10 individual counts for each concn) Ingredient concn Plating medium Initial count/ml No. of survivors at indicated times (min) Skim milk alone.... SPC agar 1,134,000 18,590 2, Fat (6 %).... SPC agar 1,316,000 8, Fat (10%).... SPC agar 1,316,000 5, Fat (14%).... SPC agar 1,316,000 4, Serum solids (9 %)... S-llOt 960,000 1, Serum solids (20%)... S-11O 960,000 3, Serum solids (30%)... S-11O 960,000 10, Standard Plate Count Agar (BBL). t Staphylococcus Medium No. 110 (BBL). It may be observed in Table 2 that the regression solids concentration. The b and D values obtained from coefficient (b value), with concentrations above 6 %, these data are shown in Table 4. decreased with the increase in sugar concentration, signify- A comparison of the b and D values in the case of serum ing that an increased sugar concentration made the solids showed that b values decreased; the D values trend line less steep. When b values were plotted with their increased with the increase in serum-solids concentration. corresponding sugar concentrations (Fig. 1), they showed The b values are plotted against their respective serumthat the protective action increased linearly with the solids concentrations in Fig. 1. Analysis of variance of the increase in sugar concentration. The D values also showed three concentrations showed a significant effect from similar results, though the relationship was not linear; serum-solids concentration. The gradual and appreciable they increased at an increasing rate with the corresponding decrease in b values and a significant serum-solids consugar concentration after a 14 % sugar concentration was centration effect indicate that increasing concentrations of reached. No obvious reason is offered for the greater D serum solids apparently protect this strain of staphyvalue for skim milk alone and the lower D values for lococcus. 6, 10, and 14 % sugar. The analysis of variance on the TABLE 4. Effect of serum solids or fat concentrations on different sugar concentrations also showed a strong sugar- b and D values concentration effect. It is evident, therefore, that the I protective action of sugar starts at or about 14 %, in- Ingredient concn medium vations creases consistently with the increase in sugar concentration, and is maximal at 5 Skim milk alone.. SPC agar Effect of serutm solids. The three serum-solids concen- Fat (6%).. SPC agar trations studied were 9, 20, and 30%. The data on staphy- Fat (10%).. SPC agar lococci are shown in Table 3. The number of survivals Fat (14%).. SPC agar increased in serum-solids concentrations. The organisms Serum solids (9%). S-1O were killed in 30 min with 9 % serum solids, whereas it Serum solids (20%) S-1O took 35 min in 20 % serum solids, and they were not S completely eliminated even after 35 min in a 30 % serum- Compiled from Table 3. TABLE 5. Effect of stabilizers or emulsifiers in skim milk on the survival of Staphylococcuts aureus (196E) at 60 C Stabilizers or emulsifiers Initial count/ml Survivors/ml at indicated times (min) Skim milk... 1,134,000 18,590 2, Dariloid kb (0.5%)... 1,079,000 15,911 1, Egg yolk (0.5%)... 1,079,000 13, Gelatin (0.5%)... 1,022,000 11, Gelox (0.5%)... 1,440, S-105 (0.5%)... 1,440,000 3, Mixifier (0.5%)... 1,024,000 6, Pectin (0.5%)... 1,024,000 3, Tween Moss (0.5%)... 1,024,000 1, Velvatex (0.5%)... 1,440,000 7, Vesterine (0.5%)... 1,440,000 7, Plated on Standard Plate Count agar. Results expressed as average of five individual counts for each ingredient.

5 VOL. 11, 1963 THI,'ERM\IAIL RESISTANCE OF STAIPHYLOCOCCI IN DAIRY PRODItCTS 4i9 Effect of fat. The number of sturviving organisms in three concentrations of fat are shown in Table 3. Bacterial counts decreased with increases in fat concentration, with no surrvival after 20 min. The b and D values for these concentrations are given in Table 4, which also shows a slight, gradual increase in b values and a decrease in D values with increases in fat concentration. The b values plotted against fat concentrations (Fig. 1) showed that increasing fat concentrations were increasingly lethal to the organisms. Analysis of variance of the three concentrations failed to show a significant effect and confirmed the supposition that increasing concentrations of fat have no protective action. Effect of stabilizers and ecmiulsifiers. The stabilizers and emulsifiers were studied in only 0.5 % concentration. Their effect on the survival of this strain of S. aureus is showvn in Table 5. In all samples except one, the staphylococci were eliminated within 25 min. The number of survrivals in all stabilizers and emulsifiers was fewer than in skim milk alone. No statistical analysis was necessary to show that the products sttudied offered no protective action. DISCUSSION Among the various ingredients studied, sugar in high concentration gave the greatest protective action on the survival of S. aureus (196E); however, below about 14% sugar concentration, thermal resistance was less than in skim milk alone. Earlier workers (Fay, 1934; Robertson, 1927) studied the protective action of sugar in media other than milk. Their findings of protective action are only partly confirmed by the results of this study. That equtiimolal solutions of different sugars do not have the same protective action agrees closely with Fay's (1934) findings. Serum solids were found to be next to fat in their protective action. The results were not exactly comparable because a different recovery medium (S-1O) was employed. The medium was reported to be inhibitory to heatshocked staphylococci by Busta and Jezeski (1961). That fat offered no protective action but tended to increase thermal destruction with increasing concentrations is contrary to the concept of Anzulovic (1932), but agrees with results by Read et al. (1961) and Daoust et al. (1961) for Escherichia coli and Corynebacteriunt diphtheriae, respectively. The very low thermal death rates in some of the stabilizer and emulsifier trials may result from changes during autoclaving of these products. For example, samples of Dariloid kb, Gelox, and S-105, after autoclaving, were destabilized and separated into serum and solid portions. Of necessity, some conditions of this investigation differed from those encouintered in commercial practices; for example, autoclaved skim milk was used as a base in place of raw skim milk. Autoclaving may change the nature of the milk. The autoclaved milk was used becauise no suitable recovery meditum was available for heatshocked staphylococci. The S-110 medium was reported by Busta and Jezeski (1961), and also found to be inhibitory in preliminary investigations. However, no significant difference in protective action, for this strain of staphylococcuis, between autoclaved and unautoclaved milk was observed. The ph of milk tends to decrease after atutoclaving; it is assumed that the ph relationship wouild be similar to commercial preheating of skim milk. Secondly, the possibility of aerosol formation during inoculations and uneven mixing of the inocula with the ingredient samples in the test tube could also cause variations in results. However, each test with a different concentration was repeated a number of times with conisistent resuilts, so these factors appear to be of minor significance. ACKNOWLEDGMENT These studies were suipported in part by grant E3157 from the National Institutes of Health, U.S. Public Health Service. LITERATURE CITED ANDERSON, P. H. R., AND 1). M. STONE Staphylococcal food poisoning witth spray-dr ied milk. J. Hyg. 53: ANZUI.OVIC, J. V A study of Escherichia coli in ice cream. J. Bacter iol. 23: ARMIJoR R., 1). A. HENDERSON, R. TIMOTHEE, AND H. B. ROBI NS'ON Food poisoning outbreaks associated withi spray-dried mnilk, and epidemiologic study. AmlA. J. PUb. Health 47: B31TSTA, F. F., AND J. _. JEZESKI Effect of sodiumii clhlolride concentration in an agai- media on growth of heat shocked Staphylococcus aurleuis, strain IIo. 196 E. J. Dairy Sci. 44:1160. COLIAINS, E. B., AND W. L. 1)ITNKLEY A critical look at thle pasteurization standards. J. Milk Food Technol. 19: i)aoust, D. R., R. B. REAI), JR., AND W. LITSKY Thlermal studies of pathogenic bacteria in milk and various milk products. I. Corynebacteritim diphtheriae ATCC no J. Dairy Sci. 44: FAY, A. C The effect of hypertonic sugar solution on thiermal resistance of bacteria. J. Agr. Res. 48: GIROSCHE, C. A., H. L. LuCAS, AND M. L. SPECK Pasteurization requiiremlents for concentrated wlhole milk. J. l)airy Sci. 35: HENDRICKS, S. L., R. A. BELKNAP, AND W. J. HAIUSLER, JR Staphlylococcus food imttoxication due to cheddar cheese. J. Milk Food Technol. 22: ItEAD, R. B., JR., C. SCHWARTZ, ANI) W. LITSKY Studies on the rnal destruction of Escherichia coli in milk and milk products. Appl. Microbiol. 9: ROBERTSON, A. H Thlermophilic and thermoduric microorganisms with special references to species isolated from milk. II. The theimal resistanice of tmicroorganisms. Vt. Agr. Exptl. Sta. Buill. 274:1-27. SNEDECOR, G. W Statistical nmethods, 5tlh ed., p , Iowa State tjniverslty Press, Amnes.