Effects of Sub-Lethal Heat-Shock, i -Alanine, and L-Alanine on Germination and Subsequent Destruction of Bacillus Spores by Pasteurization 1

J. H. MARTIN and P. W. BLACKWOOD Dairy Science Department, University of Georgia, Athens 30601

Abstract

The purpose of this investigation was to stimulate the germination of Bacillus cereus and Bacillus licherdformis spores with sub-lethal heat-shock and chemical germinants so the germinated spores might be destroyed by conventional pasteurization. Heat-shock was at 65, 75, and 85 C with no hold. Effects of L-alanine, fl-alanine, and a combination of heat-shock plus either amino acid were also determined.

fl-Alanine alone stimulated germination of some spores of both organisms in skim- milk, but its effectiveness was much less than that of n-alanine. Heat-shock alone was more effective as a germination stimulant than was fl-alanine, and a combination of the two produced no more germination than either alone, n-Alanine alone or combined with heat-shock caused almost complete germination and subsequent destruetion of the spores by pasteurization, especially i f the treated product was held 15 min at 37 C.

Introduction

Modern technology has progressed in the development of new equipment and technics to improve milk processing. However, total destruction of heat resistant spores without undesirable heat-induced changes in some of the milk components is still a problem.

Bacterial spores can germinate and complete a transition from spore to vegetative state with loss of heat resistance (1), and to induce this transition would be a simple way to solve the problem of spores in dairy foods. However, it has been reported that in any population of dormant spores incubated in a suitable germina- tion environment, a small number of the spores fail to germinate (4), and i t is these "super dormant" spores which preclude the develop-

Received for publication April 30, 1971. 1Journal Series Paper 1056. University of

Georgia College of Agriculture Experiment Stations, College Station, Athens.

ment of a method for low-heat sterilization of dairy foods.

I t is possible, however, to stimulate loss of heat resistance in much of a spore population in a dairy product by the addition of certain amino acids (7). Also, the stimulatory effect of sub-lethal heating on spore germination is known (2, 3). The cumulative effect of such treatments might be sufficient to allow con- ventional processing procedures to reduce spores to insignificant levels in dairy foods. Therefore, this study was undertaken to evaluate the effects of sub-lethal heat-shock, fl-alanine, L- alanine, and the cumulative effect of various combinations thereof on the germination and subsequent destruction of germinated spores of B. cereus and B. licheniformis by low-heat pasteurization.

Experimental Procedure

Selection of organisms and preparation of spores. Bacillus cereus and B. licherdfornvis were selected because of their prevalence in raw milk (6). Stock cultures of vegetative cells of the organisms were maintained by semi- monthly transfers in trypticase soy broth. When spores were needed for an experiment, 1% of the stock culture of vegetative cells was inoculated into trypticase soy broth and in- cubated for 18 hr at 37 C. The 18-hour vegeta- tive cells were then inoculated (2 ml) onto 125- ml nutrient agar slants containing 1.0% starch and 0.01% manganese sulfate (7). During incubation at 37 C, approximately 95% sporu- lation of both organisms occurred within 7 to 9 days. Extent of sporulation was verified microscopically by observing crystal violet stained smears from the slants daffy after the first 5 days. Spores were harvested from the slants as previously described (7).

Stanc~ardization of spores. Spore suspensions of B. cereus in 0.01 ~ phosphate buffer (pH 7.2) were standardized turbidimetrically to 30% light transmittance in a Bausch and Lomb Spectronic-20 C olorimeter at wavelength 625 m~. This provided suspensions of approxi- mately 50 million spores/ml. F o r B. licherd- formis, a wavelength of 725 m/z and 58%

577

5 7 8 ~ARTIN AND BLACKWOOD

transmittance provided suspensions of approxi- mately 100 million spores/ml. Spore suspen- sions were refrigerated at 4.4 C until used.

Determination of effects of heat-shock, fl- alanine, and L-alanine. Standardized spore sus- pensions were inoculated (1 ml) into 50-ml portions of 10% solids reconstituted skimmilk which had been autoclaved for 6 rain at 121 C. Buffer controls of 50-ml were inoculated at the same time. fl-Alanine or L-alanine were added immediately after heat-shock at 0.1%. Heat- shock was at 65, 75, and 85 C with no holding time in test tubes immersed in a water bath. The time to reach the desired temperature was ap- proximately one minute in all tubes. When the samples had reached the heat-shock temperature, the tubes were immediately placed in ice water and cooled to 37 C. The amino acid was added at 37 C.

Controls consisted of : a) buffer plus spores, and b) skimmilk plus spores. With the ex- ception of heat-shocking or incorporation of the respective amino acid or both, controls were treated exactly like the experimental samples. The buffer control was included to detect any spontaneous germination, and the skimmilk con- trol to verify germination which might be in- duced by the constituents of skimmilk. Ex- perimental samples were: a) skimmilk plus spores plus B-alanine, b) skimmilk plus spores plus L-alanine, c) skimmilk plus spores, which was subsequently subjected to heat-shock, d) skimmilk plus spores, which was subsequently heat-shocked followed by addition of B-alanine, and e) skimmilk plus spores, which was sub- sequently heat-shocked followed by addition of L-alanine.

Total spore counts were first made on the inoculated sample with plate count agar (Difco) plus 0.1% starch (7). Three samples were then heat-shocked, fl-Alanine and L-alanine were added to each one of the heat-shocked samples at 0.1%. Plate counts were made to determine the initial count. Then, 10-ml portions of each of the controls and samples were placed into sterile screwcapped test tubes and laboratory pasteurized in a water bath at 63 C for 30 min, and the number of spores destroyed by pasteurization immediately after the treatments was determined by plate counts of survivors. The remaining portions of the 50-ml aliquots were incubated at 37 C for 15, 30, and 60 rain. At the end of each incubation period, portions were laboratory pasteurized and plate counts of survivors determined the effects of pas- teurization af ter incubation under conditions conducive to spore germination. At least 4 JOURI~AL OF DAIRY SOIEI~CE VOW. 55, NO.

trials were completed with each treatment and average values are reported.

Results

The data in l~igure 1 show the effect of heat- shock at 65 C in conjunction with fl-alanine and L-alanine on the subsequent destruction of spores of B. cereus by pasteurization at 63 C for 30 min. Evidently, some components of the reconstituted skimmilk stimulated germination, because af ter 60 rain at 37 G, 69.7% of the spores were destroyed by pasteurization in the skimmilk control which received no heat-shock and to which no germinant was added. Heat- shock alone followed immediately by pasteuri- zation resulted in destruction of 20.9% of spores in skimmilk, and 73.2 and 80% were destroyed af ter 30 and 60 rain of incubation at 37 C. Immediately after addition of fl-alanine, 10% of the spores were destroyed by pasteuri- zation, whereas af ter incubation of the skimmilk containing fl-alanine for 60 rain at 37 C, approximately 75% of the spores were de- stroyed by pasteurization. The inclusion of both heat-shock and fl-alanine did not increase destruction, and after 30 and 60 rain of incuba- tion, pasteurization effectiveness actually de- creased in comparison to the heat-shocked samples. L-alanine alone resulted in 55% destruction by pasteurization immediately after addition of a 0.1% concentration, and L-aIanine in combination with heat-shock at 65 C caused 80% of the spores to be destroyed by pasteuri- zation immediately after treatment. During 60 rain of incubation, more than 99% of the spores became heat sensitive and were destroyed by low-temperature pasteurization. During the en-

/

IR u,r 7 T ~ i 0 15 50 45 60

INCUBATION T IME (MIN.) Fro. 1. Effect of fl-alanine, T,-a]anine, and

heat shock at 65 C on destruction of B. cereus spores by conventional pasteurization. A, Heat shock; B, fl-alanine; C, heat shock plus fl-alanine; D, L-alanine; E, heat shock plus L-alanlne; F, skimmilk control; and G, buffer control.

S P O R E S U R V I V A L AND P A S T E U R I Z A T I O N 579

tire experiment, none of the spores in the phosphate buffer control were destroyed by pasteurization, revealing that destruction of spores in the milk samples resulted from the treatments.

Since B. licheniformis is one of the most common sporeformers in milk (6), the second par t of the study was concerned with the effects of heat-shock, fl-alanine, and L-alanine on the heat resistance of spores of this organism. The results obtained by heat-shock at 65 C are in Figure 2.

The results with B. licheniformis spores were somewhat different from those reported for B. cereus. For example, in none of the experi- ments with fl-alanine and heat-shock did the percentage destruction by pasteurization reach 70%, whereas with spores of B. cereus, de- struction was consistently in this range after 60 min at 37 C. As was the ease with B. cereus, a limited amount of germination was stimulated by constituents of the reconstituted skimmilk, resulting in 24.6% destruction by pasteurization af ter incubation of the spore-containing milk for 60 rain at 37 C. However, this is less than one-third the destruction with spores of B. cereus under the same conditions. Pasteuri- zation immediately after heat-shocking produced almost 25% destruction, and this percentage increased to 50.6 and 51.5% af ter 30 and 60 rain.

fl-Alanine alone did not stimulate much germination of spores of B. licheniforn~is initially nor during the early stages of incuba- tion, with less than 10% of the spores destroyed by pasteurization immediately after addition of fl-alanine, and only 27.7 and 34.4% destruction after 15 and 30 rain of incubation. However, af ter 60 rain of incubation, the response to

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FIG. 2. Effect of fl-alanine, L-alanine, and heat shock at 65 C on destruction of B. ~iehe~ifor¢~is spores by conventional pasteurization. A, Heat shock; B, ~-a]anine; C, heat shock plus fl-alanine ; D, L-alanine; E, heat shock plus L-a]anine; F, skim- milk control; and G, buffer control.

B-alanine was approximately the same as for heat-shocking. A combination of both ~-alanine and heat-shock resulted in 65.4% destruction of B. lichen~formis spores by pasteurization after 60 rain at 37 C. L-Alanine alone caused loss of heat resistance of about 30% of the spores immediately af ter addition to the spore-con- taining milk, and of more than 90% throughout the remainder of the incubation period. L - Alanine coupled with heat-shock at 65 C resulted in more than 99% of the spores being de- stroyed by low-temperature pasteurization. The effect was evident immediately, and no change occurred during 60 rain of incubation at 37 C.

Similar experiments with both B. cereus and B. Iiehe~iformis were conducted at heat-shock temperatures of 75 and 85 C with essentially the same results as at 65 C.

Discussion Results from this study reveal that sub-

lethal heating and small quantities of chemical germinants stimulate germination and thereby predispose some of the spores of both B. cereus and B. liche~dformis to subsequent destruction by low-heat pasteurization. To destroy most of the spores from these two species during pasteurization of raw milk would minimize problems which might result from sporeformers in pasteurized dairy foods, since these organisms are the predominant species in raw milk (6).

Extensive germination of B. licheniformis spores can be induced in phosphate buffer by L-alanine or L-cysteine (7), and in our study, r,-alanine stimulated a similar response in skim- milk. However, i t is doubtful that either of these germinants would be economical in milk processing because of their high cost. This study also revealed that one of their more economical analogs, fl-alanine, also stimulated germination, but was somewhat less effective than L-alanine. Apparent ly the position of the amino group on the fl-carbon alters the ability of the analogous structure to induce spore germination.

Heat-shock combined with L-alanine resulted in almost complete germination of spores of both organisms in skimmilk, especially if the spore-containing milk was held for 15 min or more at 37 C. However, this cumulative effect was not observed with heat-shock and fl-alanine. Germination resulting from fl-alanine alone was no greater than from heat-shock alone, and a combination of the two decreased germination in some instances. This agrees with results re- ported by Keynan, et al. (5), that heat-shock does not enhance the germination induced by all germination stimulants. However, incorporation

JOURI~A T, OF DAIRY SCIEI~CE ~OL, 55, NO, 5

580 ~[ARTIN AND BLACKWOOD

of germinat ion stimulants and pre-heat ing milk at some germinat ion s t imulat ing tempera ture before pasteurizat ion may be justified in milk processing, especially in ul tra-high tempera ture pasteurized products where pract ical steri l i ty is desirable. Results of this s tudy indicate that such treatment would at least be par t ia l ly suc- cessful in reducing the spore content of such products.

Acknowledgment The technical assistance of Mrs. Lesley Ogrosky

is gratefully acknowledged.

(3) Evans, F. R., and H. R. Curran. 1943. The accelerating effect of sub-lethal heat on spore germination in mesophilic aerobic bacteria. J. Baeteriol., 46: 513.

(4) Gould, G. W., A. Jones, and C. Wrighton. 1968. Limitations of the initiation of germination of bacterial spores as a spore control procedure. J. Appl. Bacteriol., 31: 357.

(5) Keynan, A., W. G. Murrel], and H. O. Halvorson. 1961. Diplcolinic acid content, heat activation, and the concept of dormancy in the bacterial endospore. Nature, 292: 1211.

References (1) Campbell, L. L., J r . 1957. Bacterial spore

germination---definitions and methods of study. Spores. p. 33. H. O. Halvorson, ed., Amer. Inst. Biol. Sci., Washington, D.C.

(2) Curran, H. R., and F. R. Evans. 1945. Heat activation inducing germination in the spores of thermotolerant and thermophilic aerobic bacteria. J. Bacteriol.. 49: 335.

(6) Martin, J. H., W. J. Harper, D. P. Stably, and I. A. Gould. 1962. Sporeforming microorganisms in selected milk supplies. Proc. XVI Int. Dairy Congr., Copenhagen, p. 295.

(7) Martin, J. H., and W. J. Harper. 1963. Germination response of Bacillus lichcni- formis spores to amino acids. J. Dairy Sci., 46: 663.

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