EFFECIS OF CARBOHYDRATES, PROTEINS, AND BACTERIAL CELLS INTHE HEATING MEDIA ON THE HEAT RESISTANCE OF

CLOSTRIDIUM SPOROGENES

MIKIO AMAHA AND KIN-ICHIRO SAKAGUCHIDepartment of Agricultural Chemistry, University of Tokyo, Bunkyo-ku, Tokyo, Japan

Received for publication March 8, 1954

It is well known that the heat resistance ofbacterial spores is influenced markedly by thenature of the medium in which the spores areheated. This fact is an important problemrelating to the practical sterilization in bac-teriology and in the food industry. Hencevoluminous data on the heat resistance ofClostridium botulinum and Clostridium porogenesin various kinds of foods or food juices have beenpublished (Townsend et al., 1938; Reed et al.,1951; Reynolds et al., 1952). From these data,it can be easily pointed out that the resistancevalue of a strain in a food is larger than that inphosphate buffer, whereas in another food theresistance value of the same strain is smallerthan that in neutral phosphate buffer. Althoughthe pH of the heating media has a marked in-fluence on the thermal death rate of bacteria,it appears that the cause of such a difference inresistance values of a strain cannot be attributedwholly to the variation of pH values of the foods(Sognefest et al., 1948).In fact, there are evidences that some of the

ingredients of food might exert a protectiveeffect or a death accelerating effect for the or-ganism during the heating. Weiss (1921) reportedthat the longer survival time was obtained whenthe spores of C. botulinum were suspended andheated in the foods with the higher sugar con-centration. The similar protective effect of sugarson the bacterial spores has been demonstratedby Anderson et al. (1949) and Sugiyama (1951).Murray and Headlee (1931) found that thepresence of peptone in the heating medium hadshown sometimes a protective effect for thespores of C. tetani. Recently, Kawai and Tanaka(1952) have shown that several kinds of highermolecular substances, such as gelatin, starch,gum arabic, and alginic acid, have the protectiveeffects for the spores of Bacillus mesentericusand Bacillus brevis. The test organisms used bythese workers are relatively less resistant thanthe most resistant food spoilage bacteria, and

the kinds of the substances tested on each strainare rather limited. Thus it seemed important tostudy the effects of various substances on themost resistant strain.Using the spores of Cameron's putrefactive

anaerobe 3679 (Clostridium sporogenes, ATCC7955), the effects of various carbohydrates, pro-teins, and bacterial cells were studied. In theprevious papers Amaha (1952a,b, 1953) hasclearly indicated that the thermal death timeof spores of certain strains is influenced mark-edly by the nature of the recovery media. Ingeneral the more favorable was the nature of amedium for the development of heated spores,the longer was the survival time. Hence, itshould be considered that the substances addedin the basal heating medium have the two stagesin their action to the spores; the first, during theheating time, and the second, after the substanceswere transferred in the subculture medium. Inthis paper an attempt was made to distinguishthe two effects, i.e., the first effect, a true protec-tion, and the second effect, a false protection, ofeach of substances tested.

MATERIAL AND METHODS

Strain. A putrefactive anaerobe, N.C.A.3679 (listed by the American Type CultureCollection as C. sporogenes, ATCC 7955), wasobtained from the National Canners Associa-tion. This strain is one of the most resistantamong the food spoilage bacteria, and its re-sistance values are used frequently as the basisof standardizing the thermal processing ofnonacid canned food (Stumbo, 1949).

Culture medium. A liver broth prepared in thefollowing way was used as the sporulating me-dium and the basal subculture medium. Thechopped beef liver was added to the nutrientbroth of pH 7.0 in the amount of 330 g of liverper liter of broth. The mixture was heated to100 C and allowed to boil gently for one hour.The boiled material was filtered through a filter

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paper until the broth was brought up to 1,000ml. To the broth two g of glucose and one g ofdipotassium phosphate were added, and thepH was adjusted to approximately 7.2. In tub-ing, one-half inch of the previously boiled groundbeef liver was introduced into each tube beforeadding the broth. The tubes were autoclavedfor thirty minutes at 121 C. The final pH aftersterilization was about 7.0.

Preparation of spore suspension. The tubesof medium prepared as described above wereinoculated with 0.2 ml of the stock culture ofthe test organism. The inoculated tubes wereheated then for 15 minutes in a boiling waterbath to remove the entrapped air and subse-quently incubated at 30 C for a period of 30days. The resulting culture was strained asepti-cally through a gauze pad to remove liver par-ticles, and then centrifuged. The supernatantbroth was decanted, and the residual spore cropwas suspended again in sterile phosphate bufferof pH 7.0. The suspension was recentrifuged towash, and again the supernatant was decantedand suspended in the phosphate buffer. Thenthe spore suspension was filtered through afilter paper (no. 2 paper, Tohyo-Roshi Co.)to remove spore clumps. The filtrates (sporesuspensions) then were heated for 5 minutesin boiling water to kill the vegetative cells andstored in a refrigerator at 3 to 4 C. The sporeconcentration was determined by a haemacy-tometer (Thoma)bA stock spore suspension which contained

12.4 million spores per ml and 7.8 million (heatkilled) vegetative cells per ml was used through-out this work. It was confirmed that no ap-preciable variations in heat resistance occurredduring the storage for 60 days in the case of thisstrain of C. sporogenes.Detmination of heat revsitance. It has long

been known that the thermal death time ofthe spores of aerobic and anaerobic bacteria varieswith the spore concentration, having a definiterelationship between them (Amaha and Saka-guchi, 1951; Amaha, 1953). Therefore, all experi-ments were performed in a concentration of1 X 104 spores per ml of the heating medium.The basal heating was the sterile neutral phos-phate buffer. The test substances were dissolvedin the buffer and the pH of the solution (suspen-sion) was adjusted to 7.0 by the small amount ofa dilute solution of HCl or NaOH. An aliquotof the stock spore suspension was added to the

solution' prepared as above to give a final con-centration of 1 X 104 spores per ml.One ml of the suspension was pipetted into

each of the thermal death time tubes; these wereof hard gla, 8 mm outside diameter, 7 mminside diameter, and about 70 mm in length.The tubes then were sealed with an oxygenflame, and sets of 20 to 30 tubes were placed ina basket, made of copper screening, for the heattreatment. The basket was immersed completelyin a constant temperature oil bath (=0.15 C)equipped with a power stirrer. A correction of3.5 minutes was allowed for the lag period forthe contents of the tubes to reach the tempera-ture of the heating bath (115 C).At intervals of either 3 or 4 minutes, the

basket was opened and tubes in groups of fourwere removed from the bath and immediatelyimmersed in an ice water bath. The cooled tubeswere wiped clean, flamed, and opened; and thetube contents then were transferred asepticallyto a freshly exhausted tube of liver broth. Thebroth was covered then with thioglycolate agarand the tubes were incubated at 30 C. Growthwas evidenced by the gas production and thecharacteristic odor. Since one ml of heatingmedium is transferred to a liver broth of about9 ml, the final concentration of each test sub-stance in the subculture medium is about one-tenth of that in the heating medium.

Differentiation of the true protective effect and thefalse protective effect. In order to differentiatethe true and the false protective effects of theadded substances, the following two experimentsof each of the test substances were conductedfurther in parallel with the first experimentdescribed above. The second experiment wasas follows. The 1 X 101 spores of P.A. 3679were suspended and heated in the neutral phos-phate buffer (without any added substance)and then transferred into the liver broth to which

1 These media containing the test substancesdid not need further sterilization before addingspores since it could be proved that the samesurvival times could be obtained with and withoutthe previous sterilization of the media to whicheach of such substances as starch, glucose, pectin,and albumin was added. However, this fact will berather exceptional, which seems due to the extra-ordinary resistance of the test organism P.A. 3679compared with that of the other bacteria which areusually brought into the medium by the testsubstances.

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the same amount of the test substances as inthe first experiment had been added aseptically,and were incubated at 30 C. If the test sub-stance exerts its effect only in the subculturemedium stimulating the growth of the heatedspores and so gives rise to an elongation of thesurvival time, the same length of the survaltime ought to be obtained in both of the firstand second experiments.The third experiment is the control in which

the 1 X 104 spores of P.A. 3679 were heated inthe phosphate buffer and then subcultured inliver broth without adding any substance. Thiscontrol should attain to a survival time of 25minutes at 115 C as reported in a previouspaper (Amaha, 1953). When this control valuecould not be obtained, all the data in parallelwere abandoned, and the experiments were con-ducted anew. Comparing the three survival timevalues thus obtained, it could be determinedeasily whether the added substance acts pro-tective in the heating medium (this is a trueprotective effect), or the substance exerts agrowth stimulating effect in the subculturemedium (this is a false protective effect), or thesubstance has no effect on the spores.

RESULTS

Effects of carbohydrates. The effects of presenceof various kinds of carbohydrates on the survivaltime of P.A. 3679 spores were examined. Con-trary to the several workers' results cited beforethat the higher concentration of sugars has theprotective effects, the presence of sucrose, glucose,and glycerol in concentrations from 10 to 50per cent in the heating medium did not affectthe survival time of spores of P.A. 3679. Theother carbohydrates such as pectin, gum arabic,glycogen, and dextrin with a concentration of2.5 per cent in the heating medium also did notshow any protective effect. Only the presence ofstarch in concentrations from 0.25 to 2.5 percent in the heating medium has elongated slightlythe survival time from 25 minutes (control) to28 minutes. But the same length of survival timecould be attained when the spores were heatedin the phosphate buffer without any added sub-stance and then subcultured in the liver brothto which the same amount of starch had beenadded previously.

Consequently, it was decided that the effect ofstarch was not a true protective effect which

should be exerted on the spores during theheating but was a false protective effect whichwas not exerted in the heating but exerted inthe subculture on the heated spores. Foster andWynne (1948) have found a spore-germinationaccelerating effect of starch for C. botulinumand have proved that the starch adsorbed theunsaturated fatty acids which were inhibitivefor the germination of the spores. Therefore,the effect of starch in this experiment similarlymight be a growth stimulating effect for theheated spores of P.A. 3679.

Reynolds and Lichtenstein (1950) have re-ported that the addition of 100 ppm i-ascorbicacid to the heating medium elongated the sur-vival time of Ciostridium sp. In this experiment,however, the presence of i-ascorbic acid asmuch as 2,500 ppm did not show any effect.Sugiyama (1951) indicated that the spores ofC. botulinum could acquire a stronger heat re-sistance when they were held in 50 per centsucrose solution for a few miniutes. In the presentexperiment, though the spores of P.A. 3679were held in 50 per cent sucrose solution for 10minutes to 15 hours in an icebox, any variationin the survival time of 1 X 10' spores could notbe demonstrated.

Effects of proteins, peptones, amino acids, anda nucleic acid. In this experin;ent, seven pro-teins, two peptones, two amino acids, a mixtureof eighteen amino acids, and a yeast nucleicacid were tested with concentrations of 0.25 to5.0 per cent of the heating medium. The re-sults are shown in table 1.When each of the proteins was added to the

heating medium in a concentration of 2.5 percent, gelatin, casein (from milk), globulin(from egg), glutelin (from wheat), and fibrin(from blood) did not show any effect for thesurvival times of 1 X 104 spores of P.A. 3679,but both of the albumins, serum albumin andovalbumin, showed a marked effect of elongat-ing the survival time, giving the followingsurvival times, 40 minutes and 36 minutes,respectively.

Likewise, two peptones, polypeptone (TakedaCo.) and Arei-peptone (Kyoei Seiyaku Co.), weresimilarly effective in elongating the survivaltime when they were present with a concentra-tion of 2.5 per cent in the heating medium. How-ever, the effects of albumins and peptonesdecreased as their concentrations in the heating

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TABLE 1Effects of adding protein, peptone, amino acid, or nucleic acid to the heating medium on

the survival time of P.A. 3679 spores(10,000 spores per ml)

SURVIVAL TIM AT 115 C

KIND 01 SUJBSTANCE ADDEDCONCENTRATION When added to the When added to theSKFsTANDEsD heating medium subculture medium

Survived j Killed Survived Killed

per cen min min

None (phosphate buffer only) 25 28Gelatin 2.5 25 28 25 28Casein from milk 2.5 25 28 25 28Glutelin from wheat 2.5 25 28 25 28Fibrin from blood 2.5 25 28 25 28Globulin from egg 2.5 25 28 25 28Serum albumin (Merck) 2.5 40 45 25 28

1.0 35 38 25 280.25 32 35 25 28

Ovalbumin (Merck) 2.5 36 40 25 281.0 30 33 25 280.25 25 28 25 28

Polypeptone (Takeda) 5.0 35 38 25 282.5 35 38 25 281.0 25 28 25 28

Peptone (Arei) 2.5 35 40 25 281.0 25 28 25 28

Yeast nucleic acid (Kirin) 2.5 32 35 25 281.0 32 35 25 280.25 25 28 25 28

L-Glutamic acid 2.5 25 28 25 281.0 25 28 25 28

DL-Alanine 1.0 25 28 25 28Mixture of eighteen amino acids 2.0 22 25 22 25

* The figures in this column show the concentration in the heating medium. In the subculture me-dium, the concentration of each substance was about one-tenth of these figures.

medium decreased. Serum albumin and oval-bumin gave the following smaller survival timesin the tested two concentrations of 1.0 and 0.25per cent: serum albun, 35 and 32 minutes;ovalbumin, 30 and 25 minutes, respectively.The effectiveness of the peptones disappearedwhen their concentration in the heating mediumwas reduced to 1.0 per cent. The effect of an

yeast nucleic acid (Kirin Brewing Co., proteinfree, mol wt - 30,000 to 56,000) also was tested.

When the yeast nucleic acid was added to theheating medium with the concentration of 2.5and 1.0 per cent, the elongated survival time of32 minutes was obtained. But when the con-

centration was reduced to 0.25 per cent; theelongating effect disappeared. In the comparisonof these results, it can be said that serum al-

bumin is the most effective in elongating thesurvival time of P.A. 3679 spores.On the other hand, when the spores of P.A.

3679 were heated in plain phosphate buffer andthen subcultured in the liver broth which con-tained the same amount of each of the aboveeffective substances and was sterilized previouslyby autoclaving for 10 minutes at 15 poundssteam presure, the survival times obtained didnot indicate any increase compared with thecontrol value of 25 minutes at 115 C. Therefore,the effect of albumin, peptone, and nucleic acidcan be thought to be a true protective effectwhich exerts during the heating period. Additionof 1.0 to 2.5 per cent L-glutamic acid and alsothe addition of 1.0 per cent DL-alanine did notshow any effect. However, when a mixture of

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TABLE 2Effects of the addition of living and dead cell8 to the heating medium on the survival

time of P.A. 8679 spores(10,000 spores per ml)

SURVIVAL TIM AT 115 C

SPECIS O TlE CONCENTRATION OF CELLS When added to the When added to theORGAISMS ADDED ADDED, NO. OF CELLS PR ML heating medium subculture medium

Survived I Killed Survived I Kied

min minNone (phosphate buffer) _ 25 28

Torula utilis 1 X 107 living cells 25 28 255 X 107 living cells 38 40 25 28

- 5 X 107 killed cells 25 28 25 28S X 107 killed cells* 38 40 25 28

Escherichia coli 1 X 10' living cells 25 28 25 284 X 109 living cells 25 28 25 28

Staphylococcus aureus 1 X 109 living cells 25 28 25 284 X 109 living cells 32 35 25 28

Pseudomonas fluorescen8 4 X 109 living cells 25 28 25 28

Sarcina lutea 1 X 109 living cells 25 28 25 284 X 109 living cells 32 35 25 284 X 109 killed cells 25 28 25 28

Proteus vulgaris 4 X 109 living cells 25 28 25 28Spores of Bacillus natto 1 X 109 living spores 25 28

3 X 109 living spores 35 38 25 283 X 109 killed spores 25 28 25 28

* Killed by grinding with sand in a mortar.

eighteen amino acids (the composition of whichis shown in the previous paper, Amaha andSakaguchi, 1952) was added with a concentra-tion of 2.0 per cent to the heating medium, thesurvival time of P.A. 3679 spores decreasedslightly from 25 minutes to 22 minutes. Sincethe same shortened survival time was obtainedwhen the spores were heated in phosphate bufferand then subcultured in the liver broth to whichthe same amount of the amino acid mixturehad been added, this effect of the mixture seemedto be due to its slight inhibitive effect duringthe subculture for the development of the heatedspores.

Effects of the presence of bacterial cells. Lange(1922) found that the survival time of staphylo-cocci and Escherichia coli subjected to heat canbe doubled or even tripled by the addition ofheat killed cells of the same species. The similarprotective effect of dead celLs for living cells

also was observed by Behrens (1923), Htickel(1926), and Kimata and Nito (1934). All of thesedata, however, were of nonsporeforming bac-teria. Recently, Sugiyama (1951) reported thatthe addition of large numbers of living vegeta-tive cells of C. botulinum, either heat killed orunheated, and the addition of heat killed sporesof the same strain did not affect the heat re-sistance of C. botulinum spores. Experimentswere conducted to examine the effect of thepresence of the bacterial cells of the other speciesupon the survival time of P.A. 3679 spores. Thefive aerobic nonsporeforming bacteria werecultured on nutrient agar slants for 3 days atthe optimum temperature for the growth ofeach. The spores of BaciUus natto (a variant ofB. subtil) were harvested from the nutrientagar slants after incubating for 7 days at 37 C.Cells of Torula utilis were obtained by culturingthe organism on "Koji" agar slants for 3 days

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at 30 C. The cells obtained were suspended inneutral phosphate buffer, centrifuged and re-

suspended in the buffer, and then the cell con-

centration of each suspension was determinedby means of a haemacytometer. To the livingcell suspensions the spores of P.A. 3679 were

added so as to give a concentration of 1 X 104spores per ml, and the survival times of thespores were measured at 115 C. At first, theeffects of the presence of 1 x 108 cells of theorganisms were tested. But no effect could beobserved except with the cells of torula whichis about 100 times as large in cell volume as thecells of the other five noneffective bacteria.Then, increasing the concentration of living cellsto be added to the heating medium to a very

large concentration, as large as several billioncells per ml, the protective effects for P.A.3679 spores were demonstrated by the cells ofthe following three gram positive bacteria,Staphylococcus aureu-s (Micrococcus pyogenes var.

aureus), Sarcina lutea, and spores of Bacillusnatto. Three gram negative bacteria, E. coli,Pseudomonas fluorescenm, and Proteus vulgaris,with the same order of cell concentration in theheating medium did not show any effect. Theresults are shown in table 2. Although the effec-tiveness of gram positive bacteria and ineffec-tiveness of gram negative bacteria were thusapparently indicated in these experiments, itseemed not to be said conclusively from theserather few examples. The effects of the presence

of heat killed cells also were examined. Theliving cells of Torula utilis and Sarcina luteaand living spores of B. natto were killed by heat-ing in neutral phosphate buffer for 5 minutes at90 C, for 10 minutes at 100 C, and for 15 minutesat 120 C. The presence of the heat killed cellsin the heating medium in the concentrationswhich were found to be effective in case ofliving cells of each of these organisms did notshow any effect.However, the yeast cells which were killed

by grinding thoroughly in a mortar were foundto be as effective as the living cells (see table2). The protective effects of these living cellscan be admitted as the true protection sincewhen the spores of P.A. 3679 were heated inthe phosphate buffer and then suboultured inthe liver broth to which the same amount ofliving cells effective in elongating the survivaltime of P.A. 3679 spores was added and wassterilized previously by autoclaving, any varia-tion in the survival time of the spores was notobserved as compared with the control.

Effects of the pre8ence of heat denatured pro-teins. In the previous experiments, albuminshave shown a marked protective effect at 115 C,but the albumins appeared to have been de-natured (coagulated) already in the heatingmedium at the time when the temperature ofthe medium had reached to 115 C. Hence, thealbumins previously heat denatured also seemedto have the protective effects. To clarify this

TABLE 3Effects of the presence of heat denatured proteins in the heating medium on the survival

time of P.A. 8679 spores(10,000 spores per ml)

CONDITION OF NEAT SURVIVAL TIM AT 115 C RATIO OFPROTEINS TREATMENT OF TEE NITROGEN

PROTEIN SURVIVAL Survived Killed CONTNT*

mm ~per cc,$None (control) _ 25 28Serum albumin, 2.5%native 40 45 100heat denatured, I at 80 C for 5 mn 35 38heat denatured, II at 120 C for 15 min 32 35filtrate at 120 C for 15 min 25 28 2

Ovalbumin, 2.5%native 36 40 100heat denatured at 120 C for 15 min 28 32filtrate at 120 C for 15 min 25 28 17

* Determined by Kjeldahl method.

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point, the effects of the denatured proteinsprepared as follows were tested. The 5 per centsolutions of serum albmin and ovalbumin inphosphate buffer (pH 7.0) were heated for 5minutes at 80 C or for 15 minutes at 120 C.The two kinds of the coagulum of each albuminwere resuspended in the phosphate buffer afterthey were ground in a mortar. In these suspen-sions the survival time of 1 X 101 spores ofP.A. 3679 per ml was measured at 115 C. Thepresence of 2.5 per cent serum albumin de-natured by heating for 5 minutes at 80 C or byheating 15 minutes at 120 C has shown protectiveeffects giving an elongated survival time of 35and 32 minutes, respectively. The similar pro-tective effects, though less marked, were demon-strated also by the presence of the denaturedovalbumin. The results are shown in table 3.On the other hand, the filtrates which were

obtained from the heat coagulated albuminsolutions and might include some of the effectivesubstances also were tested. But no protectiveeffects were observed.

All these data indicate clearly that the heatdenatured albumins, though their effectivenessdecreases with increasing degree of the heattreatment, also have the protective effects forP.A. 3679 spores. This fact will be of importancein the processing of foods, especially of fish andmeat products.

DISCUSSION

It can be concluded that among the variouskinds of substances tested only the four, albumin,peptone, nucleic acid, and living cells of grampositive bacteria, have the true protectiveeffect for the 1 X 101 spores of P.A. 3679 in theheating medium. The protective effect of sugarsagaint heat destruction which had been observedby many workers for the various organisms couldnot be demonstrated in this paper for the sporesof P.A. 3679. But it might be possible that amaterial which exerts a protective effect for thenonresistant forms of bacteria or for the rela-tively weak ones does not have the protectiveeffect for the more resistant bacteria as P.A.3679. Such might be the case of casein, pectin,dextrin, and sugars.The amount of each of the effective substances

needed for elongating the survival time of 1 X104 spores of P.A. 3679 in the neutral phosphatebuffer at 115 C, from 25 minutes (control) toabout 35 minutes, was as follows: Serum al-

bumin, native, 1.0 per cent (35 min); serumalbumin, heated for 5 min at 80 C, 2.5 per cent(35 min); ovalbumin, native, 2.5 per cent (36min); peptone, 2.5 per cent (35 mim); nucleicacid, from yeast, 1.0 per cent (32 min); livingcells of Torula utili8, 5 X 107 cells per ml (36mi); living cells of Sarcina lutea, 4 X l10 cellsper ml (32 mim); living spores of BaciUus naUo,3 x 109 cells per ml (35 min); living spores ofP.A. 3679, 1 X 106 cells per ml (35 min). Asdescribed in detail in a previous paper (Amaha,1953), the same length of survival time (35min) can be obtained when the spore concentra-tion of P.A. 3679 in the heating is increased to1 X 106 spores per ml. The facts seem to givesome clue to the cause of increasing surv'ivaltime with the increasing spore concentration.

In the previous papers, Amaha (1952a,b,c,1953) has confirmed that the cause of death ofspores by heat is the destruction of a proteinmolecule in the cell which controls each met-abolic system to synthesize cell materials fromthe nutrients given. The mechanism of protectionby the three effective substances, albumin,peptone, and nucleic acid, and by living bacterialcells can be assumed tentatively as follows.The substance added in the heating mediumdenatures with the heating, consuming in partthe energy of activation (or the activated watermolecules) which otherwise directly gives riseto the denaturation of protein molecule(s)of the spores, and consequently the rate of thedenaturation of the protein molecule(s) of thespore cells, to some extent, decreases and soresults in a longer survival time of the spores.This supposition seems to be supported by thefact that the protective effect of albumin de-creased when the albumin had been previouslyheat denatured and also by the fact that thebacterial cells previously heat killed were lostof their protective effects, but the cells killed bygrinding could show the same protective effectas the living cells.

SUMMARY

The effects of the presence of various sub-stances and bacterial cells in the heating mediumupon the thermal death time of 1 X 101 sporesof Clostridium 8porogenes (P.A. 3679) werestudied. Among the nine carbohydrates tested,only starch showed a slight effect in elongatingthe survival time of the spores. But the effectof starch was not a true protective effect.

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Among the nitrogen compounds tested,albunmin, peptone, and nucleic acid showed atrue protective effect, but the protective effectsdecreased or disappeared as their concentrationwas reduced from 2.5 per cent to 0.25 per cent.The presence of the living cells of some of theother bacteria also could exert a protectiveeffect. However, a very large concentration ofliving cells, as large as 4 X 109 cells of Staphylo-coccus aureus or 5 X 107 cells of Torul utilisper ml, was needed to elongate the survival timeof 1 X 104 spores of P.A. 3679. The heat killedcells of the same species did not show the pro-tective effect. The heat denatured albumin wasfound to have also the protective effect, thoughit was not so marked as the native albumin.The mechanism of the protective action ofeffective substances was discussed.

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AMAHA, M. 1952b Studies on the heat resistanceof bacterial spores. 4. Kinetics of the deathreaction of spores by heat. J. Agr. Chem. Soc.Japan, 26, 339-346.

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REED, J. M., BOHRER, C. W., AND CAMERON, E. J.1951 Spore destruction rate studies onorganisms of significance in the processing ofcanned foods. Food Research, 16, 383-408.

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