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Conditions for the Preservation of Gum Tragacanth Jellies*
Conditions for the Preservation of Gum Tragacanth Jellies* By ABRAHAM TAUB, WILLIAM A. MEER,t and LUCY W. CLAUSEN The implication that the effectiveness of certain preservatives for tragacanth jelly is reduced by either adsorption or chemical interaction between the gum and the preservative is subjected to further experimentation in this study. Results indicate that for the organisms selected in this study, effective preservation may be attained if adequate concentration of preservative and optimum pH range is selected. The organisms selected were Micrococcus pyogenes var. aureus, Bacillns subtilis, Eschericbia coli, and Candida albicans. The preservatives studied were a combination of methyl and propyl parabens, cholorobutanol, and benzoic acid. The optimum pH range was found to be below five, but under certain conditions even seven proved to be satisfactory.
study (1) it is concluded that in gurn tragacanth jellies buffered at i , the bactericidal activities of some conimonly used preservative agents are neutralized by the gurn either at the time of manufacture or upon storage. Since pharmaceutical jellies containing gum tragacanth have been marketed for many years with apparent satisfactory preservation, i t was the object of this study to determine under what conditions such preservation could be attained, and also to examine further the possible inactivation of the preservative agent by the gurn. The study was limited to a selection of three preservative agents in jellies buffered a t different pH’s; activity was tested against three bacterial organisms and one fungus. Gum tragacanth has been widely investigated with respect to its physical and chemical properties. A recent review (2) comprehensively covers the subject. Powdered rihbon tragacanth, U. S. P., type E 1 (Meer Corp., N. Y. C.) was used in the preparation of all jellies. The gum is a fine, white, freely flowing powder, 100% of which passes through a 150-mesh screen. I t originated in Iran from the 1956 crop. Various concentrations of gum were tested in preparing a suitable jelly. Two per cent w/w gum tragacanth was found to produce a satisfactory jelly of good consistency. The jellies prepared neither flowed too freely nor were they extremely viscous. The jellies were prepared using 5% w/w propylene glycol as a dispersing agent for the gum. N A RECENTLY REPORTED
I pH
* Received May 1, 1957, from the College of Pharmacy, Columbia University, New York, N. Y. Presented to the Scientific Section, A. Pn. A., New York Meeting, April 1957. Abstracted from a thesis submitted by William A. Meer in partial fulfillment of the requirements for the degree of &laster of Science. t Present Address: School of Pharmacy, University of Connecticut, Storrs, Conn.
This concentration was found not to exhibit any inhibitory effect on bacterial and mold growth. The use of McIlvaine’s buffer in a threefold dilution with distilled water enabled jellies ranging from pH 3 to pH i to be prepared. A combination of the methyl (0.2%) and propyl (0.05%) esters of para-hydroxybenzoic acid, benzoic acid 0.2% alone, and chlorobutano10.5yo alone, were used in the preservation of the prepared jellies. These agents were chosen due to their widespread usage and effectiveness, their general compatibility, and their nontoxicity (2-5). The role of pH in preservation was also considered. Benzoic acid is an excellent preservative in media which are strongly acidic. It is, however, much less effective in weakly acidic or neutral compositions. The optimum pH range for chlorobutanol is also on the acid side, pH 3-6. The parabens are active in acid, neutral, and alkaline media (6). Moreover, for any given bacterial culture there exists a pH range permitting growth, beyond which the hydrogen or hydroxyl ion concentration exhibits bactericidal action. Thus pH alone may impart preservative qualities. Many bacteria do not grow at pH 5 , and with very few exceptions, bacteria do not survive and grow at pH 3. This is an important consideration, for a jelly may thus be preserved for a greater length of time by the use of low pH in conjunction with a preservative ( i ) . It may, therefore, be unrealistic to base a study solely on jellies at pH 7 , especially since many tragacanth jellies containing therapeutic agents now in use are on the acid side. In order to determine the efficacy of preservative action, test organisms were added to jellies and controls in known quantities, and samples were taken a t predetermined time intervals to determine the effects of the preservative.
235
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to be necessary after preliminary testing gave erratic results showing overgrowth and contamination. Determinations of the efficacy of the various preservatives were then carried out by withdrawing samples at time intervals ranging from ten minutes to one week and inoculating in culture media. After plating, direct colony counts were taken as indications of the number of organisms present in the samples tested. In trausfer and plating operation, suitable dilutions were carried out t o insure that no bacteriostatic concentrations of preservative would be carried over into the plating medium. This was verified by the results obtained. Transfers were made in nutrient broth, and plating was accomplished by the use of a layering technique employing soft agar poured on nutrient agar. This method provided for evenly distributed colonies which remained on the surface of the agar rather than being diffused throughout, enabling more simplified and accurate counts t o be made. The nutrient broth,' soft agar, and nutrient agar' used were found to be suitable for the growth of all the test organisms. The media utilized for transfer and plating was at pH 7. The jellies were tested, buffered over a pH range of 3 to 7. This range was selected since many commercially prepared jellies employing tragacanth are on the acid side. For control purposes the effect of pH on inoculating organisms in unpreserved jellies and in aqueous solutions was also determined. I n order to determine whether a reduction in bacteriostatic activity took place in the presence of gum tragacanth, aqueous solutions having the same formulations but omitting the gum were tested. 1x1 Table I is listed the influence of pH on colony
To obtain evidence of preservation, representative contaminating organisms were chosen. Organisms naturally present in the gum or at the site of use of tragacanth jellies, in addition t o those demonstrating different bacterial characteristics were utilized. Micrococcus $yogenes var. a w e u s , Bacillzis subtilis, Eschericlzia coli, and Candida olbicans were selected as covering a fairly wide range of usage. EXPERIMENTAL The following general formula and method of preparation was employed for the jellies. Two per cent w/w of gum was wetted with 5% w/w propylene glycol arid thoroughly mixed. The diluted buffer solution containing the total amount of water and the selected preservative, when used, was then added a t one time with constant stirring until a gel formed. Duiing the entire procedure, as far as possible, aseptic conditions were maintamed. The finished jellies were translucent and free from lump formation, and the incorporation of air bubbles was a t a minimum. Uniform viscosities were obtained and the jellies were of good ointment-like consistency. Viscosity was also used as a criterion for standardizing the finished product. Viscosity measurements were taken using a Brookfield Model LVF viscosimeter with Helipath stand and spindle attachments. Jellies having a viscositv of 45,000 centipoises were used in testing. The testing procedure consisted of inoculating previously sterilized jellies and sterile aqueous control solutions with a standard number (approximately 100,000) of organisms per 10 Gm. of jelly or 10 ml. of solution. Sterilization of the jellies was found
1
Difco formulas.
TABLE I.-THE INFLUENCE OF pH
O N UNPRESERVED, STERILE JELLIES COSTAININC 2 AND ON CONTROL .4QUEOUS SOLUTIONS 1 / t Hour Jelly Sol.
Colony Count after1 Hour 1 Day Tellv Sol. Tellv Sol. . .
223 190 321 323 379
0 2 108 102 166
96 189 204 267 306
0 0 52 88 115
- 6
34 81 356 559
0
0
0
70 256 561 436
0 0
0
358 0 381 348 446 388 385 370
0
361 390 385
33 426 586 636
7
115 128 126 137 120
8 47 85 109 127
23 121 135 130 135
3 4 5 6 7
320 245 472 255 440 356 395 229 338 329
140 240 160 242 25F
7
Orranism
Micrococcus pyogenes var. aureiis 209
Bacillus subtilis 9524
Escherichia coli 9637
Cundidaalbicans 10259
a
pH .
10 Min. Jelly Sol. . .
3 4 5 6 7
231 0 267 86 345 156 374 178 391 235
3 4 5 6 7 3 4 5 6
0
0 0
7 GUM ~ TRAGACANTH,
2 Days Sol.
Jelly . .
0 49 59
0 27 57 1290 4320
5 17
0 0
0 0
0 0
0 0
540 676 520
695 693 712
127 133 740
827 952 801
1 10 1421
0 19 70 66 138
1 0 120 14 191 100 53 140 140 174
0 65
0 0
0 18
4
y
0 0 3
152 235 315 331 243
207 267 346 456 508
155 326 318 449 278
a
0
0 0
0 0
0
0 0 0 0 0
0 0
0 0
1153 0 2131 1609 0 4 "
0 0 0
a
a
a
S
"
2730 2380 4094 4261 3244
Too many organisms per plate for an accurate count to be taken: over 20,000
b No counts taken since counts at previous time period were very high.
1 Week Sol.
Telly . .
385 569 369 636 421
5780 6441 6238 5699 5292
1841 3014 2608 2385 2033
' *'
April 1958
SCIENTIFIC EDITION
counts of 2c)/, tragacanth jellies, and of control aqueous solutions, which contain no added preservatives and which have been inoculated after sterilization with selected organisms. In all the following tables the colony counts represent the number of organisms isolated from 0.1 Gm. of jelly or 0.1 ml. of solution. After examining the effect of pH on the test organisms, similar jellies and solutions, with preservatives added, were studied. Where p H alone was sufficient to inhibit growth, no experiments were set up with added preservatives a t these pH’s. ThF results obtained appear in Table 11. The results in Table I1 indicate that the presence of gum tragacanth reduces to some extent the activity of the preservatives tested. This retardant action of the gum is more noticeable a t higher pH’s. I t is evidenced by longer periods o f time necessary for inhibition of growth, and by higher colony counts than those obtained in the control aqueous solution; such reduction, however, is not sufficiently great to interfere with t&ir usefulness as preservatives of tragacanth jellies.
237
The observation that growth a t times occurs after an initial period of partial reduction also indicates that the inactivation of the preservative is a process which may be influenced b y aging. Thus experiments were set up so that preserved jellies and solutions might be tested after storage for 28 days. At the end of this period the jellies were inoculated with the specific organisms and tested similarly to the unstored jellies. The results are summarized in Table 111. The data presented thus far are in reference to sterilized jellies. Since most jellies are not usually sterilized prior t o the addition of preservatives, a series was made up to determine whether bacteriostatic agents are effective in nonsterile jellies. The results obtained are shown in Table IV.
DISCUSSION AND CONCLUSIONS The results of this study show that gum tragacanth jellies can be effectively preserved. Benzoic acid was found t o be ineffective as a
TABLE II.-THE BACTERIOSTATIC ACTIVITY OF FRESHLY PREPARED, PRESERVED, STERILE JELLIES CONTAINING 2% GUMTRAGACANTH, AND OF CONTROL PRESERVED AQUEOUSSOLUTIONS Preservative
Benzoic Acid 0.2%
Organism
Micrococcus Aureus 209 Bacillus subtilis 9524 Escherichia coli 9637 Candida Albicans 10259
ChlorobutA4icrococcus ano10.570 Aureus 209 Bacillus subtilis 9524 Escherichia coli 9637
Candida albicans 10259 Methyl 0.2y0 and Propyl 0.057, Parabens
Micrococcus aureus 209 Bacillus
subtilis 9524 Escherichia coli 9837 Cand ida albicans
10259
‘/% Hour
10 Min. Jelly Sol.
Jelly
7
208 60 320 312
204 0 267 221
5 7
153 134 138 127
135 95 129 110
4 5 7 3
129 69 214 116 163 124
96 5 185 86 112 102
pH
5
-Colony Count after1 Hour 1 Day Sol. Jelly Sol. Jelly
2 Days Jelly Sol.
1 Week Sol. Jelly
64 305
0 0
0 330
0 0
0 1275
0 0
9 19
78 90
3 11
6 4
2 12
39 49
2 8
9 0 111 73 74 100 0 0 7 0 41 52
0 21 238 0
23
0 0 34 0 0 22
127 0 153 157 86 92
0
0
0
0
0
0
0
0
211 83 0 0 0 0 20 17
234
196 0 0 0
0
0
0
0
7
141 7 161 133
20 85
0 64
5 7
203 332
204 326
0 0
33 254
0 0
16 114
0 0
0 60
0 0
0 0
0
5 7
167 114 186 186
50 41 116 108
42 47
8 22
1 9
4 4
2 3
2 5
30 30
3 3
4 5 7 3 5 7
229 0 259 5 233 213 0 0 13 0 29 0
157 0 125 0 202 192 0 0 0 0 25 0
0 0 93 0 146 101
0 17 51
0 0
0
7
0 27
0 0 0
n
o
0 10
0 0 1 2 0
5
5 7 5 7
4 5 7 3 5 7
0
Sol.
0
76
0 30
66 0 0 107 214 165 0 0
0
0
0
0
0
0
0
0
21
20
0
4922
0
0 0
0 0
20 0 0
0
0 0
0 0
0 0
0 0
143 152 155 124
2 2
2 9
2 3
2 6
1 1
1 4
3 1
2 2
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0
0 0 0 0
0
0
0 0 0 0 0 0
0
0 0 0 0
0
0 0 0 0
0
0 0 0
0 0
0 0 0
0 3887
0
3 0 9 3 5 4 0
0
0 0 0 0 0
0
0 0 0 0
JOURNAL OF
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THE
AMERICAN PHARMACEUTICAL ASSOCIATION VOl. XLVII, NO.4
TABLEIII.-THE BACTERIOSTATIC ACTIVITYO F PRESERVED, STERILE JELLIES CONTAINING 2% GUM TRAGACANTH, AND OF CONTROL AQUEOUSSOLUTIONS, AFTERTWENTY-EIGHT DAYSTORAGE PERIOD Colony Count a f t e r 1 Hour 1 Day Jelly Sol. Jelly Sol.
c
Preservative
Benzoic Acid 0.2%
Chlorobutanol 0.5%
Organism
pH
10 Min. Jelly Sol.
1/2 Hour Jelly Sol.
Micrococcus
5 7
218 135 313 335
210 83 287 227
5 7
150 141 129 132
141 102 132 121
72 94
2 27
22 75
1 4
4 5 7 3 5
7
135 190 160 0 150 155
85 159 116 0 122 127
19 62 121 140 108 106 0 0 30 97 100 99
8 0 87 118 83 75 0 0 11 0 52 777
0 11 142 0 0 23
0 0 59 0 0 62
5 7
235 329
0 86
250 318
0 0
180 324
0 0
31 254
0 0
0 134
0 0
0 120
0 0
5
7
137 124 128 122
132 118
34 93
28 57
0 19
2 8
2 2
1 2
1 I
24 18
5 6
4 5 7 3 5 7
160 1 159 20 183 220 160 97 175 135 159 137
145 0 111 0 174 212 122 91 126 127 162 125
aureus
209 Bacillus subtilis 9524 Escherichia coli 9637 Candida albicans 10259
Micrococcus aureus 209 Bacillus subtilis 9524 Escherichia coli9637
Candida albicans 10259 Micrococcus Methyl aureus 0.2y0and Propyl 209 0.05y0 Bacillus Parabens subtilis 9524 Escherichia coli9637
Candida albicans 10259
2 Days Jelly Sol.
71 0 293 113
136 5 216 121
46 0 105 0 199 118 134 96 104 82 151 172
1 Week Jelly Sol.
0 350
0 22
0 1780
0 0
5 9
1
7
28 40
1 5
0 0 0 0 171 58 0 0 0 0 20 111
0 0 88 0 140 96 171 0 100 12 163 149
0 0 0 0 257 128 0 0 0 0 1878 204
0 0 0 0 23 0 1306 0 6360 0 9953 197
0 0 11 0 73 0 172 0 845 3 941 126
0 18
0 20
0 0
0 0
0 0
0 0
0 0
0
0
0
0
0 0
0 0
5 1 3 1 1 2 1 7 147 126
3 0
2 3
2 3
0 1
5 1
1 2
6 8
3 1
23 32
6 3
0
0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
5 7
4 5 7 3 5
7
0 81
0
0 0 0 0
0 0 0 0 0 0
0
0
0
0
0 0
0 0
0 0 0
0 0 0
0 0 0 0
0
0
TABLEIV.-THE BACTERIOSTATIC ACTIVITY OF UNPRESERVED AND PRESERVED, NONSTERILE JELLIES CONTAINING 2(% GUMTRAGACANTH 7
1 Hour
3 4 5 7
13 18 15 571
5 6 4 596
6 5 7 583
Chlorobutanol0.5yo
3 4 5 7
36 24 22 25
7
4 7 9
8 6 5 7
9 Mold growth 0 1 2 4
Methyl 0.2% and Propyl o.0570 Parabens
3 4 5 7
12 16 14 17
2 3 9 4
3 4 3 5
0 2 2 3
Unpreserved jellies
3 4 5
Visible mold growth after 2 weeks
ti
Liquefaction after 2 weeks
Benzoic Acid 0.2%
7 5
Colony Count" after 1 Day 2 Days
PH
Preservative
Organism representing naturally occurring rontaminants.
28 Days
0 1
April 1958
SCIENTIFIC EDITION
preservative at pH 7 for the organisms tested, but at pH 5 it demonstrated good qualities. This was to be expected as benzoic acid at pH i is highly ionized losing much of its activity. Chlorobutanol proved to exert a measurable preservative action at pH 5 against all four organisms. At pH 7 it was effective in inhibiting three of the organisms, but in the case of Candidu albicans growth was manifested at pH 7. After 28 days storage this preservative was similarly effective against Micrococcus aureus, Bacillus subtilis, and Escherichia coli. A marked decrease in bacteriostatic activity a t pH’s 3,5 , and 7 against Candida albicans, however, was noted. This inactivation was much greater in the presence of gum tragacanth than in the aqueous control solutions. The breakdown of effectiveness at all pH’s, particularly at pH 7 , however, was most likely due to the instability of chlorobutanol especially after being subjected to autoclaving temperatures and storage. The influence of the gum on the bacteriostatic activity of chlorobutanol, which was not noticeable against the organisms other than Candida albicans, may have been due to a reduction of the concentration of this preservative to below an effective level. Enough loss of chlorobutanol may have resulted upon aging to reduce the amount of preservative to a point below bacteriostatic effectiveness against this particular organism. Gum tragacanth exerted no marked deleterious effect on the bacteriostatic activity of the parabens. It appears probable that an effective concentration of the parabens still remained in the jelly even after any possible slight adsorption by the gum. As evidenced by the tests on the nonsterilized jellies, containing only the naturally occurring organisms, effective bacteriostasis may still be maintained even after aging, in the cases of chlorobutanol and the parabens, and to a limited extent in the case of benzoic acid. The preserved sterile jellies after inoculation exhibited some killing power, but the preservative agents were mainly effective in keeping the number of organisms to a minimum. This was most noticeable in the case of Bacillus subtilis where complete destruction was never recorded. Bactericidal activity is not requisite for an agent used for the preservation of a jelly; a jelly may be well preserved by a bacteriostatic agent in adequate concentration. The fact that preservation was effective after a short period of time in nonsterile jellies, 1 to 2 days, at pH 5 is also an important consideration,
239
for industrial practices do not usually call for sterilization of jellies. Complete inhibition at pH 3 also demonstrates that self sterilization may be possible. Where jellies are to be used shortly after preparation, sterilization may be necessary, as the time of contact may be insufficient to effectively control any contaminating organisms. In most instances, however, a storage period of several hours to two days would be within the scope of standard manufacturing practices and under such conditions jellies could be preserved by the use of bacteriostatic agents alone without the need of autoclaving techniques. The effect of pH is important in preparing jellies. Of the preservatives employed, benzoic a d d proved ineffective, and chlorobutanol only partially effective at neutral pH. Since most pharmaceutical products having a tragacanth jelly base are prepared at pH’s below 7 , the selection of pH 7 for evaluating the preservative action of bacteriostatic agents incorporated in such jellies is unduly limited and may result in conclusions which may not truly reflect the usefulness of this widely applicable gum. The combination of methyl and propyl parabens was much more effective than either chlorobutanol or benzoic acid. The parabens possessed good antibacterial and antifungal activity at pH 7 and below. T o the extent that the specific organisms used in this study as well as the organisms commonly present in this natural gum are indicative of the factors causing spoilage of tragacanth jellies, it is possible to preserve the jellies against such decomposition by suitable selection of pH buffer and adequate concentration of preservative. Benzoic acid, at pH 5 or below, at a concentration of 0.2% is effective but to a lesser degree than the parabens. Chlorobutanol is only partly effective at a concentration of 0.5% a t pH 5 or below. After storage, however, it is not effective against Candida albicans. Optimum conditions were obtained with a combination of methyl and propyl parabens at a concentration of 0.2% and 0.05% respectively, a t a pH of i or below. REFERENCES (1) Bisman, P. O . , Cooper, J., and Jaconia. D.. TRIS JOURNAL, 46, 144(1957). (2) Sokol, H . . Duirn Standards. 20, 89(1952). (3) Goshorn. R. H . , Degeriog, E. F . , and Tetratall, P. A. I n d . E n g . Chcm. 30,640(11)88). ?4) Taub.. A , . a d Luckev. 32, 28 - . W. H . . THISTWJRNAL. (1943). ( 5 ) Gershenfeld, I . . , A m . J . P h n r n f . . 124, 3(iR(lY5?). (1;) Aalto. T. R . , Firman. M. C.,and Riqler, N. E.,THIS J O U R N A L , 42, 449(195R). (7) Fredenburgh, E. J., and Hecht, €3. P . , i b i d . , 40, 12B (1951).
study (1) it is concluded that in gurn tragacanth jellies buffered at i , the bactericidal activities of some conimonly used preservative agents are neutralized by the gurn either at the time of manufacture or upon storage. Since pharmaceutical jellies containing gum tragacanth have been marketed for many years with apparent satisfactory preservation, i t was the object of this study to determine under what conditions such preservation could be attained, and also to examine further the possible inactivation of the preservative agent by the gurn. The study was limited to a selection of three preservative agents in jellies buffered a t different pH’s; activity was tested against three bacterial organisms and one fungus. Gum tragacanth has been widely investigated with respect to its physical and chemical properties. A recent review (2) comprehensively covers the subject. Powdered rihbon tragacanth, U. S. P., type E 1 (Meer Corp., N. Y. C.) was used in the preparation of all jellies. The gum is a fine, white, freely flowing powder, 100% of which passes through a 150-mesh screen. I t originated in Iran from the 1956 crop. Various concentrations of gum were tested in preparing a suitable jelly. Two per cent w/w gum tragacanth was found to produce a satisfactory jelly of good consistency. The jellies prepared neither flowed too freely nor were they extremely viscous. The jellies were prepared using 5% w/w propylene glycol as a dispersing agent for the gum. N A RECENTLY REPORTED
I pH
* Received May 1, 1957, from the College of Pharmacy, Columbia University, New York, N. Y. Presented to the Scientific Section, A. Pn. A., New York Meeting, April 1957. Abstracted from a thesis submitted by William A. Meer in partial fulfillment of the requirements for the degree of &laster of Science. t Present Address: School of Pharmacy, University of Connecticut, Storrs, Conn.
This concentration was found not to exhibit any inhibitory effect on bacterial and mold growth. The use of McIlvaine’s buffer in a threefold dilution with distilled water enabled jellies ranging from pH 3 to pH i to be prepared. A combination of the methyl (0.2%) and propyl (0.05%) esters of para-hydroxybenzoic acid, benzoic acid 0.2% alone, and chlorobutano10.5yo alone, were used in the preservation of the prepared jellies. These agents were chosen due to their widespread usage and effectiveness, their general compatibility, and their nontoxicity (2-5). The role of pH in preservation was also considered. Benzoic acid is an excellent preservative in media which are strongly acidic. It is, however, much less effective in weakly acidic or neutral compositions. The optimum pH range for chlorobutanol is also on the acid side, pH 3-6. The parabens are active in acid, neutral, and alkaline media (6). Moreover, for any given bacterial culture there exists a pH range permitting growth, beyond which the hydrogen or hydroxyl ion concentration exhibits bactericidal action. Thus pH alone may impart preservative qualities. Many bacteria do not grow at pH 5 , and with very few exceptions, bacteria do not survive and grow at pH 3. This is an important consideration, for a jelly may thus be preserved for a greater length of time by the use of low pH in conjunction with a preservative ( i ) . It may, therefore, be unrealistic to base a study solely on jellies at pH 7 , especially since many tragacanth jellies containing therapeutic agents now in use are on the acid side. In order to determine the efficacy of preservative action, test organisms were added to jellies and controls in known quantities, and samples were taken a t predetermined time intervals to determine the effects of the preservative.
235
236
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
\'ol. XLVII, No. 4
to be necessary after preliminary testing gave erratic results showing overgrowth and contamination. Determinations of the efficacy of the various preservatives were then carried out by withdrawing samples at time intervals ranging from ten minutes to one week and inoculating in culture media. After plating, direct colony counts were taken as indications of the number of organisms present in the samples tested. In trausfer and plating operation, suitable dilutions were carried out t o insure that no bacteriostatic concentrations of preservative would be carried over into the plating medium. This was verified by the results obtained. Transfers were made in nutrient broth, and plating was accomplished by the use of a layering technique employing soft agar poured on nutrient agar. This method provided for evenly distributed colonies which remained on the surface of the agar rather than being diffused throughout, enabling more simplified and accurate counts t o be made. The nutrient broth,' soft agar, and nutrient agar' used were found to be suitable for the growth of all the test organisms. The media utilized for transfer and plating was at pH 7. The jellies were tested, buffered over a pH range of 3 to 7. This range was selected since many commercially prepared jellies employing tragacanth are on the acid side. For control purposes the effect of pH on inoculating organisms in unpreserved jellies and in aqueous solutions was also determined. I n order to determine whether a reduction in bacteriostatic activity took place in the presence of gum tragacanth, aqueous solutions having the same formulations but omitting the gum were tested. 1x1 Table I is listed the influence of pH on colony
To obtain evidence of preservation, representative contaminating organisms were chosen. Organisms naturally present in the gum or at the site of use of tragacanth jellies, in addition t o those demonstrating different bacterial characteristics were utilized. Micrococcus $yogenes var. a w e u s , Bacillzis subtilis, Eschericlzia coli, and Candida olbicans were selected as covering a fairly wide range of usage. EXPERIMENTAL The following general formula and method of preparation was employed for the jellies. Two per cent w/w of gum was wetted with 5% w/w propylene glycol arid thoroughly mixed. The diluted buffer solution containing the total amount of water and the selected preservative, when used, was then added a t one time with constant stirring until a gel formed. Duiing the entire procedure, as far as possible, aseptic conditions were maintamed. The finished jellies were translucent and free from lump formation, and the incorporation of air bubbles was a t a minimum. Uniform viscosities were obtained and the jellies were of good ointment-like consistency. Viscosity was also used as a criterion for standardizing the finished product. Viscosity measurements were taken using a Brookfield Model LVF viscosimeter with Helipath stand and spindle attachments. Jellies having a viscositv of 45,000 centipoises were used in testing. The testing procedure consisted of inoculating previously sterilized jellies and sterile aqueous control solutions with a standard number (approximately 100,000) of organisms per 10 Gm. of jelly or 10 ml. of solution. Sterilization of the jellies was found
1
Difco formulas.
TABLE I.-THE INFLUENCE OF pH
O N UNPRESERVED, STERILE JELLIES COSTAININC 2 AND ON CONTROL .4QUEOUS SOLUTIONS 1 / t Hour Jelly Sol.
Colony Count after1 Hour 1 Day Tellv Sol. Tellv Sol. . .
223 190 321 323 379
0 2 108 102 166
96 189 204 267 306
0 0 52 88 115
- 6
34 81 356 559
0
0
0
70 256 561 436
0 0
0
358 0 381 348 446 388 385 370
0
361 390 385
33 426 586 636
7
115 128 126 137 120
8 47 85 109 127
23 121 135 130 135
3 4 5 6 7
320 245 472 255 440 356 395 229 338 329
140 240 160 242 25F
7
Orranism
Micrococcus pyogenes var. aureiis 209
Bacillus subtilis 9524
Escherichia coli 9637
Cundidaalbicans 10259
a
pH .
10 Min. Jelly Sol. . .
3 4 5 6 7
231 0 267 86 345 156 374 178 391 235
3 4 5 6 7 3 4 5 6
0
0 0
7 GUM ~ TRAGACANTH,
2 Days Sol.
Jelly . .
0 49 59
0 27 57 1290 4320
5 17
0 0
0 0
0 0
0 0
540 676 520
695 693 712
127 133 740
827 952 801
1 10 1421
0 19 70 66 138
1 0 120 14 191 100 53 140 140 174
0 65
0 0
0 18
4
y
0 0 3
152 235 315 331 243
207 267 346 456 508
155 326 318 449 278
a
0
0 0
0 0
0
0 0 0 0 0
0 0
0 0
1153 0 2131 1609 0 4 "
0 0 0
a
a
a
S
"
2730 2380 4094 4261 3244
Too many organisms per plate for an accurate count to be taken: over 20,000
b No counts taken since counts at previous time period were very high.
1 Week Sol.
Telly . .
385 569 369 636 421
5780 6441 6238 5699 5292
1841 3014 2608 2385 2033
' *'
April 1958
SCIENTIFIC EDITION
counts of 2c)/, tragacanth jellies, and of control aqueous solutions, which contain no added preservatives and which have been inoculated after sterilization with selected organisms. In all the following tables the colony counts represent the number of organisms isolated from 0.1 Gm. of jelly or 0.1 ml. of solution. After examining the effect of pH on the test organisms, similar jellies and solutions, with preservatives added, were studied. Where p H alone was sufficient to inhibit growth, no experiments were set up with added preservatives a t these pH’s. ThF results obtained appear in Table 11. The results in Table I1 indicate that the presence of gum tragacanth reduces to some extent the activity of the preservatives tested. This retardant action of the gum is more noticeable a t higher pH’s. I t is evidenced by longer periods o f time necessary for inhibition of growth, and by higher colony counts than those obtained in the control aqueous solution; such reduction, however, is not sufficiently great to interfere with t&ir usefulness as preservatives of tragacanth jellies.
237
The observation that growth a t times occurs after an initial period of partial reduction also indicates that the inactivation of the preservative is a process which may be influenced b y aging. Thus experiments were set up so that preserved jellies and solutions might be tested after storage for 28 days. At the end of this period the jellies were inoculated with the specific organisms and tested similarly to the unstored jellies. The results are summarized in Table 111. The data presented thus far are in reference to sterilized jellies. Since most jellies are not usually sterilized prior t o the addition of preservatives, a series was made up to determine whether bacteriostatic agents are effective in nonsterile jellies. The results obtained are shown in Table IV.
DISCUSSION AND CONCLUSIONS The results of this study show that gum tragacanth jellies can be effectively preserved. Benzoic acid was found t o be ineffective as a
TABLE II.-THE BACTERIOSTATIC ACTIVITY OF FRESHLY PREPARED, PRESERVED, STERILE JELLIES CONTAINING 2% GUMTRAGACANTH, AND OF CONTROL PRESERVED AQUEOUSSOLUTIONS Preservative
Benzoic Acid 0.2%
Organism
Micrococcus Aureus 209 Bacillus subtilis 9524 Escherichia coli 9637 Candida Albicans 10259
ChlorobutA4icrococcus ano10.570 Aureus 209 Bacillus subtilis 9524 Escherichia coli 9637
Candida albicans 10259 Methyl 0.2y0 and Propyl 0.057, Parabens
Micrococcus aureus 209 Bacillus
subtilis 9524 Escherichia coli 9837 Cand ida albicans
10259
‘/% Hour
10 Min. Jelly Sol.
Jelly
7
208 60 320 312
204 0 267 221
5 7
153 134 138 127
135 95 129 110
4 5 7 3
129 69 214 116 163 124
96 5 185 86 112 102
pH
5
-Colony Count after1 Hour 1 Day Sol. Jelly Sol. Jelly
2 Days Jelly Sol.
1 Week Sol. Jelly
64 305
0 0
0 330
0 0
0 1275
0 0
9 19
78 90
3 11
6 4
2 12
39 49
2 8
9 0 111 73 74 100 0 0 7 0 41 52
0 21 238 0
23
0 0 34 0 0 22
127 0 153 157 86 92
0
0
0
0
0
0
0
0
211 83 0 0 0 0 20 17
234
196 0 0 0
0
0
0
0
7
141 7 161 133
20 85
0 64
5 7
203 332
204 326
0 0
33 254
0 0
16 114
0 0
0 60
0 0
0 0
0
5 7
167 114 186 186
50 41 116 108
42 47
8 22
1 9
4 4
2 3
2 5
30 30
3 3
4 5 7 3 5 7
229 0 259 5 233 213 0 0 13 0 29 0
157 0 125 0 202 192 0 0 0 0 25 0
0 0 93 0 146 101
0 17 51
0 0
0
7
0 27
0 0 0
n
o
0 10
0 0 1 2 0
5
5 7 5 7
4 5 7 3 5 7
0
Sol.
0
76
0 30
66 0 0 107 214 165 0 0
0
0
0
0
0
0
0
0
21
20
0
4922
0
0 0
0 0
20 0 0
0
0 0
0 0
0 0
0 0
143 152 155 124
2 2
2 9
2 3
2 6
1 1
1 4
3 1
2 2
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0
0 0 0 0
0
0
0 0 0 0 0 0
0
0 0 0 0
0
0 0 0 0
0
0 0 0
0 0
0 0 0
0 3887
0
3 0 9 3 5 4 0
0
0 0 0 0 0
0
0 0 0 0
JOURNAL OF
238
THE
AMERICAN PHARMACEUTICAL ASSOCIATION VOl. XLVII, NO.4
TABLEIII.-THE BACTERIOSTATIC ACTIVITYO F PRESERVED, STERILE JELLIES CONTAINING 2% GUM TRAGACANTH, AND OF CONTROL AQUEOUSSOLUTIONS, AFTERTWENTY-EIGHT DAYSTORAGE PERIOD Colony Count a f t e r 1 Hour 1 Day Jelly Sol. Jelly Sol.
c
Preservative
Benzoic Acid 0.2%
Chlorobutanol 0.5%
Organism
pH
10 Min. Jelly Sol.
1/2 Hour Jelly Sol.
Micrococcus
5 7
218 135 313 335
210 83 287 227
5 7
150 141 129 132
141 102 132 121
72 94
2 27
22 75
1 4
4 5 7 3 5
7
135 190 160 0 150 155
85 159 116 0 122 127
19 62 121 140 108 106 0 0 30 97 100 99
8 0 87 118 83 75 0 0 11 0 52 777
0 11 142 0 0 23
0 0 59 0 0 62
5 7
235 329
0 86
250 318
0 0
180 324
0 0
31 254
0 0
0 134
0 0
0 120
0 0
5
7
137 124 128 122
132 118
34 93
28 57
0 19
2 8
2 2
1 2
1 I
24 18
5 6
4 5 7 3 5 7
160 1 159 20 183 220 160 97 175 135 159 137
145 0 111 0 174 212 122 91 126 127 162 125
aureus
209 Bacillus subtilis 9524 Escherichia coli 9637 Candida albicans 10259
Micrococcus aureus 209 Bacillus subtilis 9524 Escherichia coli9637
Candida albicans 10259 Micrococcus Methyl aureus 0.2y0and Propyl 209 0.05y0 Bacillus Parabens subtilis 9524 Escherichia coli9637
Candida albicans 10259
2 Days Jelly Sol.
71 0 293 113
136 5 216 121
46 0 105 0 199 118 134 96 104 82 151 172
1 Week Jelly Sol.
0 350
0 22
0 1780
0 0
5 9
1
7
28 40
1 5
0 0 0 0 171 58 0 0 0 0 20 111
0 0 88 0 140 96 171 0 100 12 163 149
0 0 0 0 257 128 0 0 0 0 1878 204
0 0 0 0 23 0 1306 0 6360 0 9953 197
0 0 11 0 73 0 172 0 845 3 941 126
0 18
0 20
0 0
0 0
0 0
0 0
0 0
0
0
0
0
0 0
0 0
5 1 3 1 1 2 1 7 147 126
3 0
2 3
2 3
0 1
5 1
1 2
6 8
3 1
23 32
6 3
0
0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
5 7
4 5 7 3 5
7
0 81
0
0 0 0 0
0 0 0 0 0 0
0
0
0
0
0 0
0 0
0 0 0
0 0 0
0 0 0 0
0
0
TABLEIV.-THE BACTERIOSTATIC ACTIVITY OF UNPRESERVED AND PRESERVED, NONSTERILE JELLIES CONTAINING 2(% GUMTRAGACANTH 7
1 Hour
3 4 5 7
13 18 15 571
5 6 4 596
6 5 7 583
Chlorobutanol0.5yo
3 4 5 7
36 24 22 25
7
4 7 9
8 6 5 7
9 Mold growth 0 1 2 4
Methyl 0.2% and Propyl o.0570 Parabens
3 4 5 7
12 16 14 17
2 3 9 4
3 4 3 5
0 2 2 3
Unpreserved jellies
3 4 5
Visible mold growth after 2 weeks
ti
Liquefaction after 2 weeks
Benzoic Acid 0.2%
7 5
Colony Count" after 1 Day 2 Days
PH
Preservative
Organism representing naturally occurring rontaminants.
28 Days
0 1
April 1958
SCIENTIFIC EDITION
preservative at pH 7 for the organisms tested, but at pH 5 it demonstrated good qualities. This was to be expected as benzoic acid at pH i is highly ionized losing much of its activity. Chlorobutanol proved to exert a measurable preservative action at pH 5 against all four organisms. At pH 7 it was effective in inhibiting three of the organisms, but in the case of Candidu albicans growth was manifested at pH 7. After 28 days storage this preservative was similarly effective against Micrococcus aureus, Bacillus subtilis, and Escherichia coli. A marked decrease in bacteriostatic activity a t pH’s 3,5 , and 7 against Candida albicans, however, was noted. This inactivation was much greater in the presence of gum tragacanth than in the aqueous control solutions. The breakdown of effectiveness at all pH’s, particularly at pH 7 , however, was most likely due to the instability of chlorobutanol especially after being subjected to autoclaving temperatures and storage. The influence of the gum on the bacteriostatic activity of chlorobutanol, which was not noticeable against the organisms other than Candida albicans, may have been due to a reduction of the concentration of this preservative to below an effective level. Enough loss of chlorobutanol may have resulted upon aging to reduce the amount of preservative to a point below bacteriostatic effectiveness against this particular organism. Gum tragacanth exerted no marked deleterious effect on the bacteriostatic activity of the parabens. It appears probable that an effective concentration of the parabens still remained in the jelly even after any possible slight adsorption by the gum. As evidenced by the tests on the nonsterilized jellies, containing only the naturally occurring organisms, effective bacteriostasis may still be maintained even after aging, in the cases of chlorobutanol and the parabens, and to a limited extent in the case of benzoic acid. The preserved sterile jellies after inoculation exhibited some killing power, but the preservative agents were mainly effective in keeping the number of organisms to a minimum. This was most noticeable in the case of Bacillus subtilis where complete destruction was never recorded. Bactericidal activity is not requisite for an agent used for the preservation of a jelly; a jelly may be well preserved by a bacteriostatic agent in adequate concentration. The fact that preservation was effective after a short period of time in nonsterile jellies, 1 to 2 days, at pH 5 is also an important consideration,
239
for industrial practices do not usually call for sterilization of jellies. Complete inhibition at pH 3 also demonstrates that self sterilization may be possible. Where jellies are to be used shortly after preparation, sterilization may be necessary, as the time of contact may be insufficient to effectively control any contaminating organisms. In most instances, however, a storage period of several hours to two days would be within the scope of standard manufacturing practices and under such conditions jellies could be preserved by the use of bacteriostatic agents alone without the need of autoclaving techniques. The effect of pH is important in preparing jellies. Of the preservatives employed, benzoic a d d proved ineffective, and chlorobutanol only partially effective at neutral pH. Since most pharmaceutical products having a tragacanth jelly base are prepared at pH’s below 7 , the selection of pH 7 for evaluating the preservative action of bacteriostatic agents incorporated in such jellies is unduly limited and may result in conclusions which may not truly reflect the usefulness of this widely applicable gum. The combination of methyl and propyl parabens was much more effective than either chlorobutanol or benzoic acid. The parabens possessed good antibacterial and antifungal activity at pH 7 and below. T o the extent that the specific organisms used in this study as well as the organisms commonly present in this natural gum are indicative of the factors causing spoilage of tragacanth jellies, it is possible to preserve the jellies against such decomposition by suitable selection of pH buffer and adequate concentration of preservative. Benzoic acid, at pH 5 or below, at a concentration of 0.2% is effective but to a lesser degree than the parabens. Chlorobutanol is only partly effective at a concentration of 0.5% a t pH 5 or below. After storage, however, it is not effective against Candida albicans. Optimum conditions were obtained with a combination of methyl and propyl parabens at a concentration of 0.2% and 0.05% respectively, a t a pH of i or below. REFERENCES (1) Bisman, P. O . , Cooper, J., and Jaconia. D.. TRIS JOURNAL, 46, 144(1957). (2) Sokol, H . . Duirn Standards. 20, 89(1952). (3) Goshorn. R. H . , Degeriog, E. F . , and Tetratall, P. A. I n d . E n g . Chcm. 30,640(11)88). ?4) Taub.. A , . a d Luckev. 32, 28 - . W. H . . THISTWJRNAL. (1943). ( 5 ) Gershenfeld, I . . , A m . J . P h n r n f . . 124, 3(iR(lY5?). (1;) Aalto. T. R . , Firman. M. C.,and Riqler, N. E.,THIS J O U R N A L , 42, 449(195R). (7) Fredenburgh, E. J., and Hecht, €3. P . , i b i d . , 40, 12B (1951).