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The Preservation of Aqueous Preparations Containing Nonionic Surfactants I.*
442
JOURNAL OF THE
AMERICANPHARMACEUTICAL ASSOCIATION
VOl. XLVI, NO. 7
TABLE IIT.-STERILIZATIONOF VENOSETS WITH ETHYLENE OXIDEVAPOR Process Time, HI.
Temperature, F.
EtOp Gas Pressure in Atmosphere
2
73
0 50
78
4
+
= contaminated,
-
Plate Count, Spores/Strip Cap Adaptor Assembly Hood
0.50 =
Sterility Test Cap Adaptor Assembly Hood
-
293
0 0 0 0
203 300 0
-
++ + -
sterile; 0 = no organisms detected.
tained with contaminated Venosets t o establish the minimum process time provides a margin of safety. The larger sterilization equipment which was subsequently set up for production-scale operation was designed to operate with a controlled pressure of ethylene oxide vapor of 0.5 atmosphere. Since the smaller experimental equipment used more ethylene oxide, 0.63 t o 0.73 atmosphere of pressure, two additional experimental trials were made with a gas pressure of 0.5 atmosphere to duplicate plant conditions. The experiments were conducted in the same manner as before. A summary of these results is shown in Table 111. The data indicates that under the prescribed conditions a sterilization time of four hours is adequate t o achieve sterility in the Vcnoset. In actual production practice, however, a longer process time has been found more convenient and is currently in use. Two additional safety features pertain under production-scale operations. The Venosets normally carry a much lower degree of contamination than was used in these studies. Secondly, the marked sterilizing effect of
thc ethylene oxide adsorbed on the plastic and the package is operative in the plant process.
REFERENCES (1) Anonymous Chcm. Week., 7 5 , 96(0ctober 2, 1954). (2) Cotton, R. T., and Roark, R.C., Ind. Eng. Chcm., 20, 805( 1928). (3) Engley, F. B., Jr., Bact. Proc., p. 22 28(1951). (4) Ginsberg, H. S., and Wilson. A. T., PYOL. SOL.Erptl. B i d . Mcd. 73,614(1950). (5) Griffith, C. I.., and Hall, L. A,, U. S. Patent 2,189,947 (1940). (6) Gross, P. M., and Dixon, L. F., U. S. Patent 2,075,845 11”77\ \ ‘ m u‘ , .
(7) Kaye, S., Irminger, H. F.. and Phillips, C. R.. J . Lab. Cli7r. M c d . , 40, 67(1952). ( 8 ) $aye, S.,rbid., 35, 823(1950). (9) klarenbeek, A,, and Tongern, H. A,. J . H y g . (London) 52,525(1954). (10) Phillips, C. R., Bacleriol. Proc.. p. 23 23(1950). ( I I ) Phillips, C. R., and Kaye, S., Am. J. H y g . , 50, 270 (1949). (12) Sagen, H. E., A m . Phariir. hf/gs. Assoc., Sci. Coxf. Proc., p. 87 87(1954). (13) Schrader, H., and Bossert, E., U. S. Patent 2,037,439 114Wi)
( t t ) Reddish, G. F., “Antiseptics. Disinfectants, Fungicides, and Chemical and Physical Sterilization,” Lea and Febiger, Philadelphia, 1954, p. 647. (15) Roberts J. L. Allison L. E. Prickett, P. A,. and Riddle, K . B , j . Bact;riul., 45,’40(19i3).
The Preservation of Aqueous Preparations Containing Nonionic Surfactants I.* Growth of Microorganisms in Solutions and Dispersions of Nonionic Surfactants By MARTIN BARR and LINWOOD F. TICEt Studies have shown that various microorganisms, including Pse~domonnsaeruginosa, Aspergillus niger, Peniciliium notaturn, and Monilia albicans are capable of growing in solutions and dispersions of nonionic surfactants of the fatty acid ester type. It was observed that these organisms are capable of splitting the ester Iinkages of these surfactants. This is first evidenced visually by the appearance of free fatty acids in the inoculated surfactant solutions. Further study revealed that these test organisms produce esterases which split the fatty acid from the surfactant molecule. The presence of esterases was demonstrated using a tributyrin testing procedure. The liberation of free fatty acid from polyoxyethylene 2 0 sorbitan monostearate (Tween@60) solutions IS reported. SURFACTANTS of t h e sorbitan partial ester type’ and their polyoxyethyl-
ONIONIC
*Received August 27 1956, from the Department of Pharmacy, Philadelphia College of Pharmacy and Science. This work was supported by a research grant from the Atlas Powder Company, Wilmington, Del. t The authors wish t o thank Dr. Louis Gershenfeld and Dr. Bernard W i t h of the Department of Bacteriology, Philadelphia College of Pharmacy and Science, for their supgestions during this study. Atlas Powder Company, Wilmington, Del
ene derivatives have found wide and increasing use in various products. Because of their unique properties, they have found wide applications as emulsifiers, solubilizers, dispersants, detergents, and as spreading, wetting, and antifoaming agents. Engler (1) and Bolle and Mirimanoff ( 2 ) have reported that certain nonionic surfactants promote t h e growth of molds. N o studv has as
July 1957
443
SCIENTIFIC EDITION
TABLE I.-TI[E GROWTH OF MICROORGANISMS IN 1 AND 5 9 w/v SOLUTIONS AND DISPERSIONS OF NONIONIC SURFACTANTS OF THE SORBITAN PARTIAL ESTERA N D POLYOXYETHYLENE ESTERTYPES __ Control"
Surfactant
No inoculum.
+ = growth of microorganisms observed.
aersginosa
..
Polyoxyethylene 20 sorbitan monolaurate Polyoxyethylene 20 sorbitan monopalmitate Polyoxyethylene 20 sorbitan monostearate Polyoxyethylene 20 sorbitan monooleate Sorbitan monolaurate Sorbitan monopalmitate Sorbitan monostearate Sorbitan monooleate Polyoxyl 40 stearate Q
Psendomonas
.. ..
..
.. ..
.. .. .. s
+s
+" ++ +++" $8
Monitia aibicans
+" +" +" +" ++ ++ +"
Asfiergillns
nigev
+" ++"" +++" ++ +a
Penicillircm nolatrim
+* +s
+" ++ ++ +s
+s
= splitting of ester observed.
ruplicate. Two tubes of each inoculated surfactant solution or dispersion were placed in an incubator (37") and two similar tubes were stored a t room temperature (25" f lo). All tubes were observed for a period of two months for evidence of growth. The results are expressed in Table I. Examination of Table I reveals that the four microorganisms utilized in this study are capable of growing in all the surfactants employed. Growth was evidenced in the surfactant solutions and dispersions within several days after inoculation. It was noted that the microorganisms are capable of splitting the molecules of some of the surfactants, all of which are esters. The splitting of the ester EXPERIMENTAL was determined by visual examination of the inocuGrowth of Microorganisms in Aqueous Solutions lated solutions for free fatty acid formation. Thus, ester splitting was observed in solutions of the polyand Dispersions of Nonionic Surfactants.-The oxyethylene sorbitan fatty acid esters and polyoxyl purpose of this study was to determine whether various microorganisms are capable of growing in 40 stearate by the appearance of insoluble lauric, aqueous solutions and dispersions of the nonionic palmitic, stearic, or oleic acid, depending on the acid surfactants of the sorbitan partial ester type. One present in the ester. Since the sorbitan partial fatty and 5y0w/v solutions or dispersions of the following acid esters (Span products) are not soluble in water, nonionic surfactants were prepared and inoculated it was not possible t o observe visually ester splitting with the test organisms: polyoxyethylene 20 sor- by the presence of insoluble fatty acids in these susbitan monolaurate,2 polyoxyethylene 20 sorbitan pensions or dispersions. Acid number determinations were carried out on non no palm it ate,^ polyoxyethylene 20 sorbitan monostearate,* polyoxyethylene 20 sorbitan mono- the surfactant solutions and dispersions before and oleate.5 sorbitan monolaurate,6 sorbitan monopalmi- after sterilization and the determinations indicated tate.? sorbitan monostearate,s sorbitan mono- no change in the free fatty acid concentrations. All sterile controls remained stable for the duration of ole at^,^ and polyoxyl 40 stearate.1° The latter substance is not of the sorbitan partial ester type the test period. Experiments were carried out in which the numbut is a polyoxyethylene ester made by direct reaction of fatty acid with alkylene oxide. It was ber of organisms in the inoculum were reduced and, included in this study, however, as it is recognized in in others, where washed organisms were used as the U. S. P. XV (3) and is the surfactant in Hydrophilic inoculum. Results indicated that the organisms Ointment (4). To 10 ml. quantities of the sterilized were capable of growing in the surfactant esters solutions and dispersions, previously sterilized at and splitting them. The rate of growth and ester 121.3" for twenty minutes and contained in test splitting was reduced, however, when the number tubes, were added 0.1 ml. of a 24-hour thioglycollate of organisms in the inoculum was reduced and when broth suspension of Pseudomonas aeruginosa" or a washed organisms were used as the inoculum. A Quantitative Determination of the Splitting of 72-hour thioglycollate broth suspension of Monilia albicans,ia Aspergillus niger,I3 and Penicillium Tributyrin a s a Result of Esterase Activity.-Since notatum.'4 All inoculations were made in quad- the microorganisms employed in this study are known t o produce esterases (€I), i t was decided to a Tween@ 20; 8 Tween@ 40; ' Tween@ 60; 5 Tween@ 80; carry out an experiment in order t o obtain further 6 Span@ 20. 7 Span@ 40. * Span@ 6 0 . 9 Span@ 80; 10 Myrj@ information on the role esterase production by 52; registeied trademark names of the Atlas Powder Company. microorganisms plays in the splitting of molecules 11 Pseudomonas aeruginosa, isolated from a patient at the of the surfactant. An experiment was, therefore, University of Pennsylvania Hospital. 12 Monilia albicans, isolated from a patient at Children's devised t o measure quantitatively the free acid reHospital, Philadelphia. leased from an ester, tributyrin, during organism 13. 14 Asaergillus nigev and Penicillium notatum, isolated from contaminated products in the laboratory of the Departgrowth. ment of Pharmacy, Philadelphia College o f Pharmacy and The three organisms studied in this experiment S ci eac e.
yet been reported o n the growth of microorganisms other t h a n molds in solutions and dispersions of nonionic surfactants of the fatty acid ester type. No mention has been made of t h e splitting of the molecules of the nonionic surfactants by microorganisms. Such a study, is reported in P a r t I of this paper. The effectiveness of various substances as preservatives i n products containing these surfactants is reported in P a r t 11.
444
JOURNAL OF THE
140-
120
AMERICANPHARMACEUTICAL ASSOCIATION
----
Psbudomonas asrugin osa ------Aspsrgillus nigsr -- Honilio olbicans
f
-
VOl. XLVI, NO. 7
t --.-
'0°
0
4
5 Y. Surfoctont- broth inoculum SY. Surtoc?ont- wosnsd inoculum I % Surtoclont- brolh inoculum IY. Surfoctont- washed inoculum
8
12 16 20 doyr a f t e r inoculofion
A
24
28
Fig. 2.-The splitting of polyoxyethylene 20 sorbitan monostearate (Tween@60) by Aspergillzis niger. days
Fig. 1.-The
after
inoculation
esterase activity of three organisms on tributyrin.
* MEQ NaOH required t o neutralize free acids
due to the esterase activity of the organisms on 2 ml. of a 50y0 v/v tributyrin emulsion (1 ml. tributyrin).
were Pseudomonas aeruginosa, Monilia albicans, and A spergillus niger. The production of esterases by the strains of organisms employed was confirmed using the test method of Silver ( 5 ) which is based on Anderson's observation (6). The principle of the experiment was to titrate the free fatty acid liberated from a tributyrin emulsion which had been inoculated with the organisms under consideration. The procedure was as follows: A 50% v/v emulsion of tributyrin was prepared using the following formula:
.
Tributyrin16. . . . . . . . . . . . . . . . 50% Nutrient broth.. . . . . . . . . . . . . . 25y0 1% Methyl cellulose 4000. . . . . 2570 The emulsion was prepared with the aid of a Waring Blendor. Two-milliliter quantities of this emulsion were transferred to test tubes and sterilized in a n autoclave. Inocula consisting of 0.1 ml. of a 24hour nutrient broth culture of Pseudomonas aeruginosa or a 72-hour nutrient broth culture of Monilia nlbicans and Aspergilliu niger were placed in their respective tubes. One day after inoculation and on alternate days for a period of eleven days, each tube was titrated with 0.01091 N NaOH to a phenolphthalein end point after the addition of 10 ml. of neutralized alcohol. The milliequivalents of sodium hydroxide required t o neutralize the fatty acid released from 1 ml. of tributyrin (contained in 2 ml. o f a 5oy0 v/v emulsion) were calculated and are reported in Fig. 1. Uninoculated controls were titrated with the alkali solution and it was found 15
* MEQ NaOH required to neutralize the free acids due to the esterase activity of Aspergillus niger on 25 ml. surfactant solutions.
The Matheson Company, Inc., East Rutherford, N. J.
that 25.1 milliequivalents o f sodium hydroxide were necessary to neutralize 2 ml. of the 50y0 tributyrin emulsion. It is apparent from the examination of Fig. 1 that Pseudomonas aeruginosa, Aspergillus niger, and Monilia albicans are capable of producing esterases which result in the splitting of tributyrin. In this study, the bacterium, Pseudomonas aeruginosa, appeared to be a greater producer of esterase than molds. The faster initial rate of esterase production by bacteria is probably due to the fact that bacteria are faster initial growers than the moldsthe greater the number of organisms, the greater the esterase production and, hence, the faster the ester is split. The Splitting of Polyoxyethlene 20 Sorbitan Monostearate by Aspergillus niger.-In an effort t o understand better the phenomena which occur when microorganisms are inoculated into surfactant solutions and dispersions, a study was carried out to determine quantitatively the splitting of polyoxyethylene 20 sorbitan monostearate by Aspergillus niger. I t was decided to study quantitatively the effect of the test organism on 1 and 57' w/v aqueous solutions of the surfactant, both with and without other added nutrient. This was done in order t o determine what roles the concentration of surfactant and other nutrient play in the splitting of the surfactant. Twenty-five milliliters of 1 and 5y0 w/v sterile solutions of polyoxyethylene 20 sorbitan nionostearate were placed in sterile 60-ml. bottles. Onetenth milliliter of a 72-hour thioglycollate broth inoculum of Aspergillus niger was added t o both 1 and 5% w/v solutions of the surfactant and a similar procedure carried out using a washed inoculum of Aspergillus niger; i. e., free of thioglycollate medium. The bottles were placed in an incubator (37') and the solutions were titrated a t regular intervals with alcoholic 0.12 N NaOH to a phenolphthalein end
SCIENTIFIC EDITION
July 1957
point after the addition of 25 ml. of neutralized alcohol. The milliequivalents of sodium hydroxide necessary to neutralize the free fatty acid released in 25 ml. of the surfactant solutions due to the splitting of the surfactant by the organism are reported in Fig. 2. Uninoculated solutions of the surfactant were titrated with the alkali solution in a similar manner as a control. A total of 12 milliequivalents of sodium hydroxide was necessary to neutralize the 25 ml. of 1% w/v surfactant solution. A similar volume of a 5% w/v solution required 24 milliequivalents for neutralization. The amount of sodium hydroxide necessary for neutralization of the uninoculated surfactant control solutions remained constant during a 28-day period, showing that there was no hydrolysis of the ester within that time in the control, From the data in Fig. 2, it is obvious that Aspergillus niger is capable of growing in solutions of polyoxyethylene 20 sorbitan monostearate and splitting the ester. The addition of a nutrient substance to the surfactant solution (such as the 0.1 ml. of thioglycollate broth in the broth inoculum) was found t o stimulate organism growth. The faster the rate of growth, the more rapidly was the ester split.
DISCUSSION It is quite evident that the microorganisms employed in this study are capable of growing in solutions of nonionic surfactants of the sorbitan partial
445
ester and polyoxyethylene ester types. I t is difficult to believe that microorganisms can utilize these surfactants for nutritive purposes directly, especially substances of the polyoxyethylene sorbitan partial ester type which have such large molecules. Studies are now in progress t o ascertain what the nutrient is for the growth observed in the inoculated surfactant solutions. The fact that the microorganisms employed in this study are capable of growing in surfactants and splitting their molecules becomes a real problem when one considers that these same microorganisms are among the most common contaminants in pharmaceutical products. The splitting of the surfactant t o any extensive degree most probably would result in the destruction of its emulsifying and stabilizing properties and would likely result in the breakdown of the product. It is, therefore, necessary to add a preservative to products containing these surfactants in order to restrain the growth of microorganisms.
REFERENCES (1) Engler, These de Doctorat, No. 1149, Geneva, p. 30 (1950) as reported by Bolle, A,, and Mirimanoff, A,, J . Ph rm. and Pharmacol., 2 , 685(1950). Bolle, A., and Mirimanoff, A.. J . Pharm. and Pharmacol. 2 685-691(1950). (5) “The United States Pharmacopeia,” 15th rev., Mack Publishing Co., Easton, Pa., 1955, pp. 555-556. (4) Ibid.. p. 469. (6) Silver, M. J. “Bacterial Esterases and Lipases,” a thesid, Philadelphia kollege of Pharmacy and Science, 1953. ( 6 ) Anderson, J . A., J . Bnct., 27, 69(1934), an abstract.
(1)
The Preservation of Aqueous Preparations Containing Nonionic Surfactants II.* Preservative Studies in Solutions and Products Containing Nonionic Surfactants By MARTIN BARR and LINWOOD F. TICEt Forty-eight substances and combinations were studied for their possible effectiveness as preservatives for solutions of nonionic surfactants of the sorbitan partial fatty acid ester and polyoxyethylene ester types. Results indicated that sorbic acid (0.2 per cent w/v); phenylmercuric borate (0.007 per cent w/v); phenylmercuric nitrate (0.01 per cent w/v); phenylmercuric acetate (0.007 per cent w/v); n-trichloromethylmercapto-4-cyclohexene-1,2-dicarboximide (Vancides 89 RE, 0.04 per cent w/v); diethylene glycol monomethyl ether (methyl Carbitol, 3 per cent w/v); hexylene glycol (3 per cent w/v); and benzalkonium chloride (0.1 per cent w/v) are effective in preserving these solutions. The failure of various phenolic preservatives to preserve these surfactant solutions is attributedto complex formation between phenolic and pol yether groups. Studies on various cosmetic formulations containing nonionic surfactants indicate that sorbic acid (0.2 per cent w/w) and combinations of sorbic acid and hexylene glycol are effective preservatives. These preservatives were also found effective in products containing sorbitol as well as nonionic surfactants. in a previous paper (1) t h a t microorganisms are capable of growing in
T WAS REPORTED
*Received August 27, 1956, from the Department of Pharmacy, Philadelphia College of Pharmacy and Science. Work supported by a research grant from the Atlas Powder Company, Wilmington, Del. f The authors wish to thank Dr. Louis Gershenfeld and Dr. Bernard W i t h of the Department of Bacteriology, Philadelphia College of Pharmacy and Science, for their suggestions during this study.
solutions or dispersions of nonionic surfactants of the sorbitan partial fatty acid ester and polyoxyethylene ester types. It is, therefore, necessary t o preserve products containing these surfactantk in order to insure stability. This study is concerned with an invcstigation of t h e effectiveness of various substances and
JOURNAL OF THE
AMERICANPHARMACEUTICAL ASSOCIATION
VOl. XLVI, NO. 7
TABLE IIT.-STERILIZATIONOF VENOSETS WITH ETHYLENE OXIDEVAPOR Process Time, HI.
Temperature, F.
EtOp Gas Pressure in Atmosphere
2
73
0 50
78
4
+
= contaminated,
-
Plate Count, Spores/Strip Cap Adaptor Assembly Hood
0.50 =
Sterility Test Cap Adaptor Assembly Hood
-
293
0 0 0 0
203 300 0
-
++ + -
sterile; 0 = no organisms detected.
tained with contaminated Venosets t o establish the minimum process time provides a margin of safety. The larger sterilization equipment which was subsequently set up for production-scale operation was designed to operate with a controlled pressure of ethylene oxide vapor of 0.5 atmosphere. Since the smaller experimental equipment used more ethylene oxide, 0.63 t o 0.73 atmosphere of pressure, two additional experimental trials were made with a gas pressure of 0.5 atmosphere to duplicate plant conditions. The experiments were conducted in the same manner as before. A summary of these results is shown in Table 111. The data indicates that under the prescribed conditions a sterilization time of four hours is adequate t o achieve sterility in the Vcnoset. In actual production practice, however, a longer process time has been found more convenient and is currently in use. Two additional safety features pertain under production-scale operations. The Venosets normally carry a much lower degree of contamination than was used in these studies. Secondly, the marked sterilizing effect of
thc ethylene oxide adsorbed on the plastic and the package is operative in the plant process.
REFERENCES (1) Anonymous Chcm. Week., 7 5 , 96(0ctober 2, 1954). (2) Cotton, R. T., and Roark, R.C., Ind. Eng. Chcm., 20, 805( 1928). (3) Engley, F. B., Jr., Bact. Proc., p. 22 28(1951). (4) Ginsberg, H. S., and Wilson. A. T., PYOL. SOL.Erptl. B i d . Mcd. 73,614(1950). (5) Griffith, C. I.., and Hall, L. A,, U. S. Patent 2,189,947 (1940). (6) Gross, P. M., and Dixon, L. F., U. S. Patent 2,075,845 11”77\ \ ‘ m u‘ , .
(7) Kaye, S., Irminger, H. F.. and Phillips, C. R.. J . Lab. Cli7r. M c d . , 40, 67(1952). ( 8 ) $aye, S.,rbid., 35, 823(1950). (9) klarenbeek, A,, and Tongern, H. A,. J . H y g . (London) 52,525(1954). (10) Phillips, C. R., Bacleriol. Proc.. p. 23 23(1950). ( I I ) Phillips, C. R., and Kaye, S., Am. J. H y g . , 50, 270 (1949). (12) Sagen, H. E., A m . Phariir. hf/gs. Assoc., Sci. Coxf. Proc., p. 87 87(1954). (13) Schrader, H., and Bossert, E., U. S. Patent 2,037,439 114Wi)
( t t ) Reddish, G. F., “Antiseptics. Disinfectants, Fungicides, and Chemical and Physical Sterilization,” Lea and Febiger, Philadelphia, 1954, p. 647. (15) Roberts J. L. Allison L. E. Prickett, P. A,. and Riddle, K . B , j . Bact;riul., 45,’40(19i3).
The Preservation of Aqueous Preparations Containing Nonionic Surfactants I.* Growth of Microorganisms in Solutions and Dispersions of Nonionic Surfactants By MARTIN BARR and LINWOOD F. TICEt Studies have shown that various microorganisms, including Pse~domonnsaeruginosa, Aspergillus niger, Peniciliium notaturn, and Monilia albicans are capable of growing in solutions and dispersions of nonionic surfactants of the fatty acid ester type. It was observed that these organisms are capable of splitting the ester Iinkages of these surfactants. This is first evidenced visually by the appearance of free fatty acids in the inoculated surfactant solutions. Further study revealed that these test organisms produce esterases which split the fatty acid from the surfactant molecule. The presence of esterases was demonstrated using a tributyrin testing procedure. The liberation of free fatty acid from polyoxyethylene 2 0 sorbitan monostearate (Tween@60) solutions IS reported. SURFACTANTS of t h e sorbitan partial ester type’ and their polyoxyethyl-
ONIONIC
*Received August 27 1956, from the Department of Pharmacy, Philadelphia College of Pharmacy and Science. This work was supported by a research grant from the Atlas Powder Company, Wilmington, Del. t The authors wish t o thank Dr. Louis Gershenfeld and Dr. Bernard W i t h of the Department of Bacteriology, Philadelphia College of Pharmacy and Science, for their supgestions during this study. Atlas Powder Company, Wilmington, Del
ene derivatives have found wide and increasing use in various products. Because of their unique properties, they have found wide applications as emulsifiers, solubilizers, dispersants, detergents, and as spreading, wetting, and antifoaming agents. Engler (1) and Bolle and Mirimanoff ( 2 ) have reported that certain nonionic surfactants promote t h e growth of molds. N o studv has as
July 1957
443
SCIENTIFIC EDITION
TABLE I.-TI[E GROWTH OF MICROORGANISMS IN 1 AND 5 9 w/v SOLUTIONS AND DISPERSIONS OF NONIONIC SURFACTANTS OF THE SORBITAN PARTIAL ESTERA N D POLYOXYETHYLENE ESTERTYPES __ Control"
Surfactant
No inoculum.
+ = growth of microorganisms observed.
aersginosa
..
Polyoxyethylene 20 sorbitan monolaurate Polyoxyethylene 20 sorbitan monopalmitate Polyoxyethylene 20 sorbitan monostearate Polyoxyethylene 20 sorbitan monooleate Sorbitan monolaurate Sorbitan monopalmitate Sorbitan monostearate Sorbitan monooleate Polyoxyl 40 stearate Q
Psendomonas
.. ..
..
.. ..
.. .. .. s
+s
+" ++ +++" $8
Monitia aibicans
+" +" +" +" ++ ++ +"
Asfiergillns
nigev
+" ++"" +++" ++ +a
Penicillircm nolatrim
+* +s
+" ++ ++ +s
+s
= splitting of ester observed.
ruplicate. Two tubes of each inoculated surfactant solution or dispersion were placed in an incubator (37") and two similar tubes were stored a t room temperature (25" f lo). All tubes were observed for a period of two months for evidence of growth. The results are expressed in Table I. Examination of Table I reveals that the four microorganisms utilized in this study are capable of growing in all the surfactants employed. Growth was evidenced in the surfactant solutions and dispersions within several days after inoculation. It was noted that the microorganisms are capable of splitting the molecules of some of the surfactants, all of which are esters. The splitting of the ester EXPERIMENTAL was determined by visual examination of the inocuGrowth of Microorganisms in Aqueous Solutions lated solutions for free fatty acid formation. Thus, ester splitting was observed in solutions of the polyand Dispersions of Nonionic Surfactants.-The oxyethylene sorbitan fatty acid esters and polyoxyl purpose of this study was to determine whether various microorganisms are capable of growing in 40 stearate by the appearance of insoluble lauric, aqueous solutions and dispersions of the nonionic palmitic, stearic, or oleic acid, depending on the acid surfactants of the sorbitan partial ester type. One present in the ester. Since the sorbitan partial fatty and 5y0w/v solutions or dispersions of the following acid esters (Span products) are not soluble in water, nonionic surfactants were prepared and inoculated it was not possible t o observe visually ester splitting with the test organisms: polyoxyethylene 20 sor- by the presence of insoluble fatty acids in these susbitan monolaurate,2 polyoxyethylene 20 sorbitan pensions or dispersions. Acid number determinations were carried out on non no palm it ate,^ polyoxyethylene 20 sorbitan monostearate,* polyoxyethylene 20 sorbitan mono- the surfactant solutions and dispersions before and oleate.5 sorbitan monolaurate,6 sorbitan monopalmi- after sterilization and the determinations indicated tate.? sorbitan monostearate,s sorbitan mono- no change in the free fatty acid concentrations. All sterile controls remained stable for the duration of ole at^,^ and polyoxyl 40 stearate.1° The latter substance is not of the sorbitan partial ester type the test period. Experiments were carried out in which the numbut is a polyoxyethylene ester made by direct reaction of fatty acid with alkylene oxide. It was ber of organisms in the inoculum were reduced and, included in this study, however, as it is recognized in in others, where washed organisms were used as the U. S. P. XV (3) and is the surfactant in Hydrophilic inoculum. Results indicated that the organisms Ointment (4). To 10 ml. quantities of the sterilized were capable of growing in the surfactant esters solutions and dispersions, previously sterilized at and splitting them. The rate of growth and ester 121.3" for twenty minutes and contained in test splitting was reduced, however, when the number tubes, were added 0.1 ml. of a 24-hour thioglycollate of organisms in the inoculum was reduced and when broth suspension of Pseudomonas aeruginosa" or a washed organisms were used as the inoculum. A Quantitative Determination of the Splitting of 72-hour thioglycollate broth suspension of Monilia albicans,ia Aspergillus niger,I3 and Penicillium Tributyrin a s a Result of Esterase Activity.-Since notatum.'4 All inoculations were made in quad- the microorganisms employed in this study are known t o produce esterases (€I), i t was decided to a Tween@ 20; 8 Tween@ 40; ' Tween@ 60; 5 Tween@ 80; carry out an experiment in order t o obtain further 6 Span@ 20. 7 Span@ 40. * Span@ 6 0 . 9 Span@ 80; 10 Myrj@ information on the role esterase production by 52; registeied trademark names of the Atlas Powder Company. microorganisms plays in the splitting of molecules 11 Pseudomonas aeruginosa, isolated from a patient at the of the surfactant. An experiment was, therefore, University of Pennsylvania Hospital. 12 Monilia albicans, isolated from a patient at Children's devised t o measure quantitatively the free acid reHospital, Philadelphia. leased from an ester, tributyrin, during organism 13. 14 Asaergillus nigev and Penicillium notatum, isolated from contaminated products in the laboratory of the Departgrowth. ment of Pharmacy, Philadelphia College o f Pharmacy and The three organisms studied in this experiment S ci eac e.
yet been reported o n the growth of microorganisms other t h a n molds in solutions and dispersions of nonionic surfactants of the fatty acid ester type. No mention has been made of t h e splitting of the molecules of the nonionic surfactants by microorganisms. Such a study, is reported in P a r t I of this paper. The effectiveness of various substances as preservatives i n products containing these surfactants is reported in P a r t 11.
444
JOURNAL OF THE
140-
120
AMERICANPHARMACEUTICAL ASSOCIATION
----
Psbudomonas asrugin osa ------Aspsrgillus nigsr -- Honilio olbicans
f
-
VOl. XLVI, NO. 7
t --.-
'0°
0
4
5 Y. Surfoctont- broth inoculum SY. Surtoc?ont- wosnsd inoculum I % Surtoclont- brolh inoculum IY. Surfoctont- washed inoculum
8
12 16 20 doyr a f t e r inoculofion
A
24
28
Fig. 2.-The splitting of polyoxyethylene 20 sorbitan monostearate (Tween@60) by Aspergillzis niger. days
Fig. 1.-The
after
inoculation
esterase activity of three organisms on tributyrin.
* MEQ NaOH required t o neutralize free acids
due to the esterase activity of the organisms on 2 ml. of a 50y0 v/v tributyrin emulsion (1 ml. tributyrin).
were Pseudomonas aeruginosa, Monilia albicans, and A spergillus niger. The production of esterases by the strains of organisms employed was confirmed using the test method of Silver ( 5 ) which is based on Anderson's observation (6). The principle of the experiment was to titrate the free fatty acid liberated from a tributyrin emulsion which had been inoculated with the organisms under consideration. The procedure was as follows: A 50% v/v emulsion of tributyrin was prepared using the following formula:
.
Tributyrin16. . . . . . . . . . . . . . . . 50% Nutrient broth.. . . . . . . . . . . . . . 25y0 1% Methyl cellulose 4000. . . . . 2570 The emulsion was prepared with the aid of a Waring Blendor. Two-milliliter quantities of this emulsion were transferred to test tubes and sterilized in a n autoclave. Inocula consisting of 0.1 ml. of a 24hour nutrient broth culture of Pseudomonas aeruginosa or a 72-hour nutrient broth culture of Monilia nlbicans and Aspergilliu niger were placed in their respective tubes. One day after inoculation and on alternate days for a period of eleven days, each tube was titrated with 0.01091 N NaOH to a phenolphthalein end point after the addition of 10 ml. of neutralized alcohol. The milliequivalents of sodium hydroxide required t o neutralize the fatty acid released from 1 ml. of tributyrin (contained in 2 ml. o f a 5oy0 v/v emulsion) were calculated and are reported in Fig. 1. Uninoculated controls were titrated with the alkali solution and it was found 15
* MEQ NaOH required to neutralize the free acids due to the esterase activity of Aspergillus niger on 25 ml. surfactant solutions.
The Matheson Company, Inc., East Rutherford, N. J.
that 25.1 milliequivalents o f sodium hydroxide were necessary to neutralize 2 ml. of the 50y0 tributyrin emulsion. It is apparent from the examination of Fig. 1 that Pseudomonas aeruginosa, Aspergillus niger, and Monilia albicans are capable of producing esterases which result in the splitting of tributyrin. In this study, the bacterium, Pseudomonas aeruginosa, appeared to be a greater producer of esterase than molds. The faster initial rate of esterase production by bacteria is probably due to the fact that bacteria are faster initial growers than the moldsthe greater the number of organisms, the greater the esterase production and, hence, the faster the ester is split. The Splitting of Polyoxyethlene 20 Sorbitan Monostearate by Aspergillus niger.-In an effort t o understand better the phenomena which occur when microorganisms are inoculated into surfactant solutions and dispersions, a study was carried out to determine quantitatively the splitting of polyoxyethylene 20 sorbitan monostearate by Aspergillus niger. I t was decided to study quantitatively the effect of the test organism on 1 and 57' w/v aqueous solutions of the surfactant, both with and without other added nutrient. This was done in order t o determine what roles the concentration of surfactant and other nutrient play in the splitting of the surfactant. Twenty-five milliliters of 1 and 5y0 w/v sterile solutions of polyoxyethylene 20 sorbitan nionostearate were placed in sterile 60-ml. bottles. Onetenth milliliter of a 72-hour thioglycollate broth inoculum of Aspergillus niger was added t o both 1 and 5% w/v solutions of the surfactant and a similar procedure carried out using a washed inoculum of Aspergillus niger; i. e., free of thioglycollate medium. The bottles were placed in an incubator (37') and the solutions were titrated a t regular intervals with alcoholic 0.12 N NaOH to a phenolphthalein end
SCIENTIFIC EDITION
July 1957
point after the addition of 25 ml. of neutralized alcohol. The milliequivalents of sodium hydroxide necessary to neutralize the free fatty acid released in 25 ml. of the surfactant solutions due to the splitting of the surfactant by the organism are reported in Fig. 2. Uninoculated solutions of the surfactant were titrated with the alkali solution in a similar manner as a control. A total of 12 milliequivalents of sodium hydroxide was necessary to neutralize the 25 ml. of 1% w/v surfactant solution. A similar volume of a 5% w/v solution required 24 milliequivalents for neutralization. The amount of sodium hydroxide necessary for neutralization of the uninoculated surfactant control solutions remained constant during a 28-day period, showing that there was no hydrolysis of the ester within that time in the control, From the data in Fig. 2, it is obvious that Aspergillus niger is capable of growing in solutions of polyoxyethylene 20 sorbitan monostearate and splitting the ester. The addition of a nutrient substance to the surfactant solution (such as the 0.1 ml. of thioglycollate broth in the broth inoculum) was found t o stimulate organism growth. The faster the rate of growth, the more rapidly was the ester split.
DISCUSSION It is quite evident that the microorganisms employed in this study are capable of growing in solutions of nonionic surfactants of the sorbitan partial
445
ester and polyoxyethylene ester types. I t is difficult to believe that microorganisms can utilize these surfactants for nutritive purposes directly, especially substances of the polyoxyethylene sorbitan partial ester type which have such large molecules. Studies are now in progress t o ascertain what the nutrient is for the growth observed in the inoculated surfactant solutions. The fact that the microorganisms employed in this study are capable of growing in surfactants and splitting their molecules becomes a real problem when one considers that these same microorganisms are among the most common contaminants in pharmaceutical products. The splitting of the surfactant t o any extensive degree most probably would result in the destruction of its emulsifying and stabilizing properties and would likely result in the breakdown of the product. It is, therefore, necessary to add a preservative to products containing these surfactants in order to restrain the growth of microorganisms.
REFERENCES (1) Engler, These de Doctorat, No. 1149, Geneva, p. 30 (1950) as reported by Bolle, A,, and Mirimanoff, A,, J . Ph rm. and Pharmacol., 2 , 685(1950). Bolle, A., and Mirimanoff, A.. J . Pharm. and Pharmacol. 2 685-691(1950). (5) “The United States Pharmacopeia,” 15th rev., Mack Publishing Co., Easton, Pa., 1955, pp. 555-556. (4) Ibid.. p. 469. (6) Silver, M. J. “Bacterial Esterases and Lipases,” a thesid, Philadelphia kollege of Pharmacy and Science, 1953. ( 6 ) Anderson, J . A., J . Bnct., 27, 69(1934), an abstract.
(1)
The Preservation of Aqueous Preparations Containing Nonionic Surfactants II.* Preservative Studies in Solutions and Products Containing Nonionic Surfactants By MARTIN BARR and LINWOOD F. TICEt Forty-eight substances and combinations were studied for their possible effectiveness as preservatives for solutions of nonionic surfactants of the sorbitan partial fatty acid ester and polyoxyethylene ester types. Results indicated that sorbic acid (0.2 per cent w/v); phenylmercuric borate (0.007 per cent w/v); phenylmercuric nitrate (0.01 per cent w/v); phenylmercuric acetate (0.007 per cent w/v); n-trichloromethylmercapto-4-cyclohexene-1,2-dicarboximide (Vancides 89 RE, 0.04 per cent w/v); diethylene glycol monomethyl ether (methyl Carbitol, 3 per cent w/v); hexylene glycol (3 per cent w/v); and benzalkonium chloride (0.1 per cent w/v) are effective in preserving these solutions. The failure of various phenolic preservatives to preserve these surfactant solutions is attributedto complex formation between phenolic and pol yether groups. Studies on various cosmetic formulations containing nonionic surfactants indicate that sorbic acid (0.2 per cent w/w) and combinations of sorbic acid and hexylene glycol are effective preservatives. These preservatives were also found effective in products containing sorbitol as well as nonionic surfactants. in a previous paper (1) t h a t microorganisms are capable of growing in
T WAS REPORTED
*Received August 27, 1956, from the Department of Pharmacy, Philadelphia College of Pharmacy and Science. Work supported by a research grant from the Atlas Powder Company, Wilmington, Del. f The authors wish to thank Dr. Louis Gershenfeld and Dr. Bernard W i t h of the Department of Bacteriology, Philadelphia College of Pharmacy and Science, for their suggestions during this study.
solutions or dispersions of nonionic surfactants of the sorbitan partial fatty acid ester and polyoxyethylene ester types. It is, therefore, necessary t o preserve products containing these surfactantk in order to insure stability. This study is concerned with an invcstigation of t h e effectiveness of various substances and