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T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
THE INHIBITING ACTION OF CERTAIN SPICES ON THE GROWTH OF MICROORGANISMS’ By FREDA M. BACHMANN
Received April 14, 1916
A laboratory study of t h e preservative action of spices was first made b y Hoffman and Evans in 191I . They reported t h a t as a result of their experiments they found certain spices t o have considerable antiseptic properties. They found cinnamon most effective in inhibiting t h e growth of organisms; cloves and allspices next in effectiveness; and t h e other spices of little value as preservatives. They used pure cultures of some of the bacteria and yeasts, b u t depended on chance inoculation for mold growth. Apple sauce, t o which varying amounts of spice were added, was exposed for several days. T h e yeasts and bacteria were grown in sterile broth t o which cinnamic aldehyde, eugenol, or benzoic acid was added. T h e prese n t work was undertaken t o confirm t h e results of Hoffman and Evans and t o collect more d a t a on t h e subject. N E TI30 D
For the greater part of t h e work, pure cultures of organisms were used. Species of t h e common molds, Rhizopus, Penicillium, Aspergillus, and Alternaria, which are frequently found on spoiled preserves, and of t h e bacteria, B . coli, B. prodigiosus, and B. subtilis, mere the organisms used in most of the experiments. T h e Aspergillus was obtained from tomato butter which had spoiled; t h e Penicillium. from a growth on canned peaches. For t h e greater part of t h e work, agar has been used as a nutrient medium. T h e molds grow best on Thaxter’s potato hard agar, which consists of potato broth with 3 per cent agar and z per cent glucose. The bacteria were grown on ordinary nutrient agar. T h e spice in some form was introduced into the agar before it solidified. T o compare readily the growth on a spiced medium with t h a t on a medium without t h e spice, it was found convenient t o use some kind of a double plate. T h e double plate described b y FrostZ in his studies on antagonism of organisms was first used. Here t h e Petri dish is divided through t h e center b y a glass rod which is sealed t o t h e bottom of t h e dish b y means of collodion. It is then sterilized. The one objection t o this method for the present studies is that in sterilizati.on the collod’’on sometimes shrivels t o such an extent t h a t t h e rod is no longer well sealed t o t h e plate, so t h a t some of t h e agar poured in on one side is likely t o run underneath t h e rod into t h e opposite side of t h e d k h . A modification of this method seemed desirable.3 For studying t h e amount of diffusion, it was found satisfactory t o cover one-half of the bottom of the Petri dish with a piece of cheese-cloth and then sterilize t h e dishes. Agar containing no spice was then poured into the dish and when solid, the cloth with t h e ad1 Published with permission of the Director, Wisconsin Experiment Station. 2 William D. Frost. “ T h e Antagonism Exhibited by Certain Saprophytic Bacteria apainst the Bacillus T y p k o s u s Gaffky,” J . I n f . D L , ,1
(1914), 641. 8 W. D Frost and Freda M. Bachmann, “A4notherUse of the DoublePlate Method,” Science, N. 43 (1916).
s.,
Vol. 8, No. 7
herent agar removed b y means of a sterile forceps, t h u s leaving agar on only one-half of t h e plate. T h e spiced agar mas then poured into t h e side opposite t h e plain agar. I n such plates, whatever inhibition of g r o y t h there is on the side where there is no spice is due t o the amount of spice which diffuses through t h e medium and t o whatever volatilizes. T o distinguish between diffusion and rolatilization in inhibiting growth, it was necessary t o use still another kind of double plate, one in which there could be no diffusion through t h e meiium. Small, thin glass watch crystals were sterilized in the Petri dishes a n d later the spiced agar poured into the watch crystal and the plain agar in t h e Petri dish. I n plates prepared in this way t h e inhibition of growth on t h e plain agar could be due only t o t h e amount of spice which volatilizes. The organisms have also been grown on spiced agar slants and in spiced bouillon. T h e latter is not alvays very desirable because t h e spice may cause a cloudiness which makes a very small amount of growth difficult t o detect. Cooked apples t o which spice, or vinegar, or both, were added, have also been used as a medium. Spice was introduced in t h e form of ground spice, the alcoholic extracts, t h e essential oils, and the active principles. The ground spice and the alcoholic extracts were introduced directly into a definite amount of agar, The essential oils and the active principles were diluted with water. Amounts of this emulsion were introduced into the liquefied agar. The emulsion was very thoroughly shaken each time before transferring a n y of it t o the agar. T o inoculate the plates with molds, a suspension of the mold spores in sterile water was streaked across t h e agar with t h e platinum loop. I n this way a very even amount oE growth was obtained all along t h e streak in t h e control plates. However, in order t o make certain t h a t a greater amount of growth on t h e side farthest from the spiced agar would not be due t o heavier inoculation, t h e streak was made b y drawing the loop from the center t o t h e circumference, thus placing t h e greater number of spores near t h e spice. All the cultures have been grown a t room temperature. EFFECT 0 P ALCOHOLIC EXTRACTS O P SPICE
The alcoholic extracts were prepared b y t h e laboratory of pharmacy. T h e spices were extracted with alcohol and the extract evaporated so t h a t x cc. of the extract represented I g. of the spice. Controls in which the same amount of 95 per cent alcohol was used were made for comparison. The results given in Table I are from plates where t h e spiced and u n spiced agar were in contact and were taken one week after inoculation. There is considerable difference in t h e amount of spice which prevents mycelial growth and t h e amount pres-enting germination of spores. Frequently in plates in which t h e agar in one-half of the dlsh contained suEcient alcoholic extract of a spice t o prevent germination of t h e spores, the mycelium which had covered the side without spice grew readily across
T H E J O U R N A L OF I N D U S T R I A L ‘ A N D ENGINEERING C H E M I S T R Y
July, 19 16
t h e center over t h e spiced agar. This is indicated in t h e accompanying tables b y t h e abbreviation “myc.” The a m o u n t a n d kind of growth on t h e spiced agar is TABLE I The plus8!+) sign indicates growth, and fer0 (0) no growth. The ahbrevitltion myc” indicates t h a t when myceltal filaments were used for inoculation a growth of the mold resulted but that the same amount of spice prevented ger‘yination of the spores.’ The negative results in the columns marked No” are due to the diffusion and volatilization of the spice on the opposite side of the plate, i. e . , on the side containing thespice. CINKAMON CLOVES MUSTARDALCOHOL ORGANISM DILUTION KO Yes No Yes No Yes KO Yes Rhizopus . . .. . . 1 : 7 0 0 0 0 + o $ 0 1 : 14 +myc 0
..
.
dspevgiilus., , , . Penicillium..
., , ,
1 : 70 1 : 140 1:iOO 1 : 10
1:lOO 1:zoo 1:7 1 :i o
+4- ++ + + ++0 +t0 0 0 +t m y+c
++ o ++ myc + + o ++ ++ Q O +f 00
++ ++ +++ ++ +
++ ++ + ++ ++ +
++
++ ++ i- + ++ +t
1:140 4- 4entirely in proportion t o t h e amount of spice used. Where t h e mycelium has grown over t h e spiced agar from t h e side without t h e spice, t h e growth is frequently not so thick, b u t otherwise appears t o be fairly healthy a n d produces abundant fruiting branches. Where there is more spice t h e growth is t h i n , much stunted a n d in Rhizopus, no sporanges are formed. T h a t there is considerable diffusion of t h e spice in t h e form of alco~holic extracts is evidenced b y t h e amount a n d shape of t h e growth on t h e non-spice side. It will be seen from Table I t h a t where a I : 7 or I : I O dilution of cinnamon or cloves in agar was used, there was no germination of spores even on t h e side containing no spice. With I : 14 dilution of cloves on one side, there was germination of t h e spores of Rhizopus only farthest from t h e spice agar a n d t h e resulting growth was crowded near t h e rim of t h e Petri dish (Fig. I). With somewhat less spice, a s in a dilution of I : 7 0 of extract of cloves, t h e growth
be of little value in inhibiting the growth of a n y of these molds. There was very little difference between t h e plates in which alcohol a n d those in which alcoholic extract of mustard was used. It is also evident from Table I t h a t there is considerable difference in the sensitiveness of different molds to t h e alcoholic extract of any one spice. Rhizopus is very much less sensitive t h a n either Aspergillus or Penicillium t o t h e extract of cinnamon. Penicillium, on t h e other hand, is more sensitive t o t h e extract of cloves t h a n Rhizopus. E F F E C T OF G R O U X D S P I C E
T o determine t h e effect of ground spice, a weighed amount of t h e spice was introduced into a definite quantity of agar a n d t h e n sterilized. The,spiced agar was t h e n poured into a watch glass set in a Petri dish. Agar without spice was poured into t h e Petri dish around t h e watch glass. Bacteria or mold spores were placed on t h e spiced agar and on t h a t without spice. The antiseptic properties of cinnamon, cloves, allspice, a n d nutmeg were tested with t h e organisms noted in Table 11: TABLE 11-RESULTS ONE WEEK AFTER IXOCULATION CINNAMON CLOVES ORGANISMDILUTION No Yes No Yes AZternaria. 1 : 25 0 0 1:50 0 1:lOO f 0 Penicillium.. 1 : 25 0 0
,.
Rhizopus ...
.
S-side contains spiced
m , mold growth
of Rhizopus on t h e non-spiced side is triangular, tapering toward t h e center as shown in Fig. 11. If t h e interference with growth on t h e non-spiced side were due for t h e most part t o t h e volatilization of t h e spice on t h e opposite side, it would seem t h a t t h e limited growth which does occur would not always have this triangular shape, nor would it be so crowded toward t h e rim of t h e Petri dish farthest from t h e spiced agar. There is considerable difference in t h e sensitiveness of a n y one mold t o . t h e different spices. T h e extract of cloves, according t o Table I, is seen t o be t e n times as efficient as cinnamon in inhibiting t h e growth of Rhizopus. Mustard in alcoholic extract appears t o
1:50 1:lOO
I
: 25
1:50
1:lOO A s p e w i l l u s . . 1 : 25 1 3 0 1:lOO B. pvodigiosus 1 : 25 1:50
B. c o l i . .
....
B. subtilis . . .
Petri dishes. Each contains two kinds of agar. agar. Broken line shows line of inoculation
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1:lOO 1 : 25 1:50 1:lOO 1 : 75 1:50 1:lOO
++ + + + + + + ++ + + ++ ++ + ++ +
+++ ++
0 0
++ + 0 0 0
+ + + ++0 0
+0
+ ++ + + + ++ + + ++ ++ + + .+ +
F 0 T
+ ++0
++0 ++0 0 0 0
ALLSPICE NUTMEG No Yes No Yes
++0 +00 ++ 0 + T +0 + 0 0 + ++ m+w + $ +++ : t ++ 00 ++ + 4++ 00 ++ + 0 ++ 0 ++ + 0 0
0
++
++ ++ $
++ ++ ++
Cinnamon appears t o have more antiseptic properties generally t h a n either allspice or cloves. Cinnamon has little antiseptic value toward Rhizopus, b u t is valuable in preventing t h e growth of Alternaria, Penicillium, a n d Aspergillus. Cloves are about as effective as allspice for Rhizopus a n d Aspergidus b u t somewhat less so for Alternaria a n d Penicillium. Nutmeg has little or no value as a preservative even in large amounts. B . prodigiosus’ appears t o be only slightly sensitive t o spices, while B. subtilis did not grow on even a I : I O O dilution of cloves and of allspice. E F F E C T OF S P I C E AND V I N E G A R
T o determine t h e effect of vinegar a n d vinegar a n d spice a n d sugar, steamed apples were half covered with: (1) Vinegar, Sugar, and Cinnamon in such amounts as could be used in spicing apples (2) Vinegar and Cinnamon (3) Water. Sugar, and Cinnamon (4) Water and Cinnamon (5) Water (6) Vinegar (7) Vinegar, Sugar, Cinnamon, and Cloves (8) Vinegar, Cinnamon, and Cloves (9) Water, Sugar, Cinnamon, and Cloves
T H E J O U R N A L O F I l T D C S T R I A L AATD ENGINEERZIVG C H E M I S T R Y
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The same proportion of spice and vinegar or spice and water was used in each tube. T h e material was then inoculated with mold or yeast. After one week there was a good growth of mold in 3 , 4 , 5 and g, t h a t is, in all t h e tubes except those containing the vinegar. The vinegar was not diluted and contained j . 9 per cent acetic acid. The apples, after standing in t h e vinegar, spice, and sugar for 6 weeks, were not too sharply acid t o taste well, so it does not seem t h a t t h e vinegar was stronger t h a n might often be used in spicing fruit. Howerer, it is evident t h a t in these tubes the vinegar was t h e preservative. rather t h a n the spice. The formation of gas bubbles in the tubes without vinegar indicated t h e growth of t h e yeast in those tubes. The amount of cinnamon and cloves used, although sufficient for flavoring purposes, served t o retard only slightly the first gro-xth of molds a n d yeasts. 21IC ALDEHYDE A K D E U G E K O L
I n Table I11 are the results of experiments using the active principles of cinnamon and cloves, cinnamic aldehyde and eugenol. These substances were diluted with water, t h e mixture thoroughly shaken and some of this emulsion introduced into melted agar, which was then poured into plates. The d a t a are based on growth on spiced agar slants in test tubes and in plates divided with watch glasses. The grovrth on t h e spiced agar in test tubes and in plates was t h e same in each experiment and hence b u t one column is necessary t o indicate the growth in both. I n t h e first column under each spice are the results of inoculating the agar nithout spice in the Petri dish. T h e negative results in these columns are due t o volatilization of t h e spice in t h e watch glasses. TABLE 111-RBSULTS ORGANISV Rhisopus. . . . . . .
S I X E D A Y S AFTER I I O C U L A T I O N CINAAMIC ALDEHYDE
DILLTION
A-o
Yes
EUGENOL
KO
+ ++ + T
Penicillium. . Aspergillus.. . . Alternaria.,
.
....
B, subtilis..
,,
..
0
0 0 0
t
myc
0
0 0
i
.
B. coli.. . . .
0 0
0 0
., . ,
B. prodigiosus.,
0 0
1 I
0
0
0 0
0 0
0
0
Yes
+
i+
++ + t;+
-
-,-
$
++ ++ +
i +t 0
It is quite evident from t h e above t h a t cinnamic aldehyde has strong antiseptic properties. It is not only effectire when directly introduced into a medium, b u t t h e amount which volatilizes in dilutions u p t o I : 2 0 0 0 is sufficient t o inhibit t h e gromth of P e n i c i l l i u m , A s p e r g i l l u s , B . coli and B . saibtilis on the agar without spice, There was no growth of B . prodigioszis on the agar containing cinnamic aldehyde in a dilution of I : 2 0 0 0 , yet after one week enough of the spice had volatilized so t h a t when the plate was reinoculated a good growth resulted. The organism failed, however, t o produce a n y pigment. T h e spores of As;bergillus failed t o germinate on the agar without spice, b u t when after two days t h e spiced agar, dilution I : 5 0 0 0 : was reinoculated with mycelial filament, a fair amount of growth appeared. Eugenol
I v ~ 8, l , NO.7
has much less value as an antiseptic. R h i z o p u s and A l t e r n a r i a are more sensitive t o it than a n y of t h e other organisms studied. EPFECT OF THE
OILS O F ALLSPICE A K D &-LTTZIEG A N D
T H E O L E O R E S I N O F BLACK P E P P E R
The oleoresin of black pepper was found t o have no antiseptic value in dilutions of I : zoo for t h e molds and in dilutions of I : 1000 for the bacteria. The same is true of the oil of nutmeg except for R h i z o p u s , which did not grow in a dilution of I : zoo b u t grew well in a dilution of I : joo. The oil of allspice appears t o have considerable value: R h i z o p u s spores failed t o germinate in a dilution of I : j o o o b u t grew readily in I : 10000 dilution; B . p r o d i g i o s u s and B . coli were not affected b y it in dilutions of I : r o o o ; B . s u b t i l i s is more sensitive t o it and did not grow when t h e dilution was I : 1000. G E N E R A L 0B S E R V .A T I 0 K S A XD C 0 K C I,U S I 0 N S
When plain and spiced agar are in contact there is always some diffusion of the spice, whether it is in t h e form of alcoholic extract, essential oil, or ground spice. There is also always a certain amount which volatilizes. I n plates where the plain and spiced agar are in contact, t h e inhibition of growth of the organisms on the plain agar is partly due t o both diffusion and volatilization of the spice. I n such plates, when the amount of spice is great enough, the growth on t h e agar without spice is crowded near the edge of the Petri dish farthest from t h e spice, tapering along the streak TTith the broadest part of the growth farthest from the spice. This is true of both molds and bacteria b u t is more striking in the molds. The shape of this growth is due entirely t o t h e diffusion of the spice. I n plates where t h e spice is in watch glasses, so there can be no diffusion, t h e growth on the agar without spice may be more or less stunted b u t it is evenly distributed along the line of inoculation. The amount of growth is dependent on the amount of spice used and in the case of molds on t h e material used for inoculation. The spores of P e n i c i l l i u m , A s p e r g i l l u s , A l t e r n a r i a and R h i z o p u s are much more sensitive t o spice t h a n the mycelium and fail t o germinate, where if mycelial filaments are used for inoculation, a good growth results. The spores may push out stunted germ tubes no longer t h a n t h e width of the spore or very often they fail t o give a n y of the first indications of germination. There is no enlarging of t h e spore due t o taking u p water. From the practical side of food preservation this fact is of interest since chance inoculation is probably almost al\\Tays due t o spores. However. if the spice is not evenly distributed and t h e spores germinate in some part of a food material where there is less spice, the mycelium v-ill readily grow over other parts and the food become covered with mold. Frequently the amount of spice used is found t o allow a good growth of t h e mycelium of molds, b u t t o prevent sporulation, or a t least. only a few spores may be produced. The mycelium with more spice may be very poorly developed. This is very often found in R h i z o p u s . I n such a groTTth there are man!?
July, 1916
T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
protuberances on a hypha as if it had started t o branch a t various places: these are very irregular in shape b u t usually quite short and broad a t some places and narrower in others: there is little protoplasmic content in t h e hyphae where t h e growth is considerably stunted. il number of scattered oil globules may be all t h a t can be seen. There is no slimy protoplasm in many of t h e hyphae and the branches often appear t o be merely e m p t y cell walls. I n other hyphae there is evident plasmolysis resulting in a shrunken protoplast. I t has not been determined whether the morphology of t h e bacteria is changed when these are grown on or near a spiced medium. B . prodigiosus fails t o produce a n y pigment if t h e amount of spice is large. Although several transfers of this colorless growth are grown on spice, when the organism is transferred again t o a medium without spice, t h e pigment is produced as before. It does not appear from the extent of t h e present s t u d y t h a t spices, as used in t h e kitchen, in t h e usual amounts for flavoring purposes in spiced cakes, exert a very considerable preservative effect. Where cinnamon, cloves, and allspice are used in large amounts, t h e growth of molds may be retarded. I n spiced fruit where a large amount of t h e spice is used, the preservative effect may be much greater. This effect may be greater too when t h e spice is combined with vinegar. I t seems entirely possible, as suggested b y Hoffman and Evans, t h a t t h e active principles, a t least cinnamic aldehyde, could be used in such dilutions as t o prevent t h e growth of many microorganisms and yet in small enough quantities not t o spoil t h e flavor of .the product. Further studies on t h e action of spices on microorganisms are necessary before very positive a n d general statements of t h e practical value of t h e m as preservatives can be made. Pepper a n d nutmeg have little effect on t h e growth of microorganisms. A mixture of nutmeg and water boiled for a half hour and left exposed t o t h e air for chance inoculation was covered with various molds in less t h a n a week. Cloves and allspice in large amounts are quite effective in preventing t h e growth of molds and bacteria, a n d cinnamon is the most effective of t h e spices; this is true of t h e ground spices, their essential oils, a n d t h e alcoholic extracts. T h e bacteria used in this s t u d y are less sensitive for t h e most part t h a n t h e molds, b u t there is evidently considerable difference in t h e sensitiveness of various species of bacteria just as there is a difference in t h e sensitive,ness of molds. BACTERIOLOGY LABORATORY, AGRICULTURAL COLLEGE MADISON,WISCONSIN
SEPARATION AND ESTIMATION OF POLYSULFIDES AND THIOSULFATE IN LIME-SULFUR SOLUTIONS By S. D. AVERITT Received March 27, 1916
I n recent years there have been quite a number of articles written on t h e composition and analysis of lime-sulfur solution. The writer first became interested in t h e problem in 1 9 1 1 when he was Associate Referee on Insecticides for t h e Association of Official
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Agricultural Chemists. Methods for t h e analysis of lime-sulfur solution were then being considered a n d cooperative work being done. The methods' proposed b y t h e Referee a t t h a t time have since become known as the Zinc Chloride Methods. I n January of t h a t year there was issued by t h e Michigan Agricultural Experiment Station a bullet i n b y J . E. Harris in which were published methods for the analysis of lime-sulfur solution since become known as t h e Iodine Methods.2 After having worked t h e two samples of lime-sulfur submitted for cooperative work b y both methods it was found t h a t there was a marked discrepancy between t h e thiosulfate figures b y t h e t w o methods. One of t h e samples submitted was a straight limesulfur solution made with pure chemicals and the calcium calculated from the iodine titrations checked t h e determined calcium. The other sample had sodium thiosulfate added a n d the calculated a n d determined calcium would not check until t h e added sodium thiosulfate had been deducted from t h e thiosulfate figure. I n the case of the straight lime-sulfur solution, using the thiosulfate as determined b y t h e proposed methods (zinc chloride) the calculated calcium would not check t h e determined calcium. The proposed methods gave concordant results, however, and it was recommended t h a t they be made official. This recommendation was approved for final action b y t h e Association the following year. T h e next year (1912), as Referee, t h e writer in his report3 brought out t h e facts in regard t o t h e t w o methods, having first satisfied himself t h a t t h e iodine titration methods gave accurate results for thiosulfate. The Association instructed t h e Referee t o compare t h e two methods in 1913. This was done, and in his next report4 more evidence was submitted in favor of t h e iodine titration methods. There was strong opposition5 t o t h e iodine methods, however, with t h e result t h a t there are a t this time no official methods for t h e analysis of commercial lime-sulfur solutions. Taking t h e position t h a t it is desirable t h a t methods of analysis should not only be accurate b u t t h a t t h e y should possess in addition, workableness, freedom from tediousness, and as far as possible give a t least a measure of insight into the character of the substance under examination, t h e writer in this paper hopes t o make a contribution t h a t will a t least tend t o sol~7et h e problems in the analysis of commercial lime-sulfur solutions. The direct iodine titration of a lime-sulfur solution meets all of the above requirements, and while t h e zinc chloride methods may be so modified as t o give accurate results, they can never be freed from tediousness and t o the busy analyst this is a n objection t h a t 1 f
3
4
3
p. 76.
Bureau of Chemistry, Bull. 152, p. 68. Michigan Experiment Station, Tech. Bull. 6. Bureau of Chemistry, Bull. 162, p. 27. Jour. A . 0 . A . C., 1, h'o. 1, p . 59. Bureau of Chemistry, Bul!. 162, p. 38; Jour. A . 0. A . C . , I, So. I,
