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Effects of essential oils and oleoresins of plants on ethanol production, respiration and sporulation of yeasts
International Journal of Food Microbiology, 1 (1984) 63-74
63
Elsevier JFM 00007
Effects of essential oils and oleoresins of plants on ethanol production, respiration and sporulation of yeasts D.E. Conner, L.R. Beuchat, R.E. Worthington and H.L. Hitchcock Department of Food Science, University of Georgia, Agricultural Experiment Station. Experiment. GA 30212. U.S.A.
(Received 25 October 1983; accepted 3 February 1984)
Ethanol production, respiration, and sporulation of yeasts as affected by essential oils and oleoresins of allspice, cinnamon, clove, garlic, onion, oregano, savory, and thyme were investigated. Essential oils of allspice, cinnamon, and clove had little or no effect on ethanol production by Saccharomyces cerevisiae. Oils of onion, oregano, savory, and thyme delayed and/or reduced the production of ethanol. Overall, essential oils effectively suppressed ethanol production by Hansenula anomala. At the highest concentrations tested (500/ag/ml), only cinnamon, clove, garlic and thyme oleoresins substantially delayed and/or reduced ethanol production by S. cereoisiae. Most of the essential oils (100 #g/ml) impaired the respiratory activity of S. cerevisiae as evidenced by a reduction in CO2 production. Thyme oleoresin was the strongest inhibitor. Allspice and garlic oils impaired sporulation by H. anomala. All oils delayed sporulation of Lodderomyces elongisporus. Key words: Essential oils; Hansenula anomala," Lodderomyces elongisporus; Oleoresins; Saccharom.vces cereoisiae; Sporulation; Yeasts
Introduction T h e m e d i c i n a l activities of p l a n t extracts have been recognized for m a n y years. P l a n t s of the Allium species, p a r t i c u l a r l y garlic a n d onion, have been s t u d i e d most extensively ( C a v a l l i t o a n d Bailey, 1944; C a v a l l i t o et al., 1945). G r o w t h of f o o d - b o r n e p o i s o n i n g bacteria, e.g., Staphylococcus aureus ( M a n t i s et al., 1978), Salmonella typhimurium ( J o h n s o n a n d Vaughn, 1969) a n d Clostridium botulinum ( D e W i t et al., 1979), are i n h i b i t e d b y extracts of garlic a n d onion. G r o w t h of the p a t h o g e n i c yeast, Candida albicans ( T y n e c k a a n d Gos, 1973), a n d m y c o t o x i g e n i c Aspergillus species ( H i t o k o t o et al., 1980) are also i n h i b i t e d b y p l a n t extracts, i n c l u d i n g those from garlic. Little is k n o w n a b o u t the effects of essential oils a n d oleoresins on yeasts which c o m m o n l y cause f o o d spoilage or which are used in f o o d a n d b e v e r a g e f e r m e n t a tions. Pioneer studies have shown that spices have an i n h i b i t o r y effect on yeast f e r m e n t a t i o n ( C o r r a n a n d Edgar, 1933; W r i g h t et al., 1954). In o t h e r studies ( C o n n e r 0168-1605/84/$03.00 © 1984 Elsevier Science Publishers B.V.
64 and Beuchat 1984a, b) we reported that growth of several genera of yeasts is inhibited by essential oils and oleoresins of plants. Heat-stressed cells have an increased sensitivity to oils and oleoresins, indicating that these plant components inhibit structural a n d / o r metabolic repair of injured cells (Conner and Beuchat, 1984c). The study reported herein was designed to determine the effects of oils and oleoresins on ethanol production, respiration and sporulation of yeasts important to the food industry.
Materials and Methods
Evaluation of thirty-two essential oils and eight oleoresins in an earlier study (Conner and Beuchat 1984a) revealed a wide range of inhibitory activity on food spoilage and industrially important yeasts. Since the essential oils of allspice, cinnamon, clove, garlic, onion, oregano, savory and thyme exhibited the highest anti-yeast activities, these were selected for further evaluation. Oleoresins of allspice, cinnamon, clove, garlic, onion, oregano, and thyme were al~o investigated. The species of plant material from which essential oil was extracted was not in all instances the same species used to obtain the corresponding oleoresin. All oils and oleoresins were provided by Fritzsche, Dodge and Olcott, Inc., New York, N Y . Yeast evaluated and sources were Debaryomyces hansenii N R R L Y-7268 (steer dung), Hansenula anomala 67-455 (tree exudate), Lodderomvces elongisporus N R R L YB-4239 (concentrated orange juice) and Saccharomyces cerevisiae UGA-102 (Chablis wine). Test cells were prepared by culturing the yeasts in sterile yeast extract/malt extract/peptone/glucose (YMPG) broth (pH 5.5; 100 ml per 250-ml Erlenmeyer flask). The medium consisted of 3.0 g yeast extract, 3.0 g malt extract, 5.0 g peptone and 10 g glucose per 1000 ml of distilled water. Cultures incubated at 30°C on a rotary shaker for 44-48 h and diluted 10 -2 in 0.1 M potassium phosphate buffer (pH 7.0) served as inocula for experiments conducted to determine the effects of oils and oleoresins on ethanol production and sporulation. The procedure for preparing cells for respiration studies was modified as indicated below. Two yeasts, S. cerevisiae and H. anomala, were tested for ethanol production in modified Y M P G broth. Essential oils were diluted in 95% ethanol and dispersed in sterile YMPG broth containing 5% (w/v) glucose (pH 5.5; 100 ml per 250-ml Erlenmeyer flask) to give final concentrations of 25, 50, 100, and 200/.tg/ml. Two controls, one containing no added ethanol or essential oil and the other containing 1% ethanol (the highest level present in any of the test broths containing essential oils), but no oil, were included as ethanol production media. Two ethanol-producing yeasts, S. cerevisiae and H. anomala, were tested. Inoculum (1.0 ml) was dispensed into 100 ml of sterile control and test broths. Cultures were incubated statically at 30°C under a carbon dioxide gas atmosphere; at 2, 4, and 7 days, the flasks were vigorously swirled, and a 5 ml aliquot was withdrawn and centrifuged at 10000 × g for 8 rain. The supernatant fluid was carefully decanted into a sterile test tube, sealed, and stored at - 2 8 ° C until analysis could be performed.
65 Supernates were analyzed for ethanol content using a gas liquid chromatograph. Three aliquots (2.0/~1) of supernatant fluid were injected into a MicroTek 200 gas chromatograph (Tracor, Inc., Austin, TX) equipped with a flame ionization detector. The 1.83 m x 0.32 cm (ID) glass column was packed with 10% SP-1200/1% H3PO 4 on 80/100 Chromasorb WAW (Supelco, Bellefonte, PA). The oven temperature was 190°C; detector temperature, 240°C; and injector temperature, 240°C. The carrier gas was helium adjusted at a flow rate of 50 m l / m i n and the detector gases were hydrogen (65 c c / m i n ) and air (12 SCFH). A standard curve was made by plotting integral areas against respective ethanol standard solution concentrations of 0.1, 0.25, 0.50, 1.0, 2.5, 5.0, and 10.0%. Linear regression of data was determined and an equation was developed for calculating the percentage ( v / v ) of ethanol in samples based on the peak area given by each sample. The investigation of the effect of oleoresins on ethanol production was conducted using the same procedure and conditions as those used in the study using essential oils. However, the oleoresins were diluted in dimethyl sulfoxide (DMSO) instead of ethanol and dispersed into the test media to give final concentrations of 50, 100. 250, and 500 ~tg/ml. A control broth containing 1% D M S O was included. The production of CO2 by S. cerevisiae and H. anomala was investigated in Y M P G broth containing 7.5% glucose and 0, 75 and 150 /~g/ml of essential oils. Three controls were included in the test, one containing no added ethanol or essential oil, one containing 1.5% ethanol, but no oil (the level of ethanol present in the 150 ~ g / m l test broths) and the third containing 0.75% ethanol, but no oil (the level of ethanol present in the test broths containing 7 5 / ~ g / m l of essential oils). S. cerevisiae was cultured in Y M P G broth under constant agitation at 30°C for 4 4 - 4 8 h and centrifuged at 10000 x g. The supernatant fluid was discarded and the cells were suspended in 0.1 M potassium phosphate buffer (pH 7.0). This washing procedure was done twice. After the second washing, the cells were resuspended in buffer to give an absorbance of 0.4 as measured by a Spectronic 20 spectrophotometer (Bausch and Lomb, Rochester, NY) set at 600 nm. 1 ml of this cell suspension was added to the side-arm chamber of a respiration reaction flask containing 2.0 ml of the test broth in the main chamber. The flasks were then connected to a single valve differential respirometer (Gilson Medical Electronics, Inc., Middleton, WI) and allowed to equilibrate at 35°C for 20 min. After equilibration, the cell suspensions and test broths were thoroughly mixed. The addition of the cell suspensions to the test broths diluted the essential oils to give final concentrations of 50 and 100 /~g/ml, while the ethanol-supplemented controls were similarily diluted to yield 1.0 and 0.5% of 95% ethanol in the actual reaction mixture. Once the cells and broths were mixed, gas (CO 2) evolution at 35°C was manometrically determined at 15-rain intervals for 3 h. Determination of the effects of oleoresins on CO z evolution by S. cerevisiae was made using the same experimental procedures and conditions as those used in the study of the essential oils. However, the oleoresins were diluted in D M S O and dispersed to give final concentrations of 0.025 and 0.050% in the reaction fluid. A control broth containing 1% D M S O was included. Experiments were conducted to determine the effects of the eight essential oils on
66 the production of ascospores by D. hansenii and H. anomala, L. elongisporus, and S. cerevisiae. The sporulation medium, V-8 juice agar, consisted of 350 ml of V-8 juice (Campbell's Soup Co., Camden, N J), 5.0 g yeast extract, 650 ml of water, and 14 g of agar. The pH wa~ adjusted to 5.7 with 2 N NaOH. After the V-8 juice agar was autoclaved at 121°C for 15 min, it was supplemented with essential oils of allspice, cinnamon, clove, onion, oregano, savory and thyme at concentrations of 100 # g / m l and garlic oil 25 # g / m l . V-8 agar containing no added essential oil served as a control. Plates were surface-inoculated, wrapped in plastic sleeves to prevent moisture loss, and incubated at 21°C. At various intervals during a 65-day period, cells were picked from plates and observed microscopically under an oil-immersion objective for the presence of ascospores. If cells of a sample consisted of 5% or more asci, the culture was scored positive for ascospore production.
Results
The effects of essential oils on ethanol production by S. cerevisiae and H. anornala are listed in Table I. In the control broth containing no added supplements, a maximum ethanol level was observed at 168 h, while in the control broth initially containing 1% ethanol, the ethanol concentration reached 3.3% at 96 h, and then declined to 2.7% at 168 h. The decrease in ethanol content may have been due, in part, to evaporation as well as assimilation during prolonged incubation. It is estimated that less than 5% of the ethanol initially present in control flasks was lost to evaporation after 168 h. At 25 /~g/ml, allspice oil had little effect on ethanol production, while at 100 and 200/~g/ml, ethanol production was slightly delayed. Cinnamon and clove oils exhibited no effect on ethanol production. Garlic oil was lethal to S. cerevisiae, so consequently no ethanol was produced. The yeast did not overcome the inhibitory effects of this oil during the 168 h test period. Onion oil totally inhibited growth and ethanol production by S. cerevisiae at 100 and 50 /~g/ml, respectively, with lesser effects at lower concentrations. The thymol/carvacrol containing oils (oregano, savory and thyme) were similar in their effects on ethanol production by S. cerevisiae. Levels of ethanol comparable to that of the unsupplemented control were observed in broths containing 25 and 50/zg/ml of these oils. At 50/xg/ml, thyme oil did slightly depress production as compared to the control. At the 100/~g/ml level of these oils, a significant amount of ethanol was produced at 48 h; however, peak levels were not achieved until 96-168 h. The ethanol production mechanism of H. anomala was more sensitive than that of S. cerevisiae to the essential oils (Table I). In the control broths, H. anomala produced 2.4% ethanol in a 96 h period, after which the concentration declined. The levels of ethanol produced in the presence of allspice, cinnamon and clove oils were lower than those found in the control broths. Garlic oil was completely inhibitory to growth of H. anomala and onion oil had a pronounced delay effect. Increased concentrations of oregano, savory and thyme oils caused increasing delays in ethanol production. Table II summarizes the results of tests conducted to determine the effects of
67 TABLE I Effect of essential oils on ethanol production by Saccharomvces cereoisiae and Hansenula anomala Essential oil
Concn. (#g/ml)
Ethanol (%, v / v ) produced by S. cerevisiae
Ethanol (%, v / v ) produced by H. anomala
48 h
96 h
168 h
48 h
96 h
168 h
0 0
2.8 2.9
2.9 3.3
2.9 2.7
0.5 0.4
2.4 2.4
2.1 2.1
Allspice
25 50 100 200
2.8 3.2 2.7 2.5
3.0 2.7 2.9 2.8
2.4 _ a 2.5 2.9
0.4 0.6 0.6 0.5
2.1 2.2 1.8 1.3
1.9 1.9 1.0 0.9
Cinnamon
25 50 100 200
3.1 2.8 3.0 2.9
2.9 2.7 3.0 2.8
0.7 0.6 0.5 0.3
2.2 2.3 1.4 1.3
2.2 1.7 1.0 0.9
Clove
25 50 100 200
2.9 3.3 2.9 2.9
2.8 2.8 2.9 2.9
-
0.6 0.6 0.4 0.5
2.3 2.4 1.8 1.4
1.8 2.1 1.5 0.9
Garlic
25 50 100 200
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
Onion
25 50 100 200
0 0 0 0
0.2 0 0 0
3.0 0.1 0 0
0 0 0 0
1.6 0.9 0.1 0.1
0.9 1.6 2.2 0.4
Oregano
25 50 100 200
2.9 2.8 1.9 0
2.8 2.9 2.7 0.4
2.9 3.1
0.5 0.5 0.1 0
2.1 2.1 1.1 0.1
1.4 1.2 0.6 0.4
25 50 100 200
2.6 3.0 1.7 0
2.5 2.6 2.4 2.3
2.8 2.6
0.4 0.2 0.1 0
2,4 1.9 1.4 0.5
1.5 1.5 1.2 1.4
25 50 100 200
2.6 2.5 1.7 0
2.6 2.7 2.5 1.8
2.8 2.9 2.4 2.8
0.6 0.3 0 0
2.1 1.9 1.2 0.2
1.9 1.4 1.1 1.2
Control Control + 1% ethanol
Savory
Thyme
-
a Not analyzed. oleoresins
on ethanol
the respective inhibitory
production
b y S. cereoisiae a n d H. a n o m a l a . A s c o m p a r e d
essential oils at similar concentrations,
to ethanol
production.
t i o n b y S. c e r e v i s i a e o c c u r r e d
In most instances,
at 48-96
the oleoresins near maximum
h of incubation.
were much ethanol
to less
produc-
At the highest concentration
68
TABLE II Effect of oleoresins on ethanol production by Saccharomyces cerevisiae and Hansenula anomala Oleoresin
Ethanol (%, v/v) produced by S. cerevisiae
Ethanol (%, v/v) produced by H. anomala
48 h
96 h
168 h
48 h
96 h
168 h
0 0
3.1 3.0
3.1 3.0
2.3 2.2
0.6 0.9
2.3 2.2
2.1 1.8
Allspice
50 100 250 500
2.8 3.0 2.6 2.9
3.0 3.1 2.8 3.2
2.2 t.9 2.1 2.3
0.7 0.6 0.6 0.6
2.1 2.0 2.5 1.6
1.9 1.8 1.7 1.0
Cinnamon
50 100 250 500
2.8 2.8 2.6 2.3
2.8 2.8 3.2 2.9
_a 2.4 2.2
0.6 0.6 0.6 0.5
2.3 2.1 2.2 1.8
1.8 2.0 1.8 2.1
Clove
50 100 250 500
3.0 2.9 3.0 2.0
3.1 2.9 2.8 2.6
2.5 2.5 2.4 2.4
0.7 0.7 0.7 0.3
1.9 2.0 1.5 1.2
1.8 1.8 0.7 1.4
Garlic
50 100 250 500
2.8 2.7 2.7 1.7
2.8 3.2 2.8 2.9
2.2 2.2 2.1
0.5 0.6 0.4 0.4
2.1 2.1 2.1 1.9
1.9 2.1 1.5 1.6
Onion
50 100 250 500
2.9 3.1 2.9 2.7
3.1 3.3 3.2 3.0
2.2 2.3 2.2 2.5
0.5 0.6 0.6 0.5
2.3 2.2 2.1 2.3
2.0 1.7 2.0 1.8
Oregano
50 100 250 500
2.7 2.9 3.0 3.1
3.0 2.9 2.8 2.8
2.2 -
0.6 0.6 0.6 0.6
2.5 2.5 1.5 1.4
1.5 1.4 0.7 0.6
Thyme
50 100 250 500
3.0 2.9 3.0 0.1
3.0 3.0 2.5 0.2
2.4 2.4
0.6 0.7 0.2 0
2.0 1.8 1.2 0.1
1.8 1.0 0.8 0.4
Control Control + 1% DMSO
Concn. (#g/ml)
a Not analyzed.
t e s t e d (500 ~ g / m l ) , o n l y c i n n a m o n (50 # g / m l ) , c l o v e , garlic, a n d t h y m e o l e o r e s i n s s u b s t a n t i a l l y d e l a y e d a n d / o r r e d u c e d e t h a n o l liberation. In m o s t instances, the h i g h e s t e t h a n o l levels w e r e o b s e r v e d i n t h e 96 h c u l t u r e s o f H. a n o m a l a . T h e 500 / z g / m l ( 5 0 / ~ g / m l o f c i n n a m o n ) levels o f m o s t o f t h e o l e o r e s i n s ( e x c e p t o n i o n ) w e r e slightly i n h i b i t o r y to e t h a n o l p r o d u c t i o n . T h y m e oleoresin again was the m o s t i n h i b i t o r y . All levels o f t h y m e a n d c l o v e o l e o r e s i n s s l i g h t l y s u p p r e s s e d e t h a n o l p r o d u c t i o n . Interestingly, the large r e d u c t i o n s in e t h a n o l c o n t e n t s o b s e r v e d in tests
69
involving essential oil were also observed here. Again, oregano caused the largest reductions. The data illustrated in Figs. 1 and 2 from tests conducted to determine the effects of the essential oils on respiration (CO 2 evolutiop) by S. cereoisiae indicate that all of the oils reduced .the respiratory activity of the yeast as compared to the control. Ethanol also had a pronounced influence on respiration during the 3 h incubation period. The essential oils of clove, allspice, and cinnamon gave similar results (Fig. 1). The oils of garlic (Fig. 1) and onion (Fig. 2) were tested at the low concentrations of 5 and 10 # g / m l because of their strong inhibitory action against S. cerevisiae observed in previous experiments. Compared to the broth containing no essential oil or ethanol, production of CO 2 was slightly reduced over the 3 h period in the presence of low concentrations of garlic and onion oils. Oil of oregano (Fig. 2) at 50 # g / m l caused a stimulation of CO 2 evolution as compared to that observed for the 0.5% ethanol control. The presence of 100 /~g/ml oregano oil had no substantial effect on the yeast's respiration when compared to the ethanol control. Thyme oil, at both concentrations tested, slightly retarded CO 2 evolution as compared to the 500 CLOVE
CINNAMON
/-
400 300
" .-
200
,-a
lOO
°/
._.=
~
o
,
500
I
ALLSPICE 0
>
,,,
I
GARLIC
400
/
300 200
•o
/ /°
100
/0/0/o/°
0
~ 0
I 30
~
I eO
, 90
n 120
I 160
t
I 180
J 0
I 30
I0
~
120
160
180
TIME (min)
Fig. 1. Effects of 50 # g / m l (e) and 100 # g / m l (O) of clove, cinnamon, and allspice essential oils, and 5 # g / m l (e) and 10 # g / m l ( O ) of garlic oil on CO 2 production by S. cereoisiae at 35°C in YMPG broth (pH 5.5) containing 7.5% glucose. Control broths contained no added ethanol (m), 1% ethanol (*), and 0.5% ethanol (13).
70 500 THYME
SAVORY
/
400
300 200
.,.,'/;:,
100
i
i/"
.~'1o~I~ ~ 0
,
I
i
~li~j~
I
I
I
I
I
I
i
I
I~- ~'/
1
,
I
500 ONION >
OREGANO
400
/o./
300
200
/
/ /
.,./i// /
/
100
././i/o/° ,
'
--,~/°s~/,
30
60
I 90
120
o/
,
J 160
,
I 180
[ 30
i
[ 60
all
/ o
o/
o~ • o/ o~ o/ , I ~ I 120 150 180
oll 90
TIME (min)
Fig. 2. Effects of 50/~g/ml (e) and 100/Lg/ml (O) of thyme, savory, and oregano essential oils, and 5 t~g/ml (e) and 10 t~g/ml (O) of onion oil on CO 2 production by S. cerevisiae at 35°C in YMPG broth (pH 5.5) containing 7.5% glucose. Control broths contained no added ethanol (m), 1% ethanol (-), and 0.5% ethanol (m).
corresponding controls. Interestingly, savory oil (50 / l g / m l ) slightly reduced CO 2 production while 1 0 0 / z g / m l had little or no effect. Data illustrating the effects of the oleoresins on the respiration of S. cerevisiae are shown in Figs. 3 and 4. The level of D M S O contained in each of the test broths had no effect on CO 2 production in this test. All of the oleoresins at 500 ~tg/ml, and cinnamon oleoresin at 50 t~g/ml, effectively inhibited CO 2 production by S. cerevisiae. Overall, excluding garlic and onion oleoresins, oleoresins were much more inhibitory to CO 2 production (respiration) by S. cerevisiae than were any of the tested concentrations of essential oils. The suspected modes of action of the oleoresins on the respiratory activities of S. cerevisiae are the same as those for the essential oils. Sporulation data are listed in Table III. D. hansenii did not sporulate very well in this experiment; ascospores were not observed in the control culture until 65 days of incubation, and the percentage of asci was low. Poor sporulation was observed in D. hansenii cells grown in the presence of the oils of allspice, clove and garlic, and no sporulation was detected in the presence of other essential oils. Rapid sporulation
71
500
CINNAMON
ALLSPICE
/
400
,SII/
300
200
/
100
, ,.~
,
,
,
,
,
,
I
/o
"~ ~'~8~
I
,
J
500
GARLIC
CLOVE
/
400
300
200 /"
/
/
//° •
.//o~o/OI°/°/°
100
0
, o
I 3o
o~
_Z - ,
I10
I 90
t
I 120
,
I 1S0
,
I 180
o
/o /
I 0
[ 30
i~
, 60
1 90
,
I 120
I 16o
,
I 18o
TIME (rain) Fig. 3. Effects of 250 p . g / m l (o) a n d 5 0 0 / L g / m l ( O ) of allspice, clove, a n d garlic oleoresins a n d 2 5 / , t g / m l (O) a n d 50 ~ g / m l ( O ) of c i n n a m o n oleoresin on C O 2 p r o d u c t i o n by S. cerevisiae at 35°C in Y M P G b r o t h ( p H 5.5) c o n t a i n i n g 7.5% glucose. The controls c o n t a i n e d no a d d e d oleoresin or D M S O ( i ) and 1% D M S O 03).
was observed for H. anomala. Garlic oil increased the time required for sporulation to 14 days, and allspice oil delayed sporulation until 9 days. Sporulation of L. elongisporus occurred in 14 days under the control conditions. Clove oil had the least effect, delaying sporulation until day 17; allspice, cinnamon, garlic, oregano, savory, and thyme oils delayed sporulation to 27-41 days, and onion oil completely inhibited spore production. S. cerevisiae did not sporulate well in the control conditions; sporulation occurred in 65 days. An enhancement of sporulation was observed in the presence of allspice and savory oils but no sporulation occurred in the presence of the other essential oils.
Discussion
Corran and Edgar (1933) reported that cinnamon oil, and to a lesser extent clove oil, were inhibitory to yeast fermentations. The results here do not indicate an inhibitory action by these two oils, at least with regard to ethanol production.
?2 500 THYME
/S.
400
300
200
f/
100
~
o
~u 500 :S
=, >
ONION
OREGANO
/
400
3o0
/ 20o
./ / •/ •
lOO e / Io~O
0
I 0
3O
i,~? •o
~o
12o
1so
1~o
o
30
I 60
'~"
L o/, 90
/
o
o/O o.-/
oI o/
I 120
J
I aso
J
I 100
TIME (rain)
Fig. 4. Effects of 250/~g/ml (O) and 500/xg/ml (O) of thyme, onion and oregano oleoresins on CO 2 production by S. cerevisiae at 35°C in YMPG broth (pH 5.5) containing 7.5% glucose. The controls contained no added oleoresin or DMSO (i) and 1% DMSO ([3).
Wright et al. (1954) reported that cinnamon oil retarded CO 2 evolution by I~aker's yeast in simple sugar solutions. If CO 2 evolution is retarded, then ethanol production would be retarded also. Cinnamon oil did not show this effect on the strain of S. cerevisiae examined in this experiment. The inhibitory action of onion oil observed at the lower test concentrations may have been caused by an inhibition of alcohol dehydrogenase. Willis (1954) reported that 0.0005 M allicin, the active component of onion oil, inhibited the activity of alcohol dehydrogenase. Wright et al. (1954) reported that thyme enhanced CO 2 production by yeasts in simple sugar solutions. Thus, it could be expected that thyme would also enhance ethanol production; however, this phenomenon was not observed here. In contrast, Corran and Edgar (1933) reported that thyme oil was somewhat inhibitory to fermentation by yeasts, which is in agreement with the results reported here. The decrease in ethanol content of the culture media in some instances may be due to an assimilation of ethanol into other biochemical pathways. For example, H. anornala can produce large amounts of acetic acid which may be converted to ethyl acetate by the organism. Very high yields of ethyl acetate can be synthesized from glucose or ethanol by H. anornala (Phaff et al., 1978).
73 TABLE III Effects of essential oils and oleoresins on sporulation of ~our ascosporogenous yeasts Oil/oleoresin Control
Conch. (/.tg/ml)
Number of days required for sporulation a D. hansenii
H. anomala
L. elonglsporus
S. cerevisiae
0
65
6
14
65
Essential oil allspice cinnamon clove garlic onion oregano savory thyme
100 100 100 25 100 100 100 100
65 > 65 65 65 > 65 > 65 > 65 > 65
9 6 6 14 6 6 6 6
27 34 17 27 > 65 41 34 34
34 > 65 > 65 > 65 > 65 > 65 34 > 65
Oleoresin allspice cinnamon clove garlic onion oregano thyme
500 50 500 500 500 500 500
> 65 > 65 > 65 > 65 > 65 65 > 65
> 65 6 > 65 9 6 14 > 65
27 > 65 > 65 27 27 27 34
> 65 > 65 > 65 > 65 > 65 27 > 65
a Cultures were examined after various periods of incubation at 21°C on V-8 agar (pH 5.7); experiments were terminated after 65 days.
C i n n a m o n oil caused n o significant r e d u c t i o n in CO 2 evolution over that observed in the e t h a n o l controls. This is in contrast to o b s e r v a t i o n s b y Wright et al. (1954) who reported that the essential oil of c i n n a m o n retarded C O 2 evolution by b a k e r ' s yeast in simple sugar solutions, while low levels of whole c i n n a m o n e n h a n c e d or accelerated C O 2 evolution. C o r r a n a n d Edgar (1933) reported that g r o u n d cloves and, to a lesser extent, c i n n a m o n inhibited the f e r m e n t a t i v e activity a n d utilization of glucose b y yeasts. They also f o u n d that thyme, savory, a n d m a r j o r a m (similar to oregano) s t i m u l a t e d fermentative activity by b a k e r ' s yeast. A similar p h e n o m e n o n m a y have occurred here with o r e g a n o oil. The p r o d u c t i o n of C O 2 b y S. cerevisiae is a n i n d i c a t i o n of the o r g a n i s m ' s ability to f e r m e n t glucose. As the respiration or utilization of glucose into cellular energy decreases, the a m o u n t s of C O 2 a n d ethanol will be c o m p a r a b l y reduced. Baranowski a n d Nagel (1983) suggested that alkyl h y d r o x y c i n n a m a t e s , which are quite similar to the phenolic derivatives f o u n d as m a j o r c o n s t i t u e n t s in the essential oils of allspice (eugenol), clove (eugenol), c i n n a m o n ( c i n n a m i c aldehyde, eugenol), o r e g a n o (carvacrol), thyme (thymol), a n d savory (thymol), i n h i b i t e d growth of P s e u d o m o n a s f l u o r e s c e n s b y a specific m o d e of action related in part to depletion of cellular energy. A similar p h e n o m e n o n m a y have been occurring with S. cerevisiae. T h e i n h i b i t i o n of s p o r u l a t i o n by yeasts m a y be related to a n imp/fired energy
74 s y n t h e s i s in y e a s t s c a u s e d b y the p r e s e n c e of t h e s e e s s e n t i a l oils. K u e n z i et al. (1974) r e p o r t e d t h a t h i g h r e s p i r a t o r y activity, w h i c h r e q u i r e s e l e v a t e d s u c c i n a t e d e h y d r o g e n a s e a n d c y t o c h r o m e o x i d a s e activity, w a s r e q u i r e d for the s p o r u l a t i o n of S. cerevisme. Willis (1954) r e p o r t e d t h a t 0.0005 M allicin i n h i b i t e d the a c t i v i t y o f s u c c i n i c d e h y d r o g e n a s e . T h e r e f o r e , garlic oil m a y h a v e i n h i b i t e d H. a n o m a l a s p o r u l a t i o n b y i n h i b i t i n g r e s p i r a t i o n n e c e s s a r y for a s c o s p o r e f o r m a t i o n . In s u m m a r y , the e s s e n t i a l oils a n d o l e o r e s i n s o f p l a n t s c o m m o n l y u s e d as s e a s o n i n g a g e n t s in f o o d s c a n s u b s t a n t i a l l y i n h i b i t e t h a n o l p r o d u c t i o n , r e s p i r a t i o n a n d s p o r u l a t i o n o f yeasts. T h e e x t e n t o f i n h i b i t i o n in f o o d s y s t e m s r e m a i n s to be d e m o n s t r a t e d , h o w e v e r , a n d f u r t h e r i n v e s t i g a t i o n s are n e e d e d to d e t e r m i n e the full s i g n i f i c a n c e o f a n t i - y e a s t activities o f oils a n d o l e o r e s i n s in r e t a r d i n g or p r e v e n t i n g spoilage or production of foods and beverages.
References Baranowski, J.D. and C.W. Nagel, 1983. Properties of alkyl hydroxy-cinnamates and effects on Pseudomonasfluorescens. Appl. Environ. Microbiol. 45, 218-222. Cavallito, C.J. and J.H. Bailey, 1944. Allicin, the antibacterial principle of Allium satwum. I. Isolation, physical properties and antibacterial action. J. Am. Chem. Soc. 66, 1950-1951. Cavallito, C.J., J.H. Bailey and J.S. Buck, 1945. The antibacterial principle of Allium satwum. III. Its precursor and 'essential oil of garlic'. J. Am. Chem. Soc. 67, 1032-1033. Conner, D.E. and L.R. Beuchat, 1984a. Inhibitory effects of essential oils from plants on food spoilage yeasts. J. Food Sei. 49, (in press). Conner, D.E. and L.R. Beuchat, 1984b. Inhibitory effects of plant oleoresins on yeasts. Proceedings of the 12th International Symposium, International Union of Microbiological Societies, Microbial Associations and Interactions in Food, July 12-15, 1983, Budapest. Conner, D.E. and L.R. Beuchat, 1984c. Sensitivity of heat-stressed yeasts to essential oils of plants. Appl. Environ. Microbiol. 47, 229-233. Corran, J.W. and S.H, Edgar, 193. Preservation action of spices and related compounds against yeast fermentation. J. Soc. Chem. Ind. 52, 149T-152T. DeWit, J.C., S. Notermans, N. Gorin and E.H. Kampelmacher, 1979. Effect of garlic or onion oil on toxin production by Clostridium botulinum in meat slurry. J. Food Protect. 42, 222-224. Hitokoto, H., S. Morozumi, T. Wauke, S. Sakai and H. Kurata, 1980. Inhibitory effects of spices on growth and toxin production of toxigenic fungi. Appl. Environ. Microbiol. 39, 818-822. Johnson, M.G. and R.H. Vaughn, 1969. Death of Salmonella typhimurium and Escherichia coli in the presence of freshly reconstituted dehydrated garlic and onion. AppI. Environ. Microbiol. 17, 903-905. Kuenzi, M.T., M.A. Tingle and H.O. Halvorson, 1974. Sporulation of Saccharornyces cerevisiae in the absence of a functional mitochondrial genome. J. Bacteriol. 117, 80-88. Mantis, A.J., P.G. Karaioannoglou, G.P. Spanos and A.G. Panetsos, 1978. The effect of garlic extract on food poisoning bacteria in culture media. I. Staphylococcus aureus. Lebensm. Wiss. Technol. 11, 26-28. Phaff, H.J., M.W. Miller and E.M. Mrak, 1978. The Life of Yeasts, Harvard University Press, Cambridge, MA. Tynecka, Z. and Z. Gos, 1973. Inhibitory action of garlic (Allium sativum) on growth and respiration of some microorganisms. Acta Microbiol., Series B, 5, 51-62. Willis, E.D., 1954. Enzyme Inhibition by allicin, the active principle of garlic. Biochemistry 63, 514-520. Wright, W.J., C.W. Bice and J.M. Fogelberg, 1954. The effect of spices on yeast fermentation. Cereal Chem. 31,100-112.
63
Elsevier JFM 00007
Effects of essential oils and oleoresins of plants on ethanol production, respiration and sporulation of yeasts D.E. Conner, L.R. Beuchat, R.E. Worthington and H.L. Hitchcock Department of Food Science, University of Georgia, Agricultural Experiment Station. Experiment. GA 30212. U.S.A.
(Received 25 October 1983; accepted 3 February 1984)
Ethanol production, respiration, and sporulation of yeasts as affected by essential oils and oleoresins of allspice, cinnamon, clove, garlic, onion, oregano, savory, and thyme were investigated. Essential oils of allspice, cinnamon, and clove had little or no effect on ethanol production by Saccharomyces cerevisiae. Oils of onion, oregano, savory, and thyme delayed and/or reduced the production of ethanol. Overall, essential oils effectively suppressed ethanol production by Hansenula anomala. At the highest concentrations tested (500/ag/ml), only cinnamon, clove, garlic and thyme oleoresins substantially delayed and/or reduced ethanol production by S. cereoisiae. Most of the essential oils (100 #g/ml) impaired the respiratory activity of S. cerevisiae as evidenced by a reduction in CO2 production. Thyme oleoresin was the strongest inhibitor. Allspice and garlic oils impaired sporulation by H. anomala. All oils delayed sporulation of Lodderomyces elongisporus. Key words: Essential oils; Hansenula anomala," Lodderomyces elongisporus; Oleoresins; Saccharom.vces cereoisiae; Sporulation; Yeasts
Introduction T h e m e d i c i n a l activities of p l a n t extracts have been recognized for m a n y years. P l a n t s of the Allium species, p a r t i c u l a r l y garlic a n d onion, have been s t u d i e d most extensively ( C a v a l l i t o a n d Bailey, 1944; C a v a l l i t o et al., 1945). G r o w t h of f o o d - b o r n e p o i s o n i n g bacteria, e.g., Staphylococcus aureus ( M a n t i s et al., 1978), Salmonella typhimurium ( J o h n s o n a n d Vaughn, 1969) a n d Clostridium botulinum ( D e W i t et al., 1979), are i n h i b i t e d b y extracts of garlic a n d onion. G r o w t h of the p a t h o g e n i c yeast, Candida albicans ( T y n e c k a a n d Gos, 1973), a n d m y c o t o x i g e n i c Aspergillus species ( H i t o k o t o et al., 1980) are also i n h i b i t e d b y p l a n t extracts, i n c l u d i n g those from garlic. Little is k n o w n a b o u t the effects of essential oils a n d oleoresins on yeasts which c o m m o n l y cause f o o d spoilage or which are used in f o o d a n d b e v e r a g e f e r m e n t a tions. Pioneer studies have shown that spices have an i n h i b i t o r y effect on yeast f e r m e n t a t i o n ( C o r r a n a n d Edgar, 1933; W r i g h t et al., 1954). In o t h e r studies ( C o n n e r 0168-1605/84/$03.00 © 1984 Elsevier Science Publishers B.V.
64 and Beuchat 1984a, b) we reported that growth of several genera of yeasts is inhibited by essential oils and oleoresins of plants. Heat-stressed cells have an increased sensitivity to oils and oleoresins, indicating that these plant components inhibit structural a n d / o r metabolic repair of injured cells (Conner and Beuchat, 1984c). The study reported herein was designed to determine the effects of oils and oleoresins on ethanol production, respiration and sporulation of yeasts important to the food industry.
Materials and Methods
Evaluation of thirty-two essential oils and eight oleoresins in an earlier study (Conner and Beuchat 1984a) revealed a wide range of inhibitory activity on food spoilage and industrially important yeasts. Since the essential oils of allspice, cinnamon, clove, garlic, onion, oregano, savory and thyme exhibited the highest anti-yeast activities, these were selected for further evaluation. Oleoresins of allspice, cinnamon, clove, garlic, onion, oregano, and thyme were al~o investigated. The species of plant material from which essential oil was extracted was not in all instances the same species used to obtain the corresponding oleoresin. All oils and oleoresins were provided by Fritzsche, Dodge and Olcott, Inc., New York, N Y . Yeast evaluated and sources were Debaryomyces hansenii N R R L Y-7268 (steer dung), Hansenula anomala 67-455 (tree exudate), Lodderomvces elongisporus N R R L YB-4239 (concentrated orange juice) and Saccharomyces cerevisiae UGA-102 (Chablis wine). Test cells were prepared by culturing the yeasts in sterile yeast extract/malt extract/peptone/glucose (YMPG) broth (pH 5.5; 100 ml per 250-ml Erlenmeyer flask). The medium consisted of 3.0 g yeast extract, 3.0 g malt extract, 5.0 g peptone and 10 g glucose per 1000 ml of distilled water. Cultures incubated at 30°C on a rotary shaker for 44-48 h and diluted 10 -2 in 0.1 M potassium phosphate buffer (pH 7.0) served as inocula for experiments conducted to determine the effects of oils and oleoresins on ethanol production and sporulation. The procedure for preparing cells for respiration studies was modified as indicated below. Two yeasts, S. cerevisiae and H. anomala, were tested for ethanol production in modified Y M P G broth. Essential oils were diluted in 95% ethanol and dispersed in sterile YMPG broth containing 5% (w/v) glucose (pH 5.5; 100 ml per 250-ml Erlenmeyer flask) to give final concentrations of 25, 50, 100, and 200/.tg/ml. Two controls, one containing no added ethanol or essential oil and the other containing 1% ethanol (the highest level present in any of the test broths containing essential oils), but no oil, were included as ethanol production media. Two ethanol-producing yeasts, S. cerevisiae and H. anomala, were tested. Inoculum (1.0 ml) was dispensed into 100 ml of sterile control and test broths. Cultures were incubated statically at 30°C under a carbon dioxide gas atmosphere; at 2, 4, and 7 days, the flasks were vigorously swirled, and a 5 ml aliquot was withdrawn and centrifuged at 10000 × g for 8 rain. The supernatant fluid was carefully decanted into a sterile test tube, sealed, and stored at - 2 8 ° C until analysis could be performed.
65 Supernates were analyzed for ethanol content using a gas liquid chromatograph. Three aliquots (2.0/~1) of supernatant fluid were injected into a MicroTek 200 gas chromatograph (Tracor, Inc., Austin, TX) equipped with a flame ionization detector. The 1.83 m x 0.32 cm (ID) glass column was packed with 10% SP-1200/1% H3PO 4 on 80/100 Chromasorb WAW (Supelco, Bellefonte, PA). The oven temperature was 190°C; detector temperature, 240°C; and injector temperature, 240°C. The carrier gas was helium adjusted at a flow rate of 50 m l / m i n and the detector gases were hydrogen (65 c c / m i n ) and air (12 SCFH). A standard curve was made by plotting integral areas against respective ethanol standard solution concentrations of 0.1, 0.25, 0.50, 1.0, 2.5, 5.0, and 10.0%. Linear regression of data was determined and an equation was developed for calculating the percentage ( v / v ) of ethanol in samples based on the peak area given by each sample. The investigation of the effect of oleoresins on ethanol production was conducted using the same procedure and conditions as those used in the study using essential oils. However, the oleoresins were diluted in dimethyl sulfoxide (DMSO) instead of ethanol and dispersed into the test media to give final concentrations of 50, 100. 250, and 500 ~tg/ml. A control broth containing 1% D M S O was included. The production of CO2 by S. cerevisiae and H. anomala was investigated in Y M P G broth containing 7.5% glucose and 0, 75 and 150 /~g/ml of essential oils. Three controls were included in the test, one containing no added ethanol or essential oil, one containing 1.5% ethanol, but no oil (the level of ethanol present in the 150 ~ g / m l test broths) and the third containing 0.75% ethanol, but no oil (the level of ethanol present in the test broths containing 7 5 / ~ g / m l of essential oils). S. cerevisiae was cultured in Y M P G broth under constant agitation at 30°C for 4 4 - 4 8 h and centrifuged at 10000 x g. The supernatant fluid was discarded and the cells were suspended in 0.1 M potassium phosphate buffer (pH 7.0). This washing procedure was done twice. After the second washing, the cells were resuspended in buffer to give an absorbance of 0.4 as measured by a Spectronic 20 spectrophotometer (Bausch and Lomb, Rochester, NY) set at 600 nm. 1 ml of this cell suspension was added to the side-arm chamber of a respiration reaction flask containing 2.0 ml of the test broth in the main chamber. The flasks were then connected to a single valve differential respirometer (Gilson Medical Electronics, Inc., Middleton, WI) and allowed to equilibrate at 35°C for 20 min. After equilibration, the cell suspensions and test broths were thoroughly mixed. The addition of the cell suspensions to the test broths diluted the essential oils to give final concentrations of 50 and 100 /~g/ml, while the ethanol-supplemented controls were similarily diluted to yield 1.0 and 0.5% of 95% ethanol in the actual reaction mixture. Once the cells and broths were mixed, gas (CO 2) evolution at 35°C was manometrically determined at 15-rain intervals for 3 h. Determination of the effects of oleoresins on CO z evolution by S. cerevisiae was made using the same experimental procedures and conditions as those used in the study of the essential oils. However, the oleoresins were diluted in D M S O and dispersed to give final concentrations of 0.025 and 0.050% in the reaction fluid. A control broth containing 1% D M S O was included. Experiments were conducted to determine the effects of the eight essential oils on
66 the production of ascospores by D. hansenii and H. anomala, L. elongisporus, and S. cerevisiae. The sporulation medium, V-8 juice agar, consisted of 350 ml of V-8 juice (Campbell's Soup Co., Camden, N J), 5.0 g yeast extract, 650 ml of water, and 14 g of agar. The pH wa~ adjusted to 5.7 with 2 N NaOH. After the V-8 juice agar was autoclaved at 121°C for 15 min, it was supplemented with essential oils of allspice, cinnamon, clove, onion, oregano, savory and thyme at concentrations of 100 # g / m l and garlic oil 25 # g / m l . V-8 agar containing no added essential oil served as a control. Plates were surface-inoculated, wrapped in plastic sleeves to prevent moisture loss, and incubated at 21°C. At various intervals during a 65-day period, cells were picked from plates and observed microscopically under an oil-immersion objective for the presence of ascospores. If cells of a sample consisted of 5% or more asci, the culture was scored positive for ascospore production.
Results
The effects of essential oils on ethanol production by S. cerevisiae and H. anornala are listed in Table I. In the control broth containing no added supplements, a maximum ethanol level was observed at 168 h, while in the control broth initially containing 1% ethanol, the ethanol concentration reached 3.3% at 96 h, and then declined to 2.7% at 168 h. The decrease in ethanol content may have been due, in part, to evaporation as well as assimilation during prolonged incubation. It is estimated that less than 5% of the ethanol initially present in control flasks was lost to evaporation after 168 h. At 25 /~g/ml, allspice oil had little effect on ethanol production, while at 100 and 200/~g/ml, ethanol production was slightly delayed. Cinnamon and clove oils exhibited no effect on ethanol production. Garlic oil was lethal to S. cerevisiae, so consequently no ethanol was produced. The yeast did not overcome the inhibitory effects of this oil during the 168 h test period. Onion oil totally inhibited growth and ethanol production by S. cerevisiae at 100 and 50 /~g/ml, respectively, with lesser effects at lower concentrations. The thymol/carvacrol containing oils (oregano, savory and thyme) were similar in their effects on ethanol production by S. cerevisiae. Levels of ethanol comparable to that of the unsupplemented control were observed in broths containing 25 and 50/zg/ml of these oils. At 50/xg/ml, thyme oil did slightly depress production as compared to the control. At the 100/~g/ml level of these oils, a significant amount of ethanol was produced at 48 h; however, peak levels were not achieved until 96-168 h. The ethanol production mechanism of H. anomala was more sensitive than that of S. cerevisiae to the essential oils (Table I). In the control broths, H. anomala produced 2.4% ethanol in a 96 h period, after which the concentration declined. The levels of ethanol produced in the presence of allspice, cinnamon and clove oils were lower than those found in the control broths. Garlic oil was completely inhibitory to growth of H. anomala and onion oil had a pronounced delay effect. Increased concentrations of oregano, savory and thyme oils caused increasing delays in ethanol production. Table II summarizes the results of tests conducted to determine the effects of
67 TABLE I Effect of essential oils on ethanol production by Saccharomvces cereoisiae and Hansenula anomala Essential oil
Concn. (#g/ml)
Ethanol (%, v / v ) produced by S. cerevisiae
Ethanol (%, v / v ) produced by H. anomala
48 h
96 h
168 h
48 h
96 h
168 h
0 0
2.8 2.9
2.9 3.3
2.9 2.7
0.5 0.4
2.4 2.4
2.1 2.1
Allspice
25 50 100 200
2.8 3.2 2.7 2.5
3.0 2.7 2.9 2.8
2.4 _ a 2.5 2.9
0.4 0.6 0.6 0.5
2.1 2.2 1.8 1.3
1.9 1.9 1.0 0.9
Cinnamon
25 50 100 200
3.1 2.8 3.0 2.9
2.9 2.7 3.0 2.8
0.7 0.6 0.5 0.3
2.2 2.3 1.4 1.3
2.2 1.7 1.0 0.9
Clove
25 50 100 200
2.9 3.3 2.9 2.9
2.8 2.8 2.9 2.9
-
0.6 0.6 0.4 0.5
2.3 2.4 1.8 1.4
1.8 2.1 1.5 0.9
Garlic
25 50 100 200
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
Onion
25 50 100 200
0 0 0 0
0.2 0 0 0
3.0 0.1 0 0
0 0 0 0
1.6 0.9 0.1 0.1
0.9 1.6 2.2 0.4
Oregano
25 50 100 200
2.9 2.8 1.9 0
2.8 2.9 2.7 0.4
2.9 3.1
0.5 0.5 0.1 0
2.1 2.1 1.1 0.1
1.4 1.2 0.6 0.4
25 50 100 200
2.6 3.0 1.7 0
2.5 2.6 2.4 2.3
2.8 2.6
0.4 0.2 0.1 0
2,4 1.9 1.4 0.5
1.5 1.5 1.2 1.4
25 50 100 200
2.6 2.5 1.7 0
2.6 2.7 2.5 1.8
2.8 2.9 2.4 2.8
0.6 0.3 0 0
2.1 1.9 1.2 0.2
1.9 1.4 1.1 1.2
Control Control + 1% ethanol
Savory
Thyme
-
a Not analyzed. oleoresins
on ethanol
the respective inhibitory
production
b y S. cereoisiae a n d H. a n o m a l a . A s c o m p a r e d
essential oils at similar concentrations,
to ethanol
production.
t i o n b y S. c e r e v i s i a e o c c u r r e d
In most instances,
at 48-96
the oleoresins near maximum
h of incubation.
were much ethanol
to less
produc-
At the highest concentration
68
TABLE II Effect of oleoresins on ethanol production by Saccharomyces cerevisiae and Hansenula anomala Oleoresin
Ethanol (%, v/v) produced by S. cerevisiae
Ethanol (%, v/v) produced by H. anomala
48 h
96 h
168 h
48 h
96 h
168 h
0 0
3.1 3.0
3.1 3.0
2.3 2.2
0.6 0.9
2.3 2.2
2.1 1.8
Allspice
50 100 250 500
2.8 3.0 2.6 2.9
3.0 3.1 2.8 3.2
2.2 t.9 2.1 2.3
0.7 0.6 0.6 0.6
2.1 2.0 2.5 1.6
1.9 1.8 1.7 1.0
Cinnamon
50 100 250 500
2.8 2.8 2.6 2.3
2.8 2.8 3.2 2.9
_a 2.4 2.2
0.6 0.6 0.6 0.5
2.3 2.1 2.2 1.8
1.8 2.0 1.8 2.1
Clove
50 100 250 500
3.0 2.9 3.0 2.0
3.1 2.9 2.8 2.6
2.5 2.5 2.4 2.4
0.7 0.7 0.7 0.3
1.9 2.0 1.5 1.2
1.8 1.8 0.7 1.4
Garlic
50 100 250 500
2.8 2.7 2.7 1.7
2.8 3.2 2.8 2.9
2.2 2.2 2.1
0.5 0.6 0.4 0.4
2.1 2.1 2.1 1.9
1.9 2.1 1.5 1.6
Onion
50 100 250 500
2.9 3.1 2.9 2.7
3.1 3.3 3.2 3.0
2.2 2.3 2.2 2.5
0.5 0.6 0.6 0.5
2.3 2.2 2.1 2.3
2.0 1.7 2.0 1.8
Oregano
50 100 250 500
2.7 2.9 3.0 3.1
3.0 2.9 2.8 2.8
2.2 -
0.6 0.6 0.6 0.6
2.5 2.5 1.5 1.4
1.5 1.4 0.7 0.6
Thyme
50 100 250 500
3.0 2.9 3.0 0.1
3.0 3.0 2.5 0.2
2.4 2.4
0.6 0.7 0.2 0
2.0 1.8 1.2 0.1
1.8 1.0 0.8 0.4
Control Control + 1% DMSO
Concn. (#g/ml)
a Not analyzed.
t e s t e d (500 ~ g / m l ) , o n l y c i n n a m o n (50 # g / m l ) , c l o v e , garlic, a n d t h y m e o l e o r e s i n s s u b s t a n t i a l l y d e l a y e d a n d / o r r e d u c e d e t h a n o l liberation. In m o s t instances, the h i g h e s t e t h a n o l levels w e r e o b s e r v e d i n t h e 96 h c u l t u r e s o f H. a n o m a l a . T h e 500 / z g / m l ( 5 0 / ~ g / m l o f c i n n a m o n ) levels o f m o s t o f t h e o l e o r e s i n s ( e x c e p t o n i o n ) w e r e slightly i n h i b i t o r y to e t h a n o l p r o d u c t i o n . T h y m e oleoresin again was the m o s t i n h i b i t o r y . All levels o f t h y m e a n d c l o v e o l e o r e s i n s s l i g h t l y s u p p r e s s e d e t h a n o l p r o d u c t i o n . Interestingly, the large r e d u c t i o n s in e t h a n o l c o n t e n t s o b s e r v e d in tests
69
involving essential oil were also observed here. Again, oregano caused the largest reductions. The data illustrated in Figs. 1 and 2 from tests conducted to determine the effects of the essential oils on respiration (CO 2 evolutiop) by S. cereoisiae indicate that all of the oils reduced .the respiratory activity of the yeast as compared to the control. Ethanol also had a pronounced influence on respiration during the 3 h incubation period. The essential oils of clove, allspice, and cinnamon gave similar results (Fig. 1). The oils of garlic (Fig. 1) and onion (Fig. 2) were tested at the low concentrations of 5 and 10 # g / m l because of their strong inhibitory action against S. cerevisiae observed in previous experiments. Compared to the broth containing no essential oil or ethanol, production of CO 2 was slightly reduced over the 3 h period in the presence of low concentrations of garlic and onion oils. Oil of oregano (Fig. 2) at 50 # g / m l caused a stimulation of CO 2 evolution as compared to that observed for the 0.5% ethanol control. The presence of 100 /~g/ml oregano oil had no substantial effect on the yeast's respiration when compared to the ethanol control. Thyme oil, at both concentrations tested, slightly retarded CO 2 evolution as compared to the 500 CLOVE
CINNAMON
/-
400 300
" .-
200
,-a
lOO
°/
._.=
~
o
,
500
I
ALLSPICE 0
>
,,,
I
GARLIC
400
/
300 200
•o
/ /°
100
/0/0/o/°
0
~ 0
I 30
~
I eO
, 90
n 120
I 160
t
I 180
J 0
I 30
I0
~
120
160
180
TIME (min)
Fig. 1. Effects of 50 # g / m l (e) and 100 # g / m l (O) of clove, cinnamon, and allspice essential oils, and 5 # g / m l (e) and 10 # g / m l ( O ) of garlic oil on CO 2 production by S. cereoisiae at 35°C in YMPG broth (pH 5.5) containing 7.5% glucose. Control broths contained no added ethanol (m), 1% ethanol (*), and 0.5% ethanol (13).
70 500 THYME
SAVORY
/
400
300 200
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oll 90
TIME (min)
Fig. 2. Effects of 50/~g/ml (e) and 100/Lg/ml (O) of thyme, savory, and oregano essential oils, and 5 t~g/ml (e) and 10 t~g/ml (O) of onion oil on CO 2 production by S. cerevisiae at 35°C in YMPG broth (pH 5.5) containing 7.5% glucose. Control broths contained no added ethanol (m), 1% ethanol (-), and 0.5% ethanol (m).
corresponding controls. Interestingly, savory oil (50 / l g / m l ) slightly reduced CO 2 production while 1 0 0 / z g / m l had little or no effect. Data illustrating the effects of the oleoresins on the respiration of S. cerevisiae are shown in Figs. 3 and 4. The level of D M S O contained in each of the test broths had no effect on CO 2 production in this test. All of the oleoresins at 500 ~tg/ml, and cinnamon oleoresin at 50 t~g/ml, effectively inhibited CO 2 production by S. cerevisiae. Overall, excluding garlic and onion oleoresins, oleoresins were much more inhibitory to CO 2 production (respiration) by S. cerevisiae than were any of the tested concentrations of essential oils. The suspected modes of action of the oleoresins on the respiratory activities of S. cerevisiae are the same as those for the essential oils. Sporulation data are listed in Table III. D. hansenii did not sporulate very well in this experiment; ascospores were not observed in the control culture until 65 days of incubation, and the percentage of asci was low. Poor sporulation was observed in D. hansenii cells grown in the presence of the oils of allspice, clove and garlic, and no sporulation was detected in the presence of other essential oils. Rapid sporulation
71
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CINNAMON
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TIME (rain) Fig. 3. Effects of 250 p . g / m l (o) a n d 5 0 0 / L g / m l ( O ) of allspice, clove, a n d garlic oleoresins a n d 2 5 / , t g / m l (O) a n d 50 ~ g / m l ( O ) of c i n n a m o n oleoresin on C O 2 p r o d u c t i o n by S. cerevisiae at 35°C in Y M P G b r o t h ( p H 5.5) c o n t a i n i n g 7.5% glucose. The controls c o n t a i n e d no a d d e d oleoresin or D M S O ( i ) and 1% D M S O 03).
was observed for H. anomala. Garlic oil increased the time required for sporulation to 14 days, and allspice oil delayed sporulation until 9 days. Sporulation of L. elongisporus occurred in 14 days under the control conditions. Clove oil had the least effect, delaying sporulation until day 17; allspice, cinnamon, garlic, oregano, savory, and thyme oils delayed sporulation to 27-41 days, and onion oil completely inhibited spore production. S. cerevisiae did not sporulate well in the control conditions; sporulation occurred in 65 days. An enhancement of sporulation was observed in the presence of allspice and savory oils but no sporulation occurred in the presence of the other essential oils.
Discussion
Corran and Edgar (1933) reported that cinnamon oil, and to a lesser extent clove oil, were inhibitory to yeast fermentations. The results here do not indicate an inhibitory action by these two oils, at least with regard to ethanol production.
?2 500 THYME
/S.
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Fig. 4. Effects of 250/~g/ml (O) and 500/xg/ml (O) of thyme, onion and oregano oleoresins on CO 2 production by S. cerevisiae at 35°C in YMPG broth (pH 5.5) containing 7.5% glucose. The controls contained no added oleoresin or DMSO (i) and 1% DMSO ([3).
Wright et al. (1954) reported that cinnamon oil retarded CO 2 evolution by I~aker's yeast in simple sugar solutions. If CO 2 evolution is retarded, then ethanol production would be retarded also. Cinnamon oil did not show this effect on the strain of S. cerevisiae examined in this experiment. The inhibitory action of onion oil observed at the lower test concentrations may have been caused by an inhibition of alcohol dehydrogenase. Willis (1954) reported that 0.0005 M allicin, the active component of onion oil, inhibited the activity of alcohol dehydrogenase. Wright et al. (1954) reported that thyme enhanced CO 2 production by yeasts in simple sugar solutions. Thus, it could be expected that thyme would also enhance ethanol production; however, this phenomenon was not observed here. In contrast, Corran and Edgar (1933) reported that thyme oil was somewhat inhibitory to fermentation by yeasts, which is in agreement with the results reported here. The decrease in ethanol content of the culture media in some instances may be due to an assimilation of ethanol into other biochemical pathways. For example, H. anornala can produce large amounts of acetic acid which may be converted to ethyl acetate by the organism. Very high yields of ethyl acetate can be synthesized from glucose or ethanol by H. anornala (Phaff et al., 1978).
73 TABLE III Effects of essential oils and oleoresins on sporulation of ~our ascosporogenous yeasts Oil/oleoresin Control
Conch. (/.tg/ml)
Number of days required for sporulation a D. hansenii
H. anomala
L. elonglsporus
S. cerevisiae
0
65
6
14
65
Essential oil allspice cinnamon clove garlic onion oregano savory thyme
100 100 100 25 100 100 100 100
65 > 65 65 65 > 65 > 65 > 65 > 65
9 6 6 14 6 6 6 6
27 34 17 27 > 65 41 34 34
34 > 65 > 65 > 65 > 65 > 65 34 > 65
Oleoresin allspice cinnamon clove garlic onion oregano thyme
500 50 500 500 500 500 500
> 65 > 65 > 65 > 65 > 65 65 > 65
> 65 6 > 65 9 6 14 > 65
27 > 65 > 65 27 27 27 34
> 65 > 65 > 65 > 65 > 65 27 > 65
a Cultures were examined after various periods of incubation at 21°C on V-8 agar (pH 5.7); experiments were terminated after 65 days.
C i n n a m o n oil caused n o significant r e d u c t i o n in CO 2 evolution over that observed in the e t h a n o l controls. This is in contrast to o b s e r v a t i o n s b y Wright et al. (1954) who reported that the essential oil of c i n n a m o n retarded C O 2 evolution by b a k e r ' s yeast in simple sugar solutions, while low levels of whole c i n n a m o n e n h a n c e d or accelerated C O 2 evolution. C o r r a n a n d Edgar (1933) reported that g r o u n d cloves and, to a lesser extent, c i n n a m o n inhibited the f e r m e n t a t i v e activity a n d utilization of glucose b y yeasts. They also f o u n d that thyme, savory, a n d m a r j o r a m (similar to oregano) s t i m u l a t e d fermentative activity by b a k e r ' s yeast. A similar p h e n o m e n o n m a y have occurred here with o r e g a n o oil. The p r o d u c t i o n of C O 2 b y S. cerevisiae is a n i n d i c a t i o n of the o r g a n i s m ' s ability to f e r m e n t glucose. As the respiration or utilization of glucose into cellular energy decreases, the a m o u n t s of C O 2 a n d ethanol will be c o m p a r a b l y reduced. Baranowski a n d Nagel (1983) suggested that alkyl h y d r o x y c i n n a m a t e s , which are quite similar to the phenolic derivatives f o u n d as m a j o r c o n s t i t u e n t s in the essential oils of allspice (eugenol), clove (eugenol), c i n n a m o n ( c i n n a m i c aldehyde, eugenol), o r e g a n o (carvacrol), thyme (thymol), a n d savory (thymol), i n h i b i t e d growth of P s e u d o m o n a s f l u o r e s c e n s b y a specific m o d e of action related in part to depletion of cellular energy. A similar p h e n o m e n o n m a y have been occurring with S. cerevisiae. T h e i n h i b i t i o n of s p o r u l a t i o n by yeasts m a y be related to a n imp/fired energy
74 s y n t h e s i s in y e a s t s c a u s e d b y the p r e s e n c e of t h e s e e s s e n t i a l oils. K u e n z i et al. (1974) r e p o r t e d t h a t h i g h r e s p i r a t o r y activity, w h i c h r e q u i r e s e l e v a t e d s u c c i n a t e d e h y d r o g e n a s e a n d c y t o c h r o m e o x i d a s e activity, w a s r e q u i r e d for the s p o r u l a t i o n of S. cerevisme. Willis (1954) r e p o r t e d t h a t 0.0005 M allicin i n h i b i t e d the a c t i v i t y o f s u c c i n i c d e h y d r o g e n a s e . T h e r e f o r e , garlic oil m a y h a v e i n h i b i t e d H. a n o m a l a s p o r u l a t i o n b y i n h i b i t i n g r e s p i r a t i o n n e c e s s a r y for a s c o s p o r e f o r m a t i o n . In s u m m a r y , the e s s e n t i a l oils a n d o l e o r e s i n s o f p l a n t s c o m m o n l y u s e d as s e a s o n i n g a g e n t s in f o o d s c a n s u b s t a n t i a l l y i n h i b i t e t h a n o l p r o d u c t i o n , r e s p i r a t i o n a n d s p o r u l a t i o n o f yeasts. T h e e x t e n t o f i n h i b i t i o n in f o o d s y s t e m s r e m a i n s to be d e m o n s t r a t e d , h o w e v e r , a n d f u r t h e r i n v e s t i g a t i o n s are n e e d e d to d e t e r m i n e the full s i g n i f i c a n c e o f a n t i - y e a s t activities o f oils a n d o l e o r e s i n s in r e t a r d i n g or p r e v e n t i n g spoilage or production of foods and beverages.
References Baranowski, J.D. and C.W. Nagel, 1983. Properties of alkyl hydroxy-cinnamates and effects on Pseudomonasfluorescens. Appl. Environ. Microbiol. 45, 218-222. Cavallito, C.J. and J.H. Bailey, 1944. Allicin, the antibacterial principle of Allium satwum. I. Isolation, physical properties and antibacterial action. J. Am. Chem. Soc. 66, 1950-1951. Cavallito, C.J., J.H. Bailey and J.S. Buck, 1945. The antibacterial principle of Allium satwum. III. Its precursor and 'essential oil of garlic'. J. Am. Chem. Soc. 67, 1032-1033. Conner, D.E. and L.R. Beuchat, 1984a. Inhibitory effects of essential oils from plants on food spoilage yeasts. J. Food Sei. 49, (in press). Conner, D.E. and L.R. Beuchat, 1984b. Inhibitory effects of plant oleoresins on yeasts. Proceedings of the 12th International Symposium, International Union of Microbiological Societies, Microbial Associations and Interactions in Food, July 12-15, 1983, Budapest. Conner, D.E. and L.R. Beuchat, 1984c. Sensitivity of heat-stressed yeasts to essential oils of plants. Appl. Environ. Microbiol. 47, 229-233. Corran, J.W. and S.H, Edgar, 193. Preservation action of spices and related compounds against yeast fermentation. J. Soc. Chem. Ind. 52, 149T-152T. DeWit, J.C., S. Notermans, N. Gorin and E.H. Kampelmacher, 1979. Effect of garlic or onion oil on toxin production by Clostridium botulinum in meat slurry. J. Food Protect. 42, 222-224. Hitokoto, H., S. Morozumi, T. Wauke, S. Sakai and H. Kurata, 1980. Inhibitory effects of spices on growth and toxin production of toxigenic fungi. Appl. Environ. Microbiol. 39, 818-822. Johnson, M.G. and R.H. Vaughn, 1969. Death of Salmonella typhimurium and Escherichia coli in the presence of freshly reconstituted dehydrated garlic and onion. AppI. Environ. Microbiol. 17, 903-905. Kuenzi, M.T., M.A. Tingle and H.O. Halvorson, 1974. Sporulation of Saccharornyces cerevisiae in the absence of a functional mitochondrial genome. J. Bacteriol. 117, 80-88. Mantis, A.J., P.G. Karaioannoglou, G.P. Spanos and A.G. Panetsos, 1978. The effect of garlic extract on food poisoning bacteria in culture media. I. Staphylococcus aureus. Lebensm. Wiss. Technol. 11, 26-28. Phaff, H.J., M.W. Miller and E.M. Mrak, 1978. The Life of Yeasts, Harvard University Press, Cambridge, MA. Tynecka, Z. and Z. Gos, 1973. Inhibitory action of garlic (Allium sativum) on growth and respiration of some microorganisms. Acta Microbiol., Series B, 5, 51-62. Willis, E.D., 1954. Enzyme Inhibition by allicin, the active principle of garlic. Biochemistry 63, 514-520. Wright, W.J., C.W. Bice and J.M. Fogelberg, 1954. The effect of spices on yeast fermentation. Cereal Chem. 31,100-112.