TECHNICAL
NOTES
F a t t y A c i d s of V e g e t a t i v e Cells and Spores of Bacillus l i c h e n i f o r m i s 1, 2 J. H. MARTIN Dairy Science Department South Dakota State University Brookings, SO 57006 and
P. E. SWENSON Borden Research Centre Syracuse, NY
Bacillus organisms has been reported. However, Kaneda (3) reported the lipid content of vegetative cells of several Bacillus organisms. He found that 54 to 85% of the total fatty acids of several Bacillus organisms were branched chain compounds, and he concluded that a characteristic of the genus Bacillus was an abundance of branched chain fatty acids. Kimble et al. (4) compared the fatty acids of proteolytic and nonproteolytic types of Clostridium botulinum. The total lipid was about 3.8% of the dry weight of vegetative cells and 2.5% of the dry weight of spores. Hexadecanoic and tetradecanoic acids comprised over 50% of the total fatty acid content of both spores and vegetative cells of Clostridium botulinura. That total lipids of spores were less than that of vegetative cells led us to believe that the type of lipid might play a part in the heat resistance of bacterial spores. We decided to determine the fatty acid content of spores and vegetative cells of Bacillus licbeni]brmis, hoping that some relationship to the heat resistance mechanism of the spores could be established.
ABST RACT
Lipids were extracted from vegetative cells and spores of Bacillus licbeniformis. Vegetative cells were grown in nutrient broth and spores on nutrient agar. Total lipid approximated 2.89% of the dry weight of vegetative cells and 2.09% of the dry weight of spores. The fatty acids were prepared as methyl esters and analyzed by gas chromatography and mass spectrometry. There were six fatty acids in concentrations greater than 5% of the total lipid in both spores and vegetative cells, but only palmitic acid was common to both. Fatty acids from vegetative cells in quantities of 5% or more of the total lipid material were lauric, myristic, palmitic, palmitoleic, and linoleic acids. Fatty acids from spores in concentrations greater than 5% of the total lipid were isopentadecylic, palmitic, Carbon-17 iso, and three other long or branched chain fatty acids which were not identified. Spores contained more long and branched chain fatty acids with odd numbers of carbon atoms than did vegetative cells.
MATERIALS AND METHODS Microorganisms
INTRODUCTION
Several components of spores have been suggested contributors to heat resistance; and minerals, especially calcium, are most often mentioned (5, 7). The lipid content of sporeforming microorganisms has been unique, but no direct relationship to heat resistance of
Received March 15, 1976. 1Published with the approval of the Director, South Dakota Agricultural Experiment Station as Publication No. 1410 of the Journal series. 2Portions of this research were conducted at the University of Georgia and the Pennsylvania State University.
Spores and vegetative ceils were propagated and harvested from B. licbeniformis, a sporeforming microorganism which was isolated from raw milk (6). This organism was one of two (the other was B. cereus) sporeforming Bacillus organisms which comprise approximately 80% of the total spore flora of raw milk. Propagation and Harvestingof Sporesand Vegetative Cells
A common growth medium (Difco Nutrient Broth) was used for propagation of both spores and vegetative cells. For propagation of spores, agar was added at 1.5% along with .1% soluble
1830
TECHNICAL NOTE starch and .01% manganese sulfate, both of which aid in sporulation (1, 2). The spores were grown on agar slants at 37 C. The length of incubation was 5 to 7 days, depending on the percentage of sporulation after the 5th day. Spores were harvested when approximately 90% of the cells had formed spores. Spores were harvested by removing the spore growth from nutrient agar slants with sterile glass beads, followed by centrifuging and washing with M/100 phosphate buffer, and recentrifuging and rewashing for a total of six times. Vegetative cells were grown in nutrient broth for 18 h at 37 C. The same procedure for washing and centrifuging spores was used for the vegetative ceils. Both spores and vegetative cells were then lyophilized for subsequent analysis for total lipid and fatty acids. Lipid Extraction
Cells or spores were put into a weighed 250 ml, ground glass joint (24/40), boiling flasks, and accurate weights were obtained. To the flask were added 100 ml of chloroform-methanol (2:1), five boiling chips, and a Teflon coated stirring bar. The flask was hooked to a water cooled condenser, and the entire apparatus placed on a 130 C hot plate stirrer and refluxed for 30 rain. After refluxing, the solution was cooled, and the solvents removed by a rotary vacuum evaporator at 40 C. The lipids then were dissolved in redistilled diethyl ether. This solution was filtered through Whatman #2 filter paper into a weighed 125 ml ground glass joint boiling flask, and the sample again taken to dryness on the rotary evaporator. A known weight of lipid was obtained. The lipid was then dissolved in redistilled diethyl ether and transferred to a ground glass joint 20 mm × 150 mm test tube. The sample was taken to dryness under a stream of nitrogen. Five boiling chips and 3 ml of .5 N NaOH in methanol were added. A 30 m m funnel containing a marble was placed on the test tube, and the apparatus placed on a 130 C hot plate and refluxed for 20 min. Two milliliters of boron trifluoride-methanot were added, and refluxing was continued for 10 more rain. Six milliliters of saturated NaCI were added, and the solution was cooled under tap water. Methyl esters were extracted by adding 3 ml
1831
of reidstilled diethyl ether, inverting five times; and after separation of the phases, the ether was removed with a disposable pipet. The extraction step then was repeated, and the sample was concentrated under a stream of nitrogen. Gas-liquid Chromatographic and Mass Spectral Analysis
The chromatograph was a Hewlett-Packard Model 5750. The operating parameters are in Table 1. A LKB 9000 combined gas chromatograph mass spectrometer also was used. The column and operating conditions used for the separation of the methyl esters on the mass spectrometer were identical to those used for the gas chromatograph (see Table 1). Flash heater and molecular separator temperatures were 280 C. Mass spectra were obtained with a constant accelerating voltage of 3500 volts with 70 ev energy and a scanning time of 5 s over a m/e range of 4 to 400. Identification of unknowns was accomplished by comparison of sample spectra to spectra of authentic compounds (Applied Science Laboratory, State College, Pennsylvania), and by gas liquid cochromatography. RESULTS AND DISCUSSION
The chromatographic profile of the fatty acids from vegetative cells of B. licbeniformis is in Fig. 1. There were 16 distinct peaks, all
TABLE 1. Operating parameters for the gas-liquid chromatography for fatty acid analysis. Controlled factor
Operating condition
Column
1.83 m X 3.17 mm Stainless Steel Tubing 10% Dega + 2% H3PO4 80/1OO Mesh Gas Chrom A Nitrogen 50 ml/min 40 ml/min 450 ml/min
Stationary phase Support phase Carrier gas Flow rate Hydrogen Air Temperatures Detector Injection port Column Chart speed Sample size Detector sensitivity
250 C 250 C 130 C to 180 C @ 1 C/rain .25 in/rain 10.0 ~1 800 (10~ × 8)
Journal of Dairy Science Vol. 59, No. 10
1832
MARTIN AND SWENSON
T A B L E 2. Fatty acids in vegetative cells of Bacillus licbeniformis. Peak number
Percent by weight
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
3.0 6.1 1.5 6.1 1.0 1.0 1.0 2.1 21.2 8.5 3.3 3.3 3.8 11.4 25.6 2.2 2.2
F a t t y acid
Common name
Identity
10:0 12:0 13 :iso 14:0 . . . . . .
Capric Lauric Isoundecylic Myristic
Positive Positive Tentative Positive Unknown Unknown Unknown Positive Positive Positive Tentative Tentative Positive Positive Positive Positive Unknown
. .
15~0 16:0 16 : 1 17 :iso 17 :anteiso 18:0 18 : 1 18 : 2 18: 3 . . . .
O t h e r f a t t y a c i d s in s p o r e s in c o n c e n t r a t i o n s g r e a t e r t h a n 5% o f t h e t o t a l l i p i d w e r e i s o p e n t a decylic (14.5%), C-17-iso (15-methyl-hexade-
. .
. . Pentadecylic Palmitic Palmitoleic ... . .. Stearic Oleic Linoleic Linolenic
.
.
canoic) (5.1%), and three acids of unknown i d e n t i t y r e p r e s e n t e d b y p e a k s 23 a n d 2 4 o n t h e c h r o m a t o g r a p h i c p r o f i l e (Fig. 1), a n d b y p e a k
T A B L E 3. F a t t y acids in spores of Bacillus licbeniformis. Peak number
Percent by weight
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
1.0 1.0 1.0 1.0 1.0 1.4 14.5 1.0 1.6 6.9 3.1 5.1 2.9 1.0 1.3 2.1 4.0 1.2
4.8 4.1 1.3 2.6 16.0 7.3 3.3 13.0
Journal of Dairy Science Vol. 59, No. 10
F a t t y acid
Common name
Identity
11:0 12:0
Undecylic Lauric
Tentative Tentative Unknown Tentative Tentative Positive Positive Positive Tentative Positive Positive Positive Positive Positive Tentative Positive Positive Positive Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
.
13 :iso 14:iso 14:0 15:iso 15:0 16:iso 16:0 16:1 17:iso 17: anteiso 17:0 18:iso 18:0 18:1 18:2
.
.
Isoundecylic lsomyristic Myristic Isopentadecylic Pentadecylic Isopalmitic Palmitic Palmitoleic
Margaric lsostearic Stearic Oleic Linoleic
TECHNICAL NOTE
1833
Vegetative Ceils
I
I0
8
I
FIG. 1. Chromatographic profile of the fatty acids from vegetative ceils of Bacillus licbeniformis. weight of the total lipid. These were lauric (6.1%), myristic (6.1%), palmitic (21.2%), palmitoleic (8.5%), oleic (11.4%), and linoleic (25.6%). The remaining acids were in concentrations comprising less than 4% by weight of the total lipid content of vegetative cells. The fatty acid profile of spore lipids was different from that of vegetative cells. Table 3 contains similar data on the identity of the fatty acids from spores of B. licbeniformis. There were 26 distinct chromatographic peaks representing fatty acids from spores. Efforts to identify fatty acids responsible for peak 3 and all those above peak 19 were unsuccessful. The remaining 15 fatty acids were identified either tentatively or positively by GLC and mass spectrometry. Those acids comprising 5% or more of the total lipid content were different from in vegetative cells, with the exception of palmitic acid, which comprised 6.9% by weight of the total lipid content of spores, and 21.2% (Table 2) of the total lipid of vegetative cells.
representing fatty acids with more than 10 carbon atoms. Although conditions were employed which would have detected fatty acids having less than 10 carbon atoms, none were found. Figure 2 shows the chromatographic profile of the fatty acids from spores of B. licbeniformis. There were more fatty acids in spores than in vegetative cells, but as in the vegetative cells, no fatty acids were detected which had less than 10 carbon atoms. The identity of the fatty acids from vegetative cells of B. licbeniforrnis is in Table 2. Efforts to identify fatty acids represented by Peaks 5, 6, 7, and 17 were unsuccessful. Either tentative or positive identification was accomplished with the remaining fatty acids by a combination of gas liquid chromatography and mass spectrometry. Although 17 peaks were apparent from vegetative cells, indicating 17 different fatty acids, only 6 fatty acids were in sufficient concentrations to exceed 5% by
Spores
10
12
17
I
FIG. 2. Chromatographic profile of the fatty acids from spores of Bacillus licbeniformis. Journal of Dairy Science Vol. 59, No. 10
1834
MARTIN AND SWENSON
26, w h i c h c o u l d n o t b e s h o w n o n t h e c h a r t because o f t h e l i m i t a t i o n s o f t h e camera. T h e f a t t y acids f r o m s p o r e s o f B. licbeniforrnis are m o r e c o m p l e x a n d m u c h less c o m m o n in n a t u r e t h a n t h o s e in vegetative ceils. Further study may determine a relationship b e t w e e n this p h e n o m e n o n a n d d i f f e r e n c e s in h e a t resistance b e t w e e n spores a n d vegetative cells o f B. licbeniformis. ACKNOWLEDGMENT
T h e a u t h o r s express a p p r e c i a t i o n t o Gary Reineccius, c u r r e n t l y a t t h e U n i v e r s i t y o f Minnesota, f o r mass s p e c t r o m e t r y analyses. REFERENCES
1 Charney, J., W. P. Fisher, and C. P. Hegarty. 1951. Manganese as an essential for sporulation in the genus Bacillus. J. Bacteriol. 62:145.
Journal of Dairy Science Vol. 59, No. 10
2
Foster, J. W., W. A. Hardwick, and B. M. Guirard. 1950. Anti-spornlation factors in complex organic media. I. Growth and spornlation studies on Bacillus larvae. J. Bacteriol. 59:463. 3 Kaneda, T. 1969. Fatty acids in Bacillus larvae,
Bacillus lentimorbus, and Bacillus popilliae. J. Bacteriol. 98:143. Kimble, C, E., M. L. McCollough, V. A. Paterno, and A. W. Anderson. 1969. Comparison of the fatty acids of proteolytic type B and nonproteolyric types E and F of Clostridium botulinum. Appl. Microbiol. 18:883. 5 Levinson, H. S., and M. T. Hyatt. 1964. Effect of sporulatlon medium on heat resistance, chemical composition, and germination of Bacillus megaterium spores. J. Bacteriol. 87:876. 6 Martin, J. H., D. P. Stahly, W. J. Harper, and I. A. Gould. 1962. Sporeforming microorganisms in selected milk supplies. Proc. XVIth Intern. Dairy Congr. 2:295. 7 Slepecky, IL, and J. W. Foster. 1959. Alterations in metal content of spores of Bacillus raegateriurn and the effect on some spore properties. J. Bacteriol. 78:117.
4
NOTES
F a t t y A c i d s of V e g e t a t i v e Cells and Spores of Bacillus l i c h e n i f o r m i s 1, 2 J. H. MARTIN Dairy Science Department South Dakota State University Brookings, SO 57006 and
P. E. SWENSON Borden Research Centre Syracuse, NY
Bacillus organisms has been reported. However, Kaneda (3) reported the lipid content of vegetative cells of several Bacillus organisms. He found that 54 to 85% of the total fatty acids of several Bacillus organisms were branched chain compounds, and he concluded that a characteristic of the genus Bacillus was an abundance of branched chain fatty acids. Kimble et al. (4) compared the fatty acids of proteolytic and nonproteolytic types of Clostridium botulinum. The total lipid was about 3.8% of the dry weight of vegetative cells and 2.5% of the dry weight of spores. Hexadecanoic and tetradecanoic acids comprised over 50% of the total fatty acid content of both spores and vegetative cells of Clostridium botulinura. That total lipids of spores were less than that of vegetative cells led us to believe that the type of lipid might play a part in the heat resistance of bacterial spores. We decided to determine the fatty acid content of spores and vegetative cells of Bacillus licbeni]brmis, hoping that some relationship to the heat resistance mechanism of the spores could be established.
ABST RACT
Lipids were extracted from vegetative cells and spores of Bacillus licbeniformis. Vegetative cells were grown in nutrient broth and spores on nutrient agar. Total lipid approximated 2.89% of the dry weight of vegetative cells and 2.09% of the dry weight of spores. The fatty acids were prepared as methyl esters and analyzed by gas chromatography and mass spectrometry. There were six fatty acids in concentrations greater than 5% of the total lipid in both spores and vegetative cells, but only palmitic acid was common to both. Fatty acids from vegetative cells in quantities of 5% or more of the total lipid material were lauric, myristic, palmitic, palmitoleic, and linoleic acids. Fatty acids from spores in concentrations greater than 5% of the total lipid were isopentadecylic, palmitic, Carbon-17 iso, and three other long or branched chain fatty acids which were not identified. Spores contained more long and branched chain fatty acids with odd numbers of carbon atoms than did vegetative cells.
MATERIALS AND METHODS Microorganisms
INTRODUCTION
Several components of spores have been suggested contributors to heat resistance; and minerals, especially calcium, are most often mentioned (5, 7). The lipid content of sporeforming microorganisms has been unique, but no direct relationship to heat resistance of
Received March 15, 1976. 1Published with the approval of the Director, South Dakota Agricultural Experiment Station as Publication No. 1410 of the Journal series. 2Portions of this research were conducted at the University of Georgia and the Pennsylvania State University.
Spores and vegetative ceils were propagated and harvested from B. licbeniformis, a sporeforming microorganism which was isolated from raw milk (6). This organism was one of two (the other was B. cereus) sporeforming Bacillus organisms which comprise approximately 80% of the total spore flora of raw milk. Propagation and Harvestingof Sporesand Vegetative Cells
A common growth medium (Difco Nutrient Broth) was used for propagation of both spores and vegetative cells. For propagation of spores, agar was added at 1.5% along with .1% soluble
1830
TECHNICAL NOTE starch and .01% manganese sulfate, both of which aid in sporulation (1, 2). The spores were grown on agar slants at 37 C. The length of incubation was 5 to 7 days, depending on the percentage of sporulation after the 5th day. Spores were harvested when approximately 90% of the cells had formed spores. Spores were harvested by removing the spore growth from nutrient agar slants with sterile glass beads, followed by centrifuging and washing with M/100 phosphate buffer, and recentrifuging and rewashing for a total of six times. Vegetative cells were grown in nutrient broth for 18 h at 37 C. The same procedure for washing and centrifuging spores was used for the vegetative ceils. Both spores and vegetative cells were then lyophilized for subsequent analysis for total lipid and fatty acids. Lipid Extraction
Cells or spores were put into a weighed 250 ml, ground glass joint (24/40), boiling flasks, and accurate weights were obtained. To the flask were added 100 ml of chloroform-methanol (2:1), five boiling chips, and a Teflon coated stirring bar. The flask was hooked to a water cooled condenser, and the entire apparatus placed on a 130 C hot plate stirrer and refluxed for 30 rain. After refluxing, the solution was cooled, and the solvents removed by a rotary vacuum evaporator at 40 C. The lipids then were dissolved in redistilled diethyl ether. This solution was filtered through Whatman #2 filter paper into a weighed 125 ml ground glass joint boiling flask, and the sample again taken to dryness on the rotary evaporator. A known weight of lipid was obtained. The lipid was then dissolved in redistilled diethyl ether and transferred to a ground glass joint 20 mm × 150 mm test tube. The sample was taken to dryness under a stream of nitrogen. Five boiling chips and 3 ml of .5 N NaOH in methanol were added. A 30 m m funnel containing a marble was placed on the test tube, and the apparatus placed on a 130 C hot plate and refluxed for 20 min. Two milliliters of boron trifluoride-methanot were added, and refluxing was continued for 10 more rain. Six milliliters of saturated NaCI were added, and the solution was cooled under tap water. Methyl esters were extracted by adding 3 ml
1831
of reidstilled diethyl ether, inverting five times; and after separation of the phases, the ether was removed with a disposable pipet. The extraction step then was repeated, and the sample was concentrated under a stream of nitrogen. Gas-liquid Chromatographic and Mass Spectral Analysis
The chromatograph was a Hewlett-Packard Model 5750. The operating parameters are in Table 1. A LKB 9000 combined gas chromatograph mass spectrometer also was used. The column and operating conditions used for the separation of the methyl esters on the mass spectrometer were identical to those used for the gas chromatograph (see Table 1). Flash heater and molecular separator temperatures were 280 C. Mass spectra were obtained with a constant accelerating voltage of 3500 volts with 70 ev energy and a scanning time of 5 s over a m/e range of 4 to 400. Identification of unknowns was accomplished by comparison of sample spectra to spectra of authentic compounds (Applied Science Laboratory, State College, Pennsylvania), and by gas liquid cochromatography. RESULTS AND DISCUSSION
The chromatographic profile of the fatty acids from vegetative cells of B. licbeniformis is in Fig. 1. There were 16 distinct peaks, all
TABLE 1. Operating parameters for the gas-liquid chromatography for fatty acid analysis. Controlled factor
Operating condition
Column
1.83 m X 3.17 mm Stainless Steel Tubing 10% Dega + 2% H3PO4 80/1OO Mesh Gas Chrom A Nitrogen 50 ml/min 40 ml/min 450 ml/min
Stationary phase Support phase Carrier gas Flow rate Hydrogen Air Temperatures Detector Injection port Column Chart speed Sample size Detector sensitivity
250 C 250 C 130 C to 180 C @ 1 C/rain .25 in/rain 10.0 ~1 800 (10~ × 8)
Journal of Dairy Science Vol. 59, No. 10
1832
MARTIN AND SWENSON
T A B L E 2. Fatty acids in vegetative cells of Bacillus licbeniformis. Peak number
Percent by weight
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
3.0 6.1 1.5 6.1 1.0 1.0 1.0 2.1 21.2 8.5 3.3 3.3 3.8 11.4 25.6 2.2 2.2
F a t t y acid
Common name
Identity
10:0 12:0 13 :iso 14:0 . . . . . .
Capric Lauric Isoundecylic Myristic
Positive Positive Tentative Positive Unknown Unknown Unknown Positive Positive Positive Tentative Tentative Positive Positive Positive Positive Unknown
. .
15~0 16:0 16 : 1 17 :iso 17 :anteiso 18:0 18 : 1 18 : 2 18: 3 . . . .
O t h e r f a t t y a c i d s in s p o r e s in c o n c e n t r a t i o n s g r e a t e r t h a n 5% o f t h e t o t a l l i p i d w e r e i s o p e n t a decylic (14.5%), C-17-iso (15-methyl-hexade-
. .
. . Pentadecylic Palmitic Palmitoleic ... . .. Stearic Oleic Linoleic Linolenic
.
.
canoic) (5.1%), and three acids of unknown i d e n t i t y r e p r e s e n t e d b y p e a k s 23 a n d 2 4 o n t h e c h r o m a t o g r a p h i c p r o f i l e (Fig. 1), a n d b y p e a k
T A B L E 3. F a t t y acids in spores of Bacillus licbeniformis. Peak number
Percent by weight
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
1.0 1.0 1.0 1.0 1.0 1.4 14.5 1.0 1.6 6.9 3.1 5.1 2.9 1.0 1.3 2.1 4.0 1.2
4.8 4.1 1.3 2.6 16.0 7.3 3.3 13.0
Journal of Dairy Science Vol. 59, No. 10
F a t t y acid
Common name
Identity
11:0 12:0
Undecylic Lauric
Tentative Tentative Unknown Tentative Tentative Positive Positive Positive Tentative Positive Positive Positive Positive Positive Tentative Positive Positive Positive Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
.
13 :iso 14:iso 14:0 15:iso 15:0 16:iso 16:0 16:1 17:iso 17: anteiso 17:0 18:iso 18:0 18:1 18:2
.
.
Isoundecylic lsomyristic Myristic Isopentadecylic Pentadecylic Isopalmitic Palmitic Palmitoleic
Margaric lsostearic Stearic Oleic Linoleic
TECHNICAL NOTE
1833
Vegetative Ceils
I
I0
8
I
FIG. 1. Chromatographic profile of the fatty acids from vegetative ceils of Bacillus licbeniformis. weight of the total lipid. These were lauric (6.1%), myristic (6.1%), palmitic (21.2%), palmitoleic (8.5%), oleic (11.4%), and linoleic (25.6%). The remaining acids were in concentrations comprising less than 4% by weight of the total lipid content of vegetative cells. The fatty acid profile of spore lipids was different from that of vegetative cells. Table 3 contains similar data on the identity of the fatty acids from spores of B. licbeniformis. There were 26 distinct chromatographic peaks representing fatty acids from spores. Efforts to identify fatty acids responsible for peak 3 and all those above peak 19 were unsuccessful. The remaining 15 fatty acids were identified either tentatively or positively by GLC and mass spectrometry. Those acids comprising 5% or more of the total lipid content were different from in vegetative cells, with the exception of palmitic acid, which comprised 6.9% by weight of the total lipid content of spores, and 21.2% (Table 2) of the total lipid of vegetative cells.
representing fatty acids with more than 10 carbon atoms. Although conditions were employed which would have detected fatty acids having less than 10 carbon atoms, none were found. Figure 2 shows the chromatographic profile of the fatty acids from spores of B. licbeniformis. There were more fatty acids in spores than in vegetative cells, but as in the vegetative cells, no fatty acids were detected which had less than 10 carbon atoms. The identity of the fatty acids from vegetative cells of B. licbeniforrnis is in Table 2. Efforts to identify fatty acids represented by Peaks 5, 6, 7, and 17 were unsuccessful. Either tentative or positive identification was accomplished with the remaining fatty acids by a combination of gas liquid chromatography and mass spectrometry. Although 17 peaks were apparent from vegetative cells, indicating 17 different fatty acids, only 6 fatty acids were in sufficient concentrations to exceed 5% by
Spores
10
12
17
I
FIG. 2. Chromatographic profile of the fatty acids from spores of Bacillus licbeniformis. Journal of Dairy Science Vol. 59, No. 10
1834
MARTIN AND SWENSON
26, w h i c h c o u l d n o t b e s h o w n o n t h e c h a r t because o f t h e l i m i t a t i o n s o f t h e camera. T h e f a t t y acids f r o m s p o r e s o f B. licbeniforrnis are m o r e c o m p l e x a n d m u c h less c o m m o n in n a t u r e t h a n t h o s e in vegetative ceils. Further study may determine a relationship b e t w e e n this p h e n o m e n o n a n d d i f f e r e n c e s in h e a t resistance b e t w e e n spores a n d vegetative cells o f B. licbeniformis. ACKNOWLEDGMENT
T h e a u t h o r s express a p p r e c i a t i o n t o Gary Reineccius, c u r r e n t l y a t t h e U n i v e r s i t y o f Minnesota, f o r mass s p e c t r o m e t r y analyses. REFERENCES
1 Charney, J., W. P. Fisher, and C. P. Hegarty. 1951. Manganese as an essential for sporulation in the genus Bacillus. J. Bacteriol. 62:145.
Journal of Dairy Science Vol. 59, No. 10
2
Foster, J. W., W. A. Hardwick, and B. M. Guirard. 1950. Anti-spornlation factors in complex organic media. I. Growth and spornlation studies on Bacillus larvae. J. Bacteriol. 59:463. 3 Kaneda, T. 1969. Fatty acids in Bacillus larvae,
Bacillus lentimorbus, and Bacillus popilliae. J. Bacteriol. 98:143. Kimble, C, E., M. L. McCollough, V. A. Paterno, and A. W. Anderson. 1969. Comparison of the fatty acids of proteolytic type B and nonproteolyric types E and F of Clostridium botulinum. Appl. Microbiol. 18:883. 5 Levinson, H. S., and M. T. Hyatt. 1964. Effect of sporulatlon medium on heat resistance, chemical composition, and germination of Bacillus megaterium spores. J. Bacteriol. 87:876. 6 Martin, J. H., D. P. Stahly, W. J. Harper, and I. A. Gould. 1962. Sporeforming microorganisms in selected milk supplies. Proc. XVIth Intern. Dairy Congr. 2:295. 7 Slepecky, IL, and J. W. Foster. 1959. Alterations in metal content of spores of Bacillus raegateriurn and the effect on some spore properties. J. Bacteriol. 78:117.
4
