THE EFFECT OF TWEEN 80 ON THE FATTY ACID COMPOSITION OF LACTOBACILLUS BUCHNERI AND LACTOBAGILL US BREVIS

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1 J. Gen. App!. Microbiol., 41, (1995) THE EFFECT OF TWEEN 80 ON THE FATTY ACID COMPOSITION OF LACTOBACILLUS BUCHNERI AND LACTOBAGILL US BREVIS PEKKA NIKKILA,* TOM JOHNSSON, LEENA TOIVONEN, AND HEIKKI ROSENQVIST Laboratory of Biochemistry and Microbiology, Department of Chemical Engineering, Helsinki University of Technology, Espoo, Finland (Received January 11, 1995; Accepted July 27, 1995) The effect of Tween 80 on the cellular fatty acid spectra of different strains of two closely related Lactobacillus species, Lactobacillus buchneri and Lactobacillus brevis, was studied. When cultivated in MRS-broth containing Tween 80 all L. buchneri strains contained two different cyclopropane fatty acids, dihydrosterculic and lactobacillic acid, while L. brevis strains contained only dihydrosterculic acid. In contrast, when the cells were cultivated in a modified MRS-broth without Tween 80, only lactobacillic acid was found in the bacteria except in one L. brevis strain that did not contain either lactobacillic acid or dihydrosterculic acid. This L, brevis strain was in contrast to most Lactobacillus strains unable to cyclisize cis-vaccenic acid (cis- l l-octadecenoic acid) produced by its own biosynthetic machinery. Still it was able to cyclisize oleic acid (cis-9-octadecenoic acid) supplemented by its growth medium. Characterization of microbes based on their fatty acid composition has attracted much interest during the past decade (1, 8). Among others, cellular fatty acid spectra of several lactic acid bacteria have been analyzed by gas chromatography, and these have been used to characterize different Lactobacillus, Lactococcus and Leuconostoc strains (6, 9,11,12). The identification of microbes based on their fatty acid composition should be carried out using strictly standardized growth conditions and analytical methods, as both are known to affect significantly the results of fatty acid analysis. However, when characterizing lactic acid bacteria from foods and fermentation processes based on their fatty acid composition many different media have been used to cultivate the bacteria before fatty acid analyses (2, 7,10). MRS-medium * Address reprint requests to: biology, Department of Chemical Espoo, Finland. Dr. Pekka Nikkila, Engineering, Helsinki Laboratory of Biochemistry and Micro- University of Technology, Kemistintie 1, 327

2 328 NIKKILA et al. VOL. 41 (4) that contains Tween 80 (polyoxyethylene sorbitanmonooleate) has frequently been used for standardized cultivation of lactobacilli, although the oleic acid of Tween 80 is known to be incorporated into the lipids of lactic acid bacteria and thus to alter the fatty acid composition of the cells (5). The occurrence of cyclopropane fatty acids is characteristic of many lactic acid bacteria (6). The main cyclopropane fatty acids in lactobacilli are lactobacillic acid (11,12-methyleneoctadecanoic acid; cyc-19 : 0 (1 lc)) and dihydrosterculic acid (9,10-methyleneoctadecanoic acid; cyc-19 :0 (9c)). These acids are formed by methylenation of olefinic fatty acids: lactobacillic acid is synthesized by methylenation of cis-vaccenic acid (cis- l l-octadecenoic acid; 18: 1 (1 lc)) produced by the lactic acid bacteria, while dihydrosterculic acid is synthesized by methylenation of oleic acid (cis-9-octadecenoic acid; 18:1 (9c)) supplemented by the culture medium (3). In this paper, the effect of Tween 80 on the cellular fatty acid spectra of different strains of two closely related Lactobacillus species, L. buchneri and L. brevis, was studied. We were particularly interested whether Tween 80 has any effects on the identification of these Lactobacillus strains based on their fatty acid profiles. MATERIALS AND METHODS Microorganisms and culture conditions. The Lactobacillus strains examined in this work are listed in Table 1. The bacteria were maintained in MRS-agar at 4 C and subcultured once a month. The composition of MRS-broth is given in Table 2. For fatty acid analysis, all strains were cultured in two different media: in MRSbroth and in a modified MRS-broth (without Tween 80). To prepare the inoculum each strain was transferred to 10 ml of fresh broth and cultivated at 30 C to the late exponential phase. One to five percent (v/v) of this actively growing culture was transferred to 5 ml of broth and allowed to grow to the exponential phase. Five hundreds microliters of this culture was inoculated into two parallel flasks containing 50 ml of broth and cultivated until the late stationary phase of growth was Table 1. List of Lactobacillus strains examined.

3 1995 Effect of Tween 80 on Bacterial Fatty Acids 329 Table 2. Composition of MRS-broth (4). reached. The temperature was maintained at 30 C during the cultivations and the flasks were shaken at 50 rpm in an orbital shaker/incubator. The growth of the bacteria was monitored with a Klett-Summerson colorimeter (filter no. 66, Klett Manufacturing Co., Inc., New York, U.S.A.). Fatty acid analysis. For fatty acid analysis two 5-ml samples of each culture at the late stationary phase were centrifuged (15 min, 6,000 X g) and the cells were washed once with tap water. After centrifugation the cells were freeze-dried and weighed for cell dry weight determination. The dry cells were suspended with 1 ml of saponification reagent (3.7 M NaOH in 50% (v/v) methanol). The samples were flushed with N2, mixed and the tubes were placed in a boiling water-bath at 100 C for 5 min. The tubes were then mixed again, returned to the water-bath for 25 min and cooled to room temperature. The samples were methylenated by adding 4 ml of methylenation reagent (3.3 M HCl in 50% (v/v) methanol), mixed and placed in a water-bath at 80 C for 10 min. The samples were cooled to room temperature and the fatty acid methyl esters were extracted with 1.5 ml of hexane : methyl tent-butyl ether solution (1:1, v/v) by shaking vigorously for 10 min. The lower aqueous phase was removed and the organic phase containing the fatty acid methyl esters was washed with 3 ml of 0.3 M NaOH by shaking for 5 min to remove any free fatty acids. If necessary, the samples were centrifuged (3,000 X g) and the upper organic phase was collected, dried with Na2SO4 and transferred to separate vials. Heptadecanoic acid methyl ester (Sigma, St. Louis, MO, U.S.A.) was used as the internal standard. The major fatty acids were identified from their GC peak retention times relative to fatty acid methyl ester standards (Sigma) and GC-MS. The cyclopropane compounds were identified by GC-MS and by the occurrence of two or one cyclopropane peaks when the bacteria were grown on media with or without Tween 80. GC was done using a Hewlett-Packard (Avondale, PE, U.S.A.) model 5890A gas chromatograph equipped with a flame ionization detector, a capillary inlet system and a model 7673A automatic liquid sampler (Hewlett- Packard). The GC conditions used were: HP-FFAP (25 m 0.2 mm 0.3,um) column, helium as carrier gas and column flow rate 1 ml/min, split-ratio 1: 20, injector and detector temperature 250 C. The oven was programmed to warm up

4 330 NIKKILA et al. VOL. 41 from an initial temperature of 70 to 200 C at the rate of 25 C/min. The peak areas were measured using the HP-ChemStation software. The combined GC-MS was carried out using the GC-chromatograph described above equipped with a Hewlett- Packard mass selective detector 5971A. The conditions were as above except that column inlet pressure was set to 70 kpa. Mass spectra were recorded at an electron energy of 70 ev, trap current 300,uA, ion source temperature 180 C and molecular separator temperature 135 C. RESULTS The cellular fatty acid composition of the Lactobacillus strains cultivated in MRS-broth are presented in Table 3. Eight major fatty acids accounted for over 95% of total fatty acids. Three of them were straight-chain saturated acids (myristic acid, 14:0; palmitic acid, 16:0; and stearic acid, 18 :0), three straightchain monounsaturated acids (hexadecenoic acid, 16: 1, position and configuration of the double bond not determined; oleic acid, 18: 1 (9c); and cis-vaccenic acid, 18: 1 (llc)) and two cyclopropane acids (dihydrosterculic acid, cyc-19:0 (9c); and lactobacillic acid, cyc-19 :0 (l lc)). These fatty acids were all found in L. buchneri strains, while in L. brevis strains lactobacillic acid could not be detected at all. Table 4 shows the cellular fatty acid spectra of the Lactobacillus strains cultivated in a modified MRS-broth (without Tween 80). Here the relative amounts of palmitic and stearic acids in L, buchneri strains were greater than when grown in a medium with Tween 80 (Table 2). Correspondingly, the amounts of oleic and cis-vaccenic acids were smaller. This was because the relative amount of oleic acid in the cells was very small when no Tween 80 was added to the growth medium. Furthermore, no dihydrosterculic acid could be found in the L. buchneri strains grown without Tween 80. However, in most L, buchneri strains the proportion of cyclopropane fatty acids was greater when cultivated without Tween 80. In the two L. brevis strains examined, the proportion of unsaturated oleic and cis-vaccenic acids was greater when cultivated without Tween 80 (Table 3) than in Table 3. Fatty acid composition of Lactobacillus strains grown in MRS-broth.

1Q4c r'rnart of Tcvaon Q(1 nn Rontari~l ~i'n#v A nirlo 211 Table 4. Fatty acid composition of Lactobacillus strains grown in modifi ed MRS-broth (without Tween 80). the standard MRS-broth.

5 1Q4c r'rnart of Tcvaon Q(1 nn Rontari~l ~i'n#v A nirlo 211 Table 4. Fatty acid composition of Lactobacillus strains grown in modifi ed MRS-broth (without Tween 80). the standard MRS-broth. Strain Valio 2001 did not contain any cyclopropyl fatty acids when grown in medium in which Tween 80 was omitted, and also in strain VTT E the cyclopropane fatty acid content was smaller in this medium. DISCUSSION In L. buchneri strains, the effect of Tween 80 on fatty acid composition of the cells was quite straightforward. The oleic acid of Tween 80 was incorporated into the membranes of the cells and the relative amount of cis-vaccenic acid in the cells was smaller compared to that in cells grown without Tween 80. At the same time, part of the oleic acid incorporated into the cells was methylenated to dihydrosterculic acid. As a consequence, the cyclopropane fatty acid content of the cells consisted mostly of dihydrosterculic acid. Similar results on the effect of Tween 80 on fatty acid composition have been previously observed in a Leuconostoc oenos strain (5). The effect of Tween 80 on the fatty acid composition of L. brevis strains was less straightforward. The oleic acid of Tween 80 was incorporated into the cells and some of it was methylenated as in L. buchneri strains. However, the cis-vaccenic acid produced by the cells was not methylenated at all, and consequently dihydrosterculic acid was the only cyclopropane fatty acid present in L. brevis cells when the culture medium contained Tween 80. According to these results, L. buchneri and L. brevis strains can be identified and distinguished from each other when grown in standard MRS-medium containing Tween 80 based on the amount of cyclopropane fatty acids present in the cells. All L. buchneri strains contained two different cyclopropyl fatty acids, dihydrosterculic and lactobacillic acid, while L. brevis strains contained only dihydrosterculic acid. When cultivated in medium without Tween 80, no clear difference between the cyclopropane fatty acid content of L. buchneri and L. brevis strains could be observed and only lactobacillic acid was found in all strains except for one L, brevis

6 332 NIKKILA et al. VOL. 41 strain that did not contain any cyclopropyl fatty acids. This L. brevis strain was, in contrast to most Lactobacillus strains, unable to cyclisize cis-vaccenic acid (18: 1 (1 lc)) produced by its own biosynthetic machinery. However, it was still capable of cyclisizing oleic acid (18:1 (9c)) supplemented by growth medium. This phenomenon has been reported before in a Pediococcus pen tosaceus strain (S). In conclusion we found that cis-vaccenic acid and oleic acid are not equally methylenated to their corresponding cyclopropane fatty acids. This has also been reported by Buist and Pon (3) who studied the methylenation fluorinated substituents of cis-vaccenic and oleic acids. The results here and those of Arigoni's (3) showing that the absolute configuration of lactobacillic acid and dihydrosterculic acid produced by Lactobacillus plantarum are 11R,12S and 9S, IOR, respectively, raise new questions concerning the function and mechanism of formation of C19 cyclopropane fatty acids in lactic acid bacteria. These results also support the theory of the existence of two separate isoenzymes for cyclisizing the two positional isomers of olefinic fatty acids (3). We thank TEKES (Technology Development Centre of Finland) for financial support and Annika Mayra-Makinen (Valio Ltd.) for kindly providing the Valio strains. REFERENCES 1) Axelsson, L. T., Lactic acid bacteria: Classification and physiology. In Lactic Acid Bacteria, ed. by Salminen, S. and von Wright, A., Marcel Dekker Inc., New York (1993), p ) Blank, G., Adejumo, A. A., and Zawistowski, J., Eugenol induced changes in the fatty acid content of two Lactobacillus species. Lebensm.-bliss. Technol., 24, (1991). 3) Buist, P. H. and Pon, R. A., An unexpected reversal of fluorine substituent effects in the biomethylation of two positional isomers: A serendipitous discovery. J. Org. Chem., 55, (1990). 4) de Man, J. C., Rogosa, M., and Sharpe, M. E., A medium for the cultivation of lactobacilli. J. Appl. Bacteriol., 23, (1960). S) Lonvaud-Funel, A. and Desens, C., Constitution en acides gras des membranes des bacteries lactiques du vin, incidences des conditions de culture. Sci. Aliments, 10, (1990). 6) O'Leary, W. M. and Wilkinson, S. G., Gram-positive bacteria. In Microbial Lipids, Vol. 1, ed. by Ratledge, C. and Wilkinson, S. G., Academic Press, London (1988), p ) Richter, K., Becker, U., and Mai, W., Characterization of lactic acid bacteria by means of gas chromatography. Biotechnol. Food Ind. Proc. Int. Symp., Budapest, p (1988). 8) Rizzo, A. F., Korkeala, H., and Mononen, I., Gas chromatography analysis of cellular fatty acids and neutral monosaccharides in the identification of lactobacilli. Appl. Environ. Microbiol., 53, (1987). 9) Schmitt, P., Mathot, A. G., and Divies, C., Fatty acid composition of the genus Leuconostoc. Milchwissenschaft, 44, (1989). 10) Tanasupawat, S., Ezaki, T., Suzuki, K.-I., Okada, S., Komagata, K., and Kozaki, M., Characterization and identification of Lactobacillus pentosus and Lactobacillus plantarum strains from fermented foods in Thailand. J. Gen. Appl. Microbiol., 38, (1992). 11) Uchida, K. and Mogi, K., Cellular fatty acid spectra of hiochi bacteria, alcohol-tolerant lactobacilli, and their group separation. J. Gen. Appl. Microbiol., 19, (1973). 12) Veerkamp, J. H., Fatty acid composition of Bifdobacterium and Lactobacillus strains. J. Bacteriol., 108, (1971).

Automated Sample Preparation for Profiling Fatty Acids in Blood and Plasma using the Agilent 7693 ALS

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