Effects of Cultural Conditions on the Cellular Fatty Acid. Composition of Lactobacillus heterohiochii, an Alcoholophilic Bacterium õ

Transcription

1 Agr. Biol. Chem., 39 (4), 837 `842, 1975 Effects of Cultural Conditions on the Cellular Fatty Acid Composition of Lactobacillus heterohiochii, an Alcoholophilic Bacterium õ Kinji UCHIDA Central Research Laboratories, Kikkoman Shoyu Co., Ltd., Noda, Chiba-ken 278 Received October 17, 1974 Effects of cultural conditions on the fatty acid composition of Lactobacillus heterohiochii were studied with the special reference to the formation of unusually long chain fatty acids (C20 and longer ones). The fatty acid composition did not significantly alter throughout cultural ages, and the acids with unusually long chains were synthesized under a variety of conditions. The proportion of the unusual acids rose with an increase in the concentra tion of yeast-extract in media. No unequivocal relationship between alcohol concentration of media and the chain lengths of the fatty acids was observed. The raise in alcohol concentration resulted in a significant increase of the ratio of total unsaturated to saturated acids (U/S ratio). The U/S ratio decreased with the rise of growth temperature below 25 Ž, but it stayed constant at temperature higher than 28 Ž. Lactobacillus heterohiochii, an alcoholo philic spoilage bacterium of Sake (Japanese rice wine), was found to produce fatty acids with unusually long chains (C20 `C30) along with those of normal chain lengths known for most lactobacilli.1) It has been also found that all the unsaturated acids are higher homologues of the cis-vaccenic acid series2) and that the unusually long chain acids are located almost exclusively in polar glycerolipids of this organism.3) For further understanding of the synthesis and the physiological significance of the unusu ally long chain acids, it might be useful to explore the effects of various environmental conditions on the formation of the unusual acids in this bacterium. It has been commonly observed, in various kinds of microbes, that the proportion of the unsaturated fatty acids decreased as the growth temperature was raised.4 `7) However, this õ Lipids of Alcoholophilic Lactobacilli. Part V. See References 2,3). The major part of this work was presented at the 14th annual meeting of Japanese Con ference on the Biochemistry of Lipids held on June 10, 1972, in Osaka. See Reference 19). relationship is found not to be applicable for L. heterohiochii, at least over a certain range of temperature. While a large interest has been taken in the action of alcohols on biomembranes8,9) or physical state of membrane lipids,10) very few reports have been appeared as to the effects of alcohols on lipid metabolism in bacteria, probably because of the absence of organisms having such a high alcohol-tolerance as shown in the hiochi-bacteria.11) In the present paper, effects of cultural tem perature, ethanol concentration and some components of media on the fatty acid com position of this alcoholophilic lactobacillus are described. MATERIALS AND METHODS Microorganisms. Lactobacillus heterohiochii H-1, S-14, S-41, and S-56 was used as described previous ly.1) L. fermentum H-34, a heterofermentative hiochi lactobacillus, was also used for comparative purposes. Media and cultural methods. The basal medium used here contained the following components per liter; yeast extract (Difco), 5.0g; polypeptone (Daigo), 5.0g; glucose, 10.0g; KCl, 0.5g; KH2PO4, 0.2g; CaCl2.

2 838 K. UCHIDA 2H2O, 0.1g; MgSO4 7H2O, 0.1g ; FeCl3 6H2O, 0.001g; MnSO4 5H2O, 0.001g; sodium acetate, 20.0g; DL mevalonolactone, 0.005g; and ethanol, 60ml. The ph was adjusted to 5.0. Methods for the preparation of media and the conditions for the cultivation and harvest of the bacteria were the same as previously de scribed1) unless otherwise specified. Extraction and analysis of fatty acids. Cellular fatty acids were extracted from alkali-hydrolyzates of lyo philized cells, methylated with diazomethane, and ana lyzed by gas-liquid chromatography as previously reported.1) RESULTS 1) Cultural age and cellular fatty acid com position Cellular fatty acid compositions of the strain S-56 at various growth phases were examined. (Fig. 1) Except for some increases of cyclopropane acid content at the expense of cis-vaccenic acid during growth, the cellular fatty acid composition of this organism did not significantly change throughout the cultural ages. Fatty acids with unusually long chains were present in the earlier phase of the growth in almost the same proportions as in later phase. 2) Effect of cultural temperature Cellular fatty acid compositions of L. heterohiochii S-56 grown at various tempera tures are shown in Table T. Duplicate deter minations of the fatty acids of the cells harvest ed at different ages agreed well with each other. The proportion of the fatty acids with unusually long chains (C20 and longer ones) showed the largest value at 25 Ž (36%) and decreased gradually at higher temperature. The proportions of total unsaturated acids to total saturated acids (U/S ratio) in the cellular fatty acids of L. heterohiochii S-56 and those of L. fermentum H-34 were plotted against growth temperature in Fig. 2. The U/S ratio of L. fermentum fatty acids declined almost linearly with increasing temperature. The proportion of total unsaturated acids (including cyclopropane acids) of L. heterohiochii S-56 decreased with an increase of temperature from 19 to 25 Ž. However, the U/S ratio stays constant over a range from 28 to 39 Ž. FIG. 1. Fatty Acid Composition of L. heterohiochii S-56 at Various Growth Ages. The growth was traced by determining the weights of the lyophilized cells grown in 300ml of culture. Data for minor components are not plotted. œ \ œ, 16:0; \, 18:0; ---, 18:1; \, 20:0; ---, 20:1; \, 22:0; ---, 22:1; \, 24:0; \, 19-cyclopropane acid. FIG. 2. Effect of Growth Temperature on the Pro portions of Total Unsaturated Acids to Total Saturat ed Acids (U/S ratio) in the Cellular Fatty Acids of L. heterohiochii S-56 and L. fermentum H-34. Cyclo propane acids are included in the unsaturated acid. \, L. heterohiochii S-56; œ \ œ, L. fermentum H-34

3 Fatty Acids of Alcoholophilic Lactobacilli 839 TABLE T. EFFECT OF CULTURAL TEMPERATURE ON THE CELLULAR FATTY ACID COMPOSITION OF L. heterohiochii S-56 a) Per cent of the total fatty acid; tr refers to less than 0.1%. b) cy denotes cyclopropane acid.c) Ratio of total unsaturated fatty acids to total saturated acid. Cyclopropane acids were included in the total unsaturated fatty acids. This relationship between growth temperature and the U/S ratio of the cellular fatty acids was also observed in the another strain (H-1). 3) Effect of ethanol concentration of the medium L. heterohiochii S-56 and S-41 were cultured in media containing zero, 5, 10 and 15% (v/v) ethanol, and the fatty acids of the cells were analyzed. Alteration of the fatty acid com position of the strain S-41 is observed as shown in Fig. 3. Although the content of the unusually long chain acids was affected to some extent by ethanol in the medium, there is no clear relationship between the amounts of unusually long chain acids and the ethanol concentra tion of media. The strain S-41 produced the unusually long chain acids in the largest proportion without ethanol, while the strain S-56 did in the medium containing 5% ethanol. On the other hand, it was observed that all the saturated acids decreased with a parallel increase in the unsaturated acids when ethanol concentration in media was raised. The U/S ratio of the cells grown with 15% ethanol reached nearly 3 fold values as compared with the cells grown without ethanol. In the case of the strain S-56, per cent composition of each saturated or unsaturated acid did not shift all alike with the increase of the ethanol concentration, but U/S ratio rose linearly as observed in the strain S-41. (Fig. 4) 4) Effect of main constituents of the medium L. heterohiochii S-56 was cultivated in 9

4 840 K. UCHIDA FIG. 4. Effect of Ethanol Concentration on the Pro portions of Total Unsaturated Acids to Total Saturat ed Acids (U/S ratio) in the Cellular Fatty Acids of L. heterohiochii. \, S-41; \, S-56. sented in Table U. Effects of glucose, yeast extract and polypeptone can be seen by com paring the data shown in the columns of medium A-B-C, A-D-E and A-F-G in FIG. 3. Effect of Ethanol Concentration in Media on the Proportions of Fatty Acids of L. heterohiochii S-41. Data for minor components are not plotted. œ \ œ, 16:0; œ--- œ, 16:1; \, 18:0; ---, 18:1; \, 20:0; ---, 20: 1; \, 22:0 ; ---, 22:1; \, 24:0; ---, 19-cyclopropane acid; ~- E- ~, Sum of the unusually long chain acids (C20 and longer ones). different media (Table U) and fatty acids of the cells grown in each medium were analyzed. Total cell growth and per cent proportions of the unusually long chain acids (sum of C20 and longer ones) in the total fatty acids are pre- Table U, respectively. Reduction of the glucose concentration di minished almost proportionally the final cell growth, but caused no significant alteration of the fatty acid composition. In contrast, yeast extract affected appearently the proportion of the acids with unusually long chains inde pendent of the total cell growth. The effect of polypeptone was somewhat intermediate of those of the formers. 5) Effect of gylcerol and 1-phosphoglycerol Effects of the supplementation of glycerol TABLE U. EFFECTS OF THE CONCENTRATIONS OF GLUCOSE, POLYPEPTONE AND YEAST EXTRACT ON THE FORMATION OF THE UNUSUALLY LONG CHAIN ACIDS BY L. heterohiochii S-56 a) Other constituents were the same as the basal medium.b ) Dry weight of the cells grown in 300ml culture.c) Sum of the unusually long chain fatty acids (C20 and longer ones) in the total fatty acids.

5 Fatty Acids of Alcoholophilic Lactobacilli 841 TABLE V. EFFECT OF THE ADDITION OF GLYCEROL OR 1-PHOSPHOGLYCEROL TO THE MEDIUM ON THE FORMATION OF THE UNUSUALLY LONG CHAIN ACIDS BY L. heterohiochii STRAINS present as acyl moieties of glycerolipids.3) And, in the present experiments, supplement ing of glycerol or 1-phosphoglycerol in the culture showed no significant decrease in the synthesis of the unusually long chain acids. Therefore, the mechanism of formation of the unusual acids by this alcoholophilic microbe is thought not to be the same as observed in a) DL, 1-Phosphoglycerol sodium salt. b) Sum of the percentages of the unusually long chain acids (C20 and longer ones) in the total fatty acids. and of 1-phosphoglycerol to the basal medium on the formation of the unusually long chain fatty acids were presented in Table V. No significant reduction of the amount of the un usually long chain acids was observed. DISCUSSION It has been confirmed that the fatty acids with unusually long chains occurred in L. heterohiochii cells throughout whole stages of the growth, and that they were synthesized either under various environmental conditions or in a variety of media. This finding indicates that the formation of these unusual fatty acids is an inherent character of this species, although their relative amounts are affected under some cultural conditions. Only one factor, the amount of yeast ex tract in media, has been hitherto shown to promote the production of the unusually long chain acids of this organism. Some vita mins or some active substances in yeast extract might be involved in the production of the unusual fatty acids. In has been reported12) that when a glycerol requiring auxotroph of Bacillus subtilis was deprived of glycerol, some extra homologous fatty acids longer than those usually occurring in glycerol-supplemented cultures were syn thesized. These unusually elongated acids were present as free fatty acids in the bacterial lipids. In the case of this alcoholophilic species, as described before, most of the unusually long chain acids were found to be the glycerol-requiring mutant of B. subtilis. The effect of temperature on the cellular fatty acids of many kinds of microorganisms has been studied4 `7) and it has been com monly observed that the proportion of un saturated acids decreased as the growth tem perature was raised, so that an adequate physico-chemical state of the membrane lipids was maintained at each growth tem perature.13) L. fermentum H-34, a hiochi lactobacillus strain, showed a typical reduction of the U/S ratio of cellular fatty acids as the growth temperature increased, over the whole range of temperature examined. In the case of L. heterohiochii, however, at temperature higher than 28 Ž, the over all U/S ratio of the fatty acids remained constant. Over the same higher temperature range, the proportion of longer chain acids, which should have higher melting points, was found to decrease with the increasing temperature. The shift of the cellular fatty acid composition due to the alteration of growth temperature as observed with this bacterium may cause some appreciable change in over all rigidity of the membrane lipids. Occurrence of polar glycerolipids with unusually long acyl chains in this alcoholo philic bacterium suggests some probable correlations between its alcoholophilism or alcohol-tolerance and the unusual acyl moie ties of the membrane lipids.3) However, there is no clear relationship between the ethanol concentration of cultural media and the chain lengths of the fatty acids. Unex pectedly, an elevation of the ethanol concen tration resulted in a significant increase of the over all U/S ratio of the fatty acids. The reverse relationship between growth tempera ture and the proportion of unsaturated acids

6 842 K. UCHIDA has been well established and many papers on the physiological significance of this phenomenon14 `16) as well as its control mechanism in lipid biosynthesis 17,18) have been published. On the contrary, alteration of the U/S ratio of the microbial fatty acids by alcohol concentration in media has not so far been known. Further studies on this unusual behavior as to the control of the fatty acid composition of these alcoholophilic lac tobacilli are in progress. In the present study, it has been established that L. heterohiochii, a spoilage bacterium of Sake, shows an unusual behavior in con trolling the unsaturation of the fatty acids, in addition to the unusual nature in regulating their chain lengths. Acknowledgements. The author wishes to express his thanks to Prof. K. Arima of Tokyo University for the guidance in this work. The author also thanks Drs. T. Yokotsuka and T. Mizunuma for their support and encouragement, and Miss K. Nagura for her technical assistance. REFERENCES 1) K. Uchida and K. Mogi, J. Gen. Appl. Micro biol., 19, 233 (1973). 2) K. Uchida, Biochim. Biophys. Acta, 348, 86(1974). 3) K. Uchida, Biochim. Biophys. Acta, 369,146(1974). 4) A. G. Marr and J. L. Ingraham, J. Bacterial., 84,1260 (1962). 5) D. G. Bishop- and J. L. Still, J. Lipid Res., 4, 87 (1963). 6) A. J. Fulco, Biochim. Biophys. Acta, 144, 701 (1967). 7) R. A. Levin, J. Bacteriol., 112, 903 (1972). 8) M. Kondo and M. Kasai, Biochim. Biophys. Acta, 311, 391 (1973). 9) H. Ishii, J. G. Joly and C. S. Lieber, Biochim. Biophys. Acta, 291, 411 (1973). 10) S. J. Paterson, K. W. Butler, P. Huang, J. Labelle, T. C. P. Smith and H. Schneider, Biochim. Bio phys. Acta, 266, 597 (1972). 11) H. Momose, S. Miyake, and K. Noshiro, Jozo kyokaishi, 65, 999 (1970). 12) L. Mindich, J. Bacterial., 110, 96 (1972). 13) C. W. M. Haest, J. De Gier and L.L.M. van Deenen, Chem. Phys. Lipids, 3, 413 (1969). 14) "Current Trends in the Biochemistry of Lipids," ed. by J. Ganguly and R. M. S. Smellie, Academic Press, London, 1972, p ) J. E. Cronan, Jr. and P. R. Vagelos, Biochim. Biophys. Acta, 265, 25 (1972). 16) M. Kito, Tanpakushitsu-Kakusan-Koso, 17, 500 (1972). 17) M. Sinensky, J. Bacterial., 106, 449 (1971). 18) H. Okuyama, Tanpakushitsu-Kakusan-Koso, 18, 114 (1973). 19) K. Uchida, Proc. Japan. Conf. Biochim. Lipids, 14,1(1972)