requirements and the ease of measuring the extent of their growth or acid

THE PANTOTHENIC ACID REQUIREMENTS OF LACTIC ACID BACTERIA' VERNON H. CHELDELIN, EDWARD H. HOAG, AND HERBERT P. SARETT Department of Chemistry, Oregon State College, Corvallis, Oregon Received for publication August 5, 1944 Advances in vitamin and amino acid research are being speeded by the successful use of lactic acid bacteria as assay organisms. Their fastidious growth requirements and the ease of measuring the extent of their growth or acid production render them ideally suited for studies in this field. Interest in these organisms has centered about the nutritional requirements of Lactobacillus casei and Lactobacillus arabinosus, which have been used extensively for the measurement of several B vitamins and amino acids. Research on the growth requirements of other species of lactic acid bacteria has been extended for other vitamin problems. Special studies have been made of Streptococcus lactis R (Mitchell, Snell, and Williams, 1941), Leuconostoc mesenteroides (Gaines and Stahly, 1943), and Lactobacillus fermentum2 36 (Sarett and Cheldelin, 1944). The present paper is a survey of the pantothenic acid nutrition of 33 species and strains of these bacteria. Growth comparisons are compiled using different media, and evaluations are made of the various organisms for possible use in the assay of pantothenic acid. EXPERIMENTAL Culture media. Four culture media have been used in this study. Their components are listed in table 1. Medium A is a synthetic type to which acidhydrolyzed vitamin-free casein is added. Media B, C, and D contain in addition pantothenic-acid-free extracts of peptone or yeast. All of the known B vitamins and several other growth-stimulating substances are present in all media except C. The latter is essentially that of Pennington, Snell, and Williams (1940) with the exception that the glucose and sodium acetate concentrations of the original medium have been increased to the amounts recommended by Stokes and Martin (1943). Cultures. Cultures were generously supplied to us from various laboratories and included members of the genera Lactobacillus, Streptococcus, and Leuconostoc, 1 Presented at the second meeting of the Oregon Academy of Science, Portland, 1944. Published with the approval of the Monographs Publications Committee, Oregon State College, Research Paper No. 85, School of Science, Department of Chemistry. This work was supported by the Nutrition Foundation, Inc., New York, and the General Research Council, Oregon State System of Higher Education, Corvallis. 2The correct name of this organism has been suggested by Dr. J. M. Sherman to be Lactobacillus fermenti. 41

42 V. H. CEELDELIN, E. H. HOAG AND H. P. 5AREIT as follows:3 Lactobacillus casei, L. acidophilus (2 strains), L. plantarum, L. arabinosus (2 strains), L. pentosus (2 strains), L. delbrueckii (2 strains), L. brevis (2 strains), L. buchneri (2 strains), L. pentoaceticus, L. lycopersici, L. mannitopoeus, L. fermenti (2 stiain), L. gayoni, L. brassicae, Streptococcus lactis (4 strains), S. liquefaciens, S. duraus, S. zymogenes, Leuconostoc mesenteroides, anrd Leuconostoc dextranicus. TABLE 1 Ingredients of growth media A B C D Acid-hydrolyzed technical casein,*g...4...4 Acid-hydrolyzed vitamin-free casein, g... 10 10 10 Alkali-treated peptone,* g 10 10 Glucose, g... 40 40 40 40 Sodium acetate, anhydrous, g... 36 36 24 24 Alkali-treated Difoo yeast extract,* g... 2 2 2 Cystine hydrochloride, mg... 200 200 200 200 Tryptophane, mg... 200 200 200 Asparagine, mg... 1 1 1 Adenine sulfate, mg... 20 20 20 Guanine hydrochloride, mg... 20 20 20 Xanthine, mg... 20 20 20 Uracil, mg... 20 20 20 Thiamin hydrochloride, pug... 200 200 200 Riboflavin, Ag... 400 400 200 400 Nicotinic acid, pg... 200 200 200 Pyridoxine hydrochloride, jug... 200 200 200 Biotin, (free acid),,ug... 0.8 0.8 0.8 Inositol, mg... 5 5 5 p-aminobenzoic acid,,ug...... 200 200 200 Choline chloride, mg... 2 2 2 Folic acid,$jg... 5 5 15 Inorganic salts, A and B, ml... 10 10 10 10 Distilled water to 1 liter; ph 6.6-6.8. * Pennington, Snell, and Williams (1940). t Snell and Wright (1941). A folic acid concentrate was kindly furnished by Dr. R. J. Williams of the University of Texas, and is used here in terms of 40,000 potency. Snell and Strong (1939). Technique of testing responses. Experiments are carried out in 20 x 150 mm, lipless, pyrex test tubes to which the substances to be tested are added, diluted to 5 i1, and 5 ml of the appropriate medium added. The organisms for inocula are grown similarly in medium C to which 0.2,ug of pantothenic acid, 5 mg of 'We are indebted to the following persons for several of the bacterial cultures used: W. B. Bollen and J. E. Simmons, Oregon State College; R. J. Williams and E. E. Snell, University of Texas; E. McCoy and W. H. Peterson, University of Wisconsin; L. A. Burkey, U. S. Dept4 of Agriculture; C. S. Pederson, N. Y. Agricultural Expt. Station; B. W. Hammer, Iowa State College.

PANTOTHENIC ACID AND LACTIC ACID BACTERIA liver extract4 and 5 mg of Difco yeast extract have been added. These are centrifuged after 16 to 24 hours of growth, resuspended, and washed twice with sterile 0.9 per cent NaCl solution. One drop of each final suspension is added to the appropriate tubes for testing. Cultures of L. brevis L35, L. gayoni F20, L. brassicae, Leuconostoc dextranicus, Leuconostoc mesenteroides, S. lactis R8043, S. lactis RG1A, S. lactis 374, S. liquefaciens (2 strains), and S. zymogenes are grown at 30 C. All others are grown at 37 C. Turbidity readings are observed in a Pfaltz and Bauer fluorophotometer equipped with a special holder for the tubes which have been used throughout the study. With uniform tubes it is possible to make several turbidity measurements during the course of each experiment. TABLE 2 Response of lactic acid bacteria to pantothenic acid and its constituent moieties TURBIDITY* AFTER 24 HOuRS ML 0.1 N ACID PRODUCED TN 72 HOURS,ug Pantothenic acid per pg Pantothenic acid per S g ORGANISM 0 10 ml culturet 10 ml culturet a-a 0 0.2 1 1+ 0 0.2 1 nnetesp YL YL lact. L. delbrueckii 72 A 0.01 0.53 0.80 1.00 0.1 6.0 14.0 16.4 C 1.3 1.5 1.4 (Group I) B 0.01 0.61 0.92 1.00 0.2 6.1 14.9 16.4 C 1.3 1.4 1.4 L. arabinosus 17-5 A 0.04 0.32 0.56 0.96 C 1.8 1.8 1.8 (Group II) B 0.06 0.41 0.75 1.00 1.0 12.5 14.5 18.5 C 0.07 0.64 1.35 1.50 1.8 15.4 18.6 19.9 L. casei (Group A 0.02 0.05 0.06 0.64 0.1 3.3 6.6 15.6 C 2.0 2.0 2.0 III) B 0.02 0.06 0.08 0.89 1.1 5.4 9.8 18.5 C 0.07 0.35 0.55 0.73 1.9 13.8 17.6 20.0 D 0.07 0.76 1.22 1.25 1.9 13.6 19.9 19.1 * Turbidity is given in terms of optical density. t Pantothenic acid quantities are given in terms of calcium pantothenate. $ YL = 5 mg yeast extract plus 5 mg liver extract. Group numbers are described in the text, along with the group classification of the other 30 cultures studied. Turbidity values after 24 hours' growth are reported in terms of optical density (log 100 minus log galvanometer reading). Production of lactic acid after 3 days' growth has been determined in all experiments by titration with 0.1 N alkali. Turbidity readings after 3 days are not shown as they correlate generally with the amount of acid produced. Since it has been impossible to perform the experiments on all of the organisms at one time, L. casei is included in every set for purposes of comparison. Results. The response to pantothenic acid by the different organisms in the four growth media is summarized in table 2. All of the organisms tested are 4We are indebted to Dr. T. H. Jukes of the Lederle Laboratories, Pearl River, New York, for the liver extract used. 43

44 44V. H. CHELDELIN, E. H. HOAG AND H. P, SARETT seen to require pantothenic acid for growth, but they are unable to utilize the f3-alanine and lactone5 moieties. The results confirm and extend earlier observations (Snell, 1941; Snell, Strong, and Peterson, 1939; and several others). Tubes containing added yeast and liver extracts are included with each series for comparison. It is assumed that growth ii the presence of these extracts is the maximum for each organism, and a medium is adjudged to satisfy the nutrient requirements of the organism if the response to added pantothenic acid is essentially equal to that obtained with these added extracts. On this basis the organisms have been classified into three groups, which are described below. None of the 33 cultures are able to reach maximum growth in the absence of peptone, yeast, and liver extracts (medium A). Those organisms which attain maximum growth with pantothenic acid added to a medium containing alkalitreated yeast (medium B) are classified in group I. The four organisms in this group are L. delbrueckii 72, L. delbrueckii 3, L.fermenti 76, and S. durans. These organisms attain as much growth and produce as much acid (16 to 18 ml) on medium B with 1 ug of pantothenic acid as with added yeast and liver extracts. For S. durans, maximum acid production is only 9 to 10 ml. Of these four organisms, L. delbrueckii 72 shows the best relative response on medium A. In group II are organisms which grow optimally in the presence of alkalitreated yeast and peptone with no added B vitamins except riboflavin (medium C). Most of the organisms tested may be placed in this group. Some of these grow extremely rapidly. The cultures become very turbid in 10 to 14 hours and produce 18 to 20 ml of acid in three days. These are L. arabinosus 17-5, L. (irabinosus 8014, L. pentosus 124-2, L. pentosus 8041, L. plantarum 8292, L. pentoaceticus 367, and L. buchneri K14. Other organisms in this classification which produce less acid (up to 10 ml) are L. brevis 118-8, L. brevis L35, L. lycopersici 4005, S. lactis RG1A, S. lactis 374, S. lactis R8043, S. zymogenes, Leuconostoc dextranicus, Leuconostoc mesenteroides Pd6O, S. liquefaciens (Oregon State College), and L. gayoni F20. Organisms which grow optimally only if the medium is supplied with some or all of the known B vitamins in addition to alkali-treated yeast and peptone extracts are included in group III. In this group are L. acidophilus 832, L. acidophilus (Oregon State College), L. brevis, L. casei, L. buchneri, L. fermenti 86, L. brassicae, S. tiquefaciens (Iowa State College), and S. lactis 125. The first 4 of these organisms produce approximately 20 ml of acid per culture; the others produce 8 to 10 ml. In addition, one organism, L. mannitopoeus, has been observed to reach maximum growth on medium D only when untreated yeast and liver extracts are included. Acid production in the absence of these extracts is only about 60 per cent of the maximum obtainable. DISCUSSION The type of medium represented by C appears well suited for routine work, since it contains relatively few ingredients and is easy to prepare. Moreover, 6 Throughout this paper, the lactone moiety of the pantothenic acid -molecule refers to (-)a-hydroxy-,b, j-dimethyl-'y-butyrolactone. This compound was kindly supplied by Dr. J. C. Keresztesy, Merck & Co., Rahway, New Jersey.

PANTOTHENIC ACID AND LACTIC ACID BACTERIA the pantothenic-acid-free extracts of peptone and yeast supply the organisms with a number of growth-promoting substances, some of which are as yet uncharacterized. The first seven cultures listed in group II possess desirable features for use as assay organisms for pantothenic acid. They grow and produce acid more rapidly than does L. casei, a group III organism, which is generally used for assay purposes. Further experiments with these group II organisms have shown L. arabinosus 17-5 to be useful for pantothenic acid assay (Hoag, Sarett, and Cheldelin, 1944). For the study of vitamins other than pantothenic acid, the special growth requirements of the Group III organisms may be useful. Thus, L. casei is stimulated by folic acid. Thiamin is necessary for growth of L. fermenti 36, and its use for assay purposes is described elsewhere (Sarett and Cheldelin, 1944). The remaining organisms are not stimulated by folic acid or thiamin. SUMMARY Pantothenic acid has been found to be a growth determinant for 33 strains of lactic acid bacteria. The f3-alanine and lactone moieties of pantothenic acid are not utilized by these organisms. Several organisms which produce large amounts of lactic acid on a relatively simple medium appear well suited for the assay of pantothenic acid. Growth and acid production have been compared using 4 media. Optimum response of most species can be obtained in the presence of added pantothenic acid by supplementing a simple growth medium with extracts of peptone and yeast which are free of pantothenic acid. The addition of other B vitamins to this medium is utilized in studying the other requirements of some species. REFERENCES GAINES, S., AND STAHLY, G. L. 1943 The growth requirements of Leuconostoc mesenteroides and preliminary studies on its use as an assay agent for several members of the vitamin B complex. J. Bact., 46, 441-449. HOAG, E. H., SARETT, H. P., AND CHELDELIN, V. H. 1944 Paper presented before the American Chemical Society, Cleveland, Ohio. MITCHELL, H. K., SNELL, E. E., AND WILLIAMS, R. J. 1941 The concentration of "folic acid." J. Am. Chem. Soc., 63, 2284. PENNINGTON, D., SNELL, E. E., AND WILLIAMS, R. J. 1940 An assay method for pantothenic acid. J. Biol. Chem., 135, 213-222. SARETT, H. P., AND CHELDELIN, V. H. 1944 The use of Lactobacillus fermentum 36 for thiamin assay. J. Biol. Chem., 106, 153-160. SNELL, E. E. 1941 Growth inhibition by N-(a,y-dihydroxy-j3,,B-dimethylbutyryl) taurine and its reversal by pantothenic acid. J. Biol. Chem., 141, 121-128. SNELL, E. E., STRONG, F. M., AND PETERSON, W. H. 1939 Growth factors for bacteria. VIII. Pantothenic acid and nicotinic acid as essential growth factors for lactic and propionic acid bacteria. J. Bact., 38, 293-308. STOKES, J. L., AND MARTIN, B. B. 1943 Influence of buffer and glucose in the Lactobacillus casei assay for pantothenic acid. J. Biol. Chem., 147, 483-484. 45