showed that the culture could grow in micro

Transcription

1 NUTRITIONAL STUDIES ON LACTOBACILLUS HETEROHIOCHI A. L. DEMAIN, E. L. RICKES, D. HENDLIN, AND EVELYN C. BARNES Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey Received for publication June 15, 196 The hiochi bacteria are lactic acid bacteria known for their ability to spoil Japanese rice wine (sak6). Recent taxonomic studies by Kitahara, Kaneko, and Goto (1957a, b) show that those members which are not dependent on mevalonic (hiochic) acid (Tamura and Folkers, 1958), i.e., the hiochi lactobacilli, can be classified into known species of the genera Lactobacillus and Leuconostoc. The dependent forms (true hiochi bacilli), however, do not correlate with recognized species and are given the new names Lactobacillus heterohiochi and Lactobacillus homohiochi, according to their type of fermentation. Both of these species were reported to require a peptidic factor (factor L) which could be supplied by liver or peptone. When Lactobacillus heterohiochi strain H-1 was inoculated into a mevalonic acid-containing medium which was chemically defined with the exception of its vitamin-free acid-hydrolyzed casein component, we found that it failed to grow unless supplemented with yeast extract. Although enzyme-hydrolyzed casein (a source of factor L) spared the yeast extract requirement, the latter was still required for good growth indicating that factors other than peptides were required. Our aim was to determine whether known compounds could replace yeast extract and enzyme-hydrolyzed casein or whether unidentified growth factors were involved. The present paper describes our experiments which show that it is possible to substitute compounds for these crude materials. Our studies have resulted in the formulation of a medium composed solely of vitamin-free acid-hydrolyzed casein and known compounds which supports more extensive growth than has been previously reported with L. heterohiochi. MATERIALS AND METHODS Organism. Lactobacillus heterohiochi strain H-i was supplied by Dr. G. Tamura of the University of Tokyo in Japan. It was preserved in the lyophilized state. Inoculum development. Preliminary studies 147 showed that the culture could grow in micro inoculum broth (Difco) containing.4,ug per ml of the dibenzylethylene diammonium salt of mevalonic acid' at ph 5 and 28 C. Adjustment of the ph to 6.7 or the temperature to 37 C inhibited growth. Better growth was achieved when the yeast extract content of the broth was raised from 2 per cent to 5 per cent (w/v). Incubation was stationary in colorimeter tubes (2 by 175 mm) covered with Morton stainless steel closures (Bellco Glass Company, Vineland, New Jersey). No effect on growth was encountered by shaking or by replacing these closures with rubber stoppers or cotton. The favorable conditions mentioned above were adopted for the preparation of inoculum for the nutritional experiments. The, cells were grown in this modified micro inoculum broth for several weeks, centrifuged, washed once with a mineral salts mixture (Demain, 1958), and made up in the salts mixture at a concentration which exhibited a percentage light transmission (%T) of 5 to 1 per cent at 66 m,u in a Lumetron colorimeter (model 42-E). One drop of this heavy suspension (about.5 ml) was added to each experimental tube. Growth experiments. The initial medium for these experiments was similar to that of Skeggs et al. (1956) except that the enzymatic digest of casein was omitted, the Tween 8 (polyoxyethylene sorbitan monooleate) concentration was decreased, and the ph was adjusted to 5. instead of 6.5 (table 1). Thus, the medium was chemically defined with the exception of the acid-hydrolyzed casein component. The medium was prepared at double strength, and 5 ml were added to the colorimeter tubes. An additional 5 ml of glass-distilled water or an additive were added, and the tubes were covered with stainless steel closures. After autoclaving at 12 C for 15 min, the tubes were cooled, inoculated, and incubated at 28 C without agitation. Duplicate 1 The dibenzylethylene diammonium salt of mevalonic acid will be referred to as mevalonate.

2 148 TABLE 1 Composition of media DEMAIN, RICKES, HENDLIN, AND BARNES Component Initial Medium Medium E [VOL. 81 Glucose... L-Asparagine, L-glUtamic acid, L-glutamine, DL-serine... L-Cysteine- HCl... DL-Alanine... DL-Tryptophan... L-Cystine... Adenine, guanine, xanthine, uracil... Thymine, orotic acid... Salts A (Snell and Wright, 1941)... Salts B (Snell and Wright, 1941)... Ascorbic acid... Calcium pantothenate.. Nicotinic acid... Folic acid, pyridoxal, p-aminobenzoic acid. Pyridoxine. Thiamine, riboflavin... B12, lipoic acid... Biotin... Mevalonate... Tween 8... Absolute ethanol (aseptic addition)... Salt-free, vitamin-free acid-hydrolyzed casein*... Final ph... amri/liter 2 g 5 mllg 2 mg ami/liter 2 g 3 g eaecii 5 mg 5 mg 2 mg 5 mg each 5 mg each 2 mg each 2 mg each 1 ml 5 ml 1 pg 1 pg 5 ug each 2 pig 1p,g each 1 ug each 5 pg 7.5 g 5. 1 ml 5 ml 4 mg 1 mg 5 pg each 2 pg 1 ug each 1,ug each 5,ug 5 mg 5 ml 7.5 g 5. * Nutritional Biochemicals Corporation. tubes were used throughout the study. The extent of growth was measured in the Lumetron colorimeter and recorded as optical density. When densities were below 3, suspensions were diluted and reread. Thus, the recorded density values represent the observed optical density in the range where Beer's Law was obeyed, multiplied by the dilution factor. Volatile additives were sterilized by Seitz filtration and aseptically added to the tubes of autoclaved medium. RESULTS Preliminary experiments. When the initial medium described above was supplemented with.4 ug per ml of mevalonate, it supported the z uj 4% ~ Medium A + Yeast Extract Medium A Figure 1. Growth of Lactobacillus heterohiochi strain H-1 in medium A (see "text") in the presence and absence of 5 mg per ml yeast extract and in medium B (= medium A + N-Z-Case). growth of the mevalonic acid-dependent Lactobacillus acidophilus strain ATCC-4963 (Skeggs et al., 1956) at its optimal ph of 6.5. However, such supplementation was insufficient to allow growth of L. heterohiochi at its optimum, ph 5. Some growth did occur when yeast extract was added. Increasing the concentration of mevalonate 1-fold had only a slight effect in the absence of yeast extract but showed a marked stimulation of both rate and extent of growth in its presence. Excellent growth with yeast extract was obtained at an optimal concentration of 5,ug per ml of mevalonate. Thus, L. heterohiochi requires over 1 times the concentration of mevalonic acid which is optimal for L. acidophilus strain Figure 1 shows the effect of yeast extract on the growth of L. heterohiochi in the initial medium supplemented with 5,ug mevalonate per ml (medium A). Effect of enzyme-digested casein in medium A. The addition of 2 mg per ml of enzyme-hydrolyzed casein (N-Z-Case, Sheffield Chemicals, Norwich, New York), a source of peptides, to medium A stimulated the onset of growth but could not replace yeast extract with regard to extent of growth (figure 1). Higher concentrations of N-Z-Case failed to raise the optical

3 1961] NUTRITION OF L. HETEROHIOCHI 149 l-- ( zw-aj 49 3.[ X..5 Yeast Extract + 2.5mg lml Cosein Digest / -< Cosein Digest Casein Digest /4 2mg /ml Yeast t Extract + 2.5mg iml Cossi4n Digest o TOTAL mg /ml CASEIN DIGEST + YEAST EXTRACT Figure 2. Response of Lactobacillus heterohiochi strain H-1 in medium A to increasing concentrations of enzyme-hydrolyzed casein, to increasing concentrations of yeast extract in the presence of 2.5 mg per ml of enzyme-hydrolyzed casein, and to increasing concentrations of enzyme-hydrolyzed casein in the presence of 2 mg per ml of yeast extract plus 2.5 mg per ml of enzyme-hydrolyzed casein. Duration of experiment, 6 days. Nutritional Biochemical Corporation vitamin-free casein hydrolyzate (enzyme) used. density beyond the level observed with 2 mg per ml. Addition of enzyme-hydrolyzed casein to the medium spared the yeast extract requirement as shown in table 2. It appeared that the factor(s) supplied by the casein digest were present to a limited extent in yeast extract, but that yeast extract also contained an additional factor(s). This can be seen clearly in figure 2. The lower curve shows the response to the digest alone. At 2.5 mg per ml, the dosage-response curve is leveling off due to the lack of yeast extract factor(s). Addition of yeast extract gives a burst of growth but at 25 mg per ml of yeast extract, the casein factor(s) is evidently in very low supply. Thus, the further addition of enzymehydrolyzed casein markedly stimulates growth once again. We decided to include N-Z-Case in the medium TABLE 2 Sparing of yeast extract requirement by addition of enzyme-hydrolyzed casein to medium A Yeast Extract No casein hydrolyzate Optical Density (2.5 mg per ml) Casein hydrolyzate mg/mi Nutritional Biochemical Corporation vitamitsfree casein-hydrolyzate (enzyme) used. Similar results were obtained with N-Z-Case. Duration of experiment, 6 days. at 2 mg per ml in order to study the yeast extract factor(s). Growth in this medium (medium B) was markedly stimulated by yeast extract and provided an excellent assay medium for its factor(s). Effect of pantothenic and nicotinic acids in medium B. The marked effect of yeast extract upon growth in medium B was destroyed by ashing and lost upon dialysis with the activity being recoverable in the dialyzate. Passage of yeast extract through a column of Amberlite IR-12 cation exchange resin in the hydrogen form removed its activity. Although an ammonia eluate had some activity, it only approached the activity of yeast extract when the effluent (which had no activity alone) was included in the medium. In the presence of the effluent, the eluate could be replaced by 1 pg per ml of calcium pantothenate. Since the initial medium contained.1,ug per ml, a conventional amount for lactic acid bacteria, it was obvious that L. heterohiochi required higher concentrations of this vitamin than most lactobacilli. Pantetheine was only about one-fourth as potent as calcium pantothenate, whereas coenzyme A, pantoyl lactone, f3-alanine, and ethanolamine were inactive. By supplementation of medium B with excess calcium pantothenate (4 pag per ml), we were able to focus our attention on the effluent factor. This was found to be replaceable by 1 pg per ml of nicotinic acid. Again, this was 1 times the level present in the initial medium which indicated that L. heterohiochi has a higher nicotinic acid requirement than other lactic acid organisms. Nicotinamide was no more active than nicotinic

4 15 DEMAIN, RICKES, HENDLIN, AND BARNES [VOL. 81 acid, whereas diphosphopyridine nucleotide and triphosphopyridine nucleotide were less active. Effect of removal of N-Z-Case and addition of asparagine. In medium B supplemented with calcium pantothenate and nicotinic acid, rapid growth ensued which was not further stimulated by yeast extract. The aim now was to replace the N-Z-Case. WNhen this was removed, no growth was evident. However, it was discovered that low concentrations of L-asparagine could substitute for part of the N-Z-Case requirement. Medium C was thus devised by removing N-Z- Case from medium B and supplementing with 4,ug per ml of calcium pantothenate, 1,ug per ml of nicotinic acid, and 1,ug per ml of L-asparagine. In figure 3, growth in medium B, in medium B less N-Z-Case, and in medium C are compared. It can be seen that although medium C gives more total growth than medium B3, we still lack a component of N-Z-Case responsible for early growth. Effect of amino acids and ascorbic acid. It was felt that the missing component was the peptidic factor L and attempts were made to replace this material with high levels of individual amino acids (Peters, Prescott, and Snell, 1953; Demain and Hendlin, 1958). Those that showed stimulation at 3 mg per ml were L-glutamine, L-asparagine, L-glutamic acid, and DL-serine. A combination of the four gave a greater stimulation than any of the amino acids alone. Since reducing agents had been shown to increase the growth of the peptide-requiring Lactobacillus casei in the presence of high levels of amino acids (Rickes, Koch, and Wood, 1949), we tested the effect of ascorbic acid. It was found to be stimulatory at.1 mg per ml and toxic at 1 mg per ml. Further addition of L-cysteine at.5 mg per ml increased growth even more. These observations, depicted in the curves of figure 4, resulted in the formulation of medium D which was composed of medium C plus L-glutamine, L-asparagine, L-glutamic acid, and DL-serine, each at 3 mg per ml, ascorbic acid at.1 mg per ml, and L-cysteine at.5 mg per ml. Growth in medium D not only occurred at the same rate as in medium A supplemented with yeast extract but, in addition, the lag phase in medium D was 2 days shorter (compare figure 4 with figure 1). Effect of ethanol and related compounds. The results with medium D showed that known chemical compounds were capable of duplicating 9 a 7 6 z 4 14 C-) Medium C Medium E / / / bmedium B Loss N-Z- Case a L- x o a Figure 3. Growth of Lactobacillus heterohiochi strain H-1 in medium B, in medium B less N-Z- Case, and in medium C (= medium B less N-Z- Case + calciumi pantothenate + nicotinic acid + L-asparagine). 9 a 8 A Medium D 7 / 6 Medium C + L Glutamine + L Asparogine + z 5-/ L Glufamic Acid + a / L Serino -i 4% 4. ~~~~~~~ o ac 2train H-1 in medium C, inm Medium C Figure 4. Growth of Lactobacillus heterohiochi strain H-i in medium C, in medium C + 3 mg per ml each of L-glutamine, L-asparagine, L-glutamic acid and DL-serine, and in medium D (= medium C + the 4 amino acids + ascorbic acid + L-cysteine).

5 1961] NUTRITION OF L. HETEROHIOCHI 151 TABLE 3 Effect of arginyl-leucine in the absence of acid-hydrolyzed casein Medium Optical Density r 4 / X; \ \~~~~~~~ - B utanol 2.5 o obutyric Acid Pyruvic Acid a mg /ml Figure 5. Response of Lactobacillus heterohiochi strain H-1 in medium D to increasing concentrations of ethanol, n-propanol, n-butanol, butyric acid, and pyruvic acid. Duration of experiment, 5 days. Studies with higher concentrations showed that n-propanol becomes toxic at much lower concentrations than does ethanol. I.- z w 4-J 8 9- X IJ Figure 6. Growth of Lactobacillus heterohiochi strain H-1 in medium E (= medium D + ethanol). the growth effects of yeast extract in medium A. However, the work was extended by testing for further stimulation by adding crude materials to medium D; one of these, sak6, the natural habitat (1) Medium E (2) As 1 less acid-hydrolyzed casein.2 (3) As 2 plus mixture of amino acids..7 (4) As 3 plus.5 mg per ml L- arginine * HCl (5) As 3 plus 1 mg per ml L- arginine- HCl (6) As 3 plus 2 mg per ml L- arginine* HCl (7) As 3 plus 3 mg per ml L- arginine * HCl (8) As 3 plus 1 ug per ml L-arginyl-Lleucine * HCl (9) As 3 plus 5 pg per ml L-arginyl-Lleucine HCl (1) As 3 plus 1 pg per ml L-arginyl-Lleucine * HCl (11) As 3 plus 5 pg per ml L-arginyl-Lleucine * HCl Duration of experiment, 4 days. of L. heterohiochi, was active. We found the activity of sake to be due solely to its alcohol content. Absolute ethanol stimulated growth at.5 ml (4 mg) per ml. An examination of related compounds indicated that n-propanol, n-butanol, and pyruvic and butyric acids also showed this effect, but all were much more toxic than ethanol at concentrations greater than that of maximal stimulation (figure 5). Acetic acid showed a. slight stimulation while methanol, acetaldehyde, glycerol, mannitol, lactic acid, formic acid, and propionic acid were either inactive or toxic. Composition of medium E. The final medium developed in this study was medium E which was prepared by adding.5 ml ethanol aseptically to tubes of medium D after autoclaving. It is capable of supporting heavy growth of L. heterohiochi strain H-1 (optical densities approaching 9) as shown in figure 6. Examination of its composition (table 1) shows that it is chemically defined with the exception of the vitamin-free acid hydrolyzate of casein. We have not done extensive work on this component of the medium, but some of our results are of interest. When the acid hydrolyzate was removed from the medium, no growth occurred. Only

6 152 DEMAIN, RICKES, HENDLIN, AND BARNES [VOL. 81 slight growth took place when a mixture of amino acids simulating the composition of casein was added. In view of the replacement of acidhydrolyzed casein by arginine peptides for the growth of avian L. bifidus (Hendlin et al., 1955), we tested high levels of arginine and low levels of arginyl-leucine in medium E modified by substituting the mixture of amino acids for acid-hydrolyzed casein. The results in table 3 show a stimulation of growth but only a partial replacement of the hydrolyzed casein. DISCUSSION The yeast extract requirement of L. heterohiochi in medium A can be divided into two parts for the sake of discussion. The first is that portion of the requirement which can be satisfied by enzyme-hydrolyzed casein and presumably includes the peptidic factor L. We have found that it is possible to replace the enzyme digest with a mixture of ascorbic acid, L-cysteine, and high concentrations of L-asparagine, L-glutamic acid, L-glutamine, and DL-serine. It is of interest in this regard that Kihara and Snell (1959) have recently reported that the strepogenin requirement of L. casei can be replaced by cysteine, glutamine, and serine at high levels. The remainder of the yeast extract requirement was replaceable with increased concentrations of calcium pantothenate and nicotinic acid. These vitamins are required at a concentration of 1 jig per ml which is at least 1 times the level required by most lactic acid bacteria. L. heterohiochi, interestingly enough, also requires ten times as much mevalonic acid as does L. acidophilus strain The recent report of Tamura and Suzuki (1958) on the nutrition of L. heterohiochi, which appeared during our studies, is of interest for comparative purposes. These workers found enzymatic digests of casein to be rich sources of factor L and yeast extract to be poorer. Our results are in agreement. In addition to noting the essentiality of nicotinic acid and pantothenic acid, they also found riboflavin, thiamine, folic acid, and Tween 8 to be absolute requirements and purines and pyrimidines to be stimulatory. They failed to note any effect of ascorbic acid or alcohol, but their basal medium contained a crude casein digest which may have obscured the stimulatory response. They did, however, report a small growth promoting effect of low concentrations of asparagine. Whereas the maximal growth observed by Tamura and Suzuki corresponded to an optical density of.8 in the presence of a saturating concentration of mevalonic acid, medium E is capable of supporting growth to an optical density 1 times as great. SUMMARY Lactobacillus heterohiochi strain H-1, responsible for the spoilage of Japanese rice wine, failed to grow in a mevalonic acid-containing medium (typical for lactic acid bacteria) unless the mevalonic acid concentration was increased 1-fold and yeast extract was added. The yeast extract requirement could be subdivided into one part replaceable by enzyme-digests of casein and another part not replaceable by such supplementation. It was possible to substitute a mixture of ascorbic acid, L-cysteine, and high concentrations of L-asparagine, L-glutamic acid, L- glutamine, and DL-serine for the casein digest. The remainder of the yeast extract requirement was eliminated by increasing the nicotinic acid and calcium pantothenate concentrations at least 1-fold. A further stimulation was obtained by adding ethanol to the medium. The final medium (medium E) was chemically defined with the exception of the vitamin-free acid-hydrolyzed casein component. Such a medium was capable of supporting much denser growth than has been previously reported with this organism and, in addition, does not require the presence of enzymatic digests of casein. REFERENCES DEMAIN, A. L Minimal media for quantitative studies with Bacillus subtilis. J. Bacteriol., 75, DEMAIN, A. L., AND D. HENDLIN 1958 Growth stimulation of a strain of Bacillus subtilis by glycine peptides. J. Bacteriol., 75, HENDLIN, D., P. L. WILLIAMS, J. WALL, AND R. B. WALTON 1955 The nutrition of avian Lactobacillus bifidus. Federation Proc., 14, 225. KIHARA, H., AND E. E. SNELL 1959 The nature of the "strepogenin" requirement of Lactobacillus casei. Federation Proc., 18, 532. KITAHARA, K., T. KANEKO, AND. GOTO 1957a Taxonomic studies on the hiochi-bacteria, specific saprophytes of sak6. I. Isolation and grouping of bacterial strains. J. Gen. Appl. Microbiol. (Japan), 3, KITAHARA, K., T. KANEKO, AND. GOTO 1957b Taxonomic studies on the hiochi-bacteria,

7 19611 NUTRITION OF L. HETEROHIOCHI 153 specific saprophytes of sak6. II. Identification and classification of hiochi-bacteria. J. Gen. Appl. Microbiol. (Japan), 3, PETERS, V. J., J. M. PRESCOTT, AND E. E. SNELL 1953 Peptides and bacterial growth. IV. Histidine peptides as growth factors for Lactobacillus delbrueckii J. Biol. Chem., 22, RICKES, E. L., P. J. KOCH, AND T. R. WOOD 1949 Additional observations on the rate of growth of Lactobacillus casei. J. Biol. Chem., 178, SKEGGS, H. R., L. D. WRIGHT, E. L. CRESSON, G. D. E. MACRAE, C. H. HOFFMAN, D. E. WOLF, AND K. F. FOLKERS 1956 Discovery of a new acetate-replacing factor. J. Bacteriol., 72, SNELL. E. E., AND L. D. WRIGHT 1941 A microbiological method for the determination of nicotinic acid. J. Biol. Chem., 139, TAMURA, G., AND K. FOLKERS 1958 Identity of mevalonic and hiochic acids. J. Org. Chem., 23, 772. TAMURA, G., AND Y. SUZUKI 1958 Hiochic acid, a new growth factor for hiochi bacteria. III. Nutritional requirements of hiochi bacteria. J. Agr. Chem. Soc. Japan, 32,