2. Studies on Lactic Acid Fermentation.

Part 1. Metabolism of Pyruvic Acid by Hetero-type Lactic Acid Bacteria.

By Hideo KATAGIRI and Kazutami IMAI

The metabolism of pyruvate by hetero-fermenters was found to vary according to experimental conditions; acetic acid formation was increased under aerobic conditions, while anaerobically acetoin formation was accelerated. When pyruvate was added to the growing culture during the early stage of growth, it was reduced to lactic acid, while acetoin was obtained from pyruvate when it was added during the later stage of the growing culture. No relationship of acetaldehyde to acetoin formation was ascer tained, and no evidence was obtained on ethanol formation from pyruvate.

mentation, we have studied the meta Introduction bolism of pyruvate by hetero-type lactic In nature, formation of lactic acid acid bacteria. is commonl seen, namely in muscle or in various microorganisms. As for Experimental and Results

lactic acid bacteria, they are divided The microorganism used in this into two groups; one is a homotype report isisolated from cheese by us,. which produces lactic acid as the sole and identifiedas Lactobacillus fermen product of sugar fermentation, and the tum ƒÀ(hetero-type). other is a hetero-type which produces Pyruvic and lactic acid were deter carbon dioxide and ethanol or acetic mined by the methods of Friedemann, acid in addition to lactic acid. The and Haugen2) and of Friedemann et mechanism of lactic acid fermentation al.,3) respectively. Acetoin was deter by bacteria has not yet been clarified. mined by the method of Van Niel.4) It has been shown by De Moss et al.,1) The bacterial cells grown in malt ex that the fermentation of glucose by a tract (containing 5% CaCO3) for 48 hetero-type lactic acid bacteria would hours were harvested by Sharpless not proceed via the Embden-Meyerhof . ultracentrifuge. These intact cells were scheme, but no other scheme of the used for experiments. mechanism has yet been put forward. Pyruvic acid is considered the mother-

In order to get some evidence on 2) T. E. Friedemann and G. E. Haugen, J. Biol. chemical changes involved in the fer- Chem., 147, 415 (1943). 3) T. E. Friedemann, M. Cotonio and P. A. Shaf 1) D. De Moss, R. C. Bard and L C. Gunsalus, fer, J. Biol. Chem., 73, 335 (1927). J Bast., 62, 499 (1951). 4) Van Niel, Biochem. Z., 187, 472 (1927)

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.substance of lactic acid, but controver ascertained by yeast. Several reaction sialm discussions are put forward as t formulas have hitherto been proposed whether ethanol is derived from pyruvic for bacterial metabolism of pyruvate. acideven in the case of hetero-type For instance: lactic acid bacteria as was already

(1) CH3•ECO•ECOOH•¨CH3•ECHO+C02 Carboxylase (2) CH3•ECO•ECOO-+I/2O2•¨CH•ECOO-+CO2 Pr. vulgaris5) (3) CH3•ECO•ECOO-+(OH)2•EPO2-+O2•¨CH3• COOPO3--+CO2 L. delbreeckii5) (4) CH3•ECO•0COO-+(OH)2.P02•¨CH3 COOP03--+HCOO-+H+ E. coli7) (5) CH3•EC0•EC00-+(OH)2•P02•¨CH3• COOPO3--+H2+C02 Cl, butylicum8) (6) CH3•ECO•ECOOH+DPN+CoA•¨Acetylphosphate +CO2+DPNH2 E. colic) (7) 2CH3•ECO•.COOH+H20•¨CH3•CHOH•ECOOH+CH•ECOOH+C02 Gonococcus and Staphylococcus10) (8) CH3•ECO•ECOOH+H2•¨CH,CHOH•ECOOH Lactic dehydrogenase (9) 2 CH3•ECO•ECOOH•¨2CO2+CH3•ECHOH•ECO•ECH3 A. aerogenes11) The formula (1) is a well known out that there are some reaction systems reaction occuring in yeast, but no con other than these listed above, for ex vincing demonstration of this reaction ample, Wood-Werkman's reaction or was obtained by ethanol forming malic enzyme system of fixing carbon bacteria, except Termobacteriummobile dioxide to pyruvate. and Sarcina ventriculi,12)since presence 1. Manometric experiments of carboxolase in lactic acid bacteria Using the Warburg manometer, we was never verified. observed the pyruvate metabolism of Formulas (2) to (7) are the reactions cell suspensions. The composition of producing acetic acid, and are divided the reaction mixture is as follows: Na- into two types: one needs phosphate and the other proceeds without phos phate. Formula (7) represents the re action in which hydrogen generated from pyruvate is transported to the other pyruvate molecule and thence lactic acid is formed. It is noteworthy .that in formula (9) of the acetoin forming system shown by Silverman and Werkman,11) carboxylase is not implicated. It is indisputable to point 5) P. K. Stumpf, J. Biol. Chem., 159, 529 (1945). 6) F. Lipmann, Nature, 144, 381 (1939). 7) G. Kalnitsky and C. H. Werkman, Arch. Bio chem., 2, 113 (1943), 8) H. J. Koepsell and M. J. Johnson, J. Biol. Chem., 145. 379 (1951). 9) S. Korkes, A. del Campillo, I.C. Gunsalus and S. Ochoa, J. Biol. C&em., 193, 721 (1951) . 10) H. A. Krebs, Biochem J., 31, 661 (1937). 11) M. Silverman and C. H. Werkman , J. Biol. Fig. 1. Gaseous Metabolism of Pyru1 ate by Chem., 138, 35 (1941). cell Suspensions 12) J.B. Sumner and K. Myrback, The Enzymes , V -• Carbon dioxide evolution ol. 11, 824 (1952). Oxygen u,take

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pyruvate 0.08 M, Mn++27.5.ƒÁ.Mg++ Necessity of phosphate in the pyruvic 610ƒÁ(as chloride), cocarboxylase 40ƒÁ, oxidation system is again ascertained by M/15 phosphate buffer (pH 6.4) of the results shown in Fig. 3, in which 0.8 ml and cell suspension (intact cells of 20mg dry weight); total volume is

2.5ml, the temperature kept at 37•Ž and the gas phase is air. The results are shown in Fig. 1 in which we observed oxygen uptake and carbon dioxide evolution. In the reaction mixture we were able to determine acetoin as Ni-dimethylglyoxime, and to detect lactic13) and acetic acids14) by

paper partitionchromatography. From these results, decomposition of pyruvate by a cell suspension was clearly demon

strated, however, at least three reactions such as pyruvic oxidation system, the acetoin forming system and the lactic Fig. 3. Effect of Citrate•E dehydrogenase system would be suppos -• Carbon dioxide evolution •B....•B Oxygen uptake ed to be related to the decomposition I...... Phosphate plus citrate buffer of pyruvate. When phosphate buffer II...... Phosphate buffer is substituted by citrate, carbon dioxide citrate has no inhibitory effect, but evolution and oxygen uptake are greately reveals slight activation of this system. diminished as shown in Fig. 2, so that It is worthwhile to point out that no noticeable effect on this system was ever detected with Ca-pantothenate of

50ƒÁ/2.5ml nor 2,4-dinitrophenol of 1/1000M.

2. Macro experiments As was shown in the preceding // ex

periments we deduced the existence of three systems, in the pyruvate metabol ism; macro experiments are carried out in order to get a clear idea of the relationship among these systems. The scale of the reaction mixture is Fig. 2. Necessity of Phosphate •E -• Carbon dioxide evolution about twenty times as great as that of •B....•B Oxgen uptake the manometric experiments and it con I...... Phosphate buffet II...... Citrate buffer tained Napyruvate 400mg, cocarboxyla

phosphate would play an important se 800ƒÁ, Mg++12mg, Mn++0.55mg, role in the pyruvic oxidation system. 15ml of M/15 phosphate buffer (pH 13) J. W. H. Lugg and B. T. Overell, Nature, 160, 87 (1947). 6.4) and the cell suspension (intact cells 14) E. P. Kennedy and H. A. Barker, Anal. Cbem., of 355mg dry weight) in a total volume 28, 1033 (1951).

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of 50ml. Incubation period was three At the end of reaction the mixture hours at 37•Ž. was analyzed, and pyruvic, acetic, and A) Reaction under aerobic and an lactic acids, and acetoin were deter aerobic conditions. mined. The results are shown in Table The reactions were proceeded as I in which it is clearly pointed out, that follows: under aerobic.conditions (No. 2), acetic No. 1-anaerobic (filled with H2 gas acid formation is increased, while for and incubated statically). mation of lactic acid and acetoin is No. 2-aerobic (kept in the air and decreased. agitated).

Table I Metabolism of Pyruvate under Aerobic and Anaerobic Conditions

B) Effect of riboflavin blue (1/10,000 M) reveals almost no Under aerobic conditions we tested effect, but the addition of riboflavin the effect of riboflavin and of methyl- (1/10,000M) results in an increasing ene blue. In this case anaerabic condi effect on acetic and lactic acid formation tions are brought about by evacuation. and a noticeable decrease of acetoin As is shown in Table II, methylene formation.

Table II Effect of Riboflavin

C) // Acetoin metabolism acid and ethanol, so that the reaction As was shown in the preceding ex mixture was kept for as long as 48 periment, this microorganism forms a hours in order to find out whether large quantity of acetoin. It is important the formed acetoin would be decom to investigate whether acetoin is a posed in the course of time. The results normal intermediate product of hetero are shown in Table III in which there type lactic acid fermentation. is no evidence of degradation of acetoin Tomiyasu15) reported that certain since the yield of acetoin was found bacteria could split acetoin into acetic to be constant (29.2 and 25.3 %) 15) Y. Tomiyasu, J. Agr. Chem. Soc. Japan, 14, 634 (1938). despiteincubation periods (3 and 48hrs.

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Table III Consumption of Acetoin

respectively). The mechanism of bac or dimedon was added to the reaction terial formation of acetoin is not yet mixture containing Na-pyruvate 240mg, clear.Silverman and WerkmanlV re cocarboxylase 800ƒÁ, Mg++12mg, ported that acetaldehyde is not an Mn++0.55mg, 15ml of M/15 phos intermediate product in the reaction of phate buffer (pH 6.4) and the cell Aerobacter, while Happold and Spen suspension (intact cells of 355mg dry cer 16) deduced a reaction in which weight) in a total volume of 15ml. acetaldehyde-enzymecomplex and ace The mixture was placed in a 55ml tate will condence to diacetyl and then Erlenmeyer flask and incubated statical will be reduced to acetoin. No relation ly (not strictly anaerobic conditions), ship of acetaldehyde to acetoin forma acetaldehyde or dimedon was added, tion was ascertained by the results shown 130 or 100mg/50ml respectively. in Table IV, in which acetaldehyde

Table IV Effect of Dimedon and Acetaldehyde

3. Experiments on growing culture culture, one of them was added to the To investigate the effect of acetalde cultures (malt extract, containing 5 per hyde or alcohol on a normal growing cent CaCO3) of 24 hours' incubation.

Table V Effect of Acetaldehyde, Ethanol, and Dimedon on a Growing Culture

16) F. C. Happotd and C. P. Spencer, Biochim. et Biophys. Acta, 8, 543 (1952).

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At the same time we inoculated the CaCOa) after requisite period of incuba seed of bacteria to the malt extract tion, and incubated again for an addi containing dimedon and incubated at tional three hours at 37•Ž and then 37•Ž for 72 hours in each case. At analyzed, were carried out. These re the end of fermentation, cultures . were sults are shown in Table VI in which analyzed, and the results are shown it will be seen that almost all the in Table V. From the Table it is pyruvate added was consumed even in observed that alcohol has no effect three hours and was converted solely to on the ratio of fermentation products, lactate when pyruvate is added to a while acetaldehyde reveals an increase young culture of 24 hours' incubation on the formation of alcohol and acetate. The pyruvate which was added to However, it is difficult to conclude the culture of fourtyeight hours' incu that acetaldehyde is a precursor of bation was converted mainly to lactate ethanol or acetate, because dimedon and then to acetoin, about 45 per cent has no effect on the fermentation of of the consumed pyruvate is considered sugar, and furthermore the subsequent, to be converted to lactate and about 35 experiment does not show any evidence per cent is suggested to be used for for formation of alcohol from pyruvate. the formation of acetoin (assuming that In order to investigate pyruvate zmol. of acetoin is formed from 2mol. metabolism in a growing culture, expe of pyruvate; riments in which Na-pyruvate (320mg 2 CH3.CO.COOH•¨ CH3•CHOH• CO•ECH3+2C02). per 50ml) was added to the culture medium (malt extract containing 4%/ On the other hand, consumption of

Table VI Pyruvate Metabolism in Growing Culture

-14- Vol. 19, No. 1, 1955] Studies on Lactic Acid Termeutation. pyruvate added to seventy-two hours, through out the experiments. From culture is found to be only 57 per these results we deduced that pyruvate cent, and the majority of the consumed cannot act as a precursor of alcohol in pyruvate is used for the formation of hetero type lactic acid fermentation. acetoin (about 60 per cent). It is important to point out that added Department of Agriculture, pyruvate is never converted to alcohol, Kyoto University since formation of alcohol is constant [Received,Oct. 13, 1954]

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