Method for Assaying Volatile Compounds by Headspace Gas Chromatography and Application to Growing Starter Cultures

Method for Assaying Volatile Compounds by Headspace Gas Chromatography and Application to Growing Starter Cultures C. MONNET, P. SCHMTTT, and C. DlVlES Laboratoire de Microbiologie Ecole Nationale Superieure de Bidogie Appliquee & la Nutrition et a I'Alimentation Universit6 de Bourgogne 21000 Dijon, France ABSTRACT

Automatic headspace gas chromatography was used to assay volatile compounds in fermented milks, including diacetyl, which is of particular interest because of its aromatic properties. aAcetolactic acid, produced by lactic acid bacteria, is an unstable compound that is chemically transformed to acetoin and diacetyl during incubation at 85'C in the headspace sampler, leading to an overestimation of diacetyl concentrations in fermented milks. The oxidative decarboxylation of synthetic a-acetolactic acid to diacetyl during the assay decreased as the pH increased: at pH 4.0, 4096 of the a-acetolactic acid present was transformed to diacetyl, but this reaction was limited to 6% at pH 7.0. When the assay mixture was degassed and heated to 1WC before analysis, the reaction was limited to 2%, leading to a more precise assay of diacetyl in the presence of aacetolactic acid. The method for assaying diacetyl was applied to a mixed culture of Leuconostoc mesenteroides ssp. cremoris and Luctococcus Iactis ssp. lactis in milk. Acetoin (1.07 mM) and aacetolactic acid (.18 mM) were produced, but not diacetyl. In our culture conditions, the redox potential dropped rapidly at the beginning of fermentation, which prevented diacetyl production by the oxidative decarboxylation of a-acetolactic acid. When the same fermentation was carried out with agitation, the redox potential remained high, and diacetyl production was significant, reaching .032 mM (2.8 m a ) .

Received November 3. 1993. Accepted February 14. 1994. 1994 J Dairy Sci 77:1809-1815

(Key words: headspace gas chromatography, diacetyl, leuconostoc, mixed culture) Abbreviation key: ALA = a-acetolactic acid. INTRODUCTION

Mesophilic starters in fermented milks are responsible for the synthesis of flavor compounds, the most important of which are diacetyl and acetaldehyde. Among the numerous methods described for assaying diacetyl, that of Westerfeld (23) assays the sum of diacetyl and acetoin, but the diacetyl concentration in milk products is much lower (0 to 8 m a )than that of acetoin (0 to 300 m&) (2). The methods used by Walsh and Cogan (22), by Veringa et al. (21), and by Jonsson and Pettersson (7) are based on the separation of diacetyl and acetoin by distillation. Direct analysis by gas chromatography (20) is difficult because water and other components of dairy products have an unfavorable effect on the reliability of the method. The use of an automatic headspace sampler eliminates this disadvantage because only vapor in equilibrium with the previously heated liquid sample is injected in the column. a-Acetolactic acid (ALA) is an unstable compound that spontaneously decarboxylates to acetoin and is also transformed to diacetyl in oxidizing conditions. Veringa et al. (21) and Jordan and Cogan (8) showed that assays of diacetyl in the presence of ALA are overestimated because the compound is oxidized during analysis. Lactic acid bacteria that utilize citrate, in particular Leuconostoc sp., often accumulate ALA in the culture medium (6, 8, 18); therefore, we studied the influence of ALA on the assay of diacetyl using headspace gas chromatography. Headspace gas chromatography was used for the study of mixed cultures of Leuconostoc mesenteroides ssp. cremoris and Luctococcus

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MONNET ET AL.

lactis ssp. lactis. Leuconostoc mesenteroides ssp. cremoris can transform citric acid in milk into diacetyl and acetoin at acid pH (3) but is not very acidifying and must thus be combined with an acidifying strain such as L. luctis ssp. lactis in order to produce a lactic curd. The production of diacetyl was investigated with respect to the redox potential and the presence of ALA. MATERIAL AND METHODS Strains and Culture Condltlons

Leuconostoc mesenteroides ssp. cremoris C1 and L. lactis ssp. lactis S3 were obtained from the Centre de Recherche International Andrk Gaillard-Yoplait (Ivry sur Seine, France). Strains C1 and S3 were grown at 30'C in MRS (5) and M17 media (19), respectively. Cells were harvested at the end of exponential growth phase, washed twice with .85% NaCl, and inoculated in UHT-sterilized skim milk (Candia, Lyon, France). For cultures that were not agitated, 20 sterile 35-ml flasks were filled with 30 ml of inoculated milk and hermetically sealed. Flasks were incubated without agitation at 23°C and sampled regularly during growth. Agitated fermentation was carried out in a 1-L cylindrical bottle containing .5 L of medium that was open to the atmosphere. A magnetic stirring bar was used at 250 rpm to ensure that the redox potential always remained >O mV. Chemical Analyseo

Samples removed during growth were rapidly frozen in 50% CaC12 (wt/vol) at -30'C and were analyzed immediately after thawing. Citrate was determined with an enzymatic method (Boehringer Mannheim, Mannheim, Germany). The ALA and the sum of diacetyl plus acetoin were assayed with the method of Westerfeld (23), as modified by Veringa et al. (21). The redox potential was measured during growth with a combination platinum electrode using the xerolyt Ag:AgC1 reference system (Ingold A.G., Urdorf, Switzerland). Preparation of ALA

The ester of ALA (a-methyl-a-acetoxyethyl acetoacetate; Oxford Chemicals Ltd., Journal of Dairy Science Vol. 77. No. 7, 1994

Brackley, Northants, England) was transformed to ALA, ethanol, and acetate by addition of two equivalents of NaOH. The saponification reaction was performed at 20'C for 30 min. Efficiency of the reaction was verified by assay of the ALA and ethanol produced. Chromatography

The chromatograph was equipped with a flame ionization detector @ani 8500; Dani S.p.A., Monza, Italy) with a 25-m CP WAX 52 CB capillary column (inner diameter, .32 mm; film thickness, 1.2 pm; Chrompack, Middelburg, The Netherlands). The chromatograph was connected to a Dani 3950 automatic headspace sampler. Operating parameters of the chromatograph were the following: injector and detector temperature, 200'C; N2 flow rate in the column, 1.3 mumin; column inlet split, 1:7; H2 flow rate in the detector, 25 mumin; and air flow rate in the detector, 200 mumin. The oven heating cycle was 60°C for 2 min, then a temperature increase at 30"C/min up to 95°C. followed by 95°C for 5 min, then a temperature increase at 30"C/min up to 15OoC, and finally 150'C for 1 min, for a total cycle time of 20 min. Parameters of the headspace sampler were the following: heating temperature, 85'C; heating time, 40 min; sample pressurization, 60 kPa; pressurization time, 20 s; and injection loop filling time, 15 s. Samples (10 ml each) were transferred to a 20-ml headspace vial @ani) that was hermetically sealed with crimped cap and an aluminum capsule. One vial was put in the headspace sampler every 20 min and then heated for 40 min before being automatically injected into the chromatograph. Acetaldehyde, acetone, ethanol, diacetyl, and acetoin were calibrated with external standards in UHT skim milk. The pH of the sample was adjusted in the headspace vial to the desired value with NaOH (3 M) and L-lactic acid (90g/L) under conditions of slow stirring with a magnetic bar. RESULTS AND DISCUSSION Analysls by Headapace Gas Chromatography

Acetaldehyde, acetone, ethanol, diacetyl, and acetoin were assayed in UHT skim milk; retention times and detection limits are in Ta-

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DIACETYL. MEASUREMENT IN FERMENTED MILK TABLE 1. CbaraCteri~tic~ of headSpaCe gaS Chromat~mphy response of volatile comp0unds.l Compound Acetaldehyde Acetone Ethanol Diacetyl Acetoin

Retention time

Detection limit

Coefficient of variation2

(h)

(mg/L)

(%)

2.49

.2 .2 1.O .2

7.13

10.0

2.5 7.7 1.6 3.4 3.2

.94 1.30

2.07

lVolatile compounds were added to UHT skim milk. Talculated from five repetitions.

ble 1. The detection limits of acetaldehyde and diacetyl were .2 mg/L, which was sufficiently low to enable use of the method with dairy products. Calibration was linear up to 100 mg/ L for ethanol, 250 mg/L for acetoin, and 10 mg/L for acetaldehyde, acetone, and diacetyl. The coefficients of variation for peak areas generally were low (1.6 to 7.7% for five repetitions). Effect of pH on Peak Areas. The five volatile compounds assayed were prepared in UHT

160-

Q (A

g

Y

(A

2

8l

120.

40 0

3

4

5

6

8 PH

7

91011

Figure 1. Effect of pH on the peak areas of 4.4 mgiL of acetaldehyde (A), 4.3 mgiL of acetone (e), 39.5 mgL of ethanol P), 4.0 m f l of diacetyl p),and 97.6 mgR. of acetoin (0). The compounds were prepared in UHT skim milk and analyzed by headspace chromatography.

I

PH Figure 2. Effect of pH on the transformation of .5 mM a-acetolactic acid (ALA) to diacetyl in UHT skim milk during incubation in the headspace sampler.

skim milk at different pH. The peak areas (Figure 1) showed practically no variation for acetone, ethanol, and acetoin. However, peak areas of acetaldehyde and diacetyl dropped at pH >7.0. This drop resulted from an irreversible chemical reaction of these compounds at basic pH, thus explaining why the headspace assay must be carried out at pH 17.0. Effect of pH on the Transformation of ALA to Diacetyl. The samples were heated to 85'C during incubation in the headspace sampler. In order to detect the possible release of diacetyl from ALA during the heating, ALA was prepared by saponification and added to UHT skim milk at the concentration of .5 mM at different pH (Figure 2). The samples were put in the headspace sampler as soon as the ALA was added. At pH 4.0, ~ 4 0 %of the ALA was transformed to diacetyl during incubation, and the remainder of the ALA was transformed to acetoin because ALA assays after headspace analysis showed that this compound disappeared totally and that the sum of diacetyl plus acetoin (assayed by gas chromatography) corresponded to the initial ALA concentration. Oxidative decarboxylation (diacetyl produced: initial ALA, expressed in millimoles) decreased as the pH increased, becoming 6.3% at Journal of Dairy Science Vol. 77, No. 7, 1994

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MONNET ET AL.

pH 7.0; the remainder of ALA was trans- TABLE 2. Effect of a prior treatment1 on the transformaformed to acetoin. Results were similar for tion of a-acetolacticacid (ALA) (.5 mM) to diacetyl during other ALA concentrations: at .I, .2, 1, 1.5, and the diacetyl assay. 2 mM, oxidative decarboxylation at pH 7.0 Conversion Prior of ALA was 7.4, 8.0, 7.4, 6.3, and 7.5%. respectively. treatment to diacetyl For the assay of diacetyl, headspace analysis Samples must thus be performed at pH 7.0to minimize the conversion of ALA to diacetyl. UHT Skim milk. pH 7.0 6.3 Effect of Oxygen on the Transformation of + 2.2 + 2.4 ALA lo Diucetyl. To eliminate air from the + 2.1 flask, samples adjusted to pH 7.0 and containing .5 mM ALA were bubbled with 10 ml of Lactic curd? neutralized 9.4 N2, sealed, and immersed in a boiling water to pH 7.0 + 2.2 bath for 15 min before being put in the head+ 2.2 space sampler. The results (Table 2) show that, + 1.9 for UHT skim milk and the fermented milk, UHT Part-skim milk, the treatment as described reduced the conver- pH 7.0 5.5 sion of ALA to diacetyl to a mean near 2%. + 2.5 + 2.4 Under these conditions, the overestimation of + 2.5 diacetyl is therefore considerably limited. When UHT part-skim milk or air-saturated Air-saturated' UHT 9.9 UHT skim milk (oxygenated by vigorous shak- skim milk, pH 7.0 + 2.6 ing in air) was used, the conversion of ALA to + 2.5 diacetyl was limited to a mean near 2.5% with + 2.6 the prior treatment. The treatment had no ef'In the prior treatment, the sample was degassed with fect on the peak areas of the five volatile 10 ml of N, sealed, and heated for IS min at 1WC; + = compounds prepared in UHT skim milk at pH with prior treatment; - = without prior treatment. 7.0 (data not shown). Heating at 1 W C also 'Obtained by culm of Lucfococcus &tis ssp. lactis prevented changes in the sample caused by the s3 in UHT skim milk. activity of lactic acid bacteria between thawing 3Milk was oxygenated by vigorous shaking in air for 1 and analysis. In samples containing ALA, the h so that the dissolved oxygen concentration in the real diacetyl concentration was calculated to d u m was between 80 and 100% of that of fully airaccount for the 2% oxidative decarboxylation saturattd medium. of ALA:

D = d - .02 x A where (expressed as millimolar) D = true diacetyl content, d = measured diacetyl, and A = ALA. Because most of the ALA was transformed to acetoin when the assay was performed at pH 7.0, the acetoin peak represented the concentration of acetoin and ALA. Thus, to obtain the exact concentration of acetoin in fermented milks, the total concentration of diacetyl plus acetoin was determined using the method of Westerfeld (23). as modified by Veringa et al. (21), from which the diacetyl concentration assayed by headspace chromatography was subtracted. Using a distillation assay, Jordan and Cogan (8) obtained a 2 to 7% oxidative decarboxylation of ALA to diacetyl, depending on the pH. Jansson and Pettersson (7) reported that the oxidative decarboxylation of ALA did not ocJournal of Dairy Science Vol. 77, No. 7, 1994

cur ( 4 % ) during distillation under nitrogen because of the very low redox potential of fermented milks. Veringa et al. (21), however, thought that this affirmation was not demonstrated and proposed distillation at pH 9.0, which would limit oxidative decarboxylation to 2%. The method proposed herein also enabled this conversion to be limited to 2% but has the additional advantage of being rapid and enables other volatile compounds that are present in fermented milks to be simultaneously assayed. Mixed Cultures of Leuc. mesenteroides u p . crewnoria and L. lactis up. lads

Unugitated Culture. Leuconostoc mesenteroides ssp. cremoris C1 and L luctis ssp.

DIACETYL MEASUREMENT IN FERMENTED MILK

lactis S3 were inoculated at .5 x 106 and 9.5 x 106 cfu/ml, respectively, in UHT skim milk at 23'C in unagitated conditions. Lactococcus lactis ssp. lactis was used to acidify the medium; this strain cannot consume citrate in the milk or produce diacetyl, acetoin, or ALA (data not shown). A low inoculation ratio of Leuconostoc compared with Lactococcus was used, which causes citrate to be used later during fermentation and, thus, to be consumed at low pH, 4 0 , that favors its transformation to diacetyl, acetoin, or ALA (3). The pH, the redox potential, the use of citrate, and the production of diacetyl, acetoin, ALA, ethanol, and acetone are shown in Figure 3A. Acetaldehyde production could not be detected in this culture (concentration <.2 m a ) , probably because Leuconostoc can transform acetaldehyde produced by Lactococcus to ethanol (13, 15). Ethanol was produced by the heterofermentation metabolism of Leuconostoc, and its maximal concentration was 1.06 mM. Acetone was not produced, and the residual value was that of UHT skim milk (.11 mM); mesophilic lactic acid bacteria are incapable of producing acetone (12). Citrate was used starting at pH 6.5, but acetoin production started only at pH 5.0. The production of acetoin was greater than that of ALA or of diacetyl, and its concentration (1.07 mM) was maximal when about 80% of the citrate had been consumed and decreased thereafter, probably because of the removal of acetoin reductase repression by citrate (14, 16). The ALA was produced at a maximal concentration of .18 mM at 17 h of culture. Changes in the redox potential were comparable with that described by Jansson and Pettersson 0; redox potential dropped to -345 mV after 2 h 0 of culture and then remained at ~ 3 0 mV. When diacetyl was assayed without special precautions (sample was not neutralized), the concentration reached .027 mM (2.3 mgiL), which is overestimated as a result of the interference by ALA in the assay of diacetyl. When the sample was neutralized, when the pretreatment was conducted (by degassing and boiling), and when the transformation of 2% of the ALA to diacetyl was considered, the concentration obtained (corresponding to the real concentration of diacetyl) was .002 mM (.2 m@), which is a value at the lower limit of detection. This result shows the importance of employing special precautions for the assay of diacetyl. At

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low redox potential, Leuconostoc thus produces acetoin and ALA, but not diacetyl. Agitated Culture. The aeration of a mixed culture should favor diacetyl production by enabling the oxidative decarboxylation of ALA during fermentation. The pH, substrates, and metabolites were followed during an agitated mixed culture of h u e . mesenteroides ssp. cremoris and L luctis ssp. lactis (Figure 3B). Agitation ensures a supply of oxygen, which maintains the redox potential at >O mV. Acidification was slower than in unagitated cultures because oxygen probably inhibited the growth of Lactococcus. The production of ethanol (.64 mM) was lower than in the unagitated culture. The production of ALA (.18 mM) was similar to that in the unagitated culture. Production of acetoin was lower (.38 mM) than in the unagitated culture (1.07 mM). which can be explained by the larger portion of citrate consumed before the pH reached 5.0 because of the lower rate of acidification in the agitated culture. Acetoin is produced from citrate only at acid pH. Diacetyl production was nevertheless significant, reaching .032 mM (2.8 mg/L) versus .002 mM (.2 mgL) in unagitated culture. This difference may be explained by the high redox potential in agitated culture, which enables a part of the ALA produced by Leuconostoc to undergo chemical oxidative decarboxylation to diacetyl. Two mechanisms have been proposed for the synthesis of diacetyl by aroma bacteria. Speckman and Collins (17) thought that diacetyl arose from the condensation of one molecule of acetyl-coenzyme A with active acetaldehyde (hydroxyethylthiamine pyrophosphate) by diacetyl synthase; de Man (4) suggested that diacetyl is produced by the chemical oxidative decarboxylation of ALA. The results in the present work are in good agreement with the second hypothesis. The lack of diacetyl production in the unagitated culture, even though acetoin and ALA were produced, can be explained by the negative value of the redox potential that prevented the production of diacetyl by the oxidative decarboxylation of ALA. Therefore, the ALA content must be considered for two reasons: 1) when ALA is not considered, the diacetyl concentration is overestimated] and, 2) when the redox potential is Journal of Dairy Science Vol. 77, No. 7, 1994

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MONNET ET AL.

high, ALA is partially transfomed to diacetyl, which explains the considerable increase in the diacetyl concentration in aerobic conditions (1, 9, 10, 11).

7.0

ACKNOWLEDGMENTS

We are grateful to P. Ramos (Yoplait, Ivry sur Seine, France) for the gift of the strains and

7.0

I

8 7

6.5

6 6.0 5

5 5.5

-

9 3 5

5.0

2 4.5

f

4

.

0

1

1

4.0

E i

0

.4

.2 0

C

0

Figure 3. Unagitated (A) and agitated (B) mixed cultures of Leuconostoc mesenteroides ssp. cremoris C1 and Lactococcus lactis ssp. lactis S3 inoculated at .5 x 106 and 9.5 x 1Oa cfidml. respectively, in UHT skim milk at 23'C. a) pH (0).redox potential Q, citrate (0); b) acetoin (01 a-acetolactic acid (ALA) (0).ethanol p),acetone @); and c) diacetyl without assay precautions 0,and diafetyl assayed at pH 7.0 after degassing and heating at 1 W C and by taking into account the oxidative decarboxylation of a-acttolactic acid C).

Journal of Dairy Science Vol. 77. No. 7. 1994

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DIACETYL MEASuRwlENT IN FERMENTED MILK

her participation in discussion. We thank Yoplait for its participation in financial support. Grant Number 9160558 Agrobio 2002 (French Ministry of Research and Technology). REFERENCES 1 Bassit. N., C. Y.Boquien, D. Pique, and G.Comeu. 1993. Effect of initial oxygen concentration on diacetyl and acetoin production by Lactococcus lactis subsp. lactis biovar. diaceiylactis. Appl. Environ. Microbiol. 59:1893. 2Cogan. T. M. 1975. Citrate utilization in milk by Leuconostoc cremoris and Streptococcus diacerylactis. J. Dairy Res. 42:139. 3Cogan. T. M., M. O’Dowd, and D. Mellerick. 1981. Effects of pH and sugar on acetoin production from citrate by Leuconostoc lactis. Appl. Environ. Microbiol. 41:l. 4de Man, J. C. 1959. The formation of diacetyl and acetoin from a-acetolactic acid. Recl. Trav. Chim. Pays-Bas Belg. 78:480. 5 de Man, J. C., M. Rogosa, and M. E. Sharpe. 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23:130. 6Hugenholtz. J., and M.J.C. Starrenburg. 1992. Diacetyl production by different strains of Lactococcw kactis subsp. kactis VBT. diacerykactis and Leuconostoc spp. Appl. Microbiol. Biotechnol. 38:17. 7 JBnsson, H., and H. E. Pettersson. 1977. Studies on the citric acid fermentation in lactic starter cultures with special interest in a-acetolactic acid. 2. Metabolic studies. Milchwissenschaft 32:587. 8 Jordan, K. N., and T. M. Cogan. 1988. Production of acetolactate by Streptococcus diaceryluctis and Leuconostoc spp. J. Dairy Res. 55:227. 9Kaneko. T., M. Takahashi, and H. Suzuki. 1990. Acetoin fermentation by citrate-positive Lactococcus luctis subsp. lactis 3022 grown aerobically in the presence of Hemin or Cu2+. Appl. Environ. Microbiol. 56:2644. 10Kaneko. T., Y.Watanabe, and H. Suzuki. 1990. Enhancement of diacetyl production by a diacetylresistant mutant of citrate-positive Lactococcus lactis ssp. lactis 3022 and by aerobic conditions of growth. J. Dairy Sci. 73:291.

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11 Kaneko, T., Y. Watanabe, and H. Suzuki. 1991. Differences between Lacrobocillus casei subsp. cmei 2206 and citrate-positive Lactococcus lactis subsp. lactis 3022 in the characteristics of diacetyl production. Appl. Environ. Microbiol. 57:3040. 12 Kcenan, T. W., D. D. Bill, and R. C. Lindsay. 1967. Acetone in milk cultures of lactic streptococci and Leuconostoc citrovorum. Can. J. Microbiol. 13:1118. 13 Keenan, T. W., R. C. Lindsay, and E. A. Day. 1966. Acetaldehyde utilization by Leuconostoc species. Appl. Microbiol. 14:802. I4Mellerick, D., and T. M. Cogan. 1981. Induction of some enzymes of citrate metabolism in Leuconostoc lactis and other heterofennentative lactic acid bacteria. J. Dauy Res. 48:497. 15 Schmitt. P., and C. Divibs. 1990. Effect of acetaldehyde on growth, substrates and products by Leuconostoc mesenteroides ssp. cremoris. Biotechnol. Rog. 6:421. 16Schmitt, P., and C. Divih. 1992. Effect of varying citrate levels on C4 compound formation and on enzyme levels in Leuconostoc mesenteroides subsp. cremoris grown in continuous culture. Appl. Microbiol. Biotechnol. 37:426. 17 Speckman, R. A,, and E. B. Collins. 1968. Diacetyl biosynthesis in Streptococcus diuceiyluctis and Leuconostoc citrovorum. J. Bacteriol. 95:174. 18 Starrenburg, M.J.C.. and J. Hugenholtz. 1991. Citrate fermentation by Lactococcu and Leuconostoc spp. Appl. Environ. Microbiol. 57:3535. 19Terzagh1, B. E.,and W. E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29:807. 20Th0mhill, P. J., and T. M. Cogan. 1984. Use O f gasliquid chromatography to determine the end products of growth of lactic acid bacteria. Appl. Environ. Microbiol. 47:1250. 21 Veringa, H. A,. E. H. Verburg, and J. Stadhouders. 1984. Determination of diacetyl in dairy products containing a-acetolactic acid. Neth. Milk Dairy J. 38: 251. 22Walsh. B., and T. M. Cogan. 1974. Separation and estimation of diacetyl and acetoin in milk. J. Dairy Res. 41:25. 23 Westerfeld, W.W. 1945. A colorimetric determination of blood acetoin. J. Biol. Chem. 16:495.

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