Z Lebensm Unters Forsch (1983) 176:440M43

LebensmittelUntersuchung und-Forschung
@J. F. Bergmann Verlag 1983

Zeitschrift flJr

HPLC Analysis of Organic Acids in Lactic Acid Fermented Vegetables

R o l f A n d e r s s o n and Bruno H e d l u n d SIK - The Swedish Food Institute, Box 5401, S-402 29 G6teborg, Sweden

Hochdruck-fliissigchromatographische Analyse der organischen Siiuren von milchsauren Gemiisen Zusammenfassung. Ein H P L C - V e r f a h r e n zur Analyse von organischen S/iuren in Gemfisen nach Milchsfiuregfirung wird beschrieben. Eine H P X - 8 7 - K o l o n n e mit 0.013 m - H 2 S O 4 als mobile Phase wurde verwendet. Die Ergebnisse der T r e n n u n g y o n Oxalsfiure, Weins/iure, )~pfels~iure, Bernsteinsfiure, Milchsfiure, Ameisens~iure, Essigs~iure, Propions/iure u n d Butters~iure werden wiedergegeben. D u r c h Zuckerarten verursachte St6rungen werden besprochen. Summary. A n H P L C technique is described for the
analysis o f organic acids in lactic acid fermented vegetables. A n Aminex H P X - 8 7 c o l u m n with 0.013 M H2SO4 as mobile phase was used. Separation data are presented for oxalic, citric, tartaric, malic, succinic, lactic, formic, acetic, propionic, and butyric acids. Interference problems f r o m sugars are also reported.

The present investigation was carried out in order to develop an H P L C - m e t h o d for the analysis o f organic acids, primarily p r o d u c e d by lactic acid bacteria in fermented vegetables. Analytical results are presented for oxalic, citric, tartaric, malic, succinic, lactic, formic, acetic, propionic and butyric acids.

Materials and Methods

Sample Preparation

Aqueous solutions of organic acids were analyzed directly by HPLC.Cell- and particle-free solutions from bacteriological cultivations and vegetable fermentations were obtained by centrifugation of the samples followed by a sterile filtration using a membrane filter (0.22 gin; Millipore Corp., Bedford, Mass.). The solutions obtained were analyzed directly by the HPLC method, as described below.
Apparatus and Operating Conditions

Introduction
Organic acids occur in fermented p r o d u c t s as a result o f hydrolysis, biochemical metabolism, and microbial activity. Quantitative determination o f these acids is i m p o r t a n t in fermented foods for technical, nutritional, sensorial, and microbial reasons. Titrimetric methods, gas c h r o m a t o g r a p h y , colorimetric analysis and enzymatic m e t h o d s are examples o f techniques that are used for analyses o f organic acids in foods [1-4]. However, because o f simplicity and speed o f analysis, the H P L C technique is an attractive method, which requires a m i n i m u m o f sample preparation prior to separation and permits quantitative determination o f organic acids in a short time [571.

The HPLC system used included a Milton Roy 711 pump (Milton Roy, U.K., Ltd.), a Valco CV-6-UHP injection valve, and an R 401 differential refraetometer (Waters Assoc., USA). A 300 7.8 mm stainless-steel column, packed with Aminex HPX-87 (Bio-Rad Lab., Calif., USA), and maintained at constant temperature by a water jacket was used as the separation column. In order to protect the separation column from contaminants, it was equipped with a MicroGuard pre-column (Bio-Rad). The conditions for the HPLC-analysis were:mobile phase: 0.013 M-H2SO4, flow rate: 0.5 ml/min, column temperature: 40 C.
Calibration Graphs and Calculations

Standard solutions of ten organic acids in water were analyzed individually and mixed in concentration of 0.05 to 4.0% (w/w). The resulting peak heights were determined and plotted against the concentration of the acids. The slope of each individual line is the response factor for each acid, and the recti linear correlation coefficient is a measure of the analytical precision.
Cultivation of Micro-Organism

Twenty-four isolates of lactic acid bacteria were cultivated in 300-mlflasks containing 150 ml of MRS-broth (Oxoid Ltd., England). The cultures were incubated at 20 C, and samples were withdrawn repeatedly for the determination of organic aicds.

R. Andersson and B. Hedlund: HPLC Analysis of Organic Acids

Table 1. Data for aqueous calibration standards
23

441

Acid

Retention time min 7.0 8.4 9.0 10.2 12.8 13.1 14.4 15.6 18.7 23.3

Response factor, slope mm/% 797 1,632 1,397 1,398 831 810 679 644 563 316

Correlation coefficients, r 2

Oxalic Citric Tartaric Malic Succinic Lactic Formic Acetic Propionic Butyric

0.9984 ~ 0.9998 0.9997 0.9999 1.0000 0.9999 0.9996 1.0000 0.9999 0.9980

i
, , | |

r 2 is determined from 0.05 to 2.0%

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Fermentation of Carrots
Carrots were fermented, using Lactobacillus plantarum as a starter culture (Chr. Hansen's Lab., Copenhagen, Denmark) in the following way. The carrots were washed, sliced, and placed in a glass jar together with 1.5 % (w/w) NaC1 and inoculated with the starter culture to a final concentration of about 5 10 6 bacteria/g. The fermentation was performed at 20 C. Samples were withdrawn at regular intervals for the determination of lactic bacteria, pH and organic acids.

10 TIME

15 (min)

20

25

Fig. 1. Chromatogram of a standard solution containing the following acids: oxalic (1), citric (2), tartaric (3), malic (4), succinic (5), lactic (6), formic (7), acetic (8), propionic (9), and butyric (10)

Results and Discussion

Standard Calibration Graphs

Aqueous standards of individual acids were chromatographed separately and mixed to determine the retention times and the response factors of each acid (Table 1). Figure 1 is a standard chromatogram of a mixture of the acids.
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The calibration graphs for each acid are shown in Fig. 2. The peak height values are average values from at least three determinations. The calibration graph data are presented in Table 1. The correlation coefficients are close to unity in the concentration range studied with one exception, oxalic acid. The slope of the graph for this acid is significantly nonlinear at concentrations above 2%. This might be due to nonlinearity for the detector response in this region or to decarboxylation of the acid.
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Fig. 2. Calibration graphs for organic acids

442 Table 2. Production of organic acids from 24 isolates cultivated in MRS broth at 20 C for 3 days Acid Oxalic Citric Tartaric . Malic Succinic Lactic Formic Acetic Propionic Butyric Lowest conc. % 0.23 0.06 . 0.02 0 0.57 0 0.17 0 0 . Highestconc. Meanvalue % % 0.93 0.41 . 0.06 0 1.25 0 0.29 0 0 0.52 0.12 0.04 0.84 0.23

R. Andersson and B. Hedlund: HPLC Analysis of Organic Acids Table3. Retention times for sugars determined on an Aminex HPX-87 column Sugar Maltose, Sucrose Glucose Xylose Fructose Arabinose Retention time rain 7.5 9.0 9.8 10.0 10.8

See text below concerninginterference from sugars

Results and Chromatograms for Lactic Acid Bacteria

The H P L C technique was used to determine organic acids produced by isolated lactic acid bacteria cultivated in MRS broth. In this way it was possible to rapidly characterize a number of isolates with respect to their production of organic acids. The individual acids were qualitatively determined by comparison with standard chromatograms and by doping the samples with reference acids. Table 2 shows the results from 24 isolated lactic acid bacteria cultivars. It is obvious that there is a great variation in the acid profiles from different bacteria.

T I M E (min)

Interference Problems with Sugars

It should, however, be pointed out that sugars may disturb the analysis, since the retention times for some sugars are the same as for acids (see Table 3). Thus, glucose and tartaric acid appear as one peak when determined together. Furthermore, fructose interferes with the determination of malic acid. At equal concentrations the peak is split, but when the fructose content is higher than the malic acid content, one peak is not split and this makes it impossible to determine the acid, These results show that interference problems from sugars must be taken into account when analyzing organic acids on an HPX-87 column. On the other hand, it is possible to determine organic acids and non-interfering sugars at the same time.

Fig.3. Chromatogram from lactic acid fermented carrots. NaC1 (salt), maltoseand sucrose(S0, citricacid (2), tartaric acid (3) and/or glucose ($3), malic acid (4), lactic acid (6) and acetic acid (8)

Table 4. Content of organic acids in controlled fermentation of carrots using L. plantarumas a starter culture Acid Concentration % Fermentation time (days) 0 Citric Malic Lactic Acetic 0.54 0A2 0 0 1 0.61 0.46 0 0 2 0.20 0.73 0.21 0.07 3 0.21 0.90 0.36 0.12 6 0.25 0.92 0.57 0.12

Organic Acids in Fermented Carrots - an Application Example

A representative chromatogram from fermented carrots is presented in Fig. 3, and the results are summarized in Table 4. Normally, fermented vegetables contain 1.5-2.0% acids. Fermentation processing of vegetables often in-

cludes determinations of pH and a titrimetric value of the total acid content. Lactic and acetic acids are considered to be the most important acids. It is, however, important to know the exact composition of all the acids present. Further studies of lactic acid fermented vegetables concerning the influence of the organic acids produced on the sensory quality, nutritional value and storage stability are therefore needed.
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R. Andersson and B. Hedlund: HPLC Analysis of Organic Acids I n this paper we have s h o w n that H P L C is a fast a n d simple m e t h o d for the d e t e r m i n a t i o n of organic acids in fermented vegetables. However, the possibility of interference from sugars m u s t be considered.

443 2. Bohannon TE, Manius G, Mamaril F, Li Wen LF (1978)J Chrom Sci 16:28 3. Drucker DB (1981) J Chrom 208:279 4. Siebert F (1974) In: Bergmeyer HU (ed) Methods of enzymatic analysis. 2nd ed., vol 2, p 1570-1573 Academic Press, New York 5. Marsili RT, Ostapenko H, SimmonsRE, Green DE (1981)J Food Sci 46:52 6. Palmer JK, List DM (1973) J Agr Food Chem 21:903 7. Turkelson VT, Richards M (1978) Anal Chem 50:1420

Acknowledgments. We gratefully acknowledge the technical assistance of Inger /~kesson and Petr Adamek.

References
1. AOAC (1975) OfficiaImethods of analysis, 12th ed. (Horwitz W ed). Association of Official Agricultural Chemists, Washington

RecNved January25, 1983