The use of ionic chromatography in determining the contamination of apple juice by lactic acid

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contamination of apple juice by lactic acid

Maciej Wojtczak, Aneta Antczak, Malgorzata Przybyt

To cite this version:

Maciej Wojtczak, Aneta Antczak, Malgorzata Przybyt. The use of ionic chromatography in determin- ing the contamination of apple juice by lactic acid. Food Additives and Contaminants, 2010, 27 (06), pp.817-824. �10.1080/19440041003664143�. �hal-00593894�

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The use of ionic chromatography in determining the contamination of apple juice by lactic acid

Journal: Food Additives and Contaminants Manuscript ID: TFAC-2009-365.R1

Manuscript Type: Original Research Paper Date Submitted by the

Author: 26-Jan-2010

Complete List of Authors: Wojtczak, Maciej; Technical University of Lodz, Institut of Chemical Technology of Food

Antczak, Aneta; Technical University of Lodz, Institut of Chemical Technology of Food

Przybyt, Małgorzata; Technical University of Lodz, Institute of General Food Chemistry

Methods/Techniques: Chromatographic analysis, Screening - biosensor Additives/Contaminants: Process contaminants

Food Types: Fruit juices

For Peer Review Only

The use of ionic chromatography in determining the contamination of apple juice by lactic acid

Maciej Wojtczak

, Aneta Antczak

, Małgorzata Przybyt

a

Technical University of Lodz, Institute of Chemical Technology of Food, 90-924 Lodz, Stefanowskiego 4/10, Poland

b

Technical University of Lodz, Institute of General Food Chemistry, 90-924 Lodz, Stefanowskiego 4/10, Poland

Corresponding author:- e-mail: maciej.wojtczak@plodz.pl

Abstract

In this study, high performance anion exchange chromatography (HPAEC) with conductometric detection was used for determining lactic acid content of apple juice subjected to fermentation with the strains of Lactobacillus brevis and Lactobacillus plantarum, obtained from a collection, at 20 and 30

C. At the same time, lactate content was determined by means of enzymatic tests and biosensors. Lactic acid concentrations determined by the chromatographic method are similar to those obtained during analysis by enzymatic tests. However, acid concentrations determined by means of biosensors substantially diverge from these results.

Keywords: lactic acid, lactic fermentation, ion chromatography, apple juice

Introduction

Lactic fermentation is one of the key processes in the food industry. It helps to provide products with the right aroma, taste and consistency, increases their shelf life, improves their nutritive value, and sometimes also contributes to probiotic properties of the final product (Feord 2002). Apart from the food industry, it is also widely used in the chemical, pharmaceutical, cosmetic, leather, and textile industries (Rojan et al. 2007, Zhang et al. 2007).

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Lactic fermentation is a process of anaerobic conversion of saccharides to lactic acid. It is one of the ways of obtaining the energy necessary for maintaining the life processes of microorganisms under anaerobic conditions. Lactic fermentation can be either homofermentative, where two molecules of lactic acid are formed from one molecule of glucose (Li et al. 2004), or heterofermentative, where one molecule of glucose is converted to one molecule of lactic acid, one molecule of acetic acid (under aerobic conditions) or one molecule of ethanol (under anaerobic conditions) and one molecule of carbon dioxide (Kandler, 1983).

There are two optically active stereoisomers of lactic acid: isomer L(+) and isomer D(-). Lactic acid may be produced by microbiological fermentation, which leads to either L- or D- lactic acid or racemate, depending on microorganisms, substrates and fermentation conditions employed in the production process. Renewable resources including sugars, starch and lignocellulose are abundant substrates for fermentative production. A racemate of both isomers is produced by chemical synthesis. The most commonly used synthetic method for chemical production of lactic acid is the hydrolysis of lactonitrile derived from acetaldehyde and hydrogen cyanide, which are produced by petrochemical process (Oh et al. 2005, Reddy et al. 2008, Zhang et al. 2007).

Lactic acid can be produced using bacteria or fungi such as Rhizopus oryzae in submerged culture. Lactic acid producing bacteria (LAB) have received wide interest because of their high growth rate and product yield (Chopin, 1993, Zhang et al. 2007). LAB are generally mesophilic but they can grow at temperatures as low as 5°C or as high as 45°C. Majority of strains of LAB grow at pH 4,0 – 4,5, but some are active at pH 3,2 and others at pH 9,6 (Caplice & Fitzerald, 1999).

High production of lactic acid is normally achieved by selecting suitable microbial strains in the process of fermentation. Strains of Lactobacillus can be homofermentative or heterofermentative. However, the strains of Lactobacillus brevis used in this study belong to a group of lactic acid bacteria obligately heterofermentative which ferment hexose in the pentose phosphoketolaze pathway. Lactobacillus plantarum is a group of facultatively heterofermentative microorganisms that metabolize hexose in the Embden-Meyerhof-Parnas pathway and pentose and some other substrates in the pentose phosphoketolaze pathway (Stiles & Holzapfel, 1997; Libudzisz et al. 2008). Among lactic fermentation bacteria, typical homofermentative species include:

Lactobacillus delbrueckii, L. casei, L. acidophilus, L. bulgaricus (Hickey et al. 1986, Vodnar &

Socaciu, 2008). While L. delbrueckii uses glucose, fructose, and sucrose for the production of lactic acid by homofermentation, the other bacteria additionally use lactose and galactose (Chang et al.

1999, Vodnar & Socaciu, 2008).

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Lactic acid bacteria are used in the food industry for several reasons. Their growth lowers both the carbohydrate content of the foods that they ferment, and the pH due to lactic acid production. It is this acidification process which is one of the most desirable side-effects of their growth. The pH may drop to as low as 4.0, low enough to inhibit the growth of most other microorganisms including the most common human pathogens, thus allowing these foods prolonged shelf life (Libudzisz et al. 2008). The desirable characteristics of industrial microorganisms are their ability to rapidly and completely ferment cheap raw materials, requiring minimal amount of nitrogenous substances, providing high yields of preferred stereo specific lactic acid under conditions of low pH and high temperature production of low amounts of cell mass and negligible amounts of other by-products (Narayanan et. al. 2004).

Too high a content of lactic acid can be critical in the fruit and vegetable industry, and in particular in the juice industry, where a major problem for fruit juice manufacturers is product spoilage by lactic acid bacteria which cause undesirable fermentation processes (Trifirò et al. 1997).

Their presence is often detected after 4-5 days or later, which leads to contamination of a large volume of juice at various stages of the manufacturing process. This in turn may result in decreasing the quality of the final product or even in rendering it undrinkable and necessitating its disposal. Thus, the bacteria cause immense financial losses in manufacturing plants. The Code of Practice of AIJN (European Fruit Juice Association) established the maximum permissible concentration of lactates in fruit juices, which is 0.5 g l

(Trifirò et al. 1997).

The determination of lactic acid content in fruit juices may be conducted by a variety of methods. The most selective and sensitive methods are enzymatic tests, which, however, involve a substantial amount of work, as it is necessary to perform a separate analysis for every measured component, and the measurement itself requires a tight time schedule to be observed painstakingly (Cunha et al. 2002, Trifirò et al. 1997). Analysis of lactic acid content may be also conducted by the chromatographic method using different separation techniques and detectors like HPLC/UV-VIS (Vodnar & Socaciu C. 2008) or with the use of biosensors (Przybyt & Biernasiak 2008). However, recent studies have shown the HPLC method with UV-VIS detection to be inexact as it results in significant differences in the determined concentrations as compared to results obtained by means of biosensors, which in most cases substantially overstated the result largely due to insufficient separation of the lactic acid peak (Vodnar & Socaciu, 2008).

Therefore, the authors decided to use high performance anion exchange chromatography with conductometric detection for determining lactic acid concentration. Ion chromatography is an instrumental technique used for the separation and determination of organic and inorganic anions and cations and other substances after their prior conversion to the ion form. Its advantages include:

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quick analysis, efficiency, high sensitivity and very good repeatability of the results. Furthermore, the short measurement time and small samples required for analysis make it possible to detect and quantify an analyte at the level of µg l

. Using a single analytical technique, it is possible to obtain complete information on the ion composition of the analyzed sample. (Fritz, 2000, Michalski, 2005).

Materials and methods

The material for analysis was apple juice containing 11.2% of dry matter prepared by diluting concentrated apple juice containing 68.9% of dry matter. The concentrate was supplied by the company VINKON S.A., and the study was conducted as part of the collective research project Quali-Juice “Quality assurance and development of an early warning system for microbial spoilage for the European fruit juice industry”.

Apple juice was subjected to fermentation with the strains of Lactobacillus plantarum (ŁOCK 0864) and Lactobacillus brevis (ŁOCK 0845). 699 mL of dissolved and sterilized juice was inoculated with 1 mL of bacteria suspension. The study consisted of measurement of changes in the concentration of lactic acid by the high performance anion exchange chromatography method with conductometric detection. In all the assays, for comparative reasons, the concentration of L-lactic acid anions was quantified by means of biosensors: Biosen C Line sport (EKF, Germany) and LactatProfi 3000 (ABT, Germany) as well as enzymatic tests Megazyme (Ireland), which were also used for determining the concentration of D-lactic acid anions. Apple juice fermentation was conducted at two temperatures: 20 and 30

C. The samples were taken at 0, 24, 48, 72, 96 and 168 hours of the fermentation process. In order to stop the fermentation each sample was immediately frozen and stored until the moment of analyzing.

For enzymatic assay (spectrophotometric) of lactate 1 mL of sample was added to 100 mg of polyvinylpyrrolidone (PVPP), shaked vigorously, left for 5 minutes and then centrifuged at 14 000 rpm for 15 minutes. The assay was done according to the procedure given by the producer of kits.

Each assay was triplicated. The absorbance was measured at 340 nm in disposable PMMA Plastibrand® cuvettes (Sigma, Germany) with UV spectrophotometer Nicolet Evolution 300 from Thermo Electron Corporation (MA, USA).

The assay of lactate concentration with the use of biosensors was done according to the producer manual with standards and buffers purchased from them.

Chromatographic analysis was performed with an ion chromatograph DIONEX ICS-3000 with a conductivity detector. Separation was conducted with the use of the column Ion Pac AS 11- HC 4x250mm and a conductivity suppressor ASRS- ULTRA II 4mm produced by the company

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DIONEX (USA). Chromatographic analysis was recorded with the use of the programme Chromeleon

, version 6.70.

Juice samples were diluted with distilled water (18MΩ) at a ratio of 1:10, and before injection they were filtered through membrane filters with a pore diameter of 0.45 µm.

The separation of acids contained in apple juices was conducted in the following conditions: flow:

1.5 ml min

; column temperature: 30

C; injection: 10µL. Gradient separation was used with the following eluents being used as the mobile phase: 5 mM NaOH (eluent 1), 100 mM NaOH (eluent 2) and distilled water 18MΩ (eluent 3) according to the following time and composition program (eluent 1, 2 and 3 were expressed as percent [v/v]): -5 to 0 min, 20, 0 and 80; 0 to 17 min, 90, 0 and 10; 17 to 21 min, 0, 15 and 95; 21 to 30 min, 0, 30 and 70; 30 to 31 min, 20, 0 and 80.

A validation procedure was conducted for the method of quantifying lactic acid standard (Sigma-Aldrich

, Europe) content by means of HPAEC with conductivity detection by determining two ranges of linearity: from 0.001 to 0.1 mg ml

, where the equation was f(c) = 25,196 c; and from 0.1 to 0.6 mg ml

, where the equation was f(c) = 19,021 c + 0,684. The lower limit of detection was established at LOD = 60 mg ml

and the limit of quantification at LOQ = 180 mg ml

1

. The precision of the method was expressed as standard deviation of repeatability S

at 0.0045 mg ml

and the repeatability limit r at 0.012 mg ml

.

Discussion

Sample chromatograms of apple juice at various temperatures during fermentation conducted with the use of the strains of Lactobacillus brevis and Lactobacillus plantarum at various stages of the fermentation process are presented in Figure 1 and Figure 2.

Results for lactic acid content obtained with the use of HPAEC, enzymatic tests and biosensors on particular days of fermentation at both temperatures are shown in Figure 3a and 3b for Lactobacillus brevis at 20 and 30

C, respectively, and Figure 4a and 4b for Lactobacillus plantarum at 20 and 30

C. All the analyzed samples of apple juice contained lactic acid. The concentration of L-lactic acid anions increased during fermentation conducted at both 20 and 30

C but significantly higher content of lactic acid was noted at 30

C. A greater increase in lactic acid was observed in the case of fermentation conducted with the use of Lactobacillus brevis strains. At 20

C, an increase in acid concentration was observed from 0.10 g l

at 0 h to 0.22 g l

at 96 h of fermentation, while at 30

C from 0.13 to 0.30 g l

at 0h and 96h, respectively. At the same time, results obtained by means of enzymatic tests were at a similar level: from 0.12 to 0.19 g l

(at 0 and 96 h of fermentation) at 20

C and from 0.10 to 0.25 g l

at 30

C, respectively. Biosensors were used only to determine the level of the L-lactic acid isomer. In the case of the ABT biosensor, the concentration of L-lactates in

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samples inoculated with Lactobacillus brevis was 0.05 g l

at 0 h to 0.16 g l

at 96 h (at 20

C) and was similar to that of L-lactates quantified by means of enzymatic tests, which were 0.06 – 0.13 g l

1

, respectively. The EKF biosensor, as compared to enzymatic tests, produced lower values for L- lactic acid, which were from 0.03 g l

at 0 h to 0.1 g l

at 96 h of fermentation at 20

C, while at 30

C these were from 0.02 to 0.16 g l

, respectively. In the case of fermentation by Lactobacillus brevis it was not possible to determine lactic acid content at 168 h, as the samples became moldy.

Lactobacillus plantarum strains caused a much lower increase in the acid at either temperature. In the case of juices inoculated with Lactobacillus plantarum at 20

C, no significant increase in lactic acid concentration was observed. Acid content produced by Lactobacillus plantarum measured by means of the HPAEC technique and the ABT biosensor was similar and corresponded to L-lactic acid content determined by means of enzymatic tests. There wos no significant increase of lactic acid content during fermentation at 20

C. At 30

C the concentration of lactic acid measured by HPAEC increased from 0.11 to 0.18 g l

. In the case of the EKF biosensor, at both temperatures acid concentration was below the quantification limit of the apparatus.

Enzymatic tests made it possible to determine particular isomers of lactic acid: D- and L- lactates but during the process of fermentation, only the concentration of the L-isomer changed.

Enzymatic tests are a popular method for determining the content of various components that is used in the industry; however, they are a time consuming technique as it is necessary to perform a separate analysis for every component to be quantified. The use of biosensors also involves some inconvenience, and during analyses it was observed that PVPP sample discoloring at low concentrations led to overestimated results in the case of ABT. During EKF analyses, subsequent repetitions of determinations resulted in increasing figures (Przybyt & Biernasiak 2008). The HPAEC technique with conductivity detection used in the research produces results that are comparable with popularly used reliable enzymatic tests. It is a simple technique which does not take much time or a large number of tests for analysis. HPAEC seems to be an extremely useful method for exact determination of lactic acid content in fruit juices. However, due to the high cost of an ion chromatograph, it is hard to unequivocally decide the viability of this method for on-line control of the technological process. However, it seems to be a very good, exact, and sensitive method for periodical control of organic acid concentration in fruit juices.

Conclusions

On the basis of the results obtained, it was found that:

1. During fermentation caused by lactic bacteria (at both 20 and 30°C), L-lactic acid content

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increases, while the concentration of D-lactates remains at the same level. However, in the case fermentation by Lactobacillus brevis the increase of lactic acid was significantly higher than in the case of Lactobacillus plantarum.

2. High performance anion exchange chromatography with conductivity detection makes it possible to precisely determine lactic acid in juices as it gives results comparable to the popularly used reliable enzymatic tests, and thus it allows for on-line monitoring of contamination of fruit juices with lactic acid. Additionally, the simplicity of the method, the small volume of samples and short time of analysis to obtain information about the ion composition of the material studied, makes the HPAEC technique particularly suitable for periodic control of organic acid concentration in fruit juices.

3. The final choice of the device (HPAEC, enzymatic tests or biosensor) by the future user (juice producing company) should be determined by its particular demands (simplicity of the measurements, possibility of usage at line) and economical impact (price of the device and consumables).

Acknowledgements.

This work was supported partially from the EU-FP6 project Quali - Juice /COLL-CT-2005-012461/ and Ministry of Science and Higher Education in Poland.

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Przybyt M., Biernasiak J.: Zastosowanie biosensorów do oznaczania mleczanów w owocowych sokach komercyjnych i koncentratach, śywność. Nauka. Technologia. Jakość, 5(60):168-177, 2008 (in Polish).

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Figure Captions:

Figure 1. Sample chromatograms of apple juice inoculated with Lactobacillus brevis after 0h, 48h and 96h.

Figure 2. Sample chromatograms of apple juice inoculated with Lactobacillus plantarum after 0h, 48h and 96h.

Figure 3a. Lactic acid content determined by means of ion chromatography, enzymatic assays and biosensors (ABT and EKF) during fermentation conducted with Lactobacillus brevis strains at 20°C.

Figure 3b. Lactic acid content determined by means of ion chromatography, enzymatic assays and biosensors (ABT and EKF) during fermentation conducted with Lactobacillus brevis strains at 30°C.

Figure 4a. Lactic acid content determined by means of ion chromatography, enzymatic tests and biosensors (ABT and EKF) during fermentation conducted with Lactobacillus plantarum strains at 20°C.

Figure 4b. Lactic acid content determined by means of ion chromatography, enzymatic tests and biosensors (ABT and EKF) during fermentation conducted with Lactobacillus plantarum strains at 30°C.

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Figure 1. Sample chromatograms of apple juice inoculated with Lactobacillus brevis after 0h, 48h and 96h.

230x481mm (96 x 96 DPI)

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Figure 2. Sample chromatograms of apple juice inoculated with Lactobacillus plantarum after 0h, 48h and 96h.

228x474mm (96 x 96 DPI)

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Figure 3a. Lactic acid content determined by means of ion chromatography, enzymatic assays and biosensors (ABT and EKF) during fermentation conducted with Lactobacillus brevis strains at 20°C.

259x169mm (96 x 96 DPI)

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Figure 3b. Lactic acid content determined by means of ion chromatography, enzymatic assays and biosensors (ABT and EKF) during fermentation conducted with Lactobacillus brevis strains at 30°C.

259x169mm (96 x 96 DPI)

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Figure 4a. Lactic acid content determined by means of ion chromatography, enzymatic tests and biosensors (ABT and EKF) during fermentation conducted with Lactobacillus plantarum strains at

20°C.

253x169mm (96 x 96 DPI)

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Figure 4b. Lactic acid content determined by means of ion chromatography, enzymatic tests and biosensors (ABT and EKF) during fermentation conducted with Lactobacillus plantarum strains at

30°C.

258x169mm (96 x 96 DPI)

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