The use of ionic chromatography in determining the

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The use of ionic chromatography in determining thecontamination 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

http://mc.manuscriptcentral.com/tfac Email: [email protected]

Food Additives and Contaminants

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The use of ionic chromatography in determining the contamination of apple

juice by lactic acid

Maciej Wojtczaka, Aneta Antczaka, Małgorzata Przybytb

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

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

Corresponding author:- e-mail: [email protected]

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 300C. 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-1 (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-1. 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 300C. 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-1; column temperature: 300C; 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-1, where the equation was f(c) = 25,196 c; and

from 0.1 to 0.6 mg ml-1, where the equation was f(c) = 19,021 c + 0,684. The lower limit of

detection was established at LOD = 60 mg ml-1 and the limit of quantification at LOQ = 180 mg ml-

1. The precision of the method was expressed as standard deviation of repeatability Sr at 0.0045 mg

ml-1 and the repeatability limit r at 0.012 mg ml-1.

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 300C, respectively, and Figure 4a and 4b for Lactobacillus plantarum

at 20 and 300C. 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 300C but significantly

higher content of lactic acid was noted at 300C. A greater increase in lactic acid was observed in the

case of fermentation conducted with the use of Lactobacillus brevis strains. At 200C, an increase in

acid concentration was observed from 0.10 g l-1 at 0 h to 0.22 g l-1 at 96 h of fermentation, while at

300C from 0.13 to 0.30 g l-1 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-1 (at 0 and 96 h of fermentation) at

200C and from 0.10 to 0.25 g l-1 at 300C, 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-1 at 0 h to 0.16 g l-1 at 96 h (at 200C) 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-1 at 0 h to 0.1 g l-1 at 96 h of fermentation at 200C, while at

300C these were from 0.02 to 0.16 g l-1, 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 200C, 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 200C. At 300C the concentration of

lactic acid measured by HPAEC increased from 0.11 to 0.18 g l-1. 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.

References:

Caplice E., Fitzgerald G. F.: Food fermrntations: role of microorganisms in food production and

preservation; International Journal of Food Microbiology 50: 131 – 149, 1999.

Chang D.E., Jung H.C., Rhee J.S., Pan J.G.: Homofermentative Production of D- or L-Lactate in

Metabolically Engineered Escherichia coli RR1, Applied and Environmental Microbiology, 65(4):

1384-1389, 1999.

Chopin A.: Organization and regulation of genes for amino acid biosynthesis in lactic acid bacteria;

FEMS Microbiol. Rev. 12: 21–38, 1993.

Code of Practice for Evaluation of Fruit and Vegetable Juices. Association of the Industries of Juice

and Nectars from Fruits and Vegetables of the Europe Economic Community, Brussels, Belgium,

2001.

Cunha S. C., Fernandes J. O., Ferreira I. M.P.L.V.O.:”HPLC/UV determination of organic acids in

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fruit juices and nectars”, European Food Research and Technology, 214: 67–71, 2002.

Feord J.: Lactic acid bacteria in a changing legislative environment; Antonie van Leeuwenhoek, 82:

353–360, 2002.

Fritz J.S., Gjerde D.T.: Ion Chromatography, 3rd edition, Wiley-VCH, ISBN 3-527-29914-9.

Hickey M. W., Hillier A.J., Jago G.R.: Transport and Metabolism of Lactose, Glucose, and

Galactose in Homofermentative Lactobacilli, Applied and Environmental Microbiology 51(4): 825-

831, 1986.

Kandler O.: Carbohydrate metabolizm in lactic acid bacteria, Antonie van Leeuwenhoek, 49: 209-

224, 1983.

Li H., Mustacchi R., Knowles C. J., Skibar W., Sunderland G., Dalrymple I, Jackman S.A.: An

electrokinetic bioreactor: using direct electric current for enhanced lactic acid fermentation and

product recovery; Tetrahedron, 60: 655-661, 2004.

Libudzisz Z., Kowal K., śakowska Z.: Mikrobiologia techniczna. Mikroorganizmy w

biotechnologii, ochronie środowiska i produkcji Ŝywności t.2, Wydawnictwo Naukowe PWN, ISBN

978-83-01-15223-0 (in Polish).

Michalski R.: Chromatografia jonowa, podstawy i zastosowania; Wydawnictwa Naukowo-

Techniczne, Warszawa 2005 (in Polish).

Narayanan N., Roychoudhury P. K., Srivastava A.: L (+) lactic acid fermentation and its product

polymerization; Electronic Journal of Biotechnology, 8: 167–179, 2004.

Oh H., Wee Y. J., Yun J. S., Han S. H., Jung S., Ryu H. W.: Lactic acid production from agricultural

resources as cheap raw materials, Bioresource technology, 96: 1492-1498, 2005.

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).

Reddy G.,. Altaf Md, Naveena B.J., Venkateshwar M., Vijay Kumar E.: Amylolytic bacterial lactic

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acid fermentation — A review, Biotechnology Advances, 26: 22-34, 2008.

Rojan P. John, Rajeev K. Sukumaran, K. Madhavan Nampoothiri, Ashok Pandey: Statistical

optimization of simultaneous saccharification and L(+)-lactic acid fermentation from cassava

bagasse using mixed culture of lactobacilli by response surface methodology; Biochemical

Engineering Journal, 36: 262-267, 2007.

Stiles M. E,, Holzapfel W. H.: Lactic acid bacteria of foods and their current taxonomy;

International Journal of Food Microbiology, 36: 1–29, 1997.

Trifirò A., Saccani G., Gherardi S., Vicini E., Spotti E., Previdi M.P., Ndagijimana M., Cavalli S.,

Reschiotto C.: Use of ion chromatography for monitoring microbial spoilage in the fruit juice

industry, Journal of Chromatography A, 770: 243-252, 1997.

Vodnar D. and C.: Comparative analysis of lactic acid production by apple fruits and juice, using

HPLC analysis and the tectronik senzytec biosensor, Bulletin UASVM, Agriculture (2): 444 - 449,

2008.

Zhang Z. Y., Jin B., Kelly J.M.: Production of lactic acid from renewable materials by Rhizopus

fungi, Biochemical Engineering Journal 35: 251-263, 2007.

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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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