J. Sep. Sci. 2008, 31, 2707 – 2713 M. Ligoret al. 2707 Magdalena Ligor 1 Reda Jarmalaviciene 2 Michal Szumski 1 Audrius MaruÐka 2 Boguslaw Buszewski 1 1 Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Torun, Poland 2 Department of Chemistry, Faculty of Natural Sciences, Vytautas Magnus University, Kaunas, Lithuania Original Paper Determination of volatile and non-volatile products of milk fermentation processes using capillary zone electrophoresis and solid phase microextraction coupled to gas chromatography The aim of the investigations was to develop analytical methods for the determina- tion of selected volatile and non-volatile organic compounds numbering among the final products of milk fermentation. The analyzed compounds were as follows: bia- cetyl and carboxylic acids (formic, acetic, citric, and lactic). The model yogurt was prepared under controlled conditions in our laboratory by addition of the selected bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus) to the milk sample. The temperature, time, and stirring were controlled during the fermentation process. Factors considered in SPMPE – GC – FID method development included fiber expo- sure time, salt addition, temperature of extraction, and temperature of desorption. Various SPME fibers, for example with PDMS, CAR/PDMS, PA, and PDMS/DVB coat- ings, were tested to obtain the highest recovery of the investigated compounds extracted from yogurt samples. Based on these preliminary experiments, qualitative and quantitative analyses for the determination of biacetyl were performed by SPME – GC – FID. Moreover, a capillary zone electrophoresis method was developed for the determination of carboxylic acids in the yogurt samples. The buffer composi- tion as well as deproteinization by acetonitrile were found to have a crucial effect on the analysis. Keywords: Biacetyl / Carboxylic acids / CZE / GC / SPME / Received: April 2, 2008; revised: May 10, 2008; accepted: May 24, 2008 DOI 10.1002/jssc.200800183 1 Introduction Yogurt is one of the products of the milk fermentation processes occurring on addition of Lactobacillus bulgaricus and Streptococcus thermophilus bacteria to pasteurized and homogenized milk at elevated temperature. Some inves- tigations showed that the bacteria present in yogurt have a positive effect on the presence and balanced con- centration of the physiological bacteria (Lactobacillus acid- ophilus) as well as pathogenic bacteria (e. g. Helicobacter pylori) in the digestive tract [1]. Therefore, the consump- tion of yogurt is highly recommended as a means of sup- plying the organism with these so-called probiotic bacteria (e. g. Lactobacillus, Bifidobacterium) and exploiting their ben- eficial influence on the digestive tract and overall body condition including the immune system [2 – 9]. The bacteria present in milk kept under appropriate thermal conditions produce a wide range of organic com- pounds such as: non-volatile acids (lactic, pyruvic, etc.), volatile acids (formic, acetic, propionic, etc.), carbonyl compounds (acetaldehyde, acetone, acetoin, biacetyl, etc.) as well as a heterogeneous group of substances formed during thermal degradation of proteins, fats, and lactose [10, 11]. The final flavor of yogurt, which is related to the con- sumer's acceptance of the final product, is connected with the presence of several compounds, namely: biace- tyl, acetoin, lactic acid, and acetaldehyde. The buttery- nutty flavor of yogurt mainly comes from biacetyl. The presence of biacetyl is related to the biochemical path- way of flavors in yogurt [12 – 15]. The parent substance for the synthesis of biacetyl is pyruvic acid, which is the product of glucose metabolism by a pathway known as glycolysis. Then the bacteria reduce the ketone groups to form acetoin and 2,3-butanediol, relatively flavorless compounds. The scheme of biosynthesis of biacetyl and Correspondence: Professor Boguslaw Buszewski, Chair of Envi- ronmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin St, PL-87 100 Torun, Poland E-mail: bbusz@chem.uni.torun.pl Fax: +48 56 6114837 Abbreviations: SPME, solid phase microextraction i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com