"FT-IR spectroscopy is a powerful tool for the characterization of microflora and exopolysaccharides recovered from kefir grains

BOSCH A; GOLOWCZYC M; REY F; ROMANIN D; BORST, N; WENNING M; ABRAHAM A. G.; GARROTE G L; DE ANTONI G L; SCHERRER S, YANTORNO O

Berlin. Alemania

Congreso; FTIR Spectroscopy in Microbiological and Medical Diagnostics Congress,; 2007

Kefir is fermented milk with a slightly acidic taste obtained by incubating milk withkefir grains. These grains are white or lightly yellow gelatinous masses characterized byirregular forms, constituted by a complex mixture of lactococci, homofermentative andheterofermentative lactobacilli, yeasts and acetic acid bacteria living in a strong and specificsymbiosis. This complex microflora is enclosed in a matrix containing mainly proteins andpolysaccharides, known as kefiran.Current investigations for the improvement of probiotic products based on kefir grainsand kefiran have been focused on the study of both polysaccharides and microfloracomposition of the grains. Different grains obtained from various Argentinean sources wererecently chemically and microbiologically analysed. Microbiological characterization atspecie level was carried out applying biochemical tests, whole-cell protein profiles,hydrophobicity, and cellular aggregation (1). Although the results obtained in such study weresuitable to distinguish the species isolated, these analyses are time consuming, expensive andsometimes of limited reliability. Therefore, in this work a different approach based on Fouriertransform infrared (FT-IR) spectroscopy and Hierarchical Cluster Analysis (HCA) wasapplied to analyse entire grains, purified polysaccharides, and for the discrimination andidentification of lactobacilli and yeasts recovered from local kefir grains.Forty-four lactobacilli and 37 yeasts isolated from 7 different kefir grains and 23reference strains were included in this study. First derivative spectra obtained from the abovementioned organisms were analyzed by HCA using Pearson’s product-moment correlationcoefficient and Ward’s algorithm (2). Lactobacilli identification was performed by a sequentialidentification scheme based on HCA(3). We proposed a first level of discrimination betweenhomo and hetero-fermentative lactobacilli using 4 windows: 1789-1700, 1059-935, 3000-2927 and 896-833 cm-1. Lactobacilli classified as heterofermentative were then discriminatedby a 2nd HCA model, which identified all these isolates as L. kefir (20 isolates) except 1 thatwas discriminated as L. parakefir. The 3rd HCA model developed for the discriminationamong homofermentative lactobacilli grouped all these isolates as L. plantarum (23 isolates).Finally, we developed and independent model for the identification of yeasts isolates whichindicated that three main genera were present in the grains analysed hereby: Saccharomyces,Kluyveromyces and Issatchenkia. Eighteen isolates were identified as S. cereviciae, 6 as S.unisporus, 10 as K. marxianus and 4 as belonging to Issatchenkia genera. Additionally, yeastand lactobacilli identified by the developed models were tested against the correspondingspectral libraries of the Abteilung Mikrobiologie, Zentralinstitut für Ernährungs-undLebensmittelforschung (ZIEL), Technical University of Munich, Germany.FT-IR proved to be a rapid and powerful toll for the discrimination and identificationat the species level of lactobacilli and yeasts isolated from kefir grains.