Structural investigation and response surface optimisation for improvement of kefiran production yield from a low-cost culture medium Mehran Ghasemlou a,⇑ , Faramarz Khodaiyan a , Kambiz Jahanbin b , Seyed Mohammad Taghi Gharibzahedi a , Salman Taheri c a Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran, Iran b Shahrood University of Technology, Faculty of Agriculture, School of Agricultural Engineering, Shahrood, Iran c Chemistry and Chemical Engineering Research Center of Iran, P.O. Box 14335-186, Tehran, Iran article info Article history: Received 25 July 2011 Received in revised form 5 December 2011 Accepted 17 January 2012 Available online 25 January 2012 Keywords: Kefiran Cheese whey Exopolysaccharide Structural analysis Response surface methodology abstract Kefiran, a water-soluble heteropolysaccharide with molecular weight of 1.35  10 6 Da and a specific opti- cal rotation of +64° (c 1.0, H 2 O), was isolated from kefir grains grown in cheese whey and further purified through DEAE-Sepharose XK26. Response surface methodology was employed to optimise the culture conditions for kefiran production from kefir grains to be lactose concentration 67 g/l, yeast extract 13 g/l, pH 5.7 and temperature 24 °C. Intrinsic viscosity was 5.84 dl/g using the Huggins extrapolation and 5.53 dl/g using the Kramer extrapolation. Monosaccharide analysis revealed that kefiran is composed of glucose (Glc) and galactose (Gal) in a relative molar ratio of 1.0:1.1. Its structural features were eluci- dated by a combination of FT-IR, methylation and GC–MS analysis, periodate oxidation–Smith degrada- tion, partial acid hydrolysis and NMR spectroscopy ( 1 H, 13 C and HMBC). The data obtained indicated that kefiran possessed a backbone of (1 ? 6)-linked Glc, (1 ? 3)-linked Gal, (1 ? 4)-linked Gal, (1 ? 4)-linked Glc and (1 ? 2,6)-linked Gal, with a branch attached to O-2 of Gal residues and terminated with Glc residues. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The gradually increasing demand for natural polymers in vari- ous industrial applications has led in recent years to the develop- ment of exopolysaccharides produced by microorganisms. These exopolysaccharides (EPS) are important because they can impart functional properties to food and confer health benefits (Laws, Gu, & Marshall, 2001). Kefir is a refreshing and acidic fermented beverage that origi- nated in the Caucasus Mountains. Traditionally, it was prepared by fermenting fresh milk with kefir grains, which consist mainly of a complex and variable population of lactic-acid bacteria (LAB), yeasts and sometimes acetic-acid bacteria (Laws et al., 2001). These grains are small and whitish or slightly yellowish, with irregular shapes kept together in a slimy polysaccharide matrix commonly called kefiran. Kefiran is finding increasing use in the food industry as a texturing and gelling agent. It is a water-soluble polysaccharide containing approximately equal amounts of glucose and galactose residues in a chain sequence (Micheli, Uccelletti, Palleschi, & Crescenzi, 1999; Mukai, Toba, Itoh, & Adachi, 1988). The structure of the repeating unit of this EPS has been elucidated mainly by methylation analysis and NMR data (Maeda, Zhu, Omura, Suzuki, & Kitamura, 2004; Mukai, Toba, Itoh, Nimura, & Adachi, 1990; Yokoi, Watanabe, Fujii, Mukai, Toba, & Adachi, 1991). However, few works relating to the structure of the kefiran obtained from kefir grains grown in cheese whey are available in the literature. This polysac- charide has attracted considerable interest, because several func- tional properties such as antibacterial, antifungal and antitumour activities have been reported for it (Murofushi, Shiomi, & Aibara, 1983). Moreover, there has been growing research interest towards the isolation and purification of kefiran with the high yield, high concentration and high productivity needed for its use in the food industry (Cheirsilp, Shoji, Shimizu, & Shioya, 2003; Micheli et al., 1999; Mitsue, Tachibana, Hara, & Fujio, 1998). However, little is known about polysaccharide production in cheese whey, which might be an economic alternative to milk. Whey is produced in abundance as a by-product of the cheese- making industry, and its disposal represents a serious environmen- tal problem. However, it has potential for use as a substrate for the production of biopolymers (Zafar & Owais, 2006). Response surface methodology (RSM) is a well-known method applied in the optimisation of culture conditions and other critical 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.01.046 ⇑ Corresponding author. Address: Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran, P.O. Box 4111, Karaj 31587-77871, Iran. Tel.: +98 912 598 7860; fax: +98 261 2248804. E-mail address: mghasemlou@ut.ac.ir (M. Ghasemlou). Food Chemistry 133 (2012) 383–389 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem