Short communication Study of kefir grains application in sourdough bread regarding rope spoilage caused by Bacillus spp. I. Mantzourani a , S. Plessas a,⇑ , G. Saxami b , A. Alexopoulos a , A. Galanis b , E. Bezirtzoglou a a Democritus University of Thrace, Faculty of Agriculture Development, Laboratory of Microbiology, Biotechnology and Hygiene, 193 Pantazidou str., GR-68200 Orestiada, Greece b Democritus University of Thrace, Department of Molecular Biology and Genetics, Applied Microbiology and Molecular Biotechnology Research Group, Dragana, GR-68100 Alexandroupolis, Greece article info Article history: Received 18 March 2013 Received in revised form 1 July 2013 Accepted 19 July 2013 Available online 27 July 2013 Keywords: Kefir grains Rope spoilage Sourdough DGGE analysis abstract Sourdough breads prepared with kefir grains resulted in appearance of rope spoilage at the 15th day of bread storage, while the control samples (sourdough breads prepared with wild microflora) were spoiled approximately at the 7th day. Denaturing Gradient Gel Electrophoresis (DGGE) analysis confirmed the above macroscopic observation since Bacillus spp. were detected on sourdough breads prepared with kefir grains at the 15th day of bread storage. The content of organic acids that play synergistic role regard- ing the enhancement of bread self life was also determined. Lactic acid concentration of sourdough breads prepared with kefir grains were approximately 41–82% higher than the control samples, while acetic acid concentration was about 0.5–1-fold higher respectively. The concentration of some other organic acids studied was also found in higher levels (up to 0.06 lg/g) than the control samples. These findings could probably explain the stability of breads prepared with kefir grains against rope spoilage. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Rope and mould spoilage is the most significant cause for bread deterioration besides staling and result in respectable pecuniary losses for producers (Simsek, Hilmi Con, & Tulumoglu, 2006; Vale- rio, De Bellis, Lonigro, Visconti, & Lavermicocca, 2008). In the case of rope spoilage the main strains involved are members ofBacillus genus mainlyBacillussubtilis, Bacilluslicheniformis, and occasionally Bacilluspumilus, Bacillusmegaterium, Bacilluscereus, Bacillusclausii andBacillusfirmus (Katina, Sauri, Alakomi, & Mattila-Sandholm, 2002). However, the discrimination within the genus Bacillus is very difficult due to the large number of species (Pepe, Blaiotta, Moschetti, Greco, & Villani, 2003). Likewise, the applications of molecular methods have been used in order to provide rapid and reliable identification. In this manner, the 16S ribosomal DNA (rDNA) PCR-denaturing gradient gel electrophoresis (DGGE) analy- sis is considered a reliable approach to overcome most of the draw- backs described above. In particular, DGGE analysis of the V3 region of 16S rDNA, is a powerful method for differentiating micro- organisms at the species level and has already applied successfully for the identification of Bacillus spp. in rope bread (Sadeghi, Mort- azavi, Bahrami, & Sadeghi, 2012). ‘‘Ropey’’ bread has a distinct flavor close to ripe cantaloupe and becomes discoloured and sticky. Sourdough seems to offer an alternative for the attitude towards spoilage, since there are cor- roborating reports revealing positive effects (Choi, Kim, Hwang, Kim, & Yoon, 2012). On the other hand, in order to optimize the sourdough bread making process, the control of fermentation pH and temperature and the selection of a defined starter culture with specific and desirable properties should be assessed. Especially, the proper selection of starter culture is an indispensable issue. For this reason the use of kefir grains seems to be an interesting perspective. Kefir is a mixed culture contains primarily LAB, yeasts and sometimes acetic acid bacteria. Kefir has been applied in straight bread and sourdough bread making, improving the sensorial characteristics, self-life and general properties of breads (Filipcev, Simurina, & Bodroza-Solarov, 2007; Plessas et al., 2011). However, there is no published data in the literature concerning the examination of sourdough breads prepared with kefir grains versus rope spoilage. The aim of the present study was to investigate the occurrence of rope-producing Bacillus spp. in sourdough breads prepared with kefir grains during storage, through the application of PCR-DGGE analysis. 2. Materials and methods 2.1. Kefir grains production from cheese whey Kefir grains were isolated from Russian kefir drink and were produced through anaerobic fermentation for 48 h, at 30 °C in cheese whey (Plessas, Alexopoulos, Bekatorou, & Bezirtzoglou, 2012). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.07.098 ⇑ Corresponding author. Tel./fax: +30 2552041141. E-mail address: splessas@agro.duth.gr (S. Plessas). Food Chemistry 143 (2014) 17–21 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem