   “ ” 22-23,  2011 38 COMPARISON OF THE FERMENTATION PROCESS AND ACIDIFICATION ACTIVITY OF STREPTOCOCCUS THERMOPHILUS AND ITS UREASE-DEFICIENT DERIVATE N. NINOVA-NIKOLOVA*, Z. URSHEV ELBY Bulgaricum PLC, 12A Malashevska str, Sofia * corresponding author: ninova.n@lbbulgaricum.bg Key words: acidification rate, Streptococcus thermophilus, urease, urease-deficient mutant ABSTRACT Streptococcus thermophilus strain Yt3 was isolated from yoghurt starter BY145-18, strongly influenced by urea in milk. Its urease-deficient spontaneous mutant Yt3uD3-1 showed lack of urease activity. S. thermophilus Yt3 and its urease negative mutant Yt3uD3-1 were compared for their growth at different fermentation conditions of varying pH and temperature during the process of pH-controlled fermentation. The optimal pH and temperature resulting in maximal cell numbers of S. thermophilus Yt3 and its urease-deficient derivative Yt3uD3-1 were determined. A concentrated culture prepared from the urease-deficient mutant Yt3uD3-1 showed faster acidification rate in milk medium than the mother strain Yt3. The basic fermentation characteristics for the two bacterial strains determined in this study permit the large-scale production of concentrated cultures with a high number of viable cells in the bacterial concentrate. INTRODUCTION Streptococcus thermophilus is the most important strain industrially used as a part of starter cultures for yoghurt and cheese production. The optimal parameters for the preparation of bacterial concentrate and the acidification rate of the particular strain are substantial technological characteristics of starter culture. The acidification rate of S. thermophilus strains is related to the rate of lactic acid production by the culture and is influenced by the urease activity of this microorganism. Urease activity of S. thermophilus leads to a delay of pH dynamics in milk and cheese due to the ammonia production [1; 2]. To achieve a faster acidification rate urease-deficient S. thermophilus mutants are used. In all other aspects urease-deficient strains are expected to be identical to the mother strain. An urease-deficient strain Yt3uD3-1 was obtained from an industrial S. thermophilus strain Yt3 and the two cultures were compared for their growth at different fermentation conditions with varying pH and temperature during the process of pH-controlled fermentations. The comparison of the viability and the acidification activity of the concentrated parent strain and its urease-deficient derivate was the main objective of this study. MATERIALS AND METHODS Bacterial strains Strain S. thermophilus Yt3 was isolated from starter culture BY145-18 maintained in the LBB culture collection (LB Bulgaricum PLC, Sofia, Bulgaria). Strain Yt3uD3-1 is a spontaneous urease-deficient mutant isolated from S. thermophilus Yt3 as described previously [3]. Preparation of concentrated bacterial cultures The concentration of bacterial strains was performed in lab-scale (2 L medium) bioreactor New Brunswick Scientific, USA BIOFLO 2000. The milk-based medium contained skim milk powder, lactose, whey, yeast extract, peptone from casein, peptone from soya and was sterilized at 118 0 C for 6 minutes. The fermentation process was carried out at low oxygen conditions supported by constant flow of N 2 . Sodium hydroxide (2M) was used as a neutralizing agent. The agitation was strictly maintained at 150 rpm. The fermentation was conducted at automatically controlled temperature and pH which were preliminarily set up at 39 0 C and 43 0 C and pH 5.9, 6.0 and 6.2, respectively. Acidifying activity of concentrated bacterial cultures The measurement of the acidifying activity of the concentrated bacterial preparations was carried out using a pH meter. Sterile 10% reconstituted skim milk, supplemented with 4M urea was inoculated with 5% culture of Yt3 or Yt3uD3-1 obtained by pH-controlled fermentation. The cultures were incubated at 43 0 C. pH was continuously measured. The time necessary to reach pH 4,8-4,7 was used to compare the acidification activity of cultures. Cell counting, viability assessment Viable cell counts at the end of the fermentation were determined by the plate count method. Ten- fold serial of dilutions of the culture were plated into solid M17 agar and incubated at 37 0 C for 72 hours.