Biochemical Engineering Journal 52 (2010) 65–70 Contents lists available at ScienceDirect Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej Influence of fermentation on the acid tolerance and freeze drying survival of Lactobacillus rhamnosus GG Antonis Ampatzoglou, Benjamin Schurr, Gurjot Deepika, Sasitorn Baipong, Dimitris Charalampopoulos ∗ Department of Food and Nutritional Sciences, The University of Reading, P.O. BOX 226, Reading RG6 6AP, Berkshire, England, UK article info Article history: Received 1 March 2010 Received in revised form 2 July 2010 Accepted 5 July 2010 Keywords: Fermentation Lactobacillus rhamnosus GG Freeze drying Acid tolerance Survival Probiotics abstract The aim of this work was to investigate the effect of fermentation pH and time on the acid tolerance, glycolytic activity, and survival during freeze drying of Lactobacillus rhamnosus GG. Two fermentations were performed, one was pH controlled (pH 6.8), and the other was pH uncontrolled. Cells were col- lected after 10 h (late exponential phase), 25 h (mid stationary phase) and 37 h (late stationary phase) of fermentation. In the case of the pH controlled fermentation, the acid tolerance of the cells collected at 10 h was remarkably high, as the viability decreased only by 0.2 logs; in the case of the 25 h and 37 h, it decreased 1.5 and 6.0 logs, respectively. In the case of the pH uncontrolled fermentation, for all three time points the cell reduction was high, ranging between 3 and 4 logs. The cells from the pH controlled fermentation, and in particular the cells from the 10 h sample, demonstrated considerably higher gly- colytic activity compared to the cells from the uncontrolled fermentation. The type of fermentation had a lesser effect on the ability of the cells to survive during freeze drying, as in all cases the cell reduction was small (<1 log). The cells from the 10 h sample from the pH controlled fermentation survived significantly (P < 0.05) better than the cells from the 25 and 37 h sample; the opposite was the case for the cells from the pH uncontrolled fermentation. However, in all cases, the differences between the samples were less than 0.5 log. In conclusion, both the fermentation pH and time were found to have a significant impact on the glycolytic activity of L. rhamnosus GG, and on its ability to survive under acidic conditions and during freeze drying. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Beneficial lactic acid bacteria, prior to their use as probiotics, have to satisfy certain safety, functional and technological criteria. From a technological point of view, it is important that the probiotic strains are able to grow to high cell densities in the fermentation medium, survive during the preservation process, remain viable during storage, and survive the adverse conditions of the gastroin- testinal tract [1]. Most commercial probiotics are produced by batch fermenta- tion performed under optimal conditions for pH and temperature. In the case of both probiotic and non-probiotic Lactobacillus strains, the optimum pH is usually between 6.0 and 7.0 [2,3] and the temperature between 37 and 42 ◦ C [3,4]. In terms of the growth medium, lactobacilli in general have complex nutrient require- ments, including fermentable carbohydrates, proteins and amino acids, vitamins of the B complex, nucleic acid derivatives, unsatu- rated free fatty acids and various minerals [5,6]. The most common ∗ Corresponding author. Tel.: +44 0118 3788216; fax: +44 0118 9310080. E-mail address: d.charalampopoulos@reading.ac.uk (D. Charalampopoulos). preservation method used is freeze drying, a process that can affect significantly cell viability. In order to improve cell viability, a lot of research is carried out on the use of cryoprotective compounds, such as sucrose, lactose, trehalose, sorbitol, proteins and skim milk [7–10]. A small number of studies have shown that a relationship exists between the survival of the strains either freezing or freeze drying and the fermentation conditions, in particular the pH and temperature. More specifically, it has been shown that a more acidic fermentation pH (pH 5 vs pH 6) has a positive effect on the cell sur- vival of L. reuteri [11] and the cryotolerance, defined as the ability of the cells to recover their acidification activity, of L. acidophilus [12]. Moreover, a fermentation temperature of 30 ◦ C increased the cryotolerance of L. acidophilus compared to 37 and 42 ◦ C. Another study however, using Bifidobacterium cells, showed no relationship between other fermentation parameters, such as the fermentation time, and the freeze drying survival [13]. After ingestion, probiotics have to survive the adverse gastric environment in order to reach the intestine and exert their bene- ficial effects. Several studies have been conducted using simulated gastric juices in order to select acid tolerant strains and understand the mechanisms behind their acid tolerance [14–16]. In order to improve the survival of strains the research has mainly focused 1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.07.005