Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

Transcription

1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying A. Siaterlis, G. Deepika and D. Charalampopoulos Department of Food Biosciences, The University of Reading, Berkshire, Reading, UK Keywords freeze drying, growth medium, Lactobacillus, probiotics, vegetable peptone. Correspondence Dimitris Charalampopoulos, Department of Food Biosciences, The University of Reading, PO Box 226, Whiteknights, Reading RG6 6AP, UK : received 19 May 2008, revised 3 September 2008 and accepted 22 September 2008 doi: /j x x Abstract Aims: To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying. Methods and Results: A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased. Conclusions: A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process. Significance and Impact of the Study: The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium. Introduction During recent years, there has been an increasing interest in incorporating probiotic bacteria into non dairy foods and in developing dried formulations for nutraceutical applications. As a result, emphasis is put by manufacturers and researchers on developing technologies to enhance the fermentation productivities, improve cell survival during drying and storage, and maintain probiotic functionality throughout the process. The choice of growth medium for producing commercial amounts of probiotics is constrained by costs, production yields and product type (Champagne et al. 2005). For dairy-based probiotic applications, probiotic strains are grown in milk-based media supplemented with a variety of complex ingredients, such as yeast extract, malt extract, meat peptones, and casein and milk hydrolysates (Gomes et al. 1998; Avonts et al. 2004; Burns et al. 2008). For nondairy applications, the growth media should contain nonanimal derived components, such as yeast extract and vegetable-derived peptones (Heenan et al. 2002; Matto et al. 2006). Fermentation is usually followed by freeze drying, a process that can affect significantly cell viability. For dairy probiotic applications, complex ingredients, such as skim milk are commonly used as cryoprotectants (Carvalho et al. 2004a; Otero et al. 2007). However, alternative cryoprotectants can be used, such as disaccharides (e.g. sucrose, lactose and trehalose), polyalcohols (e.g. mannitol and sorbitol), amino acids and proteins (Hubalek 2003; Carvalho et al. 2004a). Besides exploring the suitability of various cryoprotectants for probiotic applications, it is important to understand the possible relationship between the fermentation process and cell Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009)

2 Survival of Lactobacillus spp. during freeze drying A. Siaterlis et al. survival during freeze drying. Key parameters likely to play a role include the growth phase, the composition of the growth medium and the process conditions, such as ph (Carvalho et al. 2004b; Saarela et al. 2005; Meng et al. 2008). The aim of this work was to evaluate the effects of the culture medium and the cryoprotectant used on the cell growth of human-derived Lactobacillus rhamnosus GG and Lactobacillus plantarum strains and on their survival during freeze-drying. More specifically, the objectives were to (i) develop a growth medium based on glucose, yeast extract and vegetable-derived peptone that supports high cell densities, (ii) compare sucrose, trehalose and sorbitol in terms of their ability to protect the cells during freeze drying and (iii) investigate the effect on growth medium on cell survival during freeze drying. Materials and methods Bacterial strains Lactobacillus rhamnosus GG (ATCC 53103) and L. plantarum (NCIMB 8826) were used in this study. For preparation of the cell banks, the cells were grown in MRS broth (Oxoid) at 37 C for approximately 15 h. The cultures were then diluted with sterile glycerol (Sigma) at 20% (v v), aliquoted into 1Æ8-ml cryovials, and stored at )80 C. Growth media and culture conditions The strains were grown in various complex media, according to a full factorial experimental design with three factors, i.e. glucose (Sigma), vegetable peptone No. 1 (Oxoid) and yeast extract (Oxoid), each tested at two different concentrations. The basic components of all media were: 0Æ2 gl )1 MgSO 4 Æ7H 2 O, 0Æ05 g l )1 MnSO 4 Æ4H 2 O,1mll )1 Tween 80, 11Æ3 gl )1 K 2 HPO 4 and 4Æ6 gl )1 KH 2 PO 4. The composition of the eight media tested, which were designated as M1, M2, M3, M4, M5, M6, M7 and M8, is presented in Table 1. MRS broth was used as control. The inoculum was prepared by growing the bacterial cells into 100 ml of MRS broth in a 500-ml shake flask. The culture was incubated at 37 C for approximately 15 h. The cells were harvested by centrifugation at 3200 g and re-suspended in an equal volume of PBS (Oxoid). The complex media were inoculated with an appropriate volume of the PBS inoculum suspension so that the starting optical density (OD 600 ) was approximately 0Æ2. Each growth experiment was conducted in triplicate. Table 1 Composition of growth media used for the response surface design Medium Freeze drying Late exponential phase cells (12 h) grown in medium M1 were freeze dried using a variety of cryoprotective agents at various concentrations, including sucrose, trehalose and sorbitol [1%, 5% and 10% (w v)]. The bacterial cultures were centrifuged for 15 min at 3200 g and 4 C. The supernatants were discarded and the cells were re-suspended in 5 ml of drying medium. The suspensions were incubated at room temperature for 1 h and then frozen at )80 C for 24 h. The frozen cultures were then freeze dried in a IEC Lyoprep 3000 freeze drier (Lyoprep, Dunstable, UK) for approximately 45 h. The OD 600 and the cell viability were measured both pre- and post-freeze drying. Each experiment was conducted in triplicate. In order to evaluate the effects of the growth media on the ability of the cells to survive the freeze drying process, standard amounts of cells grown in the nine growth media (M1 M8 plus MRS) were collected and freezedried in a 5% sucrose, in the case of L. rhamnosus GG, and in 10% sucrose in the case of L. plantarum, under the conditions described above. The OD 600 and cell viability were measured both pre- and post-freeze-drying. Each experiment was conducted in triplicate. Cell concentration Glucose (g l )1 ) Yeast extract (g l )1 ) M M M M M M M M Peptone (g l )1 ) For OD measurements, 1 ml of the culture was diluted in 9 ml of distilled water and the absorbance measured at 600 nm using a Thermo Biomate 3 spectrophotometer (Thermo, Hemel Hempstead, UK). The measurements were performed in triplicate. The plate count method was used to determine the number of viable bacterial cells. The culture was serially diluted in sterile PBS, and 100 ll of the suspension was subsequently spread onto a MRS agar plate, in triplicate. The plates were incubated at 37 C for about 2 days, after which they were counted and expressed as colony forming units (CFU) per millilitre. 296 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009)

3 A. Siaterlis et al. Survival of Lactobacillus spp. during freeze drying Reducing sugar analysis The dinitro salicylic acid (DNS) assay was used to measure glucose concentration in culture supernatants, according to Chaplin and Kennedy (1994). Briefly, the concentration of reducing sugars was measured by adding 1 ml of the DNS reagent to 100 ll of the sample in a capped test tube. The mixture was well mixed and heated at 100 C for 10 min. It was then cooled to room temperature and its absorbance measured at 570 nm. A standard curve was constructed using glucose at various concentrations. Residual moisture analysis The residual moisture was estimated using a vacuum oven (Gallenkamp). The freeze dried cultures were weighed and dried at 100 C until no further change in the weight was observed. A small amount of air, desiccated by passing through concentrated H 2 SO 4, was let in the oven while drying. The percentage moisture was estimated by: % moisture = (W 0 ) W 1 ) W 0 100, where W 0 the initial weight of the sample and W 1 the weight of the sample after drying. The measurements were performed in triplicate. was around 10 9 CFU ml )1 and the OD 600 around 8Æ5 (data not shown). Media M2 and M3, which contained lower amounts of yeast extract, did not support as well the growth of L. rhamnosus GG. In media M5 M8, which contained 10 g l )1 of glucose, the viable cell counts were lower than in M1 M4. In commercial MRS, containing 20 g l )1 of glucose, the maximum cell concentration was approximately 10 9 CFU ml )1. For media M1 M4, as well as MRS, the ph dropped down to approximately 4, whereas for media M5 M8, it dropped down to 4Æ5 and then remained constant (data not shown). Lactobacillus plantarum grew for approximately 12 h, after which it entered into the stationary phase (Fig. 2). The best growth was obtained with MRS (10 10 CFU ml )1, OD 600 approximately 7Æ5). For the complex media M1 M4, the maximum cell concentrations were slightly lower than CFU ml )1 (Fig 2a), whereas for media M5 M8, they were even lower than that (Fig 2b). For media M1 M4 and MRS, the ph dropped down to approximately 4, whereas for media M5 M8, it dropped down to 4Æ5 and then remained constant. For both strains, glucose was completely consumed after approximately 10 h in media M1 M4, whereas in media M5 M8 and MRS, glucose was still present after 35 h, at levels between 4 and 6 g l )1 (data not shown). Results Growth of Lactobacillus plantarum and Lactobacillus rhamnosus GG in complex media Figure 1 shows the growth curves of L. rhamnosus GG in media M1 M8. In all the media tested, the cells grew for approximately 12 h, after which they entered into the stationary phase. The maximum cell concentration in terms of both viable cell counts and OD 600 was obtained with media M1 and M4. In both media, the viable cell counts Effect of cryoprotective agents on cell survival during freeze drying For the freeze drying experiments, both strains were grown in medium M1. Lactobacillus plantarum was more resistant to freeze drying compared to L. rhamnosus GG, regardless of the cryoprotective agent used (Fig. 3). Among the three sugars tested, sucrose, at a concentration between 5% and 10% (w v) offered better protection during freeze drying compared with trehalose and sorbitol, for both strains. Lactobacillus rhamnosus GG (a) (b) 9 9 Figure 1 Growth curves of Lactobacillus rhamnosus GG in complex media. (a) Media containing 20 g l )1 of glucose, M1 (r), M2 ( ), M3 ( ), M4 ()), MRS (h). (b) Media containing 10 g l )1 of glucose, M5 (r), M6 ( ), M7 ( ), M8 ()), MRS (h) Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009)

4 Survival of Lactobacillus spp. during freeze drying A. Siaterlis et al. (a) 10 0 (b) Figure 2 Growth curves of Lactobacillus plantarum in complex media. (a) Media containing 20 g l )1 of glucose, M1 (r), M2 ( ), M3 ( ), M4 ()), MRS (h). (b) Media containing 10 g l )1 of glucose, M5 (r), M6 ( ), M7 ( ), M8 ()), MRS (h). demonstrated a survival of around 60% with sucrose 10% (w v), whereas in the case of the L. plantarum strain, sucrose 5% (w v) resulted to a survival of approximately 90%. Depending on the freeze drying medium, the residual moisture ranged between 0Æ1% and 0Æ5% for L. rhamnosus GG and 0Æ1% and 0Æ4% for L. plantarum. Effect of media composition on survival during freeze drying In this set of experiments, the aim was to investigate whether the growth media affected the ability of the cells to survive the freeze drying process. The cells were grown in media M1 M8 and freeze dried in 5% sucrose in the case of L. rhamnosus GG and 10% sucrose in the case of L. plantarum (Fig. 4). The percntage survival of L. rhamnosus GG ranged between 75% and 85% in media M1 M4, whereas in the case of media M5 M8 it was lower, ranging between 40% and 60%. In the case of L. plantarum, the cell survival in media M1 M4 and MRS Survival (%) Sucrose 10% Sucrose 5% Sucrose 1% Trehalose 10% Trehalose 5% Trehalose 1% Sorbitol 5% Sorbitol 1% PBS Figure 3 Survival (%) of ( ) Lact. plantarum and (h) Lact. rhamnosus GG after freeze drying in various media. ranged from 60 to 90%, whereas in media M5 M8 it was significantly lower, ranging from 2 to 6%. Discussion There are many technological challenges in producing probiotic cultures. From a culture s supplier perspective key targets include culturing the micro-organism to high cell concentration, minimizing cell death during freezing or freeze drying, and maintaining the functional properties of the cells during the production process and storage (Saarela et al. 2000; Lacroix and Yildirim 2007). The aim of this work was to develop a simple growth medium based on non-animal derived components for the growth of human derived L. rhamnosus GG and L. plantarum, evaluate the protective effect of trehalose, sucrose and sorbitol during freeze drying of the cells, and investigate whether a relationship exists between the composition of the growth medium and the ability of the cells to survive the freeze drying process. The results from the media development studies suggest that the growth of L. rhamnosus GG and L. plantarum in the media containing 10 g l )1 glucose was most likely glucose limited, as in all cases glucose was consumed within 6 8 h. It is interesting however to note that the cell concentration continued to increase for up to 2 h after glucose was consumed. This was most likely due to the fact that the cells were utilizing alternative carbon sources present in yeast extract and peptone. In the media containing 20 g l )1 glucose, the growth was most likely limited by nitrogen or another growth factor, because glucose was still present after 35 h and the ph was 4, higher than in MRS, where it dropped down to approximately 3Æ5. In the case of L. rhamnosus GG, the presence of yeast extract in the medium seemed to improve more the growth of the cells than peptone did. Yeast extract has been shown to improve the growth of lactobacilli when added on basal complex media, most likely due to 298 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009)

5 A. Siaterlis et al. Survival of Lactobacillus spp. during freeze drying Figure 4 Survival (%) of (a) Lact. plantarum and (b) Lact. rhamnosus GG strains freeze dried in 5%, and 10% (w v) sucrose, respectively. The strains were grown in media M1 M8. (a) 120 Survival (%) M1 M2 M3 M4 0 M5 M6 M7 M8 Growth medium MRS (b) 100 Survival (%) M1 M2 M3 M4 M5 M6 M7 M8 Growth medium MRS its B vitamin and nucleotides content (Nancib et al. 2005; Xu et al. 2008). Among different nitrogen sources, including yeast extract, ammonium sulphate, urea, peptone and casein hydrolysates, yeast extract was found to be the most effective additive to date juice for the growth of L. rhamnosus (Nancib et al. 2005). Burns et al. (2008) reported that the highest acidification activity among various nitrogen sources was observed when yeast extract was added to a whey-based culture medium (concentrations ranged between 3 and 10 g l )1 ), used for the growth of Lactobacillus acidophilus and Lactobacillus paracasei. Similarly, Avonts et al. (2004) showed that increasing the amount of yeast extract in a milk-based medium (from 2 to 10 g l )1 ) enhanced the growth of Lactobacillus johnsonii and Lactobacillus gasseri. In the case of L. plantarum, MRS supported better the cell growth than the rest of the media. In order to examine whether L. plantarum strain was able to utilize acetate, which is present in commercial MRS at a concentration of 5 g l )1, the strain was grown in the best growth medium (M1) supplemented with 5 g l )1 of acetate. It was observed that the final cell concentration dropped down to approximately 10 9 CFU ml )1, indicating that the 5gl )1 of acetate present in MRS should actually be inhibiting cell growth. Therefore, the most likely explanation of the increased cell growth in MRS is that the animal-derived nitrogen sources present in MRS, such as Lab Lemco, provide nutrients that stimulate cell growth, and thus the cells are able to overcome the acetate inhibition. This finding highlights the species- and straindependency of certain nutritional requirements, which has been observed in other works too (Lechiancole et al. 2002; Burns et al. 2008). The results from the freeze drying studies showed that the choice of the cryoprotectant used and its concentration significantly affected the survival of L. rhamnosus GG and L. plantarum. Among sucrose, trehalose and sorbitol, sucrose at 5% and 10% offered the best protection to the cells whereas sorbitol the least. Similar observations were reported by Zhao and Zhang (2005) for Lactobacillus brevis. In general, sucrose and trehalose are considered the best cryoprotectants for various types of bacteria (Leslie et al. 1995), including lactobacilli (Conrad et al. 2000; Zayed and Roos 2004; Meng et al. 2008), although skim milk is the main ingredient that is most commonly used industrially and the one that most information is available for (Hubalek 2003; Carvalho et al. 2004a; Otero et al. 2007). Damage to biological systems derived from freeze drying has been attributed to changes in the physical state of the membrane and to changes in the structure of sensitive proteins in the cell (Leslie et al. 1995). A possible reason for the significant protective effect of disaccharides, such as trehalose and sucrose, is that they are able to lower the transition temperature of dry membranes via replacement of water between the lipid headgroups and prevent unfolding and aggregation of proteins by hydrogen bonding with polar groups of proteins. (Crowe et al. 1988; Carpenter et al. 1990; Leslie et al. 1995). The results from the studies investigating the effect of the growth medium on the freeze drying ability of the cells demonstrated that there was a clear relationship between the two. It must be noted that in order to eliminate the effect of other factors, the starting cell concentration prefreeze drying as well as the inoculum preparation procedure were standardized. When the cells were starved for glucose (i.e. in the case of the media with 10 g l )1 glucose) cell survival decreased significantly, especially in the case of L. plantarum (Fig. 4a) and to a lesser extent in the case of L. rhamnosus GG (Fig. 4b). This was most likely due to the fact that glucose was consumed before the end of the exponential phase, which meant that the cells were glucose limited for approximately 2 3 h. In contrast, when the cells were grown in media containing 20 g l )1 of glucose, cell survival was much higher. Furthermore, it seems from the data in Fig. 4(a,b) that cell survival was enhanced as the levels of yeast extract and peptone increased, although concrete conclusions are difficult to make because of the relatively large errors. It is a Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009)

6 Survival of Lactobacillus spp. during freeze drying A. Siaterlis et al. possibility though that the presence of nitrogenous components influence cell survival, albeit to a lesser extent than sugar. In accordance with our results, it has been suggested that the physiological status of the cells, which can be influenced by several physicochemical factors during growth affects cell survival; however, there are not many studies postulating this. Among these, most have looked at the effect of fermentation time (Saarela et al. 2005), ph (Palmfeldt and Hahn-Hagerdal 2000) and the type of sugar used as energy source (Carvalho et al. 2004b). In a similar study to ours, conducted by Carvalho et al. (2003), it was observed that starvation of Lactobacillus delbrueckii ssp. bulgaricus cultures resulted in improved survival during freeze drying. Starvation though was achieved by re-suspending stationary phase cells into water and keeping them at 20 C for 20 min, which might have resulted to a stronger stress response than in our case. Nevertheless, our results show that from a fermentation point of view, the cells should be harvested before the sugar is completely consumed. In conclusion, glucose and to a lesser extent yeast extract, are the most important factors influencing the growth of human-derived L. plantarum and L. rhamnosus GG in a complex medium, consisting entirely of nonanimal derived components. In addition, the concentrations of these particular components in the growth medium affected the ability of the cells to survive the freeze drying process, indicating that there is a link between the physiological status of the cells and their resistance towards adverse environmental conditions. In terms of the cryoprotectants tested, sucrose offered better protection than trehalose and sorbitol for both strains. Acknowledgement The authors would like to acknowledge the financial contribution to this project by the Food Processing Knowledge Transfer Network (UK). References Avonts, L., Van Uytven, E. and De Vuyst, L. (2004) Cell growth and bacteriocin production of probiotic Lactobacillus strains in different media. Int Dairy J 14, Burns, P., Vinderola, G., Molinari, F. and Reinheimer, J. (2008) Suitability of whey and buttermilk for the growth and frozen storage of probiotic lactobacilli. Int J Dairy Technol 61, Carpenter, J.F., Crowe, J.H. and Arakawa, T. (1990) Comparison of solute-induced protein stabilization in aqueoussolution and in frozen and dried states. J Dairy Sci 73, Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X. and Gibbs, P. (2003) Effects of addition of sucrose and salt, and of starvation upon thermotolerance and survival during storage of freeze-dried Lactobacillus delbrueckii ssp bulgaricus. J Food Sci 68, Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X. and Gibbs, P. (2004a) Effects of various sugars added to growth and drying media upon thermotolerance and survival throughout storage of freeze-dried Lactobacillus delbrueckii ssp bulgaricus. Biotechnol Progr 20, Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X. and Gibbs, P. (2004b) Relevant factors for the preparation of freeze-dried lactic acid bacteria. Int Dairy J 14, Champagne, C.P., Gardner, N.J. and Roy, D. (2005) Challenges in the addition of probiotic cultures to foods. Crit Rev Food Sci 45, Chaplin, M.F. and Kennedy, J.F. (1994) Carbohydrate Analysis: a Practical Approach. Oxford: Oxford University Press. Conrad, P.B., Miller, D.P., Cielenski, P.R. and de Pablo, J.J. (2000) Stabilization and preservation of Lactobacillus acidophilus in saccharide matrices. Cryobiology 41, Crowe, J.H., Crowe, L.M., Carpenter, J.F., Rudolph, A.S., Wistrom, C.A., Spargo, B.J. and Anchordoguy, T.J. (1988) Interactions of sugars with membranes. Biochim Biophys Acta 947, Gomes, A.M.P., Malcata, F.X. and Klaver, F.A.M. (1998) Growth enhancement of Bifidobacterium lactis Bo and Lactobacillus acidophilus Ki by milk hydrolyzates. J Dairy Sci 81, Heenan, C.N., Adams, M.C., Hosken, R.W. and Fleet, G.H. (2002) Growth medium for culturing probiotic bacteria for applications in vegetarian food products. LWT-Food Sci Technol 35, Hubalek, Z. (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology 46, Lacroix, C. and Yildirim, S. (2007) Fermentation technologies for the production of probiotics with high viability and functionality. Curr Opin Biotech 18, Lechiancole, T., Ricciardi, A. and Parente, E. (2002) Optimization of media and fermentation conditions for the growth of Lactobacillus sakei. Ann Microbiol 52, Leslie, S.B., Israeli, E., Lighthart, B., Crowe, J.H. and Crowe, L.M. (1995) Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microb 61, Matto, J., Alakomi, H.L., Vaari, A., Virkajarvi, K. and Saarela, M. (2006) Influence of processing conditions on Bifidobacterium animalis subsp lactis functionality with a special focus on acid tolerance and factors affecting it. Int Dairy J 16, Meng, X.C., Stanton, C., Fitzgerald, G.F., Daly, C. and Ross, R.P. (2008) Anhydrobiotics: the challenges of drying probiotic cultures. Food Chem 106, Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009)

7 A. Siaterlis et al. Survival of Lactobacillus spp. during freeze drying Nancib, A., Nancib, N., Meziane-Cherif, D., Boubendir, A., Fick, M. and Boudrant, J. (2005) Joint effect of nitrogen sources and B vitamin supplementation of date juice on lactic acid production by Lactobacillus casei subsp rhamnosus. Bioresource Technol 96, Otero, M.C., Espeche, M.C. and Nader-Macias, M.E. (2007) Optimization of the freeze-drying media and survival throughout storage of freeze-dried Lactobacillus gasseri and Lactobacillus delbrueckii subsp delbrueckii for veterinarian probiotic applications. Process Biochem 42, Palmfeldt, J. and Hahn-Hagerdal, B. (2000) Influence of culture ph on survival of Lactobacillus reuteri subjected to freeze-drying. Int J Food Microbiol 55, Saarela, M., Mogensen, G., Fonden, R., Matto, J. and Mattila- Sandholm, T. (2000) Probiotic bacteria: safety, functional and technological properties. J Biotechnol 84, Saarela, M., Virkajarvi, I., Alakomi, H.L., Mattila-Sandholm, T., Vaari, A., Suomalainen, T. and Matto, J. (2005) Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp lactis cells produced without milk-based ingredients. J Appl Microbiol 99, Xu, G.Q., Chu, J., Zhuang, Y.P., Wang, Y.H. and Zhang, S.L. (2008) Effects of vitamins on the lactic acid biosynthesis of Lactobacillus paracasei NERCB Biochem Eng J 38, Zayed, G. and Roos, Y.H. (2004) Influence of trehalose and moisture content on survival of Lactobacillus salivarius subjected to freeze-drying and storage. Process Biochem 39, Zhao, G. and Zhang, G. (2005) Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying. J Appl Microbiol 99, Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009)