Spirulina enhances the viability of Lactobacillus rhamnosus E N after freeze-drying in a protective medium of sucrose and lactulose

Letters in Applied Microbiology ISSN 0266-8254 ORIGINAL ARTICLE Spirulina enhances the viability of Lactobacillus rhamnosus E N after freeze-drying in a protective medium of sucrose and lactulose M. Kordowska-Wiater 1, A. Waśko 1, M. Polak-Berecka 1, A. Kubik-Komar 2 and Z. Targoński 1 1 Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences in Lublin, Lublin, Poland 2 Department of Applied Mathematics and Computer Science, University of Life Sciences in Lublin, Lublin, Poland Keywords freeze-drying, Lactobacillus, protection, response surface methodology, Spirulina, viability. Correspondence Monika Kordowska-Wiater, Department of Biotechnology, Human Nutrition and Food Commodities, University of Life Sciences in Lublin, Skromna 8, 20-704 Lublin, Poland. E-mail: monika.kordowska-wiater@up.lublin.pl 2010 2347: received 29 December 2010, revised 19 April 2011 and accepted 19 April 2011 doi:10.1111/j.72-765x.2011.03068.x Abstract Aims: Response surface methodology (RSM) was used to optimize a protective medium for enhancing the viability of Lactobacillus rhamnosus E N cells during lyophilization. Methods and Results: Spirulina, sucrose and lactulose were selected, on the basis of a Plackett-Burman factorial design, as important protectants having the following protective effects on cell viability: 102Æ025, 36Æ885 and )34Æ42, respectively. A full-factorial central composite design was applied to determine optimal levels of three used agents. Conclusion: The optimal protective medium composition was determined to be: Spirulina 1Æ304% (w v), lactulose 5Æ48% (w v), and sucrose 13Æ04% (w v) (Polish Patent P-393189). The predictive value of cell viability in this medium was 89Æ619%, and experimental viability obtained during freeze-drying was 87Æ5%. Significance and Impact of the Study: In this study, Spirulina was used for the first time as the protective agent in freeze-drying medium, significantly increasing lactobacilli viability and giving synbiotic character of the final product. Introduction Viability of Lactobacillus cells is commercially important. One of the main methods of bacterial preservation used on an industrial scale is lyophilization (freeze-drying). This process, however, gives rise to side effects that cause loss of cell viability (Huang et al. 2006). To eliminate some of those undesirable effects, factors such as freezedrying parameters, the kind of protective medium with an addition of cryoprotectants (e.g. skim milk, polyols, carbohydrates), the physiological state of the cells and rehydration conditions can be modified (Huang et al. 2006). A large number of viable cells in a probiotic product are a guarantee of their positive action on human health during shelf-live. It is known that the substances, called prebiotics, stimulate the growth and viability of lactic acid bacteria (Saarela et al. 2000), which is why they are commonly added to probiotic food to get a synbiotic product (Aryana and McGrew 2007). In this context, it seems interesting to explore the use of prebiotics as cryoprotectants for enhancing the viability of cells during lyophilization. Spirulina is a prebiotic obtained from dried biomass of the cyanobacterium, Arthrospira platensis. It is a rich source of proteins, vitamins, essential amino acids, and other phytochemicals. Spirulina has the positive effect on human health and has the GRAS (generally recognized as safe) status for use in food and dietary supplements. It also positively affects the growth of lactic acid bacteria (Akalin et al. 2009), but until now, no information has been published about the application of Spirulina powder as a protectant for enhancing Lactobacillus viability during lyophilization. Response surface methodology (RSM) is an efficient statistical technique to explore nonlinear relationships among factors and dependent variables. RSM includes factorial design and regression analyses (Harahan & Lu 2006; Xiaobo et al. 2006) and is commonly preceded by primary screening to limit the number of depended vari- Letters in Applied Microbiology 53, 79 83 ª 2011 The Society for Applied Microbiology 79

A protective medium with Spirulina M. Kordowska-Wiater et al. ables (Preetha et al. 2007). With this end in view, a Plackett-Burman design for screening n variables using only n + 1 experiments was applied (Myers and Montgomery 1995). There is a little information about the RSM application to studying the effects of the factors on bacterial viability during lyophilization (Fonseca et al. 2001; Huang et al. 2006). In the present article, RSM was applied for the first time in experiments with Spirulina, sucrose and lactulose to determine an optimal protective medium for the lyophilization of Lact. rhamnosus E N. Material and methods Microorganism and culture conditions Lact. rhamnosus strain E N (Biomed Serum and Vaccine Production Plant Ltd. in Lublin, Poland) was used in all experiments. After revitalization and two passages in MRS medium at 37 C for 24 h, the culture was used to prepare 24-h inoculum for bioreactor culture of Lact. rhamnosus E N. The working volume was 500 ml in a 1-l vessel. Rods were incubated at 37 C (using a water coat) for 18 h in relatively anaerobic conditions with stirring (100 rev min )1 ). Preparation procedure Cells in an early stage of the stationary phase were harvested by centrifugation at 12879 g for 20 min at 4 C and next, the biomass was washed twice in aseptic physiological saline with the addition of 0Æ2% (v v) Tween 80 and sonicated on ice in five cycles (20 s 30 s) to disrupt cell chains. After a last centrifugation, pellets were resuspended in one ml of protective media and incubated at appropriate cryoprotective temperatures and times in accordance with the results of Plackett-Burman design analysis (Table 1). The following protectansts, selected in primary screening (A.W., M.K.-W. and M.P.-K., unpublished data), were used: Spirulina (100% dried powder of Arthrospira platensis; ªOcean Star International, Inc., Snowville, UT, USA), lactose, sucrose, lactulose, and trehalose. In the next stage of study (RSM), the procedure of cell preparation was the same as mentioned earlier, but combinations of protectants were composed on the basis of a central composite design (CCD) matrix for three factors (Table 2). As an additional control, a standard protective mixture containing 8% (w v) skim milk and 6% (w v) sucrose was also used for the lyophilization of investigated bacteria. Freeze-drying Half of the samples of bacterial suspensions in protective media were frozen at )80 C for one h and then freezedried in freeze-dryer (Labconco, Kansas City, MO) at temperature )50 C and pressure 0Æ024 mbar for 18 h. The remaining, control samples were not frozen nor freeze-dried. Determination of bacteria viability The number of viable cells before (control samples) and after lyophilization was determined by the plate method. Suitable decimal dilutions of control and rehydrated samples (0Æ1 ml) were plated and overlaid with sterilized MRS agar. After incubation colonies were counted, the numbers of viable cells were determined as colony forming units (CFU) ml )1, and the viability of the cells for each protective combination was calculated (Huang et al. 2006). Statistical analysis CCD was used to estimate response surfaces, following the general model equation: Table 1 Plackett-Burman design for seven variables Temperature Time Spirulina Lactose Sucrose Lactulose Trehalose Run f.l.* C f.l. h f.l. % f.l. % f.l. % f.l. % f.l. % 1 )1 4 )1 0Æ5 )1 0 1 5 1 5 1 5 )1 0 2 1 25 )1 0Æ5 )1 0 )1 0 )1 0 1 5 1 5 3 )1 4 1 2 )1 0 )1 0 1 5 )1 0 1 5 4 1 25 1 2 )1 0 1 5 )1 0 )1 0 )1 0 5 )1 4 )1 0Æ5 1 0Æ2 1 5 )1 0 )1 0 1 5 6 1 25 )1 0Æ5 1 0Æ2 )1 0 1 5 )1 0 )1 0 7 )1 4 1 2 1 0Æ2 )1 0 )1 0 1 5 )1 0 8 1 25 1 2 1 0Æ2 1 5 1 5 1 5 1 5 *Factor level. Concentration in % (w v). 80 Letters in Applied Microbiology 53, 79 83 ª 2011 The Society for Applied Microbiology

M. Kordowska-Wiater et al. A protective medium with Spirulina Table 2 Central composite design matrix with values of Lactobacillus rhamnosus viability Run Spirulina Sucrose Lactulose f.l.* % f.l.* % f.l.* % Mean viability (%) 1 )1 0Æ5 )1 5 )1 5 39Æ22 2 )1 0Æ5 )1 5 1 11 29Æ54 3 )1 0Æ5 1 11 )1 5 48Æ32 4 )1 0Æ5 1 11 1 11 41Æ96 5 1 1Æ1 )1 5 )1 5 27Æ89 6 1 1Æ1 )1 5 1 11 42Æ6 7 1 1Æ1 1 11 )1 5 96Æ04 8 1 1Æ1 1 11 1 11 99Æ13 9 )1Æ682 0Æ296 0 8 0 8 12Æ59 10 1Æ682 1Æ304 0 8 0 8 15 11 0 0Æ8 )1Æ682 2Æ96 0 8 62Æ15 12 0 0Æ8 1Æ682 13Æ04 0 8 13 0 0Æ8 0 8 )1Æ682 2Æ96 76Æ6 0 0Æ8 0 8 1Æ682 13Æ04 50Æ86 15 0 0Æ8 0 8 0 8 58Æ56 16 0 0Æ8 0 8 0 8 58Æ5 17 0 0Æ8 0 8 0 8 58Æ3 18 0 0Æ8 0 8 0 8 52Æ49 19 0 0Æ8 0 8 0 8 59Æ25 20 0 0Æ8 0 8 0 8 Æ64 *Factor level. Concentration in % (w v). Y ¼ b 0 þ X b i X i þ X b ii X 2 i þ X b ij X i X j ; ð1þ where Y represents a response variable, b 0 is the interception, b i is linear effect, b ii is quadratic effect, and b ij are interaction effect coefficients. X i, and X j, are coded values of the factors used in the model. A formula for the variables encoding can be found in Elibol (2004) and Huang et al. (2006). The following factors, preselected using a Plackett-Burman design, were included in the CCD model: Spirulina (X 1 ), sucrose (X 2 ) and lactulose (X 3 ). The 2 3 full-factorial CCD for three independent variables with six star points and the same number of replicates at centre points was used to maximize the percentage of cells after lyophilization. The goodness of fit of the obtained model was measured by determination coefficients R 2. Response surface 3-D plots were shown to illustrate the relationships between the experimental factors and the predicted values. The figures and the results of the analyses were obtained using Statistica version 7.0 (Stat Soft Inc., Tulsa, OK). Results Cryoprotective agents were selected for RSM based on their effects on the viability of Lact. rhamnosus E N cells during freeze-drying, as evaluated using the Placket-Burman method. The highest positive effect (102Æ025) was obtained for Spirulina, followed by sucrose (36Æ885). The effect of lactulose was also strong but negative ()34Æ42), which means that too high concentrations of this carbohydrate can lower cell viability during lyophilization. Table 2 shows a CCD for those three factors and the survival values for Lact. rhamnosus cells obtained in each run. The highest viability (96-99%) was obtained in samples with a protective medium containing 1Æ1% (w v) Spirulina, 11% (w v) sucrose, and different concentrations of lactulose. The quadratic regression model coefficients for the coded variables were determined so the formula (1) took the following form: Y ¼ 578157 þ 62636X 1 03964X 2 1 þ 97647X 2 þ 06651X 2 2 08509X 3 06334X 2 3 þ 107538X 1 X 2 03787X 1 X 3 08413X 2 X 3 The value of R 2 was 0Æ76, meaning that 76% of total variation was explained by the model. On the basis of the obtained regression model, the optimal protective medium composition was determined to be Spirulina 1Æ304% (w v), sucrose 13Æ04% (w v), and lactulose 5Æ48% (w v). Effects of selected protective agents on Lact. rhamnosus viability are presented on Fig. 1. The Letters in Applied Microbiology 53, 79 83 ª 2011 The Society for Applied Microbiology 81

A protective medium with Spirulina M. Kordowska-Wiater et al. 120 100 80 20 70 0 20 50 30 20 10 90 80 12 10 8 6 4 2 70 0 10 50 30 Sucrose (%) 12 10 8 Lactulose (%) 12 10 8 6 Lactulose (%) 6 4 2 4 2 2 0 4 0 8 1 0 1 2 1 4 0 2 0 6 0 2 0 4 0 6 4 6 Spirulina (%) 0 8 1 0 1 2 1 4 Spirulina (%) 8 10 Sucrose (%) Figure 1 Interaction effects of Spirulina (X 1 ) and sucrose (X 2 ); Spirulina (X 1 ) and lactulose (X 3 ); sucrose (X 2 ) and lactulose (X 3 )onlactobacillus rhamnosus E N viability (%) after freeze-drying. ( ) >80; ( ) <80; ( ) <; ( ) <; ( ) <20; ( ) <0; ( )<)20; ( ) >50; ( ) <50; ( ) <; ( ) <30; ( ) <20; ( ) <10; ( ) >80; ( ) <80; ( ) <70; ( ) <; ( ) <50; ( ) <. 12 predictive cell viability in this medium was estimated at 89Æ619%. When the bacterial strain was lyophilized in the optimal protective medium, a mean cell viability of 87Æ5% was achieved. To verify the effectiveness of the new protective medium in enhancing Lact. rhamnosus E N survival, the process of lyophilization of the bacteria in a standard protective mixture containing 8% (w v) skim milk and 6% (w v) sucrose was examined. Lactobacillus viability in that medium was only 16Æ26%, which confirms the very good protective ability of the medium optimized by RSM. Discussion The viability results obtained when Lact. rhamnosus E N was lyophilized in the optimal medium are higher than those reported by other authors (Carcoba and Rodrıguez 2000; Huang et al. 2006). Skim milk has been the most frequently used protective agent for freeze drying of lactic acid bacteria. L. lactis ssp. lactis CECT 5180 cells freezedried in skim milk had a viability of 44Æ3% (Carcoba and Rodrıguez 2000). Zayed and Roos (2004) found that skim milk powder offered a 22Æ4% survival rate for freeze-dried Lact. salivarius when used alone, and a survival rate of 83 85% when supplemented with trehalose and sucrose. Huang et al. (2006) reported on the basis of RSM that the optimum values for the independent variables investigated by them were sucrose 66Æ g l )1, glycerol 101Æ20 g l )1, sorbitol 113Æ00 g l )1 and skim milk 130Æ00 g l )1, with the corresponding viability of 86Æ53%. However, the addition of skim milk to some food products could lead to allergy. Our study shows that in the case of Lact. rhamnosus E N, skim milk can be replaced with Spirulina without a loss of cell viability. This observation is consistent with another study reporting a high nutritional value of Spirulina (Varga et al. 1999). A high amino acid content in Spirulina could explain its protective function. Some amino acids like methionine, proline, asparagine and threonine have been reported to have a cryoprotective effect (Rudolph and Crowe 1985). The protective action of the disaccharides sucrose and lactulose, on the other hand, could be because of their capacity to hydrate biological structures such as proteins and membranes, referred to as the water replacement hypothesis (Crowe et al. 2001). Their role in protecting cell membranes and stabilizing proteins from denaturation had already been studied (Crowe et al. 1988). A positive effect of sucrose had been previously observed during the preservation of frozen and freeze-dried LAB (Carcoba and Rodrıguez 2000; Zayed and Roos 2004; Siaterlis et al. 2009). Perez et al. (2002) found that lactulose was efficient in membrane stabilization during lyophilization. 82 Letters in Applied Microbiology 53, 79 83 ª 2011 The Society for Applied Microbiology

M. Kordowska-Wiater et al. A protective medium with Spirulina In comparison with other results (Huang et al. 2006), the value of R 2 in our study was lower. However, the analysis of variance (anova) indicated that the model was significant at the probability level of 0Æ03. In that case, the lower value of R 2 could be explained by cell aggregation caused by high biosynthesis of exopolysaccharides by Lact. rhamnosus E N. Concluding, the present investigation shows that the new protective medium containing Spirulina, sucrose and lactulose significantly increase Lact. rhamnosus E N viability, which indicate that it can be industrially applicable. Owing to addition of Spirulina, the final product will have the properties of synbiotic and dietary supplements. Acknowledgement This work was financially supported by grant R12 06303 from the Polish Ministry of Science and Higher Education. References Akalin, A.S., Unal, G. and Dalay, M.C. (2009) Influence of Spirulina platensis biomass on microbiological viability in traditional and probiotic yogurts during refrigerated storage. Ital J Food Sci 21, 357 364. Aryana, K.J. and McGrew, P. (2007) Quality attributes of yoghurt with Lactobacillus casei and various prebiotics. LWT Food Sci Technol, 1808 18. Carcoba, R. and Rodrıguez, A. (2000) Influence of cryoprotectants on the viability and acidifying of frozen and freezedried cells of the novel starter strain Lactococcus lactis ssp. lactis CECT 5180. Eur Food Res Technol 211, 433 437. Crowe, J.H., Crowe, L.M., Carpenter, J.F., Rudolph, A.S., Winstrom, C.A., Spargo, B.J. and Anchordoguy, T.J. (1988) Interactions of sugars with membranes. Biochim Biophys Acta 947, 367 384. Crowe, J.H., Crowe, L.M., Oliver, A.E., Tsvetkova, N., Wolkers, W. and Tablin, F. (2001) The trehaloze myth revisited: introduction to a symposium on stabilization of cells in the dry state. Cryobiology 43, 89 105. Elibol, M. (2004) Optimization of medium composition for actinorhodin production by Streptomyces coelicolor A3(2) with response surface methodology. Process Biochem 39, 1057 1062. Fonseca, F., Beal, C. and Corrieu, G. (2001) Operating conditions that affect the resistance of lactic acid bacteria to freezing and frozen storage. Cryobiology 43, 189 198. Hanrahan, G. and Lu, K. (2006) Application of factorial designs and Response Surface Methodology in modern experimental design and optimization. Crit Rev Anal Chem 36, 1 151. Huang, L., Lu, Z., Yuan, Y., Lü, F. and Bie, X. (2006) Optimization of a protective medium for enhancing the viability of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus based on response surface methodology. J Ind Microbiol Biotechnol 33, 55 61. Myers, R.H. and Montgomery, D.C. (1995) Response Surface Methodology. Process and Product Optimization Using Designed Experiments. NY: John Wiley & Sons. Perez, C., De Jesus, P. and Griebenow, K. (2002) Preservation of lysozyme structure and function upon encapsulation and relaease from poly(lactic-co-glycolic) acid microspheres prepared by the water-in-oil-in-water method. Int J Pharmacol 248, 193 206. Preetha, R., Jayaprakash, N.S., Rosamma, P. and Singh, I.S. (2007) Optimization of carbon and nitrogen sources and growth factors for the production of an aquaculture probiotic (Pseudomonas MCCB 103) using response surface methodology. J Appl Microbiol 102, 1043 1051. Rudolph, A.S. and Crowe, J.H. (1985) Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline. Cryobiology 22, 367 377. Saarela, M., Mogensen, G., Fonden, R., Mättö, J. and Mattila-Sandholm, T. (2000) Probiotic bacteria: safety, functional and technological properties. J Biotechnol 84, 197 215. Siaterlis, A., Deepika, G. and Charalampopoulos, D. (2009) Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying. Lett Appl Microbiol 48, 295 301. Varga, L., Szigeti, J. and Ördög, V. (1999) Effect of Spirulina platensis biomass enriched with trace elements on combinations of starter culture strains employed in the dairy industry. Milchwissenschaft 54, 247 248. Xiaobo, Z., Haiying, W., Linyu, H., Yongcheng, L. and Zhongtao, L. (2006) Medium optimization of carbon and nitrogen sources for the production of eucalyptene A and xyloketal A from Xylaria sp. 2508 using response surface methodology. Process Biochem 41, 293 298. 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, 1081 1086. Letters in Applied Microbiology 53, 79 83 ª 2011 The Society for Applied Microbiology 83