APPLICATION OF PLACKETT-BURMAN DESIGN IN SCREENING FREEZE DRYING CRYOPROTECTANTS FOR Lactobacillus bulgaricus

Abstract

Lactobacillus bulgaricus is the bacteria commonly used in probiotic dairy product, including yogurt and cheese. The bacteria may be stored for long periods of time if it is freeze-dried. The cryoprotectant mixture for L. bulgaricus was optimized during the process of freeze-drying using a Plackett-Burman design and the steepest ascent test. In our initial tests, the cell survival rate and the number of viable cells were associated with the type of cyroprotectant used. Therefore, our optimization protocol focused on increasing survival rate. Substances that previously had a protective effect during freeze-drying were investigated, for example: sucrose, lactose, skim milk powder, sodium bicarbonate, sodium glutamate, magnesium sulfate, sodium ascorbate, yeast extract, vitamin B2, and phosphate buffer. We determined that the optimum cryoprotectant composition for L. bulgaricus consists of 28.0 g/100 mL skim milk powder, 24.0 g/100 mL lactose and 4.8 g/100 mL sodium ascorbate. The optimized cryoprotectant provides a 63.25% cell survival rate.

Headnote

Lactobacillus bulgaricus is the bacteria commonly used in probiotic dairy product, including yogurt and cheese. The bacteria may be stored for long periods of time if it is freeze-dried. The cryoprotectant mixture for L. bulgaricus was optimized during the process of freeze-drying using a Plackett-Burman design and the steepest ascent test. In our initial tests, the cell survival rate and the number of viable cells were associated with the type of cyroprotectant used. Therefore, our optimization protocol focused on increasing survival rate. Substances that previously had a protective effect during freeze-drying were investigated, for example: sucrose, lactose, skim milk powder, sodium bicarbonate, sodium glutamate, magnesium sulfate, sodium ascorbate, yeast extract, vitamin B2, and phosphate buffer. We determined that the optimum cryoprotectant composition for L. bulgaricus consists of 28.0 g/100 mL skim milk powder, 24.0 g/100 mL lactose and 4.8 g/100 mL sodium ascorbate. The optimized cryoprotectant provides a 63.25% cell survival rate.

Keywords: Lactobacillus bulgaricus, freeze-drying, cryoprotectant, bacteria survival rate, Plackett-Burman design

Introduction

The Gram-positive bacterium Lactobacillus bulgaricus is a member of the acidophilus group of lactobacilli and is widely used in probiotic cultures (van de Guchte et al., 2006). L. bulgaricus is employed worldwide because it is able to produce lactic acid in the production of yogurt, cheese and other fermented products (Guillouard et al., 2004; De Urraza et al., 1997) and is of vital importance to the food industry, in combination with Streptococcus thermophilus.

During the preparation of the starter cultures, the production and maintenance techniques that maximize the storage stability, viability and activity of the bacterial cells must be established (Passot et al., 2012). Freeze-drying is the most convenient and successful method of preserving bacteria, yeasts and sporulating fungi (Berny et al., 1991) and it has been widely used in microbiology for many decades to stabilize and store cultures (Morgan et al., 2009). However, not all bacterial strains survive the freeze-drying process and quantitative viability rates as low as 0.1% have been reported (Abadias et al., 2001). The major causes of cell death during freeze-drying are related to ice crystal formation, membrane damage from high osmolarity due to high concentrations of internal solutes, macromolecule denaturation, and the removal of water, which affects properties of many hydrophilic macromolecules in cells (Thammavongs et al., 1996; Chitra et al., 2003; De Paz et al., 2002; Allison et al., 1999). For this reason, a variety of protective agents have been added to the drying media before freeze-drying to protect the viability of probiotics during dehydration (Hubalek, 2003).

The stability of probiotic microorganisms during freeze-drying and storage may be enhanced by the addition of protective agents (Zayed and Roos, 2004). It is well documented that carbohydrates have protective effects for probiotic bacteria during freeze-drying. For example, the remarkably high glass transition temperature (Tg) of trehalose can raise the glass-phase transition temperature of cells and therefore viable cells can be protected by reaching the glassy phase without nucleating intracellular ice (Fowler et al., 2005). Apart from this, sorbitol (Linders et al., 1997; Carvalho et al., 2002), mannitol (Efiuvwevwere et al., 1999), sucrose (Carvalho et al., 2003), lactose (Higl et al., 2007), mannose (Carvalho et al., 2004a), inulin, and fructo-oligosaccharides (Schwab et al., 2007) were reported to have the same impact. Amino acids may have the same protective effects as carbohydrates. A study by Mattern et al. (1999) showed that phenylalanine, arginine, and glycine can prevent denaturation during protein vacuum-drying. Sodium glutamate can also protect the cell (de Valdez et al., 1983; Teixeira et al., 1995). Several studies have suggested that some salt buffers, such as NaCl or KCl (Carvalho et al., 2003), sodium citrate (Kets et al., 2004; Kurtmann et al., 2009), phosphate (Ohtake,2004), calcium carbonate and manganese sulfate can help to protect cells during freeze-drying if they are used with other protectants. Buitink et al. (2000) found that proteins had a higher Tg than sugar and suggested that proteins play an important role in glass formation. Hence, proteins, including skim milk, whey protein, blood serum, serum albumin and peptone are efficient desiccation protectants (Hubalek et al., 2003; Abadias et al., 2001).

The classic method of cryoprotectant optimization involves changing one ingredient at a time, keeping the other substrate components at fixed levels. This laborious and time-consuming method does not guarantee that the optimal mixture will be determined. The Plackett-Burman design is a statistical screening method where n variables are studied in n+1 experimental runs and since there are fewer experimental replicates, time and resources are saved (Srinivas et al., 1994; Carvalho et al., 1997). Moreover, the design is orthogonal in nature, implying that the effect of each variable is pure in nature and not confounded with interaction between variables. Software to plan the experimental design and to run the data analysis makes the analysis easier than other approaches (Naveena et al., 2005). Furthermore, the steepest ascent test can determine the correct dosage of the significant factors.

In our previous work, we studied the effects of sucrose, lactose, skim milk, yeast, vitamin B2, NaHCO3, MgSO4, sodium ascorbate, sodium glutamate, and phosphate buffer on the survival of Lactobacillus bulgaricus during freeze-drying in single factor experiments. We found that the optimum concentration of single protective agents for L. bulgaricus during freeze-drying was 25% (W/V) sucrose, 20% (W/V) lactose, 25% (W/V) skim milk, 20% (W/V) yeast, 25% (W/V) vitamin B2, and 4.5% (W/V) sodium ascorbate. The survival rate with these concentrations were 24.5% with sucrose, 35.6% with lactose, 64.4% with skim milk, 62.2% with yeast, 16.3% with vitamin B2, and 84.7% with sodium ascorbate (Chen et al., 2013a; Chen et al., 2013b). In this paper, we chose several protective agents based on previous studies to screen and optimize a mixture of cryoprotectant substances when freeze-drying L. bulgaricus and to measure the survival and viability of cells after freeze-drying.

Materials and Methods

Microorganism and growth medium

Lactobacillus bulgaricus was obtained from the School of Food and Biological Engineering, Shaanxi University of Science & Technology (Xi'an, China). The strain was isolated from commercial yogurt and has the ability to ferment goat milk when inoculated with 3% culture starter of Streptococcus thermophilus and Lactobacillus bulgaricus (at a ratio of 1:1.5) and added to 8% sugar, then incubated at 43°C for 4h. The total number of viable cells can reach up to 1.90×109CFU/mL, with pH 4.16 and an optimal incubation time of 18h (Wang, 2011). MRS medium was used for activation and cultivation of L. bulgaricus. Viability of cells was determined on MRS agar medium.

Lactobacillus bulgaricus culture and microorganism collection

Activated L. bulgaricus was inoculated with 4% (v/v) inoculum in MRS medium, then incubated at 37°C for 18h. The culture was centrifuged at 6000×g for 10 min to harvest the L. bulgaricus cells.

Preparation of protective agents

The protective agent solutes used in the experiment were made with distilled water and formulated into various concentrations. The sugars were sterilized at 115°C for 15 min. Because amino acids and vitamin B2 cannot tolerate high temperatures, the concentrated solution of these compounds were sterilized by filtration using a membrane with pore size of 0.22 μm, which was preliminary sterilized at 121°C for 20 min. The other protective agents were sterilized at 108°C for 15 min.

Freeze-drying

The L. bulgaricus cells were frozen at -40°C for 12-24h after protective agents were added and then frozen at -51°C, 6.93 Pa for 24h using a vacuum freeze dryer.

Viable count

The diluted bacterial suspension was aliquoted into 0.1 mL doses with a syringe and dropped into a count plate, then spread uniformly. The undercut plate was incubated at 37°C for 36-48h and then the viable L. bulgaricus cells were counted.

Calculation of survival

Survival rate was calculated as the number of viable cells after drying/number of viable cells before drying×100%

Plackett-Burman screening of protective agents and the steepest ascent test

The Plackett-Burman design used 10 factors spanning 12 runs, with each factor fixed at two levels (namely a lower level and a higher level, represented by +1 and -1) based on optimizations in previous studies (Chen et al., 2013a; Chen et al., 2013b). The protective agents tested are listed in Table 1, along with the amount used. These agents included sucrose, lactose, skim milk powder, sodium bicarbonate, sodium glutamate, magnesium sulfate, sodium ascorbate, yeast extract, vitamin B2 and phosphate buffer, which were screened for their impact on viable counts and survival rates of L. bulgaricus cells.

Statistical analysis

A statistical analysis was performed in SAS (Version, 12.0, SAS Institute Inc., Cary, NC, USA) to identify the significant variables and their corresponding coefficients. The coefficient, sum of squares (SS %), and confidence intervals (CI) were evaluated to analyze the number of viable bacteria and the survival rate from each of the trials.

Results

Plackett-Burman screening of protective agents for Lactobacillus bulgaricus

The relationship between protective agents and the survival rate of L. bulgaricus is shown in Table 2. The survival rate of freeze-dried L. bulgaricus cells is represented by Y1 (%) and the number of viable cells of freeze-dried powder is represented by Y2 (×1011 CFU/g). The survival rate was calculated by a formula containing a factor of viable cells, where the influence of protective agents was measured by the survival rate. All of the protective agents had different effects on the cells so that when the agents were changed, the survival rate of L. bulgaricus also changed.

Three variables namely lactose, sodium ascorbate and skim milk powder accounted for a large proportion of the percent sum of squares on the Pareto chart (Fig. 1). This indicates that these three variables had a significant impact on the survival rate and they protect the cells better than the other agents tested in this study. Furthermore, the trend lines of the 95% confidence interval of these factors (Fig. 2) suggested that these three variables had a positive effect. When the concentrations of these three variables were increased, the survival rate of L. bulgaricus also gradually increased.

The path of steepest ascent

The results of the Plackett-Burman design suggests that lactose, sodium ascorbate and skim milk powder can protect the cell and significantly affect the survival rate of L. bulgaricus (Fig. 1). We increased or decreased concentrations of the significant factors, according to the signs of their main effects. The path of steepest ascent design and results are shown in Table 3. The value of Y1 and Y2 represent the survival rate of freeze-dried L. bulgaricus cells and the number of viable cells in the freeze-dried powder. The number of viable cells and the cell survival rate in the freeze-dried powder was higher in group 3 than the other groups. The optimized concentrations of the three factors in group 3 were 28.0 g/100 mL of lactose, 24.0 g/100 mL of sodium ascorbate, and 4.8 g/100 mL of skim milk powder.

Discussion

Freeze-drying has been used to manufacture lactic acid bacteria powders for decades and is based upon sublimation. Typically, cells are first frozen and then dried by sublimation under high vacuum (Santivarangkna et al., 2007). It has been shown that cellular inactivation occurs mostly at the freezing step (Tsvetkov et al., 1983). During freezing, the material is gradually dehydrated and ice slowly forms outside the cell, leading to extensive cellular damage. In addition, water bound in each cell plays an important role in stabilizing the structure and function of biological macromolecules, including those present on the cell wall and cell membrane. Consequently, water removal during freeze-drying can lead to destabilization of the structural integrity of these cellular components, resulting in loss or impairment of function (Brennan et al., 1986).

Therefore, in order to protect cells during freeze-drying, many studies have focused on approaches to minimize damage by protective agents. Sugars were reported to exhibit enhanced desiccation tolerance by forming hydrogen bonds with proteins during drying, which help maintaining the tertiary protein structure in the absence of water (Leslie et al., 1995). Proteins can also decrease the injury to cells. Skim milk has been used as a drying medium because it can prevent cellular damage by stabilizing the cell membrane constituents (Castro et al., 1995). Additionally, skim milk can protect microbial cells from damage caused by the formation of ice crystals during freezing, because proteins in skim milk can form a viscous layer on the surface of the cells, which can increase the solution's viscosity and maintain amorphous ice crystals near the cell (Carvalho et al., 2004b). This indicates that skim milk is a suitable cryoprotectant for L. bulgaricus.

The loss of activity of the freeze-dried cultures can also be a consequence of cell damage and membrane lipid oxidation (Castro et al., 1997). The addition of ascorbic acid to the drying medium has already been demonstrated to have a protective effect on L. bulgaricus during storage (Teixeira et al., 1995). Kurtmann et al. (2009) observed that the detrimental effects of atmospheric oxygen was reduced by including ascorbate in the freeze drying medium for L. acidophilus. This may explain why ascorbic acid protected the cells during freeze-drying in our experiments.

Conclusions

In this study sucrose, lactose, skim milk powder, sodium bicarbonate, sodium glutamate, magnesium sulfate, sodium ascorbate, yeast extract, vitamin B2, and phosphate buffer were investigated as protective agents for freeze-dried Lactobacillus bulgaricus. Screening of cryooprotectant contents for L. bulgaricus used a Plackett-Burman design and demonstrated that skim milk powder, sodium ascorbate and lactose have a significant impact on the survival of L. bulgaricus during freeze-drying and all of the above protective agents were demonstrated to have a positive effect. We found that the optimum concentration of these three agents was 28.0 g/100 mL skim milk, 24.0 g/100 mL sodium ascorbate, and 4.8 g/100 mL lactose. These results provide the basis for the safe and effective long-term storage of Lactobacillus bulgaricus and will provide economic benefits to the yogurt and cheese industry.

Acknowledgements

The project was partly supported by the Science and Technology Research Development plan project of Shaanxi Province (No. 2014K01-17-07), Shaanxi Provincial Education Department (No. 2013JK0747) and the Key Technology Project of Xianyang city (2014K01-15).

References

References

Abadias, M., Benabarre, A., Teixido, N., Usall, J., Vinas, I. 2001. Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake. International Journal of Food Microbiology, 65, 173-82.

Abadias, M., Teixido, N., Usall, J., Benabarre, A., Vinas, I. 2001. Viability, efficacy, and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media. Journal of Food Protection, 64(6), 856-861.

Allison, S.D.,Chtang, B., Randolph, T.W., Carpenter, J.F.1999. Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding. Archives of Biochemistry and Biophysics, 365, 289-298.

Berny, J.F., Hennebert, G.L. 1991.Viability and stability of yeast cells and filamentous fungus spores during freeze-drying: effects of protectants and cooling rates. Mycologia, 83, 805-15.

Brennan, M., Wanismail, B., Johnson, M.C., Ray B. 1986. Cellular damage in dried Lactobacillus acidophilus. Journal of Food Protection, 49, 47-53.

Buitink, J., van den Dries, I.J., Hoekstra, F.A., Alberda, M., Hemminga, M.A. 2000. High critical temperature above Tg may contribute to the stability of biological systems. Biophysical Journal, 79(2), 1119-1128.

Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X., Gibbs, P. 2002. Survival of freeze-dried Lactobacillus plantarum and Lactobacillus rhamnosus during storage in the presence of protectants. Biotechnology Letters, 24, 1587-1591.

Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata F.X., 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. Journal of Food Science, 68, 2538-2541.

Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X., 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. Biotechnology Progress, 20, 248-254.

Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X., Gibbs, P. 2004b. Relevant factors for the preparation of freeze-dried lactic acid bacteria. International Dairy Journal, 14(10), 835-847.

Carvalho, C.M.L., Serralheiro, M.L.M., Cabral, J.M.S., Airebarros, M.R. 1997. Application of factorial design to the study of transesterification reactions using cutinase in AOT-reversed micelles. Enzyme and Microbial Technology, 27, 117-123.

Castro, H., Teixeira, P., Kirby, R. 1995. Storage of lyophilized cultures of Lactobacillus bulgaricus under different relative humidities and atmospheres. Applied Microbiology and Biotechnology, 44, 172-176.

Castro, H.P., Teixeira, P.M., Kirby, R. 1997. Evidence of membrane damage in Lactobacillus bulgaricus following freezedrying. Journal of Applied Microbiology, 82, 87-94.

Chen, H., Wang, J., Luo, Q., Shu, G.W. 2013a. Effect of NaHCO3, MgSO4, sodium ascorbate, sodium glutamate, phosphate buffer on survival of Lactobacillus bulgaricus during freeze-drying. Advance Journal of Food Science and Technology, 5(6), 771-774.

Chen, H., Zhang, Q.H., Luo, Q., Shu, G.W. 2013b. Effect of five materials including sucrose, lactose, skim milk, yeast, vitamin B2 on survival of Lactobacillus bulgaricus during freeze-drying. Advanced Materials Research, 700, 255-258.

Chitra, T., Lian, Y.U. 2003. Effective inhibition of mannitol crystallization in frozen solutions by sodium chloride. Pharmaceutical Research (Dordrecht), 20, 660-667.

De Paz, R.A., Dale, D.A., Barnett, C.C., Carpenter, J.F., Gaertner, A.L., Radolph T.W. 2002. Effects of drying methods and additives on the structure, function, and storage stability of subtilisin; role of protein conformation and molecular mobility. Enzyme and Microbial Technology, 31, 765-774.

De Urraza, P., De Antoni, G. 1997. Induced cryotolerance of Lactobacillus delbrueckii subsp. bulgaricus LBB by preincubation at suboptimal temperatures with a fermentable sugar. Cryobiology, 35, 159-164.

de Valdez, G.F., de Giori, G.S., de Ruiz Holgado, A.P., Oliver, G. 1983. Comparative study of the efficiency of some additives in protecting lactic acid bacteria against freeze-drying. Cryobiology, 20, 560-566.

Efiuvwevwere, B.J.O., Gorris, L.G.M., Smid, E.J., Kets, E.P.W. 1999. Mannitol-enhanced survival of Lactococcus lactis subjected to drying. Applied Microbiology and Biotechnology, 51, 100-104.

Fowler, A., Tone,r M. 2005. Cryo-injury and biopreservation. Annals of the New York Academy of Sciences, 1066, 119-135.

Guillouard, I., Lim, E., Van de Guchte, M., Grimaldi, C.,et al. 2004. Tolerance and adaptative acid stress of Lactobacillus delbrueckii ssp. bulgaricus. Lait, 84, 1-6.

Higl, B., Kurtmann, L., Carlsen, C.U., Ratjen, J., Forst, P., Skibsted, L.H., Kulozik U., Risbo, J. 2007. Impact of water activity, temperature, and physical state on the storage stability of Lactobacillus paracasei ssp. paracasei freeze-dried in a lactose matrix. Biotechnology Progress, 23, 794-800.

Hubalek, Z. 2003. Protectants used in the cryopreservation of microorganisms. Cryobiology, 46, 205-229.

Kets, E.P.W., Jpelaar, P.J.I., Hoekstra, F.A., Vromans, H. 2004. Citrate increases glass transition temperature of vitrified sucrose preparations. Cryobiology, 48, 46-54.

Kurtmann, L., Carlsen, C.U., Risbo, J., Skibsted, L.H. 2009. Storage stability of freeze- dried Lactobacillus acidophilus (La-5) in relation to water activity and presence of oxygen and ascorbate. Cryobiology, 58(2), 175-180.

Leslie, S.B., Israeli, E., Lighthart, B., Crowe, J.H. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Applied Environmental Microbiology, 61(10), 3592-3597.

Linders, J.M., de Jong, G.I.W., Meerdink, G., Vantriet, K. 1997. Carbohydrates and the dehydration inactivation of Lactobacillus plantarum: the role of moisture distribution and water activity. Journal of Food Engineering, 31, 237-250.

Mattern, M., Winter, G., Kohnert, U., Lee, G. 1999. Formulation of proteins in vacuum-dried glasses. II. Process and storage stability in sugar-free amino acid systems. Pharmaceutical Development and Technology, 4(2), 199-208.

Morgan, C., Vesey, G. 2009. Freeze-Drying of Microorganisms Encyclopaedia of Microbiology (3rd Edition). Academic Press, Oxford, UK, 162-173.

Naveena, B.J., Altaf, M., Bhadriah, K., Reddy, G. 2005. Selection of medium components by Plackett-Burman design for production of L (+) lactic acid by Lactobacillus amylophilus GV6 in SSF using wheat bran. Bioresource Technology, 96, 485-490.

Ohtake, S. 2004. Effect of sugar-phosphate mixtures on the stability of DPPC membranes in dehydrated systems. Cryobiology, 48, 81-89.

Passot, S., Cenard, S., Douania, I., Tréléa, I.C., et al. 2012. Critical water activity and amorphous state for optimal preservation of lyophilised lactic acid bacteria. Food Chemistry, 132, 1699-1705.

Santivarangkna, C., Kulozik, U., Foerst, P. 2007. Alternative drying processes for the industrial preservation of lactic acid starter cultures. Biotechnology Progress, 23, 302- 315.

Schwab, C., Vogel, R., Gänzle, M.G. 2007. Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying. Cryobiology, 55(2), 108-114.

Srinivas, M.R.S., Naginchand, Lonsane, B.K. 1994. Use of Plackett-Burman design for rapid screening of several nitrogen sources, growth/product promoters, minerals and enzyme inducers for the production of alpha-galactosidase by Aspergillus niger MRSS 234 in solid state fermentation. Bioprocess Engineering, 10, 139-144.

Teixeira, P.C., Castro, M.H., Malcata, F.X., Kirby, R.M. 1995. Survival of Lactobacillus delbruckii ssp. bulgaricus following spray drying. Journal of Dairy Science, 78, 1025- 1031.

Thammavongs, B., Corroler, D., Panoff, J.M., Auffray, Y., et al. 1996. Physiological response of Enterococcus faecalis JH2-2 to cold shock: growth at low temperatures and freezing/thawing challenge. Letters of Applied Microbiology, 23, 398-402.

Tsvetkov, T., Brankova, R. 1983. Viability of micrococci and lactobacilli upon freezing and freeze-drying in the presence of different cryoprotectants. Cryobiology, 20, 318-323.

van de Guchte, M., Penaud, S., Grimaldi, C., Barbe, V., et al. 2006. The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution. Proceedings of the National Academy of Sciences, 103, 9274-9279.

Wang, C.F. 2011. The study on preparation techology of probiotics and synbiotics goat yogurt. Shaanxi University of Science & Technology, Xi'an, China.

Zayed, G., Roos, Y.H. 2004. Influence of trehalose and moisture content on survival of Lactobacillus salivarius subjected to freeze-drying and storage. Process Biochemistry, 39, 1081-1086.