Enhacement of the Viability of Lactobacillus plantarum during the Preservation and Storage Process Based on the Response Surface Methodology


Objective: The Response Surface Methodology (RSM) is a commonly used system to optimize cell viability of probiotic strains when they are subjected to different preservation and storage processes. Methods and Results: To determine the optimal levels of incorporation of several cry oprotectants (skim milk, sucrose and trehalose) in the freeze-drying process of Lactobacillus plantarum, a range of experiments based on a Rotational Central Composite design (CCD) were conducted. The results were adjusted to a quadratic model, resulting in the presence of interaction between the different variables. Solving a regression equation, we obtained the optimum concentrations of cryoprotective agents: 24.06% milk powder, 6.22% sucrose, 5.63% trehalose. To visualize the interactions between the three variables involved in the study, Design Expert? software was used. Conclusions: The analysis reveals that while trehalose has a direct effect on the viability of L. plantarum, skim milk and sucrose exert quadratic effects. There are also interactions between cryoprotectants, which emphasize the synergies produced between milk and sucrose and between sucrose and trehalose, which allows maintaining the viability of L. plantarum. Significance and Impact of the Study: The addition of new oligosaccharides as trehalose in premixtures for functional feed can maintain the viability of L. plantarum during longer periods of time, ensuring the proper administration of probiotics to their destinations.

Share and Cite:

Gisela, G. , Leonardo, A. , Lucia, P. , Rodrigo, V. , Eduard, G. and Angeles, C. (2014) Enhacement of the Viability of Lactobacillus plantarum during the Preservation and Storage Process Based on the Response Surface Methodology. Food and Nutrition Sciences, 5, 1746-1755. doi: 10.4236/fns.2014.518188.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Parkes, G.C., Sanderson, J.D. and Whelan, K. (2009) The Mechanisms and Efficacy of Probiotics in the Prevention of Clostridium difficile-Associated Diarrhea. The Lancet Infectious Diseases, 9, 237-244.
[2] Collado, M.C., Isolauri, E., Salminen, S. and Sanz, Y. (2009) The Impact of Probiotic on Gut Health. Current Drug Metabolism, 10, 68-78.
[3] Lomax, A.R. and Calder, P.C. (2009) Probiotics, Immune Function, Infection and Inflammation: A Review of the Evidence from Studies Conducted in Humans. Current Pharmaceutical Design, 15, 1428-1518.
[4] Veerappan, G.R., Betteridge, J. and Young, P.E. (2012) Probiotics for the Treatment of Inflammatory Bowel Disease. Current Gastroenterology Reports, 14, 324-333.
[5] Vilà, B., Fontgibell, A., Badiola, I., Esteve-Garcia, E., Jiménez, G., Castillo, M. and Brufau, J. (2009) Reduction of Salmonella enterica var. enteritidis Colonization and Invasion by Bacillus cereus var. toyoi Inclusion in Poultry Feeds. Poultry Science, 88, 975-979.
[6] Pâtruicâ, S. and Mot, D. (2012) The Effect of Using Prebiotic and Probiotic Products on Intestinal Micro-Flora of the Honeybee (Apis mellifera carpatica). Bulletin of Entomological Research, 102, 619-623.
[7] Cebrián, R., Baños, A., Valdivia, E., Pérez-Pulido, R., Martínez-Bueno, M. and Maqueda, M. (2012) Characterization of Functional, Safety, and Probiotic Properties of Enterococcus faecalis UGRA10, a New AS-48-Producer Strain. Food Microbiology, 30, 59-67.
[8] Huyghebaert, G., Ducatelle, R. and Van Immerseel, F. (2011) An Update on Alternatives to Antimicrobial Growth Promoters for Broilers. The Veterinary Journal, 187, 182-188.
[9] Nagpal, R., Kumar, A., Kumar, M., Behare, P.V., Jain, S. and Yadav, H. (2012) Probiotics, Their Health Benefits and Applications for Developing Healthier Foods: A Review. FEMS Microbiology Letters, 334, 1-15.
[10] Wright, S. (2009) Homogeneity and Stability of Nutritional Premixes. www.naturalproductsinsider.com/articles/2009/07/homogeneity-and-stability-of-nutritional-premixes.aspx
[11] Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X. and Gibbs, P. (2003) Impedimetric Method for Estimating the Residual Activity of Freeze-Dried Lactobacillus delbrueckii ssp. bulgaricus. International Dairy Journal, 13, 463-468.
[12] Li, H.P., Lu, M.J., Guo, H.F., Li, W. and Zhang, H.P. (2010) Protective Effect of Sucrose on the Membrane Properties of Lactobacillus casei Zhang Subjected to Freeze-Drying. Journal of Food Protection, 73, 715-719.
[13] Selmer-Olsen, E., Birkeland, S.E. and Sørhaug, T. (1999) Effect of Protective Solutes on Leakage from and Survival of Immobilized Lactobacillus Subjected to Drying, Storage and Rehydration. Journal of Applied Microbiology, 87, 429-437.
[14] Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X. and Gibbs, P. (2004) Relevant Factors for the Preparation of Freeze-Dried Lactic Acid Bacteria. International Dairy Journal, 14, 835-847.
[15] Heckly, R.J. (1985) Principles of Preserving Bacteria by Freeze-Drying. Developments in Industrial Microbiology, 26, 379-395.
[16] Palmfeldt, J., Adsträom, P.R. and Hahn-Häagerdal, B. (2003) Optimisation of Initial Cell Concentration Enhances Freeze-Drying Tolerance of Pseudomonas chlororaphis. Cryobiology, 47, 21-29.
[17] FEFANA (2005) Probiotics in Animal Nutrition. www.fefana.org/resources/documents/publications/total%20def%20probio.pdf
[18] Font, G., Savoy, G., Pesce, A. and Oliver, G. (1983) Comparative Study of the Efficiency of Some Additives in Protecting Lactic Acid Bacteria against Freeze-Drying. Cryobiology, 20, 560-566.
[19] Schwab, C., Vogel, R. and Gänzle, M.G. (2007) Influence of Oligosaccharides on the Viability and Membrane Properties of Lactobacillus reuteri TMW1.106 during Freeze-Drying. Cryobiology, 55, 108-114.
[20] Crow, J.H., Carpenter, J.F. and Crowe, L.M. (1973) The Role of Vitrification in Anhydrobiosis. Annual Review of Physiology, 60, 73-103.
[21] Villarreal, M.A., Díaz, S.B., Disalvo, E.A. and Montich, G.G. (2004) Molecular Dynamics Simulation Study of the Interaction of Trehalose with Lipid Membranes. Langmuir, 20, 7844-7851.
[22] Hubalek, Z. (2003) Protectants Used in the Cryopreservation of Microorganisms. Cryobiology, 46, 205-229.
[23] Guilio, B.P., Orlando, P., Barba, G., Coppola, R., De Rosa, M., Sada, A., De Prisco, P. and Nazarro, F. (2005) Use of Alginate and Cryoprotective Sugars to Improve the Viability of Lactic Acid Bacteria after Freezing and Freeze-Drying. World Journal of Microbiology and Biotechnology, 21, 739-746.
[24] 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. Journal of Industrial Microbiology & Biotechnology, 33, 55-61.
[25] Khoramnia, A., Abdullah, N., Liew, S.L., Sieo, C.C., Ramasamy, K. and Ho, Y.W. (2011) Enhancement of Viability of a Probiotic Lactobacillus Strain for Poultry during Freeze-Drying and Storage Using the Response Surface Methodology. Journal of Animal Science, 82, 127-135.
[26] Pérez, L. (2011) Evaluación de nuevas cepas probióticas: Efectos sobre micotoxinas y en Helicicultura. Ph.D. Dissertation, Autonomous University of Barcelona, Bellaterra.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.