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- Bergenholtz, Sa Schoug, et al.
(författare)
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A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying
- 2012
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Ingår i: Cryobiology. - : Elsevier BV. - 0011-2240 .- 1090-2392. ; 64:3, s. 152-159
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Tidskriftsartikel (refereegranskat)abstract
- Freeze-drying of bacterial cells with retained viability and activity after storage requires appropriate formulation, i.e. mixing of physiologically adapted cell populations with suitable protective agents, and control of the freeze-drying process. Product manufacturing may alter the clinical effects of probiotics and it is essential to identify and understand possible factor co-dependencies during manufacturing. The physical solid-state behavior of the formulation and the freeze-drying parameters are critical for bacterial survival and thus process optimization is important, independent of strain. However, the maximum yield achievable is also strain-specific and strain survival is governed by e.g. medium, cell type, physiological state, excipients used, and process. The use of preferred compatible solutes for cross-protection of Lactobacilli during industrial manufacturing may be a natural step to introduce robustness, but knowledge is lacking on how compatible solutes, such as betaine, influence formulation properties and cell survival. This study characterized betaine formulations, with and without sucrose, and tested these with the model lactic acid bacteria Lactobacillus coryniformis Si3. Betaine alone did not act as a lyo-protectant and thus betaine import prior to freeze-drying should be avoided. Differences in protective agents were analyzed by calorimetry, which proved to be a suitable tool for evaluating the characteristics of the freeze-dried end products.
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| 2. |
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| 3. |
- Schoug, Åsa, et al.
(författare)
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Differential effects of polymers PVP90 and Ficoll400 on storage stability and viability of Lactobacillus coryniformis Si3 freeze-dried in sucrose
- 2010
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Ingår i: Journal of Applied Microbiology. - : Oxford University Press (OUP). - 1364-5072 .- 1365-2672. ; 108:3, s. 1032-1040
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Tidskriftsartikel (refereegranskat)abstract
- AIMS: To investigate the effect of freeze-dried Lactobacillus coryniformis Si3 on storage stability by adding polymers to sucrose-based formulations and to examine the relationship between amorphous matrix stability and cell viability. METHODS AND RESULTS: The resistance to moisture-induced sucrose crystallization and effects on the glass transition temperature (Tg) by the addition of polymers to the formulation were determined by different calorimetric techniques. Both polymers increased the amorphous matrix stability compared to the control, and poly(vinyl)pyrrolidone K90 was more effective in increasing amorphous stability than Ficoll 400. The viability of Lact. coryniformis Si3 after storage was investigated by plate counts following exposure to different moisture levels and temperatures for up to 3 months. The polymers enhanced the cellular viability to different degrees, dependent upon polymer and storage condition. CONCLUSIONS: Polymers can be used to enhance the stability of freeze-dried Lact. coryniformis Si3 products, but cell viability and matrix stability do not always correlate. The general rule of thumb to keep a highly amorphous product 50 degrees below its Tg for overall stability seemed to apply for this type of bacterial products. We showed that by combining thermal analysis with plate counts, it was possible to determine storage conditions where cell viability and matrix stability were kept high. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will aid in the rational formulation design and proper determination of storage conditions for freeze-dried and highly amorphous lactic acid bacteria formulations. We propose a hypothesis of reason for different stabilizing effects on the cells by the different polymers based on our findings and previous findings.
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| 4. |
- Schoug, Åsa, et al.
(författare)
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Freeze-drying of Lactobacillus coryniformis Si3--effects of sucrose concentration, cell density, and freezing rate on cell survival and thermophysical properties
- 2006
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Ingår i: Cryobiology. - Maryland Heights, USA : Elsevier BV. - 0011-2240 .- 1090-2392. ; 53:1, s. 119-127
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Tidskriftsartikel (refereegranskat)abstract
- Freeze-drying is commonly used to stabilize lactic acid bacteria. Many factors have been reported to influence freeze-drying survival, including bacterial species, cell density, lyoprotectant, freezing rate, and other process parameters. Lactobacillus coryniformis Si3 has broad antifungal activity and a potential use as a food and feed biopreservative. This strain is considered more stress sensitive, with a low freeze-drying survival, compared to other commercialized antifungal lactic acid bacterial strains. We used a response surface methodology to evaluate the effects of varying sucrose concentration, cell density and freezing rate on Lb. coryniformis Si3 freeze-drying survival. The water activity of the dry product, as well as selected thermophysical properties of importance for freeze-drying; degree of water crystallization and the glass transition temperature of the maximally freeze concentrated amorphous phase (Tg') were determined. The survival of Lb. coryniformis Si3 varied from less than 6% to over 70% between the different conditions. All the factors studied influenced freeze-drying survival and the most important factor for survival is the freezing rate, with an optimum at 2.8 degrees C/min. We found a co-dependency between freezing rate and formulation ingredients, indicating a complex system and the need to use statistical tools to detect important interactions. The degree of water crystallization decreased and the final water activity increased as a function of sucrose concentration. The degree of water crystallization and Tg' was not affected by the addition of 10(8)-10(10) CFU/mI. At 10(11) CFU/ml, these thermophysical values decreased possibly due to increased amounts of cell-associated unfrozen water.
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| 5. |
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| 6. |
- Schoug, Åsa, et al.
(författare)
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Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of Lactobacillus coryniformis Si3
- 2008
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Ingår i: Journal of Industrial Microbiology & Biotechnology. - Heidelberg, Germany : Springer. - 1367-5435 .- 1476-5535. ; 35:3, s. 175-181
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Tidskriftsartikel (refereegranskat)abstract
- During the industrial stabilization process, lactic acid bacteria are subjected to several stressful conditions. Tolerance to dehydration differs among lactic acid bacteria and the determining factors remain largely unknown. Lactobacillus coryniformis Si3 prevents spoilage by mold due to production of acids and specific antifungal compounds. This strain could be added as a biopreservative in feed systems, e.g. silage. We studied the survival of Lb. coryniformis Si3 after freeze-drying in a 10% skim milk and 5% sucrose formulation following different fermentation pH values and temperatures. Initially, a response surface methodology was employed to optimize final cell density and growth rate. At optimal pH and temperature (pH 5.5 and 34 degrees C, the freeze-drying survival of Lb. coryniformis Si3 was 67% (+/- 6%). The influence of temperature or pH stress in late logarithmic phase was dependent upon the nature of the stress applied. Heat stress (42 degrees C) did not influence freeze-drying survival, whereas mild cold- (26 degrees C), base(pH 6.5), and acid- (pH 4.5) stress significantly reduced survival. Freeze-drying survival rates varied fourfold, with the lowest survival following mild cold stress (26 degrees C) prior to freeze-drying and the highest survival after optimal growth or after mild heat (42 degrees C) stress. Levels of different membrane fatty acids were analyzed to determine the adaptive response in this strain. Fatty acids changed with altered fermentation conditions and the degree of membrane lipid saturation decreased when the cells were subjected to stress. This study shows the importance of selecting appropriate fermentation conditions to maximize freeze-drying viability of Lb. coryniformis as well as the effects of various unfavorable conditions during growth on freeze-drying survival.
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