PubMed · 14821057
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S FOCACCIA. 1951. [Lyophilization methods].. https://pubmed.ncbi.nlm.nih.gov/14821057/
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Maintaining the viability of lactic acid bacteria during dehydration and subsequent storage remains a significant challenge. Here, we employed single-cell RNA sequencing to reveal the heterogeneous stress responses of Lactobacillus delbrueckii subsp. bulgaricus, identifying seven distinct transcriptional clusters across the liquid culture, freeze-drying, and storage phases. The dominant clusters in the freeze-drying and storage were not completely consistent, showing significant functional differentiation. Genomic stability may be important for survival during freeze-drying and storage, while intracellular energy homeostasis appears important for viability during storage. The magnesium transporter mgtB was highly expressed in clusters tolerant to freeze-drying and storage, suggesting a critical role for Mg2+ homeostasis. Further experimental validation confirmed that Mg2+ treatment significantly bolstered stress resistance, increasing immediate post-freeze-drying survival by over 2-fold (up to 92.90%) and post-storage survival by over 5-fold (up to 5.98%). Proteomic data indicated that Mg2+ supplementation correlated with the maintenance of several biological functions potentially relevant to bacterial survival during freeze-drying and storage, including DNA repair, translation, and central carbon metabolism. These findings provide a map of microbial stress resistance through population heterogeneity and offer a potential strategy that may be adapted for enhancing the stability of other industrial lactic acid bacteria products.
PURPOSE: To link the aptness to freeze-drying and the stability under storage to the topology of lipid nanocapsules. METHODS: The aptness to freeze-drying and the stability under storage of lipid nanocapsules prepared from different compositions with a lecithin content in the 2-20% range were estimated from the preservation of the physical structure, preventing the leakage of the oily phase. The influence of the outer shell composition and of the physical characteristics (investigated by photon correlation spectroscopy and differential scanning calorimetry) on the physical stability was correlated to the topology of the nanoparticulate carrier. RESULTS: Confirming the assumption that lecithin confers hardness to the outer shell of lipid nanocapsules, this study shows that the aptness to freeze-drying and the stability under storage depend on the Solutol/lecithin (S/L) ratio in the formulation. The DSC study points out a complexation between lecithin and trehalose, the cryoprotectant, reinforcing the stabilising properties of lecithin. CONCLUSIONS: This paper is a contribution to methodological development of the formulation of lipid nanocapsules, with a special emphasis on the aptness to freeze-drying and the stability under storage.
PURPOSE: To develop a procedure based on manometric temperature measurement (MTM) and an expert system for good practices in freeze drying that will allow development of an optimized freeze-drying process during a single laboratory freeze-drying experiment. METHODS: Freeze drying was performed with a FTS Dura-Stop/Dura-Top freeze dryer with the manometric temperature measurement software installed. Five percent solutions of glycine, sucrose, or mannitol with 2 ml to 4 ml fill in 5 ml vials were used, with all vials loaded on one shelf. Details of freezing, optimization of chamber pressure, target product temperature, and some aspects of secondary drying are determined by the expert system algorithms. MTM measurements were used to select the optimum shelf temperature, to determine drying end points, and to evaluate residual moisture content in real-time. MTM measurements were made at 1 hour or half-hour intervals during primary drying and secondary drying, with a data collection frequency of 4 points per second. The improved MTM equations were fit to pressure-time data generated by the MTM procedure using Microcal Origin software to obtain product temperature and dry layer resistance. Using heat and mass transfer theory, the MTM results were used to evaluate mass and heat transfer rates and to estimate the shelf temperature required to maintain the target product temperature. RESULTS: MTM product dry layer resistance is accurate until about two-thirds of total primary drying time is over, and the MTM product temperature is normally accurate almost to the end of primary drying provided that effective thermal shielding is used in the freeze-drying process. The primary drying times can be accurately estimated from mass transfer rates calculated very early in the run, and we find the target product temperature can be achieved and maintained with only a few adjustments of shelf temperature. The freeze-dryer overload conditions can be estimated by calculation of heat/mass flow at the target product temperature. It was found that the MTM results serve as an excellent indicator of the end point of primary drying. Further, we find that the rate of water desorption during secondary drying may be accurately measured by a variation of the basic MTM procedure. Thus, both the end point of secondary drying and real-time residual moisture may be obtained during secondary drying. CONCLUSIONS: Manometric temperature measurement and the expert system for good practices in freeze drying does allow development of an optimized freeze-drying process during a single laboratory freeze-drying experiment.