Biosearch S.A.U (a Kerry® Company), R&D Department, 18004, Granada, Spain.
Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, F-91120, Palaiseau, France.
Cryobiology. 2023 Sep;112:104556. doi: 10.1016/j.cryobiol.2023.104556. Epub 2023 Jul 10.
Ligilactobacillus salivarius is a lactic acid bacterium exhibiting several health benefits. However, it is sensitive to freeze-drying and storage in the dried state, thus limiting its commercial exploitation. Our objective was to identify markers of cell resistance by applying multiscale characterization to L. salivarius CECT5713 cell populations exhibiting different resistance to freeze-dried storage. Cells were produced under two different sets of production conditions differing in the culture parameters (temperature, neutralizing solution, and harvesting time) and the protective formulation composition. The culturability, membrane integrity, and cell biochemical composition assessed by Fourier transform infrared (FTIR) micro-spectroscopy were evaluated after freezing, freeze-drying, and subsequent storage at 37 °C. Membrane properties (fatty acid composition, membrane fluidity, and phospholipid organization), as well as matrix physical properties (glass transition temperature and water activity), were determined. The most resistant cells to freeze-dried storage exhibited the highest cyclic fatty acid content and the most rigid membrane. Freeze-drying and storage induced damage to membrane integrity, proteins, nucleic acids, and constituents of the peptidoglycan cell wall. From the FTIR spectra analysis, we propose the minimization of the variations of the 1058 and 1714 cm vibration bands (that arise mainly from symmetric C-O-C stretching and CO stretching, respectively) induced by the freeze-drying process as a marker of storage stability. We confirmed that a matrix with a glass transition temperature at least 50 °C higher than the storage temperature is crucial for L. salivarius CECT5713 storage stability. In addition, this work explored promising FTIR methods for a better understanding of the protection mechanisms involved.
唾液乳杆菌是一种具有多种健康益处的乳酸杆菌。然而，它对冻干和干燥状态下的储存敏感，因此限制了其商业开发。我们的目标是通过对表现出不同冻干储存抗性的唾液乳杆菌 CECT5713 细胞群体进行多尺度表征来鉴定细胞抗性的标志物。细胞是在两种不同的生产条件下产生的，这些条件在培养参数（温度、中和溶液和收获时间）和保护剂配方组成上有所不同。在冷冻、冻干和随后在 37°C 下储存后，通过傅里叶变换红外（FTIR）微光谱评估了可培养性、膜完整性和细胞生化组成。还测定了膜性质（脂肪酸组成、膜流动性和磷脂组织）以及基质物理性质（玻璃化转变温度和水活度）。对冻干储存最具抗性的细胞表现出最高的环脂肪酸含量和最刚性的膜。冻干和储存会导致膜完整性、蛋白质、核酸和肽聚糖细胞壁成分受损。从 FTIR 光谱分析中，我们提出将冻干过程引起的 1058 和 1714cm 振动带的变化（主要来自对称 C-O-C 伸缩和 CO 伸缩）最小化作为储存稳定性的标志物。我们证实，对于唾液乳杆菌 CECT5713 的储存稳定性，具有至少比储存温度高 50°C 的玻璃化转变温度的基质至关重要。此外，这项工作探索了有前途的 FTIR 方法，以更好地理解所涉及的保护机制。
