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. 2025 Aug 5:9:100453.
doi: 10.1016/j.crmicr.2025.100453. eCollection 2025.

Cold stress enhances cryotolerance in Lacticaseibacillus rhamnosus B6 via membrane lipid remodeling and differential protein expression

Ting Yang  1 , Jia Zhang  1   2 , Ranjin Zhang  1   2 , Juan Zhang  2 , Zhenmin Liu  1 , Zhengjun Wu  1
Affiliations

Cold stress enhances cryotolerance in Lacticaseibacillus rhamnosus B6 via membrane lipid remodeling and differential protein expression

Ting Yang et al. Curr Res Microb Sci. .

Abstract

Understanding the molecular mechanisms underlying the cryotolerance of lactic acid bacteria is critical for preserving their viability in food processing. In this study, Lacticaseibacillus rhamnosus B6 and KF7 with different phenotypic properties were pretreated at 4 °C for 2 h before liquid nitrogen freezing. Cold-stressed B6 exhibited significantly higher survival (53 %) than KF7 (30 %) and untreated controls (44 % vs. 10 %, p < 0.05), with few disrupted cells observed under SEM. Cold stress altered the membrane fluidity in B6 by increasing unsaturated fatty acids (UFA) and the ratio of UFA to saturated fatty acids (UFA/SFA) from 1.36 to 1.62 (p < 0.05). 219 proteins, primarily those involved in fatty acid biosynthesis, translation and transport processes were up regulated in cold-stressed B6, compared to untreated counterparts. Noteworthy, capsules of B6 likely contributed to its higher cryotolerance. Our findings revealed a dual cryotolerance mechanism in L. rhamnosus B6, i.e., dynamic lipidome remodeling and coordinated protein expression regulation. This study highlighted the importance of the cell surface features of individual strain of L. rhamnosus and cold stress treatment in preparing probiotics as well as direct to vat starter cultures with high cell vitality employing deep freezing process.

Keywords: Cold stress; Cryotolerance; Fatty acids; Lacticaseibacillus; Proteomics.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image, graphical abstract
Graphical abstract
Fig 1
Fig. 1
Growth curve of L. rhamnosus B6 and KF7 in MRS broth at 37 °C.
Fig 2
Fig. 2
Survival rate of frozen cell of L. rhamnosus B6 and KF7 in liquid nitrogen. Results of three independent experiments were expressed as mean ± SD (n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Fig 3
Fig. 3
Flow cytometry of L. rhamnosus B6 and KF7 after freezing in liquid nitrogen before and after cold stress. (a) Flow cytometry histogram of PI staining. (b) Flow cytometry histogram of cFDA staining. (c) Flow cytometry histogram of strain B6 frozen by liquid nitrogen control (non-stressed) condition. (d) Flow Cytogram of strain B6 frozen by liquid nitrogen after cold stress. (e) Flow cytometry histogram of strain KF7 frozen by liquid nitrogen control (non-stressed) condition. (f) Flow cytometry histogram of strain KF7 frozen by liquid nitrogen after cold stress.
Fig 4
Fig. 4
Cell morphological changes of strains B6 and KF7 were observed by scanning electron microscope after liquid nitrogen freezing. (a) Morphology of strain B6 control (non-stressed) condition. (b) Morphology of strain B6 with cold stress. (c) Morphology of strain KF7 control (non-stressed) condition. (d) Morphology of strain KF7 with cold stress. The arrows indicate cells with ruptures or surface collapses.
Fig 5
Fig. 5
Pathway classification based on GOG/KOG analysis of differentially expressed protein of L. rhamnosus B6 in response to cold stress. The ordinate was the number of differentially expressed genes in this pathway. Proteins with a variation of at least 1.5 folds were selected.
Fig 6
Fig. 6
The cell structures of L. rhamnosus B6 (a) and KF7 (b) were observed by transmission electron microscope.
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