Effects of salt stress on the freeze-drying survival rate of Lactiplantibacillus plantarum LIP-1.

In this study, we examined the effects of different salt stress application methods on the Lactiplantibacillus plantarum LIP-1 freeze-drying survival rate. The application of salt stress during the stationary phase significantly improved Lactiplantibacillus plantarum LIP-1 freeze-drying survival rates (P < 0.05). The indirect application of salt stress via phosphate-buffered saline containing 0.4 mol/L NaCl (NB group) led to significantly higher freeze-drying survival rates compared to when salt stress was directly applied (NA group: the concentration of NaCl is 0.4 mol/L) (P < 0.05). Following exposure to salt stress, Lactiplantibacillus plantarum LIP-1 cells exuded excessive Na+ out of the cell and transported extracellular K+ into the cell, resulting in upregulation of the trkA gene, which is related to K+ transport, thereby significantly upregulating the expression of a lysR-type transcription factor, which increased the cell membrane unsaturated fatty acid content, reducing the degree of cell membrane damage and improving the freeze-drying survival rate. When the concentration of NaCl is 0.4 mol/L, compared with direct salt stress application, indirect application led to higher intracellular pH and ATP content, which effectively reduced DNA and cell membrane damage, respectively. Together, these results demonstrate that appropriate indirect salt stress application can improve Lactiplantibacillus plantarum LIP-1 freeze-drying resistance.

The Effect of Proline on the Freeze-Drying Survival Rate of Bifidobacterium longum CCFM 1029 and Its Inherent Mechanism.

Amino acids, which are important compatible solutes, play a significant role in probiotic lyophilization. However, studies on the functions of Bifidobacterium during freeze-drying are limited. Therefore, in this study, we compared the freeze-drying survival rate of Bifidobacterium longum CCFM 1029 cultivated in different media containing different kinds of compatible solutes. We found that the addition of 21 g/L proline to the culture media substantially improved the freeze-drying survival rate of B. longum CCFM 1029 from 18.61 ± 0.42% to 38.74 ± 1.58%. Interestingly, this change has only been observed when the osmotic pressure of the external culture environment is increased. Under these conditions, we found that proline accumulation in this strain increased significantly. This change also helped the strain to maintain its membrane integrity and the activity of some key enzymes during freeze-drying. Overall, these results show that the addition of proline can help the strain resist a tough environment during lyophilization. The findings of this study provide preliminary data for producers of probiotics who wish to achieve higher freeze-drying survival rates during industrial production.

Effect of oleic acid on the viability of different freeze-dried Lactiplantibacillus plantarum strains.

Freeze drying is one of the most convenient ways to preserve microorganisms, but in the freeze-drying process, strains will inevitably suffer varying degrees of damage under different conditions. The deterioration of cell membrane integrity is one of the main forms of damage. The type and ratio of fatty acids in the cell membrane affect its characteristics. Therefore, it is worth investigating whether certain fatty acids can increase freeze-drying resistance. In this study, we found that adding a low concentration of oleic acid to a cryoprotectant could increase survival rate of strains of Lactiplantibacillus plantarum following freeze drying, and the optimal concentration of oleic acid was determined to be 0.001%. When 0.001% oleic acid was added to phosphate-buffered saline, the freeze-drying survival rate of L. plantarum increased by up to 6.63 times. Adding 0.001% oleic acid to sorbitol, the survival rate of L. plantarum increased by as much as 3.65 times. The 0.001% oleic acid-sucrose cryoprotectant resulted in a freeze-drying survival rate of L. plantarum of about 90%, a 2.26-fold improvement compared with sucrose alone. Although the effect of oleic acid depends on the cryoprotectants used and the strain treated, addition of oleic acid showed significant improvement overall. Further experiments showed that adding a low concentration of oleic acid to the cryoprotectants improved the freeze-drying survival rate of L. plantarum by maintaining cell membrane integrity and lactate dehydrogenase activity. Our findings provide a new strategy for safeguarding bacterial viability in commonly used cryoprotectants by the addition of a common food ingredient, which may be extensively applied in the food industry.

Improving the freeze-drying survival rate of Lactobacillus plantarum LIP-1 by increasing biofilm formation based on adjusting the composition of buffer salts in medium.

Lactic acid bacteria can improve their resistance to adverse environments through the formation of biofilm. This study found that adding different buffer salts in culture medium had a great impact on the freeze-drying survival rate of the Lactobacillus plantarum LIP-1, which could be linked to biofilm formation. Transcriptome data showed that potassium ions in buffer salt increased the expression of the luxS gene in the LuxS/autoinducer-2 (AI-2) quorum sensing system and increase synthesis of the quorum sensing signal AI-2. The AI-2 signal molecules up-regulated the cysE gene, which helps to promote biofilm formation. By adding a biofilm inhibitor, d-galactose, and performing a real-time quantitative polymerase chain reaction experiment, we found that d-galactose could down-regulated the luxS and cysE genes, reduced biofilm formation, and decreased the freeze-drying survival rate. The results of this study showed that promoting biofilm formation using appropriate buffer salts may lead to better freeze-drying survival rates.

Effects of different initial pH values on freeze-drying resistance of Lactiplantibacillus plantarum LIP-1 based on transcriptomics and proteomics.

The growth and the resistance to adverse environments of lactic acid bacteria would be affected by adjusting the initial pH of the medium. In order to explore the effect of changing the initial pH of culture medium on the freeze-drying survival rate of the Lactiplantibacillus plantarum LIP-1, the effect of initial pH on cell membrane fatty acid composition and key enzyme activity were mainly determined, and the internal mechanism was studied by transcriptomics and proteomics methods. We found that compared with initial pH 7.4 group, initial pH 6.8 group could improve the freeze-drying survival rate of the L. plantarum LIP-1. It was possibly due to the lactate dehydrogenase (LDH) was upregulated in the initial pH6.8 group, which led to a rapid decrease in culture pH. To reduce the inhibitory effect of long-term acid environment on growth, the strain upregulated the expression of fatty acid synthesis-related genes and proteins, promoted the relative content of cyclopropane and unsaturated fatty acids, improved integrity of the cell membranes. The adjustment of fatty acid composition maintained the integrity of the cell membrane in a freeze-drying environment to improve the freeze-drying survival rate of the initial pH6.8 group. In addition, the long-term acid environment stimulated a cross-stress tolerance mechanism that significantly upregulated the expression of a cold stress protein. The results indicated that the optimal initial pH of the medium could improve the ability of L. plantarum LIP-1 to resist freeze-drying.

Effects of buffer salts on the freeze-drying survival rate of Lactobacillus plantarum LIP-1 based on transcriptome and proteome analyses.

Buffer salts are often added to culture medium to promote bacterial growth. However, we found that buffer salts can improve the freeze-drying survival rate. In this experiment, the mechanisms for the effects of different buffer salts on the survival rate of freeze-dried strains were examined. The results showed that buffer salts had important effects on the freeze-drying survival rate of L. plantarum LIP-1 that were related to changes in fatty acid composition. Different buffer salts affected the expression of fatty acid metabolic genes. A new gene cluster that regulates fatty acid metabolism and synthesis was discovered. Potassium ions in buffer salts upregulated the trkA gene and lysR-type transcription factor, and then upregulated the expression of fatty acid synthesis-related acc and fab family genes. These genes help to extend the fatty acid carbon chain and promote the unsaturated fatty acids content, which improves cell membrane fluidity and improves resistance to freeze-drying.