| In this study,Lactobacillus plantarum LIP-1 was investigated for its resistance to vacuum freeze-drying and its stability in storage at room temperature by two different treatments: calcium ion culture and salt stress.The effects of calcium ion culture and salt stress on the viability,survival rate and storage stability of freeze-dried strains were investigated by measuring cell wall sensitivity(penicillin,lysozyme),cell membrane sensitivity(Na Cl),cell membrane fluidity,cell membrane integrity and key enzyme activities,and the underlying mechanisms were also studied.The experimental results showed that when 0.5 mmol/L calcium ion was added to the MRS medium,the growth and freeze-drying survival rate of the strains were significantly increased compared with the control group without calcium ion addition(P < 0.05).The storage stability of the strains was improved by adding calcium ions during storage at room temperature,and the number of viable bacteria in the experimental group with calcium ions increased by 13.65 times compared with the control group(MRS)at 8 weeks,and the storage survival rate was significantly(P < 0.05)increased by increasing calcium ion concentration.Also,the addition of calcium ions significantly shortened the length of Lactobacillus plantarum LIP-1 compared to the control group.The alkaline phosphatase assay,lysozyme and penicillin susceptibility tests revealed that the cell wall of the calcium ion cultured strain suffered less damage during vacuum freeze drying as well as storage at room temperature.The cell membrane sensitivity,fluidity and integrity tests showed that the degree of damage to the cell membrane was reduced and the survival rate of the organism was increased.After the strains were treated with salt stress during the delayed,logarithmic and stable periods,it was found that the freeze-drying survival rate of the strains was increased to different degrees,among which the freeze-drying survival rate of the strains was significantly higher than that of the other groups after the single-factor stable period salt stress treatment(P < 0.05).And the stability was also significantly improved during storage at room temperature(P < 0.05).The resistance of Lactobacillus plantarum LIP-1 to freezedrying and the stability of storage at room temperature were improved after the treatment with salt stress during the stabilization period,as shown by the subsequent tests of Fourier infrared spectroscopy,key enzyme activities and cell membrane fluidity.In the subsequent crossover experiments,it was found that the freeze-drying survival rate of the crossover experimental group was not significantly different from that of the calcium ion culture,and its room temperature storage stability was significantly higher than that of the salt stress experimental group(P < 0.05).Subsequently,based on Fourier infrared spectra,it was found that the cell membrane lipids,proteins,and DNA of the cross-treated strains were less damaged after freeze-drying than those of the control group.The experimental results show that the salt-stressed group has the best survival rate of vacuum freeze-drying under the same freeze-drying and room-temperature storage conditions.The calcium ion culture strain had the best storage stability at room temperature.The crossover group had higher pre-freeze viability and significantly improved storage stability compared to the salt stress group(P < 0.05).It was found that the interaction between calcium ion culture and single-factor salt stress during the stabilization period had a certain protective effect on the cell wall,cell membrane and key enzymes during the freezedrying process,resulting in the strong resistance to freeze-drying and storage stability at room temperature. |
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