| Probiotics have been widely used in food industry,but low temperature stress during cold storage often leads to decreased cell viability of probiotics.Antifreeze peptides can noncolligatively reduce the freezing point of solution,inhibit ice recrystallization and protect cell membrane,so they can be used as a natural and safe antifreeze protectant for probiotics.In this study,the enzymatic hydrolysis of antifreeze peptides from tilapia skin was optimized to protect the frozen Lactobacillus rhamnosus ATCC7469,and the antifreeze protective effect and mechanism of the peptides were studied.The main research conclusions are as follows:Using the relative survival rate of L.rhamnosus ATCC7469 after freezing as the evaluation index of antifreeze activity,trypsin was determined as the best protease through experiments and it's relative survival rate of L.rhamnosus ATCC7469 was 49.24 ± 9.41%.The optimal conditions were determined by single factor and response surface methodology as follows: amount of enzyme 2200 U/g,solid-liquid ratio 1:10(w/v),enzymatic hydrolysis temperature 49°C,and enzymatic hydrolysis time 6.8 h.The relative survival rate of the prepared antifreeze peptide against L.rhamnosus ATCC7469 was 98.32 ± 1.79%.The molecular weight distribution analysis showed that the antifreeze peptides from tilapia skin(APT)prepared under the optimal conditions were mainly composed of small peptides from1 k Da to 2 k Da,accounting for 63.37%.Amino acid composition analysis showed that APT contained glycine(35.45%),alanine(13.29%)and proline(12.43%).The polypeptide sequence analysis showed that there were tripeptide repeats(-Gly-X-Y-)in APT,which was consistent with the characteristics of high antifreeze activity polypeptides.Under APT protection,the activities of ?-galactosidase(?-GAL)and lactate dehydrogenase(LDH)in L.rhamnosus ATCC7469 cells after freezing were significantly higher than those in saline group,which were 179.79± 11.00 U/m L and 11.41 ± 0.99 U/L(p < 0.05).The extracellular protein concentration was significantly lower than saline group,only 9.02 ± 3.39 g/m L,and APT antifreeze protection effect was better than that of 1 mg/m L sucrose,1 mg/m L skim milk and 20% glycerol positive control groups.Scanning electron microscope results showed that cells had normal,smooth and rounded surface morphology in APT group.Metabonomics results showed that the differences in intracellular metabolites of L.rhamnosus ATCC7469 in APT group and Saline group were mostly related to the maintenance of energy metabolism of cells under freezing stress.The accumulation of proline may play the same role as it does in plants,regulating cell permeability,enhancing resistance to cell dehydration,and transduction of cold stress signals.The content of palmitoleic acid was significantly decreased(p < 0.05),and the content of linolenic acid was significantly increased(p < 0.05).It may be that L.rhamnosus ATCC7469 can adapt to the low temperature anaerobic environment by changing the unsaturation of fatty acids and the length of carbon chain,maintain the fluidity of cell membrane,reduce the physical damage of ice crystals to cells,and improve the survival rate. |
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