| Freeze-drying,as the most common method of strain preservation,has been widely used in the industrial production of lactic acid bacteria culture.The drying process can result in the exposure of bacteria to a variety of adverse factors such as low temperature,acid,dehydration,and oxidation,causing damage or even death.Now,few studies have been reported on cell damage and reduced viability due to oxidative stress during freeze-drying.In this study,taking Bifidobacterium animalis subsp.lactis Probio-M8(B.lactis Probio-M8)as study objects,antioxidant peptide HP3-2 from whey protein hydrolysate has been used as a lyoprotectant to protect the cell during freeze-drying.Combined with the proteomic approach to study the differential protein expression in bacteria,and investigate the effects of the structure and biological properties of cell membranes during freeze-drying.The protective mechanism of antioxidant peptide HP3-2 on B.lactis Probio-M8 during freeze-drying was revealed from the cellular molecular level and physiological changes.The main results are as follows:(1)The hydrolysis of whey protein by alkaline protease is stronger than that by neutral protease,pepsin,trypsin,and papain.The hydrolysate of whey protein has strong antioxidant activity.Whey protein hydrolysis,as a lyoprotectant,can improve the survival rate of B.lactis Probio-M8 in freeze-drying and storage.It also reduces the accumulation of intracellular reactive oxygen species and the content of oxidation product malondialdehyde,reduces the effect of temperature on the inactivation rate of bacteria during storage,and improves the storage stability.(2)The antioxidant peptides extracted from whey protein hydrolysate(WPH)were investigated.The antioxidant activity of WPH,which was isolated and purified via ultrafiltration,cation exchange chromatography,and reversed-phase liquid chromatography,was evaluated using its antioxidant ability.It has been shown high DPPH scavenging(88.11±0.38%),oxygen radical uptake(2.73±0.10μmol TE/g),and superoxide anion uptake(62.54±3.16 U/L).Compared with whey protein,the antioxidant peptide HP3-2 has a stronger protective effect on B.lactis Probio-M8 during the freeze-drying process.(3)The antioxidant peptide HP3-2 could reduce the effect of oxidative stress on cell membrane structure,maintain the fluidity and potential of the cell membrane,and control the change ion concentration(Na~+,H~+)inside and outside the cell membrane through ion channels.It can protect the activity of intracellular hydrolases,such as F6PPK(50.41±1.68IU/g)and K~+Na~+-ATPase(5.26±0.08 U/g),and maintain the homeostasis of the intracellular environment.Antioxidant peptides HP3-2 can improve the survival of strains of B.lactis Probio-M8(80.40±3.34%)during freeze-drying.(4)Protein expression of B.lactis Probio-M8 during freeze-drying was analyzed using TMT quantitative proteomics.The results showed that lyoprotectant can protect intracellular ribosomal proteins and stimulates the translational synthesis of proteins.The antioxidant peptide HP3-2 can increase the expression of nicotinamide-nucleotide amidohydrolase family protein,ribulose-phosphate 3-epimerase,Ppx/Gpp A family phosphatases in cells,and affect various metabolic pathways,such as nucleotide metabolism,carbohydrate metabolism,and vitamin metabolism.It also promotes resistance of B.lactis Probio-M8 as well as its antioxidant capacity and reduces cellular damage from oxidative stress.This study provides a theoretical basis for the mechanism of cell damage to B.lactis Probio-M8 by oxidative stress during freeze-drying and also provides new methods and new ideas for new antioxidant lyoprotectant and the industrial production of culture and bifidobacterium products. |
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