Study On The Isolation,Identification And Antioxidant Activity Of Peptides In Fermented Sausage

The two most used starter cultures in fermented sausages are lactic acid bacteria（LAB）and coagulase-negative staphylococcus（CNS）.LAB can rapidly reduce p H value,promote the formation of unique flavor,flesh color,and taste of sausage,inhibit the growth of spoilage microorganisms,and ensure the safety of the products.CNS has protease and lipase hydrolytic activity,which can promote the formation of sausage flavor and texture.In the process of fermented sausage production,proteases and peptidases from microorganisms can hydrolyze proteins to produce small molecular polypeptides and free amino acids.These small molecules not only contribute to the formation of the unique flavor of fermented sausage,but also promote the improvement of biological activity of sausage.The antioxidant activity is one of the most widely studied.Excessive protein and fat oxidation is the main reason for the deterioration of fermented sausages during processing and storage.Natural antioxidant peptides have become a hotspot in recent years due to their high antioxidant activities and stable structure.However,there are few reports on the inhibition of protein and lipid oxidation in fermented sausages by endogenous microbial enzymes produced by inoculation and fermentation.It has been shown that Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201 have strong protease hydrolytic activity.Therefore,this research explored the effects of mixed bacteria Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201 on protein degradation and peptide antioxidant capacity in the fermented sausage.The fermented sausage hydrolysates were purified by ultrafiltration,molecular sieve chromatography,and reversed-phase high performance liquid chromatography（RP-HPLC）,and then identified by liquid chromatography-tandem mass.Peptide sequences were synthesized and their antioxidant activities were determined.The peptide antioxidant capacities were evaluated by cell toxicity,cell protection,antioxidant enzyme activities,and malondialdehyde（MDA）contents in Caco-2 cells.Specific research contents and conclusions are as follows:1.Effects of Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201 on proteolytic changes and bioactivities（antioxidant and antihypertensive activities）in fermented pork sausageThis section explored the influences of Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201 on protein degradation and bioactivities（antioxidant and antihypertensive activities）of peptides in sausages fermented for 21 days.Polyacrylamide gel electrophoresis verified the hydrolysis of sarcoplasmic and myofibrillar proteins,especially intense in inoculated group,which was evidenced by obvious degradation of myosin heavy chain（200-220 k Da）,myosin light chain（21 k Da）,actin（45 k Da）,myoglobin（17 k Da）and other bands.A significant increment（P<0.05）of hydrophobic amino acid concentration and ACE inhibitory activity was observed in inoculated group.Based on DPPH,hydroxyl radical,superoxide anion scavenging activities and Ferric-reducing antioxidant power values,the inoculated group exhibited higher antioxidant ability,but possessed lower ABTS+·scavenging activity than control group（P<0.05）.22peptides with high confidence were identified and selected by LC-MS/MS,which were mainly from troponin T,myosin,and particularly actin.These peptides shared joint characteristics with other bioactive peptides.We concluded mixed starters promoted proteolytic hydrolysis and peptide formation with high ACE inhibitory and antioxidant activities.2.Isolation,purification,and identification of peptides from inoculated fermented sausageInoculated fermented sausage was selected as the raw material to purify antioxidant peptides.The hydrolysates were first extracted and then purified by ultrafiltration,molecular sieve chromatography,and reversed-phase high performance liquid chromatography.DPPH free radical clearance rate and FRAP value were utilized to evaluate the antioxidant activity of each component.The fragments with high antioxidant activity were identified by LC-MS/MS.Fourteen antioxidant peptides were then chosen to be synthesized for activity verification.The peptides SDEEVEH and FAGDDAPR showed strong DPPH free radical scavenging activity,the peptide PPAPP had high FRAP value,and the peptide EPPVT、ALELDSNLYR and QEYDESGPSIVHR showed strong ABTS+·free radical scavenging activity.3.Study on intracellular antioxidant activity of fermented sausage peptidesIn this chapter,Caco-2 cells were used as carriers,polypeptides MWCO-1 obtained after ultrafiltration and A obtained after molecular sieve chromatography were used as research objects,and the antioxidant capacity of the two fragments in oxidized Caco-2 cells was explored by combining peptide incubation with H₂O₂ oxidative damage treatment.The results showed that peptide fragments isolated from fermented sausage had no toxic and side effects on Caco-2 cells.The activities of antioxidant enzymes were increased and lipid peroxidation were inhibited due to peptide pre-incubation.These results indicated that MWCO-1 and A from fermented sausages can be applied as natural antioxidants in the food and pharmaceutical industry.