The Screening And Antioxidation Study Of Antioxidant Lactic Acid Bacteria From Jinhua Ham

Meat products are rich in nutrients such as fats,proteins,minerals and vitamins.Processing and long term storage of meat products would cause lipid and protein oxidation resulting in the nutrition loss,flavor deterioration and decline of functional properties.The chemosynthetic antioxidants have been widely used in the meat industry for a long time.However,excessive supplementation of chemosynthetic antioxidants would cause potential harm to human.Therefore,it is of great challenge to develop and apply the natural antioxidant technology.Lactic acid bacteria are currently recognized as safe food-grade microorganism in the world.It is also one of the earliest microorganisms used in fermented meat products and widely applied in other food industry.Researches in vivo and in vitro have shown that lactic acid bacteria not only improve the sensory properties,nutritional characteristics,food edibility and safety,and preservation of products,but also possess the potential antioxidant activity.The study of microbiotic antioxidant is less mature than the chemical antioxidant.However with the improvement of people health awareness,the development and the utilization of functional lactic acid bacteria have gradually become the focuspoint.The dissertation used microbial resources from Jinhua ham to isolate and screen the lactic acid bacteria with antioxidant properties.The antioxidant activity of the lactic acid bacteria was evaluated systematically from free radical level,cellular level and biological organism level by conventional methods in vitro,cell oxidative damage model and animal aging model.In addition,the related mechanism and the application approach were also investigated.On this basis,fermented sausage was used to explore the effects of antioxidative lactic acid bacteria on the protein oxidation and the quality of fermented meat products during the ripening process.It could be implied the mechanism of regulation of the protein oxidation and the improvement of meat quality of fermented sausage by supplementing the antioxidant lactic acide bacteria.The main results are as follows:1.Analysis of bacterial community diversity in Jinhua hamThe high-throughput sequencing technology was used to study the bacterial community diversity of Jinhua hams in the early state of aging,which was selected from fermentation chamber with different years(5 th,15 th and 30 th year).Total 1070 OTUs were obtained which belonged to 18 phylums and 242 genuses.The structure and the diversity of bacterial community were different among the different hams,while the dominant genuses among hams including Staphylococcus,P.bacteria,Propionibacterium,Acinetobacter and Bacillus remained stable.The bacterial community diversity in ham decreased gradually along with the fermentation chamber years.In addition,the dominant concentration of Staphylococcus tended to increase accompanying with the deceasing proportion of other bacterial flora.2.Screening of antioxidant lactic acid bacteria and the study on their antioxidant activity in vitroNJAU-01,a lactic acid bacteria strain with high antioxidant activity,was separated and purified froru Jinhua ham according to the indicators of meat starter culture selection standard and total antioxidant capacity.The total antioxidant capacity of NJAU-01 was 55.25 U/mg protein.It was identified as Lactobacillus plantarum by characterizing the morphological,physiological and biochemical characteristics and 16S rDNA analysis and designated as Lactobacillus plantarum(NJAU-01).The antioxidant experiment in vitro indicated that the strain could tolerate 1 mM H2O2.The DPPH radical scavenging activity and lipid peroxidation scavenging capacity of the cell-free extract were signiflcantly higher than that of bacterial suspension(P<0.05)while no significant differences of·OH,02-scavenging ability and reducing ability were observed.3.Study on the antioxidant activity of L.plantarum NJAU-01 based on cell modelThe ROS level fluorescence detection results showed that fluorescence intensity of cell group incubated with bacterial fluid decreased obviously compared with that of positive cells.The results of scanning electron microscopy and transmission electron microscopy indicated that the cell membrane of the positive group was damaged,and the cells in the bacterial intervention group had the certain recovery capacity.Cell apoptosis analysis showed that the medium apoptotic rate of cells in the bacterial intervention group was 6.1%,which was much lower than that of the positive group(95.6%).SOD activity assay showed that the inhibition rate of oxidative damage and the activity of SOD in the positive group were both lower than those in the cell group incubated with1010 CFU/mL bacterial fluid(P<0.05).The results of MDA level indicated that the MDA content in the positive group was significantly higher than that in the bacterial fluid protection group(P<0.05).The above results demonstrated that the bacteria increased the antioxidant capacity of cells.On the basis of the above studies,we constructed an electrochemical cell sensor to further evaluate the antioxidant capacity of L.plantarum NJAU-01.The results showed that the peak current value Ip was linearly proportional to the concentration of H2O2 in the range of 0.05 M to 0.85 M.When the detection limit was 0.02 Ms the correlation coefficient was 0.996.The signal intensity of Ip value decreased with increasing concentration of L.Plantarum NJAU-01,and RAC(relative antioxidant capacity)value was 88.94%,indicating that 1010 CFU/mL L.plantarum NJAU-01 had the highest antioxidant activity.The results of the above methods showed that L.plantarum NJAU-01 had fairly strong antioxidant ability.The accuracy and the reliability of the results from the constructed electrochemical cell sensor were also verified.4.Study the antioxidant activity of L.plantarum NJAU-01 based on animal aging modelMice subacute aging model induced by D-galactose was established by feeding NJAU-01.The aging model group and the positive control group were set to compare with NJAU-01 treatment group.Result showed that the weight of aging model mice was significantly lower than that of other groups(P<0.05).D-galactose could significantly increase the content of MDA in mice serum,heart and liver and decrease the activity of antioxidant enzyme,which proved the effeetiveness of the aging model.In addition,the antioxidant enzyme activity of aging model group mice was significantly lower than the control while the antioxidant enzyme activity of positive control group mice was significantly higher than the control(P<0.05).When the concentration of NJAU-01 was 109 CFU/mL,the activity of SOD,GSH-Px,CAT and T-AOC of mice serum,heart and liver was significantly higher than other groups(P<0.05).Result showed that NJAU-01 could exert its antioxidant activity in vivo,which was positively correlated with the bacterial concentration.5.Effects of L.plantarum NJAU-01 on protein oxidation and quality of fermented sausages during ripeningIn order to explore the potential application value of NJAU-01 in fermented sausage,the effect of NJAU-01 selected from Jinhua ham on the protein oxidation of and the quality fermented sausage was investigated.The addition of commercial strains was regarded as positive control group while the control group was set without any strain addition.The study found that protein carbonyl content was significantly lower than the control in the 107 CFU/g and 109 CFU/g NJAU-01 addition group(P<0.05).In addition,the total sulfhydryl content was significantly higher than the control while the surface hydrophobicity of sausage was lower than the other groups(P<0.05).The bands of sarcoplasmic protein with molecular weight of 130-250 kDa,100-130 kDa and 35-25kDa were obviously changed.The results demonstrated that NJAU-01 could effectively inhibit the protein oxidation of fermented sausage.The level of protein oxidation of sausage was moderately modified by the NJAU-01 addition.In addition,the effect of NJAU-01 addition on the quality of dry fermented sausage was investigated.The results showed that NJAU-01 could quickly reduce the pH,the water activity and the moisture content of products and improve the safety and preservation of fermented sausage.Based on the analysis of volatile flavor compounds in ripened sausage,aldehydes and total volatile compounds of sausage,107 CFU/g NJAU-01 group was significantly higher than those with 105 CFU/g and 109 CFU/g NJAU-01(P<0.05).There was no significant difference in sensory score between NJAU-01 treatment group and the control(P>0.05).Taking pH,Aw value,color,texture,TBARS and flavor indicators into account,NJAU-01 is proved to be a potential fermentation strain with excellent characteristics for meat products.