| Antioxidant damage is one of the most important causes of aging and pathological changes.There are many bioactive protein fragments hidden in milk casein.Cheese,which is rich in protein,is a good carrier of probiotics,which can guarantee the survival rate and activity of the strain after digestion.At the same time,probiotic strains such as L.helveticus and L.rhamnosus have been proved to have good antioxidant activities.The antioxidant activity of cheese could be improved by adding probiotics,but the reasons and the differences among different strains were unclear.Therefore,on the premise of good acid tolerance and salt tolerance of the strains,which were suitable for cheese production,L.rhamnosus 1.0911 with the best antioxidant activity and L.helveticus 1.0612 with the most hydrolysable activity were selected from 9 experimental probiotics,and we added them to cheddar cheese respectively.Cheddar cheese with only starter was as control.The changes of the number of live bacteria,antioxidant activity and degree of protein hydrolysis in three groups of cheese during ripening were studied.The components of cheese extract were separated and purified by ultrafiltration,macroporous resin adsorption and gel chromatography,and we analyzed the amino acid of peptides.Finally,the effects of probiotics on the quality of Cheese were studied by means of cheese composition,texture and sensory evaluation.This experiment provides the theoretical and experimental basis for the development of probiotic functional cheese.(1)Studies on the functional characteristics of probiotics9 strains of probiotics all had acid and salt tolerance,but L.helveticus 1.0612 and L.rhamnosus 1.0911 had the highest survival rates in acid and salt environments,so they were more suitable for cheese production.Comparative analysis of DPPH free radical scavenging ability,hydroxyl radical scavenging ability and reducing ability,L.rhamnosus 1.0911 showed the best antioxidant activity in 9 strains(DPPH radical scavenging rate 27.35%,hydroxyl radical scavenging rate 40.96,reduction ability 0.908).And the result of proteolytic degree test and protease activity test showed that L.helveticus 1.0612 had strong hydrolytic ability,and its protein hydrolysis degree was 11.05 LLeu mmol/L.Therefore,they were added to cheddar cheese separately.(2)Studies on the number of probiotics,antioxidant activities and degree of protein hydrolysis of cheddar cheeseDuring ripening,antioxidant avtivities of three batches(Batch-1 control;Batch-2 adding L.rhamnosus 1.0911;Batch-2 adding L.helveticus 1.0612)all increased first,then decreased,and finally levelled off.DPPH free radical scavenging and hydroxyl radical scavenging rate reached the maximum at fourth month,and the reduction ability reached the maximum in fifth month.In the ninth month,DPPH free radicals in three groups of cheese were 35.36%,39.43% and 41.53%,respectively.Scavenging rates of hydroxyl free radicals were 35.12%,37.33% and 38.50% respectively.Reduction power were 0.372.0.498 and 0.537,respectively.The antioxidant activities of probiotic cheese were significantly higher than those of control(P<0.05),and L.helveticus 1.0612 cheddar cheese had the highest activities.In the ninth month,the numbers of live strains of Lactococcus in the three groups were 7.82 lg CFU / g,7.74 lg CFU/g and 7.73 lg CFU/ g,respectively.The number of Lactobacillus were 4.66 lg CFU / g,7.58 lg CFU/g and 7.53 lg CFU/ g,respectively.There were no differences between L.helveticus 1.0612 cheese and L.rhamnosus 1.0911 cheese(P>0.05),but cheese with L.helveticus 1.0612 had significantly better activities than that with L.rhamnosus 1.0911(P<0.05).This indicated that the effect of the number of lactic acid bacteria on the antioxidant activity of probiotic cheese was negligible.In the ninth month,the values of pH 4.6-SN,12% TCA-SN and 5% PTA-SN of cheddar cheese added with L.helveticus 1.0612 were 33.39%,18.38% and 9.40% respectively,cheese added with L.rhamnosus 1.0911 were 30.52%,19.64% and 11.78% respectively,and control group were 29.01%,21.54% and 13.10% respectively.There were significant differences among three groups(P<0.05).L.helveticus 1.0612 cheese had the strongest degree of hydrolysis,and its antioxidant activities were significantly higher than those of the other groups(P<0.05).Therefore,probiotics with better activity of hydrolysis can promote the hydrolysis of cheese proteins.This produced more peptides and amino acid with antioxidant activities to enhance the activity of cheese.(3)Separation and purification of cheese antioxidant peptides and determination of amino acid componentsWSE of Batch-1,Batch-2 and Batch-3 were separated by 10 kDa and 5 kDa ultrafiltration membranes after nine months.At the same time,antioxidant abilities(DPPH free radical scavenging ability,hydroxyl radical scavenging ability and reducing ability)were also measured.Fragment A3(< 5 kDa)of each sample had the smallest molecular weight,but the strongest antioxidant abilities.The free radical scavenging rates of DPPH were 67.14% and 71.30%,respectively,the scavenging rates of hydroxyl radical were 49.50% and 69.44% respectively and reducing power were were 0.45,0.68 and 0.82,respectively.There were significant differences among three samples(P<0.05),and the antioxidant activity of WSE with L.helveticus 1.0612 was the best.Macroporous resin was used to absorb salt.Further purification by Sephadex G-25 gel chromatography was carried.Three groups of fragments were isolated from each sample.And the second fragment(F2)of each sample had the best antioxidant activity.The free radical scavenging rates of DPPH were 60.22%,69.95% and 78.65%,respectively.There were significant differences among three samples(P<0.05).And WSE with L.helveticus 1.0612 still had the highest activity.Then the amino acid components of the second fragment from each sample were determined,and the results showed that the activity of antioxidant peptides in cheese was influenced by the types and contents of amino acids.The contents of the four amino acids including His,Pro,Cys and Leu in the Batch-3(WSE with L.helveticus 1.0612)were significantly higher than those in the other two batches(P<0.05).This might be due to the deep hydrolysis of casein by L.helveticus 1.0612,which produced these amino acids or short peptides with them.And they had good free radical scavenging abilities themselves,or could chelate metal ions to reflect reducibility,thereby enhancing the antioxidant capacity of cheese.(4)Effect of probiotics on the qualities of cheddar cheeseThe effects of probiotics on the qualities of cheddar cheese were evaluated by composition,texture analysis and sensory analysis.The results showed that the content of cheddar cheese in the three groups was within the standard range,and there was no significant difference(P>0.05)among three groups except the salt.After adding probiotics,the hardness and cohesion of cheddar cheese were significantly changed(P<0.05),and the elasticity of cheese was increased due to the hydrolysis abilities of L.helveticus 1.0612.But there was no significant difference in other aspects(P>0.05).In sensory evaluation,cheese with L.helveticus 1.0612 had significant bitter taste(P<0.05),but there was no significant difference among three groups in other indexes(P>0.05). |
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