Effect Of Probiotics On The Production Mechanism Of Cheese Bioactive Peptides And Their Structural Characteristics

It has been found that cheese is not only rich in many nutrient components,but also produces rich bioactive peptides during the ripening due to the casein of cheese was hydrolyzed by the protease and peptidase of lactic acid bacteria into large peptide,free amino acid and even functional active peptide,such as antioxidant peptides,immunoregulatory peptides,antimicrobial peptides,which make cheese rich in special physiological function,which has attracted close attention of scholars.Cheese contains not only lactic acid bacteria in raw milk,but also exogenous starter Lactococcus and non-starter lactic acid bacteria and some of them belong to probiotics.However,the ripening period of cheese is too long,even some of them have been ripend for several years,so that the bioactive peptides may be further hydrolyzed.To promote the production of specific bioactive peptides from cheese,it is also necessary to add probiotics with high hydrolytic activity.The addition of probiotics may change the microbial structure of cheese and affect the hydrolysis of cheese protein,thus affecting the production of active peptides and the texture of cheese,resulting in a decline in quality.Therefore,it is significance to study the structural changes of bacterial flora,formation mechanism of bioactive and its structural characteristics in order to obtain the cheese with high biological activity.The antioxidant peptides and angiotensin-converting enzyme?ACE?-inhibitory peptides of cheese were studied in our study.First,probiotics with good hydrolysis ability and potential of peptide producing were used in this study to analysis their protease properties.The probiotics,which have the potential to produce peptides were then added to Cheddar cheese.The survival and effect of probiotics on microbial structure,aminopeptidase activity,proteolytic patterns and activity of antioxidant and ACE-inhibitory during cheese ripening were investigated.The relationship between probiotics and antioxidation and ACE-inhibitory activity of cheese was found.The antioxidant and ACE-inhibitory peptides of cheese were isolated by ultrafiltration and sephadex G-25 gel,and then identified the structure with LC-MS/MS to study the hydrolysis position of casein and the structure and activity of two kinds of peptides.The main results were as follows:?1?By screening the proteolytic ability of nine strains of probiotics,results demonstrated that L.helveticus 1.0614,L.rhamnosus 1.0386,L.helveticus 1.0618 and L.rhamnosus 1.0911 had the strongest proteolytic ability among nine probiotics,the activity of these four probiotics were?0.507±0.008?,?0.481±0.006?,?0.417±0.009?and?0.320±0.006?respectively.According to the hydrolyze properties of casein of these four strains,results demonstrated that the peptide yield,protein recovery of L.helveticus 1.0614 were significantly higher than those of other strains?P<0.05?.The casein hydrolysates of these four strains had antioxidant activity and ACE-inhibitory activity,and gradually increased with the increasing of hydrolysis time.The antioxidation and ACE-inhibitory activity of hydrolyzed casein of L.rhamnosus 1.0386 and L.helveticus 1.0614 were significantly higher than those of other two strains?P<0.05?.But there was no significantly difference between these two hydrolysates in the activity of DPPH radical scavenging,reduction ability and ACE-inhibitory?P>0.05?.Therefore,L.rhamnosus 1.0386 and L.helveticus 1.0614 were selected as the target probiotics for further study.?2?The enzymatic properties of CEP,Pep N and Pep X in the proteolytic system of these two strains were analyzed.The activity of CEPs from these two probiotics were significantly higher than the other two proteases?P<0.05?.The CEPs from L.helveticus 1.0614 and L.rhamnosus1.0386 were?63.57±1.03?U/mL and?59.28±1.03?U/mL.The activity of CEP of L.helveticus1.0614 was significantly higher than that of L.rhamnosus 1.0386?P<0.05?.According to the Lineweaver-Burk plot of three proteases from two strains,the affinity of CEP to casein was the strongest,which inferred that CEP plays a major role in the hydrolysis of casein.The optimum enzyme production conditions of two probiotics were analyzed.The optimum temperature and pH of CEP,Pep N and Pep X from L.rhamnosus 1.0386 were 40?,45?and35?;pH was 6.5,7.0 and 7.0 respectively,and about L.helveticus 1.0614 were 45?,40?and45?;pH was 6.5,6.5 and 7.0 respectively.Three proteases from L.rhamnosus 1.0386 and L.helveticus 1.0614 showed good stability at 4⁴0?,pH5.0⁷.0.Na⁺,Ca²⁺and K⁺could significantly increase the activity of three proteases.The ripening temperature of the cheese is4¹2?,the pH of the cheese system is about 5.0 and cheese also certain amounts of Na⁺and Ca²⁺,which could promote the probiotic protease activity.?3?The addition of L.rhamnosus 1.0386 and L.helveticus 1.0614 had no significant effect on the composition of Cheddar cheese?P>0.05?,which met the requirements of Cheddar cheese standard.The hardness and cohesiveness of cheese with probiotics were significantly lower than those of the blank cheese after 180 d?P<0.05?,but had no effect on the overall texture of cheese.The viable count of L.rhamnosus 1.0386 and L.helveticus 1.0614 in fresh cheese were?8.03±0.06?and?7.92±0.13?log CFU/g,which showd that the cell wall protease of these two strains could keep a high content.During the ripening of cheese,these two strains also dominated all ripening stages of cheese and maintained their viability of?8.93±0.04?log CFU/g and?8.44±0.05?log CFU/g at 240 d.The addition of probiotics made the flora structure more diverse,mainly increased Lactobacillus and had no effect on the growth of Lactococcus.Because of the competition between probiotics and Enterococcus,the viable count of Enterococcus was significantly decreased?P<0.05?.By analyzing the activity of aminopeptidase during cheese ripening,results demonstrated that after 120 d,Pep N and Pep X of cheese with L.rhamnosus 1.0386 and L.helveticus 1.0614increased significantly?P<0.05?.The content of Pep N of these two cheeses reached the maximum at 150 d and 210 d.The content of Pep N reached the maximum at 150 d.The aminopeptidase activity of cheeses with probiotics was significantly higher than that of control cheese?P<0.05?.The results showed that the intracellular aminopeptidase was involved in the proteolysis of cheese with the death of probiotic bacteria.The proteolysis of cheeses with L.rhamnosus 1.0386 and L.helveticus 1.0614 showed significantly increase in primary proteolysis and secondary proteolysis of probiotic cheeses during the whole ripening and significantly higher than that of control cheese?P<0.05?.The content of pH4.6-SN of these two probiotic cheese were?26.46±1.06?%and?28.37±1.55?,the content of 12%TCA-SN were?25.64±1.14?%and?26.32±1.32?%.The 5%PTA-SN of probiotic cheeses were significantly higher than control after 180 d?P<0.05?.Cheddar cheese added L.helveticus 1.0614 had the highest degree of hydrolysis.It can be concluded that probiotics can promote the primary and second hydrolysis of cheese protein.After 180^th day of ripening,the short peptide and free amino acid soluble in 5%PTA were produced under the action of probiotic cell wall protease and aminopeptidase.The stronger the hydrolysis ability of strain was,the more obvious influence of the strain was.?4?During the ripening,the antioxidant activity of cheeses with L.rhamnosus 1.0386 and L.helveticus 1.0614 showed upward trend and reached maximum at 240 d and significantly higher than control cheese?P<0.05?.At the 240^th day of ripening,the DPPH scavenging ability of Cheddar cheeses with L.rhamnosus 1.0386 and L.helveticus 1.0614 were?74.05±1.23?%and?80.12±1.53?%;The hydroxyl radical scavenging ability were?71.2±1.33?%and?72.8±1.29?%;The reducing ability were?0.592±0.023?and?0.602±0.124?.At 0-60 d,the ACE-inhibitory activity of cheese with L.rhamnosus 1.0386 and L.helveticus 1.0614 was significantly increased by 52.2%and 57.2%?P<0.05?,and reached maximum at 240 d and the activity were?92.2±1.09?%and?95.1±1.22?%.These results suggested that the addition of probiotics could significantly increase the antioxidant activity and ACE-inhibitory activity of cheeses?P<0.05?,and the L.helveticus1.0614 with higher hydrolysis ability had the obviously influence.According to the correlation analysis,we found that the number of Lactobacillus in cheese which with L.rhamnosus 1.0386 and L.helveticus 1.0614 were positively correlated with the degree of hydrolysis,antioxidant and ACE-inhibitory activity,but not significantly?P>0.05?.Compared with blank cheese,the content of 12%TCA-SN and 5%PTA-SN of probiotic cheeses were significantly positive correlation with antioxidant and ACE-inhibitory activity?P<0.05?.5%PTA-SN of cheese with L.helveticus 1.0614 showed significantly positive correlation with antioxidant and ACE-inhibitory activity?P<0.01?,which indicated that the antioxidant and ACE-inhibitory activity of cheese was mainly related to the secondary hydrolysis of protein and the content of short peptide of cheese.Strong hydrolytic probiotics could promote the hydrolysis of protein and produce small molecular bioactive peptides in order to increased the antioxidant and ACE-inhibitory activity of cheeses significantly?P<0.05?.The stronger the hydrolysis ability of strain,the more short peptides is produced.?5?Three fractions?>10 kDa,3-10 kDa,and<3 kDa?were separated from the end of the ripened cheese by ultrafiltration.Among the cheeses polypeptides fractions,the<3 kDa exhibited the highest free radical scavenging capacity and ACE-inhibitory activity.The DPPH scavenging ability,hydroxyl radical scavenging ability,reducing ability and ACE-inhibitory activity of fraction?<3 kDa?of cheese with L.rhamnosus 1.0386 were?82.3±0.93?%??80.6±0.35?%?0.592±0.022 and?93.5±1.63?%,about cheese with L.helveticus 1.0614 were?89.4±0.32?%??82.3±0.65?%?0.501±0.031 and?96.7±0.32?%.Results demonstrated that the lower the molecular weight,the stronger the antioxidant and ACE-inhibitory activity.The results of gel chromatography and structure identification showed that the structure of antioxidant peptide and ACE-inhibitory peptide were different,but the molecular weight was less than 1000 Da and the length of peptide was 5¹0,which mainly from?-CN,follwed by?_s1-CN.Compared with L.rhamnosus 1.0386,the shorter length of peptides was obtained by adding L.helveticus 1.0614,and from variety of casein types.The antioxidant peptides and ACE-inhibitory peptides of cheeses with two probiotics had similar structural characteristics.The antioxidant peptides had higher content of hydrophobic amino acids and aromatic amino acid,such as Pro?P?and Tyr?Y?;N terminal of ACE-inhibitory peptide were Val?V?,Ile?I?,Leu?L?,C terminal were Val?V?,Ala?A?,Phe?F?or Pro?P?.The peptides with antioxidant activity may also have a certain ACE-inhibitory activity.The activity of the peptide depends on the position of key amino acids and the length of peptide chain.The C-terminal of the peptide chain is generally the binding site for capturing free radicals and the active sites of ACE.In general,when the peptide chain C-terminal is Pro and the peptide is short,it has higher antioxidant and ACE-inhibitory activity.According to the hydrolysis site of probiotics and the typical structure of active peptides,the cell wall protease of probiotics is the key protease to produce the antioxidant peptides and ACE-inhibitory peptides.At the late stage of ripening,aminopeptidase can also induce cheese to produce two active peptides.