Separation And Purification Of Metallothionein From Egg And Its Effect On Lactic Acid Bacteria

This project mainly investigated the extraction and purification of egg metallothionein and its effect on lactic acid bacteria(LAB).The extraction method and conditions were optimized.Ultrafiltration membrane was used for separation,and gel column was used for separation and purification under the optimal conditions.The effects of metallothionin on the growth of LAB in vitro and the radical scavenging effects of metallothionin on DPPH and hydroxyl free radicals were studied.Eggs rich in metallothionein were used as the main research material in this experiment,and the metallothionein extraction yield was the main indicator.The effects of homogenization and enzymatic hydrolysis on the extraction yield were compared by single factor experiments and response surface method to search for optimal conditions.They are: the ratio of egg materials and extraction liquid 1:10,protease concentration 6439 U/g,extraction temperature44 ℃ and extraction pH 8.6.Under these conditions,egg metallothionein extraction yield of96.74% was obtained.Enzymatic hydrolysis was used as the extraction method.The crude extract of metallothionein under the optimal conditions was ultrafilted by 30 KD ultrafiltration membrane under 1.5MP pressure to remove macromolecular heteroproteins.The yield of metallothionein was 91.20% and the recovery percentage of protein was 46.88%.At 1.5MP,small-molecule heteroproteins were removed and concentrated through a 2 KD ultrafiltration membrane.The yield of metallothionin was 94.73% and the recovery rate of protein was 78.95%.After ultrafiltration,dissolved metallothione was fractionated by SephadexG-50,and two major protein peaks appeared,which indicated that this gel chromatography method can effectively remove heteroproteins.After further separation by DEAE cellulose DE-23,it was found that the main metallothionein in the egg was MT2,and the purity was 91.87%.To investigate the effects of metallothionein on the growth of LAB,Bifidobacterium sp.,Lactobacillus acidophilus and their mixture were selected.The best LAB culture media were selected by measuring pH and the OD values.The results showed that egg hydrolysates(EH)as nitrogen source could promote the proliferation of LAB.EH was found as the better nitrogen source than intact proteins.The OD values of EH-supplemented fermentation broths of Bifidobacterium sp.,Lactobacillus acidophilus and their mixture are 1.58,1.32 and 1.29 times of their counterparts with unhydrolyzed egg proteins.EH has a positive effect.The supplemented contents of metallothionein was also optimized,on the basis of pH values,OD values and total numbers of bacterial colonies in the fermentation broths.The results showed that metallothionein could also promote the proliferation of LAB,and the optimal doses of metallothionein in Bifidobacterium sp.,Lactobacillus acidophilus and their mixture were found to be 50 μg/mL,37.5 μg/mL and 50 μg/mL,respectively.The radical scavenging activity against DPPH and hydroxyl free radicals of combined metallothionin and LAB was found to be higher than the ones of individual component.This project used eggs produced by hens fed with zinc-rich Chinese herbs and marine organisms,which enriches the research of metallothionein from different sources to a certain extent.The metallothionein was extracted by enzymatic hydrolysis,and an ultrafiltration membrane was added to the separation and purification of metallothionein,which provides a theoretical basis for large-scale production of metallothionein.It paves a new way for the application of metallothionein and LAB in food.