The Study Of DNA Changes During Milk Processing And Storage And Its Application In The Identification Of Adulteration

The milk quality changes during the processing and storage,but the researches about the changes in the quality of bovine DNA and its application in adulteration detection is still rare.Therefore,the DNA purification procedures of milk were optimized,then the changes in DNA quality,including DNA yield,purity,integrity,PCR detection limit and mtDNA:nDNA,were investigated during milk processing and storage.Furthermore,the application of PCR technique for the adulteration detection was also introduced.This study aimed to provide basis and standard method for adulteration detection of milk and milk products.The main results were as follows:1.The optimization of DNA purification procedures for milk.The purification efficiency of the classic organic method(PCIAA)and two commercial kits(KIT1 and KIT2)on the quality of DNA from milk were compared.The PCIAA method purified a DNA content of 97ng/mL,which was significantly higher than KIT1(25ng/mL)and KIT2(40ng/mL)(P<0.05).The PCIAA and KIT2 purified a DNA purity of 1.56 and 1.50,which were significantly higher than KIT1(1.24)(P<0.05).The mtDNA:nDNA ratio was 36 when PCIAA was adopted,which was significantly higher than KIT1(17)and KIT2(12)(P<0.05).Furthermore,there were no significant differences in the integrity of total DNA,mtDNA and nDNA among the three purification procedures.Therefore,our results suggested that the PCIAA method could give us more and purer DNA,so it should become the standard method for DNA isolation from milk.2.The effets of processing on the quality of DNA in milk.Compared with unprocessed milk samples,DNA content and mtDNA:nDNA ratio from fresh milk increased after heat treatment(P<0.05),but there were no significant changes in DNA purity during heat treatment(P>0.05);The DNA content from reconstituted milk did not change during heat treatment(P>0.05),the DNA purity significantly declined(P<0.05),while the mtDNA:nDNA increased after heat treatment(P<0.05).Furthermore,double-stranded DNA obtained from fresh and reconstituted milk increasingly degraded with the end-point core temperature of milk increase,and when heating fresh or reconstituted milk to an end-point core temperature of about 94?,double-stranded DNA fully degraded.However,heat treatment did not influence the detection limit of PCR;fresh or reconstituted milk compositions in goat milk mixtures were detectable by PCR under all treatments,and enabled a good sensitivity threshold of 0.1%.3.The effects of storage on the quality of DNA in UHT milk.There were no significant changes in DNA content and purity during the storage of UHT milk(P>0.05),but the mtDNA:nDNA ratio varied significantly during UHT milk storage(P<0.05).Furthermore,intact double-stranded DNA were not observed in all commercial UHT milk samples,which suggested that the intact DNA was degraded after UHT treatment.But storage did not influence the detection limit of UHT milk composition in goat milk,commercial UHT milk compositions in goat milk mixtures were able to be detected during their shelf life,and enabled a good detection limit of 0.1%by PCR.4.Conventional PCR and real-time PCR were developed for qualitative and quantitative detection of cow milk compositions as adulterants in commercial ground goat milk powders,respectively.The qualitative detection system could detect 0.05ng DNA template,and the detection limit was 0.1%in simulated adulterated samples.While the quantitative detection system could detect 0.005ng DNA template,and the established standard curve was:y=-3.78x+25.52,which displayed the good linear correlation(R2=0.9984)when cow milk compositions were ranged from 0.1%-50.0%,the quantification of cow milk composition in goat milk powder could be achieved in this range.