| Apple is an internationally competitive agricultural product with large-scale cultivating and many kinds of apple processing products in China.Recently,due to the increasing health awareness of consumers,the sales of NFC(Not form concentrate)apple juice are increasing day by day.At present,more than 16 million tons of fruit juices were consumed every year in the world.NFC apple juice has to be a victim of profit for adulterator owing to its high price and broad market background.Many FC(Form concentrate)juices and NFC blended FC juice products were sold as fake of NFC juices.This phenomenon has disrupts the juice product market order of fair competition,and the authenticity of NFC apple juice has became the issues of great concern to consumers.Therefore,there is an urgent need to develop a technology that can detect the authenticity of NFC apple juice and utilize this technique in practices.In this paper,NFC apple juice was prepared from different varieties of apple,on the basis of ensuring microbial safety and juice quality,carbon,hydrogen and oxygen isotopes,types and contents of polyphenols and DNA in NFC apple juice were studied by stable isotope ratio mass spectrometry,HPLC and RT-PCR.The main results are as follows:1.Isotope ratio mass spectrometry(IRMS)is an effective method in the true and false identification of fruit juice.In this study,the values of ?D,?18O and ?13C of NFC apple juice and adulterated apple juice(AAJ)were analyzed and studied by isotope ratio mass spectrometry(IRMS)using different apple cultivars.The results showed that ?D and ?18O values of water in NFC apple juice were higher than those of exogenous water in adulterated apple juice,and the ?D,?18O value of AAJ juice was negative correlation with the addition content of exogenous water.By measuring the value of ?13C,it can be seen that the content range of fructose,glucose and disaccharide in NFC apple juice was-25.64?26.83‰,-25.01?-26.36‰,and-22.41?-23.23‰ respectively.Where the percentage ranges of fructose,glucose and disaccharide in total sugar was 48.84?52.39%,14.34?28.85%,and 10.47?18.78%,respectively.The oligosaccharides were not detected in this research.But the ?13C value and percentage in total sugar of disaccharide for AAJ apple juice were not in the above range.This research showed the method of IRMS can be used to establish a database after analyzing a large number of samples of NFC and AAJ apple juice,so as to better identify the authenticity of NFC apple juice on the juice market.2.The polyphenols was extracted from 10 different varieties of NFC apple juice by ultrasonic assisted extraction with methanol used as an extraction agent in this part of experiment.The polyphenols which we suppose maybe contained in apple NFC juice was detected through high performance liquid chromatography method,the types and contents of polyphenols in different varieties of apple NFC juice was analyzed and compared with FC apple juice.The results showed that 6 kinds of polyphenols(Gallic acid,p-hydroxybenzoic acid,chlorogenic acid,caffeic acid,epicatechin,quercetin)were contained in all the 10 kinds of apple NFC juice,and the content of 6 kinds of polyphenols have significantly difference.There are two kinds of polyphenols(gallic acid and glucoside)were not contained in all the 10 kinds of apple NFC juice.The content of chlorogenic acid was highest of all the 6 kinds of polyphenols in all the 10 kinds of apple NFC juice and the most highest content of chlorogenic acid was 116.691±0.0641 mg/L in NFC fruit juice of Fuji Apples,while the lowest content of chlorogenic acid was 37.3361±0.0099 mg/L in NFC fruit juice of Han Fu apples.But the phlorizin appeared in FC juices of Hanfu apples,nd phenols of phloretin and phloreside,appeared in FC apple juice of Qianajin,Ziyou,Yanguang and Fuji apples.The research results can be used as an important basis for distinguishing adulteration of NFC juice.If phlorizin and phlorizin could be identified in NFC apple juice,this fruit juice can be defined as adulterated fruit juice with FC juice.3.A new method of extracting DNA from fresh apple juice(NFC)was established,and the real-time quantitative PCR was developed to identify NFC apple juice and adulterated apple juice(AAJ).The test results found that the extraction concentration of juice DNA can greatly improved after adding silicon based magnetic beads(Si-OH@Fe3O4 NPs)based on CTAB for DNA extraction method.Because the base magnetic beads(Si-OH@Fe3O4 NPs)can effectively adsorb DNA in juice,the extracted DNA concentration was 50?60 ng/ml under reasonable operating conditions,while the optimal conditions for extracting DN A in juice by magnetic beads were 1 mL of sample dosage and 3 min of reverse mixing.The standard curve for RT-PCR amplification of continuously diluted DNA from NFC apple juice was y=-2.0959x+30.714,and the correlation was R2=0.9985.The RT-PCR amplification curve of DNA in AAJ juice with different degrees adulterated was y=-0.9683+28.591,and the correlation was R2=0.9819.The RT-PCR based on the new DNA extraction method has good stability,repeatability and reliability in NFC apple forgery identification.In a word,the higher value of ?D,?18O and ?13C in NFC apple juice was found compared with FC apple juice,and more kinds and contents of polyphenols,and higher DNA concentration for any apple variety,while these values change regularly with the degree of adulteration.The application of IRMS,HPLC and RT-PCR(Real-time fluorescence quantitative polymerase chain reaction)analysis to the identification of NFC apple juice is helpful to the supervision of NFC juice market and establish of national juice standards.Those methods also can ensure the quality of NFC juice,and making consumers trust NFC apple juice brand and standardizing and rationalizing China's juice market. |
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