Fruit Content of Fruit Juice and Apple Juice Content of Cider Using DNA Methodology - Literature review Example

Review of Literature: Adulteration of the food is a very serious economic factor. The fruits and their products are given more importance because of the large consumption and wide applications. The guarantee that the fruit juice that we take in is very safe is the most important thing for the customers. The food safety is guaranteed by the development of the analytical techniques. The analytical techniques include multivariate compositional analysis, UV –visible, AAS, AES, IRMS, GC-IRMS, DSC etc., (Cordella et al. 2002). The Polymerase Chain Reaction and the laboratory –on-a –chip capillary electrophoresis (LOC) were used for the identification of the DNA in the fruit juices. The PCR –RFLP method was used for the analysis of the different fruits in the fruit juice. The PCR heteroduplex system was successfully applied to the LOC and was found to show greater sensitivity for the identification of the fruit juices. After this process, random reannealing of the amplified DNA was done based on Hardy- Weinberg law. The heteroduplexes obtained from these studies have proved that the heteroduplexes were underestimated at the lab-on-a-chip method. The differential data interpretation is done to obtain better results. (Scott and Knight 2009). DNA profiling method was developed for the fruit samples. Different DNA extraction methods were practiced for the isolation of the genomic DNA from the fresh fruits. Some of the DNA extraction methods are CTAB- based DNA extraction method and some kits such as Nucleon Phytopure plant DNA Extraction kit, DNeasy Plant mini kit and G2 buffer & Genomic –tip 20/G kits can be used for the extraction of the DNA from the given fruit juices. SSR markers are used for the identification of the DNA from the given samples. The SSR analysis was able to reveal that the genotypes were the same for the dried fruits and the fresh juices. The DNA samples obtained from the canned and processes foods will be very small of 500 bp. (Yamamoto et al. 2006). These DNA samples can be easily amplified using the high quality primers. In the pear fruit DNA sequence there are 15 SSR sequences and among them 9 SSR markers were obtained from the fruit juice containing pear. The reason for this could be that the DNA obtained from the fruit juices were around 150 base pairs and they were very small for amplification. The DNA profiling and the cultivar methods were found to be very useful for the identification of the DNA of the fruits from the fruit juices. (Yamamoto et al. 2006). Orange juice is one of the most popular juices of the world, consumed by the people of all ages. The orange juices are now being complained about the adulteration. Hence the qualities of the fruit juices are determined by HPLC, capillary electrophoresis and pyrolysis mass spectrometry. These methods are used to determine the chemical components of the juice. The analyses of the DNA sample are the recent methods. The use of DNA markers are increasing day by day. The DNA markers used in the analysis include Random Amplified Polymorphic DNA, (RAPD), Restriction Fragment Length Polymorphism, (RFLP), Sequence - Characterized Amplified Regions (SCARs) and Chloroplast DNA (cp DNA). (Liu and Shyu 2006). Of these analyses, the chloroplast DNA is found to be more efficient in the analysis. The Denaturing gradient gel electrophoresis (DGGE) technique helps to separate the PCR amplicons of the same length based on the nucleotide sequence using the denaturing gradient gel. Two types of DGGE system used are perpendicular and parallel DGGE. The trnL intron and trnL- trnF intergenic spacer are amplified using the specific primers and the PCR products are obtained to run the DGGE. (Liu and Shyu 2006). This method is very accurate and can be well used in the fruit industry for the estimation of the DNA in the fruit juice. (Liu and Shyu 2006). Product quality has become one of the important concept to win the customers confidence. To achieve this, the quality of the products should be assayed for purity. Tin the fruit industry, the adulteration is one of the most important things to be noted of. Hence the scientists are looking for the designing novel methods. The construction of the database for the orange and its types are thus very essential. (Liu and Shyu 2006). The DNA library for these fruits can be used for the comparison of the results of the unknown sample and can be determined easily. The near- infra red transflectance (NIR) spectroscopy was used for the identification of the apple juice adulterations in the samples. High Fructose Corn syrup and the sugar solution are used for the analysis of the NIR. The presence of the sugars can be easily found using the NIR. Thus it acts as a rapid identification technique for the presence of adulterance. (Leon, Kelly and Downey 2005). The presence of the pear juice and its content in the food can be found by using the real time PCR. In this method a fluorescence dye is used for the analysis called SYBR green. The study also aimed at the establishment of the thaumatin – like protein of pear intron sequence. (Yamamoto et al. 2006). In the pear, the thaumatin-like protein intron sequence was first sequenced and added to the data base. By using this method the pear DNA concentration as low as 10pico grams can be easily detected per micro litre. (Jianxun et al. 2010). For the quality assurance of the product, the use of the new and most advanced techniques is essential. The DNA based methods give more accuracy of the results. The food industry uses the DNA based methodologies for the analysis of the quality of the food and also to check the adulteration level in the food products. The techniques that can be used for the analysis of the products include Differential scanning calorimetry, FT-IR, Fourier-transform infrared, ELISA, enzyme-linked immuno sorbent assay, UV–vis, ultraviolet–visible, Site-specific nuclear isotopic fractionation NMR spectroscopy, FT-MIR, Fourier-transform mid-infrared, GC, gas chromatography, GC-MS, gas chromatography-mass spectroscopy, GC-TOFMS, gas chromatography-time of flight mass spectroscopy, MS, mass spectroscopy, HPLC, high performance liquid chromatography, LR-NMR, low-resolution NMR spectroscopy, NMR, nuclear magnetic resonance, PCA, principal components analysis, PCR, principal components regression, SNIF-NMR, site-specific nuclear isotopic fractionation NMR spectroscopy, PLS, partial least squares are some of the techniques that are used for the analysis. (Reid, O’Donnell and Downey 2006). Carbohydrate chromatography is also used for the analysis and estimation of fruit juices. This chromatography is used to identify the percentage of carbohydrates that is present in the sample and the ratio of the components and how much percentage the adulteration has occurred. Here the specific carbohydrates are used as markers for the analysis of the products. (Prodolliet and Hischenhuber 1998). The identification of the freshly squeezed and the reconstituted orange juice can be easily determined using the rapid and accurate molecular approaches. The primers were designed accurately according to the needs. The design is based on the 18S and ITS ( internal transcribed spacer) region. The fresh juice prepared and preheated to 80 °C for 30 seconds created damage to the DNA. It also failed in the amplification of the DNA. It was found that the juice that was reconstituted could not produce the perfect PCR product and the freshly prepared juice DNA was amplified as a whole without any modification. Thus these findings have given rise to the new DNA –integrity method which has very high potential applications for identifying the adulterations. (Ng et al. 2006). The scarcity of the analytical techniques for the determination of the fruit content in the fruit juices has made the scientists to look for the simplest and more accurate method that can also be performed very easily in a very short time. The real- time PCR is one among the analytical techniques. The DNA is extracted from the fruit juices by using the DNA extraction methods. The DNA integrity assessment is then performed. The DNA integrity will enable the scientists to get a better amplified DNA from the real –time PCR. The DNA integrity assessment is done using the molecular markers. For the real-time PCR 10 primers were designed. These primers were designed such that the conserved regions of the rbcL sequences from the apple (X69749.1), blackcurrant (L11204.2), blueberry (L12625.2, AF419837.1, AF419836.1, AF419835.1, AF421107.1, AF124576.1), raspberry (U06825.1), strawberry (U06805.1), orange (58678-60105), pineapple (L19977.1) and pomegranate (L10223.1). (Palmieri, Bozza, and Giongo 2009). These primers were used in different combinations to Amplify the DNA products obtained from the fruit juices. The amplified products ranged from 1000 to 100 pico grams. The amplification limit in this experiment was between 250- 1000 base pairs. The sequence specific amplification products were determined. (Palmieri, Bozza, and Giongo 2009). The primers also enabled to differentiate the species based on the polymorphic profiles. If there are more bands in the gel then the occurrence of more peaks were seen in the real-time PCR. The new primers designed were able to provide specific sequence amplification at smaller level only. But this little amplification was enough for the identification of that particular species. As the blueberry fruit was completely characterized, that DNA was used as the standard DNA for the comparison. The melting peak profiles were carried out at different percentages and the new species specific primers also gave better results. Thus it was concluded that multiplex PCR is a very useful technique that can provide better resolution and quick detection between the species. The detection of the fluorescence level at various amplification steps gave better results than the other analytical techniques. That was a good indicator for further quantitative analysis. The new sequences identified from these PCR gave additional information regarding the fruit species. (Palmieri, Bozza, and Giongo 2009). The Lab-on-a –chip technology, PCR-RFLP profiling approach is much suitable for the determination of fruit species. The standard fruit juices are pomegranate and blueberry juices. They are recognized as premium grade juices. This method used PCR and restriction enzyme digestion. The psbA – trnH intergenic spacer region of the chloroplast gene was determined using the lab – on – a – chip method. (Garrett, Arun and Dolley 2001). Plant barcodes were also used for the analysis of the fruit juices. Barcode is nothing but an initiative to develop a database based on the cytochrome c gene for all the living organisms that helps them in taxonomic identification. The use of the PCR-RFLP provides greater benefits for the direct determination of the species by using the universal primers. The most important requirement for the good quality results is the use of good quality DNA. The good quality DNA is obtained by using the standard DNA extraction methods. The CTAB DNA extraction method can be used for obtaining a good quality DNA form the species. (Garrett, Arun and Dolley 2001). The chloroplast genes are unique to the plants. Hence the identification of the coding and the non-coding regions are much useful for the identification of the plant species though it may be present in a very low concentration. The DNA extraction method described here is as follows: The fruit juices were obtained from the commercial juice producers. The fresh juices were mixed thoroughly by shaking and inverting. The fruit juices are then diluted with the ultra pure water. This dilution is done to reduce the percentage of sugar in the juice. The DNA was extracted from the fruit juices using the Standard CTAB DNA extraction method. One ml of the sample was suspended in the 5 ml of the CTAB buffer, 100mmTris HCl, 20mM EDTA, 1.4M NaCl and 40µl of proteinase K solution. It was vigorously shaked ad stored at 60 degree Celsius overnight. (Garrett, Arun and Dolley 2001). The clear supernatant was removed and added with equal volume of chloroform and centrifuged and the supernatant was collected. Equal volume of iso- propanol was added to precipitate DNA. The pelleted DNA was washed with ethanol and dried and stored in the 1X TE buffer. The concentration of the DNA was estimated using suitable methods. The DNA obtained was amplified before moving to the PCR. The amplified product was confirmed with the gel electrophoresis. (Garrett, Arun and Dolley 2001). The effect of the food processing on the PCR –based amplification method was determined and the genomic DNA was quantified. The small difference in the soya needs to be detected using the Roundup Ready (RR) soya which is heat treated at low pH. Agilent 2100 Bioanalyzer was used to measure the concentration and the number of different sized PCR products that were obtained after the PCR analysis. (Garrett, Arun and Dolley 2001). A simple multiplex PCR was developed and the GM soya types were analyzed. The four types of GM soya were analyzed using a single reaction mix. The maximum primer concentration was used for the analysis and the PCR product thus obtained was resolved in the gel electrophoresis. Gel electrophoresis separates the DNA based on their size and charge. (Garrett, Arun and Dolley 2001).One- dimensional and Two-dimensional gel electrophoresis are used for the analysis. The standard molecular markers are used for the assay. In the multiplex PCR, the lectin gene of 80 bp was targeted. Though they were able to get good results by this method, the post –PCR analysis was also done to calculate the size accurately. The peaks obtained for the four types of GM soya were analyzed using the Agilent 2100 Bioanalyzer. (Palmieri, Bozza, and Giongo 2009).The results from the Agilent 2100 Bioanalyzer enabled to know that the PCR product concentration was higher for the 170 bp, 150 bp, and 202 bp of the EPSPS (5-enolpyruvylshikamate-3-phosphate synthase). (Palmieri, Bozza, and Giongo 2009). And there was a slight increase in the lectin gene concentration in the PCR product. This method is very useful than the gel based analysis and they provide great potential for the analysis of the raw materials and also for the presence of the desired genotype in the foods. (Garrett, Arun and Dolley 2001). The conventional methods used for the analysis were not able to give good resolution of the food samples. The DNA based methods are more reliable and they prvde accurate results on analysis. The need for the best analytical instrument is the question in the analytical industry. The use of selected molecular markers RAPD and SCAR can provide better results. As the PCR based analysis are more sensitive and the results are based on the purity of the DNA sample. The DNA extraction methods used should be standard. Each species vary in characteristics from one another and hence the DNA isolation procedures must be slightly modified. The DNA fingerprinting methods like random amplified polymorphic DNA (RAPD), DNA amplification finger printing(DAF), arbitrarily primed PCR (AP-PCR), PCRrestriction fragment length polymorphism (PCRRFLP), intersimple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), amplification refractory mutation system (ARMS), simple sequence repeat (SSR) analysis, directed amplification of minisatellite-region DNA (DAMD) and sequence characterized amplified regions (SCAR). .(Dhanya and Sasikumar 2010). Of all these techniques, RFLP is widely used for the analysis of the adulterants in the samples because of its low cost and greater differentiating ability. The RAPD is the more fast technology that requires only very simple primers and the previous sequence information is not required for the analysis. RAPD is more sensitive to a variety of plant species. The RAPD markers can be easily converted into the SCAR markers and used for the analysis. (Palmieri, Bozza, and Giongo 2009). This method is very specific for large scale screening and is very easy. The quantitative competitive PCR (QC-PCR) and the real – time PCR leads to the better quantification and confirmation of the number of adulterants present in the sample. The sequence based methods require prior information of the species which is not practically possible for all the species identification. The hybridization methods are also useful for the identification of many species at a time. 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