Fruit Content of Cider Using DNA Methodology - Literature review Example

?Literature review: Fruit content of fruit juice and apple juice content in cider using DNA methodology Background information Fruit juice production is a high-value trade involving international beverage companies. This industry is concerned in producing juices for soft drinks (orange juice, apple juice, grape juice etc.), alcoholic drinks (wine, cider, perry etc.) and other fruit-based products such as jams, jellies, ice creams and yoghurt. Orange, apple, pear, grapes, pineapple and various types of berries (strawberry, black berry, blue berry, red berry and raspberry) are some of the leading fruit types utilized for juice production. Some varieties of a particular fruit type may have a higher demand for a particular product and thus will gain a higher price than another variety of the same fruit type. For instance, orange (Citrus sinensis) juice is more in demand and thus have a higher value than mandarin (C. reticulata) juice though both belong to the Citrus species. Fruit varieties are qualitatively graded using characters such as composition and quality of syrup and this too makes a vast difference in prices. Therefore, especially in mass-scale fruit juice production, the producers may be tempted to adulterate highly-priced products with a less expensive alternative to increase the product volume and thereby gain better profits. Such adulteration may be in the form of diluting with water, sugars, high fructose corn syrup, spent process water and addition of juices from less expensive fruit varieties (as with orange juice dilution with mandarin). Authentication is an important issue in food industry and it is essential to provide the consumer with correct information about the contents of a product. Food labeling and traceability are regulated by EU directive 2000/13/EC and its amendments and by EU regulative 178/2002 respectively (Commission Regulation (EC) No 13/2000 and Commission Regulation (EC) No 178/2002). However, with the adulterated products, the information provided in the label may not be very accurate. This will not only deceive the consumer but may be a dangerous practice as in the case of presence of allergens in food products. Therefore, it is of vital importance to develop technologies to analyze a food commodity quantitatively as well as qualitatively and determine the actual ingredients in a product to protect high quality of food products and thereby certify consumer rights. 2. Detection methods used in fruit juice industry In fruit juice industry, many technologies have being used to detect composition of a product. In the past, Analysis of total nitrogen (N), phosphorous (P) and potassium (K) was practiced to assess fruit content of (eg. orange) juice but, this method could be applied only when levels of these components are well documented. Hence it was apparent that other methods were in need and hence minerals, organic acids (citric acid, malic acid), amino acids and sugars (glucose, fructose and sucrose) were analyzed. During past years, assessment of food products has been conducted using spectroscopy (UV, NIR, MIR), isotopic analysis, methods based on chromatography (High pressure liquid chromatography (HPLC), LC, TLC, GC MC and LC MS), electronic nose, immunological methods such as ELIZA and thermal analysis (Reid, O’Donnell and Downey, 2006). Paper chromatography and HPLC has been used in detecting sugars and acids, Gas liquid chromatography (GLC) and electrophoresis for amino acids and chemical analysis for acidity are some examples for these applications. Lately, as reported by Li, Goovaerts and Meurens (1996), Near- infrared spectroscopy (near-IR) was used to detect sugars (glucose, sucrose, fructose) and acids (citric and malic acids) in orange juice. Each of these methods has their own advantages as well as limitations and thus, more refine and more sensitive technologies were in constant demand. Anthocyanin pigment analysis with HPLC and electrospray mass spectroscopy (ESMS), stable isotopic carbon analyses, NMR spectroscopy and analysis of trace oligosaccharides are some of the more recent detection methods used in food industry (Wrolstead and Durst, 2007). In fact, HPLC alone or in combination with other methods such as MS is considered as a main method in authentication of food . Peak pattern of anthocyanin is characteristic of a fruit type and fingerprinting methods based on anthocyanin profiling has being used in differentiating cranberry and elderberry. Polyphenol profiles have detected the presence of grape fruit in orange juice and sour orange in sweet orange juice. Aroma profiling is another method tested on juices. An on-line RPLC-GC-MS method has been developed for analyzing chiral compounds which are characteristic components of the aroma from fruit beverages and which could be used in assessing authenticity (Nollet, 2004). With the advent of DNA (deoxyribonucleic acid) technologies, food authentication has developed into a more efficient and a systematic procedure. This has permitted the detection of food adulterations or verifications of components in a food commodity, a more realistic, more accurate and a less cumbersome process. 3. Use of DNA technology in fruit juice industry DNA is present in every cell of an organism. Any tissue of a particular organism provides identical DNA despite age, location or development stage. Although plant DNA can get damaged due to acids and heat during processing, it is comparatively thermostable and hence withstand severe denature during food processing than chemical and biochemical compounds. As the sequencing patterns of DNA made by the four bases i.e. A G C T (Adenine, Guanine, Cytosine, Thiamin) are so diverse among individuals but unique for an individual, it makes DNA-based detection method, a powerful, efficient and a simple method for food authentication. Such molecular approaches can greatly enhance the qualitative and quantitative verification procedures that can estimate the actual components and their content in a fruit juice sample. DNA-based techniques can be broadly categorized into three sections as PCR (Polymerase Chain Reaction)-based, sequencing-based and hybridization-based techniques (Dhanya K and B. Sasikumar, 2010). DNA hybridization, Species-specific PCR primers, RFLP (Restriction Fragment Length Polymorphisms) analysis and Single strand conformational polymorphism (SSCP) are some of the DNA-based techniques applied in fruit authenticity (“What’s in your fruit juice?”, 2009). However, although DNA hybridization is a powerful technique, it is a time and labor consuming method and requires a large quantity of pure DNA. This may be the reason for the extensive use of PCR-based techniques in detection of adulterants in food industry where only a small quantity of DNA is required. PCR in combination with fluorescently labelled primers (competitive PCR and ‘Real-time’ PCR), methylation specific ‘real-time’ PCR, SSCP, RFLP, Single Strand Conformation Polymorphisms, Inter-Retrotransposon Amplified Polymorphisms and direct detection of DNA-Invader assay are some of the major DNA-based PCR techniques used in food authentication (Wiseman, 2003) . The principle of PCR is to extract a small quantity of DNA (with a specific DNA sequence) from a selected sample and multiply it many times through repeated denature and anneal so as to produce millions of identical DNA copies. This amplified quantity of DNA allows it to be analyzed to detect its composition with respect to the bases and their sequences. As the DNA composition is unique to an individual variety, it can be easily differentiated from another variety. A ‘marker’ in DNA technology is a ‘small piece of DNA with a known composition’ and such markers that are specific to variety or a species can be identified and used to identify a particular plant type, species or a variety. Thus, a marker originated from a sample of mandarin juice is unique to mandarin but differ from a sample of orange. Hence if a sample of ‘pure’ orange juices express that specific marker during PCR, it can be concluded that the orange juice is adulterated with mandarin juice. This principle is being widely applied by DNA technology in detecting adulterations, in variety identification and in assessing fruit contents in fruit juices. Differentiating pure orange juice from mandarin juice has been of interest of several workers, since pure orange seeks high price and is thus subjected to adulteration by mandarin juices. Since Citrus species have been subjected to cross breeding extensively, common DNA sequences can be present in different Citrus varieties, which in turn complicate variety identification by PCR alone. Hence PCR-based analytical techniques are modified to suit this situation. A novel heteroduplex assay was helpful in quantitative identification of mandarin (C. reticulata) juice in orange (Citrus sinensis) juice (Food composition and additives: Report, 2006). A PCR restriction fragment length polymorphism PCR-RFLP) profile was successfully developed for six main fruit types (Apple, Blueberry, Elderberry, Grape, Pear and Pomegranate) that are used in the fruit juice industry (Scott and Knight, 2008). These profiles permitted correct identification of the fruit type when tested with DNA obtained from leaf or fruit samples. It was shown that the profile variation was very less in varieties of a single species thus facilitating the differentiation process. Such clear differentiation however, could not be obtained from juice concentrates which indicated either DNA was degradated during processing or due to genetic difference of fruits used in juice preparation. It was concluded that RFLP profiles could not verify the authenticity of juice samples from concentrate, but can be used in authenticity testing of fruit- based products such as fruit puree or conserves. Similarly, a PCR-RFLP assay has been used to detect grapefruit juice in orange juice (Scott and Knight, 2009) with a detection limit of 10% v/v. Sensitivity of this method was improved to 2.5% v/v when PCR was coupled with LOC (laboratory-on-a-chip) capillary electrophoresis system in detecting mandarin juice in orange juice, indicating that LOC produce better quantitative analytical data from PCR methods. PCR coupled with Agilent 2100 capillary electrophoresis system has been applied to distinguish DNA between strawberry and raspberry. Here, microsatellite markers (Fvi11 and Fvi20) were differentiated by PCR and measured with DNA 1000 chip method (Burns, Sanders and Burrell, 2009). Although DNA is found in cell nucleus (genomic), chloroplasts and mitochondria, it has been shown that genomic DNA was the best for identification purposes. As reported by Yamamoto et al. (2006), when DNA profiling method was developed for fresh and processed pear samples using four DNA extraction methods, all four methods produced adequate quantities of genomic DNA to be identified with SSR markers. Methods based on DNA fingerprinting provide powerful means to detect the presence of different fruit types in a fruit juice. However, to quantitatively define the fruit content in a fruit drink, quantitative detection methods need to be practiced and the need of quantitative markers to determine fruit juice content in soft and alcoholic drinks has been felt. Array technologies, micro chips, fluorescence and Real time PCR are such methods that are being used in fruit industry. Work carried out by Palmieri , Bozza and Giongo (2009) indicate that the qualitative and quantitative detection for the presence of berry (strawberry, blackberry, currents and raspberry) DNA in different food matrices was possible with Real-time PCR. When pear DNA in fruit juice samples (Pear juice, Pear nectar, Apple juice, Apple nectar, Peach juice) was detected using Real time PCR with fluorescence dye SYBR Green, it was possible to detect pear DNA in these food samples at levels as low as 10 pg/?L (Scott and Knight, 2009). 4. Detection methods of apple juice content in cider Cider is made from apple and generally it is the apple juice that has not been filtered. However, during the preparation of cider, addition of other ingredients like fermentable sugar syrup (fructose, glucose) has being practiced as part of the production procedure. After fermentation and dilution (known as ‘chaptalisation’) final cider may contain around 30% apple juices. A detailed report prepared by Thomas (2004) to determine suitable chemical markers for assessment of apple content in cider describe that, out of the main mineral components in apple, potassium was the best single parameter for measuring juice content in cider and can act as a suitable primary marker while sorbitol (a major non-fermentable carbohydrate in apple) can be tested as a secondary marker. This report concludes that analyzing potassium and sorbitol as primary and secondary markers respectively is a suitable method to determine the juice content of ciders. Gomis et al. (2004) detected apple juice content in natural and sparkling cider using HPLC chemometic sugar analysis. Sugars were analyzed by reversed phase liquid chromatography and after analyzing data with Bayesean analyses, they decided that ciders could be classified according to the origin of the apple juice (as fresh, liquefaction concentrate or press concentrate). Furthermore, additions of liquefaction concentrate apple juice to natural cider in quantities above 5% and press concentrates higher than 10% could be detected from this method. Composition of apple in cider may vary according to the apple variety utilized. Campo et al. (2005) were able to differentiate 35 cider apple accessions belonging to four different varieties based on titratable acidity, malic acid, pH, glucose and glucose/fructose ratio using chemometric techniques. The first application of DNA technology to the identification of apple cultivars is reported by Melchiade et al. (2007). They have genetically authenticated two leading Italian apple varieties, ‘Annurca’ and ‘Annurca Rossa del Sud’ using fluorescence-based capillary electrophoresis and polymorphic DNA satellites. It was possible to differentiate 17 different apple accessions with four simple sequence repeats (SSRs) which could authenticate the presence of ‘Annurca’ in the apple derivatives used for analyses. Xiayan et al. (2008) tested three methods for extracting genomic DNA from apple juice and the quality and quantity of extracted DNA was analyzed by spectrophotometer, PCR amplification and RAPD. Their work proposes suitable methods of DNA extraction from apple juice for authentication purposes. 5. Summary Importance of food authentication has been well recognized and suitably documented. With the development of mass-scale business ventures in food industry, adulteration of costly food items with less expensive components has been a severe problem with many food products. An important area where adulteration is practiced to a greater scale is in the soft drink and alcohol industry. It is essential to control this practice at every possible opportunity and many analytical methods are being developed on detecting adulterants in solid food as well as in drinks and juices. The fact that the producers who involve in adulterating their products find newer and better methods of food adulteration accelerate the technology development for novel and efficient detecting methods of these adulterants. Starting from NPK analysis, fruit juice detection methods have progressed through analyzing minerals, sugars, organic acids and amino acids to modern DNA-based techniques. With the advent of spectroscopy, chromatography, immunology, isotopy and subsequently DNA-based technology, the efficacy of these analytical measures gradually increased and now the techniques permit the identification of fruit type, variety and even the geographical origin of the fruit source simply by analyzing a juice sample. Currently, quantitative detection of fruit components present in a food commodity is gaining wide importance and the development of computerized analytical techniques as well as powerful statistical methods has immensely materialized its progress. These techniques, along with LC-MS (liquid chromatography – mass spectrometry) and NMR (nuclear magnetic resonance spectroscopy) methods are predicted to be used more in future in fruit juice industry (Hammonds, 2009). Currently, HPLC technology is widely applied for detecting apple juice content in cider but methods have been developed for successful isolation of DNA from apple juices and for variety identification via detection of DNA in different apple varieties. This indicates the possibility of applying DNA technology in cider authentication in future. Cited references “What’s in your fruit juice?” (2009). Food and NHP authenticity. Technology Watch. 6(1): 5-8. WellnessWest, Canada Burns M., R. Sanders and A. Burrell (2009). Strawberry and Raspberry Fruit Differentiation Using the Agilent CE 2100. BioanalyzerAgilent Technologies, Inc., USA. [Accessed 19 April 2011] Campo G. del, J I Santos, I. Berregi and A. Munduate (2005). 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A review of DNA-based methods used in FSA project. < www.food.gov.uk/multimedia/pdfs/presentationbygwiseman.PDF> [Accessed 19 April 2011] Wrolstead, R. E. and R.W. Durst (2007). Fruit juice authentication: what have we learned? In. Authentication of food and wine. American Chemical Society symposium series. Vol. 952 Chapter 10, pp. 147-163 Xiayan, S., C. Ying, H. Wensheng, W.Yajun, Y.Fei, G.Yiqiang and D. Wen (2008). Comparison on the Extraction Method of DNA in Apple Juice and Study on the Amplification of RAPD. J. Chinese Inst. Food Sci. and Tech. (Vol. 2) Yamamoto, T. T. Kimura, T. Hayashi and Y. Ban. (2006). DNA Profiling of Fresh and Processed Fruits in Pear. Breeding Science. 56(2): 165 – 171 Read More