1. 1 Instruments used for the estimation of natural juices in nectars – Estimation of fruit content in fruit nectars Martin Kubík Czech Agricultural and Food Inspection Authority (CAFIA) Workshop on Estimation of Natural Juices in Nectars, Cairo, Egypt 10 - 11 November 2014

2. Why to measure fruit content?  To ensure compliance with current (EU) legislation QUID requirements General rules on labelling and advertising Specific requirements e.g. Fruit Juice Directive  To help the consumer make better food choices 2

3. In which products?  fruit nectars- 25 – 50% fruit juice  fruit drinks – 5 -90% fruit juice  applicable (after consideration) to other fruit and vegetable products e.g. jams, preserves etc. The calculation of the fruit juice content based on analysis of the fruit nectars and drinks is an important part of the authenticity control. 3

4. How to measure fruit content?  The basic approach : Identify one or several “index-constituent(s)” specific to a particular type of fruit Determine the concentration of each index- constituent in the product under investigation Compare this concentration with that expected for the pure fruit 4 J.F. Kelford, Analytical Problems with Fruit Products. Food Preservation Quarterly 29, 65-71, 1969

5. How to measure fruit content  Ideal properties for “index-constituent” :  Easy to measure with adequate accuracy  Present only in an fruit to be measured  Analyte in the ingredient is subject to very little natural variation  Analyte level is unaffected by food processing  Is sufficiently rare or expensive that is unlikely to be added 5

6. How to measure fruit content  Ideal “index-constituent” is not always available  Concentration in known constituents is influenced by many factors:  Variety  Maturity  Growing area  Climate  Fertilization  Irrigation  Specific markers can be influenced by processing techniques  Interference of other components in a complex food matrix 6

7. How to measure fruit content  Selection of the „best“ possible “index- constituent (s)” usually:  relevant and reliable authentic parameters as established in the Code of Practice (AIJN) for juice from one kind of fruit  Not all parameters can be used in general as a result of interference caused by the use resp. addition of - water used for dilution containing minerals as sodium, calcium … - sugars containing glucose, fructose, sucrose - acids as citric acid, malic acid, … - declared or undeclared fruit 7

8. How to measure fruit content  Currently used “index-constituent” for fruit juice content determination ( do not interfere with the water used or with the allowed ingredients ) :  Fruit acids (malic, isocitric, quinic, tartaric shikimic…)  Minerals (K, Mg, P, Mn..)  Amino acids (formol number, proline..)  Flavonoides, sugars (alcoholic sugars), anthocyanins, carotenoids…. 8

9. How to measure fruit content  2 possible situations 1. composition of raw material is known (analytical results available or raw material available) Not common situation - calculation of fruit content quite easy – just simple comparing results from raw material (fruit juice) and product (e.g. nectar) and calculation. RISK - raw material available is not really identical to raw material used for manufacturing - raw material is not authentic 100% juice WRONG RESULTS 9

10. How to measure fruit content  2 possible situations 2. no exact knowledge about composition of raw material just information from the label e.g. „50% of fruit juice“ 10

11. How to measure fruit content - calculation models  several calculation models could be find in literature for limited range of fruit  most of them based on calculation from analytical results of chosen parameters in product of interest and mean values from databases sometimes including a so-called weighting coefficient If the beverage contains more than one juice, or juice has been added to acidifying or the problem for calculation is more complex 11

12. How to measure fruit content - calculation models  results from various models do not differs dramatically up to 5% but undeclared fruits in case of adulteration or undeclared or declared fruit for highlighting of colour (e.g. red beet concentrate or elderberry concentrate) could have influence on some parameters and be taken into account so e.g. suspicious or outlined results is necessary to eliminate before calculation  knowledge about composition of possible ingredients is esential 12

13. How to measure fruit content - influence of ingredients  1% of Tri potassium citrate increases level of K for 3600 mg/kg  chokeberry increases level of sorbitol  elderberry increases level of isocitric acid detection via anthocyanins profile  addition of synthetic malic acid – detection via fumaric acid 13

14. How to measure fruit content - calculation models examples Early attempts 1940 : Hinton and Macara. The Composition of some Jam Fruits and the Determination of the Fruit Content of Jams 1949 : Steiner …. 1965 : Nehring and Klinger  Combination of two or three index constituents in a linear equation to give fruit content  Ingredient content = a 1 X 11 + a 2 X 12 + a 3 X 13 where X 1j is the analytical determination and a j is an appropriate factor Most common index-constituents  Ash, potassium, phosphate 14

15. How to measure fruit content - calculation models examples  orange nectar/drink (1): Gesellschaft Deutscher Chemiker/GDCh, Flüssiges Obst 6, 1979) orange juice content (%) = potassium (analyzed) mg/l x 20/1750 + phosphate (analyzed) mg/l x 20/460+ proline (analyzed) mg/lx 20/750+ formol number (analyzed)x 15/18+ L-malic acid (analyzed) mg/lx 15/1500+ D-isocitric acid (analyzed) mg/lx 10/90 15

16. How to measure fruit content - calculation models examples  orange nectar/drink (2): Bielig/Hofsommer (Technical University Berlin, privat, 1983) orange juice content (%) = potassium mg/l x 15/1900 + phosphate mg/l x 20/460+ proline mg/lx 20/800+ formol x 20/20+ L-malic acid mg/lx 10/1700+ D-isocitric acid mg/lx 15/90 16

17. How to measure fruit content - calculation models examples  orange nectar/drink (3): Ooghe (Zeitschrift für Lebensmittel-Untersung und – Forschung 1990:191) orange juice content (%) = potassium mg/l x 25/1900 + D-isocitric acid mg/lx 25/90+ L-lysine mmol/l x 25/0,19+ L-arginine mmol/lx 25/3,11 17

18. How to measure fruit content - calculation models examples  orange nectar/drink (4): Czech Agriculture and Food Inspection Authority (CAFIA) orange juice content (%)= average of following results: potassium mg/l x 100/1900 magnesium mg/lx 100/115 phoshorus mg/l x 100/163 formol numberx 100/20,5 L-malic acid mg/lx 100/1900 D-isocitric acid mg/lx 100/100 ash g/lx 100/3,5 18

19. How to measure fruit content - calculation models examples  Comparison of the results obtained using different formulas:  sample - orange nectar (50% of orange juice declared) GDCh – 56,7 % Bielig/Hofsommer– 53,9 % CAFIA– 52,7 % Max diference4 % 19

20. How to measure fruit content - calculation models examples  apple nectar/drink (1): Koch (Flüssiges Obst 2, 1980) apple juice content (%) = potassium mg/l x 33,3/1175 + phosphate mg/l x 33,3/200+ ash g/lx 33,3/2,5 20

21. How to measure fruit content - calculation models examples  apple nectar/drink (2): Gesellschaft Deutscher Chemiker/GDCh, Lebensmittelchemie und gerichtliche Chemie 36, 1982) apple juice content (%) = potassium mg/l x 35/1150 + phosphate mg/l x 35/210+ sorbitol mg/lx 10/4000+ L-aspartic acid mg/lx 20/100 21

22. How to measure fruit content - calculation models examples  apple nectar/drink (3): Bielig/Hofsommer (Technical University Berlin, privat, 1983) apple juice content (%) = potassium mg/l x 35/1150 + phosphate mg/l x 35/200+ sorbitol mg/l x 10/3000+ L-asp+ser+asn+glu+ala mmol/l x 20/3,32 22

23. How to measure fruit content - calculation models examples  apple nectar/drink (4): Czech Agriculture and Food Inspection authority (CAFIA) orange juice content (%)= average of following results: potassium mg/l x 100/1200 magnesium mg/lx 100/58 phosphorus mg/l x 100/58 formol numberx 100/6,5 L-malic acid mg/lx 100/3000 ash g/lx 100/2,7 sorbitol g/lx 100/4,75 (phloridzin mg/lx 100/32) 23

24. How to measure fruit content - calculation models examples  grapefruit nectar/drink: Gesellschaft Deutscher Chemiker/GDCh, Flüssiges Obst 12/1981) grapefruit juice content (%) = potassium mg/l x 20/1400+ phosphate mg/l x 20/370+ proline mg/lx 10/450+ formol numberx 30/19+ D-isocitric acid mg/lx 20/200 24

25. How to measure fruit content - calculation models examples  lemon nectar/drink (1): Gesellschaft Deutscher Chemiker/GDCh, Flüssiges Obst 12/1981) lemon juice content (%) = potassium mg/l x 20/1400 + phosphate mg/l x 20/350+ proline mg/lx 10/400+ formol numberx 15/17+ L-malic acid mg/lx 10/2700+ D-isocitric acid mg/lx 25/250 25

26. How to measure fruit content - calculation models examples  lemon nectar/drink (2): Ooghe (Zeitschrift für Lebensmittel-Untersung und – Forschung 1990:191) lemon juice content (%) = potassium mg/l x 25/1420 + D-isocitric acid mg/lx 25/355+ L-asparagine mmol/l x 25/4,05+ total amino acids mmol/lx 25/19,1 26

27. How to measure fruit content - calculation models examples  passionfruit nectar/drink: Rother / Gesellschaft Deutscher Chemiker/GDCh, 1989, not published) passionfruit juice content (%) = potassium mg/l x 20/2900 + phosphate mg/l x 10/580+ formol numberx 20/32+ L-malic acid mg/lx 10/2900+ D-isocitric acid mg/lx 20/270 + L-aspartic acid mg/lx 20/930 27

28. How to measure fruit content - calculation models examples  apricot nectar/drink : Wallrauch / Gesellschaft Deutscher Chemiker/GDCh, 1987, not published) apricot juice content (%) = potassium mg/kgx 15/2800 + magnesium mg/kg x 20/95+ formol numberx 20/26+ L-malic acid g/kgx 5/8+ citric acid g/kgx 5/8 + D-isocitric acid mg/kgx 20/110+ sorbitol g/kgx 15/4 28

29. How to measure fruit content - calculation models examples  strawberry nectar/drink : Wallrauch / Gesellschaft Deutscher Chemiker/GDCh, 1987, not published) strawberry juice content (%) = potassium mg/kgx 20/1650 + magnesium mg/kg x 25/110+ formol numbrerx 5/12+ L-malic acid g/kgx 10/2,1+ phosphate g/kgx 15/620 + D-isocitric acid mg/kgx 25/47 29

30. How to measure fruit content - calculation models examples  cranberry nectar/drink : E. Coppola, 4th European Symposium on Food Authenticity, Nantes, 1997 cranberry juice content (%) = (%)= average of following results: quinic acid %x 100/1,06 malic acid %x 100/0,78 citric acid %x 100/1,06 Additional requirements: No sorbitol or fumaric acid, Glu/Fru ratio always > 1,8, four major anthocyanins 30

31. How to measure fruit content - calculation models examples  multifruit nectars/drinks  2 kinds of fruits - some rough calculation possible  necessary to find parameters which are present in one fruits and not in the other  e.g.  combination strawberry x apple  sorbitol and phloridzin present in apple but not present in strawberry  isocitric acid present in strawberry but not present in apple  only some combination of fruits are possible  generally – lack of specific parameters  more fruits - impossible 31

32. How to measure fruit content - other techniques o FT IR Quantitative method using simple linear regression and PLS analysis Showed determination of fruit content feasible in strawberry jam Wilson et al., Food Chemistry, 47 (3), 303-306, 1993. o DNA Quantitation of the fruit species in a product requires availability of selective oligonucleotides (PCR primers and hybridisation probes). the accuracy of the method is affected by : Quality of DNA (DNA degradation!) PCR inhibitors present in the matrix DNA extraction efficiency 32

33. How to measure fruit content - other techniques o on the basis of hemicellulose (apricot strawberry) –specificity?

34. Uncertainty of fruit content estimation  every laboratory method has some uncertainty  in case of fruit content 2 sources of uncertainty methods of measurement variability of fruit In case of more parameters used for calculation some elimination of uncertainties Knechtel, EQCS-Workshop 2005  result ± 10% abs. 50% (40% – 60%) Knechtel, EQCS-Workshop 2005 CAFIA  result ± 20-25% rel. CAFIA

35. Recommendations  Calculation models have to be used with expertise  The whole analytical picture has to be considered  Consider elimination of obvious outliers  On basis of selected reliable authentic parameters calculation- models for other fruits can be developed 35

36. 36 Thank you for your attention