Estimation of Proline in Fruit and Vegetable Juices According to EN 1141 and German Food and Feed Code §64 LFBG 31.00-7

Section Overview:

• Introduction
• Reagents, Instruments, & Materials
• Sample Preparation
• Experimental Procedure
• Measurement
• Evaluation

Note: Pursuant to the valid copyright regulations, this application note contains only a rough description of the content of the official method, followed by a detailed description of the specific measurement procedure with the Spectroquant^® Prove Spectrophotometers. A detailed description of the method-specific handling steps can be found in the official method EN 1141 and German Food and Feed Code §64 LFBG 31.00-7²

Introduction

Each type of fruit and vegetable has its own specific amino acid composition characteristic. Therefore, adulteration of fruit and vegetable juices can be identified by determining the amino acid content during amino acid analysis. For example, natural orange juice has proline as the amino acid in the highest concentration. Therefore, proline determination can be used to indicate if the juice is of synthetic or natural origin³.

The proline content is determined after the extraction of a colored complex formed by the ninhydrin reaction with proline. The extract is measured photometrically at 509 nm. This method is based on the official method EN 1141¹ and German Food and Feed Code §64 LFBG 31.00-7² and describes the determination of proline in fruit and vegetable juices.

Measuring Range

• Method 2539:  Proline Juice EN 1141 (0 – 1200 mg/L)

Sample Material

• Fruit and vegetable juices

Reagents, Instruments and Materials

• For the measurement, one of the following Spectroquant^® photometers is necessary:
  - Spectroquant^® VIS Spectrophotometer Prove 100 Plus (1.73026)
  - Spectroquant^® UV/VIS Spectrophotometer Prove 300 Plus (1.73027)
  - Spectroquant^® UV/VIS Spectrophotometer Prove 600 Plus (1.73028)
  Note: Also, legacy Prove 100/300/600 instruments are suitable
  
• Rectangular cells 10 mm (1.14946)
• Ninhydrin GR for analysis (1.06762)
• Ethylene glycol monomethyl ether for analysis EMSURE^® (1.00859)
• n-Butyl acetate for analysis EMSURE^® (1.09652)
• Formic acid 98-100 % for analysis EMSURE^® (1.00264)
• Sodium sulfate anhydrous for analysis EMSURE^® (1.06649)
• L-Proline  ≥99% (HPLC), ReagentPlus^® (P0380*)
• Test tubes, closable, approx. 25 mL
• Water bath (temperature controllable)
• Funnel
• Folded filter, hydrophobic 110 mm
• Volumetric flasks, 20 mL, 500 mL
• Standard laboratory glassware (e.g. glass beakers) and pipettes
  

*In case certified reference material is required, use 93693.

Preparation of Ninhydrin Solution

The solution must be prepared according to EN 1141 resp. German Food and Feed Code §64 LFBG 31.00-7

SAMPLE PREPARATION

• Light-colored samples with proline concentration below 50 mg/L can be analyzed directly
• Deep-colored samples with proline concentration below 50 mg/L need to be diluted in a range of 1 part sample + 1 part distilled water up to 1 part sample + 4 parts distilled water
• Samples with Proline concentration 50 mg/L to 499 mg/L needs to be diluted with distilled water 1+9 (1 part sample + 9 parts distilled water)
• Samples with Proline concentration 499 mg/L to 1200 mg/L needs to be diluted with distilled water 1+19 (1 part sample + 19 parts distilled water)
• Note the dilution ratio

EXPERIMENTAL PROCEDURE: NINHYDRIN TEST

Hydroxyproline determination

1. Reagent blank

• Mix 1.0 mL of the prepared sample with 1.0 mL formic and 2.0 mL ethylene glycol monomethyl ether in a closable test tube.
• Incubate the mixture in a water bath according to EN 1141 resp. German Food and Feed Code §64 LFBG 31.00-7.
• Cool down the mixture according to EN 1141 resp. German Food and Feed Code §64 LFBG 31.00-7.
• Add 10 mL n-butyl acetate and extract the colored complex into the organic phase according to EN 1141 resp. German Food and Feed Code §64 LFBG 31.00-7.
• Filtrate the total solution through a hydrophobic folded filter according to EN 1141 resp. German Food and Feed Code §64 LFBG 31.00-7.
• Use the filtrate for the photometric measurement.

2. Sample

• Mix 1.0 mL of the prepared sample with 1.0 mL formic and 2.0 mL ninhydrin solution in a closable test tube.
• Incubate the mixture in a water bath according to EN 1141 resp. German Food and Feed Code §64 LFBG 31.00-7.
• Cool down the mixture according to EN 1141 resp. German Food and Feed Code §64 LFBG 31.00-7.
• Add 10 mL n-butyl acetate and extract the colored complex into the organic phase according to EN 1141 resp. German Food and Feed Code §64 LFBG 31.00-7.
• Filtrate the total solution through a hydrophobic folded filter according to EN 1141 resp. German Food and Feed Code §64 LFBG 31.00-7.
• Use the filtrate for the photometric measurement.

MEASUREMENT

Note: It is advisable to measure the reagent blank and the sample using the same cell as the one used for the zero adjustment or else a cell with identical optical characteristics and an identical absorption (matched pair).

• Open the methods list (<Methods>) and select Method No. 2539 „Proline Juice EN 1141”.
• The instrument automatically prompts a “Zero adjustment”.
• For the zero adjustment fill a clean and dry 10 mm rectangular cell with distilled water.
• After prompting, insert the filled rectangular cell into the cell compartment. The zero adjustment is performed automatically.
• Confirm the performance of the zero-adjustment procedure by clicking on <OK>
• A window with an input field to enter the dilution ratio (1+x) pops up.
• Enter the number of used parts of distilled water for the sample dilution, confirm with <OK>, and click on <START> to switch to the measurement procedure.
  For example:
  1. For undiluted samples, the dilution ratio is 1+0, enter “0”
  2. For samples with a dilution ratio of 1+9 enter “9”
  3. For samples with a dilution ratio of 1+19 enter “19”
• Fill the prepared reagent blank into a clean and dry 10 mm rectangular cell. Insert the cell into the cell compartment. The measurement is performed automatically. A (✓) symbol appears behind the cue “Insert Reagent Blank”.
• Confirm the measurement by clicking on <OK>.
• Finally, fill the prepared sample solution into a clean and dry 10 mm rectangular cell. Insert the cell into the cell compartment. The measurement is performed automatically. A (✓) appears behind the cue “Insert Sample”.
• Confirm the measurement by clicking on <OK>.
• Read off the result in mg/l and the absorption for the reagent blank (A_RB) and the sample (A_Sample) from the display.
• Tap the <START> button to start the measurement procedure for the next sample.

EVALUATION

Statement of the results: 

• Proline [mg/L]
• Absorbance of reagent blank A_RB
• Absorbance of sample A_Sample

Method Control

• The method can be checked using L-proline ≥99% (HPLC), ReagentPlus^® (P0380)  as a standard substance, or in case  a certified reference material is required, use the TraceCERT ^® product (93693)
• Prepare a stock solution with 100 mg/L L-proline by dissolving 50.0 mg L-proline in approx. 450 mL distilled water. Transfer the solution completely to a 500 mL volumetric flask and fill it up to the mark with distilled water
• Dilute the stock solution to 25 mg/L L-proline with distilled water (5 mL stock solution and 20 mL in a 20 mL volumetric flask).
• Analyze the prepared standard as described in sections “Procedure” and “Measurement”
• Hereby enter a value of “0” for the dilution ratio of 1+0

Adjustment

• In case of significant deviations in the method control procedure the preprogrammed factor of 5.612 or the current factor used in the calculation of the displayed results can be adjusted by the user.
• The corrected factor must be recalculated as follows:
  Factor corrected = Current factor x (target value standard / measured and recalculated value standard)
  
• To edit the preprogrammed factor, select method 2539 from <Methods>.
• Close the window for the “Zero adjustment” by clicking on <X>.
• Close the input field for the dilution by clicking on <X>
• Click <Settings> and select the list “FACTORS”.
• Tip on the input field “Factor”, enter the corrected factor, and confirm by clicking on <OK>.
• Close the window for the “Zero adjustment” by clicking on <X>.
• For the next measurement, restart the method by selecting the method a new from <Methods>.

Note

• To find the used factor, select Method 2539 from <Methods>.
• Close the window for the “Zero adjustment” by clicking on <X>.
• Close the input field for the dilution by clicking on <X>.
• Click <Settings> and select the list “FACTORS”.

See more applications for photometry at Protocols and Application Notes

 

References

1. DIN EN 1141:1994-12, Frucht- und Gemüsesäfte_- Spektralphotometrische Bestimmung des Prolingehaltes; Deutsche Fassung EN_1141:1994. https://doi.org/10.31030/2737171

2. German Food and Feed Code §64 LFBG 31.00-7:1997 Spektralphotometrische Bestimmung des Prolingehaltes in Frucht- und Gemüsesäften. [Internet]. Available from: https://www.dinmedia.de/en/technical-rule/bvl-l-31-00-7/1251060

3. Matissek R, Fischer M, Steiner G. 2010. Lebensmittelanalytik. 4th ed. Springer; 2009.