ifu methods & recommendations...use of dna methods in the analysis of fruit juices, purees and...
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IFU methods & recommendations
For controlling & testing the quality and authenticity of juices, purees and concentrates and related products
John Collins
Executive Director
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IFU and methods
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Won’t any method do?
• It is true you might get the result you want with an “in house” method
• However can you be sure?
• IFU methods are prepared by:-
• Methods of Analysis Commission (MAC) – Chemical methods
• Microbiological Working Group (MWG) – Microbiological methods
What is the IFU MAC and MWG?
• A group of experts from the worldwide juice industry tasked with:-
• Providing the juice industry with “fit for purpose" analytical procedures for the assessment of quality and authenticity of juices
• Offer advice on suitable methods for juice analysis
Chemical methods
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What does IFU “MAC” do?
• Develops a consistent approach to a particular component’s analysis
• Prepares the written procedure
• Assess its performance using the principles of the ISO standard (5725) for method validation via inter-laboratory studies on fruit juices.
• This makes them suitable for adoption by the Codex Committee of Methods of Analysis and Sampling (CCMAS) for inclusion in the Codex FJ standard
IFU chemical methods
• Shown to produce reliable results
• Implementation only requires verification
• Operational range on your instrumentation
• Checking that your performance characteristics are within the method’s defined range
• Methods contain the
• expected variation seen between two results conducted within a laboratory in short time (method repeatability r)
• expected variation seen between two laboratories conducted at a similar time (method reproducibility R)
Catalogue of methods
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01a Relative density
02 Ethanol GC
03 Titratable acidity
05 Volatile acids
07a Sulphur Dioxide
08 Soluble solids
09 Ash
10 Ash Alkalinity
11 pH Value
12 Hydroxymethylfurfural
13 Copper
17a Ascorbic Acid
18 Fermentation test
20a Tartaric Acid Rapid
21 Determination of L-malic acid
22 Determination of citric acid
24 Detection of Artificial Colours
25 Organoleptic Evaluation
27 Pectin
28 Determination of Total Nitrogen
30 Determination of Formol Numbers
33 Determination of Sodium, Potassium
36 Determination of Sulphates
37 Determination of Chloride
45 Determination of Essential Oils
46 Detection of Pectinesterase (PE)
49 Determination of Proline
50 Determination of Phosphate
52 Determination of Alcohol
53 Determination of Lactic Acid
54 Determination of D-Isocitric Acid
55 Determination of Glucose and Fructose
56 Determination of Sucrose
57 Determination of Free Amino Acids
58 Determination of Hesperidine
59 Total Carotenoids and Individual Groups
60 Determination of Centrifugal Pulp
61 Determination of Total Dry Matter
62 D-Sorbitol (enzymatic)
63 Preservatives (HPLC)
64 D Malic Acid (enzymatic)
Catalogue of methods
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65 Tartaric Acid in Grape Juice
66 Acetic Acid
67 Determination of Sugars and Sorbitol (HPLC)
68 Test for Pectin
66 Determination of Hydroxymethylfurfural
70 Cell Content of Pulps and Juices
71 Anthocyanins by HPLC
72 Fumaric Acid HPLC
73 Detection of Starch
74 Nitrate IC
75 Haze Instability Test
76 Gluconic Acid in Grape Juice
77 Glycerol
78 Galacturonic Acid HP AEC
79 Polyols in Fruit Juices Using Electrochemical Det.
80 Spectrophotometric Colour Measurement
81 Ergosterol
82 Nitrate
83 Colour measurement in blood orange juice
84 Stability tests for clarified juices
85 Net volume
R01 Detection of Invert Sugar
R02 Determination of Patulin
R03 Use of Isotopic Procedures
R04 Detection of Syrup Addition CapGC
R05 Vitamin C Analysis
R06 Determination of Heavy Metals
R07 Turbidity Measurements
R08 Analysis of High Intensity Sweeteners
R10 Ochratoxin
R12 Confirmation of country of origin
R13Use of DNA methods in the analysis of fruit juices,
purees and concentrates
R14 Determination of Oranic
R15 Quality systems for juice labs
R16 Methods for Pesticide residue
Updates
• #3 Titrateable acidity (2015)• QA check. Standardisation of Sodium Hydroxide
• Also an e-learning video
• #7a Sulphur Dioxide (2015)• Problem close to the limit of 10ppm
• Reduce interlaboratory variability (R)
• Standardisation of the heating stage• Also an e-learning video
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Updates
• #8 Refractometric soluble solids (2017)
• updated to give a broader acid correction and the conversion tables have been revised
• Addition of a weight per litre table
• Also and e-learning video
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Updates
• #71 for the analysis of anthocyanins in FJ has recently been updated and published to include:-
• a) The use of modern HPLC columns that offer much better resolution (sharper peaks)
• b) Additional fruits which have become commonly used since the original procedure was published in the 90’s
• c) Peak assignment in the chromatograms
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IFU 71 (revised)
Why are these important developments?
a. New columns offer better chromatography, particularly useful in juices with complex profiles (e.g. blueberry) that offers a better possibility to detect undeclared blending
b. AIJN has recently added information to its reference guides for red black juices as to which anthocyanins would be expected in the product
c. With the new peak identification given in the method’s “reference library” this will assist sample assessment for non-experts
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New Chromatogram from 71 (revised)
Updates
• #81 Ergosterol (2015)• Substitute for the Howard Mould Count in Tomato products
• Marker for moulds
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Source Dr. Achim Gessler
Ergosterol is a specific constituent of the fungal cell wall
Updates
• #83 Colour of Blood Orange (2017)
• # 80 Spectrophotometric – not suitable for cloudy juices
• # R09 Tristimulus
• # 83 Red colourabsorbance at 520nm
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Updates
• #84 Stability tests for clarified juices (2017)
• The main stability tests are:• Detection of pectin in fruit juices (“Ethanol
test”)• Detection of starch in fruit juices (“Iodine
test”)• “Hot/cold stability test” (test for
phenolic/protein compounds)• “Acetone test”• “Bento-test” for assessing the dosages of
bentonite and gelatin required to give a “good” fining
• Determination of gelatin bentonite demand for proper fining (Proteins)
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“Acetone test” for proteins
Right tube: clear solution no
problems, good clarification.
Left: Cloudy solution showing that
there is a problem in with the
clarification step of the juice.
Updates
• #82 Nitrate (2016)• #74 Ion chromatography
method
• #82 simple colourmetricmethod
• Call for ring test
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Updates
• #85 The net volume determination of consumer packaged juices, nectars and related products (2019)
• Based upon measurement of weight
• Calculation to volume using updated with per litre tables in IFU method #8
• Routine procedure for all bottlers
• Essential for yield management
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Under review
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• #17 Ascorbic acid• Indophenol and Iodine method
• 17a by HPLC
• R05 Vitamin C analysis
• #70 Cell content of pulps
and juices• Cells packed in juices
• Juices containing cells
• #43 Velcorin• 2 options
• Ethyl methyl carbonate
• Dimethyl carbonate
• Working group is evaluating a suitable procedure
• Limonin• Planned ring test
Recommendations• A method that could not be approved in an inter-laboratory study
• Lack of members capable of running them or, • Method did not return “good” enough data
• Review of an analytical topic
• General analytical systems/good practice
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R01 Detection of Invert Sugar
R02 Determination of Patulin
R03 Use of Isotopic Procedures
R04 Detection of Syrup Addition CapGC
R05 Vitamin C Analysis
R06 Determination of Heavy Metals
R07 Turbidity Measurements
R08 Analysis of High Intensity Sweeteners
R10 Ochratoxin
R13Use of DNA methods in the analysis of fruit juices,
purees and concentrates
R12 Confirmation of country of origin
R15 Quality systems for juice labs
R16 Revew of pesticide measurement
Recommendations
• Rec 2. Determination of patulin - (Revised 2019)
• Update with techniques since Rec was first published
• SPE extraction
• DAD
• MS
• Rec 16. Review of methods of analysis for pesticide residues (2019)
• Discussion on MRL setting
• Historical methods
• QuEChERS Method
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Recommendations
Rec 3: Use of Isotopic procedures (Revised 2017)
• First published in 1996
• Since then the use of internal referencing has improved detection limits
• internal carbon isotope ratios of individual sugars,
• relative carbon isotope ratios seen for the sugars, acids,
individual acids, pulp and pectin,
• positional carbon isotope ratios of malic and ascorbic acids,
• internal deuterium and carbon isotope ratios of citric acid
• internal oxygen isotope ratios of juice water and ethanol derived from the sugars
• These and other developments are reviewed
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Thanks to Eurofins for permission to reproduce this figure
Intra-molecular referencing for citric acid from lemon juice & commercial sources
Recommendations
Rec 12: Confirmation of country of origin (2018)
• There is a growing interest/legal requirement to label the country of origin of some products in some countries.
• This leads to the need for methods to validate the claim.
• Although it is not likely to be mandatory for FJ in many markets method for this assessment are discussed in this recommendation
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Figure 6:- 1H-NMR screening of an apple juice and assessment of it likely
country of origin
Recommendations
Rec 13: The use of DNA methods (2018)
• Traditional methods to investigate the presence of one fruit juice in another would look for chemical markers such as sorbitol for apple/pear addition in a non-sorbitol fruit juice such as strawberry
• DNA testing has significantly developed and improved over recent years
• DNA can degrade under elevated temperatures in acidic conditions , so it may not be suitable in all juice types, such as some clarified juices
• This recommendation provides the principles of the test, methods available and applications in juices, for example mango variety differentiation
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Organisation of genotypic SNP diversity for the four original
citrus species and other species after analysis of SNP data by
Principal Components Analysis (PCA).
Recommendations
• Rec 14: Methods to assess “organic” or “Bio” nature of Fruit Juices
• There is a growing consumer interest in organic products for a number of reasons.
• Perceived healthily nature, “easier” on the environment, better taste etc. In most cases the “organic” nature is “proven” via a paper trail from a registration body.
• However, there have been a number of method proposed to provide analytical verification of this classification.
• These are discussed with a number of their limitations highlighted
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Recommendations
Rec 15: Quality systems for juice laboratories
• Published September 2017
• To assist Laboratory Managers as to the type of checks and controls they should consider adopting to ensure their lab produce reliable data
• It includes topics on:
• Documentation
• Training records
• Servicing / calibration
• Quality Assurance samples
• QA sample analysis and charts
• Performance analysis schemes
• Shewartcharts
• Data management
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Figure 6: Inconsistent trend data with 10 out of 11 points below the dataset’s mean
Recommendations
Rec 15: Quality systems for juice laboratories
•Translated• Japanese
• Chinese
• Arabic
• Spanish
• French
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Under development
• Aroma analysis
• Viscosity
• Conversion of analytical results
• Manual methods vs enzymatic methods
• H-NMR (Rec 19)
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Microbiological methods
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Micro methods
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MM1Microbiological examination of fruits, vegetables and related products: General
laboratory instructions
MM2 Total Count of Potential Spoilage Microorganisms of Fruits and related Products
MM3 Yeasts Count Procedure
MM4 Moulds Count Procedure
MM5 Lactic acid bacteria, count procedure
MM6 Mesophilic & Thermoduric - Thermophilic Bacteria : Spores Count
MM7Sterility Testing of "Aseptic Filled Products", "Commercial Sterile Products",
"Preserved Products"
MM8Alternative Rapid Microbiological Methods in the Fruit Juices and Related
Products Industry
MM9Microbiological Examination of Potential Spoilage Microorganisms of Tomato
products
MM10Microbiological Examination of Potential Spoilage Microorganisms of Low Acid
and High pH Vegetable Products
MM11 Fruits Juices and Pathogenic Micro-Organisms
MM12Method on the Detection and Enumeration of Spore-forming Thermo-Acidophilic Spoilage bacteria (Alicyclobacillus spp.)
• Make it simpler
• Name change
• For the detection of main product spoiler
• Media – BAT broth or BAT agar
• Pour plating technique for enumeration in 1g.
• Filter type
• Trouble shooting guide
• Optional pre-incubation
• Performance characteristics
• Final validation completed in line with ISO stds.
IFU 12. Method on the detection and enumeration of spore-forming thermo-acidophilic spoilage bacteria (Alicyclobacillus sp.)
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• Micro methods 1 to 6
• New micro method 13
• Detection of Clostridia spores
• Low pH products with acid tolerant species
Under review – Micro methods
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How can I access the methods?
37Month 20XX, Title
• As a member you have free access (subscription) for a specified number of users (4 to unlimited)
• Non-member subscriptions are available
• Subscriptions last for 12 months
• username and password
• Access via the website
Access to IFU methods
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39Month 20XX, Title
Front page of the website
www.ifu-fruitjuice.com
Conclusions
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Conclusions
1. The MAC and MWG offers the fruit juice industry an invaluable resource due to its collective expertise
2. Provides procedures that return reliable results to limit any disputes between parties when analysing a product against a “juice specification”
3. Provides a balanced view of expert’s on new methods of analysis
4. Can react quickly to a new analytical issue “hitting” the juice industry via recommendation preparation
5. In the longer term can issue a “new” tried and tested method
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Thank you for your attention.
If you have any further questions you may contact
John Collins
IFU Executive Director
or chairman of the MAC
Mikko Hofsommer
GfL Berlin