Tiêu chuẩn iso 08292 1 2008

Microsoft Word C041256e doc Reference number ISO 8292 1 2008(E) © ISO 2008 INTERNATIONAL STANDARD ISO 8292 1 First edition 2008 04 01 Animal and vegetable fats and oils — Determination of solid fat co[.]

Measurement protocol and test sample

Select the appropriate protocol from Table 1 based on the sample type and specific requirements For certain fat types or application scenarios, the protocols in Table 1 may not be suitable, and the measurement protocols detailed in Annex C could be more appropriate for accurate results.

Prepare the test sample in accordance with ISO 661

Table 1 — Measurement protocols Measurement protocol Tempering Measurement conditions First time at 0 °C Time Temp.

Second time at 0 °C Time No NameApplicable to Instrumental conditions min h ºCmin Type min 1DNon- stabilizing direct

Fats and blends primarily composed of vegetable fats, hydrogenated, and/or interesterified fats crystallize in the β'- polymorph, making them suitable for margarines, spreads, shortenings, and other food applications The crystallization process typically features a fat crystal network with a crystalline melting point (f) ranging from 1.4 to 1.45, ensuring desirable texture and stability Key processing parameters include a repetition time (a, t rep) of 2 seconds and a pulse number (n p) of 3, optimized for 60 ± 2 seconds Additionally, the fats are stabilized in the β-phase through a parallel (30 ± 1) 2D β-stabilizing direct mechanism, contributing to product consistency and quality.

Cocoa butter, cocoa butter equivalents, and similar fats with high levels of 2-oleo-di-saturated triacylglycerols tend to crystallize in the β-polymorph, which requires a repetition time (t_rep) of 6 seconds for optimal analysis Accurate measurement relies on averaging pulse data, typically using three pulses; however, some older instruments may only be capable of one or four pulses, affecting data consistency Proper selection of pulse parameters is essential to prevent partial melting of the test portion during measurement, ensuring reliable results and avoiding reductions in the SFC (saturated fat content).

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Oven, water baths and temperature-controlled blocks

Set this equipment up for the required temperatures as specified in the protocol.

Determination of the conversion factor (where necessary)

The first measurement can only be taken after the signal from the solid phase has decreased significantly, due to the instrument's dead time To account for this delay, a conversion factor is applied, ensuring accurate correction of the measurement results.

Calibration tubes with plastic-in-oil standards provide a reproducible but approximate conversion factor for β′-polymorphic, general-purpose fats across the relevant temperature range However, these standards do not offer accurate conversion factors for β-polymorphic fats like cocoa butter To prevent systematic errors when working with such fats or unknown fat blends, it is essential to determine a more precise and reliable conversion factor tailored specifically to the fat's polymorphic behavior.

To determine the saturation fatty acid content (SFC) of fats or fat blends, follow the procedures outlined in ISO 8289-2, which involves measuring a liquid oil reference Additionally, perform the measurement according to this part of ISO 8292, recording the SFC as obtained It is also essential to document the values of S1 and S2 associated with this measurement, consulting the spectrometer manual for proper procedures.

For each test sample, calculate the “true” SFC, w SFC,i , using ISO 8292-2

For each test portion, work out the extrapolation factor, f, required to equate the indirect and direct SFC determinations, and given by Equation (1):

− × − (1) where w SFC,i is the “true” SFC;

S 1 is the magnetization decay signal measured at about 11 às;

S 2 is the magnetization decay signal measured at about 70 às

Calculated factors for sample blends vary depending on temperature, with temperature differences often overlooked by the direct method To achieve accurate results, it is recommended to average the factors within the 20 °C to 30 °C temperature range, where solids are closest to a 50% mass fraction and the factor variation has the greatest impact For fats like cocoa butter that crystallize in a β-polymorph, the factor typically ranges from 1.6 to 1.7.

Due to the difficulty in determining the precise optimal ratio for blends of β-polymorphic fats like cocoa butter with β′-polymorphic fats such as milk fat or palm fractions, it is advisable to adopt alternative formulation approaches Understanding the complex polymorphic behavior of these fats is essential for achieving desired texture and stability in fat-based products, making empirical testing and industry best practices key to successful blending.

ISO 8292-2 for all such blends to determine the true SFC

Should the results be measured using an incorrect factor, they can easily be recalculated using Equation (2):

SFC err err err corr

ISO 2008 © emphasizes the importance of accurate measurement and reporting, with superscripts “err” and “corr” indicating erroneous and corrected values For example, if a cocoa butter sample is initially measured at 49.0% mass fraction using a correction factor of 1.41, the corrected value ensures precise and reliable test results Adhering to these standards enhances the credibility and consistency of analytical data in food testing.

SFC err w = 49,0), but it is known that the correct value for the instrument is f = 1,64, then Equation (2) gives

Variations in the measurement parameter f across different instruments and sites are unavoidable, as f depends partly on the specific instrument used To ensure consistency, it is essential to exchange reference samples during the establishment of commercial contracts, allowing both parties to agree on the solids content and the appropriate f value For instance, when following measurement protocol 2D, exchanging a standard reference cocoa butter sample is recommended to accurately determine the correct f value.

NMR spectrometer

Using the calibration tubes (6.2.2), calibrate the spectrometer according to the manufacturer's instructions and at the intervals recommended by the manufacturer

Set the conditions for the spectrometer according to the measurement protocol chosen in 8.1

Before each direct method determination, ensure accurate spectrometer calibration by inserting each of the three calibration tubes into the spectrometer and recording the SFC measurements Repeat these measurements to verify consistency The measured SFC values for each tube must not deviate by more than 0.3% absolute from their known calibration values to ensure reliable results.

If any SFC deviates during calibration, the parameter 'f' must be adjusted, and the calibration process repeated until all three calibration tubes show deviations of no more than 0.3% If necessary, recalibration of the spectrometer may be required to ensure accuracy (refer to section 8.4.1).

Filling the measurement tubes

Fill the tubes with approximately 2 ml of fat or a depth of 30 to 50 mm, as specified by the instrument manufacturer Cap the tubes securely and place them in racks that maintain a vertical position Using metal racks allows for direct placement of filled tubes, saving time and enhancing convenience This setup facilitates easy transfer of the test samples to the oven and water baths without additional handling or transfers.

For measurements in parallel, fill one measurement tube from each test sample for each measurement temperature; for measurements in series, fill a single measurement tube sequentially from each test sample.

Removing the thermal history

When all the required tubes have been filled, transfer them to the oven (6.4) Hold at the oven temperature for a minimum of 15 min.

Equilibrating at the initial temperature

Transfer all tubes to a 60 °C water bath or block, and hold for at least 15 minutes to ensure proper equilibration Although longer incubation times are acceptable, do not reduce the duration below 15 minutes, as this may prevent complete sample equilibration.

Crystallization and tempering

From this stage onwards, all the times shall be maintained within the tolerances specified here or in the measurement protocol

Transfer the tubes into a 0 °C bath if the selected measurement protocol requires it Keep them in the 0 °C bath for the duration indicated in the “First time at 0 °C” column of Table 1 or Annex C to ensure accurate measurement conditions.

If required by the chosen measurement protocol, transfer the tubes into the tempering bath set to the specified temperature Leave in the tempering bath for the specified time

Measuring the SFC

In most circumstances and as given in Table 1, make the measurements in parallel

Series measurement is ideal when only small test samples are available or minimal preparation time is required It is also suitable according to measurement protocol 4D (see Annex C) for achieving the best comparability with traditional dilatometric methods used to determine solid content.

At (1,0 ± 0,5) min or (2,0 ± 0,5) min intervals, transfer the tubes for each test portion to each of the measurement temperature water baths (6.3.2) or blocks (6.3.3) The number of tubes transferred at each

The recommended measurement interval of 1 to 2 minutes should be tailored based on the number of temperature points, operator expertise, and apparatus layout Selecting the appropriate time interval ensures accurate readings, especially when monitoring multiple tubes or temperatures simultaneously Ultimately, the interval choice depends on the specific measurement setup and operational conditions for optimal results.

Experience demonstrates that transferring a tube from the bath or block to the spectrometer takes less than 15 seconds, enabling rapid measurements This efficiency allows for the processing of six tubes at different temperatures comfortably within a short timeframe, ensuring high throughput and reliable temperature control for accurate spectroscopic analysis.

To ensure accurate spectrometer measurements, transfer the tubes to the spectrometer in the same sequence as they were placed in the temperature baths or blocks, following the specified timing intervals of either approximately 1 minute or 2 minutes, with a tolerance of ±0.5 minutes Before placing each tube in the measurement cell, briefly wipe it with a soft tissue to remove water residues Record the SFC reading after each measurement, and note a zero reading if the test sample appears completely clear.

At (1,0 ± 0,5) min intervals, transfer the tube containing a test portion to the first (lowest) of the measurement temperature water baths (6.3.2) or blocks (6.3.3)

After the designated measurement time, transfer the tubes to the spectrometer in the same order they were placed in the temperature baths or blocks Briefly wipe each tube with a soft tissue to remove water, then position it in the measurement cell Finally, record the Spectral Feature Count (SFC) for accurate and reliable results.

Transfer the tubes containing each test portion to the second (next lowest) of the measurement temperature baths or blocks at (1,0 ± 0,5) min intervals

Repeat the procedures from the second paragraph until all tubes have been measured

If the NMR spectrometer is not equipped with a computer or other automatic calculation device, then record the signals manually and compute the SFC according Equation (3) (see Clause 9)

IMPORTANT — For reliable and reproducible results, adhere to the times and tolerances specified

This is easily achieved using a laboratory stop-clock (6.5), preferably an analogue clock with a large sweep second hand, moving the tubes as the clock moves round to the appropriate time

Alternatively, if a digital clock is used, it is convenient to set it to 0:00 or 12:00 at the start.

Number of determinations

Carry out one determination on each of two test portions in separate tubes taken from the same test sample.

Cleaning the measurement tubes

To ensure accurate measurements, measurement tubes must be clean, dry, and free from any residual fat prior to filling with the test sample Due to their narrow diameter, cleaning these tubes can be challenging, often requiring the use of solvents or specialized narrow brushes Alternatively, tubes can be effectively cleaned using laboratory automatic washers or standard domestic dishwashers, provided they are kept in a more or less vertical position during the cleaning process to remove fats thoroughly Proper cleaning and maintenance of measurement tubes are essential for reliable test results and adherence to quality standards.

Either use a laboratory washer, equipped with special support “fingers” which can just fit into the tube and inject hot detergent solution inside

Use a washer without specialized “fingers” by supporting tubes in a wire-mesh rack equipped with appropriately sized slots for each tube Ensure the rack has a wire-mesh lid to securely hold the tubes when inverted This type of rack offers the advantage of allowing tubes to be placed upside down directly in the rack once the measurement sequence is complete, facilitating easy handling and transfer of filled tubes.

Preheat the oven to 80°C and heat the racks to allow the fat to melt and drain away effectively After heating, invert the racks and transfer them to the dishwasher for thorough cleaning Once washed and dried, the tubes racks can be used as convenient holders or the tubes can be removed and stored for reuse, ensuring optimal hygiene and practicality.

When an NMR spectrometer lacks a computer or automatic calculation device, manually recorded signals are used to determine the SFC at a specific temperature, denoted as w SFC,T This value represents the percentage mass fraction of the component The calculation is performed using Equation (3), enabling accurate measurement of the SFC without automated tools.

− + (3) where f is the conversion (extrapolation) factor to correct the NMR signal observed at 11 às to that at time zero;

S 1 is the magnetization decay signal measured at about 11 às;

S 2 is the magnetization decay signal measured at about 70 às

See Annex B for more details of the theory

To ensure accurate reporting, express the test result as the arithmetic mean of the two determinations outlined in equation (8.10), provided that the repeatability criterion specified in section 10.2 for each w SFC,T value is met Report the final calculated mean to one decimal place for clarity and precision.

Interlaboratory test

Interlaboratory tests assessing the precision of the method are detailed in Annex A Please note that the test results may only be applicable to specific SFC ranges and fats included in the study, and may not be generalizable to other fats or SFC ranges.

Repeatability

The absolute difference between two independent single test results—performed on identical test material, within the same laboratory, by the same operator, using the same equipment, and within a short interval—will rarely exceed the repeatability limit, r Specifically, this occurs in no more than 5% of cases, as defined in or derived from Tables 2 and 3.

Reproducibility

The absolute difference between two single test results—performed with the same method on identical test materials in different laboratories by different operators using varying materials—generally does not exceed the reproducibility limit, R, in more than 5% of cases This limit, R, is provided in or derived from Tables 2 and 3, ensuring consistent and reliable test performance across different testing environments.

Table 2 — Repeatability limit, r , and reproducibility limit, R , for measurement protocol 1D

Temperature Repeatability limit, r Reproducibility limit, R °C Minimum Maximum Mean Minimum Maximum Mean

Table 3 — Repeatability limit, r , and reproducibility limit, R , for measurement protocol 2D

Temperature Repeatability limit, r Reproducibility limit, R ºC Minimum Maximum Mean Minimum Maximum Mean

NOTE Details of the fats used in the collaborative study are given in Annex A Statistics are not given where only one test sample was measured at a temperature

The test report must include essential information such as complete sample identification, details of the NMR spectrometer used, and the measurement method with reference to ISO 8292 It should also specify the measurement protocol, testing temperatures, and the type of temperature control employed, whether a water bath with aluminium blocks, metal racks, or temperature-controlled blocks Additionally, the report must detail the obtained results and any relevant operating parameters not covered by ISO 8292, including any incidents that could have impacted the test outcomes.

Table A.1 — Summary of statistical evaluation — Measurement protocol 1D

Shortening blend, interesterified hardstock (C)

Palm oil/palm stearin blend (F)

No laboratories retained after eliminating outliers

No test results, all labs 42 44 44 42 44 40

Coefficient of variation of repeatability, CV(r)

Coefficient of variation of reproducibility, CV(R)

Coefficient of variation of repeatability,

Coefficient of variation of reproducibility,

No test results, all labs 38 32 32 38 32 34 Mean 40,78 29,74 40,62 86,11 1,73 23,76

No test results, all labs 20

Table A.2 — Summary of statistical evaluation — Measurement protocol 2D

Cocoa butter, soft Brazilian type (G)

Cocoa butter, standard West African type (H)

Illipe butter (Borneo tallow, Tengkawang fat) (I)

Cocoa butter equivalent, standard type, w SFC,30 ≈

Palm mid- fraction, hard/CBE grade, (K)

Theory of the direct method

A short radio-frequency pulse is applied to rotate the magnetic field by 90°, aligning it perpendicular to the static magnetic field generated by the permanent magnet This RF pulse induces magnetization perpendicular to the original field, which then decays over hundreds of milliseconds primarily due to spin-spin relaxation As a result, the magnetization signal detected diminishes gradually, as illustrated in Figure B.1.

S 1 magnetization decay signal measured at about 11 às

S 2 magnetization decay signal measured at about 70 às

S L magnetization decay signal corresponding to liquid phase after about 70 às

S S magnetization decay signal corresponding to solid phase at time 0

S S ′ magnetization decay signal corresponding to solid phase after about 11 às

S S+L magnetization decay signal corresponding to both solid and liquid phases at time 0

S S+L ′ magnetization decay signal corresponding to both solid and liquid phases after about 11 às t time

Figure B.1 — Decay of magnetization signal from a fat sample after application of a single 90° radio-frequency pulse

In solid-state NMR, proton signals decay rapidly within tens of microseconds, whereas in liquids, they decay much more slowly over tens to hundreds of milliseconds; on commercial bench-top instruments, the liquid signal typically decays in just a few milliseconds Advanced electronics enable the separate measurement of solid and liquid signals, allowing for the determination of the solid-liquid fraction (SFC) However, instrument dead time after the pulse, as shown in Figure B.1, prevents immediate measurement, meaning the total signal (S + L) cannot be directly recorded—only the modified signal (S + L') after approximately 11 microseconds The NMR spectrometer captures two signals, S1 and S2, at specific times to analyze the sample's composition effectively.

11 às and 70 às, corresponding to S S+L ′ and S L , respectively

For the direct method, a linear extrapolation from S S+L ′ at about 11 às to S S+L at the unmeasurable time 0 is assumed so that:

S S = f S S ′ (B.1) where f is an extrapolation factor to be determined empirically

− + (B.4) which is another expression of Equation (3)

The direct method provides only an approximate value of the SFC due to its limitations This is because linear extrapolation is inherently inaccurate when applied to non-linear decay curves Additionally, the SFC value varies depending on molecular mobility factors such as temperature and the specific packing arrangement of protons, which influence the measurement's precision.

(in other words, the polymorphism), as well as the crystal size — for values given by a typical spectrometer, see Table B.1; c) f varies with temperature as the liquid phase expands

Table B.1 — Values of f according to polymorphism

Polymorphism Value range for factor, f α 1,10 to 1,30 β′ 1,40 to 1,50 β 1,60 to 2,00

The direct method is commonly preferred for routine analysis due to its excellent reproducibility and straightforward calibration process Since most fats used in practical applications predominantly exist in the β′-polymorph form, a consistent f-value between 1.40 and 1.45 is typically employed across various temperatures This value is established through calibration with plastic-in-oil standards, ensuring accuracy and consistency in measurements.

First time at 0 °C Tempering Measurement conditions Time Temp No Name

Applicable toInstrumental conditions min min ºC

This study examines the second crystallization cycle of fats at 0 °C, focusing on slow-crystallizing milk fats, their fractions, and blends containing predominantly milk fat It also includes tallows, their fractions, and blends mainly composed of tallow, as well as other slow-crystallizing fats with a crystallization factor (f) between 1.40 and 1.45 The experimental parameters used are a repetition time (t_rep) of 2 seconds and three pulses (n_p = 3), with the process optimized for understanding the crystallization behavior of these fats under controlled conditions.

This article discusses the crystallization properties of AOCS solid fat index cFats and blends, primarily composed of vegetable fats that are hydrogenated or interesterified These fats crystallize in the β′−polymorph and are commonly used in margarines, spreads, shortenings, and various food applications The typical solid fat content ranges from 1.40 to 1.45, with a repetition time of 2 seconds and three pulses during measurement.

(15 ± 1) d(30 ± 1)26,7 (15 ± 1) minSeries or parallel at 10,0 °C, 21,1 °C, 26,7 °C, 33,3 °C and 37,8 °C only

(45 ± 2) 5 Rapid As for 1D, but where a faster method is required for production control purposesf = 1,40 to 1,45; repetition time a, t rep = 2 s; No pulses b, n p = 3

— — (30 ± 1) minParallel (15 ± 1) 6 Ultra-rapid As for 1D, but where a very fast method is required for production control purposes f = 1,40 to 1,45; repetition time a, t rep = 2 s; No pulses b, n p = 3

— — Substitute 1 min in liquid nitrogen e

The article discusses the specific parameters and procedures for measuring solid fat content (SFC), highlighting that parallel measurements should be conducted at (30 ± 1) seconds to ensure accuracy for fats in the β-polymorph form It emphasizes the importance of averaging data collected from multiple pulses—preferably three—to improve reliability, noting that older instruments may only support one or four pulses The method follows the AOCS dilatometric approach (Cd 10-57) for determining the solid fat index (SFI), which provides the most accurate correlation between SFC and SFI Prior to the initial crystallization at 0 °C, samples should be held at 26.7 °C for 15 ± 1 minutes to ensure proper crystallization Caution is advised when handling liquid nitrogen due to its potential hazards, including severe frostbite; proper safety protocols must be strictly followed.

[1] ISO 5555, Animal and vegetable fats and oils — Sampling

[2] ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results — Part 1: General principles and definitions

[3] ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results — Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method

[4] GRIBNAU, M.C.M Determination of solid/liquid ratios of fats and oils by low-resolution pulsed NMR

[5] S HUKLA , V.K.S Studies on the crystallization behaviour of the cocoa butter equivalents by pulsed nuclear magnetic resonance — Part I Fette Seifen Anstrichmittel, 1983, 85, p 467-471

[6] TIMMS, R.E Chapter 4, Section A In: Confectionery fats handbook, p 63-78 Oily Press, Bridgwater,

[7] VAN DUYNHOVEN, J.,DUBOURG, I., G-J GOUDAPPEL, G.-J.,ROIJERS, E Determination of MG and TG phase composition by time-domain NMR J Am Oil Chem Soc 2002, 79, p 383-388

[8] WADDINGTON, D Some applications of wide-line NMR in the oils and fats industry In: HAMILTON,R.J.,

BHATI, A., editors Fats and oils: Chemistry and technology, p 25-45 Applied Science Publishers, London, 1980

[9] AOCS method Cd 10-57, Solid fat index

Tiêu đề	Animal And Vegetable Fats And Oils — Determination Of Solid Fat Content By Pulsed NMR — Part 1: Direct Method
Trường học	International Organization for Standardization
Chuyên ngành	Animal and Vegetable Fats and Oils
Thể loại	tiêu chuẩn
Năm xuất bản	2008
Thành phố	Geneva

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Số trang	32
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