Method of Analysis Library
Determination of Undenatured Whey Protein Nitrogen (WPN) in Nonfat Dry Milk
v 2.0 | Effective 09/06/2023
1.0 Purpose
This Analytical Method defines the standard operating procedures for determination of undenatured whey protein nitrogen (WPN) content in nonfat dry milk.
2.0 Scope
This SOP is applicable to determination of WPN in nonfat dry milk. The analogous product outside the U.S., skimmed milk powder (SMP), may also be tested according to this procedure.
3.0 Definitions
| 3.1 | Whey protein (or “serum protein”) is actually a complex collection of proteins which are not coagulated by cheesemaking enzymes (e.g., rennet, zymogen) and which are consequently drained away from the curd in the liquid whey during the cheesemaking process. Whey proteins are more susceptible to denaturation by heat during processing than the casein proteins are, so they can be used as an indicator of the degree of heat exposure experienced by nonfat dry milk during its manufacture. |
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| 3.2 | Nitrogen is the elemental constituent of dairy which is presumed to be directly correlated to protein content by the application of an established conversion factor. Multiplying the nitrogen content of a dairy sample by a constant value of 6.38 yields a value for the protein content of that sample. There are low levels of other non-protein constituents which contribute to the overall nitrogen content of a dairy sample, and which must be separated in some fashion to arrive at a true protein measurement. |
| 3.3 | The Kjeldahl method for determination of nitrogen is the well-established global reference method for analysis of protein content in many foodstuffs, including dairy. This wet chemistry method relies on complete chemical digestion of the sample into its elemental constituents using concentrated mineral acid, followed by recovery of the nitrogen which is then measured indirectly but quantitatively by titration. An appropriate modification of the classical Kjeldahl method is the technique by which the WPN values are determined for the ADPI Certified Reference Standards which form the basis of the calibration curve for this spectrophotometric WPN method. |
| 3.4 | The property of nonfat dry milk known as whey protein nitrogen (WPN) or sometimes whey protein nitrogen index (WPNI) is a value representing the amount of undenatured whey protein in the material. WPN is expressed in milligrams of undenatured whey protein nitrogen per 1 gram of sample, normalized to a sample moisture content of 3.16% for the sake of consistency. The larger the WPN value, the more undenatured whey protein is present and therefore less overall exposure to heat during manufacture; conversely, the smaller the value, the greater the exposure to heat. Nonfat dry milk is classified as to the degree of heat exposure using these WPN values as follows: |
| Classification | WPN (mg per 1g of sample) |
|---|---|
| Low heat | ≥ 6.0 |
| Medium heat | 1.5 < value < 6.0 |
| High heat | ≤ 1.5 |
| A further classification that is not formally defined in the U.S. is that of “high heat, heat stable” which is typically associated with a WPN less than or equal to 1.0. | |
| 3.5 | A spectrophotometer is an analytical laboratory instrument which provides a consistent source of energy across a range of the electromagnetic spectrum, is able to illuminate a sample with one or more specific wavelengths provided by that energy source, and then can measure the absorbance of that energy by the sample. This absorbance can be correlated to various physical or chemical characteristics of the sample, and the utilization of reference standards with known absorbance values allows for calculation of quantitative results for the sample. |
| 3.6 | Absorbance is the amount that the source energy decreases when passing through the sample, at a given wavelength and path length as set for the spectrophotometer. This may be expressed in established absorbance units (AU) or as a percentage relative to the absorbance of a defined reference such as the empty sample cell or the cell filled with a reference solution such as a blank. |
| 3.7 | Transmittance is the difference between the percentage absorbance of the sample and 100%, at a given wavelength and path length as set for the spectrophotometer. If a sample absorbs 10% of the spectrophotometer’s source energy under given conditions, then the corresponding transmittance is 100% - 10% = 90%. |
| 3.8 | Turbidity is the degree to which a suspension of droplets or particles in a solution will scatter light that is passed through it. A turbid suspension may appear uniformly “cloudy” or the suspended particles may be visually distinguishable from the liquid phase, yielding a more translucent effect. Suspensions of this nature may tend to settle into distinct layers or phases if given time and left undisturbed. While due to a different principle than true absorbance, turbidity has the same effect of reducing the amount of electromagnetic energy that will pass through a sample. As such, turbidity can be interpreted in terms of absorbance or transmittance using a spectrophotometer or even a colorimeter, if the wavelength for examination is properly chosen. Specialized instruments called turbidimeters are also available for the singular determination of turbidity. |
| 3.9 | A blank is a reference preparation which defines zero absorbance (or 100% transmittance) for a given analysis. In the case of this method, 100% transmittance is established by using a casein-free preparation of the sample, without acid addition to precipitate the undenatured whey protein, adjusting the instrument reading with that preparation at the time of use. |
4.0 Principle
Undenatured whey protein is isolated from a reconstituted nonfat dry milk sample by incubating with saturated sodium chloride solution followed by filtration to remove casein and any whey proteins which are already denatured. Once isolated, the undenatured whey proteins in the filtrate are then denatured with hydrochloric acid, and the turbidity which results from the precipitation of these proteins is determined spectrophotometrically by measuring transmittance of the turbid suspension at 420 nm. Sample transmittance is correlated to undenatured whey protein nitrogen via a calibration curve prepared from reference standards of known WPN content.
5.0 Reagents and Materials
As with any instrument-based analytical method, the results obtained by this procedure are dependent on proper care and maintenance of the spectrophotometer. Follow the manufacturer’s recommendations and make any necessary system checks before proceeding to calibrate and/or to test samples.
The calibration curve depends on suitable reference standard materials with known values for WPN. We recommend ADPI Certified Reference Standards, both low heat (LH) and high heat (HH), for calibration. Note that the certified WPN values for these Standards is already normalized to 3.16% moisture, using the actual moisture content for these powders as determined at the time of certification. If the moisture content deviates substantially from original levels, the WPN values will also be affected. Take care to store and use the Standards properly such that their moisture levels are not compromised; replace with new Standards as often as necessary and no later than the “Valid until” date on the label, in any case.
Cuvettes for the spectrophotometer must be optically clear and without visible defects such as scratches in the optical path. Inspect carefully before use and replace as necessary.
Adhere to the following requirements for consistent and accurate results:
Figure 1: ADPI Certified Reference Standard Nonfat Dry Milk (Low heat variant shown)
| 5.1 | ADPI Certified Reference Standard Nonfat Dry Milk, Low Heat and High Heat, available for order through the blue button; |
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| 5.2 | Laboratory balance(s), with sensitivity of ± 0.0001 grams for sample weighing and ± 0.1 grams or better for all other weighing; |
| 5.3 | Spatula, or equivalent, suitable for weighing the samples and other materials required by this procedure; |
| 5.4 | Water, sediment-free, distilled or efficiently filtered; |
| 5.5 | Test tube, 25 x 150 mm, or equivalent, suitable for reconstituting the sample; |
| 5.6 | Stopper, or other suitable closure, compatible with the test tube; |
| 5.7 | Hot stir plate or equivalent, suitable for heating water to near boiling; |
| 5.8 | Graduated cylinder or equivalent, suitable for measuring the 2 liter quantity of water required for preparation of the saturated sodium chloride solution below; |
| 5.9 | Beaker or equivalent, with sufficient capacity for preparing the saturated sodium chloride solution below; |
| 5.10 | Water bath, capable of holding a consistent temperature of 37 ± 1°C; |
| 5.11 | Filter paper, Schleicher & Schuell No. 605, 15 cm pleated, or equivalent; |
| 5.12 | Filter paper, Schleicher & Schuell No. 602, 9 cm, or equivalent; |
| 5.13 | Sodium chloride, reagent grade or better, free from anti-caking agents (which are commonly added to ensure flowability); |
| 5.14 | Sodium chloride solution, saturated: |
| a. Combine 1000 grams of sodium chloride with 2 liters of water; | |
| b. Heat to near boiling, with constant stirring, to achieve a saturated solution; | |
| c. Allow to cool to ambient temperature, taking care to avoid disturbing the solution which can cause precipitation in some cases; | |
| d. Filter the saturated solution through a No. 605 pleated filter paper into a suitable container for storage. | |
| 5.15 | Lab timer, or equivalent, capable of measuring intervals as required below; |
| 5.16 | Funnels, glass, or equivalent, compatible with the required filtration steps; |
| 5.17 | Volumetric pipettes, Class A, calibrated “to deliver” or equivalent, in volumes of 1.0, 2.0, 3.0, 4.0, 5.0, 10.0, 20.0, and 100.0 mL; |
| 5.18 | Hydrochloric acid, concentrated, reagent grade or better; |
| 5.19 | Graduated cylinder or equivalent, suitable for measuring the quantities of hydrochloric acid and water required to prepare the hydrochloric acid solution below; |
| 5.20 | Beaker or equivalent, with sufficient capacity for preparing the hydrochloric acid solution below; |
| 5.21 | Hydrochloric acid solution, 10% (w/v): |
| a. Slowly add 23 mL of concentrated hydrochloric acid to 77 mL of water in a beaker while stirring; substantial heat will evolve as the acid dissociates, so combine with caution; | |
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b. Allow to cool to ambient temperature before using.
(Alternatively, many 10% solutions of hydrochloric acid are commercially available and may be substituted, assuming the appropriate grade of materials used.) |
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| 5.22 | Spectrophotometer or equivalent instrument, equipped with a cuvette holder, capable of measuring absorbance / transmittance at 420 nm, ideally providing not less than 35% difference in transmittance between the low heat and high heat calibration preparations. |
6.0 Personal Safety Precautions
In all cases, the practitioner’s company’s internal policies and procedures regarding personal safety supersede the following ADPI recommendations:
| 6.1 | Milk (dairy) is globally classified as an allergen and should be properly handled with personal safety needs in mind. |
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| 6.2 | Read and understand all precautions for safe handling and disposal shown in the reagent Safety Data Sheet (SDS), including use of any prescribed Personal Protective Equipment (PPE). |
| 6.3 | Dairy ingredients are foods and as such are exempt from U.S. requirements regarding Safety Data Sheets (SDSs), where ingredient-specific safe handling instructions would be provided. Despite this exemption, many dairy ingredients are manufactured and marketed in powder form, and powders should be recognized as potential physical irritants, such as to the eyes, nose, and if inhaled. |
| 6.4 | Concentrated hydrochloric acid is a hazardous fuming mineral acid; its vapors are also corrosive and harmful. Exercise appropriate care, including handling in an appropriately rated fume hood when in its concentrated form. |
| 6.5 | Some testing apparatus described above may be susceptible to breakage, therefore be aware of associated personal risks. Inspect apparatus before use and replace any items which are compromised. |
| 6.6 | Exercise care when using hot water baths and heated testing materials. Read and understand the bath manufacturer’s warnings and instructions for safe use and handle any heated materials with tongs or other suitable utensil. |
7.0 General Considerations
| 7.1 | The moisture level of the sample must be known in order to normalize the raw WPN result to 3.16% moisture. Use ADPI Analytical Method #004a: Vacuum Oven Method (or a secondary method which has been validated against this vacuum oven method) for the determination. |
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| Using the raw result as-is will be increasingly inaccurate as the sample moisture strays further from the reference value of 3.16%. This inaccuracy becomes of increasing importance when powder is being produced near the boundaries which distinguish between classifications, and it is especially problematic when striving to produce high heat, heat stable powder, where the WPN must be accurately determined at or below the level of 1.0 mg per 1 gram of sample. | |
| 7.2 | Modern spectrophotometers (as well as colorimeters, turbidimeters, etc.) are customarily capable of developing multi-point calibration curves with linear regressions, thus automating the calibration process and the calculation and reporting of sample results. Alternative methods for developing the geometric relationship between concentration and transmittance are completely acceptable, for example: |
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a. Using spreadsheet applications for processing the calibration data; b. Manual generation of a calibration curve graph and interpolating sample results visually from that graph. |
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| 7.3 | Wherever a volume is expressed with a decimal place (e.g., 10.0 mL) then the use of Class A volumetric glassware (or the equivalent) is implied. Accuracy of this method depends on compliance with this requirement. |
| 7.4 | Samples should be homogeneous, representative, and equilibrated to ambient temperature before handling, in a manner which will not compromise their suitability for moisture testing. |
| 7.5 | Follow Good Laboratory Practices (GLPs) wherever applicable. |
8.0 Generating the Calibration Curve
| 8.1 | Low heat stock solution: |
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| a. Accurately weigh 20.0 grams of ADPI Certified Reference Standard Nonfat Dry Milk, Low Heat, recording the weight to the nearest 0.1 mg, and transfer the powder quantitatively to a 500 mL Erlenmeyer flask; | |
| b. Add 20.0 mL of water to the flask; | |
| c. Add 80 grams ± 0.1 grams of sodium chloride to the flask; | |
| d. Stopper, shake for 1 minute, and place in a water bath at 37°C to incubate for 30 minutes; | |
| e. During the first 15 minutes of the 30-minute incubation period, shake the flask 8-10 times to ensure complete saturation with sodium chloride; but allow the flasks to incubate undisturbed for the latter 15 minutes of the period; | |
| f. Immediately on completion of the incubation period, and without cooling or any further agitation of the saturated preparation, gently pour through an S&S No. 605 pleated filter paper, allowing to gravity filter, and collecting the filtrate; cover the funnel with a watch glass during filtration to help mitigate evaporation; and reprocess the filtrate through the same paper if necessary until about 100 mL of clear filtrate has been collected. | |
| 8.2 | High heat stock solution: Proceed as described above for the low heat stock solution, instead using ADPI Certified Reference Standard Nonfat Dry Milk, High Heat. |
| 8.3 |
Calibration standard solutions: a. Combine the following portions of low heat stock solution and high heat stock solution in separate test tubes, as described below: |
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Standard Solution # |
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|---|---|---|---|---|
| Low heat stock | High heat stock | |||
| 1 | 10.0 | plus | 0.0 | |
| 2 | 8.0 | plus | 2.0 | |
| 3 | 6.0 | plus | 4.0 | |
| 4 | 4.0 | plus | 6.0 | |
| 5 | 2.0 | plus | 8.0 | |
| 6 | 0.0 | plus | 10.0 | |
| b. Stopper each test tube and invert slowly to mix the contents. | |
| 8.4 | Transfer 1.0 mL of each calibration standard solution into a cuvette; |
| 8.5 | Add 10.0 mL of saturated sodium chloride solution to each cuvette; |
| 8.6 | Stopper the cuvettes and mix by slowly inverting; |
| 8.7 | One at a time, process each cuvette to develop turbidity of its undenatured whey protein, as follows: |
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a. Add 2 drops of 10% hydrochloric acid, using a 5 mL volumetric pipette for the addition; b. Stopper the cuvette and mix by inverting slowly, two times; do not shake, which can cause foaming and/or may introduce fine bubbles which can be misinterpreted by the spectrophotometer as turbidity; allow to stand for 5 minutes; c. At the 5-minute mark, again invert the cuvette slowly, one time, to ensure its contents are homogeneous, and measure the percent transmittance with the spectrophotometer at a wavelength of 420 nm; d. Repeat until all six cuvettes are processed and their corresponding percent transmittance values are recorded. |
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| 8.8 | Calculate the actual WPN for each of the six standard solutions, based on the certified values for the low heat and high heat reference standard materials, as follows: |
| 8.9 | Construct the calibration curve, using the calculated concentration values and corresponding percent transmittance measurements for all six standard solutions; |
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| 8.10 | Percent transmittance values for the new calibration curve should fall within measurement tolerances for the previously prepared curve; if they fall outside established tolerances, it is possible that one or both reference standard powders has become invalid, and/or that the preparations were made in error; investigate and correct as necessary, potentially including replacement of the ADPI Certified Reference Standards; |
| 8.11 | Linearity for the calibration curve (R2) should be ≥ 0.99; if this acceptance criterion is not met, examine the curve for outliers, using a student’s t-test or similar statistical tool; if your laboratory policy permits, exclude the outlier(s) and reevaluate the linearity; if unable to achieve this minimum requirement, repeat the calibration; |
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| 8.12 | Range between the percent transmittance for standard solution #1 and standard solution #2 should be approximately 35%; monitor this range over time, as a decrease in range may indicate an instrument issue such as loss of source lamp intensity; |
| 8.13 | When consistency, linearity, and percent transmittance range requirements have been met, proceed to prepare and test samples. |
9.0 Sample Reconstitution and Filtration
| 9.1 | Accurately weigh 2.0 grams of nonfat dry milk, recording the weight to the nearest 0.1 mg, and transfer the powder quantitatively to a test tube; |
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| 9.2 | Add 20.0 mL of water to the test tube; |
| 9.3 | Add 8 grams ± 0.1 grams of sodium chloride to the tube; |
| 9.4 | Stopper, shake for 1 minute, and place in a water bath at 37°C to incubate for 30 minutes; |
| 9.5 | During the first 15 minutes of the 30-minute incubation period, shake the tube 8-10 times to ensure complete saturation with sodium chloride; but allow the tube to incubate undisturbed for the latter 15 minutes of the period; |
| 9.6 | Immediately on completion of the incubation period, and without cooling or any further agitation of the saturated preparation, gently pour through an S&S No. 602 filter paper, allowing to gravity filter, and collecting the filtrate; cover the funnel with a watch glass during filtration to help mitigate evaporation; and reprocess the filtrate through the same paper if necessary until about 5 mL of clear filtrate has been collected. |
10.0 Blank Preparation and Measurement
| 10.1 | Transfer 1.0 mL of the clear filtrate from 9.6 into a cuvette; |
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| 10.2 | Add 10.0 mL of saturated sodium chloride solution to the cuvette; |
| 10.3 | Stopper the cuvette and mix by inverting slowly: this is the blank preparation; |
| 10.4 | “Blank” or “zero” the spectrophotometer with the blank preparation, equivalent to setting its value to 100% transmittance at the measurement wavelength of 420 nm. |
11.0 Sample Preparation and Measurement
| 11.1 | Transfer 1.0 mL of the clear filtrate from 9.6 into a cuvette; |
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| 11.2 | Add 10.0 mL of saturated sodium chloride solution to the cuvette; |
| 11.3 | Stopper the cuvette and mix by inverting slowly; |
| 11.4 | Add 2 drops of 10% hydrochloric acid, using a 5 mL volumetric pipette for the addition; |
| 11.5 | Stopper the cuvette and mix by inverting slowly, two times; do not shake, which can cause foaming and/or may introduce fine bubbles which can be misinterpreted by the spectrophotometer as turbidity; allow to stand for 5 minutes; |
| 11.6 | At the 5-minute mark, again invert the cuvette slowly, one time, to ensure its contents are homogeneous; |
| 11.7 | Measure the percent transmittance of the sample preparation at the measurement wavelength of 420 nm; |
| 11.8 | Repeat steps 11.1 through 11.7 to create and measure a duplicate sample preparation; |
| 11.9 | Compare the percent transmittance measurements of the two duplicate sample preparations: they should agree with one another within a tolerance of 2% transmittance, in which case, proceed to section 12.0 to calculate sample results; but if this tolerance requirement is not met, then complete steps 11.1 through 11.8 again to create and measure two more sample preparations, for a total of four sample measurements; and then proceed to section 12.0. |
12.0 Results Calculation
Using the slope and y-intercept values from the regression equation obtained in section 8 above, calculate the raw (uncorrected) sample result as follows:
Alternatively, the internal quantitative results features of the spectrophotometer may be used to arrive at the WPN results for each of the replicate sample preparations, in which case the final uncorrected result for the sample is reported as the arithmetic mean of the replicate results.
As described in 7.1, the best practice is to correct the raw (uncorrected) sample result to a standard moisture content of 3.16%, to normalize the sample result to the same moisture content as that of the ADPI Certified Reference Standards from which the calibration curve was prepared. The effect of this correction will be lesser when sample moisture content is close to 3.16% but conversely will be greater as the sample moisture level deviates from this reference value, either higher or lower. Calculate the corrected WPN result as follows:
13.0 External References
| 13.1 | Modification of the Harland-Ashworth method, "Journal of Dairy Science", Kuramoto et al, 42:28, 1959; |
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| 13.2 | "Standard Methods for the Examination of Dairy Products (“SMEDP”)", 17th edition, Ch. 15 – Chemical and Physical Methods, section 15.134 – Undenatured Whey Protein Nitrogen (Class O). |
14.0 ADPI Document Linkages
In no event shall ADPI be liable for any direct, indirect, incidental, special, exemplary, or consequential damages (including, but not limited to: the need to procure substitute goods or services; loss of use, data, or profits; or business interruption) however caused and on any theory of liability, whether in contract, strict liability, or tort (including negligence or otherwise) arising in any way out of the publication, use of, or reliance upon any standard, even if advised of the possibility of such damage and regardless of whether such damage was foreseeable.
