Isolation of casein for stable isotope ratio analysis of butter, cheese, and milk powder

Rationale Stable isotope ratio analysis (SIRA) is commonly used for the authentication of dairy commodities, providing evidence to support the geographical origin and production background of products. We set out to optimise methods for the isolation of a common constituent (casein) from three dairy commodities, which would permit easier inter‐ and intra‐commodity comparisons following SIRA. Methods Three published methods for isolation of protein (from cheese, milk, and butter) were adapted to yield protein (casein) fractions from commercial cheddar cheese, whole milk powder (WMP), and butter samples with a high degree of purity for subsequent SIRA. The casein fractions isolated underwent elemental analysis (H, C, and N), protein determination, and some also underwent SIRA of O and S. Two‐way analysis of variance and Tukey post hoc comparisons tested differences between methods. Results For each product, an optimised casein isolation method was chosen based on the C/N ratio and protein content. An optimum solvent lipid extraction (petroleum spirit–diethyl ether (2:1)) and casein precipitation method was chosen for cheddar cheese casein. A final solvent lipid extraction (heptane–isopropanol (3:2)) was necessary for WMP and butter casein extraction. δ13C and δ2H values validated the methods' abilities to remove contaminating lipid and isolate pure casein. Conclusions Casein of high purity, for subsequent SIRA, can be isolated from cheddar cheese, WMP, and butter following modifications of previously published methods.

contribution of other fractions to variation in the stable isotope values between products. For example, the isotopic values of protein and lipid fractions differ substantially 12,19 ; therefore, differences in the isotopic values of whole dairy products could be due to differences in lipid concentration between the products as opposed to factors such as geographical origin or production parameters. A second advantage of using a protein fraction such as casein is that, because of its elemental composition, it is possible to measure the isotopic values of all the light elements common to foods (H, C, N, O, S); this is not the case with lipid fractions, in which N and S are limiting.
For milk samples, centrifugation has been commonly used to remove the lipid fraction, followed by acidification to pH 4.6 of the aqueous fraction to precipitate the casein fraction. 1,5,14,20 For cheese, the methods of Manca et al 21 17,18 For butter, 3 bulk butter or butter protein obtained following solvent extraction of the lipid fraction has been used for SIRA. For milk powder, bulk whole milk powder (WMP) 25 and bulk skimmed milk powder have been used. 4 Thus, depending on the preparatory methods used, the proportions of lipid, protein, and other constituents may vary in the fractions used for SIRA, making it difficult to make comparisons between products based on SIRA data.
The objective of the study presented here was to develop methods for the isolation of a casein fraction of high purity from three different dairy products (cheddar cheese, WMP, and butter) that would enable subsequent intra-and inter-product SIRA comparisons.

| Isolation of casein fraction from cheddar cheese
The methods used to isolate the casein fraction from cheddar cheese are summarised in Figure 1. Each method was carried out in triplicate.

In Method A (an adaptation of the methods of Camin et al 2 and
Manca et al 21 ), grated cheese (6 g) was freeze-dried (Buchi Lyovapor L-200) for 24 h in 10 cm Â 10 cm mini-grip resealable sample bags (VWR, APHA121-086). Using an Ultra-Turrax (IKA DI 25 basic homogeniser; 13 500 rpm, 3 min), 4 g of the freeze-dried cheese was homogenised with petroleum spirit-diethyl ether (2:1, v/v) (30 mL) in a 50 mL centrifuge tube (Corning ® , 430828), centrifuged (Beckman F I G U R E 1 Summary of methods A, B, C, and D and of A+, B+, C+, and D+ (additional lipid extraction) for cheddar cheese casein isolation Coulter Avanti J-E centrifuge; 2500g, 6 min), and the supernatant was decanted. This step was repeated twice. The pellet was vortexed (Stuart Scientific SA8 vortex mixer) (30 s) with deionised water (30 mL) and acidified to pH 4.6 by dropwise addition of 1 M HCl using a pH meter (Mettler Toledo FE20/EL20). After gently agitating in a 50 mL glass beaker using a magnetic stir bar for 30 min, and centrifugation (1200g, 8 min), the supernatant was decanted. The casein pellet was suspended in 10 mL of deionised water, vortexed for 30 s, and centrifuged (1200g, 4 min). The water was decanted, and the remaining pellet was retained in a 6 cm Â 6 cm sample bag and freeze-dried. Freeze-dried powder was stored in a desiccator before elemental (Section 2.6) and protein (Section 2.7) analysis and SIRA (Section 2.8).
In Method B the procedure outlined for Method A was followed with an additional lipid extraction step. Following the final freezedrying step, the resulting casein powder was homogenised using an Ultra-Turrax (13 500 rpm, 1 min) with 10 mL of petroleum spiritdiethyl ether (2:1, v/v) in a 50 mL centrifuge tube, centrifuged (2500g, 6 min), and the supernatant was decanted. The remaining casein pellet was placed into a 6 cm Â 6 cm open sample bag and freezedried. Freeze-dried powder was stored in a desiccator before elemental (Section 2.6) and protein (Section 2.7) analysis and SIRA (Section 2.8).
In Method C the procedure outlined in Method A was following with additional temperature adjustments to improve final casein yield. 26 Prior to acidification, the pellet was cooled to approx. 4 C in a refrigerator and, after acidification, was held in a water bath at approx. 35 C for at least 30 min.
In Method D the procedure outlined for Method C was followed with an additional lipid extraction step. Following the final freezedrying step, the resulting casein powder was homogenised using an Ultra-Turrax (13 500 rpm, 1 min) with 10 mL of petroleum spirit-diethyl ether (2:1, v/v) in a 50 mL centrifuge tube, centrifuged (2500g, 6 min), and the supernatant was decanted. The remaining casein pellet was placed into a 6 cm Â 6 cm open sample bag and freezedried. Freeze-dried powder was stored in a desiccator before elemental (Section 2.6) and protein (Section 2.7) analysis and SIRA (Section 2.8).

| Isolation of casein fraction from WMP
The methods used to isolate the casein fraction from WMP are summarised in Figure 2. Each method was carried out in triplicate.
Previous studies conducting SIRA of milk powders have used the bulk milk powder rather than casein isolated from the milk powder. 4,25,27 Therefore, Method A was adapted from the methods in Freeze-dried powder was stored in a desiccator before elemental (Section 2.6) and protein (Section 2.7) analysis and SIRA (Section 2.8).
In Method B the procedure outlined for Method A was followed with an additional lipid extraction step. Following the final freezedrying step, the resulting casein powder was weighed into a 50 mL F I G U R E 2 Summary of methods A, B, C, and D and of A+, B+, C+, and D+ (additional lipid extraction) for WMP casein isolation glass beaker and reconstituted using a magnetic stir bar with 10 mL of warm water (approx. 40-50 C) for a minimum of 1 h. Petroleum spirit-diethyl ether (2:1, v/v) (10 mL) was added and left to stir for a further minimum of 15 min. Following centrifugation (5000g, 15 min), the supernatant was decanted, and the defatted casein pellet was placed in an open 6 cm Â 6 cm sample bag and freeze-dried. Freezedried powder was stored in a desiccator before elemental (Section 2.6) and protein (Section 2.7) analysis and SIRA (Section 2.8).
In Method C the procedure outlined for Method A was followed with an additional solvent lipid extraction step. Petroleum spiritdiethyl ether (2:1, v/v) (10 mL) was added to reconstituted WMP which was agitated for 15 min (in a 50 mL glass beaker using a stir bar and magnetic stir plate) prior to centrifugation (5000g, 15 min) and removal of the upper lipid layer.
In Method D the procedure outlined in Method C was followed with additional temperature adjustments to improve final casein yield. 26 Prior to acidification, the pellet was cooled to approx. 4 C in a refrigerator and, after acidification, was held in a water bath at approx. 35 C for at least 30 min.

| Isolation of casein fraction from butter
The methods applied to the isolation of casein from butter are summarised in Figure 3. Each method was carried out in triplicate. In In Method B the procedure outlined for Method A was followed with an additional lipid extraction step. Following the final freezedrying step, the resulting casein powder was weighed into a 50 mL centrifuge tube with 30 mL of petroleum spirit-diethyl ether (2:1, v/v), placed in a shaking water bath at room temperature for approx. 30 min, and centrifuged (1200g, 20 min). The supernatant was removed, and the washed casein was placed into a 6 cm Â 6 cm sample bag and freeze-dried. Freeze-dried powder was stored in a desiccator before elemental (Section 2.6) and protein (Section 2.7) analysis and SIRA (Section 2.8).
In Method C the procedure outlined for Method A was followed with an additional lipid extraction step and additional temperature adjustments to improve final casein yield. 26 Petroleum spirit-diethyl ether (2:1, v/v) (30 mL) was added to the aqueous phase following separation in the separating funnel, centrifuged (1200g, 10 min), and the supernatant was decanted. This step was repeated with 15 mL of petroleum spirit-diethyl ether (2:1, v/v) (15 mL). The aqueous layer was then cooled to approx. 4 C in a refrigerator before acidification and after which it was held in a water bath at approx. 35 C for at least 30 min.
F I G U R E 3 Summary of methods A, B, C, and D and of A+, B+, C+, and D+ (additional lipid extraction) for butter casein isolation In Method D the procedure outlined for Method C was followed with an additional lipid extraction step. Following the final freezedrying step, the resulting casein powder was weighed into a 15 mL centrifuge tube with 10 mL of petroleum spirit-diethyl ether (2:1, v/v), placed in a shaking water bath at room temperature for approx. 30 min, and centrifuged (1200g, 20 min). The supernatant was removed, and the washed casein was placed into a 6 cm Â 6 cm sample bag and freeze-dried. Freeze-dried powder was stored in a desiccator before elemental (Section 2.6) and protein (Section 2.7) analysis and SIRA (Section 2.8).

| Additional heptane-isopropanol (3:2, v/v) extraction
As a result of the data obtained following the elemental and protein analyses of casein fractions from cheese, WMP, and butter following methods Methods A-D above, an additional lipid extraction step, using a more polar solvent (heptane-isopropanol (3:2, v/v)), was applied. This additional modification was based in the procedure described in Radin, 28 although hexane was replaced with heptane for safety reasons. 29 The freeze-dried cheese, WMP, and butter casein fractions collected following Methods A, B, C, and D were transferred from sample bags to 50 mL centrifuge tubes and 5 mL of heptaneisopropanol (3:2, v/v) was added. The suspension was homogenised using an Ultra-Turrax (9500 rpm for 1 min) and centrifuged (5400g, 10 min) at room temperature (approx. 20 C). The supernatant was decanted and a further 2.5 mL of heptane-isopropanol (3:2, v/v) was added, followed by homogenisation, centrifugation, and removal of the supernatant. This step was repeated once again, and any remaining solvent was evaporated from the defatted casein by leaving in a fume hood overnight. The casein fraction was collected and prepared for elemental (Section 2.6) and protein (Section 2.7) analysis and SIRA (Section 2.8).

| Elemental analysis
Hydrogen, carbon, and nitrogen analysis was conducted with an Exeter CE440 CHN analyser. The standard used was acetanilide. Nitrogen is measured with reference to pure helium carrier gas, the difference in thermal conductivity being proportional to nitrogen content. Typically, the precision is ±0.3% for homogeneous, stable, solid samples.

| Statistical analysis
The effect of the use of different methods of casein extraction on the C/N ratio, protein (%) and stable isotope ratio values of the products was tested using a two-way analysis of variance (ANOVA) followed by a Tukey test for post-hoc comparisons. The statistical analysis of the data was performed using R 31

| RESULTS AND DISCUSSION
The three products analysed in this study (cheddar cheese, WMP, and butter) have undergone various levels of processing during manufacture and may be described as complex food matrices.
Isolating a common constituent (casein) from each is therefore useful in permitting stable isotope ratio comparisons both within and between product categories. However, differences in the products' compositions and manufacturing methods pose challenges in terms of extraction of the casein fraction because of the potential for interference from other constituents, most notably lipid. Milk fat constitutes a minimum (on dry matter basis) of 22% of cheddar cheese, 32 26% of WMP, 33 and 80% of butter. 34 However, a further challenge is the low casein content of butter, with casein estimated to be about 0.48% by fresh weight of butter. Therefore, extraction methods which optimise lipid removal and yield a casein fraction of high purity, following extraction, are desirable.
A positive linear relationship between C/N ratio values and lipid content has been recorded in muscle tissue samples. 35 The C/N ratio for milk casein is approximately 3.5, 14 with higher values being indicative of interference from lipid. For this reason, elemental analysis together with protein determination were used to evaluate the purity of the casein fractions extracted from cheese, WMP, and butter using the various methods outlined in Section 2.1.

| Cheddar cheese: elemental and protein analyses of casein fraction
The C/N ratio of casein extracted from cheddar cheese ranged from 3.50 to 3.55 (Table 1)   T A B L E 3 Hydrogen, carbon, and nitrogen content, C/N ratio, protein content, and isotope ratio values of butter casein obtained following each of methods A, B, C, and D alone or with an additional heptane-isopropanol extraction step (methods A+, B+, C+, D+)

| Butter: elemental and protein analyses of casein fraction
Butter C/N ratios varied across isolation methods (range 3.71 to 7.58) and lower protein contents were associated with higher C/N ratios (

| CONCLUSIONS
Through the systematic adoption of a number of approaches, involving centrifugation, pH and temperature adjustment, and solvent extraction, it is possible to obtain from different dairy products a casein fraction with a high degree of purity suitable for SIRA. The approaches adopted are more complex, requiring additional solvent extraction, in the case of WMP and butter. The advantage of isolating a casein fraction with a high degree of purity lies in the potential to make meaningful inter-and intra-product comparisons of the effects of geographical origin, animal production system, or processing on stable isotope composition and to provide evidence to support authenticity claims.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/rcm.9402.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.