1 3

Eur Food Res Technol (2014) 238:819–827DOI 10.1007/s00217-014-2158-3

ORIgInal PaPER

δ13C values and phytanic acid diastereomer ratios: combined evaluation of two markers suggested for authentication of organic milk and dairy products

Stefanie Kaffarnik · Markus Schröder · Katja Lehnert · Ton Baars · Walter Vetter

Received: 12 april 2013 / Revised: 19 December 2013 / accepted: 4 January 2014 / Published online: 23 January 2014 © Springer-Verlag Berlin Heidelberg 2014

Keywords Food authentication · Organic food · Ea-IRMS · δ13C values · Milk fat · Dairy products · Phytanic acid

Introduction

Food quality in terms of ingredient levels and the pro-duction system is considered of general importance for a growing number of consumers. For instance, the mar-ket share of organic milk and dairy products is increas-ing despite significantly higher retail prices compared to conventional products [1, 2]. accordingly, authentication of highly rated food such as organic milk by analytical markers is more and more becoming a crucial issue in food control in order to protect consumers from fraud [3, 4]. a key parameter in this context is the feed supplied to the cows, which depends largely on the farming system. For instance, pasture and hay are essential principal food items supplied in organic milk production [5, 6]. By contrast, conventional farmers take advantage of higher proportions of concentrate and maize silage. grass-based feed consists of C3-plant, which are more depleted in 13C (δ13C values ranging from −23 to −30 ‰) than the C4-plant maize (δ13C values of maize silage ranging from −12 to −13 ‰) [7, 8]. These differences in the 13C content of feed of the cows are reflected in the milk and the milk fat they pro-duce [7–13]. For instance, Boutton et al. [11] switched the diet of isotopically equilibrated alfalfa hay diet (C3-plant feed) to maize (C4-plant) and observed a fast increase in the δ13C values. accordingly, δ13C values have been sug-gested as an authentication marker for organic milk (i.e., δ13C value of −28 ‰ and more negative in organic milk compared to −26.6 ‰ or more positive in conventional milk) [9].

Abstract authentication of organic milk by suitable markers is currently attracting more and more interests in food control. In this study, we aimed to compare the effi-ciency of the markers stable carbon isotope ratio (δ13C value) with the SRR/RRR phytanic acid diastereomer ratio (SRR/RRR) of milk fat for distinguishing different feed-ing systems. For stable carbon isotope ratio analysis by elemental analysis–isotope ratio mass spectrometry (Ea-IRMS), we first developed a simple sample preparation method based on milk fat extracts allowed to evaporate the solvent in tin capsules for liquid samples. The δ13C values and reproducibility measured with this alternative sample pre-treatment method excellently matched those obtained with the current standard method. applied to milk sam-ples, the results of the Ea-IRMS analysis were linked to the SRR/RRR, and both markers allowed to distinguish milk from cows fed with hay (δ13C value > −28.0 ‰, SRR/RRR <1.5) from feed used in conventional milk production which contained maize silage (C4-plants) (p < 0.001). Milk fat of organic retail cheese samples was also highly depleted in 13C (δ13C value −30.0 ‰ ± 1.1), and the SRR/RRR was low (<1.5). However, seven cheese samples showed incon-sistent δ13C values and SRR/RRR, most likely due to the feeding of grass silage. Both parameters (δ13C values and SRR/RRR), together with the phytanic acid content of milk fat, also allowed distinguishing between the feeding of high amounts of pasture, hay, and/or grass silage.

S. Kaffarnik · M. Schröder · K. lehnert · W. Vetter (*) Institute of Food Chemistry, University of Hohenheim, 70953 Stuttgart, germanye-mail: walter.vetter@uni-hohenheim.de

T. Baars Research Institute of Organic agriculture (FIBl), 5070 Frick, Switzerland

820 Eur Food Res Technol (2014) 238:819–827

1 3

Further potential analytical markers of organic milk are the ratio of specific groups of fatty acids along with specific fatty acids such as α-linolenic acid (18:3n-3), eicosapentae-noic acid (20:5n-3), conjugated linoleic acids (Cla), and phytanic acid, all of which were found at higher quantities in organic milk fat [5, 9, 14–22].

Our recent attempts at the authentication of organic milk were based on its higher phytanic acid content and the lower SRR/RRR phytanic acid diastereomer ratio (hereafter, SRR/RRR) [17, 18, 21]. This non-natural fatty acid is formed by ruminants (and fish) from phytol, i.e., the side chain of chlorophyll [23]. an SRR/RRR of >1.5 in conventional milk compared to equal amounts of both diastereomers (SRR/RRR ~1) in organic milk indicated the marker’s dependency on the feed supplied and probably the bacterial composition in the rumen [18, 24]. The marker SRR/RRR could also be success-fully applied to cheeses, except for three conventional cheese samples bought retail which rather showed the characteris-tics of organic cheese [18]. The low SRR/RRR in these three samples indicated limitations in the unequivocal authentica-tion of organic milk. Evidence for this issue was recently pro-duced by Baars et al. [20] who grouped organic and conven-tional milk into high and low input systems. By means of the SRR/RRR, they found remarkable similarities between milk samples from conventional and organic low input systems, both in summer and winter milk [20].

The goal of the study was to determine the δ13C values (‰) of bovine milk samples from different feeding stud-ies and cheese samples bought retail in germany and to combine these results with the SRR/RRR and phytanic acid concentrations for a clearer differentiation of organic and conventional milk. For this purpose, we wished to establish an alternative and convenient sample preparation method for stable carbon isotope ratio analysis (δ13C values) by elemental analysis–isotope ratio mass spectrometry (Ea-IRMS) of milk fat. The method was based on the pipet-ting of small aliquots of milk fat extract into tin capsules for liquid samples. Storing the open tin capsules overnight at room temperature was accompanied with the removal of the solvent, and the homogeneous milk fat samples could be directly analyzed. The results obtained with this Ea-IRMS method were compared with standard procedures using solid milk fat.

Materials and methods

Milk and cheese samples

Study 1: Whole milk samples (pools of morning and even-ing milk samples from 2 days) from three Holstein cows were collected for at least 100 days (between October 2009 and February 2010) at the agricultural Experiment

Station of the University of Hohenheim (location Meierei-hof, Stuttgart, germany) [24]. The experiment started with typical conventional, concentrate-based diet (phase 1) con-sisting of 16.0–20.4 % C4-plants (maize silage) and 77.6–82.4 % C3-plants (grass silage, hay, 2nd cut hay, concen-trate), and 1.6–2.0 % mineral feed, expressed at dry matter basis. In phase 2, the hay ration was gradually raised within 7 days to 100 %. In phase 3, only hay was fed for 44 days (“organic feed”). Within 1 day, the feed was changed back from hay to the initial feed (“conventional feed,” phase 4). For cow 3, a subsequent phase 5 was added in which the cow received a diet consisting of 99 % grass silage (C3-plant diet) with 1 % mineral feed for 14 days. Between one and five milk samples per feeding phase and cow were ana-lyzed by Ea-IRMS.

Study 2: In addition, pool milk samples (90 dairy cows: 60 Simmental and 30 offspring) were collected every fourth day between March and June 2010 at a dairy farm (Scharenstetten, germany) in transition from conventional to organic farming [24]. Three samples before transition took place, including the sample from the previous year (“conventional”), three samples of the transition phase, and additional four of the constant phase (“organic”) after tran-sition to organic farming were analyzed and compared with data on phytanic acid [24].

Study 3: Complementary milk samples (n = 5) were obtained from one cow from an organic farm located in the surrounding area of Stuttgart (five morning milk samples daily milked by hand collected within 15 days in January and February 2009 [25]). This cow was feed with organic items containing 100 % C3-plants (about 85.7 % hay, grass silage, and clover silage and about 14.3 % concentrate from oat, peas, and clover). In addition, five conventional milk samples (morning samples daily milked mechanically) from a further cow raised at the agricultural Experiment Station of the University of Hohenheim (location Meierei-hof, Stuttgart, germany) collected within 16 days in March 2009 were analyzed as well. The conventionally fed cow got 71.5 % C3-plant (grass silage, clover silage, hay, after grass, mineral feed, wheat, defatted rape shred, barley, field beans, and rapeseed oil) and 28.5 % C4-plant (maize silage, maize, maize gluten) forage [18].

Fat extracts of 14 organic cheese (Edam cheese, gouda cheese (2×), butter cheese (2×), allgäuer Emmental cheese, Emmental cheese, Romadur cheese, camembert, cow milk mozzarella, sliced cheese, brie, Bavaria blue, mountain cheese), and ten conventional cheese (Edam cheese, gouda cheese, butter cheese, Emmental cheese, Romadur cheese, limburg cheese, cow milk mozzarella, cow milk feta, Harzer cheese, hand cheese) samples previ-ously analyzed on phytanic acid [17, 18], were analyzed by Ea-IRMS. One organic (Bioland, Schwarzwälder Sau-errahmbutter) and one conventional (Ja!, Markenbutter)

821Eur Food Res Technol (2014) 238:819–827

1 3

butter samples were used for gas chromatography with iso-tope ratio mass spectrometry (gC-IRMS) analysis.

Standards, chemicals, and materials

Tripalmitin was from Fluka (Buchs, Switzerland), and USgS40 was from the United States geological Survey (Reston Stable Isotope laboratory, Reston, Va, USa). Methanol and n-hexane (both HPlC gradient grade) were from Th. geyer (Renningen, germany). Cyclohexane and ethyl acetate (both purest, Sigma-aldrich, Taufkirchen, germany) were combined and distilled to give the azeo-tropic mixture of 46:54 (w/w).

Borontrifluoride-methanol-complex solution (13–15 % BF3 in methanol) was from Sigma-aldrich (Taufkirchen, germany). Concentrated sulfuric acid (BaSF, ludwig-shafen, germany) was added to methanol to give the 1 % sulfuric acid in methanol solution. Sodium chloride (99.8 %) was from Carl Roth (Karlsruhe, germany). High-purity helium (5.0) and carbon dioxide gas (IRMS refer-ence gas, 4.5) were from Westfalen (Münster, germany).

Tin capsules for solids (Sn purity 97.5 %, 0.07 ml vol-ume, 4.0 × 6.0 mm size) and tin capsules for liquids (Sn purity 99.9 %, 0.04 ml volume, 3.5 × 5.5 mm size) were from IVa analysentechnik (Meerbusch, germany). Tin capsules for liquids differ from those for solids; in that, liq-uid samples or sample solutions do not leak out.

Milk fat extraction

Homogenized whole milk samples were lyophilized to give whole milk powder. approximately 1 g whole milk powder or homogenized and lyophilized cheese was used for accel-erated solvent extraction (aSE 200, Dionex, Idstein, ger-many) [17, 26, 27]. The gained lipids were set to a defined solution volume of 10 ml cyclohexane/ethyl acetate (46:54, w/w). The fat content of the extract solutions was determined by weighing the dry fat obtained from 1 ml solution (containing ~25–45 mg ml−1 milk fat).

Stable carbon isotope ratio analyses

Elemental analyzer–isotope ratio mass spectrometry (Ea-IRMS) analyses were performed with a Euro Ea 3000 ele-mental analyzer (Hekatech, Wegberg, germany) coupled to a Delta plus XP isotope ratio mass spectrometer (Thermo Finnigan MaT, Bremen, germany) using the parameters described elsewhere [28]. In brief, the furnace temperatures for oxidation and reduction were set to 1,000 and 650 °C. a gC column filled with 5 Å molecular sieve heated to 90 °C was used for the removal of undesired gases. The helium carrier gas pressure was set at 40 kPa and purge flow at 80 ml min−1.

Relative 13C/12C ratios were expressed in δ13C notation using Vienna PeeDee belemnite (VPDB) as the reference standard according to the International atomic Energy agency (IaEa, Vienna, austria) (Eq. 1):

USgS40 (certified δ13C value of −26.39 ± 0.04 ‰ rela-tive to VPDB [29]) was used as secondary standard to cali-brate the CO2 obtained from tripalmitin (−24.40 ± 0.04 ‰) which was used as external working standard for the meas-urements. Each Ea-IRMS standard or sample run started and ended by measuring twice the reference CO2 gas. Each sequence, the external working standard tripalmitin was measured in the beginning and in the end. In case of long sequences (>20 measurements), the working standard tri-palmitin was additionally measured in the middle of the sequence. This procedure served to eliminate variations in the δ13C values from run to run. Day-to-day variations were corrected by the δ13C value of the calibrated working stand-ard and each isotopic measurement were compensated by adjusting the δ13C values of the sample against it. Isodat 2.0 (Thermo Finnigan MaT, Bremen, germany) software was used for processing of results and for analysis.

Ea-IRMS analyses were performed with dry milk fat (method a) and milk fat solutions (method B). Method a: Milk fat extract (1 ml; ~30 mg) was liberated from the solvent with a gentle stream of nitrogen. about 1 mg of dry milk fat (s) was transferred by means of a spatula into tin capsules for solids and weighed. Method B: Milk fat extract (~30 μl; fat content ~1 mg) was filled with a 50-μl glass syringe in pre-weighed tin capsules for liq-uids. The samples were stored overnight at room tempera-ture for evaporation of the solvent. The sample amount was determined by back weighing of the fat containing capsules after preparation. Repeatability of the methods was tested in triplicate with three milk samples from different feeding periods (phase 1: day 4/5, phase 3: day 50/51, and phase 4: day 85/86) and in addition with three different cheese sam-ples (organic butter cheese, organic mountain cheese, and conventional cow milk feta). after this measure, all sam-ples were prepared with method B in duplication.

gC/MS analysis of phytanic acid as methyl ester

Samples previously analyzed on phytanic acid content and the SRR/RRR were processed according to Vetter and Schröder [17, 18]. In brief, fatty acids in the aSE milk extracts (see above) were converted into methyl esters by means of BF3-methanol complex [30, 31]. FaMEs were extracted from the aqueous solution with 2 ml n-hexane. The use of internal standards and the quantification mode,

(1)δ

13C = [(13C/12C)sample − (13C/12C)reference]/

(13C/12C)reference × 1, 000(

0/00

)

822 Eur Food Res Technol (2014) 238:819–827

1 3

as well as the determination of the SRR/RRR by gC/EI-MS-SIM, was presented elsewhere [18, 27].

Enrichment of phytanic acid methyl ester for compound-specific isotope ratio analysis

Fatty acids of one organic and one conventional butter sam-ple (~5 g butter fat) were transferred into FaME by means of 100 ml of 1 % sulfuric acid in methanol solution [32]. The resulting FaMEs were separated by urea complexation [32]. The filtrate of urea complexation was fractionated by high-speed counter-current chromatography (HSCCC) [33]. The fraction containing pure or highly concentrated phytanic acid methyl ester was diluted to appropriate con-centration and analyzed by gC-IRMS.

gas chromatography with isotope ratio mass spectrometry (gC-IRMS) determinations of the δ13C value (‰) of phytanic acid methyl ester

gC-IRMS analyses were performed with a 6890 gas chromatograph (Hewlett-Packard/agilent, Waldbronn, germany) coupled to a Delta plus XP isotope ratio mass spectrometer (Thermo Finnigan MaT, Bremen, germany). Fractions prepared according to the previous step were ana-lyzed by gC-IRMS in combination with a nonpolar column using the parameters described elsewhere in details [34]. Previous studies have shown that methylation of fatty acids was not accompanied with isotope fractionation [34–36]. However, the methanol used in our study for methylation was strongly depleted in 13C (δ13C value −38.9 ‰ ± 0.2). Thus, conversion of the free fatty acids into fatty acid methyl esters resulted in a decrease in the 13C content, and this effect was the larger the less the fatty acid was depleted in carbon and the less the carbon number was (Eq. 2).

where x is the number of carbon in the aliphatic chain of the free fatty acid. For 18:0 methyl ester (measured δ13C

(2)

δ13C (FAMEcalc.)

=

[

(δ13C)free fatty acid × x + (δ13C)MeOH

]

/ (x + 1)

value: −28 ‰), the impact of the methanol group on the δ13C value on the free fatty acid was 0.6 ‰ which was in the same range as the effect of transesterified milk fat (~0.7 ‰, data not shown). For phytanic acid (20 carbons), the measured δ13C value of −36 ‰ of the methyl ester was only affected by about 0.1 ‰ from the methylation and thus negligible. However, the δ13C values reported in sec-tion “Results and discussion” were corrected by means of Eq. 2.

Statistical evaluation

Statistical evaluation of the δ13C values of milk fat and the SRR/RRR was performed by one-way anOVa. This anal-ysis of variance was calculated in Excel with α = 0.05. a statistical significance between two different groups was constituted by p = 0.0001. Pearson’s correlation was used to evaluate the correlation between the δ13C values and the SRR/RRR in milk and cheese milk fat samples. The cor-relation coefficient r was calculated in Excel. a high linear correlation was assigned to 0.5 < r < 0.7, and a very high correlation to 0.7 < r < 0.9.

Results and discussion

Sample preparation method for stable carbon isotope ratio analysis of milk fat by Ea-IRMS

For the six samples investigated, the δ13C values of the classic method of using solid milk fat samples (method a) were exactly matched by the alternative method based on milk fat solutions (method B). This also applies to the higher 13C content by >3 ‰ of samples 1 and 3 (conven-tional feed including maize) compared to sample 2 (hay feed) (Table 1). In addition, the low standard deviations (≤0.05 ‰, n = 6) corresponded well with those reported by Molkentin and giesemann (<0.15 ‰, n = 3) [13] and confirmed a good repeatability of method B (Table 1). This method may thus serve as alternative method for (semi-) solid samples difficult to homogenize or to pipet. a better

Table 1 Mean δ13C values and standard deviations after analysis of six milk fat samples prepared by the standard method (method a) and the modified method suggested in this study (method B)

a Obtained from a feeding study during day 4/5 (sample 1), day 50/51 (sample 2), and day 85/86 (sample 3)b Weighed into tin capsules for solidsc Pipetted into tin capsules for liquids; measured after evaporation of the solvent

Method Sample type Milk sample 1a Milk sample 2a Milk sample 3a Butter cheese Mountain cheese Cow milk feta

δ13C (‰) δ13C (‰) δ13C (‰) δ13C (‰) δ13C (‰) δ13C (‰)

a Solid milk fatb −24.53 ± 0.03 −27.99 ± 0.03 −24.95 ± 0.06 −29.80 ± 0.02 −31.29 ± 0.02 −28.20 ± 0.09

B Milk fat solutionsc −24.64 ± 0.02 −28.19 ± 0.05 −25.10 ± 0.02 −29.92 ± 0.02 −31.36 ± 0.02 −28.22 ± 0.01

823Eur Food Res Technol (2014) 238:819–827

1 3

handling of liquid samples in tin capsules for liquids had also been reported by aramendía et al. [37] while study-ing the δ18O values of olive oil. However, they received discouraging results due to sample contamination by atmospheric oxygen [37], which was not a problem in the analysis of stable carbon isotope ratios. The only additional requirement is the use of special, more expensive tin cap-sules for liquids, because the solvent and parts of the sam-ple leaked out from common capsules for solids. accord-ingly, the modified technique (method B) was subsequently used for the analysis of milk fat samples by Ea-IRMS. Due to excellent repeatability, all samples were only analyzed in duplicate and means of the δ13C values will be discussed.

Evaluation of δ13C values with SRR/RRR phytanic acid diastereomer ratio (SRR/RRR) of milk fat samples from three different feeding studies

During the gradual transition from conventional feed to hay within 7 days (phase 2), the δ13C values linearly decreased by ~0.3 ‰ per day until only hay was fed (Fig. 1, phases 2 and 3). accordingly, the isotopic signature of the feed was immediately reflected in the δ13C value of the milk fat. The direct impact of the diet on the δ13C value could also be ver-ified in an additional study in which we analyzed samples from a farm in transition from conventional to organic prac-tices. The investigated samples showed a linear decrease in the δ13C values from −24.3 ± 0.65 ‰ (“conventional farm-ing”) to −27.8 ± 0.14 ‰ (“organic farming”) with time (Fig. 2). likewise, the concentration of phytanic acid in the milk fat correlated with the amount of green items in the cow’s diet (Fig. 2). However, the SRR/RRR (conventional farming = 2.5) immediately decreased after transition to a constant SRR/RRR of 1.3 (data not shown) [24].

Throughout the hay feeding (phase 3) in study 1, the δ13C value was −28 ‰ or more depleted in 13C. The δ13C val-ues of phases 1 and 3 were typical for conventional versus organic milk as presented by Molkentin and giesemann [13].

likewise, high SRR/RRR of 2.2–3.8 in milk was typi-cal for the use of conventional feed [18]. When the diet was changed to hay, the portion of RRR diastereomer increased and the SRR/RRR dropped to ~1 (Fig. 1, Table 2). However, the change in the SRR/RRR was less linear as observed with the δ13C value (Fig. 1, day 15). The different SRR/RRR indicated that the digestion of grass or maize in the rumen involved different microorganisms. This obvious change in the microbial rumen composition could be the reason for the slight delay in shift of the SRR/RRR in the formation of phytanic acid.

Fig. 1 Development of the δ13C values (‰) of milk fat with changing diet composi-tion (phases 1 and 4: 16–20 % C4-plants, phase 2: increasing hay content to 100 %, phase 3: only hay was fed (100 % C3-plants)). Mean values (black line) from three different cows (study 1). Contribution of the SRR phytanic acid diastereomer (SRR/RRR diastereomer ratio) (dashed line) of the four phases was shown in parallel. The hori-zontal grey dashed line repre-sent the border between organic and conventional samples

ph

ase

2

ph

ase

4

20

30

40

50

60

70

80

90

100

30

29

28

27

26

25

24

230 10 20 30 40 50 60 70 80 90

(1.5)

(4.0)

(9.0)

(2.3)

(1.0)

(0.67)

(0.43)

(0.25)

13C

SRR/RRR13

Cva

lue

[‰]

contribution [%] of S

RR

diastereomer

(SR

R/R

RR

diasteromer ratio)

ph

ase

1

ph

ase

3

days

C3 plant

C4 plant

0.0

0.1

0.2

0.3

0.430

29

28

27

26

25

24

230 20 40 60 80 100

13C

days

concentrationofphytanic

acid(g/100g)

13C

phytanic acidcontent "organic“

"conventional“

valu

e [‰

]

Fig. 2 Changing of the δ13C values (‰, gray line) and phytanic acid content (g/100 g lipid, dashed black line) in milk fat of pooled milk (60 dairy cows) from a farm in transition from conventional to organic farming (study 2). The horizontal dashed line represent the border between conventional and organic values

824 Eur Food Res Technol (2014) 238:819–827

1 3

Switching back from hay to conventional feed (contain-ing C4-source maize silage) within 1 day was also mir-rored by the progression of both markers. Within 4 and 10 days, respectively, both the δ13C values (~−25 ‰) and the SRR/RRR (>2) fell back on the initial values in phase 1 (Fig. 1, phase 4). The progression of the δ13C values was similar to observations made by Metges et al. [12] for whole milk samples. Metges et al. [12] noted that chang-ing the feed from a diet rich in C4-plants to C3-plants was accompanied by a depletion of 13C from δ13C value −13.9 to −26.2 ‰, directly after the metabolic reaction. Since Metges et al. exclusively fed C4-plants to the cows, the corresponding whole milk samples in the “conventional” phase were higher in 13C than ours (−13.9 vs. −24.5 ‰).

additional samples were analyzed for verification of these observations (study 3). Organic milk samples from study 3 were highly depleted in 13C (δ13C values −31.1 ‰ ± 0.18 (n = 5); 100 % C3-plant forage), and also, the SRR/RRR was low (0.87 ± 0.06, Table 2). By con-trast, milk samples from an additional cow, which received conventional fodder, were richer in 13C (δ13C values −24.7 ‰ ± 0.15 (n = 5); 71.5 % C3-feed items), and the SRR/RRR was also higher (5.65 ± 0.08 vs. 3.8, Table 2) [18]. These results also agreed well with the previous ones.

Molkentin and giesemann determined δ13C values of −26.6 ‰ and higher in 13C for conventionally pro-duced milk, while organic milk fat was lighter in carbon (−28.0 ‰ and lighter in 13C) [8]. likewise, an SRR/RRR >1.5 was found typical of milk from cows which were conventionally fed [24]. Both markers were significantly different in the conventional feeding phases (Fig. 1; phases 1 and 4) and the “organic” phase with hay (Fig. 1, phase 3) (0.0001 < p > 0.01, Table 2) and also in organic and conventional milk fat samples from different farms (p < 0.0001, study 3, Table 2). In addition, the δ13C values of milk fat from phases 1 and 4 showed no significant dif-ference (p > 0.05). a high correlation was found between the SRR/RRR and δ13C values (Pearson correlation coef-ficient of the feeding study r = 0.80; organic and conven-tional milk r = 0.98).

analysis of the milk samples from cows fed with grass silage resulted in a mean δ13C value of −29.9 ‰, i.e., the lowest 13C content measured in any of the milk fat sample of the feeding study 1 (Table 2, phase 5). The milk fat of these samples showed no significant difference in the δ13C values compared with those measured after the feeding of hay (Table 2, p = 0.085). accordingly, the milk produced from cows fed with grass silage would be classified organic by means of the δ13C values (Table 3). These differences between δ13C value and the SRR/RRR could be used for a more detailed classification by linking them with the phytanic acid concentration in the milk fat (Table 3). The feeding of hay resulted in a low phytanic acid content in

the milk fat, and only linking the phytanic acid content with the SRR/RRR (~1) could verify that these samples (i.e., more favorable winter diet in organic farming) were produced according to organic requirements (Table 3) [20]. The combined evaluation of δ13C value, SRR/RRR, and phytanic acid content made it possible to differenti-ate between the feeding of pasture (desired organic feed in summer), hay (desired organic feed in winter), and grass silage (less favorable feed in organic low input farming) in the feed of the cows (Table 3). note, however, that these markers (δ13C value, SRR/RRR, and phytanic acid concen-tration in milk fat) are only suited to verify the authenticity of the feed used on farms and not of particular demands on housing conditions in organic farming (e.g., offering more space to individual animals; keeping individual ani-mals in groups instead of being isolated; giving free access to outdoor pasture whenever possible). These essential characteristics of organic husbandry can only be controlled onsite. Hence, the thorough authentication of organic milk must link the chemical analysis of the milk by the markers (verification of the milk itself including the handling in the filling plants) with a suited control system of the farms for husbandry practices.

δ13C values (‰) and SRR/RRR phytanic acid diastereomer ratios (SRR/RRR) of selected organic and conventional cheese samples

Previous data of SRR/RRR in cheeses were equivocal because three of ten conventional cheeses bought retail shared the characteristics of organic cheeses [18]. a total of 24 samples from this previous study—including the three equivocal conventional cheese samples—were examined by means of Ea-IRMS in this study. The milk fat of the 14 organic cheeses was highly depleted in 13C (mean δ13C value −30.0 ‰ ± 1.12, Table 2) according to Molkentin [38], and eight of the SRR/RRR were also in the expected range of organic cheese (Fig. 3, bottom right). However, the SRR/RRR in six samples was higher which indicated higher proportions of silage in the feed (Table 3). By con-trast, six of the ten conventional cheese samples featured in the corresponding levels of both markers (Fig. 3, top left, Table 2). Three further conventional cheeses were very light in carbon (δ13C value −28.9 ‰ ± 0.9), and also, the SRR/RRR (~1.0) was characteristic for organic milk fat (Fig. 3, bottom right).

However, one conventional cheese (cow milk feta) and six organic cheese (Mozzarella, Emmental, two but-ter cheeses, Romadur, and sliced cheese) samples showed inconsistent δ13C values and the SRR/RRR. These sam-ples were light in carbon (i.e., typical of organic milk), but the SRR/RRR was high (>1.5, i.e., typical of conven-tional milk). This was most likely due to a high portion of

825Eur Food Res Technol (2014) 238:819–827

1 3

grass silage in the fodder of the cows, because the feeding of grass silage was found to generate such high SRR/RRR in milk fat comparable to conventional feed cows [20]. By contrast, the δ13C value and also high phytanic acid con-tent in these samples indicated the presence of organic milk

(phytanic acid concentration >0.2 g/100 g milk fat [17]). Since grass silage is considered less favorable in organic farming, the combined evaluation of the δ13C value or phy-tanic acid content with the SRR/RRR provided additional information on the feed supplied to cows [24].

Table 2 Milk fat δ13C values (mean values) and SRR/RRR phytanic acid diastereomer ratio (SRR/RRR) of four different phases (ph1–ph5) during a feeding experiment* with three cows (top), of two

additional cows one from a organic farm (cow 4, study 3) and one conventional dairy cow (cow 5) (middle), and of organic and conven-tional cheese samples (bottom)

* Phases 1 and 4: conventional feed (containing C4-plants), phase 2: The proportion of hay gradually increased to 100 %, phase 3: 100 % hay diet (C3-plants). Cow 3: additional phase 5 with 99 % grass silage/1 % mineral feed** Significance (p value) of the results was determined by anOVa evaluation (α = 0.05)

Sample origin Samples (n)

δ13C mean value [‰]

SD p* Significance** SRR/RRR SD p* Significance*

Phase 1

Cow 1 2 −25.1 0.0045 2.1 0.05

Cow 2 3 −24.5 0.021 3.3 0.49

Cow 3 2 −23.8 0.064 1.6 0.00

all cows 7 −24.5 0.32 5.8 × 10−8 (ph1 vs. ph3)

<0.0001 2.5 0.83 0.0002 (ph1 vs. ph3)

<0.001

Phase 2

Cow 1 2 −26.1 to −26.8

– – – 3.3 – –

Cow 2 2 −25.5 to −26.9

– – – 3.2 – –

Cow 3 3 −26.2 to −26.8

– – – 1.5–1.7 – –

all cows 7 −25.9 to −26.8

– – –

Phase 3

Cow 1 2 −28.8 0.016 1.1 0.26

Cow 2 4 −28.7 0.42 1.1 0.52

Cow 3 3 −28.1 0.890 1.1 0.23

all cows 9 −28.5 0.48 3.1 × 10−9 (ph3 vs. ph4)

<0.0001 1.1 0.36 6.1 × 10−5 (ph3 vs. ph4)

<0.0001

Phase 4

Cow 1 2 −24.8 0.27 3.0 1.24

Cow 2 4 −25.2 0.23 4.2 1.26

Cow 3 2 −24.8 0.065 2.2 0.76

all cows 8 −25.0 0.20 0.078 (ph4 vs. ph1) >0.05 3.2 1.37 0.20 (ph4 vs. ph1) >0.05

Phase 5

Cow 3 2 −29.9 0.0015 0.0015 (ph5 vs. ph1) >0.001 2.4 0.29 0.015 (ph5 vs. ph1)

≥0.01

0.085 (ph5 vs. ph3) >0.05 0.0020 (ph5 vs. ph3)

>0.001

“organic farm”

Cow 4 5 −31.07 0.18 7.2 × 10−12 (org.-conv.)

<0.0001 0.87 0.06 9.3 × 10−7 (org.-conv.)

<0.0001

“Conventional farm”

Cow 5 5 −24.74 0.15 5.65 0.80

Cheese

Organic 14 −30.0 1.12 3.2 × 10−5 (org.-conv.)

<0.0001 1.55 1.12 0.02 (org.-conv.) >0.01

Conventional 10 −26.1 2.45 2.75 2.33

826 Eur Food Res Technol (2014) 238:819–827

1 3

Recently, Baars et al. [20] analyzed milk samples from four farming practices (i.e., low and high input in both con-ventional and organic milk) and found impacts of both the season and the degree of intensification on the phytanic acid content and the SRR/RRR of milk fat. These factors (season and degree of intensification) influencing the δ13C values were most likely the reason for the ambiguity in the assignment of the three conventional cheeses. Hard cheeses like mountain cheese are traditionally made from grass- and hay-fed cows, whereas semi-hard cheeses such as gouda, Edam, and butter cheese are also frequently made from milk obtained from cows fed with silages from grass plus maize. as mentioned before, fodder rich in grass silage is common practice in high input farming systems but considered less favorable in organic low input farming systems.

Compound-specific δ13C values (‰) of phytanic acid methyl ester

Finally, we studied the δ13C value of phytanic acid from an organic and a conventional butter sample by

compound-specific isotope ratio analysis (gC-IRMS). Phy-tanic acid is exclusively formed in the rumen of cows from phytol by microorganisms [39]. In view of the different SRR/RRR in organic and conventional milk in dependence of the feed, we were interested to determine whether this was also reflected in the δ13C value. Since compound-spe-cific isotope ratio analysis by gC-IRMS requires a base-line separation of the analyte from other compounds in the sample, a direct analysis of the fatty acids obtained from milk fat by transesterification was impossible. Therefore, phytanic acid was enriched from one organic and one con-ventional butter sample by urea complexation and HSCCC. The δ13C values were corrected for the impact of the methyl group according to Eq. 2 (Materials and methods). While both butter fat samples differed in the SRR/RRR, they were both extraordinarily depleted in 13C (δ13C value ~ −35.9 ‰). By contrast, stearic acid methyl ester (only available from conventional butter) showed a δ13C value of −27.4 ‰ which was similar to milk fat (as deter-mined by Ea-IRMS). The very strong depletion of 13C of phytanic acid (methyl ester) from both butter samples is a clear indication for isotope fractionation during its conver-sion in the rumen. In agreement with that, depletion in 13C compared to straight chain homologues was also observed for other microbial fatty acids (i.e., methyl-branched fatty acids) [34]. However, compound-specific isotope ratio analysis of phytanic acid methyl ester was found to be unsuited as marker for the authentication of organic milk fat because the low 13C content of phytanic acid was found in both samples.

Conclusions

an alternative sample preparation method for Ea-IRMS determinations of δ13C values was successfully applied to the analysis of milk fat from milk and cheese. The

Table 3 Classification of milk samples according to the target values of the three markers δ13C value, SRR/RRR phytanic acid diastere-omer ratio (SRR/RRR), and phytanic acid concentration in depend-

ence of the type of green fodder in the cow′s ration (upper part) and classification of the samples by the combination of two markers (lower part)

a δ13C values: conventional > −26.6 ‰ > organicb SRR/RRR: conventional > 1.5 > organicc phytanic acid concentration: conventional < 0.2 g/100 g < organic

Parameter Pasture Hay grass silage

δ13C valuea Organic (rich in C3-plants) Organic (rich in C3-plants) Organic (rich in C3-plants)

SRR/RRRb Organic Organic Conventional (no pasture or hay)

phytanic acid conc.c Organic Conventional Organic

phytanic acid conc. and SRR/RRR Rich in pasture Rich in hay Rich in grass silage

phytanic acid conc. and δ13C Rich in pasture or grass silage Rich in hay Rich in grass silage or pasture

SRR/RRR and δ13C Rich in pasture or hay Rich in pasture or hay Rich in grass silage

0.5

1.5

2.5

3.5

4.5

5.5

-31-30-29-28-27-26-25-24-23

SR

R/R

RR

δ13 C value [‰]

conventional cheeseorganic cheese "organic“

"conventional“

Fig. 3 Two-dimensional plot of the SRR/RRR phytanic acid dia-stereomer ratio (SRR/RRR) against the δ13C values (‰) of 14 organic and 10 conventional cheese samples

827Eur Food Res Technol (2014) 238:819–827

1 3

combination of δ13C value, the SRR/RRR, and phytanic acid concentration provided more detailed insights into the type of feed supplied to the cows.

Acknowledgments We are grateful to Raoul von Schmettow and Rainer Funk at the research farm of the University of Hohenheim (agricultural Experiment Station of the University of Hohenheim, location Meiereihof, Stuttgart, germany) for design and performance of the feeding study with the cows and Wolfgang armbruster for the technical support during the IRMS measurements.

Conflict of interest The authors declare that they have no conflict of interest.

Compliance with Ethics Requirements This article does not con-tain any studies with human or animal subjects.

References

1. Bund Ökologischer lebensmittelwirtschaft (2011) http://www.boelw.de/uploads/media/pdf/Dokumentation/Zahlen__Daten__Fakten/ZDF2011.pdf, accessed 9 May 2012

2. Kahl J, Baars T, Bügel S, Busscher n, Huber M, Kusche D, Rembiałkowska E, Schmid O, Seidel K, Taupier-letage B, Velimirov a, Załecka a (2012) J Sci Food agric 92:2760–2765

3. Tres a, van Ruth SM (2011) J agric Food Chem 59:8816–8821 4. Capuano E, Boerrigter-Eenling R, van der Veer g, van Ruth SM

(2013) J Sci Food agric 93:12–28 5. Molkentin J (2009) J agric Food Chem 57:785–790 6. nauta WJ, Baars T, Saatkamp H, Weenink D, Roep D (2009)

livest Sci 121:187–199 7. Camin F, Perini M, Colombari g, Bontempo l, Versini g (2008)

Rapid Commun Mass Spectrom 22:1690–1696 8. Bontempo l, lombardi g, Paoletti R, Ziller l, Camin F (2012)

Int Dairy J 23:99–104 9. Molkentin J, giesemann a (2007) anal Bioanal Chem

388:297–305 10. Wilson gF, Mackenzie DD, Brookes IM, lyon gl (1988) Br J

nutr 60:605–617 11. Boutton TW, Tyrrell HF, Patterson BW, Varga ga, Klein PD

(1988) J anim Sci 66:2636–2645 12. Metges C, Kempe K, Schmidt H (1990) Br J nutr 63:187–196 13. Molkentin J, giesemann a (2010) anal Bioanal Chem

398:1493–1500 14. Butler g, nielsen JH, larsen MK, Rehberger B, Stergiadis

S, Canever a, leifert C (2011) nJaS Wagening J life Sci 58:97–102

15. Slots T, Butler g, leifert C, Kristensen T, Skibsted lH, nielsen JH (2009) J Dairy Sci 92:2057–2066

16. Jahreis g, Fritsche J, Steinhart H (1996) lipid Fett 98:356–359 17. Vetter W, Schröder M (2010) Food Chem 119:746–752 18. Schröder M, Vetter W (2011) J am Oil Chem Soc 88:341–349 19. Ellis Ka, Innocent g, grove-White D, Cripps P, Mclean Wg,

Howard CV, Mihm M (2006) J Dairy Sci 89:1938–1950 20. Baars T, Schröder M, Kusche D, Vetter W (2012) Org agric

2:13–21 21. Che Bn, Kristensen T, nebel C, Dalsgaard TK, Hellgren lI,

Young JF, larsen MK (2013) J agric Food Chem 61:225–230 22. Collomb M, Bisig W, Bütikofer U, Sieber R, Bregy M, Etter l

(2008) Int Dairy J 18:976–982 23. van Den Brink DM, van Miert JnI, Dacremont g, Rontani J-F,

Wanders RJa (2005) J Biol Chem 280:26838–26844 24. Schröder M, lutz nl, Tangwan EC, Hajazimi E, Vetter W (2012)

Eur Food Res Technol 234:955–962 25. Schröder M, Yousefi F, Vetter W (2011) Eur Food Res Technol

232:167–174 26. Weichbrodt M, Vetter W, luckas B (2000) J aOaC Int

83:1334–1343 27. Thurnhofer S, lehnert K, Vetter W (2008) Eur Food Res Technol

226:975–983 28. Vetter W, armbruster W, Betson TR, Schleucher J, Kapp T, leh-

nert K (2006) anal Chim acta 577:250–256 29. Coplen TB, Brand Wa, gehre M, gröning M, Meijer HaJ,

Toman B, Verkouteren RM (2006) anal Chem 78:2439–2441 30. Fiebig H-J (2011) Deutsche gesellschaft für Fettwissenschaft:

Deutsche Einheitsmethoden zur Untersuchung von Fetten, Fett-produkten. Tensiden und verwandten Stoffen, Wissenschaftliche Verlagsgesellschaft, Stuttgart

31. Jalali-Heravi M, Vosough M (2004) J Chromatogr a 1024:165–176

32. Schröder M, Vetter W (2013) J am Oil Chem Soc 90:771–790 33. li D, Schröder M, Vetter W (2012) Chromatographia 75:1–6 34. Vetter W, gaul S, Thurnhofer S, Mayer K (2007) anal Bioanal

Chem 389:597–604 35. Pulchan J, abrajano Ta, Helleur R (1997) J anal appl Pyrol

42:135–150 36. naraoka H, Yamada K, Ishiwatari R (1994) J Mass Spectrom Soc

Jpn 42:315–323 37. aramendía Ma, Marinas a, Marinas JM, Moreno JM, Moalem

M, Rallo l, Urbano FJ (2007) Rapid Commun Mass Spectrom 21:487–496

38. Molkentin J (2013) Food Chem 137:25–30 39. Hansen RP (1966) nature 210:841