ARTICLE IN PRESS
JOURNAL OF
FOOD COMPOSITION
AND ANALYSIS
Journal of Food Composition and Analysis 19 (2006) 843–849
www.elsevier.com/locate/jfca
Short Communication
Characterization of buffalo milk by 31P-nuclear magnetic
resonance spectroscopy
Giuseppina Andreotti, Enrico Trivellone, Andrea Motta
Istituto di Chimica Biomolecolare del CNR, Comprensorio Olivetti, Edificio 70, Via Campi Flegrei 34, I-80078 Pozzuoli, Napoli, Italy
Received 21 November 2005; received in revised form 1 March 2006; accepted 5 March 2006
Abstract
We report a 31P nuclear magnetic resonance (31P-NMR) investigation of buffalo milk, milk ultrafiltrate, and phospholipids from milk
fat; for comparison corresponding data were also acquired for cow milk samples. In buffalo milk samples, we identified
glycerophosphorylcoline, glycerophosphorylethanolamine and inorganic orthophosphate resonances, together with a broad peak
assigned to serylphosphate residues of casein. Buffalo milk ultrafiltrate showed the presence of several phosporous signals, and ten of
them (inorganic phosphate, phosphocreatine, glycerophosphorylcoline, glycerophosphorylethanolamine, N-acetylglucosamine-1phosphate, glucose-6-phosphate, galactose-1-phosphate, phosphorylethanolamine, phosphorylcoline, and glycerol-1-phosphate) were
unambiguously identified by addition of pure standards. In phospholipids fractions from buffalo milk, we clearly identified
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and phosphatidylinositol. A preliminary estimation (mol%) of each phosphorylated compound was obtained. By comparing 31P-NMR data from milk samples from buffaloes and
cows, we concluded that these milks are rather similar as far as the phosporous distribution in small molecules is concerned.
r 2006 Elsevier Inc. All rights reserved.
Keywords: Buffalo milk; NMR spectroscopy; Phosporous NMR
1. Introduction
The composition of buffalo milk is still not as well
characterized as the bovine counterpart. The phosporous
in bovine milk is 0.9–1.0 g/kg of milk (Walstra and Jenness,
1984), and it is mostly present as inorganic orthophosphate
occurring as Ca-phosphate salt in casein micelles or in
solutions. The remaining phosporous is present as phosphate monoesters (phosphoserine residues of the caseins
and sugar phosphate), and phosphodiester (phospholipids); a small amount is also present in nucleotides.
To the best of our knowledge, the only information
regarding phosporous content in buffalo milk was reported
by Spanghero and Susmel (1996), who indicated that,
compared with cow milk, the phosporous content in
buffalo milk is 30% higher.
We report here an investigation of buffalo milk, milk
ultrafiltrate, and phospholipids by 31P-NMR spectroscopy
Corresponding author. Tel.: +39 081 8675241; fax: +39 081 8041770.
E-mail address: [email protected] (G. Andreotti).
0889-1575/$ - see front matter r 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2006.03.014
aiming at an identification and a preliminary estimation
(mol%) of phosphorylated compounds. The results are
compared with corresponding data from newly acquired
bovine milk samples.
In recent years, NMR spectroscopy has been widely used
for studying biological systems and is a technique of
increasing use in food science (Belloque and Ramos, 1999;
Belton et al., 1993; Gil et al., 1996). This is a non-invasive
technique that preserves the sample structure and extracts
useful information from chemically complex and highly
heterogeneous systems.
We have previously investigated triacylglycerols from cow
and buffalo milks by 13C-NMR spectroscopy, and showed
that, notwithstanding its limited sensitivity, NMR spectroscopy can be safely used to quantitate milk fatty acids,
providing reliable data as those obtained by gas chromatography (Andreotti et al., 2000). We have also described the
possibility to discriminate ovine, caprine and bovine milks
by using 13C-NMR parameters (Andreotti et al., 2002).
31
P high-resolution NMR spectroscopy is very interesting to study milk, although only resonances originating
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G. Andreotti et al. / Journal of Food Composition and Analysis 19 (2006) 843–849
from sufficiently mobile 31P atoms can be observed. This
means that the inorganic orthophosphate held as calcium
phosphate in casein micelles is not detectable, and the
phosphoseryl residues in macromolecular aggregates such
as casein micelles may appear as a broad signal, so broad
that it may become non-detectable.
The identification of phosphorylated compounds in
buffalo milk is interesting not only for a better characterization of this biological fluid from the nutritional point of
view, but it may also provide useful information about
molecules, such as phospholipids, very important in
medical, biological, biotechnological and industrial fields.
It is well documented the use of phospholipids 31P-NMR
analysis as a profiling tool for the determination and
characterization of tissues (Schiller and Arnold, 2002). In
addition there are commercial interests for phospholipids
because of their emulsifying properties widely used for
pharmaceutical and cosmetic applications (Gunstone,
2004).
2. Materials and methods
2.1. Milk manipulation and samples
Buffalo and cow raw milks (two individual samples of
each) were obtained from local dairy farms, stored at 4 1C
and analyzed without further manipulation within 24 h
after milking. All milks manipulation reported hereafter
were started within 24 h after milking.
The milk ultrafiltrate was obtained from milk serum
prepared by two successive centrifugations: the first, at
3800g and 5 1C for 30 min, yielded the skim milk; this was
then centrifuged at 45 000g and 10 1C for 45 min in order to
remove casein micelles. The milk serum was ultrafiltrated
on an Amicon PM-10 membrane (cut-off 10 kDa). The
ultrafiltrate was then lyophilized and dissolved in H2O to a
final volume 10 times lower than the original.
The pH of the tenfold ultrafiltrate samples was adjusted
to 9.4 by addition of 1 M NaOH or 1 M HCl, and the
precipitate formed during the pH adjustment was removed
by centrifugation (at 10 000g and 5 1C for 5 min).
Phospholipids preparation was based on Murgia et al.
(2003). Since the milk cream is essentially constituted of
triacylglycerols (about 98 wt%), it is necessary to enrich the
samples in the phospholipid fraction (about 1 wt%) by
treatment with acetone in which only apolar lipids dissolve.
Typically, 5 g of milk cream was extracted twice with
200 mL of acetone in order to eliminate most of the
triacylglycerols, and the insoluble residue was collected.
The residue was then dispersed in 60 mL chloroform/
methanol 2:1 (v/v) and filtered. After filtration, the solution
was washed with the same volume of a 0.01 M Na4-EDTA0.1 M NaCl solution, to avoid the presence of metallic
divalent cations. These cations interact with anionic
phosphates forming coordination complexes that alter the
chemical shielding of the constituent phosporous nucleus
(Costello et al., 1976). The organic phase was recovered,
dried with anhydrous Na2SO4, filtered, and then evaporated at 35 1C in a rotary evaporator. One-tenth of dried
samples were dissolved in 0.5 mL triethylamine/dimethylformamide/guanidinium hydrochloride (15, 50 mL, and 5 g,
respectively) in order to acquire 31P-NMR spectra.
2.2. High resolution
31
P-NMR
High-resolution 31P-NMR spectra, acquired at the
NMR Service of Istituto di Chimica Biomolecolare del
CNR (Pozzuoli, Italy), were obtained at 27 1C on a Bruker
Avance-400 operating at 161.97 MHz, using an inverse
probe fitted with a gradient along the Z-axis. The 1Hdecoupled, one-dimensional 31P spectra were obtained
using the following conditions: spectral width 200 ppm,
delay time 7 s, pulse width of 8.0 ms (601 spin-flip angle),
number of scans 3000, number of data points 32 K. The
inverse-gated decoupling technique was used to avoid the
nuclear Overhauser effect on the signals.
Row milk and ultrafiltrate samples contained 10% 2H2O
for internal lock, and signals were referred to internal
phosphocreatine ( 2.45 ppm at pH 6.8 for milk;
2.32 ppm at pH 9.4 for ultrafiltrate). Phospholipids
samples contained 10% dimetylformamide-d7 for internal
lock, and internal phosphatidylcholine (0 ppm) as reference. Sphingomyelin from chicken egg yolk, rac-1,2dipalmitoyl-glycero-3-phosphoethanolamine (phosphatidylethanolamine), 1,2-dipalmitoyl-sn-glycero-3-phosphocoline (phosphatidylcholine), 3-sn-phosphstidyl-L-serine
from bovine brain (phosphatidylserine), and phosphatidyl
inositol sodium salt from soybean (from Sigma-Aldrich)
were used as standards for phospholipids assignments.
Phosphocreatine, glucose 6-phosphate, fructose-6-phosphate, phosphorylethanolamine, were from Fluka. Glycerophosphorylcholine, phosphorylcholine, lactose-1-phosphate,
N-acetylglucosamine-1-phosphate, galactose-1-phosphate, glycerol-1-phosphate were from Sigma-Aldrich.
2.3. Preliminary quantitative spectral analysis
Fourier-transformed spectra were phased and then
baseline corrected by spline interpolation of 14 baseline
selected points. The spectral signals were fit to a sum of
Lorentzian curves by a non-linear least-squares algorithm.
The calculated area of each NMR signal was used to give a
preliminary estimation (mol%) of each compound previously identified by addition of standards. For each
compound the average of two samples will be reported
throughout the following paragraphs.
Data analysis was performed with the software MacFID
1D 5.3 (Tecmag Inc., Houston, TX, USA).
3. Results and discussion
In order to obtain comparable data sets, NMR experiments of cow milk samples, although previously reported
(Belton et al., 1985; Wahlgren et al., 1986; Belton and
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845
Lyster, 1991; Murgia et al., 2003), were newly acquired,
and the buffaloes’ and cows’ spectra analyzed in parallel.
agreement with the fact that in these macromolecular
aggregates the 31P nuclei are of limited mobility.
3.1. Milk
3.2. Milk ultrafiltrate
The 31P-NMR spectrum of buffalo and cow milk are
reported in Fig. 1A and B, respectively. Three sharp peaks
are observed: the tallest, at 1.77 ppm, arises from the
inorganic phosphate in solution. The two upfield peaks at
1.18 and 0.63 ppm originate from glycerophosphorylethanolamine and glycerophosphorylcoline, respectively. Except for the phosphocreatine peak at 2.45 ppm (not
shown), used as internal reference, no resonances were seen
at negative frequencies. In agreement with reported data
(Belton et al., 1985; Wahlgren et al., 1986), we also
identified glycerophosphorylcoline and glycerophosphorylethanolamine in bovine milk samples. Such a common
signal distribution suggests that pyrophosphate and its
esters, as well as triphosphates, are either absent in buffalo
milk, or present in free solution at a concentration too low
to be observed. It has been suggested that both glycerophosphorylethanolamine and phosphocreatine originate from
secretory cell leakage (Wahlgren et al., 1986).
The resonances of phosphoproteins present in casein
micelles appear as a broad signal between 2.5 and 4 ppm,
downfield to the inorganic phosphate peak. This is in
In order to identify other soluble compounds present in
buffalo milk at low concentration, we prepared a tenfold
concentrated milk ultrafiltrate at pH 9.4. Milk ultrafiltrate
was chosen to avoid the presence of phosphoproteins.
In addition to inorganic phosphate, phosphocreatine,
glycerophosphorylcoline, and glycerophosphorylethanolamine, detected in the whole milk spectrum of Fig. 1A,
we identified few other components in the buffalo
ultrafiltrate (Fig. 2A). Identification was achieved by
selective addition of standards, in agreement with previous
assignments performed on cow samples (Wahlgren et al.,
1986). Phosphocreatine ( 2.32 ppm, peak 1), glycerophosphorylcoline (0.62 ppm, peak 2), and glycerophosphorylethanolamine (1.26 ppm, peak 3) showed chemical shift
values slightly different from those measured in the milk
because of the pH difference (6.8 for milk and 9.4 for
ultrafiltrate). We further identified N-acetylglucosamine-1phosphate at 2.83 ppm (peak 4), glucose-6-phosphate at
5.21 ppm (peak 10), and galactose-1-phosphate at 3.25 ppm
(peak 5). Selective addition of lactose-1-phosphate and
glucose-1-phosphate (both expected at 3.13 ppm, as observed in cow samples showed in Fig. 2B and indicated by
an arrow) did not affect any of the signals in the buffalo
milk ultrafiltrate spectra, suggesting that none of the
signals observed were representative of these compounds
which may be absent or at a non-detectable level in
buffalo samples. Phosphorylethanolamine, fructose-6phosphate and ribose-5-phosphate all showed a chemical
shift of 4.63 ppm; however, since phosphorylethanolamine
is the most abundant species among the three compounds
in cow milk (Walstra and Jenness, 1984), we assigned
this resonance in buffalo sample to phosphorylethanolamine (peak 8). No resonances were seen at negative
frequencies in the spectra, except for the phosphocreatine
peak at 2.32 ppm (peak 1), confirming that triphosphates such as ATP may be absent or at a non-detectable
level.
Together with glycerophosphorylcoline and glycerophosphorylethanolamine, other resonances of phospholipid
precursors were detected: phosphorylcoline at 4.11 ppm
(peak 7), and glycerol-1-phosphate at 5.09 ppm (peak 9).
Unidentified resonances between 4.7 and 4.8 ppm are
assigned to casein peptides still remaining after sample
manipulation.
A preliminary estimation of each compound was
performed (Table 1). All of the peaks between 3 and
6 ppm were simulated, and the calculated area of each
identified compound was used to determine the relative
concentration. The total area was used as normalization
parameter. The data refer to the average values obtained
from two different samples for each type of milk. Although
a larger data set is required for a more accurate estimation,
Fig. 1. 31P-NMR spectra of buffalo (A) and cow (B) milks. The spectra
were referenced to phosphocreatine at 2.45 ppm. Sharp signals were
assigned as follows: inorganic phosphate (1.77 ppm), glycerophosphorylethanolamine (1.18 ppm), and glycerophosphorylcoline (0.63 ppm).
Phosphoproteins in casein micelles appeared downfield with respect to
the inorganic phosphate as a broad signal between 2.5 and 4.0 ppm.
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Fig. 2. 31P-NMR spectra of a tenfold concentrated buffalo (A) and cow (B) milk ultrafiltrates at pH 9.4. The assignments were as follow: phosphocreatine
(used as an internal reference), 2.32 ppm (peak 1); glycerophosphorylcholine, 0.62 ppm (peak 2); glycerophosphorylethanolamine, 1.26 ppm (peak 3); Nacetylglucosamine-1-phosphate, 2.83 ppm (peak 4); galactose-1-phosphate, 3.25 ppm (peak 5); inorganic phosphate, 3.44 ppm (peak 6); phosphorylcholine,
4.11 ppm (peak 7); phosphorylethanolamine, 4.63 ppm (peak 8); glycerol-1-phosphate, 5.09 ppm (peak 9); and glucose-6-phosphate, 5.21 ppm (peak 10).
The arrow in panel B indicates lactose-1-phosphate and/or glucose-1-phosphate.
in order to take into account the natural variability, it can
be noticed that except for glycerophosphorylcholine,
inorganic phosphate and glucose-6-phosphate which
showed a similar content in both types of milk, all of the
other compounds were differently represented. In particular phosphocreatine, glycerophosphorylethanolamine,
phosphorylcholine, phosphorylethanolamine were more
abundant in buffalo than in cow milk, while N-acetylglucosamine-1-phosphate, galactose-1-phosphate, and glycerol-1-phosphate were more abundant in cows than in
buffalo sample.
3.3. Phospholipids
31
P-NMR spectroscopy is an extremely powerful tool in
lipid research for the analysis of phospholipids although
the use of strictly defined experimental parameters (extraction media and solvent/detergent system for NMR) is
crucial for obtaining reliable and reproducible results
(Schiller and Arnold, 2002).
Milk phospholipids mainly occur as constituents of the
fat globule membranes. As in all the natural samples, each
class of phospholipids is a mixture of various molecular
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Table 1
Phosphorilated compounds identified in
ultrafiltrate
Compound (peak numbera)
Phosphocreatine (1)
Glycerophosphorylcholine (2)
Glycerophosphorylethanolamine (3)
N-acetylglucosamine-1-phosphate (4)
Galactose-1-phosphate (5)
Inorganic phosphate (6)
Phosphorylcholine (7)
Phosphorylethanolamine (8)
Glycerol-1-phosphate (9)
Glucose-6-phosphate (10)
31
P-NMR spectrum of milk
(mol%)b
Buffalo
Cow
2.2
9.5
2.5
4.3
1.9
57.0
6.7
2.8
0.8
1.3
1.0
10.7
1.7
6.7
3.0
50.7
1.7
0.9
1.6
1.4
a
According to Fig. 2.
A preliminary estimation was performed as follows: all of the peaks
between 3 and 6 ppm in Fig. 2 were simulated, and the calculated area of
each identified compound was used to determine the relative concentration. The total area was used as normalization parameter. Each value is a
mean of two different samples.
b
species differing in acyl side chain composition, whose
precise molecular mass is unknown. Phosphatidylcholine,
phosphatidylethanolamine, and sphingomyelin are the
most prevalent classes of bovine milk phospholipids, while
phosphatidylserine and phosphatidylinositol are present to
a lesser extent (Jensen, 2002).
Phospholipid analysis can be performed by a number of
different analytical techniques, each with its particular
advantages and disadvantages. These are chromatographic
techniques such as HPLC and TLC, or mass spectrometry
used in combination with gas chromatography, or 1H/13C
NMR spectroscopy (see Schiller and Arnold, 2002, for an
overview).
Because 31P-NMR depends upon the detection of the
single phosporous atom present in each phospholipid
molecule, 31P-NMR spectroscopy has become a useful
technique both for qualitative and quantitative analyses of
these molecules. The application of 31P-NMR spectroscopy
for phospholipids characterization has largely been improved by the use of a biphasic chloroform/methanol/
water-EDTA solvent that made it possible to narrow and
spread the 31P signals (Menses and Glonek, 1988; Glonek
and Merchant, 1996).
We used as solvent a mixture of triethylamine/dimethylformamide/guanidinium hydrochloride (Bosco et al., 1997;
Culeddu et al., 1998), which has been reported to overcome
partition problems connected with the biphasic solvent and
slightly enlarge the range of 31P-NMR chemical shifts, thus
improving the resolution. Furthermore, a lower chemical
shift dependence on the phospholipid concentration is
observed in this solution. On the other hand, the formation
of adducts between triethylamine and some phospholipids
is observed: in particular, the interaction between one or
more molecules of triethylamine and the amine protons of
847
the polar head of phosphatidylethanolamine via hydrogen
bonds has been described.
Fig. 3A shows the 1H-decoupled 31P NMR spectrum of
phospholipids obtained from buffalo milk cream, dissolved
in the triethylamine/dimethylformamide/guanidinium hydrochloride solvent. For comparison, the cow’s spectrum is
also shown (Fig. 3B). The same class of phospholipids were
detected in buffalo and cow samples. The resonances of
phosphatidylcholine (peak 1, 0 ppm), phosphatidylserine
(peak 6, 0.47 ppm), phosphatidylethanolamine (peak 7,
0.55 ppm), sphingomyelin (peak 9, 0.85 ppm), and phosphatidylinositol (peak 10, 1.07 ppm) were clearly identified
by selective addition of standard compounds. According to
Murgia et al. (2003), the signals at 0.16 ppm (peak 2) and
0.19 ppm (peak 3) were identified as two adducts formed by
phosphatidylethanolamine; the signals at 0.58 ppm (peak 8)
and 0.22 ppm (peak 4) were assigned to phosphatidylethanolamine plasmalogens and to its adduct, respectively;
finally the signal at 0.40 ppm (peak 5) was assigned to
monomethylphosphatidylethanolamine.
Phosphatidic acid gave a peak at 5.25 ppm (not shown),
and its intensity increases with the time suggesting that it
originates from modification of other phospholipids.
Phosphatidyl glycerol (expected at 1.36 ppm) was also
Fig. 3. 1H-decoupled 31P-NMR spectra of a phospholipids mixture
obtained from buffalo (A) and cow (B) milk creams. Phospholipids were
dissolved in the monophasic triethylamine/dimethylformamide/guanidinium hydrochloride solution mixture. The signals were assigned as
follows: phosphatidylcholine (used as an internal reference), 0 ppm
(peak 1); phosphatidylethanolamine adducts, 0.16 ppm (peak 2) and
0.19 ppm (peak 3); phosphatidylethanolamine plasmalogens adduct,
0.22 ppm (peak 4); monomethylphosphatidylethanolamine, 0.40 ppm
(peak 5); phosphatidylserine, 0.47 ppm (peak 6); phosphatidylethanolamine, 0.55 ppm (peak 7); phosphatidylethanolamine plasmalogens,
0.58 ppm (peak 8); sphingomyelin, 0.85 ppm (peak 9); and phosphatidylinositol 1.07 ppm (peak 10).
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Table 2
Phosphorilated compounds identified in
phospholipids
Compound (peak numbera)
Phosphatidylcholine (1)
Phosphatidylethanolamine (7+2+3)
Phosphatidylserine (6)
Sphingomyelin (9)
Phosphatidylinositol (10)
31
P-NMR spectrum of milk
(mol%)
Buffalob
Cowb (c)
21.6
21.8
5.9
22.1
17.5
24.0
23.5
3.6
24.2
12.0
(26.8)
(25.8)
(1.5)
(26.8)
(14.0)
a
According to Fig. 3.
A preliminary estimation was performed as follows: all of the
resonances in Fig. 3 were simulated, and the calculated area of each
identified compound was used to determine the relative concentration. The
total area was used as normalization parameter. Each value is a mean of
two different samples.
c
Data from Murgia et al. (2003).
b
added but none of the signals was affected suggesting that
this compound may be absent or at a non-detectable level
in both buffaloes’ and cow’s phospholipid samples.
As mentioned above, phospholipids in natural samples
occur as a mixture of various molecular species differing in
acyl chain composition, thus each 31P signal contains the
contribution of different molecular formulas and only the
molar percentage of each phospholipid class can be
evaluated. All of the resonances were simulated and the
area of each peak used for a preliminary quantitative
analysis (mol%) of the phospholipid content of buffalo
sample (Table 2). The data refer to the average values
obtained from two different samples for each type of milk.
Cow data from Murgia et al. (2003), are also reported for
comparison.
Although a larger data set is required for a more
accurate estimation of milk composition, we can remark
that similarly to cow milk, the most abundant class of
phospholipids in buffalo milk are sphingomyelin, phosphatidylethanolamine and phosphatidylcholine.
In addition to the natural variability of milk samples, an
accurate analysis should take into account that the
phospholipid content can be affected by many factors such
as the age, breed, diet, and stage of lactation of the animal.
4. Conclusions
NMR spectroscopy is a technique of increasing use for
diary research. It does not require an extensive chemical
manipulation of samples, and can easily highlight differences and/or similarities between samples as complex as
milk and its fractions. Herein, we analyzed buffalo milk by
31
P-NMR spectroscopy to investigate the phosporous
distribution in different compounds, and compared the
results with those from cow milk. Although larger data sets
are required to address the natural variability of milk
composition and the influence of diet, age, etc., it can be
safely concluded that both buffalo and cow milk contain
the same class of phosphorylated compounds. A preliminary quantification of these compounds highlighted similarities and/or differences between buffalo and cow milks.
Our investigation aimed to give a contribution to the
characterization of buffalo milk since it is used for the
production of ‘‘Mozzarella di bufala campana’’, a typical
AOP cheese produced in some of the regions of southern
Italy, and appreciated worldwide. In addition, this analysis
is useful to provide information about phospholipids from
milk because of an increasing interest in recovering new
kind of biomass as new sources of biosurfactants for
commercial utilization.
Acknowledgments
The authors wish to thank Dominique Melck and
Edoardo Pagnotta (ICB, Pozzuoli) for technical assistance.
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