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Study of the protein-bound fraction of calcium, iron, magnesium and zinc in bovine milk
Spectrochimica Acta Part B 56 Ž2001. 1909᎐1916
Study of the protein-bound fraction of calcium, iron, magnesium and zinc in bovine milk 夽 a , Fernando V. Silvaa , Gisele S. Lopes a , Joaquim A. Nobrega ´ b b,U Gilberto B. Souza , Ana Rita A. Nogueira a
Departamento de Quımica, Uni¨ ersidade Federal de Sao ´ ˜ Carlos, Sao ˜ Carlos SP, Brazil b Embrapa Pecuaria ´ Sudeste, P.O. Box 339, 13560-970 Sao ˜ Carlos SP, Brazil Received 6 December 2000; accepted 30 May 2001
Abstract Two approaches were used to study the interaction of Ca, Fe, Mg and Zn with bovine milk proteins by inductively coupled plasma optical emission spectrometry ŽICPOES.. Selective separations in bovine milk samples were accomplished employing an acid protein precipitation using 100 g ly1 trichloroacetic acid ŽTCA., and an enzymatic protein hydrolysis using 50 g ly1 pepsin ŽPEP. solution, respectively. The results were compared with total mineral contents determined after microwave-assisted acid digestion. The results obtained by enzymatic and acid precipitation evidenced the different interaction forms of Ca, Fe, Mg and Zn in the system formed by milk components. Iron was not solubilized by the TCA treatment, but was recovered completely after the enzymatic treatment. Quantitative recoveries of Ca, Mg and Zn were obtained using both approaches, showing that these analytes were bound to milk compounds affected by either treatment. Calcium, Mg and Zn are mainly associated with colloidal calcium phosphate and Fe is bound to the backbone of the casein polypeptide chain, cleaved by pepsin enzyme. The proposed approaches could be used to assess the complexity of these chemical interactions. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Milk; Casein; Trichloroacetic acid; Pepsin; ICPOES
夽
This paper was presented at the 6th Rio Symposium on Atomic Spectrometry, held in Concepcion ´ and Pucon, ´ Chile, December 2000, and is published in the Special Issue of Spectrochimica Acta Part B, dedicated to that conference. U Corresponding author. Tel.: q55-16-261-5611; fax: q55-16-261-5754. E-mail address: [email protected] ŽA.R. Nogueira.. 0584-8547r01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 8 4 - 8 5 4 7 Ž 0 1 . 0 0 3 1 3 - 5
1910
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916
1. Introduction Milk is a complex fluid that contains all nutrients required for growth and development of newborns. All types of milk are composed of specific proteins, fats designed to be easily digested, minerals, vitamins and other components that may have an important role w1x. Milk compartmentalization influences the mineral absorption process by the gastrointestinal tract. Various approaches using appropriate digestion or solubilization methods have been proposed to determine the total content of inorganic species in milk samples w2᎐6x. However, to increase knowledge about the physiological activity of milk minerals, determination of the total content is not sufficient w7᎐9x. The establishment of distinct chemical interactions among milk components has also to be appraised w1x. Monovalent ions, such as Naq, Kq and Cly, are present in milk mainly in the ionized form. Other ionic species, such as Ca2q, Mg 2q, Zn2q, Fe 3q, carbonate, citrate and phosphate ions, are distributed among the structural compartments of milk in a highly specific way, generating a complex chemical equilibrium w1x. The study of ionic distribution in the structural compartments of milk is normally accomplished employing methodologies based on physical separation processes, such as ultra-centrifugation or dialysis using suitable chelating agents w1,10,11x. The use of solidphase extraction for speciation studies in milk samples has also been reported in the literature w12x. By using these procedures different milk fractions can be isolated and their mineral content determined using proper analytical techniques. Recently, an enzymatic approach has extracted minor and trace elements from biological materials w13x, which allows the selective extraction of the elements associated with proteins that are hydrolyzed by proteolytic enzymes. The major advantage of enzymatic hydrolysis as an alternative sample treatment approach is its selectivity w13x. The major class of protein in most milk species is casein. The casein in bovine milk occurs as a colloidal complex of proteins and salts, mainly calcium salts. When the colloidal Ca is removed,
the submicelles ␣-s1, ␣-s2,  and -casein are produced, which are phosphorylated to different degrees. The aggregates of the casein protein sub-units, calcium phosphate and some other ionic constituents in the form of micelles, give milk its white appearance. These casein micelles are susceptible to the action of most proteolytic enzymes Žproteases. w14x. The casein peptide bonds can be cleaved by proteolytic enzyme action, producing soluble peptide chains or soluble amino acids, depending on the degree of hydrolysis. Solutions with low pH values, under mild conditions, can also produce changes in quaternary, tertiary and secondary casein structures. The covalent bonds of the backbone of the polypeptide chain are not cleaved. The most visible consequence of this effect is observed in the decrease of the casein solubility w15x. Considering these aspects, the use of enzymatic hydrolysis and acid protein precipitation reactions were chosen to investigate the association of Ca, Fe, Mg and Zn with bovine milk proteins. The proteolytic enzyme employed was pepsin, the major protease present in the stomach. This enzyme preferentially breaks peptide bonds involving residues of aromatic amino acids as well as methionine and leucine, to yield peptides and a few free amino acids w15x. Among the enzymes, pepsin has a distinctive behavior due to its low isoelectric point and low optimum pH value. Trichloroacetic acid ŽTCA., on the other hand, presents no specificity for cleaving protein bonds. In solution, the trichloroacetate anion, Cl 3 C᎐COOy, interacts with positive protein groups, R᎐NHq 3 , producing a white precipitate of denatured proteins. Taking into account the distinct effects of these reagents on bovine milk proteins, the different chemical interactions between Ca, Fe, Mg, Zn and protein compounds were studied monitoring the minerals released into solution after milk sample treatment with TCA and PEP solutions.
2. Experimental 2.1. Instrumentation A Vista AX axial view simultaneous inductively
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916 Table 1 ICPOES operational parameters Power ŽkW. Plasma gas flow rate Žl miny1 . Auxiliary gas flow rate Žl miny1 . Nebulizer gas flow rate Žl miny1 . Spray chamber type Nebulizer Sample flow rate Žml miny1 . Analytical wavelengths Žnm. Ca II Fe II Mg I Zn II Y II
1911
Table 2 Heating program employed in microwave-assisted acid digestion 1.1 15.0 1.5 0.80 Sturman-Masters V-groove 0.80 315.882 285.208 285.208 206.204 371.029
coupled plasma optical emission spectrometer ŽVarian, Australia., equipped with a CCD detector was used to determine Ca, Fe, Mg and Zn. The spectrometer provided full wavelength coverage from 167 to 785 nm. The optical system was purged with argon and the Echelle polychromator was thermostatted at 34⬚C. The cool plasma tail was removed from the optical path using an endon purge gas. The operating conditions are listed in Table 1.
Step
Time Žmin.
Power ŽW.
1 2 3 4 5 Ventilation
1.0 1.0 3.0 5.0 5.0 4.0
250 0 250 400 600 0
The acid digestion of the milk samples and the certified reference material ŽWhole milk powder, NIST 8435, National Institute of Standards and Technology, Gaithersburg, MD, USA. were performed in a microwave oven ŽETHOS 1600, Milestone, Italy. equipped with 10 closed vessels made of perfluoralkoxy Teflon 䊛 ŽPFA. with a calibrated resealing pressure relief mechanism Žmaximum operating pressure 110 atm.. The vessels were located on a rotating turntable inside the microwave oven cavity. Before use, the PFA vessels were cleaned with acid and rinsed with deionized water. The heating program used for digestion is shown in Table 2.
Fig. 1. Experimental approaches used to study chemical interactions of Ca, Fe, Mg, Zn and bovine milk proteins.
1912
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916
A sub-boiling still ŽsubPUR, Milestone, Italy. was used to further purify the concentrated nitric acid. 2.2. Solutions All solutions were prepared by using analytical grade reagents and Milli-Q 䊛 water Ž18 M⍀ cm.. Three different approaches Žacid precipitation, enzymatic hydrolysis and microwave-assisted acid digestion. were employed to investigate Ca, Fe, Mg and Zn chemical interactions with bovine milk proteins. In the protein acid precipitation and enzymatic hydrolysis procedures, 100 g ly1 TCA ŽNuclear, Brazil. and 50 g ly1 PEP ŽNuclear, Brazil. solutions were used, respectively. The PEP solution was prepared in 0.01 mol ly1 HNO3 medium. A mixture containing 2 ml of sub-boiling distilled concentrated nitric acid and 1 ml of 30% vrv H 2 O 2 ŽMallinckrodt, Mexico. was used for microwave-assisted acid digestion. The analytical curves were prepared after suitable dilution of 1000 mg ly1 stock solution ŽSpex, USA. of each element: blank: 0.14 mol ly1 HNO3 ; reference solution 1:10.0 mg ly1 of Ca and Mg q1.00 mg ly1 of Fe and Zn; and reference solution 2: 20.0 mg ly1 of Ca and Mgq 2.0 mg ly1 of Fe and Zn. The samples were diluted accordingly before measurements. Yttrium Ž1.0 mg ly1 . was added to all reference solutions and samples as an internal standard before ICPOES measurements.
from the storage process on the analytical results, the raw bovine milk sample was analyzed immediately after collection. The Fe-supplemented UHT milk sample, according to the information on the product label, contained 15 mg ly1 of added Fe in the form of an amino-chelate. The standard reference milk sample ŽNIST 8435 Whole milk powder. was not reconstituted before its microwaveassisted acid digestion.
3. Results and discussion 3.1. E¨ aluation of ICPOES experimental parameters and matrix effects
2.3. Sample treatment
The ICPOES operational parameters were established using a 1.0-mg ly1 multielement solution ŽCa, Fe, Mg and Zn. and the auto-optimization program provided with the spectrometer software, where the radio frequency power and nebulizer flow rate are systematically modified in order to obtain the highest signal-to-background ratios ŽSBR. for each selected wavelength. For most wavelengths the best SBR was obtained for a radio frequency power of 1.1 kW and a nebulizer flow rate of 0.80 l miny1 . Recovery tests in 50 g ly1 PEP and 100 g ly1 TCA solutions were carried out under these conditions. The results for this experiment are summarized in Table 3. The recovery experiments showed no significant interference of TCA and PEP for all analytes. The use of Y as internal standard compensated for potential interferences related to sam-
The three different approaches employed to evaluate the protein-bound fraction of Ca, Fe, Mg and Zn in bovine milk are summarized in Fig. 1.
Table 3 Recovery of Ca, Fe, Mg and Zn in 50 g ly1 pepsin ŽPEP. and 100 g ly1 trichloroacetic acid ŽTCA. solutions
2.4. Milk samples
Analyte
Recovery Ž%. TCA
The ultra high temperature treated ŽUHT. whole milk and Fe-supplemented UHT milk samples were purchased in a local market, while the raw bovine milk sample was collected from cows in the final stage of lactation in an intensive bovine milk production system ŽEmbrapa Pecuaria ´ Sudeste, Brazil.. In order to avoid any influence
a
Ca Fe Mg Zn a b
PEP b
1
2
1a
2b
100 93.0 97.0 99.0
101 98.0 98.0 100
90.0 90.3 91.5 91.8
89.2 92.0 92.5 90.1
Ca and Mg 5.0 mg ly1 ; Fe and Zn 0.50 mg ly1 . Ca and Mg 20 mg ly1 ; Fe and Zn 2.0 mg ly1 .
Raw
UHT 1.13 Ž"0.04. 1.14 Ž"0.02. 1.14 Ž"0.02.
Raw 0.92 Ž"0.02. 0.93 Ž"0.03. 0.91 Ž"0.05.
- 0.13 - 0.13 - 0.13
Fe Žmg ly1 .
Ca Žg ly1 .
Results are average and standard deviation of three determinations.
Microwave-assisted acid digestion TCA protein precipitation PEP protein hydrolysis
Sample treatment
- 0.13 - 0.13 - 0.13
UHT 101 Ž"4. 109 Ž"3. 104 Ž"6.
Raw
Mg Žmg ly1 .
109 Ž"6. 113 Ž"4. 115 Ž"2.
UHT
2.16 Ž"0.02. 2.30 Ž"0.07. 2.42 Ž"0.08.
Raw
Zn Žmg ly1 .
3.02 Ž"0.33. 3.10 Ž"0.04. 3.68 Ž"0.27.
UHT
Table 4 Determination of Ca, Fe, Mg and Zn in raw and UHT bovine whole milk samples after microwave-assisted acid digestion, TCA and PEP sample treatment
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916 1913
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F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916
ple introduction into the plasma. The analytical curve prepared in HNO3 medium could therefore be used without any adverse effect on the accuracy of the determinations carried out in TCA or PEP medium. 3.2. Bo¨ ine milk analysis: effects of TCA and PEP treatment and determination of Ca, Fe, Mg and Zn First the accuracy of the multielement determination was checked using a standard reference material ŽNIST 8435, Whole milk powder. digested as described in Section 2. The results for Ca, Fe, Mg and Zn were statistically equivalent to certified values at a 95% confidence level. After that, the extracts obtained for raw bovine and UHT whole milk samples were analyzed employing the approach described in Fig. 1, and the results are presented in Table 4. The quantitative recoveries for Ca, Mg and Zn in TCA and PEP media suggest that these elements are present in bovine milk ionized in solution or mainly associated with the colloidal calcium phosphate responsible for casein micelle stabilization. The TCA and PEP acted on bovine milk casein micelle according to different mechanisms. The precipitate produced by TCA corresponded to casein denaturated in its isoelectric point without cleavage of peptide bonds of the casein micelle amino acid sequence. The PEP enzyme destabilized the casein micelle structure through cleavage of peptide bonds involving aromatic amino acid residues. This modification in the primary structure of casein was responsible for its solubilization in PEP medium. Consequently, the ionic species associated with colloidal calcium phosphate of the casein micelle were
released into solution after milk sample treatment with TCA or PEP. The determination of iron was not possible in raw and UHT bovine whole milk owing to the low Fe content in these samples. Considering the dilution during sample preparation, the Fe concentration in the final solutions was below the detection limit of ICPOES with axial viewing. To overcome this limitation, an Fe supplemented milk sample was used to investigate the behavior of Fe in the complex system formed by milk components. A recovery of approximately 100% was observed for Fe when the PEP procedure was used ŽTable 5., suggesting that this enzyme was able to solubilize the Fe present in milk, whereas the TCA solution was not able to do so. Therefore it is possible to infer that Fe is not associated with micellar calcium phosphate w16x. The TCA treatment caused precipitation of Fe and the quantitative release of Ca, Mg and Zn to the solution. The precipitate produced after this treatment was digested using the microwaveassisted procedure described in Section 2. Less than 5% of Ca, Mg and Zn and approximately 100% of Fe were recovered for a sample of Fesupplemented UHT whole milk. The strong affinity of casein for Fe is recognized and attributed to clustered phosphoseryl residues w16x. The distinct casein fractions are phosphorylated to different degrees. The PEP enzyme cleaves the peptide bonds of the casein amino acid sequence producing soluble peptide chains in which the Fe is still associated with the phosphoseryl cluster. The total Fe recovery with the PEP treatment suggests that this element is associated with soluble peptide chains produced after enzymatic hydrolysis reaction ŽTable 5..
Table 5 Determination of Ca, Fe, Mg and Zn in Fe-supplemented UHT whole milk sample after microwave-assisted acid digestion, TCA and PEP sample treatment Sample treatment
Ca Žg ly1 .
Fe Žmg ly1 .
Mg Žmg ly1 .
Zn Žmg ly1 .
Microwave-assisted acid digestion TCA protein precipitation PEP protein hydrolysis
1.13 Ž"0.05. 1.19 Ž"0.04. 1.15 Ž"0.05.
13.5 Ž"0.6. 0.464 Ž"0.027. 13.8 Ž"0.5.
108 Ž"5. 103 Ž"6. 116 Ž"5.
6.28 Ž"0.30. 7.10 Ž"0.30. 7.20 Ž"0.42.
Results are average and standard deviation of three determinations.
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916
The same behavior presented by raw and UHT whole bovine milk samples was observed for Fe supplemented milk ŽTables 4 and 5.. This was an indication that the thermal treatment promoted by the UHT process and the milk fortification with added Fe did not affect the mineral element distribution among the milk compartments, in spite of the changes observed in the distribution of molecular masses of proteins w17x.
4. Conclusions The results presented in this work are in agreement with those described in the literature to study the complex Ca, Fe, Mg and Zn chemical interactions with bovine milk proteins. Considering the effect of the studied reagents on bovine milk casein, it was possible to observe that Ca, Mg and Zn were mainly associated with colloidal calcium phosphate of the casein micelle, while Fe was bound to amino acids present in casein micelle polypeptide chains. The pepsin was able to produce soluble peptides from casein by its proteolytic action allowing quantitative determinations of Ca, Fe, Mg and Zn in bovine milk samples without using digestion procedures to destroy organic matter. Fe determination in the supernatant after sample treatment with TCA solution was not possible owing to the strong affinity of this element to amino acid residues of the casein micelle.
w3x
w4x
w5x
w6x
w7x
w8x
w9x
w10x
w11x
Acknowledgements ao de AmThe authors are grateful to Fundac¸˜ paro ` a Pesquisa do Estado de Sao ˜ Paulo for financial support Ž98r10814-3. and research scholarships provided to F.V.S. Ž00r00997-4. and G.S.L. Ž98r 05335-9.. A.R.A.N. and J.A.N. are grateful to the CNPq for fellowships.
w12x
w13x
References w1x M.C. Neville, P. Zhang, J.C. Allen, Handbook of Milk Composition, Academic Press, London, 1995. w2x N. Campillo, P. Vinas, ˜ I. Lopez-Garcıa, ´ ´ M. Hernandez´ Cordoba, Direct determination of copper and zinc in ´
w14x
1915
cow milk, human milk and infant formula samples using electrothermal atomization atomic absorption spectrometry, Talanta 46 Ž1998. 615᎐622. J.A. Nobrega, Y. Gelinas, A. Krushevska, R.M. Barnes, ´ ´ Direct determination of major and trace elements in milk by inductively coupled plasma atomic emission and mass spectrometry, J. Anal. At. Spectrom. 12 Ž1997. 1243᎐1246. M.A. de la Fuente, B. Carazo, M. Juarez, Determination ´ of major minerals in dairy products digested in closed vessels using microwave heating, J. Dairy Sci. 80 Ž1997. 806᎐811. J. Lameiras, M.E. Soares, M.L. Bastos, M. Ferreira, Quantification of total chromium and hexavalent chromium in UHT milk by ETAAS, Analyst 123 Ž1998. 2091᎐2095. F.A. Rivero Martino, M.L. Fernandez Sanchez, A. ´ Sanz-Medel, Total determination of essential and toxic elements in milk whey by double focusing ICP-MS, J. Anal. At. Spectrom. 15 Ž2000. 163᎐168. V.E. Negretti de Bratter, S. Recknagel, D. Gawlik, Spe¨ ciation of Se, Fe, and Zn in human milk whey: The use of instrumental neutron activation analysis ŽINAA. to corroborate element profiles measured with inductively coupled plasma atomic emission spectrometry ŽICPAES., Fresenius J. Anal. Chem. 353 Ž1995. 137᎐142. B. Michalke, P. Schramel, Application of capillary zone electrophoresis-inductively coupled plasma mass spectrometry and capillary isoelectric focusing-inductively coupled plasma mass spectrometry for selenium speciation, J. Chromatogr. A 807 Ž1998. 71᎐80. P. Bratter, I.N. Blasco, V.E. Negretti de Bratter, A. ¨ ¨ Raab, Speciation as an analytical aid in trace element research in infant nutrition, Analyst 123 Ž1998. 821᎐826. P. Zhang, J.C. Allen, A novel dialysis procedure measuring free Zn2q in bovine milk and plasma, J. Nutr. 125 Ž7. Ž1995. 1904᎐1910. M.J. Roig, A. Alegrıa, ´ R. Barbera, ´ R. Farre, ´ M.J. Lagarda, Calcium dialysability as an estimation of bioavailability in human milk, cow milk and infant formulas, Food Chem. 64 Ž1999. 403᎐409. O. Abollino, M. Aceto, M.C. Bruzzoniti, E. Mentasti, C. Sarzanini, Speciation of copper and manganese in milk by solid-phase extractionrinductively coupled plasmaatomic emission spectrometry, Anal. Chim. Acta 375 Ž1998. 299᎐306. P. Bermejo-Barrera, S. Fernandez-Nocelo, A. Moreda´ Pinero, A. Bermejo-Barrera, Usefulness of enzymatic ˜ hydrolysis procedures based on the use of pronase E as sample pre-treatment for multi-element determination in biological materials, J. Anal. At. Spectrom. 14 Ž1999. 1893᎐1900. P. Ostoa-Saloma, J. Ramırez, R. Perez-Montfort, Mea´ surement of casein digestion by a fluorometric method, Anal. Biochem. 176 Ž1989. 239᎐243.
1916
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916
w15x A.L. Lehninger, Biochemistry, Worth Publishers Inc, New York, 1975. w16x F. Gaucheron, Y. le Graet, K. Raoult, M. Piot, Physicochemical characterization of iron-supplemented skim milk, Int. Dairy J. 7 Ž1997. 141᎐148. w17x E. Coni, A. Alimonti, A. Bocca, F. La Torre, D. Pizzuti,
S. Caroli, Speciation of trace elements in milk by highperformance liquid chromatography combined with inductively coupled plasma atomic emission spectrometryin: S. Caroli ŽEd.., Element Speciation in Bioinorganic Chemistry, John Wiley & Sons, New York, 1996, pp. 255᎐285.
Study of the protein-bound fraction of calcium, iron, magnesium and zinc in bovine milk 夽 a , Fernando V. Silvaa , Gisele S. Lopes a , Joaquim A. Nobrega ´ b b,U Gilberto B. Souza , Ana Rita A. Nogueira a
Departamento de Quımica, Uni¨ ersidade Federal de Sao ´ ˜ Carlos, Sao ˜ Carlos SP, Brazil b Embrapa Pecuaria ´ Sudeste, P.O. Box 339, 13560-970 Sao ˜ Carlos SP, Brazil Received 6 December 2000; accepted 30 May 2001
Abstract Two approaches were used to study the interaction of Ca, Fe, Mg and Zn with bovine milk proteins by inductively coupled plasma optical emission spectrometry ŽICPOES.. Selective separations in bovine milk samples were accomplished employing an acid protein precipitation using 100 g ly1 trichloroacetic acid ŽTCA., and an enzymatic protein hydrolysis using 50 g ly1 pepsin ŽPEP. solution, respectively. The results were compared with total mineral contents determined after microwave-assisted acid digestion. The results obtained by enzymatic and acid precipitation evidenced the different interaction forms of Ca, Fe, Mg and Zn in the system formed by milk components. Iron was not solubilized by the TCA treatment, but was recovered completely after the enzymatic treatment. Quantitative recoveries of Ca, Mg and Zn were obtained using both approaches, showing that these analytes were bound to milk compounds affected by either treatment. Calcium, Mg and Zn are mainly associated with colloidal calcium phosphate and Fe is bound to the backbone of the casein polypeptide chain, cleaved by pepsin enzyme. The proposed approaches could be used to assess the complexity of these chemical interactions. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Milk; Casein; Trichloroacetic acid; Pepsin; ICPOES
夽
This paper was presented at the 6th Rio Symposium on Atomic Spectrometry, held in Concepcion ´ and Pucon, ´ Chile, December 2000, and is published in the Special Issue of Spectrochimica Acta Part B, dedicated to that conference. U Corresponding author. Tel.: q55-16-261-5611; fax: q55-16-261-5754. E-mail address: [email protected] ŽA.R. Nogueira.. 0584-8547r01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 8 4 - 8 5 4 7 Ž 0 1 . 0 0 3 1 3 - 5
1910
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916
1. Introduction Milk is a complex fluid that contains all nutrients required for growth and development of newborns. All types of milk are composed of specific proteins, fats designed to be easily digested, minerals, vitamins and other components that may have an important role w1x. Milk compartmentalization influences the mineral absorption process by the gastrointestinal tract. Various approaches using appropriate digestion or solubilization methods have been proposed to determine the total content of inorganic species in milk samples w2᎐6x. However, to increase knowledge about the physiological activity of milk minerals, determination of the total content is not sufficient w7᎐9x. The establishment of distinct chemical interactions among milk components has also to be appraised w1x. Monovalent ions, such as Naq, Kq and Cly, are present in milk mainly in the ionized form. Other ionic species, such as Ca2q, Mg 2q, Zn2q, Fe 3q, carbonate, citrate and phosphate ions, are distributed among the structural compartments of milk in a highly specific way, generating a complex chemical equilibrium w1x. The study of ionic distribution in the structural compartments of milk is normally accomplished employing methodologies based on physical separation processes, such as ultra-centrifugation or dialysis using suitable chelating agents w1,10,11x. The use of solidphase extraction for speciation studies in milk samples has also been reported in the literature w12x. By using these procedures different milk fractions can be isolated and their mineral content determined using proper analytical techniques. Recently, an enzymatic approach has extracted minor and trace elements from biological materials w13x, which allows the selective extraction of the elements associated with proteins that are hydrolyzed by proteolytic enzymes. The major advantage of enzymatic hydrolysis as an alternative sample treatment approach is its selectivity w13x. The major class of protein in most milk species is casein. The casein in bovine milk occurs as a colloidal complex of proteins and salts, mainly calcium salts. When the colloidal Ca is removed,
the submicelles ␣-s1, ␣-s2,  and -casein are produced, which are phosphorylated to different degrees. The aggregates of the casein protein sub-units, calcium phosphate and some other ionic constituents in the form of micelles, give milk its white appearance. These casein micelles are susceptible to the action of most proteolytic enzymes Žproteases. w14x. The casein peptide bonds can be cleaved by proteolytic enzyme action, producing soluble peptide chains or soluble amino acids, depending on the degree of hydrolysis. Solutions with low pH values, under mild conditions, can also produce changes in quaternary, tertiary and secondary casein structures. The covalent bonds of the backbone of the polypeptide chain are not cleaved. The most visible consequence of this effect is observed in the decrease of the casein solubility w15x. Considering these aspects, the use of enzymatic hydrolysis and acid protein precipitation reactions were chosen to investigate the association of Ca, Fe, Mg and Zn with bovine milk proteins. The proteolytic enzyme employed was pepsin, the major protease present in the stomach. This enzyme preferentially breaks peptide bonds involving residues of aromatic amino acids as well as methionine and leucine, to yield peptides and a few free amino acids w15x. Among the enzymes, pepsin has a distinctive behavior due to its low isoelectric point and low optimum pH value. Trichloroacetic acid ŽTCA., on the other hand, presents no specificity for cleaving protein bonds. In solution, the trichloroacetate anion, Cl 3 C᎐COOy, interacts with positive protein groups, R᎐NHq 3 , producing a white precipitate of denatured proteins. Taking into account the distinct effects of these reagents on bovine milk proteins, the different chemical interactions between Ca, Fe, Mg, Zn and protein compounds were studied monitoring the minerals released into solution after milk sample treatment with TCA and PEP solutions.
2. Experimental 2.1. Instrumentation A Vista AX axial view simultaneous inductively
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916 Table 1 ICPOES operational parameters Power ŽkW. Plasma gas flow rate Žl miny1 . Auxiliary gas flow rate Žl miny1 . Nebulizer gas flow rate Žl miny1 . Spray chamber type Nebulizer Sample flow rate Žml miny1 . Analytical wavelengths Žnm. Ca II Fe II Mg I Zn II Y II
1911
Table 2 Heating program employed in microwave-assisted acid digestion 1.1 15.0 1.5 0.80 Sturman-Masters V-groove 0.80 315.882 285.208 285.208 206.204 371.029
coupled plasma optical emission spectrometer ŽVarian, Australia., equipped with a CCD detector was used to determine Ca, Fe, Mg and Zn. The spectrometer provided full wavelength coverage from 167 to 785 nm. The optical system was purged with argon and the Echelle polychromator was thermostatted at 34⬚C. The cool plasma tail was removed from the optical path using an endon purge gas. The operating conditions are listed in Table 1.
Step
Time Žmin.
Power ŽW.
1 2 3 4 5 Ventilation
1.0 1.0 3.0 5.0 5.0 4.0
250 0 250 400 600 0
The acid digestion of the milk samples and the certified reference material ŽWhole milk powder, NIST 8435, National Institute of Standards and Technology, Gaithersburg, MD, USA. were performed in a microwave oven ŽETHOS 1600, Milestone, Italy. equipped with 10 closed vessels made of perfluoralkoxy Teflon 䊛 ŽPFA. with a calibrated resealing pressure relief mechanism Žmaximum operating pressure 110 atm.. The vessels were located on a rotating turntable inside the microwave oven cavity. Before use, the PFA vessels were cleaned with acid and rinsed with deionized water. The heating program used for digestion is shown in Table 2.
Fig. 1. Experimental approaches used to study chemical interactions of Ca, Fe, Mg, Zn and bovine milk proteins.
1912
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916
A sub-boiling still ŽsubPUR, Milestone, Italy. was used to further purify the concentrated nitric acid. 2.2. Solutions All solutions were prepared by using analytical grade reagents and Milli-Q 䊛 water Ž18 M⍀ cm.. Three different approaches Žacid precipitation, enzymatic hydrolysis and microwave-assisted acid digestion. were employed to investigate Ca, Fe, Mg and Zn chemical interactions with bovine milk proteins. In the protein acid precipitation and enzymatic hydrolysis procedures, 100 g ly1 TCA ŽNuclear, Brazil. and 50 g ly1 PEP ŽNuclear, Brazil. solutions were used, respectively. The PEP solution was prepared in 0.01 mol ly1 HNO3 medium. A mixture containing 2 ml of sub-boiling distilled concentrated nitric acid and 1 ml of 30% vrv H 2 O 2 ŽMallinckrodt, Mexico. was used for microwave-assisted acid digestion. The analytical curves were prepared after suitable dilution of 1000 mg ly1 stock solution ŽSpex, USA. of each element: blank: 0.14 mol ly1 HNO3 ; reference solution 1:10.0 mg ly1 of Ca and Mg q1.00 mg ly1 of Fe and Zn; and reference solution 2: 20.0 mg ly1 of Ca and Mgq 2.0 mg ly1 of Fe and Zn. The samples were diluted accordingly before measurements. Yttrium Ž1.0 mg ly1 . was added to all reference solutions and samples as an internal standard before ICPOES measurements.
from the storage process on the analytical results, the raw bovine milk sample was analyzed immediately after collection. The Fe-supplemented UHT milk sample, according to the information on the product label, contained 15 mg ly1 of added Fe in the form of an amino-chelate. The standard reference milk sample ŽNIST 8435 Whole milk powder. was not reconstituted before its microwaveassisted acid digestion.
3. Results and discussion 3.1. E¨ aluation of ICPOES experimental parameters and matrix effects
2.3. Sample treatment
The ICPOES operational parameters were established using a 1.0-mg ly1 multielement solution ŽCa, Fe, Mg and Zn. and the auto-optimization program provided with the spectrometer software, where the radio frequency power and nebulizer flow rate are systematically modified in order to obtain the highest signal-to-background ratios ŽSBR. for each selected wavelength. For most wavelengths the best SBR was obtained for a radio frequency power of 1.1 kW and a nebulizer flow rate of 0.80 l miny1 . Recovery tests in 50 g ly1 PEP and 100 g ly1 TCA solutions were carried out under these conditions. The results for this experiment are summarized in Table 3. The recovery experiments showed no significant interference of TCA and PEP for all analytes. The use of Y as internal standard compensated for potential interferences related to sam-
The three different approaches employed to evaluate the protein-bound fraction of Ca, Fe, Mg and Zn in bovine milk are summarized in Fig. 1.
Table 3 Recovery of Ca, Fe, Mg and Zn in 50 g ly1 pepsin ŽPEP. and 100 g ly1 trichloroacetic acid ŽTCA. solutions
2.4. Milk samples
Analyte
Recovery Ž%. TCA
The ultra high temperature treated ŽUHT. whole milk and Fe-supplemented UHT milk samples were purchased in a local market, while the raw bovine milk sample was collected from cows in the final stage of lactation in an intensive bovine milk production system ŽEmbrapa Pecuaria ´ Sudeste, Brazil.. In order to avoid any influence
a
Ca Fe Mg Zn a b
PEP b
1
2
1a
2b
100 93.0 97.0 99.0
101 98.0 98.0 100
90.0 90.3 91.5 91.8
89.2 92.0 92.5 90.1
Ca and Mg 5.0 mg ly1 ; Fe and Zn 0.50 mg ly1 . Ca and Mg 20 mg ly1 ; Fe and Zn 2.0 mg ly1 .
Raw
UHT 1.13 Ž"0.04. 1.14 Ž"0.02. 1.14 Ž"0.02.
Raw 0.92 Ž"0.02. 0.93 Ž"0.03. 0.91 Ž"0.05.
- 0.13 - 0.13 - 0.13
Fe Žmg ly1 .
Ca Žg ly1 .
Results are average and standard deviation of three determinations.
Microwave-assisted acid digestion TCA protein precipitation PEP protein hydrolysis
Sample treatment
- 0.13 - 0.13 - 0.13
UHT 101 Ž"4. 109 Ž"3. 104 Ž"6.
Raw
Mg Žmg ly1 .
109 Ž"6. 113 Ž"4. 115 Ž"2.
UHT
2.16 Ž"0.02. 2.30 Ž"0.07. 2.42 Ž"0.08.
Raw
Zn Žmg ly1 .
3.02 Ž"0.33. 3.10 Ž"0.04. 3.68 Ž"0.27.
UHT
Table 4 Determination of Ca, Fe, Mg and Zn in raw and UHT bovine whole milk samples after microwave-assisted acid digestion, TCA and PEP sample treatment
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916 1913
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F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916
ple introduction into the plasma. The analytical curve prepared in HNO3 medium could therefore be used without any adverse effect on the accuracy of the determinations carried out in TCA or PEP medium. 3.2. Bo¨ ine milk analysis: effects of TCA and PEP treatment and determination of Ca, Fe, Mg and Zn First the accuracy of the multielement determination was checked using a standard reference material ŽNIST 8435, Whole milk powder. digested as described in Section 2. The results for Ca, Fe, Mg and Zn were statistically equivalent to certified values at a 95% confidence level. After that, the extracts obtained for raw bovine and UHT whole milk samples were analyzed employing the approach described in Fig. 1, and the results are presented in Table 4. The quantitative recoveries for Ca, Mg and Zn in TCA and PEP media suggest that these elements are present in bovine milk ionized in solution or mainly associated with the colloidal calcium phosphate responsible for casein micelle stabilization. The TCA and PEP acted on bovine milk casein micelle according to different mechanisms. The precipitate produced by TCA corresponded to casein denaturated in its isoelectric point without cleavage of peptide bonds of the casein micelle amino acid sequence. The PEP enzyme destabilized the casein micelle structure through cleavage of peptide bonds involving aromatic amino acid residues. This modification in the primary structure of casein was responsible for its solubilization in PEP medium. Consequently, the ionic species associated with colloidal calcium phosphate of the casein micelle were
released into solution after milk sample treatment with TCA or PEP. The determination of iron was not possible in raw and UHT bovine whole milk owing to the low Fe content in these samples. Considering the dilution during sample preparation, the Fe concentration in the final solutions was below the detection limit of ICPOES with axial viewing. To overcome this limitation, an Fe supplemented milk sample was used to investigate the behavior of Fe in the complex system formed by milk components. A recovery of approximately 100% was observed for Fe when the PEP procedure was used ŽTable 5., suggesting that this enzyme was able to solubilize the Fe present in milk, whereas the TCA solution was not able to do so. Therefore it is possible to infer that Fe is not associated with micellar calcium phosphate w16x. The TCA treatment caused precipitation of Fe and the quantitative release of Ca, Mg and Zn to the solution. The precipitate produced after this treatment was digested using the microwaveassisted procedure described in Section 2. Less than 5% of Ca, Mg and Zn and approximately 100% of Fe were recovered for a sample of Fesupplemented UHT whole milk. The strong affinity of casein for Fe is recognized and attributed to clustered phosphoseryl residues w16x. The distinct casein fractions are phosphorylated to different degrees. The PEP enzyme cleaves the peptide bonds of the casein amino acid sequence producing soluble peptide chains in which the Fe is still associated with the phosphoseryl cluster. The total Fe recovery with the PEP treatment suggests that this element is associated with soluble peptide chains produced after enzymatic hydrolysis reaction ŽTable 5..
Table 5 Determination of Ca, Fe, Mg and Zn in Fe-supplemented UHT whole milk sample after microwave-assisted acid digestion, TCA and PEP sample treatment Sample treatment
Ca Žg ly1 .
Fe Žmg ly1 .
Mg Žmg ly1 .
Zn Žmg ly1 .
Microwave-assisted acid digestion TCA protein precipitation PEP protein hydrolysis
1.13 Ž"0.05. 1.19 Ž"0.04. 1.15 Ž"0.05.
13.5 Ž"0.6. 0.464 Ž"0.027. 13.8 Ž"0.5.
108 Ž"5. 103 Ž"6. 116 Ž"5.
6.28 Ž"0.30. 7.10 Ž"0.30. 7.20 Ž"0.42.
Results are average and standard deviation of three determinations.
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916
The same behavior presented by raw and UHT whole bovine milk samples was observed for Fe supplemented milk ŽTables 4 and 5.. This was an indication that the thermal treatment promoted by the UHT process and the milk fortification with added Fe did not affect the mineral element distribution among the milk compartments, in spite of the changes observed in the distribution of molecular masses of proteins w17x.
4. Conclusions The results presented in this work are in agreement with those described in the literature to study the complex Ca, Fe, Mg and Zn chemical interactions with bovine milk proteins. Considering the effect of the studied reagents on bovine milk casein, it was possible to observe that Ca, Mg and Zn were mainly associated with colloidal calcium phosphate of the casein micelle, while Fe was bound to amino acids present in casein micelle polypeptide chains. The pepsin was able to produce soluble peptides from casein by its proteolytic action allowing quantitative determinations of Ca, Fe, Mg and Zn in bovine milk samples without using digestion procedures to destroy organic matter. Fe determination in the supernatant after sample treatment with TCA solution was not possible owing to the strong affinity of this element to amino acid residues of the casein micelle.
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Acknowledgements ao de AmThe authors are grateful to Fundac¸˜ paro ` a Pesquisa do Estado de Sao ˜ Paulo for financial support Ž98r10814-3. and research scholarships provided to F.V.S. Ž00r00997-4. and G.S.L. Ž98r 05335-9.. A.R.A.N. and J.A.N. are grateful to the CNPq for fellowships.
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References w1x M.C. Neville, P. Zhang, J.C. Allen, Handbook of Milk Composition, Academic Press, London, 1995. w2x N. Campillo, P. Vinas, ˜ I. Lopez-Garcıa, ´ ´ M. Hernandez´ Cordoba, Direct determination of copper and zinc in ´
w14x
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cow milk, human milk and infant formula samples using electrothermal atomization atomic absorption spectrometry, Talanta 46 Ž1998. 615᎐622. J.A. Nobrega, Y. Gelinas, A. Krushevska, R.M. Barnes, ´ ´ Direct determination of major and trace elements in milk by inductively coupled plasma atomic emission and mass spectrometry, J. Anal. At. Spectrom. 12 Ž1997. 1243᎐1246. M.A. de la Fuente, B. Carazo, M. Juarez, Determination ´ of major minerals in dairy products digested in closed vessels using microwave heating, J. Dairy Sci. 80 Ž1997. 806᎐811. J. Lameiras, M.E. Soares, M.L. Bastos, M. Ferreira, Quantification of total chromium and hexavalent chromium in UHT milk by ETAAS, Analyst 123 Ž1998. 2091᎐2095. F.A. Rivero Martino, M.L. Fernandez Sanchez, A. ´ Sanz-Medel, Total determination of essential and toxic elements in milk whey by double focusing ICP-MS, J. Anal. At. Spectrom. 15 Ž2000. 163᎐168. V.E. Negretti de Bratter, S. Recknagel, D. Gawlik, Spe¨ ciation of Se, Fe, and Zn in human milk whey: The use of instrumental neutron activation analysis ŽINAA. to corroborate element profiles measured with inductively coupled plasma atomic emission spectrometry ŽICPAES., Fresenius J. Anal. Chem. 353 Ž1995. 137᎐142. B. Michalke, P. Schramel, Application of capillary zone electrophoresis-inductively coupled plasma mass spectrometry and capillary isoelectric focusing-inductively coupled plasma mass spectrometry for selenium speciation, J. Chromatogr. A 807 Ž1998. 71᎐80. P. Bratter, I.N. Blasco, V.E. Negretti de Bratter, A. ¨ ¨ Raab, Speciation as an analytical aid in trace element research in infant nutrition, Analyst 123 Ž1998. 821᎐826. P. Zhang, J.C. Allen, A novel dialysis procedure measuring free Zn2q in bovine milk and plasma, J. Nutr. 125 Ž7. Ž1995. 1904᎐1910. M.J. Roig, A. Alegrıa, ´ R. Barbera, ´ R. Farre, ´ M.J. Lagarda, Calcium dialysability as an estimation of bioavailability in human milk, cow milk and infant formulas, Food Chem. 64 Ž1999. 403᎐409. O. Abollino, M. Aceto, M.C. Bruzzoniti, E. Mentasti, C. Sarzanini, Speciation of copper and manganese in milk by solid-phase extractionrinductively coupled plasmaatomic emission spectrometry, Anal. Chim. Acta 375 Ž1998. 299᎐306. P. Bermejo-Barrera, S. Fernandez-Nocelo, A. Moreda´ Pinero, A. Bermejo-Barrera, Usefulness of enzymatic ˜ hydrolysis procedures based on the use of pronase E as sample pre-treatment for multi-element determination in biological materials, J. Anal. At. Spectrom. 14 Ž1999. 1893᎐1900. P. Ostoa-Saloma, J. Ramırez, R. Perez-Montfort, Mea´ surement of casein digestion by a fluorometric method, Anal. Biochem. 176 Ž1989. 239᎐243.
1916
F.V. Sil¨ a et al. r Spectrochimica Acta Part B: Atomic Spectroscopy 56 (2001) 1909᎐1916
w15x A.L. Lehninger, Biochemistry, Worth Publishers Inc, New York, 1975. w16x F. Gaucheron, Y. le Graet, K. Raoult, M. Piot, Physicochemical characterization of iron-supplemented skim milk, Int. Dairy J. 7 Ž1997. 141᎐148. w17x E. Coni, A. Alimonti, A. Bocca, F. La Torre, D. Pizzuti,
S. Caroli, Speciation of trace elements in milk by highperformance liquid chromatography combined with inductively coupled plasma atomic emission spectrometryin: S. Caroli ŽEd.., Element Speciation in Bioinorganic Chemistry, John Wiley & Sons, New York, 1996, pp. 255᎐285.