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Optica Publishing Group
  • Applied Spectroscopy
  • Vol. 75,
  • Issue 6,
  • pp. 718-727
  • (2021)

Prediction of α-Lactalbumin and β-Lactoglobulin Composition of Aqueous Whey Solutions Using Fourier Transform Mid-Infrared Spectroscopy and Near-Infrared Spectroscopy

Open Access Open Access

Abstract

Characterization and quantification of individual whey proteins are of crucial importance to many industrial dairy processes. Labor intensive wet-chemical methods are still being used for this purpose, but a rapid quantification method for individual whey proteins is highly desired. This work investigate the utility of Fourier transform mid-infrared spectroscopy and Fourier transform near-infrared spectroscopy for rapid quantification of the two main whey proteins (β-lactoglobulin and α-lactalbumin) in complex aqueous whey solutions simulating production process streams. MIR and NIR spectra obtained on whey samples with known and varying amounts of the proteins of interest and are used to develop partial least squares prediction models. Selection of informative wavelength regions allowed for prediction of β-lactoglobulin and α-lactalbumin concentrations with very high precision and accuracy (root mean square error of cross-validation, or RMSECV, of 0.6% and R2 of 0.99 for NIR), demonstrating the potential of being implemented for rapid in-line monitoring of protein composition in industrial whey streams. Two-dimensional MIR-NIR correlation spectroscopy is used to identify the most informative parts of the NIR spectra in relation to protein secondary structure. In addition multivariate curve resolution is applied to the MIR data to resolve mixture spectra and to elucidate the spectral ranges that were most useful in distinguishing between the two whey proteins. The study concludes that NIR spectroscopy has potential for accurate in-line protein quantification and overall secondary protein structure quantification which open new possibilities for in-line industrial applications.

© 2021 The Author(s)

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References

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2020 (1)

A. Schwaighofer, M.R. Alcaraz, L. Lux, B Lendl. “pH Titration of β-lactoglobulin Monitored by Laser-based Mid-IR Transmission Spectroscopy Coupled to Chemometric Analysis”. Spectrochim. Acta, Part A. 2020; 226: 117636.

2018 (1)

A. Schwaighofer, J. Kuligowski, G. Quintás, H.K. Mayer, B. Lendl. “Fast Quantification of Bovine Milk Proteins Employing External Cavity-Quantum Cascade Laser Spectroscopy”. Food Chem. 2018; 252: 22–27.

2016 (2)

A. Sturaro, M. De Marchi, A. Masi, M. Cassandro. “Quantification of Whey Proteins by Reversed Phase-HPLC and Effectiveness of Mid-Infrared Spectroscopy for Their Rapid Prediction in Sweet Whey”. J. Dairy Sci. 2016; 99(1): 68–76.

P.D. Ingle, R. Christian, P. Purohit, V. Zarraga, et al. “Determination of Protein Content by NIR Spectroscopy in Protein Powder Mix Products”. J. AOAC Int. 2016; 99(2): 360–363.

2015 (1)

S. Kucheryaversuskiy, C. Lomborg. “Monitoring of Whey Quality with NIR Spectroscopy: A Feasibility Study”. Food Chem. 2015; 176: 271–277.

2014 (1)

A. Juan, J. Jaumot, R. Tauler. “Multivariate Curve Resolution (MCR). Solving the Mixture Analysis Problem”. Anal. Methods. 2014; 6(14): 4964–4976.

2013 (1)

F. Van den Berg, C.B. Lyndgaard, K.M. Sørensen, S.B Engelsen. “Process Analytical Technology in the Food Industry”. Trends Food Sci. Technol. 2013; 31(1): 27–35.

2011 (1)

M.J.M. Rutten, H. Bovenhuis, J.M.L. Heck, J.A.M Van Arendonk. “Predicting Bovine Milk Protein Composition Based on Fourier Transform Infrared Spectra”. J. Dairy Sci. 2011; 94(11): 5683–5690.

2009 (1)

Å. Rinnan, F. Van Den Berg, S.B Engelsen. “Review of the Most Common Pre-processing Techniques for Near-infrared Spectra”. TrAC, Trends Anal. Chem. 2009; 28(10): 1201–1222.

2007 (2)

A. Barth. “Infrared Spectroscopy of Proteins”. Biochim. Biophys. Acta. 2007; 1767(9): 1073–1101.

J. Kong S. Yu. “Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures”. Acta Biochim. Biophys. Sin. 2007; 39(8): 549–559.

2006 (1)

K. Izutsu, Y. Fujimaki, A. Kuwabara, Y. Hiyama, et al. “Near-Infrared Analysis of Protein Secondary Structure in Aqueous Solutions and Freeze-Dried Solids”. J. Pharm. Sci. 2006; 95(4): 781–789.

1998 (1)

N. Chandra, K. Brew, K.R. Acharya. “Structural Evidence for the Presence of a Secondary Calcium Binding Site in Human Alpha-lactalbumin”. Biochemistry. 1998; 37(14): 4767–4772.

1993 (3)

W.K. Surewicz, H.H. Mantsch, D. Chapman. “Determination of Protein Secondary Structure by Fourier Transform Infrared Spectroscopy: A Critical Assessment”. Biochemistry. 1993; 32(2): 389–394.

I. Noda. “Generalized Two-Dimensional Correlation Method Applicable to Infrared, Raman, and Other Types of Spectroscopy”. Appl. Spectrosc. 1993; 47(9): 1329–1336.

F.E. Barton, D.S Himmelsbach. “Two-Dimensional Vibrational Spectroscopy II: Correlation of the Absorptions of Lignins in the Mid- and Near-Infrared”. Appl. Spectrosc. 1993; 47(11): 1920–1925.

1987 (1)

S. Wold, K. Esbensen, P. Geladi. “Principal Component Analysis”. Chemom. Intell. Lab. Syst. 1987; 2(1-3): 37–52.

1983 (2)

S. Wold, H. Martens, H. Wold. “The Multivariate Calibration Problem in Chemistry Solved by the PLS Method”. Matrix Pencils. 1983; 973: 286–293.

L.K. Creamer, D.A.D. Parry, G.N. Malcolm. “Secondary Structure of Bovine β-lactoglobulin B”. Arch. Biochem. Biophys. 1983; 227(1): 98–105.

1964 (1)

A. Savitzky, M.J.E Golay. “Smoothing and Differentiation of Data by Simplified Least Squares Procedures”. Anal. Chem. 1964; 36(8): 1627–1639.

Acharya, K.R

N. Chandra, K. Brew, K.R. Acharya. “Structural Evidence for the Presence of a Secondary Calcium Binding Site in Human Alpha-lactalbumin”. Biochemistry. 1998; 37(14): 4767–4772.

Alcaraz, M.R.

A. Schwaighofer, M.R. Alcaraz, L. Lux, B Lendl. “pH Titration of β-lactoglobulin Monitored by Laser-based Mid-IR Transmission Spectroscopy Coupled to Chemometric Analysis”. Spectrochim. Acta, Part A. 2020; 226: 117636.

Barth, A

A. Barth. “Infrared Spectroscopy of Proteins”. Biochim. Biophys. Acta. 2007; 1767(9): 1073–1101.

Barton, F.E.

F.E. Barton, D.S Himmelsbach. “Two-Dimensional Vibrational Spectroscopy II: Correlation of the Absorptions of Lignins in the Mid- and Near-Infrared”. Appl. Spectrosc. 1993; 47(11): 1920–1925.

Bovenhuis, H.

M.J.M. Rutten, H. Bovenhuis, J.M.L. Heck, J.A.M Van Arendonk. “Predicting Bovine Milk Protein Composition Based on Fourier Transform Infrared Spectra”. J. Dairy Sci. 2011; 94(11): 5683–5690.

Brew, K.

N. Chandra, K. Brew, K.R. Acharya. “Structural Evidence for the Presence of a Secondary Calcium Binding Site in Human Alpha-lactalbumin”. Biochemistry. 1998; 37(14): 4767–4772.

Cassandro, M

A. Sturaro, M. De Marchi, A. Masi, M. Cassandro. “Quantification of Whey Proteins by Reversed Phase-HPLC and Effectiveness of Mid-Infrared Spectroscopy for Their Rapid Prediction in Sweet Whey”. J. Dairy Sci. 2016; 99(1): 68–76.

Chandra, N.

N. Chandra, K. Brew, K.R. Acharya. “Structural Evidence for the Presence of a Secondary Calcium Binding Site in Human Alpha-lactalbumin”. Biochemistry. 1998; 37(14): 4767–4772.

Chapman, D

W.K. Surewicz, H.H. Mantsch, D. Chapman. “Determination of Protein Secondary Structure by Fourier Transform Infrared Spectroscopy: A Critical Assessment”. Biochemistry. 1993; 32(2): 389–394.

Christian, R.

P.D. Ingle, R. Christian, P. Purohit, V. Zarraga, et al. “Determination of Protein Content by NIR Spectroscopy in Protein Powder Mix Products”. J. AOAC Int. 2016; 99(2): 360–363.

Creamer, L.K.

L.K. Creamer, D.A.D. Parry, G.N. Malcolm. “Secondary Structure of Bovine β-lactoglobulin B”. Arch. Biochem. Biophys. 1983; 227(1): 98–105.

De Marchi, M.

A. Sturaro, M. De Marchi, A. Masi, M. Cassandro. “Quantification of Whey Proteins by Reversed Phase-HPLC and Effectiveness of Mid-Infrared Spectroscopy for Their Rapid Prediction in Sweet Whey”. J. Dairy Sci. 2016; 99(1): 68–76.

Engelsen, S.B

F. Van den Berg, C.B. Lyndgaard, K.M. Sørensen, S.B Engelsen. “Process Analytical Technology in the Food Industry”. Trends Food Sci. Technol. 2013; 31(1): 27–35.

Å. Rinnan, F. Van Den Berg, S.B Engelsen. “Review of the Most Common Pre-processing Techniques for Near-infrared Spectra”. TrAC, Trends Anal. Chem. 2009; 28(10): 1201–1222.

Esbensen, K.

S. Wold, K. Esbensen, P. Geladi. “Principal Component Analysis”. Chemom. Intell. Lab. Syst. 1987; 2(1-3): 37–52.

Fujimaki, Y.

K. Izutsu, Y. Fujimaki, A. Kuwabara, Y. Hiyama, et al. “Near-Infrared Analysis of Protein Secondary Structure in Aqueous Solutions and Freeze-Dried Solids”. J. Pharm. Sci. 2006; 95(4): 781–789.

Geladi, P

S. Wold, K. Esbensen, P. Geladi. “Principal Component Analysis”. Chemom. Intell. Lab. Syst. 1987; 2(1-3): 37–52.

Golay, M.J.E

A. Savitzky, M.J.E Golay. “Smoothing and Differentiation of Data by Simplified Least Squares Procedures”. Anal. Chem. 1964; 36(8): 1627–1639.

Heck, J.M.L.

M.J.M. Rutten, H. Bovenhuis, J.M.L. Heck, J.A.M Van Arendonk. “Predicting Bovine Milk Protein Composition Based on Fourier Transform Infrared Spectra”. J. Dairy Sci. 2011; 94(11): 5683–5690.

Himmelsbach, D.S

F.E. Barton, D.S Himmelsbach. “Two-Dimensional Vibrational Spectroscopy II: Correlation of the Absorptions of Lignins in the Mid- and Near-Infrared”. Appl. Spectrosc. 1993; 47(11): 1920–1925.

Hiyama, Y.

K. Izutsu, Y. Fujimaki, A. Kuwabara, Y. Hiyama, et al. “Near-Infrared Analysis of Protein Secondary Structure in Aqueous Solutions and Freeze-Dried Solids”. J. Pharm. Sci. 2006; 95(4): 781–789.

Ingle, P.D.

P.D. Ingle, R. Christian, P. Purohit, V. Zarraga, et al. “Determination of Protein Content by NIR Spectroscopy in Protein Powder Mix Products”. J. AOAC Int. 2016; 99(2): 360–363.

Izutsu, K.

K. Izutsu, Y. Fujimaki, A. Kuwabara, Y. Hiyama, et al. “Near-Infrared Analysis of Protein Secondary Structure in Aqueous Solutions and Freeze-Dried Solids”. J. Pharm. Sci. 2006; 95(4): 781–789.

Jaumot, J.

A. Juan, J. Jaumot, R. Tauler. “Multivariate Curve Resolution (MCR). Solving the Mixture Analysis Problem”. Anal. Methods. 2014; 6(14): 4964–4976.

Juan, A.

A. Juan, J. Jaumot, R. Tauler. “Multivariate Curve Resolution (MCR). Solving the Mixture Analysis Problem”. Anal. Methods. 2014; 6(14): 4964–4976.

Kong, J.

J. Kong S. Yu. “Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures”. Acta Biochim. Biophys. Sin. 2007; 39(8): 549–559.

Kucheryaversuskiy, S.

S. Kucheryaversuskiy, C. Lomborg. “Monitoring of Whey Quality with NIR Spectroscopy: A Feasibility Study”. Food Chem. 2015; 176: 271–277.

Kuligowski, J.

A. Schwaighofer, J. Kuligowski, G. Quintás, H.K. Mayer, B. Lendl. “Fast Quantification of Bovine Milk Proteins Employing External Cavity-Quantum Cascade Laser Spectroscopy”. Food Chem. 2018; 252: 22–27.

Kuwabara, A.

K. Izutsu, Y. Fujimaki, A. Kuwabara, Y. Hiyama, et al. “Near-Infrared Analysis of Protein Secondary Structure in Aqueous Solutions and Freeze-Dried Solids”. J. Pharm. Sci. 2006; 95(4): 781–789.

Lendl, B

A. Schwaighofer, M.R. Alcaraz, L. Lux, B Lendl. “pH Titration of β-lactoglobulin Monitored by Laser-based Mid-IR Transmission Spectroscopy Coupled to Chemometric Analysis”. Spectrochim. Acta, Part A. 2020; 226: 117636.

A. Schwaighofer, J. Kuligowski, G. Quintás, H.K. Mayer, B. Lendl. “Fast Quantification of Bovine Milk Proteins Employing External Cavity-Quantum Cascade Laser Spectroscopy”. Food Chem. 2018; 252: 22–27.

Lomborg, C

S. Kucheryaversuskiy, C. Lomborg. “Monitoring of Whey Quality with NIR Spectroscopy: A Feasibility Study”. Food Chem. 2015; 176: 271–277.

Lux, L.

A. Schwaighofer, M.R. Alcaraz, L. Lux, B Lendl. “pH Titration of β-lactoglobulin Monitored by Laser-based Mid-IR Transmission Spectroscopy Coupled to Chemometric Analysis”. Spectrochim. Acta, Part A. 2020; 226: 117636.

Lyndgaard, C.B.

F. Van den Berg, C.B. Lyndgaard, K.M. Sørensen, S.B Engelsen. “Process Analytical Technology in the Food Industry”. Trends Food Sci. Technol. 2013; 31(1): 27–35.

Malcolm, G.N

L.K. Creamer, D.A.D. Parry, G.N. Malcolm. “Secondary Structure of Bovine β-lactoglobulin B”. Arch. Biochem. Biophys. 1983; 227(1): 98–105.

Mantsch, H.H.

W.K. Surewicz, H.H. Mantsch, D. Chapman. “Determination of Protein Secondary Structure by Fourier Transform Infrared Spectroscopy: A Critical Assessment”. Biochemistry. 1993; 32(2): 389–394.

Martens, H.

S. Wold, H. Martens, H. Wold. “The Multivariate Calibration Problem in Chemistry Solved by the PLS Method”. Matrix Pencils. 1983; 973: 286–293.

Masi, A.

A. Sturaro, M. De Marchi, A. Masi, M. Cassandro. “Quantification of Whey Proteins by Reversed Phase-HPLC and Effectiveness of Mid-Infrared Spectroscopy for Their Rapid Prediction in Sweet Whey”. J. Dairy Sci. 2016; 99(1): 68–76.

Mayer, H.K.

A. Schwaighofer, J. Kuligowski, G. Quintás, H.K. Mayer, B. Lendl. “Fast Quantification of Bovine Milk Proteins Employing External Cavity-Quantum Cascade Laser Spectroscopy”. Food Chem. 2018; 252: 22–27.

Noda, I

I. Noda. “Generalized Two-Dimensional Correlation Method Applicable to Infrared, Raman, and Other Types of Spectroscopy”. Appl. Spectrosc. 1993; 47(9): 1329–1336.

Parry, D.A.D.

L.K. Creamer, D.A.D. Parry, G.N. Malcolm. “Secondary Structure of Bovine β-lactoglobulin B”. Arch. Biochem. Biophys. 1983; 227(1): 98–105.

Purohit, P.

P.D. Ingle, R. Christian, P. Purohit, V. Zarraga, et al. “Determination of Protein Content by NIR Spectroscopy in Protein Powder Mix Products”. J. AOAC Int. 2016; 99(2): 360–363.

Quintás, G.

A. Schwaighofer, J. Kuligowski, G. Quintás, H.K. Mayer, B. Lendl. “Fast Quantification of Bovine Milk Proteins Employing External Cavity-Quantum Cascade Laser Spectroscopy”. Food Chem. 2018; 252: 22–27.

Rinnan, Å.

Å. Rinnan, F. Van Den Berg, S.B Engelsen. “Review of the Most Common Pre-processing Techniques for Near-infrared Spectra”. TrAC, Trends Anal. Chem. 2009; 28(10): 1201–1222.

Rutten, M.J.M.

M.J.M. Rutten, H. Bovenhuis, J.M.L. Heck, J.A.M Van Arendonk. “Predicting Bovine Milk Protein Composition Based on Fourier Transform Infrared Spectra”. J. Dairy Sci. 2011; 94(11): 5683–5690.

Savitzky, A.

A. Savitzky, M.J.E Golay. “Smoothing and Differentiation of Data by Simplified Least Squares Procedures”. Anal. Chem. 1964; 36(8): 1627–1639.

Schwaighofer, A.

A. Schwaighofer, M.R. Alcaraz, L. Lux, B Lendl. “pH Titration of β-lactoglobulin Monitored by Laser-based Mid-IR Transmission Spectroscopy Coupled to Chemometric Analysis”. Spectrochim. Acta, Part A. 2020; 226: 117636.

A. Schwaighofer, J. Kuligowski, G. Quintás, H.K. Mayer, B. Lendl. “Fast Quantification of Bovine Milk Proteins Employing External Cavity-Quantum Cascade Laser Spectroscopy”. Food Chem. 2018; 252: 22–27.

Sørensen, K.M.

F. Van den Berg, C.B. Lyndgaard, K.M. Sørensen, S.B Engelsen. “Process Analytical Technology in the Food Industry”. Trends Food Sci. Technol. 2013; 31(1): 27–35.

Sturaro, A.

A. Sturaro, M. De Marchi, A. Masi, M. Cassandro. “Quantification of Whey Proteins by Reversed Phase-HPLC and Effectiveness of Mid-Infrared Spectroscopy for Their Rapid Prediction in Sweet Whey”. J. Dairy Sci. 2016; 99(1): 68–76.

Surewicz, W.K.

W.K. Surewicz, H.H. Mantsch, D. Chapman. “Determination of Protein Secondary Structure by Fourier Transform Infrared Spectroscopy: A Critical Assessment”. Biochemistry. 1993; 32(2): 389–394.

Tauler, R

A. Juan, J. Jaumot, R. Tauler. “Multivariate Curve Resolution (MCR). Solving the Mixture Analysis Problem”. Anal. Methods. 2014; 6(14): 4964–4976.

Van Arendonk, J.A.M

M.J.M. Rutten, H. Bovenhuis, J.M.L. Heck, J.A.M Van Arendonk. “Predicting Bovine Milk Protein Composition Based on Fourier Transform Infrared Spectra”. J. Dairy Sci. 2011; 94(11): 5683–5690.

Van den Berg, F.

F. Van den Berg, C.B. Lyndgaard, K.M. Sørensen, S.B Engelsen. “Process Analytical Technology in the Food Industry”. Trends Food Sci. Technol. 2013; 31(1): 27–35.

Å. Rinnan, F. Van Den Berg, S.B Engelsen. “Review of the Most Common Pre-processing Techniques for Near-infrared Spectra”. TrAC, Trends Anal. Chem. 2009; 28(10): 1201–1222.

Wold, H

S. Wold, H. Martens, H. Wold. “The Multivariate Calibration Problem in Chemistry Solved by the PLS Method”. Matrix Pencils. 1983; 973: 286–293.

Wold, S.

S. Wold, K. Esbensen, P. Geladi. “Principal Component Analysis”. Chemom. Intell. Lab. Syst. 1987; 2(1-3): 37–52.

S. Wold, H. Martens, H. Wold. “The Multivariate Calibration Problem in Chemistry Solved by the PLS Method”. Matrix Pencils. 1983; 973: 286–293.

Yu, S

J. Kong S. Yu. “Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures”. Acta Biochim. Biophys. Sin. 2007; 39(8): 549–559.

Zarraga, V.

P.D. Ingle, R. Christian, P. Purohit, V. Zarraga, et al. “Determination of Protein Content by NIR Spectroscopy in Protein Powder Mix Products”. J. AOAC Int. 2016; 99(2): 360–363.

Acta Biochim. Biophys. Sin (1)

J. Kong S. Yu. “Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures”. Acta Biochim. Biophys. Sin. 2007; 39(8): 549–559.

Anal. Chem (1)

A. Savitzky, M.J.E Golay. “Smoothing and Differentiation of Data by Simplified Least Squares Procedures”. Anal. Chem. 1964; 36(8): 1627–1639.

Anal. Methods (1)

A. Juan, J. Jaumot, R. Tauler. “Multivariate Curve Resolution (MCR). Solving the Mixture Analysis Problem”. Anal. Methods. 2014; 6(14): 4964–4976.

Appl. Spectrosc (2)

I. Noda. “Generalized Two-Dimensional Correlation Method Applicable to Infrared, Raman, and Other Types of Spectroscopy”. Appl. Spectrosc. 1993; 47(9): 1329–1336.

F.E. Barton, D.S Himmelsbach. “Two-Dimensional Vibrational Spectroscopy II: Correlation of the Absorptions of Lignins in the Mid- and Near-Infrared”. Appl. Spectrosc. 1993; 47(11): 1920–1925.

Arch. Biochem. Biophys (1)

L.K. Creamer, D.A.D. Parry, G.N. Malcolm. “Secondary Structure of Bovine β-lactoglobulin B”. Arch. Biochem. Biophys. 1983; 227(1): 98–105.

Biochemistry (2)

N. Chandra, K. Brew, K.R. Acharya. “Structural Evidence for the Presence of a Secondary Calcium Binding Site in Human Alpha-lactalbumin”. Biochemistry. 1998; 37(14): 4767–4772.

W.K. Surewicz, H.H. Mantsch, D. Chapman. “Determination of Protein Secondary Structure by Fourier Transform Infrared Spectroscopy: A Critical Assessment”. Biochemistry. 1993; 32(2): 389–394.

Biochim. Biophys. Acta (1)

A. Barth. “Infrared Spectroscopy of Proteins”. Biochim. Biophys. Acta. 2007; 1767(9): 1073–1101.

Chemom. Intell. Lab. Syst (1)

S. Wold, K. Esbensen, P. Geladi. “Principal Component Analysis”. Chemom. Intell. Lab. Syst. 1987; 2(1-3): 37–52.

Food Chem (2)

A. Schwaighofer, J. Kuligowski, G. Quintás, H.K. Mayer, B. Lendl. “Fast Quantification of Bovine Milk Proteins Employing External Cavity-Quantum Cascade Laser Spectroscopy”. Food Chem. 2018; 252: 22–27.

S. Kucheryaversuskiy, C. Lomborg. “Monitoring of Whey Quality with NIR Spectroscopy: A Feasibility Study”. Food Chem. 2015; 176: 271–277.

J. AOAC Int (1)

P.D. Ingle, R. Christian, P. Purohit, V. Zarraga, et al. “Determination of Protein Content by NIR Spectroscopy in Protein Powder Mix Products”. J. AOAC Int. 2016; 99(2): 360–363.

J. Dairy Sci (2)

M.J.M. Rutten, H. Bovenhuis, J.M.L. Heck, J.A.M Van Arendonk. “Predicting Bovine Milk Protein Composition Based on Fourier Transform Infrared Spectra”. J. Dairy Sci. 2011; 94(11): 5683–5690.

A. Sturaro, M. De Marchi, A. Masi, M. Cassandro. “Quantification of Whey Proteins by Reversed Phase-HPLC and Effectiveness of Mid-Infrared Spectroscopy for Their Rapid Prediction in Sweet Whey”. J. Dairy Sci. 2016; 99(1): 68–76.

J. Pharm. Sci (1)

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