Characterization of spectral absorbance for determining the reduction in protein s contents due to heating (study case of cow s milk)

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1 Characterization of spectral absorbance for determining the reduction in protein s contents due to heating (study case of cow s milk) Novia D. Irianti 1). Aulia MT. Nasution 2) Photonics Engineering Laboratory Department of Engineering Physics. FTI ITS Surabaya Indonesia 1) wi2ed_ndr@ep.its.ac.id. 2) anasution@ep.its.ac.id Abstract - Protein is an important component of milk. Subjecting to heat treatment (e.g. processing or cooking) will usually cause what so called a denaturation, i.e. diminishing its content. In this work a preliminary effort to develop a customized quantification method based on spectral absorbance measurement will be devoted. Spectral absorbance of total milk s protein (after subjected to a varied heat treatment scheme) is measured using a Beckman DU UV-VIS spectrophotometer. The spectral derivative technique is then used to analyse the measured spectral data. The fourth derivative of absorbance spectra at 290 nm are found to be most suited for quantifying the progressive changes of protein s destruction. Comparison is made to standard Kjeldahl, which shows that the deviation is in the range of 10 %. This technique is also promising to be implemented in many biomedical measurements which are closely related to changes in human body s optical properties. Keyword: milk s total protein. denaturation. spectral absorbance. spectral derivative technique. 1. INTRODUCTION Milk is a daily food with high protein content that is important for human health and metabolism, due to its high nutritional value. As important protein supplier, protein content in milk needs to be kept at a useful nutritional levels for human body. Milk is a kind of emulsion, i.e. colloid globules surrounding with water-based fluid, and each fat globule is surrounded by a membrane consisting of phospholipids and proteins. The largest component in milk (up to 80 % of total protein) is casein aggregate. There are four different types of casein, i.e. mainly α- s1-casein, α-s2-casein, β-casein, κ-casein, where most of them are bound into the micelles. In addition to the caseins, there are also smaller-structure with a more water-soluble component. They are known as whey proteins, which make up to the rest 20 % of the total protein in milk [1]. time, reducing the microbial load, as well as minimizing the risk of food poisoning [2]. When milk proteins are subjected to thermal processing, the whey components may undergo a structural change, commonly known as denaturation. This changes will be accompanied by structure unfolding as well as changes in its hydrophobic properties. If its heating temperatures is above 80 o C, the whey proteins will unfold and interact among themselves and the k- casein to form a heat-induced protein aggregates. At molecular level, this changes may affect protein s functionality [3]. Rapid determination of protein content in milk is an interest for dairy industries and analytical laboratories for a long time. Total protein concentration in cow's milk is most commonly determined using the biuret reaction and Kjeldahl method. These methods are a bit complicated, time consuming, and their measurement accuracy are limited by many interfering factors. A more promising approach is offerred by taking advantage of milk s spectral absorption properties in the UV region ( nm). Under this spectral band, proteins in milk show a more pronounced absorbance properties to other non-protein molecules (e.g. fat). It is then only necessary to dilute the milk until a negligible level of fat is attainable [4]. The goal of this research is to asses whether is it possible to measure the total protein content in milk directly, as well as to detect the proteins reduction (denaturation) due to heating through its spectral absorbance measurement. The succesful of this preliminary effort will be contibutive for further research to develop customized techniques for similar measurements. It is also promising to be implemented in many biomedical measurements which are closely related to changes in human body s optical properties. Heating treatment on milk is usually used to process milk to be safely consumable for longer period of

2 2. FUNDAMENTAL THEORIES 2.1 Effect of heat treatment on milk protein denaturation Cow s milk is mainly composed of water ( % of weight), fat ( %), protein ( %). lactose ( %), minerals ( %) and organic acids ( %) [l]. Its protein composition can be classified in two major groups as presented in Table-1 [5]. The most abundant is casein, which consists of several components, i.e. the α-s1-casein, α-s2-casein, β-casein and κ-casein. Most of them exist in form of colloidal particles, known as the casein micelle. The second protein group is the whey proteins. which include heat-sensitive. globular. water soluble proteins and enzymes. Table-1: Protein composition of cow s milk [5]. Major milk proteins [gr / l] Casein Whey α-lactalbumin 1.20 α-s1-casein α-s2-casein 3.00 β-casein 9.60 κ-casein 3.60 γ-casein 1.60 imunoglobulin 0.60 lactoferin 0.30 β-lactoglobulin 3.00 albumin serum 0.40 Heating treatments, which commonly regarded as essential step in dairy product processing, will affect the rheological as well as structure of its protein emulsions. It will then determine to a large extent the consumers acceptability to the milk products. It has been documented that heating has an impact on the particle size of model emulsions stabilized with milk proteins [6]. When milk proteins solutions are subjected to thermal processing at temperatures around 80 o C or above, they will exhibit significant loss of emulsifying ability. The proteins may undergo structural changes. commonly known as denaturation. Denaturation is a process in which protein, or nucleic acid in general, will lose its tertiary, secondary, as well as quartenary structures. In this case, it will lose the properties of amino acid structures by dissolution of hydrogen bonding and other secondary forces that governing the molecule, which eventually lose many of proteins biological properties. So the extent of the heat treatment will proportional to the denaturation level of milk proteins. Following denaturation, large aggregates will be formed that unable to cover efficiently the fat droplets anymore, which leads to emulsion s instability. This behavior can be depicted in Fig. 1, which shows that the droplet size of the milk protein-stabilized emulsions do increase proportional to increasing heating treatment. Fig. 1: Emulsion droplet size distribution in fresh milk stabilized with preheated whey proteins [7] 2.2 Protein interaction with light The characteristic absorption band of milk s protein is around wavelength of 280 nm, which arises due the combined oscillator strengths of the lowest energy transitions of the three aromatic amino acids: i.e. Trp (Triptophan), Tyr (Tyrosin), and Phe (phenylalanine). They are the large aromatic residues that are normally found in the interior of a protein, and are known to be important for protein stability. The Tyrosine has special properties since its hydroxyl side chain may function as a powerful nucleophile in an enzyme active site and is a common site for phosphorylation in cell signaling cascades. Meanwhile the Tryptophan has the largest side chain and is the least common amino acid in proteins. It has spectral properties that make it the best inherent probe for following protein s folding and conformational changes associated with biochemical processes. Spectral absorptivity characteristics of these amino acids is shown in Fig. 2. Fig 2. Molar absorbtivity of amino acids: Tyrosin, Triptophan, and Phenilalanin [adapted from 8]

3 The molar absorptivity for phenylalanine is quite low in 280nm region, thus it can normally be ignored [8]. Meanwhile due to the lower molecular symmetry of Trp and Tyr, the molar absorptivities of Trp and Tyr are much higher than that of Phe, with respective maxima of 278 nm and 275 nm [8]. They are to be the main contributors to the protein s absorption spectrum. Separating the characteristic contribution of these two amino acids can be achieved by differentiation of the absorption spectrum. The peptide bond absorbs strongly in the far UV spectrum. This very strong absorption band has been used in protein determination. As shown in Fig 2. the strongest absorption band is at nm. Because of interference spectra due to absorption by oxygen as well as lower detection sensitivity below 200 nm (vacuum-uv) by most of the available conventional spectrophotometers. the measurements are more conveniently started at 210 nm. 2.3 Absorbtion Spectrophotometry Spectral absorbance can be defined as the ratio between the intensity of radiation absorbed by matter with the intensity of radiation that comes at a particular wavelength. The value of light absorption (absorbance) of atoms or molecules can be expressed as the Beer-Lambert Law. Thelaw states that absorbance of light by a mediumm is proportional to the medium s intrinsic absoption characteristics (absorbtivity), concentration, and light path in the medium, as written in the following equation:.. log (1) where: A is the absorbance is the molar absorptivity (L mol -1 cm -1 ) is the molar concentration (mol L -1 ) is the light-path in the material (cm) and are the detectedd and probing light intensities. repectively transmitted light. which is detected by photodetector. is recorded on computer in spectral basis. The respected plot of spectral absorbance can be obtained based on measured data of and and with the help of equation (1). 2.4 Spectral Derivative Technique Spectral derivative technique is in principle based on mathematical transformationn of original spectral curve (zeroth order) into its respective n th order derivative spectra. The technique of spectral derivative analysis was firstly introduced by Rutherford in the 1920s. At that time he proposed the use of first derivative to enhance the fine-structure in mass spectrometry. The second and higher order technique was then patented 30 years later by two British industrial chemists. The renaissance of interestt in derivative spectroscopy was stimulated by the work of Butler and Hopkins in the mid-1970s. Their work related particularly to UV- The advancements Vis and fluorescence spectroscopy. of computers and optical detector technologies had much contributed to the mushrooming exploitation of the technique in many branch of applications. such as analytical biochemistry, clinical chemistry, pharmaceuticals, as well as biomedical [10]. An extensive review on the recent developments in derivative spectrophotometry can be read in [11]. Typical form of derivativee spectra from its zeroth order (original absorbance function) until its higher n th -order derivatives can be schematically shown in Fig. 3. In general. the measurement of spectral absorbance using absorption spectrophotometry technique can be shown as follow: Fig 3. Principle of spectral absorbance measurement [9] A wide-spectrum of UV-VIS lightsource after passing through a monochromator component is subjected to probe the sample. The probing configuration normally adopts an in-line arrangement. The reduced Fig 3. Typical form of derivative spectra from absorbance function Compared with conventional spectrophotometric determinations, derivativee spectrophometry has

4 proved to be a great value in eliminating the unwanted interference signals arises from measurement system. Such a interfering signals might be represented, i.e. by baseline and overlapping spectra. So the derivative spectrophotometry represents an elegant approach to the problem of resolving overlap spectra in chemical analysis. It provides much better technique in finding the hidden fingerprints of analyzed substance in compare to analysis of original spectral. Derivative spectra can be obtained by optical, electronic, or mathematical methods. Optical and electronic techniques were used on early UV-VIS spectrophotometers, but nowadays it has largely superseded by mathematical techniques [10]. The advantages of the mathematical techniques are that derivative spectra may easily be calculated. Spectral absorbance function can be approximated by Gaussian fitting of the curve, so that the absorbance values contained in the spectral graph can be fitted by these curves. Derivative functions can then being derived from these fitting functions. 3. MATERIALS AND METHODS 3.1 Materials and samples The samples of fresh cow's milk were collected during April 2010 from a cows farm in Jemursari-Surabaya, and a bottle of distilled water (H 2 O) also required for samples dilution was also prepared. 3.2 Samples treatment Dillution The only big molecules in milk are fat and protein, which can cause obvious light absorbtion. The milk samples must be diluted to a concentration that is well within the detectability range of the instrument. A dilution of times with distilled water (H 2 O) prior to measurement is suggestible, since the spectrophotometer can only read transparent and completely dissolved samples. In addition. the dilution itself will reduce the excessive fat content in the sample. Heating Heating treatment of the samples was done to simulate the degree of protein destruction due to heating. This task was accomplished by using a Magnetic Stirrer, which was set to a certain temperature and stirrer speed. Samples was subjected to a heating scheme from 80C, 90C, 100C, and 110C (each at heating duration of 100s, 300s, and 1000s ). Homogenization The raw milk sample was homogenized by a magnetic stirrer devices at 2 rpm speed of stirring. The preprocessed samples that used for absorbance measurement are ones that have been separated from the sediments, and hold into transparent cuvettes for measurement. The average diameter of the protein in the milk after homogenization is 120 nm. Other particles (lactose and inorganic salts) are dissolved in milk, and their diameters are much less than that of protein. Therefore, the measured absorbance of UV light source can accurately determine the content of the protein in the milk. 3.3 Equipments In this study, the Beckman DU-7500 UV-Visible spectrophotometer was used to obtain the spectral absorbance of samples. Range of scanning wavelengths are nm with a scan rate of 100 nm/min. Besides, some additional equipments are also used, i.e. quartz cuvetes to hold samples, filter papers, bekker glasses, pipettes, erlenmeyer flasks, and measuring flasks. 3.4 Benchmarking For the purpose of referencing, absolute protein content (in mg/mg of milk) from the diluted milk sample (prior to subjection to heating scheme) was measured using Kjeldahl method. This measurement result was utilized for calculating the protein s progressive destruction due to heating scheme. which was relatively measured based spectral analysis of the absorbance data. Meanwhile similar measurements were also done for samples after underwent heating scheme treatments. These data are used to asses the accuracy of protein s contents determination using proposed spectral absorbance method 3.5 Spectral analysis To compute the derivative spectra, the software package MATHCAD 14 was used. Derivative spectra were calculated from measured absorbance spectra by approximating them using a Gaussian fitted functions (generated in MATLAB-7.1). The amplitudes of the signature s peaks and troughs were obtained directly from the derivative spectra. Spectral band which shows following criteria, i.e. regularly pattern of changes in accordance to variability of heating scheme, no shifting, and symmetrical is used for the purposed quantification. 4. RESULTS AND DISCUSSION 4.1 Measurement results The measurements were carried out in the wavelength range of nm, corresponding to the absorbance of protein molecules and aromatic amino acid. Typical measurement absorbance spectra obtained using the spectrophotometer is shown in Fig 5. The graph clearly indicates that the change of heating scheme will deminish the protein absorbance.

5 nm is used to quantify the total protein content. This makes it possible to use specific wavelength at 290 nm trough over the peak to quantify the protein content in milk with various heating treatment. 4.3 Effect of heating treatment of milk on protein content reduction Thermal processing affects the functionality of the ingredients used for emulsion formation in milk solution, which among others is the diameter of the protein, which has a close relationship with protein content in milk. When milk is heated, the change in particle size due to destruction of protein aggregate is dependent on the temperature and duration of heat treatment. An increase in temperature will increase the percentage of protein content reduction. Fig 8 shows the percentage of protein content in milk sample due to heating treatment, i.e. temperature and heating time. Fig 5: Typical plot of spectral absorbance from sample with a) various temperature and b) various heating time at T=80C 4.2 Spectral derivative of protein A higher-order derivative spectra will improve the resolution of complex spectra at the value of decreasing the amplitude. As can be seen in Fig.6, results indicates that the derivation in fourth order will show best trend in diminishing magnitude of the spectra, in accordance with changes in heating scheme. The magnitude of the spectra which are centered at 290 nm will dimisnish as the degree of protein destruction increases. In other words, as the heating scheme increases such that more proteins molecules destructed, less protein will absorb the probing light, which means lower absorbance Fig 8: The percentage of protein content in milk sample due to heating treatment. The figure shows that the highest heating scheme (i.e. temperature of 110 o C and 1000s heating time), it will reduce the protein content ca. 96.3%. 4.4 Protein content of milk samples To ascertain whether the fourth derivative method might be an alternative for direct determination of protein milk samples, a comparison was made between protein contents measured by this method and by the reference method (Kjeldahl method). The range of total protein content for cow's milk samples determined are listed in Table 1. Table 1: Comparison of milk s total protein determination by derivative spectrophotometry vs Kjeldahl Fig 6. Fourth derivative spectral absorbance of milk sample in 90 o C of protein content in the sample. The changes in magnitude of derivative spectral at 290 nm is significant, therefore the amplitude of the peak at 290 Treatment Derivative Spectra Kjeldahl scheme analysis w/o heating C. 10 s C. 10 s C. 10 s C. 10 s The comparison in the table above can be visualized in the Fig 7 below.

6 Fig 7: Graph of comparison of milk s total protein determination by derivative spectrophotometry vs Kjeldahl The values of protein concentration of milk samples determined based on derivative spectral method were not significantly different from the ones obtained by measurement using Kjeldahl method. 5. CONCLUSION [7] Millqvist-Fureby. A. M.. Elofsson. U.. & Bergenstål. B., 2001, Surface composition of spray-dried milk protein-stabilised emulsions in relation to pre-heat treatment of proteins. Colloids and Surfaces B: Biointerfaces [8] Wetlaufer. D. B., 1962, Ultraviolet spectra of proteins and amino acids. Adv. Protein Chem [9] Taiz. L. and Zeiger. E., 2006, Plant Physiology, 4 th Ed., Lincoln Taiz and Eduardo Zeiger, Sinauer Assc. Inc. Publ.Sunderland. MA-USA. [10] Fell. A.F , Biomedical applications of derivative spectroscopy, trends in analytical chemistry. vol 2(3). [11] Rojas. F.S. and Ojeda. C.B., 2004, Recent development in derivative spectrophotometry: : A Review, Analytica Chimica Acta, 635, p The fourth derivative of UV spectra in nm wavelength range can be used to determine the protein content in milk. Measurement of protein content is done based on thr magnitude of the derivative spectra centered at 290 nm. Protein denaturation is already happened at temperature 80 o C, and the largest protein denaturation occurs at temperature 110 o C with heating time 1000s, where it decrease untill 96.3%. The remaining protein concentration was 0.29 gr/ml REFERENCES [1] McGee. H., 1984, Milk and Dairy Products, On Food and Cooking: The Science and Lore of the Kitchen, Charles Scribner s Sons, New York, p [2] McKinnon. I. R., Yap. S. E., Augustin. M. A., and Hermar. Y., 2009, Diffusing-wave spectroscopy investigation of heated reconstituted skim milks containing calcium chloride, Food Hydrocolloids, 23, [3] Singh, H. and Creamer, L. K., 1992, Heat stability of milk, in Advanced Dairy Chemistry (Ed. P. F. Fox), Vol. 1 Proteins. (pp ). Springer, London. [4] S.J. Rowland, 1938, The Determination of the Nitrogen Distribution in Milk, J. Dairy Res. 9, 42. [5] Walstra, P. and Jenness, R.,1984, Dairy Chemistry and Physics, Wiley, New York. [6] McSweeney. S. L.. Mulhivill. D. M.. and O Callaghan. D. M The influence of ph on the heat-induced aggregation of model milk protein ingredient systems and model infant formula emulsions stabilized by milk protein ingredients. Food Hydrocolloids