Adulteration of goat milk is usually done using cow’s milk product. Cow milk is used as it is widely available and its price is cheaper compared to goat milk. This paper shows a development of candidate tools for milk adulteration using cow milk. A quartz crystal microbalance immunosensor was developed using commercial crystal resonator and polyclonal antibody specific to cow milk protein. A specific protein at 208 KDa is found only in cow milk and does not exist in goat milk. The existence of this protein can be used as an indicator of cow milk content in a target solution. To detect the PSS 208 kDa protein, antibody specific to the PSS 208 was developed. The purified antibody was immobilized on top of the sensor surface on a polystyrene layer. The fraction of the immobilized antibody on the sensor was found at 1.5% of the given antibody. Using a static reaction cell, the developed immunosensor could detect the specific cow milk protein in buffer solution. The detection limit is 1 ppm. A linear relationship between frequency change and specific protein of cow milk concentration is found from a concentration of 1 ppm to 120 ppm.
Milk is one of the dairy products, which is one of the most consumed products. Milk production and consumption in developing countries have been increasing significantly [
According to FAO, world goat milk production from 2009 to 2012 is only 2.4% of global milk production compared to 83% for cow milk production [
Physically, it is very difficult to distinguish between cow milk and goat milk. Differentiation is more difficult when both types of milk were mixed. Mixing practice can lead to a serious health problem to a person who has been allergic to cow milk or to those who practice a dietary treatment due to some health reason. In some countries, it is also compulsory to state the origin of the milk used to manufacture a product such as cheese to protect the consumer right [
Many methods have been developed to identify any milk adulteration in other milk products or to differentiate the milk products. Most of the methods were based on a laboratory intensive procedure such as ELISA, PCR, spectroscopy, chromatography, and HPLC [
For developing the QCM biosensor, commercially crystal resonator with silver electrode having resonance frequency of 10 MHz was purchased from Great Micro Electronic, Surabaya, Indonesia. Quartz crystal resonator disc has 8.7 mm diameter with round silver electrode of 5 mm. The quartz crystal has a standard dimension of HC-49/U. Polystyrene coating layer was made from polystyrene well plate for ELISA (Nunc MaxiSorp) which was solved using reagent grade chloroform. The chloroform was purchased from Sigma Aldrich.
The equipment used in the measurement was an oscillator circuit for the QCM sensor developed based on the circuit as described in the work of Eichelbaum et al. [
Static reaction cell and QCM sensor.
Protein specific and antibody were extracted and purified using standard equipment at Bioscience Laboratory, Brawijaya University. PBS-Tween-PMSF, Tris-HCL buffer, Biuret reagent, and other biochemical materials for the purification were purchased from Sigma Aldrich. Cow and goat milk was purchased from local farmers in Malang, Indonesia.
Antibody concentration used was ranging from 200 ppm to 2000 ppm. Each concentration was prepared from a single stock antibody with concentration of 2000 ppm. The concentration of specific cow milk protein (PSS 208) used in this experiment was 1 ppm to 120 ppm. The concentration was made from single source of 120 ppm concentration.
Specific protein used in this work was extracted using electrophoresis. The protein was confirmed using SDS-PAGE method [
Antibody for specific cow milk protein was produced by using rabbit which was treated in accordance with the animal ethics regulation set by the Brawijaya University Animal Use Ethics. New Zealand rabbit was immunized using specific protein solution. Antibody to the specific protein was collected from the blood serum of the rabbit after 8 weeks of incubation. The specificity of the antibody against specific cow milk protein was tested using Western blotting method.
Direct use of the general purpose crystal resonator with silver electrode is inappropriate for biomolecule immobilization. The silver electrode can react unexpectedly with buffer solution which can affect the resonance frequency of the QCM. Therefore, the surface of the sensor was coated with a polystyrene. Polystyrene coating was done by spin coating technique at a rotation speed of 3000 rpm by rapid dropping of polystyrene solution in amount of 50
Determination of optimum antibody concentration to be immobilized on top of the QCM sensor was done as follows. After the sensor was installed in the static reaction cell, 70
Antibody immobilization for QCM biosensor preparation to detect cow milk concentration was done by putting 30
Key factor for the immunosensor development was a selection of specific antibody against target biomolecule to be detected. A potential candidate antibody to be used as a specific bioselective layer for the QCM sensor was investigated using protein. As we developed a candidate biosensor to detect cow milk adulteration in goat milk, protein profiling was done and aimed to select a specific protein that only exists in cow’s milk.
Following the protein isolation and purification, an electroelution procedure was done to select a specific protein of the cow milk. Figure
Protein profile matrix of cow milk (CM), goat milk (GM), cow colostrum (CC), and goat colostrum (GC).
Sample source | Molecular weight (kDa) | ||||||||||||
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9.5 | 12 | 13 | 16 | 19 | 23 | 51 | 80 | 95 | 106 | 113 | 119 |
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GM | √ |
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CC | √ | √ |
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GC | √ |
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Electrophoresis gel profile of cow milk (CM), goat milk (GM), cow colostrum (CC), and goat colostrum (GC).
Different protein profile between cow milk, cow colostrum, goat milk, and goat colostrum can be seen in Table
Antibody to specific cow milk specific protein (anti-PSS 208) was produced by introducing purified specific cow milk protein PSS 208 to rabbit (conforming to the rule set by the Brawijaya University Animal Care and Use Committee). The polyclonal antibody was then extracted from the rabbit serum after nine-week incubation. Specificity of the polyclonal antibody was tested again cow milk using Western blotting. Figure
Western blotting test result of the developed polyclonal antibody.
The development of the candidate QCM biosensor to detect the presence of cow milk within milk adulteration was done by immobilizing the polyclonal antibody specific (anti-PSS 208) to the cow milk protein 208 kDa. Optimum concentration for the antibody immobilization was obtained by measuring frequency change of the 10 MHz QCM caused by different concentration of anti-PSS 208 to be immobilized. Figure
Frequency change of the 10 MHz QCM sensor caused by anti-PSS 208 immobilization from different concentration.
The increase frequency change, which reached its maximum at 260 Hz, correlates with 1000 ppm anti-PSS 208. Increasing the antibody concentration was not followed by increasing the frequency change of the sensor. This indicates that the maximum concentration of the immobilized anti-PSS 208 was 1000 ppm. This information was important to note that giving higher anti-PSS 208 concentration to be immobilized was useless and only wasting the antibody.
Based on the Sauerbrey equation, the deposited mass on the sensor surface can be calculated based on the frequency change of the QCM sensor. Figure
Percentage of immobilized anti-PSS 208 at different solution concentration.
Higher concentration resulted in a lower percentage of immobilized antibody. At 1000 ppm, only 1.5% of the antibody in the solution given on top of the sensor surface was immobilized on the sensor surface. This result suggested that the binding rate of the antibody on top of the sensor surface through physical adsorption was affected by concentration of the antibody solution.
The observation over the ability of the QCM biosensor to detect the existence of PSS 208 cow milk specific protein was conducted by measuring the frequency change of the sensor with antibody specific layer. Frequency change was recorded during an addition of specific protein solution on top of the sensor surface. Different concentrations of PSS 208 in a Tris-HCL buffer were used. Figure
Frequency change of the developed QCM biosensor caused by PSS 208 solution.
Figure
Frequency change of the QCM sensor at given PSS 208 concentration in buffer solution.
Those limits of detection were adequate to identify any milk adulteration using cow milk. With those low limits of detection, no economic advantages can be gained. It does not give any economic benefits to add cow’s milk with a concentration of less than 1% to the milk adulterator.
Using the Sauerbrey equation, by assuming that the protein behaves as a rigid material, we can convert the frequency change to mass of the protein that reacts with the sensor. By dividing the mass with a calculated given mass, we got the percentage of the protein in the solution which reacted to the immobilized antibody on top of the sensor surface. Figure
Percentage of the given protein (PSS 208) that reacts with the immobilized antibody on top of the sensor.
At concentration lower than 20 ppm, the fraction of the given PSS 208 in the solution was slightly higher compared to those of higher concentrations. At higher concentrations, ranging from 20 ppm to 120 ppm, the percentage of the protein reacted with the sensor was about 5 percent. This means that only 5% of the target PSS 208 molecule reacted with the immobilized antibody on the sensor surface. Assuming that the immobilized antibody was limited by the sensor surface area, it is expected that at a higher concentration the percentage of the target molecule (PSS 208) to the immobilized antibody will decrease. It can be explained that as the protein was suspended in the solution, only part of the protein, which touched the sensor surface, will react with the immobilized antibody. In addition the number of the immobilized antibodies is lower than the given PSS 208 protein in the solution.
We have shown that a QCM immunosensor can be used as a candidate method to detect milk adulteration using cow milk. The developed polyclonal antibody can be produced after protein profiling of the milk. The protein profiling was done by selecting a specific protein of the cow milk, which does not exist in the target milk being falsified, which, in our case, was goat milk. Optimum concentration of the polyclonal antibody for specific cow milk protein (PSS 208) was found at a concentration of 1000 ppm. Fraction of the antibody being immobilized on top of the QCM surface with polystyrene coating was just around 1.5%. In the static cell reaction, only about 5% of the given PSS 208 was reacting with the immobilized antibody. The developed QCM immunosensor for detecting cow milk existence has a detection limit of 1 ppm. These limits of detection were adequate to be used as a candidate for goat milk adulteration using cow milk detection apparatus.
The authors declare no conflict of interests.
This work was supported by a research grant funded by Directorate General of Higher Education of the Republic of Indonesia.