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When Milk Turns Sour

A miniaturised paper-based kit for milk freshness analysis by the determination of alkaline phosphatase levels.

by Kuldeep Mahato and Dr Pranjal Chandra

Milk: An Introduction

Evolution has gifted mammals with an elixir called “milk” primarily to nourish their newly born offspring, as they are not ready for the solid-based foods.1 It is a watery fluid secreted from the mammary glands,2 and contains almost all types of nutrients including amino acids, proteins, carbohydrates, essential fats, minerals, vitamins and calcium, which are essential for the offspring’s growth.3,4

The high nutritional contents make milk a complete food for all age groups. For humans, physicians often recommend a daily consumption of milk not only for infants/babies, kids, adolescents but also adults.

According to the recent guidelines for the cap of 2015 to 2020 prescribed by the U.S. Department of Agriculture, it is suggested that every American should consume “fat free or low fat-based dairy products including milk, yoghurt, cheese and/or soy beverages”.5,6 Regular consumption of milk and dairy products can offer better bone health, heart health, and manages type 2 diabetes.1 However, raw milk consumption with higher concentration of fat may elevate the risk of the coronary heart associated disorders.7


The Need for Onsite Quality Testing of Milk

Due to milk’s nutritional richness and health benefits, its commercialisation has attained rapid growth across the globe,8 where numerous activities, such as collection of raw milk, its processing, preservation and retailing are majorly employed.

In some small-scale commercial activities, the raw milk from the milkman is collected and retailed from the collection point in a very short period of time to avoid certain microbial invasions, which could harbour the potential causative agents of milk borne infections.

In order to avoid such incidences, the milk is pasteurised using heat sterilisation followed by rapid instant cooling.9 This process kills almost every microbial forms present and makes the milk safe for consumption. To ensure effective pasteurisation, alkaline phosphatase (ALP) content is used as an indicator. ALP is a milk enzyme, a thermostable metallo-protein which withstands temperatures of 72 to 75˚C before it gets denatured.10,11 High levels of ALP present after pasteurisation indicates the presence of microbes that may not have been rendered inactive during pasteurisation.

A number of methods have been reported for ALP determination including methylene blue, phenol-based tests and microbial culture-based techniques. The tests based on the chromogenic substrate methylene blue is time-consuming, lengthy, and follows multistep protocols, which takes 40 to 500 minutes to produce results depending on the sample.12

The phenol-based tests involve the estimation of liberated phenol due to the action of present ALP and the phenol is titrated against the present redox indicator (e.g. 2,6-dibromoquinonechloroimide),13 which makes it more susceptible for false positive assessments because the presence of other electron donors or acceptors can also titrate the indicator. Therefore, these techniques are not always preferred in quality controls at commercial level especially when there are large number of samples to be tested.

Another type of conventional approach is using microbial cultures, which is also a time-consuming process. This make the test non-robust, and not ideal for miniaturised version restricting the outreach for onsite usage at the point of collection.

In recent advancements, relatively faster techniques have been established using the spectroscopic, fluorometric, and electro-chemiluminescence-based methods. However, due to their requirements of sophisticated instruments, eventually limits the application only to certain collections points.10,14


Onsite Determination of ALP Using Paper Based Disposable Device

There is a need for a handy deliverable device for the onsite determination of ALP in milk sample, which can be used at collections or distribution points like household kitchens, in order to provide assurance of safer milk consumption.

In a recent finding by a group of researchers at the Indian Institute of Technology (IIT), Guwahati, India, they developed a disposable device for the determination of ALP in milk samples, which is capable of discriminating raw milk from other forms.14 The fabricated device is small with dimensions of 2cm X 2cm X 0.1 cm and is based on the colorimetric biosensing approach.

The biosensor works by detecting the target molecule (ALP) from the sample by capturing the quantifiable signal.15 In this fabricated biosensor-based kit, the antibody for ALP has been anchored by performing a series of bio-conjugation reactions on the detection zone that eventually captures the ALP when the kit is used for ALP contained milk samples. The biosensor fabrication has been verified using different characterisation techniques such as atomic force microscopy, Fourier transform infrared spectroscopy, and digital image colourimetry (DIC). The colourimetric analytical signal comes when the bioreceptors captures the ALP by the immune complexation process followed by the catalysis of the substrate 5-bromo 4-chloro indoyl phosphate (BCIP), turning the paper into a blue-green colour. The appearance of this characteristic colour is due to the end product 5,5-dichloro,4,4-dibromo indigo complex formed after the catalysis of BCIP in the presence of ALP.

This qualitative biosensor chip is then used for the quantification of the ALP present in milk using DIC, which is based on image processing. In this approach, the images of colour changing phenomenon of colourimetric reaction are correlated with the concentration of the colour-causing element (ALP). In this case, the color change of the paper substrate is processed using image analysis by RGB profiling, where red, green, and blue are the primary channels that are recorded in pixel values when the object is captured digitally using a digital camera (the researchers used smartphone-based camera). Using the pixel intensity (RGB) values of images corresponding to different concentrations, an effective intensity values was calculated from the reference (blank) and these values were plotted for obtaining the calibration plot in standard conditions.

Thereafter, raw and pasteurised milk samples were tested for ALP content using the fabricated sensor. The colour change was observed when the biosensor was treated in raw milk, signifying the presence of native ALP. However in pasteurised milk, there was no colour change.

The biosensors reported a wide dynamic range from 10-1000U/mL, and limit of detection of 0.87 U/mL. The biosensor kit is capable of delivering the determination in the presence of various coexisting molecules with negligible interference. After this, the amount of ALP was quantified using DIC techniques with the reference standard and the obtained ALP concentration in tested milk was 17.8 U/mL, which falls in the range of natively found ALP concentrations in the raw milk.16


Conclusion and Future Direction

For the safer consumption of milk, the ALP determination is of utmost need. Since milk is a widely consumed food for many people around the world, the commercialisation dividend has a large cap, which attracts various kind of malpractices. Therefore, the consumers eventually bear the negative consequences of poor-quality milk.

In these circumstances, this biosensing kit will play a major role in safeguarding consumers against such exploitation led by commercial activities or mismanagements. The kit could come handy in kitchens and milk collection centres where food safety is of prime concern.

In the future, developers are targeting to improvise the existing biosensor kit to deliver better analytical performance. The other major goal is to make it affordable for everyone, especially in developing countries where resources are still largely limited. [APBN]


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  12. Fasken, J.E., and McClure, A.D.: ‘Phosphatase Test in Pasteurization of Milk’, Canadian Journal of Comparative Medicine and Veterinary Science, 1940, 4, (5), pp. 128-137
  13. Payne, C., and Wilbey, R.A.: ‘Alkaline phosphatase activity in pasteurized milk: A quantitative comparison of Fluorophos and colourimetric procedures’, International journal of dairy technology, 2009, 62, (3), pp. 308-314
  14. Mahato, K., and Chandra, P.: ‘Paper-based miniaturized immunosensor for naked eye ALP detection based on digital image colorimetry integrated with smartphone’, Biosensors and Bioelectronics, 2018
  15. Mahato, K., Maurya, P.K., and Chandra, P.: ‘Fundamentals and commercial aspects of nanobiosensors in point-of-care clinical diagnostics’, 3 Biotech, 2018, 8, (3), pp. 149
  16. Kitchen, B.J.: ‘Bovine mastitis: milk compositional changes and related diagnostic tests’, Journal of Dairy Research, 1981, 48, (1), pp. 167-188

About the Authors

Mr. Kuldeep Mahato is a Ph.D. student at the department of biosciences and bioengineering, Indian Institute of Technology Guwahati, Guwahati, India




Dr. Pranjal Chandra is an assistant professor at the department of biosciences and bioengineering, Indian Institute of Technology Guwahati, Guwahati, India