Qualitative Assessment for Milk Adulteration: Extent, Common Adulterants, and Utility of Rapid Tests

Abstract

Background:

The adulteration of milk is an important issue that needs to be addressed because of its deleterious effects on health. Detection of adulterants in milk can be done in laboratories or by purchasing adulteration detection kits. There are few home-based rapid tests to detect common milk adulterants, which are less known by our community. Objectives of this study were to estimate the proportion of milk adulteration and to identify common adulterants, to determine the association of milk adulteration with gastrointestinal and renal diseases; and also to validate rapid home-based tests by comparing its result with standard milk adulteration detection kit.

Materials and Methods:

A community-based study was conducted on 330 medical and paramedical staff. Collected milk samples during family visits were tested by standard milk adulteration detection tests and rapid home-based tests.

Results:

Among 330 tested milk samples, 233 samples were adulterated with one or more than one adulterant. The most common adulteration observed was the addition of water to milk, followed by detergent, urea, and neutralizer. The specificity of the rapid tests was found to be almost 100% in the detection of water and detergent in milk and accuracy was above 80%; with “substantial agreement” (kappa = 0.7) between rapid tests and standard tests on kappa statistics.

Conclusion:

The present study has identified a large extent of milk adulteration and common adulterants, which may have harmful effects on health. The utility of home-based rapid tests is clearly proven to detect common adulterants.

INTRODUCTION

Milk is a complete nutrition in a balanced proportion and is a rich source of fats, carbohydrates, milk proteins, vitamins, and minerals.[1] The Food Safety and Standards Authority of India (FSSAI) prescribes specific standards for various types of milk, to ensure the best quality and safety of this essential food product for the consumers. A national survey by FSSAI in 2012 reported 68% adulterated milk samples.[2] Later in 2018, a national survey by FSSAI again on a larger sample (6,432) reported that about 41% of samples, though safe, fall short of one or another quality parameter or standard.[3]

The adulteration of milk is an important issue that has been overlooked in many countries.[4] Commonly reported milk adulterants are water, urea, starch, neutralizers, and detergents.[5,6,7] Detergents in milk can cause gastrointestinal complications, which can lead to gastritis and inflammation of the intestine.[8] Urea in milk overburdens the kidneys as they have to filter out more urea content from the body.[7]

Qualitative detection of adulterants in milk can be easily performed with chemical reactions while quantitative detections are complex and expensive.[4] Quantitative detection techniques include LC (Liquid Chromatography), ELISA (Enzyme-Linked Immunosorbent Assay), PCR (Polymerase Chain Reaction), and PAGE (Polyacrylamide Gel Electrophoresis).[4] Some of the adulterants such as water, starch, and detergent can be detected with the help of rapid tests at home.[9] In previous research, these rapid tests were not done while assessing milk adulteration qualitatively or quantitatively.

Hence, the current study was conducted with the following objectives

1. To estimate the proportion of milk adulteration using a standard milk adulteration detection kit in milk samples collected from study participants

2. To identify common adulterants in milk

3. To determine the association of milk adulteration with gastrointestinal and renal diseases among study participants who are consuming such milk

4. To validate rapid home-based tests by comparing their results with standard milk adulteration detection kits.

5. To assess the knowledge of study participants about rapid home-based tests and create awareness about the same among them.

MATERIAL AND METHODS

The current study is a community-based cross-sectional study on a burning issue of milk adulteration. A qualitative assessment of milk samples was carried out after obtaining written informed consent from study participants. The study population comprised the medical and paramedical staff of a medical college. The medical college has 243 medical staff and 464 paramedical staff, making a total of 707. The sample size is calculated considering the adulteration proportion (68%) reported by a national survey on the adulteration of milk.[2] The calculated sample size is 328, taking a 95% confidence interval and 5% allowable error. After rounding it up, the final sample size is 330. The sampling method was simple random sampling using a table of random numbers without repetition on the sampling frame of 707 staff of the medical college. Those willing to participate and available at the time of study were included as study participants.

Data collection was started after getting ethical permission from the Institutional Ethics Committee. The study period was 2 months, July and August 2022. The whole study sample families reside in urban areas. The source of milk of these families varies in the form of different varieties of packaged milk, milk from different local dairies, from local vendors, or directly from cattle owners. Taking milk samples from both medical and para-medical staff, it was desired to get different kinds of milk samples. For data collection, each staff member’s family was visited. A predesigned proforma containing details of consumed milk, history of health hazards related to adulterated milk, and awareness about rapid home-based milk adulteration detection tests; was filled up by the researcher at the time of the family visit. In addition, a sample of milk was collected in a sterile container for analysis.

Testing of milk samples was conducted by the researcher at the departmental research laboratory of the medical college on the same day within 2 h of collection to avoid spoiling of the milk sample. To serve this purpose, not more than 10 milk samples were collected daily. For detection of milk adulteration, a “Test-O-Milk” kit was used. The source of its technology is the Defence Food Research Laboratory (DFRL). The kit includes strips to detect various harmful adulterants like starch, urea, detergent, hydrogen peroxide, boric acid, and neutralizer. The Kit also contains strips to detect the microbial quality of milk. The test strips can detect an adulteration level as less as 0.5%. Reagents entrapped on small-sized paper strips give color reactions on the addition of milk. Adulterant detection is done by simple color-forming reactions resulting in the formation of complex compounds or the exchange of anions/cations between the dyes and the constituents of adulterants. This is a paper chromatographic method that can be adopted at quality control laboratories for ascertaining the quality of milk.[10] A laboratory validation study of these paper devices reports that such technology can determine if a sample of milk is adulterated with greater than 90% accuracy, sensitivity, and specificity.[11] The Lactometer provided with the kit was used to detect the specific gravity of milk. Specific gravity less than the standard range (1.028 to 1.032) was considered as adulteration with water.

For each milk sample, rapid home-based tests were also conducted. For the detection of water, a drop of milk was put on a polished slanting surface and milk adulterated with water flowed immediately without leaving a mark.[9] For the detection of detergent, a shake test was done.[9] In 5 mL of milk sample, an equal quantity of water was added. The mixture was shaken vigorously and the formation of lather was observed to detect detergent. For the detection of starch, 2–3 drops of tincture iodine were added and the formation of blue color was noted.[9] As home-based rapid tests can detect only water, detergent, and starch; a comparison with a standard testing kit was done only for these three adulterants.

Terminologies used

Standard tests: Tests done on milk samples using “Test-O-Milk” kit for detection of milk adulteration

Rapid tests: Rapid home-based tests for the detection of three adulterants water, detergent, and starch

Data were entered in a Microsoft Excel spreadsheet. For statistical analysis, SPSS version 25 (Trial version) software was used. For qualitative data, percentages were calculated and Pearson’s Chi-square test was applied to test the relationship between adverse health status and the presence of milk adulteration. If the expected number in the cell was below 5 in a table, Yate’s correction was applied. A “P” value of ≤ 0.05 was considered as significant. The validity of home-based rapid milk adulteration tests was determined by comparing their results with results obtained by standard kit tests. The sensitivity, specificity, and accuracy of rapid home-based tests were calculated. Kappa statistics were calculated to check whether there is any agreement between rapid home-based tests and standard kit tests.

RESULTS

A total of 330 staff was recruited in the study and one milk sample was obtained from each. Out of this, 104 were medical staff and 226 were paramedical staff. Among 330 milk samples, 115 were packaged milk samples and 215 were unpackaged milk samples. Among packaged milk consumers, 63 (55%) participants buy milk directly from dairy outlets and 52 (45%) buy it from other than dairy outlet sources like grocery shops or other small shops. Among unpackaged milk consumers, the majority, i.e. 167 (77.7%) participants buy milk through milkman, 40 (18.7%) buy directly from cattle owners; and only 8 (3.6%) buy from shops.

Study participants were asked about the reason for opting for one particular type of milk over another, in terms of packaged or unpackaged milk. Among packaged milk consumers, the good quality of the packaged milk was the main reason given by 84 (73%) participants. Among unpackaged milk consumers, the bad taste and smell of packaged milk was the main reason given by 138 (64%) participants for opting for unpackaged milk.

Among 330 tested milk samples, 233 (70.6%) samples were adulterated with one or more than one adulterant. The most common adulteration observed was the addition of water to milk in 193 (58.5%) samples, which was determined by low specific gravity (<1.028) on the lactometer. The rest of the sample’s specific gravity was normal, ranging from 1.028 to 1.032. The most common chemical adulterant among milk samples was detergent (23.9%), followed by urea (9.1%); which was detected by a milk adulteration testing kit. Few milk samples reported the presence of microbes and neutralizers. In terms of numbers, detergent was found in 79 milk samples, urea in 30 samples, microbes in 15 samples, and neutralizer in 11 samples. Starch, hydrogen peroxide, and boric acid were not detected in any milk samples.

Adulteration of milk was compared statistically between packaged and unpackaged milk [Figure 1]. Adulteration was significantly higher among unpackaged milk samples than packaged milk (P < 0.0001).

Figure 1.

Adulteration of milk in packaged and unpackaged sources

Milk samples were simultaneously tested by rapid tests as well. Rapid home-based tests detected the presence of water in 144 samples, detergent in 48 milk samples, and starch was not detected in any sample. The validity of rapid tests was calculated statistically by comparing its results with the results of standard milk adulteration test kit as shown in Table 1. The accuracy and specificity of home-based rapid tests are above 80% for the detection of water and detergent adulterants in milk. As none of the samples detected starch in it, the utility of rapid tests for the detection of starch adulteration in milk could not be tested.

Table 1.

Validity of home-based rapid tests for detection of milk adulteration

Home-based rapid test result	Detection of water in milk by standard kit			Detection of detergent in milk by standard kit
	Present	Absent	Total	Present	Absent	Total
Positive	144	0	144	47	1	48
Negative	49	137	186	32	250	282
Sensitivity		74.6%; (67.9–80.6%)			59.5%; (47.9%–70.4%)
Specificity		100%; (97.3%–100%)			99.6%; (97.8%–100%)
Accuracy		85.2%; (80.9%–88.8%)			90% (86.2%–93%)

Note: Figures in parentheses indicate 95% confidence interval

Cohen’s kappa for the detection of water in milk is 0.70 with 85.15% agreement and for the detection of detergent is 0.68 with 90% agreement between both methods of testing milk adulteration. In both cases, it showed “substantial agreement” between rapid tests and standard kit tests.

Knowledge about home-based rapid tests for the detection of milk adulteration was ascertained among study participants during the current study. Only eight (2.4%) study participants knew about rapid tests. Rest 322 study participants were provided with knowledge about these tests with the help of demonstration during the current study.

The presence of any gastrointestinal and renal diseases among all family members of study participants was inquired during the study. A total of 81 participants gave a history of gastrointestinal diseases, out of which 53 had a history of gastritis, 22 peptic ulcer disease, and 6 had a history of constipation among any of the family members who were consuming the same milk. The association of gastrointestinal illnesses with the presence of adulteration in milk was studied statistically as shown in Table 2.

Table 2.

Association of milk adulteration with gastrointestinal diseases

Adulteration in milk samples		Gastrointestinal diseases		Total	χ2	“P”
		Present	Absent
Presence of any adulterant	Yes	58 (24.9)	175 (75.1)	233	0.052	0.82
	No	23 (23.7)	74 (76.3)	97
Presence of detergent	Yes	30 (38.0)	49 (62.0)	79	10.114	0.001*
	No	51 (20.3)	200 (79.7)	251

Note: Figures in parentheses indicate percentages; Pearson’s Chi-square test is used. *indicates “Statistically Significant”

A specific association of detergent in milk with gastrointestinal diseases and urea in milk with renal diseases was also determined. The presence of detergent in milk samples is significantly associated with gastrointestinal disease.

Among study participants, six had a history of renal diseases among any of their family members in the form of urinary calculi. Table 3 shows the association of adulteration in milk samples with renal diseases among study participants. The presence of urea in milk samples is significantly associated with renal disease. Though specific adulterants were significantly associated with illnesses, overall adulteration was not significantly associated with gastrointestinal or renal diseases.

Table 3.

Association of milk adulteration with renal diseases

Adulteration in milk samples		Renal diseases		Total	χ2	“P”
		Present	Absent
Presence of any adulterant	Yes	6 (2.6)	227 (97.4)	233	2.544	0.111#
	No	0 (0.0)	97 (100.0)	97
Presence of urea	Yes	3 (10.0)	27 (90.0)	30	12.375	<0.0001*
	No	3 (1.0)	297 (99.0)	300

Note: Figures in parentheses indicate percentages; Pearson’s Chi-square test is used. *indicates “Statistically Significant;” ^#indicates that Yate’s correction is applied

DISCUSSION

India is the largest producer of milk in the world. In India, about 46% of the milk produced is either consumed at the producer level or sold to nonproducers in the rural area, the balance 54% of the milk is available for sale to organized and unorganized groups.[12] Possibility of adulteration of milk is higher among these unorganized groups.[12] Quality milk program was launched in 2019 to achieve Global (Codex) standard for domestic consumption and ensuring traceability and increasing share in world exports.[12]

Milk, being a complete food and required by all age groups, needs to be supplied and consumed in pure form. Milk adulteration is a dangerous practice that is harmful to consumers. In current study, detected proportion of milk adulteration is very high, 70.6% among 330 tested milk samples. Another Indian study also reported very high proportion of milk adulteration with only 8% of milk samples being adulterant-free.[5]

The more preferred milk in study area is unpackaged milk through milkman mainly for good taste and smell of milk. Another study from urban area also reported that, majority of the respondents preferred unpackaged milk because of its good taste, accessibility, and thick consistency, followed by good smell.[5] The current study found this preferred unpackaged milk is significantly more adulterated than packaged milk. Packaged milk needs to follow quality guidelines laid down by government authority, which might be the reason for its better quality as compared with unpackaged milk.

The most common adulterant detected in the current study was water (58.5%). One more study reported that 80% of study samples had low specific gravity indicating dilution of milk with water.[7] Harmful chemicals like detergent, urea, and neutralizers were also detected as milk adulterants in the current study. About one-fourth of studied milk samples detected detergent in them, which is quite high. Previous studies have also reported urea and detergent as the most common chemical adulterants in milk samples.[5,7,8,13] Present study also reported microbes in a few samples, which could be because of “un-boiled” status of milk samples or contamination of milk through faulty handling after boiling.

There are very useful and easy methods for the detection of common milk adulterants in the form of home-based rapid tests. The validity of these tests is not reported in existing literature. The present study established validity by observing the very high accuracy of these tests in detecting water and detergent adulteration in milk. High accuracy also proves the importance of creating awareness among the community regarding the utility of rapid tests. Observed knowledge about rapid tests was only 8% among study participants, which highlights the wide scope of the spread of knowledge. Affordability is an important issue in developing countries and most of the laboratory-based adulteration detection tests might not be affordable to many of the citizens. One study reported that existing common detection techniques are not always convenient and accessible in developing countries making it difficult to address the diverse ways of fraudulent adulteration in milk.[4] Hence, rapid home-based tests are accurate and free-of-cost methods for the detection of water adulteration and detergent in milk, which are common milk adulterants. For urea detection by rapid home-based tests, the required tincture iodine bottle costs around Rs. 80 for 100 mL, which can be used for multiple samples.

Any adulteration is a harmful activity. Detergent and urea in milk are associated with gastrointestinal and renal diseases, respectively.[7,8,13] Present study also found a significant association between the presence of detergent in milk and gastrointestinal diseases. Similarly, urea in milk was significantly associated with renal diseases. Still, causality of detergent and urea in milk with occurrence of gastrointestinal and renal diseases, respectively, cannot be established as the current study is a cross-sectional study.

Another limitation of this study is a subjective assessment of color change on test strips of the kit, but the information card provided with the kit gives shades of color change to be noted to minimize observer bias. The concentration of urea in natural milk ranges from 0.2 to 0.7 g/L. Though urea is a natural constituent of milk, adulterated milk contains almost 20 times its natural concentration.[13] As the current study was based on qualitative analysis of milk, future research based on quantitative analysis would throw more light on urea adulteration in milk.

CONCLUSION

The extent of adulteration is very high in consumable milk. The most common adulterant is water, followed by detergent, urea, and neutralizer. For the detection of common milk adulterants, home-based rapid tests are very useful and accurate methods. Considering the utility of rapid tests, the lack of knowledge and the harmful effects of milk adulteration; the creation of more awareness about home-based rapid tests is required.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.