IMPACT OF ADULTERATION ON PHYSICO-CHEMICAL CHARACTERISTICS OF MARKETED RAW MILK

This study was conducted to determine the impact of adulteration on physico-chemical and coliform content of marketed raw milk. For this purpose, 300 milk samples were collected from urban and peri-urban localities of Jhang, Pakistan. The results showed that 13.3%, 9.7%, 9%, 8.3% and 5.7% of the tested milk samples were positive for cane sugar, formalin, boric acid, H 2 O 2 and urea, respectively besides the highest adulteration of water (95%). Other adulterants included soap (4.7%), sodium chloride (3.7%) and quaternary ammonium compounds (3.3%). The incidence of cane sugar and starch adulteration was highest in winter while formalin and H 2 O 2 in summer season. The compositional quality of milk was also deteriorated by relatively lowered concentrations of lactose, fats, total solids, solids-not-fat (SNF) and proteins. Likewise, the pH and freezing point of contaminated milk samples were comparatively higher than the corresponding standard values. Furthermore, the adulterated milk samples were highly contaminated with E. coli (42.5%) and Enterobacter aerogenes (27.5%) as compared to non-adulterated milk. It was concluded that milk adulteration in the study area predominates round the year badly deteriorating the physico-chemical characteristic of milk besides contaminating it with injurious adulterants and water-borne pathogens posing potential health risks to the consumers.


INTRODUCTION
Milk is known as a complete diet across the world, on account of its integral components, such as proteins, lactose, milk fat, vitamins, and minerals.Being highly digestible, these ingredients are recommended as an essential part of the everyday human diet (1).The total annual production and per capita availability of milk were estimated at 54.3 million tons and 182 liters per annum, respectively in Pakistan, from 2015-to 2016 (2).Regardless of being the 4 th largest milk producing country in the world, Pakistan is deficient in a modern and well-developed marketing system for milk.Consequently, most of the milk is sold in r aw form through informal channels.Adulteration of milk is a critical problem for dairy industries in many nations of the world (3).Currently, Pakistan is confronted with this serious issue of milk adulteration posturing a major health risk for the consumers besides inflicting economic losses to the dairy industry.In short, adulteration deteriorates the quality of milk and may cause problems of grave concern for human health, such as gastrointestinal ailments including gastric ulcer, diarrhea, colon ulcer, and kidney disorders.The mortality reported in the recent past in Chinese infants has been attributed to melamine poisoning (3).Milk comprises 87.4% of water, 12.6% milk solids, 3.7% fat, 8.9% of milk solids-not-fat, 3.4% protein, 4.8% lactose and 0.7% minerals (4).Adulteration is the reduction of the

Research Article
Pak-Euro Journal of Medical and Life Sciences

MATERIALS AND METHODS
This study was approved by the institutional ethical committee, College of Veterinary & Animal Sciences; Jhang, Pakistan, and verbal consent was acquired from the milk shop owners before samples collection (November to June, 2019-2020).A total of three hundred milk samples (n=300) were randomly collected from milk sale points and shops of urban and peri-urban localities during winter, November to February, (n=150) and again during summer, March to June (n=150) months from the same points to determine any seasonal variation in the status of milk adulteration.Milk samples were aseptically collected in screw-capped glass bottles and kept at 4°C until further processing (on a weekly basis up to ten samples).
All the milk samples were screened qualitativelyfor the presence of different adulterants through commercially available MAT (Milk adulteration Test) kit manufactured by the WTO Quality Operations Laboratory (ISO-17025:2005 Accredited Lab), University of Veterinary and Animal Sciences, Lahore, Pakistan (9) following the prescribed instructions.The sensitivity for the MAT is 95% (0.05%) for all adulterants except for formalin which had a sensitivity of 1:40,000 (10).
The physico-chemical parameters of milk samples i.e., pH, lactose, freezing point, proteins, fat, total solids and solids-not-fat were evaluated using a milk analyzer (Lacto Scan SA, Milkotronic Ltd., Bulgaria) based on ultrasonic technology.
For the microbiological analysis of milk, 40 adulterated (out of 273, ones having at least 02 adulterants or more) and 20 non-adulterated milk samples (out of 27) out of total 300 were cultured on Eosin Methylene Blue (EMB, Sigma-Aldrich) agar to appraise the type and extent of bacterial contamination in the milk samples.The presence of the colorants in EMB agar obstructs the growth of most commonly associated Gram-positive bacteria (11).Subsequently, 1mL of 10 -3 (the count derived on the plate met the standard CFU which is 30 to 300) diluted milk sample was pipetted into empty sterile petri plates, 12-15mL EMB was

RESULTS
This study was undertaken to assess the status of adulteration and its impact on the physicochemical and microbiological attributes of marketed raw milk.The incidence of water adulteration was highest (95%) and significantly different in comparison with those of other adulterants (Table I).The incidence of cane sugar was highest, followed by starch, whereas QAC were minimally used for milk adulteration in winter.Overall, the trend of starch, soap, sorbitol, cane sugar, sodium chloride and carbonated adulteration increased in the winter season.In contrast, formalin was recorded as the most frequent milk adulterant followed by H2O2, during summer.Generally, the incidence rates of urea, H2O2, quaternary ammonium compounds,boric acid and formalin were higher in the summer season.The physico-chemical attributes of contaminated milk samples significantly differed from the standard values documented for non-adulterated milk (Table III).The values of tested physical (pH and Freezing point) and chemical (Lactose, Fat, SNF and protein) parameters) of milk are significantly affected due to adulteration compared with the standard reference values.
Adulterated milk samples showed relatively lower concentrations of lactose, fats, total solids, SNF and proteins than unadulterated milk.Whereas the pH and freezing point of contaminated milk samples were comparatively higher than the corresponding standard values.
Moreover, the adulterated and unadulterated milk samples also exhibited significant variation in terms of microbial incidence (Table IV).
Table IV.Microbiological status of adulterated and non-adulterated milk samples

Contamination
incidence % (±S.E.) at 95% C.I.The values with different alphabets in superscripts in the same column are significantly different (P<0.05) between adulterated and non-adulterated milk samples using Pearson Chi Square test.

Enterobacter
Adulterated milk samples were highly contaminated with E. coli and Enterobacter aerogenes as compared to non-adulterated milk.The statistical analysis of milk culture outcomes of the studied samples showed a significantly (P<0.05)higher incidence of microbial contamination among the adulterated ones.

DISCUSSION
Milk adulteration has a variety of facets and dodges starting from animal's milking to its availability to the consumers in raw form.The present study was designed to appraise the status of milk adulteration affecting milk quality in one of the dairy rich regions of the country.Generally, water adulteration represents the simplest and oldest form of milk adulteration and is typically practiced by dealers to fulfill the gap between demand and supply of milk (12).Enhanced level of extraneous water also results from the addition of ice for increasing the shelf life of milk.The estimated level of water adulteration in the current study is compliant with the incidence rate 95% (13).Few unscrupulous dealers also use the nitrate-rich, pond water for increasing the specific gravity of raw milk.The addition of water considerably affects the nutritional value of milk (3).Moreover, milk adulterated with unhygienic water also offers a potential health hazard through the transmission of water-borne diseases (14).
Conversely, the estimated incidence rates of cane sugar and starch adulteration were significantly lower in comparison with previous studies (12).Cane sugar and starch are added to increase the viscosity of milk for masking water adulteration (3).Starch accumulation in body tissues may be fatal for the diabetic patients, while the retention of undigested starch in the colon has been associated with diarrhea in the consumers (15).
poured and completely mixed by clockwise and anti-clockwise rotation of plates at the level surface, for the detection of E. coli and Enterobacter aerogenes.The plates were incubated at 35°C for 24 hours and bacterial identification was performed by means of Gram's staining and Catalase test for preliminary screening.However, confirmed identification was made by using API 20E kit (USA) as per manufacturer's instructions.The data obtained in the current study about the incidence of adulteration were analyzed by Pearson Chi-square while the physico-chemical parameters were tested throughT-test using Statistical Package for Social Sciences (SPSS) software version 21.The analysis of adulterated and unadulterated milk was aimed at evaluating any impact of adulteration and associated activities on the quality and microbial status of milk.

Table I .
Overall incidence of milk adulteration in milk samples (n=300) from Tehsil Shorkot district Jhang Values of the parameters with similar superscripts in the same column are not significantly different (P>0.01)using Pearson Chi-square test.
*QAC: Quaternary Ammonium compounds Moreover, 13.3%, 9.7%, 9% and 8.3% of the tested milk samples were positive for cane sugar, formalin, boric acid and H2O2, respectively.Starch and carbonates were found in 6.3% of the examined milk samples.Besides, 5.7%, 4.7%, 3.7% and 3.3% of milk samples were contaminated with urea, soap, sodium chloride and quaternary ammonium compounds, respectively.Whereas sorbitol adulteration exhibited the lowest incidence of 2.7%.The comparative incidence rates of various adulterants recorded during winter and summer seasons are illustrated in TableII.Table II Seasonal incidence of milk adulteration determined on (n=150 milk samples) from each of the winter and summer seasons Adulterants

tested ( n= 150) Winter (adulterant positive samples each out of 150) Incidence (%) Summer (adulterant positive samples each out of 150)
All the parameters in the table are significantly different between seasons using Pearson Chi Square Statistic P<0.05.

Table III .
Physico-chemical properties of milk samples