Assessment of Microbial Quality and Hygienic Status of Milk, Dairy Products and Dairy-Contact Surfaces in Srinagar, Kashmir

Introduction

Milk and dairy products constitute an important component of the human diet owing to their high nutritional value and contribution to food security. However, their rich nutrient composition, near-neutral pH, and high water activity make them highly susceptible to microbial contamination during production, processing, storage, transportation, and retail marketing (Quigley et al., 2013) [25] . Consequently, ensuring the microbial safety of milk and dairy products remains a major challenge for the dairy industry and public health authorities worldwide (Haug et al., 2007) [16] . Microbial contamination of dairy products represents a significant public health concern due to its association with foodborne illnesses and the transmission of zoonotic pathogens (Newell et al., 2010) [24] . Contamination may occur at multiple stages of the dairy production chain, including milking, handling, processing, storage, and distribution. Inadequate sanitation of milking equipment, poor personal hygiene, improper storage conditions, and post-processing contamination can substantially increase microbial loads and compromise product safety, thereby posing risks to consumers, particularly children, the elderly, pregnant women, and immunocompromised individuals (Jay et al., 2005) [19] . Evaluation of microbial quality is essential for assessing the hygienic status and safety of dairy products. Total viable count (TVC) is widely used as an indicator of the overall microbial burden and sanitary quality of food products, whereas coliform count (CC) serves as an important indicator of fecal contamination and unsatisfactory hygienic practices during production and handling. Elevated TVC and CC may reflect deficiencies in sanitation, inadequate cleaning and disinfection procedures, poor temperature control, or contamination during processing and distribution (APHA, 2015) [3] . Members of the coliform group, including Escherichia coli, Klebsiella spp., Enterobacter spp., and Citrobacter spp., are frequently used as indicator organisms for monitoring the quality of milk and dairy products. Their presence often signifies contamination from environmental, animal, or human sources and may indicate conditions conducive to the survival and transmission of pathogenic microorganisms. Therefore, monitoring coliform contamination constitutes an integral component of food safety surveillance and quality assurance programmes within the dairy sector (Feng et al., 2020) [10] . To safeguard consumer health, microbiological criteria and standards have been established by regulatory agencies and international organizations, including the Food Safety and Standards Authority of India (FSSAI) and the International Commission on Microbiological Specifications for Foods (ICMSF). These standards provide benchmarks for evaluating the microbiological quality and acceptability of dairy products. Exceedance of prescribed limits may indicate inadequate hygienic practices and increased risk of foodborne disease transmission, necessitating corrective interventions across the dairy production chain (ICMSF, 2005 [18] ; FSSAI, 2011 [11] . Kashmir Valley is characterized by substantial consumption of milk and traditional dairy products; however, comprehensive information regarding their microbiological quality and compliance with established food safety standards remains limited. Furthermore, contamination associated with dairy-contact surfaces such as milking cans and pails has received relatively little attention despite their potential role in microbial dissemination. Generation of region-specific data is therefore essential for evaluating food safety risks, strengthening surveillance programmes, and developing evidence-based interventions aimed at improving dairy hygiene and protecting public health. In view of the above, the present study was undertaken to assess the microbial quality and hygienic status of milk, dairy products, and dairy-contact surfaces in Srinagar, Kashmir. A total of 210 samples comprising raw milk, pooled milk, pasteurized milk, paneer, ice cream, and swab samples collected from milking cans and pails were examined for TVC and CC. In addition, coliform isolates were characterized using conventional cultural and biochemical techniques to evaluate the microbiological status of the dairy production chain and its potential implications for food safety and public health.

Materials and Methods Sampling and isolation

A total of 210 samples comprising raw milk (n=30), pooled milk (n=30), pasteurized milk (n=30), paneer (Indian soft cheese) (n=30), ice cream (n=30), milking pail swabs (n=30) and milking can swabs (n=30) were collected randomly from dairy farms, milk vendors, farmers, and retail outlets located in Srinagar, Kashmir. Samples were collected aseptically in sterile containers and transported to the Division of Veterinary Public Health Laboratory, Skuast-K under refrigerated conditions (4±1°C) using insulated ice boxes. All samples were processed immediately upon arrival at the laboratory following standard microbiological procedures (APHA, 2015) [3] . Details of the sampling are given in Table 1.

Enumeration of total viable count (TVC) and coliform count (CC)

TVC was determined using the standard pour plate technique. Briefly, ten-fold serial dilutions of each sample were prepared in sterile diluent and 1ml aliquots were inoculated into molten nutrient agar maintained at 45-50°C.

Plates were incubated aerobically at 37°C for 24 h and colonies were enumerated. Results were expressed as log 10 CFU mL -1 or log 10 CFU g -1 or log 10 CFU cm -2 (APHA, 2015) [3] . For CC, serially diluted samples were inoculated onto Violet Red Bile Agar (VRBA; Hi-Media, Mumbai, India) using the spread plate technique. Following incubation at 37°C for 24 h, characteristic lactosefermenting colonies surrounded by a reddish-purple halo were counted as coliforms. Results were expressed as log 10 CFU mL -1 or log 10 CFU g -1 or log 10 CFU cm -2 (APHA, 2015) [3] .

Isolation and characterization of coliform bacteria

Presumptive coliform colonies obtained on VRBA were sub-cultured onto MacConkey agar (Hi-Media Laboratories, Mumbai, India) and incubated aerobically at 37°C for 24 h.

Lactose-fermenting colonies were subsequently streaked onto Eosin Methylene Blue (EMB) agar and incubated under similar conditions. Colonies exhibiting characteristic metallic green sheen suggestive of Escherichia coli, along with other distinct morphotypes, were purified on nutrient agar slants. Pure isolates were subjected to Gram staining and conventional biochemical characterization, including catalase, oxidase, indole, methyl red, Voges-Proskauer, and citrate utilization tests. Identification of isolates as Escherichia coli, Klebsiella spp., Enterobacter spp., and Citrobacter spp. was carried out according to standard microbiological procedure described by Quinn et al. (2011) [26] and Bergey's Manual of Determinative Bacteriology (Holt et al., 1994) [17] .

Statistical Analysis

Microbial counts were converted to logarithmic values (log 10 CFU mL -1 or log 10 CFU g -1 or log 10 CFU cm -2 ) prior to analysis and expressed as mean ± Standard error (SE). Statistical analysis was performed using IBM SPSS Statistics version 26.0. Differences among sample categories were evaluated at a significance level of P< 0.05.

Results and Discussion

The analysis of milk and dairy product samples unveiled a diverse microbial population, underscoring potential public health concerns. The TVC differed significantly (P< 0.05) among the various sample categories (Table 2; Fig. 1), indicating differences in microbiological quality and hygienic conditions across the dairy production and distribution chain. Elevated bacterial load in milk and dairy products is generally associated with inadequate sanitation during milking, processing, transportation, storage, and retail handling, thereby adversely affecting product quality and shelf-life (Bonfoh et al., 2003 [4] ; Chye et al., 2004 [6] ). [6] ; Hasan et al. (2015) [14] and may be attributed to increased exposure to environmental contamination, extensive handling during production, and inadequate temperature control during storage and marketing. Similarly, findings have been reported in dairy products where poor hygienic practices and handling conditions contributed to elevated bacterial counts (Soomro et al., 2002 [31] ; Elmoslemany et al., 2010 [8] ; Millogo et al., 2010 [22] ; Shekhar and Joshi, 2010 [30] ). The presence of appreciable bacterial loads in pasteurized milk is of particular concern which may arise due to defective pasteurization machinery, survival of pasteurization resistant and endospore producing bacteria, post-pasteurization handling, inadequate storage conditions (Ranieri and Boor, 2009 [27] ; Hasan et al., 2015 [14] . Coliform bacteria are widely regarded as indicators of sanitary quality and their presence in milk and dairy products generally reflects inadequate hygiene during production, processing, handling, or storage (ICMSF, 2005) [18] . In this study, significant differences (P< 0.05) in total coliform counts were observed among the various sample categories (Table 3; Fig. 2), indicating the variations in hygienic quality and the degree of fecal contamination within the dairy production chain. Milking Pail 9(30%) 0.528-0.685 Absent 0.616±0.017 b Values are expressed as log10CFU mL -1 (milk), log10CFU g -1 (dairy products), and log10CFU cm -2 (swab samples)* Means bearing different superscripts within a column differ significantly (P< 0.05) The elevated coliform counts observed in raw milk and ice cream suggest contaminated water sources, environmental exposure, and improper handling practices, similar to the findings reported by Abdalla and El Zubeir (2006) [1] ; Chatterjee et al., 2006 [5] ; El-Diasty et al., 2007 [7] ; Fakruddin et al., 2011 [9] ; Vahedi et al., 2011 [34] ; Salman et al., 2013 [29] . The detection of coliforms in pasteurized milk is noteworthy and of serious concern since they are expected to be absent following effective treatment. Their occurrence may therefore indicate post-processing contamination or poor-storage conditions after pasteurization as also reported previously by several workers (Garrote et al., 2000 [12] ; Gran et al., 2003 [13] ; Tambekar and Shirsat, 2004 [33] ; Abdel All and Dardir, 2009 [2] ; Mhone et al., 2011 [21] ). Furthermore, comparison of the observed total viable and coliform counts with FSSAI standards revealed non-compliance in several sample categories, indicating deficiencies in hygienic handling and sanitation. Overall, 24.28% of samples harbored coliforms with predominance of the fecal coliforms, aligning with global contamination rates in dairy products (Hassan and Elmalt, 2008 [15] ; Kumar et al., 2011 [20] ; Mhone et al., 2011 [21] ; Salman et al., 2011 [28] ; Mohamed and Ahmed, 2018 [23] ). Higher occurrence of E. coli (fecal coliforms) observed in this study along with other previous findings indicates E. coli species as the most common bacterial enteric pathogen contaminating the food products as the organism is widely excreted through feces contaminating the environment (Zhao et al., 1995 [36] ; Strachan et al., 2001 [32] ). These findings (Table 4; Fig. 3) highlight urgent needs for stricter hygiene protocols, improved pasteurization monitoring, and standardized cleaning practices across dairy supply chains to mitigate foodborne risks.

Conclusion

The present study demonstrated widespread microbial contamination of milk, dairy products and dairy-contact surfaces marketed in Srinagar, Kashmir. Elevated TVC and coliform counts, together with the occurrence of presumptive fecal and non-fecal coliforms, indicate deficiencies in hygienic handling, processing and sanitation practices across the dairy production chain. These findings highlight the need for strict implementation of good hygienic practices, routine microbiological surveillance and adherence to FSSAI standards to improve dairy safety and protect public health.

Acknowledgement

AcknowledgmentsThe authors acknowledge the facilities provided by the Division of Veterinary Public Health, Faculty of Veterinary Sciences and Animal Husbandry, SKUAST-Kashmir.

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