Journal Healthcare Treatment Development (JHTD) ISSN 2799-1148 · Vol. 4, No. 01 (2024) · pp. 35-43
Research Paper

Isolation of bacteria from milk based Indian sweets sold in and around Kolkata using MPN method

Dr. Manoj Yogi1, Dr. Sayan Bhattacharyya2, Dr. Atul Raj2, Amit Banik3
  1. 1 MVPH student, All India Institute of Hygiene and Public Health (AIIH & PH) Kolkata, West Bengal India.
  2. 2 Associate Professor, Microbiology, AIIH & PH Kolkata, West Bengal India.
  3. 3 Head of Department, Department of Microbiology, AIIH & PH Kolkata, West Bengal India.
  4. * Corresponding author: Dr. Manoj Yogi, drmanojyogivet@gmail.com
Received12 October 2023
Revised16 December 2023
Accepted25 December 2023
Published8 February 2024
DOIhttps://doi.org/10.55529/jhtd.41.39.47
LicenceCC BY 4.0
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Abstract

Background: Milk based Indian sweets have a real cultural and culinary weight across much of the Indian subcontinent, yet their microbiological safety is still a worrying public health issue, especially in places where making, keeping, and delivery processes don’t follow consistent hygiene standards Objective: To check the microbiological quality of milk based Indian sweets that are commercially sold in and around Kolkata, India. This was done with the multiple tube test method Methods: We collected 80 samples of milk based Indian sweets from local vendors in and around Kolkata. After collection, microbiological testing was carried out using the multiple tube test in order to detect and, where possible, characterize bacterial contamination. The bacterial isolates that came up were then identified and their frequency was calculated as percentages out of the total number of samples examined. Results: From the 80 sweets analyzed, 64 samples (80%) were positive for bacterial contamination. In total, nine bacterial species were detected: Klebsiella aerogenes (28.75%), Enterobacter cloacae (15%), Escherichia coli (12.5%), Citrobacter koseri (8.75%), Klebsiella oxytoca (6.25%), Acinetobacter baumannii (2.5%), Citrobacter freundii (2.5%), Klebsiella pneumoniae (2.5%), and Aeromonas schubertii (1.25%). Overall, bacteria belonging to the Enterobacteriaceae family made up most of the isolates. Conclusion: Such a high presence of potentially pathogenic, or opportunistic bacteria in milk based Indian sweets creates serious concerns about food safety and consumer wellbeing in the Kolkata region. Also, the strong dominance of Enterobacteriaceae members suggests a notable risk of foodborne illness transmission.

Keywords: Sweets, Food Safety, Public Health, Quality, Vendors, Food Safety.

1. INTRODUCTION

Milk-based Indian sweets hold a special place in the hearts and palates of people across the Indian subcontinent,and they are an integral part of various cultural and festive celebrations. These delectable delicacies are prepared using milk as a primary ingredient, making them not only delicious but also a potential vehicle for microbial contamination [1]. Ensuring the microbiological safety of these traditional desserts is of paramount importance to safeguard public health and maintain the integrity of the food industry.

In recent times, there has been growing concern about the safety and quality of food products, including Indian sweets. The risk of foodborne illnesses caused by pathogenic microorganisms has prompted a need for comprehensive assessments of the microbiological quality of these food items [2]. Kolkata, with its rich culinary heritage and vibrant street food culture, serves as an ideal location to investigate the microbial contaminationof milk-based Indian sweets sold in and around the city.

The objective of this study was to conduct a thorough assessment of the microbiological quality of milk-based Indian sweets available in Kolkata using the multiple tube test. The multiple tube test, also known as the Most Probable Number (MPN) method, is a widely accepted and reliable technique for enumerating coliform bacteria in food samples. By employing this method, we aimed to identify and quantify the presence of various bacterialspecies, including potential pathogenic organisms, to ascertain the overall safety of these beloved confections [3].

Food safety is a crucial aspect of public health, and the findings from this research are expected to shed light on the level of microbial contamination in milk-based Indian sweets and raise awareness among consumers, vendors, and regulatory authorities. Moreover, the identification of specific bacterial strains will help in understanding the sources and contributing factors of contamination, facilitating the development of targetedintervention strategies to mitigate risks and enhance the overall quality of these cherished desserts [4].

In the subsequent sections, we will detail the methods used for sample collection and analysis, present the results obtained from the multiple tube test, and discuss the implications of our findings for the consumers, food industry, and public health authorities. Additionally, we will propose recommendations and potential measures to ensure the microbiological safety of milk-based Indian sweets, thereby safeguarding the health andwell-being of the consumers and preserving the cultural significance of these delectable treats.

2. METHODOLOGY

  • Type of Study: Public health and microbiological study.
  • Study Design: Laboratory Based observational study.
  • Place of Study: Kolkata.
  • Samples: Milk based sweets samples
  • Method of Sampling: Random Sampling.
  • Sample Size: Sample size is 80.

3. RELATED WORK

3.1. Collection of Samples

All the aseptic precautions were taken to collect the sample avoiding external contamination. Sample were collected in sweets carry box. Each of the sample were directly brought to microbiology lab and then transferredto sterilized sample collection vials. Sample collection vials were labelled indicating the serial number of samples. At least 2-4 different nos. of sample were collected from different shops and vendor on the basis of variety of Sandesh they had in their menu. Maximum of 4 samples were collected from a particular shop.

3.2. Preparation of Samples

For first 14 samples (M 01 – M 14) the complete sweets (weight of sweets ranged from 18-22 g per piece) werediluted in 125 ml of normal saline (NS). Rest sweets sample (M 15 – M 80) were weight to 1.2 g to 1.3 g and diluted in 125 ml of normal saline.

3.3. Inoculation on Culture Media

One hundred five (105) ml of diluted sample was cultured for coliform count by multiple tube tests. 50 ml diluted sample is added to one bottle containing a 50 ml double strength MacConkey broth. 10 ml diluted sample added to 5 bottles containing 10 ml double strength medium MacConkey broth. 1 ml diluted sample added to 5 bottles containing 5 ml single strength MacConkey broth. The total volume tested was now 105 ml of diluted sample. The total number of tubes used for diluted sample culture was 11. After inoculation of sample, they were incubated at 37o C for 48 hours.

4. RESULTS AND DISCUSSION

4.1. Result Interpretation of Multiple Tube Method for coliform count [5]

By referring to the McCrady’s Probability Table the MPN can be derived and the following results were noteddown.

  • Excellent: If MPN zero (no growth in all the tubes).
  • Satisfactory: If MPN is 1-5 (number of tubes showing growth).
  • Indeterminate: If MPN is 6-10 (number of tubes showing growth) and needs more treatment to purity thesources.
  • Unsatisfactory: If MPN is above 10, this needs vigorous treatment to clear the contamination.

• Identification of Isolates

All of the distinguishing isolates were subjected to additional investigation for identification. The isolates wererecognized based on physical characteristics, cultural traits, and biochemical responses.

Table 1. Different Range of Colony Forming Unit
Table 1
Figure 1
Figure 1. Pie Chart Showing No. of Positive & Negative Samples for Coliform Bacilli by MPN Method

The above chart shows that 80 samples which were cultured on milk agar shows 80% positivity which meansthat out of 80 sample 64 samples were found positive and rest 16 samples were negative which means that there was no bacterial growth on media for these 16 samples.

Figure 2
Figure 2. Bar Diagram of No. of Coliform bacilli Count for M01 – M40

The above chart represents probable no. of coliform bacilli in 100 ml of diluted sample from M01 to M40 on multiple tube test method, the highest no of coliform bacilli was 180+ and lowest was zero (0).

M41M42M43M44M45M46M47M48M49M50M51M52M53M54M55M56M57M58M59M60M61M62M63M64M65M66M67M68M69M70M71M72M73M74M75M76M77M78M79M80

Figure 3
Figure 3. Bar Diagram of No. of Coliform Count for M41 – M80

The above chart represents probable no. of coliform bacilli in 100 ml of diluted sample from M41 to M80 on multiple tube test method, the highest no of coliform bacilli was 180+ and lowest was zero (0).

Table 2. Total No. of Coliform Bacilli & Others Isolates & their Percentage
Table 2

Out of 80 samples 64 samples were positive. In those Acinetobacter baumanni (2.5%), Aeromonas schubertii (1.25%), Citrobacter freundi (2.5%), Citrobacter koseri (8.75%), Enterobacter cloacae (15%), Escherichia coli (12.5%), Klebsiella pneumoniae (2.5%), Klebsiella aerogenes (28.75%), and Klebsiella oxytoca (6.25%) were present.

4.2. Discussion

The purpose of the current study was to use the multiple tube test to evaluate the microbiological quality of Indian sweets made with milk that were sold in and around Kolkata. Our investigation's findings showed a significant amount of microbial contamination in these classic treats, suggesting possible dangers to food safetyconnected with eating them.

Alarmingly, 64 (80%) of the 80 samples that were examined revealed the presence of germs. A wide variety of bacterial species are present, which emphasizes the need for increased awareness in monitoring the hygiene and production practices used during the manufacture and preservation of these delectable delicacies.

The two most common bacterial species found in the samples were Klebsiella aerogenes and Enterobacter cloacae, which together made up a significant 28.75% of the positive samples. Both Klebsiella aerogenes and Enterobacter cloacae are known opportunistic infections linked to foodborne diseases and are members of the Enterobacteriaceae family [6]. Concerns regarding the general microbiological safety of these milk-based Indiansweets are greatly heightened by their presence in such a high percentage of the samples.

12.5% of the positive samples contained Escherichia coli, another member of the Enterobacteriaceae family. The presence of E. coli is particularly concerning since some strains of this bacterium, especially in vulnerable populations, can result in severe food poisoning and serious health issues [7].

Other potentially dangerous bacteria were found in the samples in addition to members of the Enterobacteriaceae family [8]. In 2.5% of the positive samples, the multidrug-resistant bacterium Acinetobacter baumannii was found [9]. In 1.25% of the samples, Aeromonas schubertii was discovered, which is frequently discovered in aquatic habitats [10], [11]. Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, and Citrobacter koseri were found in 2.5%, 8.75%, 2.5%, and 6.25% of the positive samples, respectively. The presence of any pathogenic microorganisms in food items poses a possible health concern, even though the prevalence of these bacteria may appear to be minimal.

There are a number of reasons why milk-based Indian sweets might become contaminated with pathogenic and opportunistic bacteria, including poor handling techniques during preparation, poor storage conditions, and cross-contamination from machinery and surfaces in the production area [12].

Strict food safety procedures must be put in place across the whole production and distribution chain in order to address the problem of microbial contamination in Indian sweets made with milk. This involves maintaining ideal storage temperatures, adhering to good hygiene and sanitation procedures during the preparation process, and routinely checking the manufacturing environment for potential sources of contamination [13], [14].

To reduce the danger of bacterial contamination, food manufacturers and vendors must get education and training on the best practices for food safety. Furthermore, regulatory agencies should create and enforce for these items’ food safety requirements, doing routine inspections to verify compliance [15].

The samples' high concentration of these bacteria highlights the requirement for meticulous attention to food safety procedures during the preparation, storage, and distribution of Indian sweets made with milk [16]. To reducethe danger of microbial contamination and ensuing foodborne illnesses, proper handling, storage, and hygienepractices should be put in place.

It is essential to inform and train food manufacturers and vendors on best practices for food safety in order to guarantee the microbiological safety of these cherished delicacies. In order to ensure compliance, regulatory bodies should create and enforce strict criteria for food safety.

5. CONCLUSION

The results of this study highlight the pressing need to address the microbiological quality of Indian sweets made with milk that are sold in and around Kolkata. The risk to one's health from consuming these common desserts is highlighted by the high frequency of pathogenic and opportunistic germs. We may work to ensure the safe consumption of these customary foods while retaining their cultural value by establishing efficient food safety procedures, increasing awareness among customers and food vendors, and enforcing regulatory standards. To follow the success of these treatments and ensure the regional supply of milk-based Indian sweets is microbiologically safe, ongoing research and observation are required. Protecting consumer health and promoting food safety should be the collective responsibility of all stakeholders involved in the production and sale of these beloved delicacies. All parties engaged in the manufacture and distribution of Indian sweets made with milk should take action in light of the study's findings. It is possible to reduce the hazards associated with microbiological contamination and improve the general quality and safety of these traditional sweets by implementing efficient food safety procedures, increasing consumer knowledge, and enforcing regulations.

The microbiological quality of Indian sweets made with milk must continue to improve, thus more investigation and monitoring are required to assess the efficacy of initiatives. By placing a high priority on food safety, we canenjoy these delectable delicacies without endangering consumer health and keep their cultural relevance for upcoming generations. Milk based sweets contains coliforms which can come from milk, milk handler or sweetsprocessing plant.

5.1. Abbreviations

  • AIIH & PH: All India Institute of Hygiene and Public Health
  • E. Coli: Escherichia coli
  • NS: Normal Saline
  • MPN: Most Probable Method M 01, M 02…...: Name of sample

Acknowledgments

The authors have no specific acknowledgments to make for this research.

Funding Information

This research received no specific grant from any funding agency in the public, commercial, or notfor-profit sectors.

Author Contributions Statement

Name of AuthorCMSoVaFoIRDOEViSuPFu
Dr. Manoj Yogi✓✓✓✓✓✓✓✓✓
Dr. Sayan Bhattacharyya✓✓✓✓✓✓
Dr. Atul Raj✓✓✓✓✓✓✓✓
Amit Banik✓✓✓✓✓✓✓✓

C: Conceptualization; M: Methodology; So: Software; Va: Validation; Fo: Formal analysis; I: Investigation; R: Resources; D: Data curation; O: Writing – original draft; E: Writing – review & editing; Vi: Visualization; Su: Supervision; P: Project administration; Fu: Funding acquisition.

Conflict of Interest Statement

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Informed Consent

All participants were informed about the purpose of the study, and their voluntary consent was obtained prior to data collection.

Ethical Approval

Not Applicable.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. [1] H. Pennington, 'Enterobacteria and bacterial food poisoning', in Oxford Textbook of Medicine, C. P. Conlon, Ed. Oxford University PressOxford, 2020, pp. 1033-1040. doi.org/10.1093/med/9780198746690.003.0111
  2. [2] M. Pal, S. Mulu, M. Tekle, S. Pinto, and J. Prajapati, 'Bacterial Contamination of Dairy Products', Beverage Food World, vol. 43, pp. 40-43, Jan. 2016.
  3. [3] 'Microbial testing - An overview of what it is and what it is for'. [Online]. Available: https://www.biosafe.fi/insight/microbial-testing.
  4. [4] 'Water Bacteriology: Introduction, testing methods, test requirements, test', universe84a, 04-Sept- 2021. [Online]. Available: https://universe84a.com/water-bacteriology-introduction/.
  5. [5] M. E. Nyenje, C. E. Odjadjare, N. F. Tanih, E. Green, and R. N. Ndip, 'Foodborne Pathogens Recovered from Ready-to-Eat Foods from Roadside Cafeterias and Retail Outlets in Alice, Eastern Cape Province, South Africa: Public Health Implications', Public Health Implications. Int J Environ Res Public Health, vol. 9, no. 8, pp. 2608-2619, Aug. 2012. doi.org/10.3390/ijerph9082608
  6. [6] Pennington H. Enterobacteria and bacterial food poisoning. In: Conlon CP, Firth J, Conlon C, Cox T, editors. Oxford Textbook of Medicine [Internet]. Oxford University Press; 2020 [cited 2023 Jul 19]. p. 0. doi.org/10.1093/med/9780198746690.003.0111
  7. [7] A. Elbehiry, E. Marzouk, I. M. Moussa, T. M. Dawoud, A. S. Mubarak, and D. Al-Sarar, 'Acinetobacter baumannii as a community foodborne pathogen: Peptide mass fingerprinting analysis, genotypic of biofilm formation and phenotypic pattern of antimicrobial resistance', Saudi J Biol Sci, vol. 28, no. 1, pp. 1158-1166, Jan. 2021. doi.org/10.1016/j.sjbs.2021.01.018
  8. [8] L. M. Martins, R. F. Marquez, and T. Yano, 'Incidence of toxic Aeromonas isolated from food and human infection', FEMS Immunol Med Microbiol, vol. 32, no. 3, pp. 237-242, Feb. 2002. doi.org/10.1111/j.1574-695X.2002.tb00559.x
  9. [9] J. M. Tomas, The Main Aeromonas Pathogenic Factors. ISRN Microbiol. 2012. doi.org/10.5402/2012/256261
  10. [10] S. P. Oliver, B. M. Jayarao, and R. A. Almeida, 'Foodborne pathogens in milk and the dairy farm environment: food safety and public health implications', Foodborne Pathog Dis, vol. 2, no. 2, pp. 115-129, 2005. doi.org/10.1089/fpd.2005.2.115
  11. [11] B. Jadranka, D. Naletina, and N. Iva, Food safety and food quality in the supply chain. 2015.
  12. [12] N. Burke, K. A. Zacharski, M. Southern, P. Hogan, M. P. Ryan, and C. C. Adley, The Dairy Industry: Process.
  13. [13] N. Burke, K. A. Zacharski, M. Southern, P. Hogan, M. P. Ryan, and C. C. Adley, 'The dairy industry: Process, monitoring, standards, and quality', in Descriptive Food Science, IntechOpen, 2018. doi.org/10.5772/intechopen.80398
  14. [14] 'Consumers and food safety: A food industry perspective'. [Online]. Available: https://www.fao.org/3/v2890t/v2890t05.htm.
  15. [15] P. Kaynar, 'Determination of microbiological properties of milk-based desserts presented for consumption in Ankara, Turkey', vol. 2020, July 2023. doi.org/10.31083/0003-925X-71-93.
  16. [16] W. J. Fischer, B. Schilter, A. Tritscher, and R. Stadler, 'Contaminants of Milk and Dairy Products: Contamination Resulting from Farm and Dairy Practices. Encycl Dairy Sci', pp. 887-897, Dec. 2011. doi.org/10.1016/B978-0-12-374407-4.00104-7

How to cite: Dr. Manoj Yogi, Dr. Sayan Bhattacharyya, Dr. Atul Raj, Amit Banik (2024). Isolation of bacteria from milk based Indian sweets sold in and around Kolkata using MPN method. Journal Healthcare Treatment Development, 4(01), 35-43. https://doi.org/10.55529/jhtd.41.39.47

Copyright © 2024 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY 4.0). Published by HM Publishers.