1. Himalayan college of agricultural science and technology, Nepal.
2. Himalayan college of agricultural science and technology, Nepal.
3. Veterinary Officer, Veterinary Laboratory, Surkhet, Nepal.
4. Himalayan college of agricultural science and technology, Nepal.
5. Himalayan college of agricultural science and technology, Nepal.

*             Correspondence: jasmineadhikary909@gmail.com

Keywords: chicken sausage, marketed frozen, microbial, Escherichia coli, bacterial isolation

1. INTRODUCTION

Normally the sausages are made from various of processes started from flaking, chopping of meats, mixing with different types of seasoning and stuffing into natural or synthetic casing and then cooking at high temperature either smoking or steaming. Convenience and diversity of products are the main reasons the sausages are commonly consumed nowadays [1]. The sausage production is divided into several steps firstly by reducing the particle size of meat. After that, the minced meat is mixed with other ingredients and seasoning [2]. Then, the paste was stuffed into specific casing and linked for specifically length and packaging finally [3,4]. Dennis and Stringer reported that, the important criteria in storing perishable products like sausages are the temperature monitoring and control during processing until storage [5]. Developed countries conduct regular surveillance studies on food borne bacterial pathogens and poultry has been identified as a major source of microorganisms leading to food borne diseases [6]. In developing countries, financial and technological constraints limit the ability to conduct regular surveillance and there is much less understanding about the causes of food borne infections, as highlighted by several authors [7,8]. Nevertheless, production and consumption of chicken meat has significantly increased in the South Asian region in the recent past [9]. The situation in Sri Lanka is no different, and according to the Department of Animal Production and Health (DAPH) poultry meat and egg production now contributes to more than 70% of the livestock sector [10,11]. Previous studies conducted in Sri Lanka have found contamination of poultry with E.coli [12,13]. Microbiological safety of food was a challenge 20 years ago and continues to be challenging with new ones emerging [14]. It was reported that  thirty-one foodborne hazards (including viruses, bacteria, protozoa, helminthes and chemicals) have caused 600 million foodborne illnesses and 420,000 deaths globally [15,16]. Among these, foodborne bacterial diseases account for the major part of the burden where Campylobacter, Salmonella and Escherichia Coli (E. coli) are reported most frequently [17]. The Centre for Disease Control and Prevention in the USA (CDC) has identified eight main pathogens as food contaminants and the three named above are the most important of these [18] .In Nepal, a very limited research work is done on the topic of microbial analysis of Chicken Sausage. Sausage, as an important vehicle associated with illnesses caused by food borne pathogens, which lead to the development of public health hazards and it is potentially zoonotic to humans [19,20] The present study was undertaken to provide a baseline for marketed frozen chicken sausages, marketed cooked chicken sausages, self-cooked chicken sausages, self-frozen chicken sausages isolated from chicken sausage markets and meat shops of Bhaktapur. Chicken meat due to its rich nutrient content is an ideal media for the growth of various microorganisms. Thus, the contaminated meat and meat products readily causes a variety of food borne disease and zoonotic diseases [21]. Chicken and Chicken sausages is the most consumed meat in the world and is one of major source of foodborne illness [22].Contamination of raw meat easily occurs from external sources. In fact, tissue from healthy animal are sterile however, it has been pointed that during slaughter, dressing and cutting, microorganisms came chiefly from the exterior of the animal and its intestinal tract but that more added from knives, cloths, air, carts and equipment in general [23]. External contamination of meat is a constant possibility from the moment of bleeding unit consumption [24]. The extent of microbial contamination and composition of microbial flora reflect the standard hygiene of meat. Food borne microbiologic hazards caused by E.coli are responsible for as many cases of illness and death as possible each year and are thus an important food safety challenge. According to the World Health Organization, 30% of inhabitants in industrialized countries suffer every year of food-borne diseases with most of the cases attributable to the consumption of meat. Non typhoid Salmonella serotypes are responsible for over 1.4 million cases, while pathogenic Escherichia coli, including E. coli O157: H7, is responsible for 270,000 cases of food borne illness in a year [25]. Moreover, with the rapid increase of meat industry, more people are involved in meat management, treatment, slaughter and handling, which increases the chances of acquiring zoonotic ally important bacterial diseases [26]. They also predicted a 67% increase in global consumption of antimicrobial in livestock production from 2010 to 2030.In context of Nepal, studies carried out on the total consumption of antimicrobial drugs in food animal found the total growth of 53.60% between 2008 and 2012, with an average annual growth of 11.40% and they concluded that the increased demand of veterinary pharmaceuticals including antimicrobials might be as a result of increasing commercialization of poultry and dairy sector [27]. Currently, approximately 80% food-producing animals receive medication for part or most of their lives [28], and hence has the potential to generate residues in animal derived products (meat, eggs and honey) and poses a health hazard to the consumer. The main objective of this study is, “is to investigate, and analysis presence of E. coli in cooked chicken sausage and frozen cooked chicken sausage”. Understanding the factors that might present provide valuable insights into improving consumer’s consuming habits of the choices of healthy chicken sausage from the different department store.

2. MATERIALS AND METHODS

The study was field-based study that’s why descriptive and lab experimental research design were adopted. Experimental studies involve an experiment of E. coli presence in the chicken sausage. As well, descriptive research described the phenomena that really happening as it exists the microbial analysis of marketed frozen chicken sausage, marketed cooked chicken sausage, self-frozen chicken sausage, self-cooked chicken sausage from different super markets, meat shops of Bhaktapur district was examine at standard laboratory of HICAST, Kirtipur, Kathmandu. The study was conducted on 110 chicken sausage meat technology samples, including 50 of marketed frozen chicken sausages (n = 50) and 50 of Marketed cooked chicken sausage (n = 50), 5 of self-frozen chicken sausages (n= 5), 5 self-cooked chicken sausages (n= 5) were purchased from sausage retail markets and meat shops as well. They were chopped into small pieces, and 25 g from each sample was transferred to 225 ml of 1% buffered peptone-water and incubated for 24 h at 25 or 37°C.Cultures were diluted to 10-4 in 0.1% peptone-water, and 100 all volumes of different dilutions were spread on different specific agar media. The plates were then incubated at 37°C for 48 h, suspected samples were confirmed by gram’s staining and biochemical testing. Samples were analyzed for Total Viable Count, the Primary data were collected from different departmental store, supermarkets, and meat shops as per the research objective. Samples were double-bagged at the source, refrigerated until delivery to the laboratory and then handled in such a manner as to prevent cross-contamination, and were examined within 1 day of purchase; they were chopped into small pieces, and 25 g from each sample was transferred to 225 ml of 1% buffered peptone-water and incubated for 24 h at 25 or 37°C Sausage preparation in Laboratory was carried out using curing method given by Frankfurter (16th Century) the recipe for sausage making is given below calculated for 5 kg bath, 50.00 % chicken meat trimmings, lean 2.5 kg, Chicken 25.00% fat emulsion 1.25 kg, 25.00% Ice (Drinking 1.25 kg water)Per kg raw materials a total for 5 kg, 18.00 g Nitrite curing salt 90.00 g, 3.00 g Phosphate PH>7.3) 15.00g, 0.30 g Ascorbic acid. 1.5 g Per kg raw materials) (Total for 5 kg), 3.00g white pepper ground 15 g, 1.00 g Nutmeg ground 5 g, 0.50 g Cardamom ground 2.5 g, 0.20 g coriander ground. 1g and, Total 10 chicken sausage was prepared. The prepared samples were cultured in Nutrient agar which were further purified in different culture media .Cultures were plated on selective and/or differential media, namely MacConkey agar, The plates were incubated at 37°C 24 h. Bacterial colonies in each medium were then characterized on the basis of colonial, cellular morphology and staining characteristics. The total viable count (TVC) was determined by standard pour plate method. One ml of each tenfold dilution were inoculated in Nutrient agar plate just before the solidification of agar. The plates were then kept in an incubator at 37°C for 24 hrs. The number of colonies in particular dilution was multiplied by the dilution factor to obtain the TVC. One lapful of pre-enriched sample in buffered peptone water was inoculated in selective media i.e. Eosin methylene blue agar and one petri plate was kept as control. Then the petri plates were incubated at 37°C for 24 hours. Appropriate biochemical tests were done for the identification of bacteria i.e. Triple Sugar Iron (TSI) Test, Indole Production Test, Citrate Utilization Test, Methyl Red (MR) Test, Vogues-Proskauer (VP) Test, Oxidase Test, Catalase Test, For the determination of E. coli, TVC method ISO 4833-1:2013 was employed using MacConkey Agar plates instead of NA as above. For identification of E. coli, the pink colony grown in MacConkey Agar plates were re-cultured in individual tubes. Various sources and techniques were used for the collection of necessary information as described under. Both primary and secondary sources of data were collected and analyzed. A total of 110 chicken sausage packet (0.5kg) samples will be collected from different sausage selling local shops and supermarkets randomly around Bhaktapur Metropolitan city. From one chicken sausage packet, only one sausage has selected randomly as a representative sample for the test. Secondary data as published articles will be collected from the publications of Department of Livestock Services (DLS), Directorate of Animal Health of DLS, Nepal Agricultural Research Council (NARC), District Livestock Services Offices (DLSOs), Central Bureau of Statistics (CBS).

3. RESULT & DISCUSSION

The collected primary data was interpreted using SPSS 25.0 software, and the findings are identified E. coli in the chicken sausages in the laboratory and isolate E. coli for quality evaluation are presented.

Table 01: Categorical percentage of different Samples

		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	Marketed Cooked	50	45.5	45.5	45.5
	Marketed Frozen	50	45.5	45.5	90.9
	Self-cooked	5	4.5	4.5	95.5
	Self -frozen	5	4.5	4.5	100.0
	Total	110	100.0	100.0

Results displays the utilization of the “Examine Variables = Category” command in SPSS, which is employed to generate descriptive statistics and graphs for a single variable. In this instance, the variable being assessed is “category” which serves as a measure of presence of data. The output of this command encompasses the following components, Table presents the number of cases that are considered valid, meaning they contain complete data, as well as the number of cases with missing data. In this specific analysis, there are 110 valid cases and no missing cases. It provides a concise overview of the distribution of the “category” variable, which represent to the survey of the different categorical data. The breakdown of responses is as mentioned below.

Table 02: Frequency & percentage of different samples

		Frequency	Percent
Valid	Marketed-cooked	50	45.5
	Marketed-frozen	50	45.5
	Self-cooked	5	4.5
	Self-frozen	5	4.5
	Total	110	100.0

Above table shows that a total of 110 samples were collected, there are four categorical date i.e. marketed-cooked, marketed-frozen, self-cooked and self-frozen. The frequency of marketed-cooked is 50, marketed-frozen is 50, self-cooked is 5 and self-frozen is 5. The percentage of marketed-cooked, marketed-frozen, self-cooked and self-frozen are respectively 45.5%, 45.5%, 5% and 5%.

Figure 01: Categorical percentage of different samples

Table 03: Category with Results

Categorical Percentage Result
			Result		Total
			Negative	Positive
Cat	Marketed Cooked	Count	50	0	50
		% within cat	100.0%	0.0%	100.0%
	Marketed Frozen	Count	45	5	50
		% within cat	90.0%	10.0%	100.0%
	Self-Cooked	Count	4	1	5
		% within cat	80.0%	20.0%	100.0%
	Self-Frozen	Count	3	2	5
		% within cat	60.0%	40.0%	100.0%
Total		Count	102	8	110
		% within cat	92.73%	7.27%	100.0%

Table shows that a total of 110 samples were collected, there are four categorical data i.e. marketed-cooked having negative result contains 100% containing 50 samples, marketed-frozen having negative result 90% containing 45 samples, self-cooked having positive result 80% containing 5 samples and self-frozen 60 % containing 3 samples. Similarly, the percentage of marketed-cooked having positive result is 0% containing 0 samples, marketed-frozen of positive result is 10% containing 5 samples, self-cooked is 20% containing 1 sample and self-frozen is 40% containing 2 samples. Also, total positive result 92.73% containing 102 samples and total positive result 7.27% containing 8 samples.

Figure 02: Categorical Percentage Result

Table 04: Result (Positive and Negative)

		Frequency	Percent
Valid	Negative	102	92.73
	Positive	8	7.27
	Total	110	100.0

Table shows that frequency of negative result is 102 samples contains 92.73% and frequency of positive result is 8 samples contains 7.27% a total of 110 samples.

Figure 03: Result of Positive & Negative

Table 05: Result of Chi-Square Test

	Value	df	Asymptotic Significance (2-sided)
Pearson Chi-Square	8.759a	3	0.033
Likelihood Ratio	7.271	3	0.064
Linear-by-Linear Association	4.273	1	0.039
N of Valid Cases	110
a. 4 cells (50.0%) have expected count less than 5. The minimum expected count is 1.14.

The Pearson Chi-Square value of the above test is 8.759 with 3 degrees of freedom. Their associated p-value is 0.033. The test examines the associations between different four categories marketed cooked, marketed frozen, self-cooked and self-frozen of the collected samples. In this case, p-value is smaller than the chosen significance level (e.g., 0.05), indicating that there is significant association between four categories i.e. marketed cooked, marketed frozen, self-cooked and self-frozen of the collected samples. It means that four categories are significantly association. Furthermore, there is significance difference between marketed frozen and self-frozen meat on sausage making. The Linear-by-Linear association chi-square value is 4.273, with 1 degrees of freedom. The associated p-value is 0.039. This test specifically examines the linear trend in the association between the four categories of samples collection.  N of Valid Cases represents the number of valid cases or observations included in the analysis, which in the case is 110.

Table 06: Report of TVC Count

The average TVC of Self cooked (SC) is 5.27, the average TVC of marketed frozen (MF) is 5.32, the average TVC of self-frozen (SF) is 5.40. Standard value of E.coli is 20 to<100 cfu/g. Our value is less than 20 which is acceptable for human consumption.

4. CONCLUSION

Chicken sausage is consumed as a common food by the non-vegetarian population in Nepal and can be considered as an important vehicle associated with foodborne pathogens, posing a public health risk. It is potentially zoonotic to human health. The production of sausages is divided into several stages by first reducing the particle size of the meat. Then, the minced meat is mixed with other ingredients and spices. After that, the dough is filled into a specific casing and finally fixed specifically in terms of length and packaging. This was conducted on broiler chicken meat samples to identify the prevalence of Escherichia coli infection study on chicken sausages. Finding of this study determined that there is high level of risk of microbiological contamination of products manufacture at meat plant and cold store plant investigated. It seems that rough handling, improper packaging and substandard storing practice may be the probable explanation for the contamination with bacteria. Raw meat used in the production of Sausage should be in compliance with the provisions of the Code of Hygienic Practice of Fresh meat. An adequate personal hygiene should be maintained at all times during sausage production. All equipments and utensils coming in contact with meat should be sanitized with chlorinated water or hot water stream after cleaning with detergent.

 REFERENCES

1. Das, A., Bhattacharya, D., Nanda, P. K., Nath, S., & Das, A. K. (2024). Biopreservation in Meat and Meat Products. In Novel Approaches in Biopreservation for Food and Clinical Purposes (pp. 66-97). CRC Press.
2. Jaradat, Z. W., Abulaila, S., Al-Rousan, E., & Ababneh, Q. O. (2024). Prevalence of Escherichia coli O157: H7 in foods in the MENA region between years 2000 and 2022: a review. Arab Journal of Basic and Applied Sciences, 31(1), 104-120.
3. Meem, F. C., Shourove, J. H., Raihan, T., Azad, A. K., & Islam, G. R. (2024). Antibiotic resistance of ESBL-producing E. coli and other gram-negative bacteria isolated from street-vended foods in Bangladesh. Journal of microbiology, biotechnology and food sciences, 13(6), e9429-e9429.
4. Chen, P., Cheng, F., Huang, Q., Dong, Y., Sun, P., & Peng, Q. (2024). Distribution and Antimicrobial Resistance Characterization of Listeria monocytogenes in Poultry Meat in Jiading District, Shanghai. Journal of Food Protection, 87(3), 100234.
5. Serter, B., Önen, A., & Ilhak, O. I. (2024). Antimicrobial efficacy of postbiotics of lactic acid bacteria and their effects on food safety and shelf life of chicken meat. Annals of Animal Science.
6. Mahgoub, S. A., Qattan, S. Y., AlMalki, F., Kamal, M., Alqurashi, A. F., Almuraee, A. A., … & Taha, A. E. (2024). Impact of packaging atmosphere, oregano essential oil, and storage temperature on cold-adapted Salmonella Enteritidis and Salmonella Typhimurium on ready-to-eat smoked turkey. Poultry Science, 103(7), 103846.
7. Douglas, S. (2024). Evaluating Cookery Characteristics, Consumer Acceptability, and Electronic Assessment of Attributes in Ground Beef Patties (Master’s thesis).
8. Grigore-Gurgu, L., Bucur, F. I., Mihalache, O. A., & Nicolau, A. I. (2024). Comprehensive Review on the Biocontrol of Listeria monocytogenes in Food Products. Foods, 13(5), 734.
9. Pegg, E., Jackson, J., Evans, C., & Cohen, V. (2024). News and alerts News and alerts.
10. Cooper, A. L., Wong, A., Tamber, S., Blais, B. W., & Carrillo, C. D. (2024). Analysis of Antimicrobial Resistance in Bacterial Pathogens Recovered from Food and Human Sources: Insights from 639,087 Bacterial Whole-Genome Sequences in the NCBI Pathogen Detection Database. Microorganisms, 12(4), 709.
11. Omara, S. T., & Eljakee, J. A. K. E. E. N. (2024). Microbiological Assessment of Various Ready-To-Eat Foods in Cairo, Egypt, and Studying the Possible Antibacterial Effects of Garlic and Cumin Oils as Food Additives. Egyptian Journal of Veterinary Sciences, 55(1), 243-257.
12. Kumari, D., Chaudhary, P., & Janmeda, P. (2024). Genetic Engineering and Designed Promising Preservative in Food Products. Nonthermal Food Processing, Safety, and Preservation, 299-324.
13. Divanshi, M., Das, A., & Hv, R. (2024). Quality Control and Risk Assessment of Food Storage and Packaging. In Food Safety (pp. 25-40). CRC Press.
14. Shaltout, F. A. (2024). Values of Essential Oils of Plant Origin on The Micro-Organisms During the Meat Storage. Clinical research and Clinical reports, 3(4).
15. Goswami, D., Mondal, S., Hor, P. K., Santra, S., Jana, H., Gauri, S. S., … & Mondal, K. C. (2024). Bioprospecting of probiotic bacteria from traditional food of high-altitude Himalayan region. Food Bioscience, 57, 103257.
16. Garofalo, G., Ponte, M., Busetta, G., Barbera, M., Tinebra, I., Piazzese, D., … & Settanni, L. (2024). Microbial dynamics and quality characteristics of spontaneously fermented salamis produced by replacing pork fat with avocado pulp. Food Microbiology, 122, 104536.
17. Shaltout, F. A. (2024). Egypfian Medicinal Plants and Respiratory Disease. Journal of Agriculture and Educafion Research. 2 (3), 1, 7.
18. Akullo, J. O. (2024). Physicochemical Characteristics, Microbial Quality and Sensory Acceptability of Cricket (Gryllus bimaculatus) Flour Preserved With Ginger, Garlic and Turmeric Extracts (Doctoral dissertation, JKUAT-CoANRE).
19. Shaltout, F. Meat Examination in the Laboratory, the Accepatablity and the Human Health.
20. Krikorian, A. (2024). Maintaining Meat Quality through the Beef Supply Chain (Master’s thesis).
21. Gao, X., Pourramezan, H., Ramezan, Y., Roy, S., Zhang, W., Assadpour, E., … & Jafari, S. M. (2024). Application of gums as techno-functional hydrocolloids in meat processing and preservation: A review. International Journal of Biological Macromolecules, 131614.
22. Hamilton, A. N. (2024). Determination of Factors Influencing Microbial Food Safety Risks of Additive Manufacturing and 3D Printing of Food.
23. Chaudhary, V., Kajla, P., Lather, D., Chaudhary, N., Dangi, P., Singh, P., & Pandiselvam, R. (2024). Bacteriophages: a potential game changer in food processing industry. Critical Reviews in Biotechnology, 1-25.
24. Chaudhary, V., Kajla, P., Lather, D., Chaudhary, N., Dangi, P., Singh, P., & Pandiselvam, R. (2024). Bacteriophages: a potential game changer in food processing industry. Critical Reviews in Biotechnology, 1-25.
25. Shaltout, F. A. (2024). Dietary Nourishment and Food Processing Techniques.
26. Je, H. J., Kim, U. I., & Koo, O. K. (2024). A comprehensive systematic review and meta-analysis of Listeria monocytogenes prevalence in food products in South Korea. International Journal of Food Microbiology, 110655.
27. Vihanová, K. (2024). Chemical composition and antimicrobial activity of essential oils and supercritical carbon dioxide extracts of Asian spices against food pathogens in liquid and vapour phase (Doctoral dissertation, CZECH UNIVERSITY OF LIFE SCIENCES PRAGUE).
28. Liu, M., Zhang, X., Wei, A., Li, H., Zhang, H., Zheng, L., … & Wang, J. (2024). Protein‐based active films: Raw materials, functions, and food applications. Comprehensive Reviews in Food Science and Food Safety, 23(2), e13302.