Research Totoo Na To!!!!!!!!! (repaired).docx

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BACTERIAL IDENTIFICATION OF STREETFOODS IN SELECTED STALLS IN BAGUIO CITY Baquiran, Jesibel Marie T. Catores, Althea Gale C. Kazmi, Nida Hyder M. Wadwadan, Justine Faith B. Yamoyam, Alyzza Twain C. Senior High School

ABSTRACT

Keywords: Bacteria, Street foods, Biochemical test, media agar, hygiene

INTRODUCTION “Street Food” refers to a vast variety of foods and quencher prepare by vendors. It is being sold in the streets, around trading centers, terminals and public places. Street food has become popular given to be affordable and as a main source of livelihood. However street food vendors overlook the importance of proper handling, safety and nutritional quality of the food they sell which causes foodborne diseases which remain notable cause of morbidity and preventable death (Buted et al., 2014). Food is a necessary part in the life of everyone; it provides the basic necessities we need in order for our human body to activate which is useful in sustaining our health. Consumption of

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food comes hand in hand in the various roles in our body. In addition, food classification varies on the type of food we are consuming daily (Concern World, 2018). Studies show that extensive number of outbreak due to foodborne diseases have been documented international. Even developing countries were affected with this type of diseases. Annually these types of illnesses affect one-third of the population which remains the notable cause of morbidity and preventable death. Due to foodborne illnesses 48 million of people acquired this sickness, among the number who had been hospitalized, total of 3,000 patients were reported deceased. Infections in most of the cases are due to variety of bacteria, viruses and parasites (CDC, 2018). Prevalence of foodborne bacteria according to Minnesota Department of Health (MDOH) such as Escherichia coli (E. coli) causes bloody diarrhea and sometimes severe abdominal cramps, and vomiting. (MDOH, 2018), Listeria monocytogenes is another bacteria which causes muscles aches, and sometimes nausea. Another is Clostridium botulinum, tricky bacteria which makes people experience early flulike symptoms such as lethargy and muscle weakness, blurry vision, difficult swallowing and dry mouth. Next is the Campylobacter associated, if the poultry is raw or uncooked which spread to the blood that may cause arthritis and Guillain-Barre’s Syndrome. Lastly, the Salmonella enteritidis generally found in meat and poultry that are undercook. It affects the body producing stomach cramps, and plaguing fever associated with diarrhea. (Eating well, 2017). Reported cases of foodborne bacteria in United States According to National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), 1,600 estimated people were affected by listeriosis and 260 cases of death was recorded annually (NCEZID, 2018). Salmonella enteritidis outbreak linked to raw frozen products produced onset dates ranged from May 9, 2015 to July 22, 2015 in Minnesota. Five people has been infected with two strains of Salmonella enteriditis, victims ranged from 14 years to 68 years old, where 30 is the median age and 60% were male. Two (40%) were hospitalized and there was no deaths reported. The U.S Department of Agriculture’s Food Safety and Inspection Service (USDA-FSIS)

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investigated this outbreak and thus on September 17, 2015 it results that Aspen Foods carries salmonella enteritidis in their food production. (NCEZID, 2015). Total of 33 people in four states: Arizona (1), California (28), Texas (1) and Washington (3) were reported due to infections regarding ready-to-eat salad contaminated with Escherichia coli O157:H7 produced by Glass and Onion Catering sold at Trader’s Joe groceries. Two ill persons developed Hemolytic Uremic Syndrome (HUS). Public health investigators used DNA “fingerprints” of E. coli bacteria obtained through diagnostic testing with pulsed-field gel electrophoresis, or PFGE to identify cases of illness that were part of the epidemic. (NCEZID, 2013) Reported case of foodborne bacteria in the Philippines January 7, 2018, a recent case of food poisoning has been reported; 21 people to be hospitalized. Feeling dizzy and nauseous are the symptoms the victims had experienced after eating ‘Ihaw- ihaw’ located in John Abad Santos Avenue in Tondo vended by a 55 year old Divina Caballero who claimed the food she sells are clean. FDA already started the investigation regarding this issue, and they had gotten product samples to test. (Balita, p.4). An article in Food Science and Technology Research in 2004 regarding ‘’kwekkwek’’was proven to have carried foodborne bacteria such as Salmonella with just testing 25g of sample. Reported case of foodborne bacteria in Baguio City

Recent news in Northbound Philippines states that 37 Benguet students downed by food poisoning in Buguias due to contaminated candies bought outside school. These contaminated candies caused severe stomach ache and vomiting to the students who were directly rushed to Atok District Hospital (Dennis, 2018) Based on statistics, the cases of Food and Waterborne Diseases (FWBD), reported by City Health Office in Baguio City, from 410 cases in 2005 hiked up to 1,938 cases in 2010. However, health workers who monitored conclude the total cases was said to be highest in 2009 and had a total reported case of 2,082 increased by 60.7 % in 2008. Foodborne bacteria such as

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Amebiasis, Leptospirosis, Thyphoid fever, Cholera, Gastroentritis and Hepatitis A are the common causes of diarrhea resulting ingestion of contaminated food. (NSCB 2011) Review of Related Literature The results of the study entitled, “Microbiological Safety of Street Vended Foods in Jigjiga City, Eastern Ethiopia” clearly demonstrated that the street-vended foods in Jigjiga were contaminated with different pathogenic bacteria. Furthermore, the existence of these bacteria in foods could induce potential health problems for consumers. Poor personal hygiene, improper handling and storage practice of foods and poor knowledge of food vendors about food borne disease were the associated risk factors to contamination of street vended foods in Jigjiga. (Bereda, Emerie, et al., 2016) A study was conducted in Kathmandu named; “Analysis of street foods of Kathmandu for bacterial contamination”. It was performed in 12 different street foods. The surveillance study was carried in 200 children of primary grade from public school and 12 street vendors for the health hygiene and hazards associated with street food. Poor hygiene practice in preparation and handling of street food has been observed in the vendors. The lack of the knowledge in vendors about the source of bacterial contamination and absence of surveillance on street food has subjected street food to the high potential for food borne illness. The inadequate safety measure adopted by the targeted consumers of street food, the children, has augmented the risk associated with street food. All the food samples analyzed were contaminated with bacteria. The mesophilic count was recorded highest in Panipuri while as coliform count was highest in Chana tarkari. The least count of both was observed in Aaloo chop. Highest number of Staphylococccus aureus was found in Kerau (1.5X103cfu/g) and lowest in Momo (8.3 cfu/g). The dominant bacteria contaminating the food was S. aureus followed by Bacillus alvei, Escherichia coli, Enterobacter aerogenes, Bacillus subtilis, Serratia sp., and S. saprophyticus. The contaminated hand and clothing of the person who prepare food are the major source of S. aureus. Highest percentage of E. coli found in Panipuri must be due to the use of contaminated water. Chana zchatpate and Chana tarkari were the foods found to be contaminated with Salmonella sp. The type of food and the degree of hygiene practice adopted by vendor reflects the type and magnitude of bacterial contamination. Implementation of hygienic practices in vendors may reduce the contamination of street food and health education of the school children will curtail

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the incidences of food borne illness. Periodical monitoring of quality of street food will avoid any future outbreaks of bacterial pathogen. (R. TULADHAR, 2012) In Calcutta, research entitled “Street Vended food in Developing World: Hazard Analyses.” Samples that were suspected of adulteration were analyzed and in 30 of the 50 samples, unauthorized food additives were detected. Similarly, pathogen like Bacillius cereus, Clostridium pertringens, Vibrio metschinikovii and E. coli were reported in raw chicken, salad and gravy raw materials. These organisms were probably present in these foods either prior to purchase by vendors or may have been introduced by cross-communication during food handling or during preparation. (Rare, 2011) Conceptual Framework A cross-sectional study was conducted on street foods in Hawassa City from May to September 2014. A total of 72 samples from six food items such as local bread, raw fish, chilli, avocado

and

cooked

potato

were

made

following

standard

microbiological

techniques.(Tesmegan et.al.,) About 31% of the samples showed total colony counts ranging from 1.7*10^5 to 6.7*10^6 colony-forming unit per gram (CFUL/g) which is beyond the acceptable limits set for microbiological quality of ready-to-eat foods. The mean coliform and Enterobacteriacea counts raw fish, ‘kita’, and ‘ambasha’ were also higher than the limits. E.coli was the most frequent isolate (29.6%) followed by Salmonella species (12.7%) and S.aureus (9.9%). All isolates were 100% sensitive to ciprofloxacin. About 89% of Salmonella sp was resistant to Chloramphenicol. Alarmingly, 14.3% of S.aerus was resistant to vancomycin. (2016) Significance of the Study This study served as a notify whether the street food we eat is contaminated with bacteria that can cause foodborne diseases to consumers of “fishball’’, “kikiam’’, and “tokneneng’’ in particular stalls in Baguio City. By discussing the results personally with the results on hand and will stay confidential. In addition, results had been disseminated to the stalls and thoroughly explain what the findings is all about wherein it can help the vendor prevent different kinds of pathogenic bacteria

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Objective of the Study This study aim to determine the presence of Escherichia Coli (E. Coli) and Salmonella in selected street foods. MATERIALS AND METHODS The details of methods and materials that had been used in conducting the research are presented in this section of the study, such as: study design, data gathering tools and procedures. Study Design This study is under the form of quantitative research and experimental in nature for it uses necessary laboratory procedures in order to achieved the objective of the study that able to identify the presence of bacteria. Sample of the Study The researchers used street foods, specifically fishball, kikiam and tokneneng that are collected in three different stalls around Baguio City. Materials

MATERIALS

DESCRIPTION

1 gram Streetfood

Samples to be used

(fishball,kikiam, tokneneng) Sterilized Container

For sample collection

Distilled Water

Medium for samples

Inoculating loops

For inoculation of food samples

Trypticase Soy Broth (TSB)

For growing present bacteria in food samples

Analytical Balance

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Watch Glass

For Media Preparation

Alcohol Lamp Screw Caps Blood Agar Plate

Enrichment medium

Mannitol Salt Agar Plate

Isolation of gram positive cocci

Eosin-Methylene Blue agar

Isolation of gram negative bacilli

Plate MacConkey Agar Plate

Isolation of gram negative bacilli

Slides

For gram staining, biochemical tests

Normal Saline Solution bottle

For emulsifying samples for gram stain

Gram Stain Set

Initial identification of bacteria

Reagents and media for Biochemical Testing 70% Alcohol 40% Alcohol 10% Phenol Solution 10% FormalinSterile Microbial Disk

For bacterial identification

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Data Gathering Procedures The researchers conducted an experiment in the University of Baguio Laboratory to detect presence of bacteria in street food.

1 gram of food samples + 9 ml of Distilled water

amam Screw cap (Mixing) m

Inoculation to TSB, then incubation at 37OC for 48 hours

Inoculation onto BAP, MSA, EMB, McConkey incubation 35-37 OC for 24-48 hours

Colony morphology and evaluation

Gram stain then biochemical testing (IMViC)

Bacterial identification and reporting

Ethical Consideration

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Food stalls had been randomly selected and the same time they were given a consent letter. In which the letter explains what the study is all about, and upon giving the consent letters the researchers thoroughly explained the significance and objective of the study. This letter also ensures the cooperation and awareness of each vendor, and that the collected foods will only be used in conducting the research. Moreover, the vendor’s name and stall location will be treated confidential and only be used for research purposes. In performing the experiment, the researchers followed the laboratory rules, such as: wearing of lab gowns, masks, gloves and tying of hair. In addition to that, the researchers was accompanied by a technical adviser who guided them on what must be done and guaranteed that there were no afflictions occurred in the process. After conducting the experiment, wastes were properly disposed of, handled with the use of biohazard plastics and reagent bottle. Furthermore the laboratory areas and apparatus have been washed and dried accordingly. Lastly for the results attained the researchers went back to notify the involved stall owners regarding the research findings.

RESULTS AND DISCUSSION Table 1

Colonial Morphologies and Characteristics in the Different Culture Media SAMPLES

BAP Grayish white,

A (k)

glistening colonies; round pinpoint colonies

EMB

MSE

Round colonies; some with

White, pink,

brilliant green

colorless round

sheen; some

colonies

No growth

colorless Brilliant greenish

A (f)

MAC

White, round

sheen colonies;

colonies

black; some pink colonies

Light pink and dark pink colonies

No growth

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White pinpoint,

A (t)

mucoid colonies;

Black colonies

some grayish

with brilliant

white dry

greenish sheen

Fuchsia pink colonies

No growth

colonies

Table 1 shows the Colonial Morphologies and Characteristics in the Different Cultured Media particularly on Stall A. In Eosin Methylene Blue (EMB) evaluation presented in the table above, there are green sheen present in Stall A kikiam (k) and fishball A (f) which is a characteristic of coliform bacteria leading to Escherichia coli, while in Manitol Salt Agar (MSA) it has no growth. As for the Mac-Conkey Agar Plate (MAC) and Blood Agar Plate BAP although there are observation stated it does not really leads to any harmful bacteria as compared to EMB. (Related to the result were a study conducted by Nick Alonzo entitled Evaluation of street food vended in Ozamis City in which 69 food samples

from examined 173 menu items,

classified as snack foods, main dishes, sauces, and cold dishes were contained by a bacteria classified as E. coli (39.9%) based on the result. In addition, microbial quality of most street foods within the acceptable limit was less than 5.0 log10 (tu/g), but samples of buko juices, fruit salads, burger bun with spaghetti, and pansit with pork had unacceptable levels of contamination with a total mean logo (tu/g of 7.1, 7.0, 6.1 and 6.0) respectively.) As for the study entitled Foodborne Pathogens Recovered from Ready to eat Foods from Roadside Cafeterias and Retail Outlets in Alice South, Africa, findings were concluded and had proven that Salmonella spp. and Escherichia coli were not isolated in any of the 252 collected samples from vended stalls that had been tested. However, other type of bacteria such as Listeria spp (22%), Enterobacter spp (18%) Aero monas hydrophila (12%), Klebsiella oxytoca (8%), Proteus mirablis (6.3%), Staphylococcus aureus (3.2%), and Pseudomonas leutola (2.4%) were present. The results indicate that most of the ready to eat food samples did not meet bacteriology quality standards, therefore it impose potential risk to consumers. Time temperature abuse, exposure of food to flies and poor hygiene practices during preparation and serving were likely the risk factors for contamination.

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Addition to Table 1 under stall A is the tokneneng which has appeared to have greenish sheen as well in agar Eosin Methylene Blue (EMB), where in the sample underwent Biochemical Testing that further proved the existence of salmonella specie. In Chitagong City a study was conducted to determine the contamination of salmonella in different types of street foods in different places. A total of 76 shops from 120 food samples of ten different types were collected. Microbiological examination of salmonella was done after dividing the food into two categories of dry and wet food where overall about 28% samples where positive. The comparatively high bacteria in wet samples indicated contamination from water, practice of inadequate hygiene measures, mishandling, improper storage, inadequate cooking and above all unhygienic condition of the retail shop.

Table 2

Colonial Morphologies and Characteristics in the Different Culture Media SAMPLES

B (k)

BAP Grayish white shiny colonies

EMB Black with Brilliant green sheen colonies

MAC Fuchsia pink colonies

MSE

No growth

Change of color B (f)

White pinpoint

Colorless

of media to

Colorless dry

colonies

pinpoint colonies

yellow; pink

colonies

colonies

B (t)

White pinpoint

Grayish with

colonies; grayish

brilliant green

white colonies

sheen

Colorless colonies; some dark pink

No growth

colonies

For Table 2 it will be focusing more on Stall B kikiam (k) and tokneneng B (t). Tokneneng which came out with a characteristic of grayish with brilliant green sheen showing that it’s bacteria is Salmonella, while in B (kikiam) coliform bacteria has been proven in Eosin

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Methylene Blue (EMB) a selective media for Enterobactericeae with the characteristic of black brilliant green sheen colonies having a result of non-pathogenic enterobacteria species. However, for the agar MSA there is no growth present. The finding of the study is in agreement with the study conducted by Y. Haryani (2008). A total of 78 samples comprising different types of street foods, sold in different locations in Malaysia, were examined for the presence of Enterobacter cloacae. E. cloacae contamination was recorded in 9% of the samples examined. Tests for susceptibility to 12 different antibiotics showed that all were resistant to six or more antibiotics, but susceptible to chloramphenicol and gentamicin. Plasmids of four different sizes were detected from the three plasmid positive isolates. RAPD analysis using four primers yielded completely different banding patterns for all E. cloacae related disease is available. However, their occurrences have provided compelling evidence that the risk of disease transmission caused by E. cloacae through street foods is moderate. Four nonpathogenic E. coli biotype I strains isolated from cattle hides (fluorescent protein–marked mutants) were compared with four E. coli O157:H7 strains isolated from cattle fecal samples and five Salmonella serovars isolated from cattle and beef (27). To validate the proposed surrogates, Cabrera-Diaz et al. (27) compared the strains for (i) growth parameters, (ii) acid resistance, (iii) thermal resistance, (iv) cell surface hydrophobicity, (v) attachment to beef carcass surfaces, and (vi) response to antimicrobial interventions on beef carcasses. No differences were observed in maximum population density and lag time between fluorescent E. coli mutant strains and target pathogens (P > 0.05). However, two Salmonella serovars, Montevideo and Typhimurium, had lower growth rates and longer doubling times than did the mutant strains (P < 0.05). Log reductions of fluorescent mutants were not different from (P > 0.05) or were lower than (P < 0.05) log reductions for E. coli O157:H7 and Salmonella strains after exposure to pH 2.5, 3.0, and 3.5 for 0.5, 1.0, 1.5, and 2.0 h. Mean D-values for the fluorescent protein–marked strains were not different (P > 0.05) or were higher (P < 0.05) than those observed for E. coli O157:H7 and Salmonella strains after hot water interventions at 55, 60, and 65°C. Hydrophobicity values for the acid-adapted mutant surrogates (25.3 to 50.1%) were not different from those of E. coli O157:H7 (25.7 to 52.5%) or Salmonella strains (19.4 to 44.9%) (P < 0.05). Loose and strong attachments to different beef carcass regions (hot-boned

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outside rounds, briskets, and clots) also were tested. Mean counts of strongly attached cells were not different between surrogates and target pathogens for the same carcass region (P > 0.05). After application of a high-pressure initial water wash, population reductions were 1.5 to 2.1 log CFU/cm2 and were not different among potential surrogates and target pathogens (P > 0.05). Microbial reductions induced by the initial water wash followed by a hot water wash (95°C) were 2.8 to 3.7 log CFU/cm2, with no significant differences between surrogates and target pathogens (P > 0.05). The initial water wash followed by a 2% L-lactic acid spray reduced bacteria by 3.2 to 5.6 log CFU/cm2, which was significantly lower for the surrogates than for the target pathogens (P > 0.05) and would provide a margin of safety when used with this inactivation intervention regimen in future confirmatory challenge studies. The authors summarized that the fluorescent protein–marked E. coli strains were appropriate surrogates for E. coli O157:H7 and Salmonella for validation of hot water washes and lactic acid sprays on beef carcasses. Another study is reported by Kawser (1997) which showed that 49.58% of their sampled street foods were contaminated and 50.52% were sterile. This low contamination of present bacteria in the study, include solely salmonella. As presented in this research, it explained that the highest rate of contamination was found in wet foods whereas in dry foods the rate of contamination was low. A study about Enterobacteriaceae was shown to support the findings in stall B kikiam (k), 46% of the isolates, Enterobacter cloacae (14%) was the most prevalent among the isolated Enterobacteriaceae. The finding agreed with the work of Falomir et.al. Who found Enterbacteria Cloacae and Klebsilla Oxytoca, to be most prevalent coliforms in ready-to-eat-salad served in the dining halls of a preschool and a primary school in Valencia City, Spain. Therefore the presence of these organisms in the food that went under a study overnight pose a health risk to children and individual with underlying conditions.

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Table 3

Colonial Morphologies and Characteristics in the Different Culture Media SAMPLES

BAP

EMB

MAC

MSE

Change of color of media to C (k)

White pinpoint

Colorless

yellow; light

shiny colonies

pinpoint colonies

pink round

No growth

glistening colonies Change of color of media to C (f)

White pinpoint

Colorless

yellow; light

shiny colonies

pinpoint colonies

pink round

No growth

glistening colonies Change of color of media to C (t)

White colorless

Colorless

yellow; light

colonies

pinpoint colonies

pink round

No growth

glistening colonies

Table 3 shows the Colonial Morphologies and Characteristics in the Different Culture Media. There is growth in Blood Agar Plate (BAP), Eosin Methylene Blue (EMB) and MacConkey Agar (MAC) but no identified harmful bacteria while in Mannitol Salt Agar (MSA), no growth at all. The sample collected in stall C is the cleanest among the other stalls that underwent bacteria identification.

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A study in Mumbai, India elaborated that coliform, E coli, salmonella, shigella, Staphylococcus aureus and pseudomonas were all absent and said yeast and mould levels were within acceptable limits. They cautioned, however, that the air, containers or plates weren’t tested. “This was close to the limit, yet not crossing the line – making it safe for consumption,” the researcher said. While it was a relief that the food did not seem to be as polluted as its surroundings, this was just one dish from one stall. Pav-bhaji is also thoroughly cooked, while other dishes like sev-puri or bhel-puri require uncooked tomatoes, onions and coriander – which may or not be washed, in water that may or may not be contaminated. The government has certainly passed enough laws about food hygiene. As Mumbaikars, it is our responsibility to follow those rules, and understand our role in the process. It will result in better living and health conditions – for Mumbaikars and visitors alike. To support this result, a research done which regards in Microbial Contamination of Street Vended Foods from a University Campus in Bangladesh (2015) which identify the presence of common pathogens. Fifty food samples included deep fried and fried snacks; quick lunch items; pickles; fruit chutney; backed items; spicy; sour; and hot snacks were collected from fixed and mobile vendors from two locations in Dhaka City, Bangladesh tested for microorganisms following conventional microbiological process. Results proved that out of 50 food samples, 12% were confirmed to contain Escherichia coli and Shigella but the absence of Salmonella (Islam, S. et.al, 2015). Pseudomas, Micrococcus, Moraxella, and Corynebacterium are the terrestrial bacteria that are most confined in the street foods and the presence of Salmonella spp., and Escherichia coli were no were not associated on the results of the study (Kok, T., et al. 2007).

CONCLUSION AND RECOMMENDATION Street foods are important ethnic foods sold in popular tourist spots all around for marginal local vendors. In which the researchers come up with this study and conduct experiment about ready-to-eat street food samples which had undergo certain methods, resulted to have carried foodborne bacteria. One of the bacteria present is Salmonella, where in it possibly carried by the vendors or food handlers and had been transmitted to their vended foods. Another is the Escherichia coli which is found from the food itself affected by the environment

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that causes infections, pneumonia, and illnesses if consumers acquire too much. Lastly the Enterobacteria species, non-photogenic bacteria that is harmless. The stalls are located at the side of the roads that has smoke coming from cars. Also the food are fully exposed to dirt, they do not have proper covering. The location of the stalls has the factors that lead to the contamination of the street foods. Practice of inadequate hygienic measures such as mishandling, improper storage, inadequate cooking and above all unhygienic condition of the retail shops are the major causes. Recommendation Based on the finding of our research experiment, researchers would like to recommend the readers in case they want to continue with our research: 

Do colony count about the identified bacteria on the study



Conduct study on the sauces of the street food

Acknowledgement The researchers acknowledge the participation of the street food vendors and the presence of our Technical Adviser all throughout the research experiment.

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