Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) 

Volume 14, Number 1, April 2025 | Pages: 37-41 | DOI: 10.14421/biomedich.2025.141.37-41 ISSN 2540-9328 (online) 
 

 

 

 

Fermented Feed for Laying Hens: Effects on Egg Quality 

and the Number of Enteric Pathogenic Bacteria Present 

 
Daimeera Anja Lulu Amantsura1, Endah Retnaningrum2,* 

1Student of Faculty of Biology; 2Microbiology Laboratory, Department of Tropical Biology, Faculty of Biology, Universitas Gadjah Mada 

Jl. Teknika Selatan Sekip Utara Yogyakarta 55281, Tel. +62-274-580839, Fax. +62-274-580839, Indonesia. 
 

 

Corresponding author* 
endahr@ugm.ac.id 

 

 

 

 

Abstract 

 

Eggs are one of the most common food sources for human consumption because they are common, easy to obtain, and nutritious. Eggs 

contain abundant vitamins and minerals that the human body needs, are a good source of protein and fat and are rich in choline. Despite 

its high nutritional value, eggs are also one of the leading causes of food poisoning and foodborne diseases in various countries. This is 

due to contamination in eggs by pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Campylobacter bacteria. 

Fermented chicken feed contains natural probiotics added to the feed, which improves gut health, better absorption of nutrients, inhibits 

the growth of pathogenic bacteria, enhances the immune response in chickens. This study is conducted to distinguish the diffrence in 

quality of eggs that can be physically observed in chicken eggs fed with fermented feed compared to chicken eggs fed regular feed, as 

well as to compare the number of total bacteria and enteric pathogenic bacteria contained in the contents of chicken eggs that includes 

Escherichia coli and Salmonella spp bacteria. The methods used in this research include observing egg quality comparisons, calculating 

and comparing the total number of bacteria, and testing for the presence of enteric pathogenic bacteria Escherichia coli and Salmonella, 

spp. using the plate count method. The resulting data was processed using the one-way ANOVA method with a confidence level of 95%. 

The results of this research show higher egg quality in fermented fed eggs which is characterized by the color of the yolk which tends to 

be darker yellow to orange and thicker egg shells. The total bacteria count showed that the bacteria was significantly lower in eggs from 

chickens fed fermented feed. Tests for the number of enteric bacteria showed positive results for E. coli in 1 out of 10 egg content samples 

from chicken fed with fermented feed, and positive results for E. coli in 2 out of 10 egg samples from chicken fed with regular feed, while 

test results for Salmonella spp. showed negative results in all samples. 

 

Keywords: fermented feed; enteric pathogenic microbia; eggs; total bacteria count. 

 

Abbreviations: LAB: Lactic Acid Bacteria; SS: Salmonella-Shigella; EMB: Eosin-Methylene Blue; PCA: Plate Count Agar; TPC: Total 

Plate Count 

 

 

INTRODUCTION 
 

Eggs are one of the most common food sources 

consumed by humans because they are a common, easy 

to obtain and nutritious food source. Eggs contain 

abundant vitamins and minerals that the human body 

needs, including calcium, iron, potassium, zinc, 

manganese, vitamin E, folate, and many more. Eggs are 

also a good source of protein and fat and rich in choline, 

which is an important nutrient for brain health. Even 

though they have high nutritional value, eggs are also 

one of the leading causes of food poisoning and 

foodborne diseases in various countries. This is caused 

by contamination that occurs in eggs by pathogenic 

bacteria during the production period. Food poisoning 

associated with egg-borne pathogens can cause high 

morbidity or mortality with symptoms such as diarrhea, 

vomiting, nausea and stomach cramps. 

Enteric pathogenic bacteria are a group of bacteria 

that can cause intestinal infections that are transmitted 

through food, water, animals, or contact with infected 

individuals. (Arumugam et al., 2015). These bacteria can 

cause various gastrointestinal disorders, from mild to 

severe, which can be transmitted via the fecal-oral route. 

Some examples of enteric pathogens include 

Clostridioides difficile, Salmonella, Shigella, Escherichia 

coli, Campylobacter, Yersinia enterocolitica, and Vibrio 

cholerae. (Salihu et al., 2015). 

To overcome pathogenic bacterial contamination in 

animal food products, especially eggs, in recent years 

many farms have added antibiotic supplements to animal 

feed. However, this gives rise to new problems, namely 

the emergence of more and more bacteria that have 

adapted to become resistant to various types of 

antibiotics. Research conducted by Jain and Yadav in 

2017 found 73.33% of isolates of mutli-drug resistant 

Manuscript received: 23 December, 2024. Revision accepted: 17 February, 2025. Published: 25 February, 2025. 

https://doi.org/10.14421/biomedich.2025.141.37-41


 

 
 

38 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 37-41 

 

 

bacteria from egg samples with a resistance percentage 

of 86.66% to Cefixme antibiotics, 80% to Amoxicillin 

antibiotics, and 73.33% to Amoxyclave antibiotics. (Jain 

and Yadav., 2017). The emergence of bacteria that are 

resistant to antibiotics can be prevented by using other 

methods to combat pathogenic bacterial contamination in 

animals, namely by applying fermented feed. Fermented 

feed uses the basic principles of the growth of good 

microorganisms, one of which is Lactobacillus type 

bacteria which is capable of producing lactic acid which 

functions to fight the growth of pathogenic bacteria 

naturally and without the side effects that arise from the 

use of antibiotics in animal feed. 

Chicken feed fermentation is a process that involves 

making a wet mixture from fermented wet food that is 

rich in probiotics for chickens. This process is believed 

to improve chicken health and egg quality. (Ibrahim et 

al., 2020). The type of microorganism selected for the 

fermentation process influences the substances produced 

after the fermentation process occurs. (Niba et al., 2009). 

Generally, the type of microorganism used for food 

fermentation is lactic acid bacteria (LAB) or 

Lactobacillus type bacteria because in the process it 

produces lactic acid which has many benefits, especially 

in the microecology of the digestive tract and has a good 

influence on the immune response. The microorganisms 

used in this research were Em4 solutions or effective 

microorganisms. The Em4 solution contains the main 

microorganisms in the form of phototrophic bacteria, 

lactic acid bacteria, yeast, actinomycetes and 

fermentation fungi. (Fajaruddin et al., 2013). 

This research was done to determine the effect of 

fermented feed on egg quality that can be observed 

physically, including the color of the egg yolk and the 

thickness of the egg shell, to determine the difference in 

the number of enteric pathogenic bacteria contained in 

chicken eggs given fermented feed and chicken eggs fed 

regular feed, and to detect and differentiate the number 

of enteric pathogenic bacteria Escrherichia coli and 

Salmonella in chicken eggs fed fermented feed from 

chicken eggs fed regular feed. 

 

 

MATERIALS AND METHODS 
 

Study area 

Samples were taken from two different chicken farms in 

the Condong Catur area of Yogyakarta city, which used 

different types of feed, which are fermented and regular 

feed. A total of 20 samples were taken, two from each 

farm, over five different periods.  

 

Procedures 

Preparation of Egg Samples and Egg Quality 

Observation 
Each egg sample was soaked in 70% alcohol for 5 

minutes to avoid contamination from bacteria on the egg 

shell. The sterile egg was cracked using a sterile spatula 

and poured into a sterilized jar. The color of the egg yolk 

was observed and documented for later comparison with 

other samples. Egg yolks and egg whites are 

homogenized by stirring using a sterile spatula until 

homogeneous. Serial dilutions were carried out for each 

sample up to a dilution of 10-5 by taking 1 ml of the 

sample and placing it in a test tube containing 9 ml of 

sterile distilled water. 

 
Enumeration of Total Bacteria in Egg Samples 

The results of the serial dilutions 10-3 to 10-5 were 

inoculated on 1 ml of PCA media using the spread plate 

method by taking 0.1 mL of suspension using a 

micropipette, all inoculated petri dishes then incubated in 

a 37°C incubator for 48 hours. The number of colonies 

was counted using the total plate count (TPC) method on 

each sample that had been incubated. 
 

Identification and Enumeration of Enteric Pathogenic 

Bacteria Present 
The same samples from the same suspensions were 

inoculated on EMB agar plate and SS agar plate using a 

pour plate method. All inoculated petri dishes then 

incubated in a 37°C incubator for 48 hours. The number 

of colonies were counted using total plate count (TPC) 

method on each sample that had been incubated. 

 

Data analysis 

All data that had been collected were analyzed using the 

one-way ANOVA method to determine the significance 

of the Difference between the two types of samples. 

 

 

RESULTS AND DISCUSSION 
 

Egg Quality Observation 

The quality of the eggs tested in this research was done 

by comparing the color of the yolk from samples of 

chicken eggs fed fermented feed and samples of chicken 

eggs fed regular feed. Observation of the color of the egg 

yolk was carried out as an indicator of the carotenoid 

content contained in the chicken eggs samples, where a 

darker yellow to orange color indicated a higher 

carotenoid content in the egg.  

 
 

Table 1. Average score value comparison of the yolk color of chicken eggs 

fed with fermented feed and chicken eggs fed with regular feed. 

 

Types of Feed Yolk Colour Score* 

Fermented Feed 2,7 

Regular Feed 1,7 

*score 1 indicates lighter yellow, score 2 indicates yellow, score 3 

indicates darker yellow, and score 4 indicates orange 

 

Based on the results that had been obtained, eggs 

from chicken that are fed with fermented food tend to 



 

 
 

 Amantsura & Retnaningrum – Fermented Feed for Laying Hens: Effects on Egg Quality … 39 
 

 

have a darker yellow to orange color of yolk, which 

indicates a high content of carotenoids in the egg, while 

eggs from chicken that are fed with regular feed has a 

yolk color that tends to be yellow to lighter yellow which 

indicates a lower content of carotenoids. The carotenoid 

content in eggs is generally in the form of lutein and 

zeaxanthin, where these types of carotenoids cannot be 

synthesized in vitro so they must be obtained through the 

feed provided, therefore the profile of carotenoid content 

in eggs depends on the chicken's feeding. (Zaheer., 

2017). 

Based on the research conducted by Martínez-

Sánchez and Pérez-Gálvez, 2023, feeding chickens with 

a wheat base such as regular chicken feed tends to result 

in eggs of lower quality, containing fewer carotenoids, 

resulting in egg yolks with a lighter color. Meanwhile, 

higher-quality pasteurized feed mixed with forages such 

as grass produces darker yellow to orange egg yolks due 

to the high carotenoid content in the eggs. (Martínez-

Sánchez, and Pérez-Gálvez., 2023). Some of the 

ingredients of the fermented feed in this research are 

various types of leaves, grass and other forages, this then 

affects the carotenoid content in eggs, so chickens fed 

fermented feed have a higher carotenoid content in their 

eggs. 

 

Total Number of Bacteria 

Fermented feed, apart from having the benefit of adding 

beneficial nutritional content for animal health, also 

produces substances that play a role in reducing 

pathogenic bacterial contamination in animals, especially 

in the digestive tract. Fermented feed uses the basic 

principles of the growth of good microorganisms, one of 

which is Lactobacillus type bacteria, which is capable of 

producing lactic acid, which functions to fight the growth 

of pathogenic bacteria naturally and without the side 

effects that arise from the use of antibiotics in animal 

feed. This research tests the total number of bacteria in 

the egg samples using the TPC method.  

 
 
 

Table 2. Total Number of Bacteria Formed on PCA media of chicken eggs 
fed with fermented feed and chicken eggs fed with regular feed (CFU/mL). 

 

Types of Feed Total Number of Bacteria 

Fermented Feed 282,4 × 104 

Regular Feed 1170,3 × 104 

 

 

 

The total number bacteria found in the egg contents 

from chickens fed with fermented feed are significantly 

less than those found in the egg contents from chickens 

fed with regular feed. This is because one of the 

microorganisms contained in the form of lactic acid 

bacteria (LAB) in the Em4 solution in fermented feed 

can suppress and inhibit the growth of pathogenic 

bacteria in the animal's digestive tract, thereby reducing 

the possibility of pathogenic bacterial contamination in 

vivo during the egg formation process. 

 

Enteric Pathogenic Bacteria Present 

Escherichia coli and Salmonella spp. are Enterobacter 

types of pathogenic bacteria that are commonly found in 

the contamination of livestock food, especially chicken 

meat and eggs. These two bacteria are generally found in 

the digestive tract of livestock animals, but sometimes in 

vivo contamination during fertilization and egg 

formation can also occur so that these two bacteria are 

found in the egg content. (Al-Bahry et al., 2012).  

Identification and enumeration of E. coli was carried out 

using EMBA selective media, where positive results 

were indicated by the formation of dark purple bacterial 

colonies with metallic green sheen. Meanwhile, the 

Identification and enumeration of Salmonella spp. 

contained in the sample was carried out using selective 

salmonella-shigella agar media where the results were 

positive for the content of salmonella spp. characterized 

by the formation of transparent colonies with black 

precipitate in the colony’s center. 

 
 

Table 3. Average total number of enteric pathogenic bacteria of e.coli and 

salmonella spp. found in egg contents. 

 

Types of Feed E.coli Salmonella spp. 

Fermented Feed 0,46 × 103 ± 2,12 0 ± 0* 

Regular Feed 11,73 × 103 ± 1292,33 0 ± 0* 

*no salmonella growth was identified, but the growth of other 

microbes was detected. 

 

 

The results of testing the number of E.coli bacteria in 

EMBA media showed that the number of enteric E.coli 

bacteria observed in sample number 7 of the egg from 

chicken with fermented feed was 4.6 × 103 CFU/mL, 

while comparison samples 6 and 7 of the egg from 

chicken with regular feed amounted to 114 × 103 and 3.3 

× 103 CFU/mL. Each E. coli positive colony that forms 

has a flat or smooth surface with a circular shape, convex 

elevation, and is dark purple in color with a metallic 

green sheen on the medium.  

The results of the tests on SS agar medium showed no 

salmonella spp. bacteria were detected. In all samples, 

however, there was a color change in the medium to 

yellow, and the formation of dense, colorless bacterial 

colonies with entire margins, and flat elevations 

indicating the growth of other types of bacteria. SS agar 

medium has selective properties that only allow certain 

types of bacteria to grow on it, due to this fact, we know 

that the other types of microbia found on this medium are 

other types of enterobacter bacteria.  

The test results of this research are in line with 
several previous studies where the contamination content 

of pathogenic bacteria E.coli and Salmonella spp. is more 

commonly found on the outside of egg shells than inside 

the egg's contents because both types of bacteria usually 



 

 
 

40 Biology, Medicine, & Natural Product Chemistry 14 (1), 2025: 37-41 

 

 

grow in the digestive tract of animals. Several studies 

that stated similar results include research conducted by 

Fardous and Suzzaman in 2015, which stated results of 

86.67 growth of pathogenic bacteria on the outside of 

egg shells, including 10 of 14 serotypes of Salmonella 

bacteria, but no contamination was found at all in the egg 

contents. (Fardous and Suzzaman., 2015). Research 

conducted by Chaemsanit et al. in 2015 showed 

relatively similar results where 118 anaerobic bacteria 

were isolated from 16 egg samples, of which 116 were 

found on the outer shell and only two from the egg’s 

contents. The isolate obtained from the contents of eggs 

in this study was identified as a member of the 

Enterobacteriaceae bacteria. (Chaemsanit et al., 2015). 

Finally, research conducted by Adesiyun et al. in 2020 

found that there were 48.7% E.coli type bacteria and 2% 

Salmonella type bacteria on the outside of egg shells, but 

only 5.1% contained E.coli type bacteria and no other 

types of bacteria. Salmonella in the eggs. (Adesiyun et 

al., 2020). The minimal content of E.coli and the absence 

of Salmonella content in the eggs are good results which 

prove that there is very minimal contamination of these 

two pathogenic bacteria during in vivo egg formation or 

external contamination during egg production. 

 

 

CONCLUSIONS 
 

The results of this research prove fermented feed 

increased the quality of chicken eggs, and furthermore 

also significantly reduces the number of total and 

Enterobacter type pathogenic bacteria in egg content. 

Moreover, this research also proves that giving 

fermented feed improves eggs’ quality and nutritional 

content. So making and feeding fermented feed is 

another better option that can be developed further and 

applied more widely as an alternative to reduce the 

growth and contamination of pathogenic bacteria without 

using antibiotics. 

 

 

Acknowledgements: Acknowledgement is expressed 

towards the Microbiology Laboratory, Faculty of 

Biology, Universitas Gadjah Mada for providing the 

appropriate setting to conduct the research and all parties 

that fall under this institution. 

 

Authors’ Contributions: Prof. Dr. Endah Retnaningrum 

contributed to this research as the lead supervisor lecturer 

which aided in the methodology of this research 

alongside narration of this article. Daimeera Anja Lulu A 

contributed by designing the study, carrying out the 

laboratory work, and narration of this article 

 

Competing Interests: The authors declare that there are 

no competing interests. 

 

Funding: The authors declare no funding. 

 

 

REFERENCES 
 
Adesiyun, A., Offiah, N., Seepersadsingh, N., Rodrigo, S., Lashley, 

V. & Musai, L., 2006. Frequency and antimicrobial resistance 
of enteric bacteria with spoilage potential isolated from table 
eggs. Food research international, 39(2), pp.212-219. 

Al-Bahry, SN, Mahmoud, IY, Al-Mushrafi, SK, & Al-Ali, MA 
2012. Penetration of Spoilage and Food Poisoning Bacteria into 
Fresh Chicken Egg: A Public Health Concern. Global Journal 
of Bio-Science & Biotechnology. 1 (1): 33-39. Biotechnology, 
1(1), pp.33-39. 

Arumugam, K., Sudigdoadi, S. & Nugraha, G.I., 2015. Enteric 
Pathogen Bacteria in Non-Broiler Chicken Egg Shells from 
Traditional Market and Supermarket, Jatinangor Subdistrict, 
West Java. Althea Medical Journal, 2(3), pp.414-417. 

Chaemsanit, S., Akbar, A. & Anal, A.K., 2015. Isolation of total 
aerobic and pathogenic bacteria from table eggs and its 
contents. Food and Applied Bioscience Journal, 3(1), pp.1-9. 

Fajaruddin, Junus M dan Setyowati E 2013 Pengaruh lama 
fermentasi EM4 terhadap kandungan protein kasar padatan 
kering lumpur organik unit gas bio (the influences of lenght 
fermentation EM4 by containing crude dry solid protein of 
organic mud of gass bio) Jurnal Ilmu-Ilmu Peternakan 23(2) 
14-18 

Fardous, J. and Shamsuzzaman, S.M., 2015. Detection of potential 
pathogenic aerobic bacteria from egg shell and egg contents of 
hen collected from poultry. Bangladesh Medical Research 
Council Bulletin, 41(2), pp.67-72. 

Ibrahim, D., Abdelfattah-Hassan, A., Arisha, A.H., Abd El-Aziz, 
R.M., Sherief, W.R., Adli, S.H., El Sayed, R. & Metwally, 
A.E., 2020. Impact of feeding anaerobically fermented feed 
supplemented with acidifiers on its quality and growth 
performance, intestinal villi and enteric pathogens of mulard 
ducks. Livestock Science, 242, p.104299. 

Jain, A.K. and Yadav, R.A.J.E.S.H., 2017. Study of antibiotic 
resistance in bacteria isolated from table egg. Int. J. Pharm. Bio 
Sci, 8(1), pp.668-674. 

Kanhar, A.R., Phulpoto, I.A., Ur-Rehman, S., Qazi, M.A., Ghumro, 
W.A., Hussain, S.F., Kanhar, A.A., Ujjan, J.A. & Hussain, A., 
2022. Isolation, molecular typing and antibiotic sensitivity 
profiling of enteric bacterial pathogen from chicken eggs. 

Martínez-Sánchez, V. and Pérez-Gálvez, A., 2023. Microalgal 
carotenoids for food and feed applications. Handbook of Food 
and Feed from Microalgae (pp. 133-145). Academic Press. 

Pijnacker, R., Dallman, T.J., Tijsma, A.S., Hawkins, G., Larkin, L., 
Kotila, S.M., Amore, G., Amato, E., Suzuki, P.M., Denayer, S. 
and Klamer, S., 2019. An international outbreak of Salmonella 
enterica serotype Enteritidis linked to eggs from Poland: a 
microbiological and epidemiological study. The Lancet 
Infectious Diseases, 19(7), pp.778-786 

Ranjitkar, S. and Engberg, R.M., 2016. The influence of feeding 
crimped kernel maize silage on growth performance and 
intestinal colonization with Campylobacter jejuni of broilers. 
Avian Pathology, 45(2), pp.253-260.  

Ranjitkar, S., Karlsson, A.H., Petersen, M.A., Bredie, W.L., 
Petersen, J.S. and Engberg, R.M., 2016. The influence of 
feeding crimped kernel maize silage on broiler production, 
nutrient digestibility and meat quality. British poultry science, 
57(1), pp.93-104.  



 

 
 

 Amantsura & Retnaningrum – Fermented Feed for Laying Hens: Effects on Egg Quality … 41 
 

 

Ranjitkar, S., Lawley, B., Tannock, G. and Engberg, R.M., 2016. 
Bacterial succession in the broiler gastrointestinal tract. 
Applied and environmental microbiology, 82(8), pp.2399-2410. 

Sugiharto, S. & Ranjitkar, S., 2019. Recent advances in fermented 

feeds towards improved broiler chicken performance, 

gastrointestinal tract microecology and immune responses: A 

review. Animal nutrition, 5(1), pp.1-10.  

 

 



 

THIS PAGE INTENTIONALLY LEFT BLANK