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Research Article | | Peer-Reviewed

Some Locally Fermented Milk Products Sold in Onitsha Main Market and Their Bacteriological Analysis

Osuji Malachy Ikeokwu*,  Nkwonta Rosemary Celestine
Published in American Journal of BioScience (Volume 13, Issue 4)
Received: 21 May 2025     Accepted: 5 June 2025     Published: 27 August 2025
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Abstract

This research was done to unveil the bacteria associated with three locally sourced fermented milk product sold at Onitsha main market in Anambra state in the South-East of Nigeria. The three locally sourced samples include; Nono, Soybean and Yoghurt. They were obtained aseptically from three different hawkers one for each. Ten-fold serial dilution was done while spread plate technique was adopted. After the inoculation of the second, forth and six diluents followed by 24 hours incubation. The some of the results obtained include; 6.0 x 104, 2.0 x 104 and 1.0 104 for NONO corresponding to nutrient agar, MaConkey and SS agar plate. For the Soybean sample, 4.0 x 104, 1.2 x 104 and 1.0 x 104 corresponding to nutrient agar, MaConkey and SS agar plate. Finally, 5.0 x 104, 1, 5 x 104 corresponding to nutrient agar, MaConkey agar plate. No growth was observed for SS agar. Streaking method was used for purification and isolates were stored in agar slants. Biochemical identification tests done revealed possible organisms like; Kebsiella spp, Bacillus spp, Salmonella spp, Escherichia coli, Shigella sp, Lactobacillussp, Vibrio sp. Further visit to the production and processing place of NONO by the research team revealed why it was more contaminated than other samples. Government should assist the locals to maintain good sanitary condition of the place or outright closure.

Published in American Journal of BioScience (Volume 13, Issue 4)
DOI 10.11648/j.ajbio.20251304.12
Page(s) 102-107
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

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Copyright © The Author(s), 2025. Published by Science Publishing Group
Previous article
Keywords

Nono, Soybean, Yoghurt, Sanitary, Contaminations, Incubation, Inoculation

1. Introduction
The fermented dairy products consumed today are generated through controlled microbial culturing and enzymatic conversions of major and minor milk components
[3]
. Fermentation improves shelf life, increases microbiological safety, adds flavor, and enhances palatability and organoleptic qualities. The fermentation process involves a series of complex reactions carried out by microorganisms, which transform milk constituents rendering new molecules of enhanced nutritive value and digestibility. Moreover, fermentation generates metabolites that can be major contributors of a daily healthful diet
[4, 7]
. The contributions of milk components and dairy products to human health have been comprehensively reviewed
[20]
.
Exploration into the microbiology of nono, soya milk and yoghurt exposed that the fermented product is a culture of Lactobacillus bulgaricus growing with Streptococcus thermophiles
[5, 2]
. These organisms are symbiotic in their relationship as they are involved in conversion of almost all the sugar to lactic-acid and yielding small amount of bye-product. S. thermophiles produce diacetyl while acetaldehyde is produced L. bulgaricus
[21]
. They are also known as starter in milk fermentation that should be ample and feasible in the end fermented milk where aside from production of acid, similarly improves the product’s flavour significantly
[17]
. Species of Lactobacillus and Streptococcus as well as Saccharomyces isolated from ‘nono’ has been used to develop starter cultures for controlled fermentation
[9, 18]
. Other bacteria belonging to the general Lactococcus, Bacillus, Leuconostoc, Propionibacterium, ediococcus, and Bifidobacterium have been linked with fermented dairy products
[8]
. Species of Klebsiella, Enterococcus, Staphylococcus. Pseudomonas, Citrobacter, Micrococcus, Proteus, and Vibrio
[10, 14]
has also been isolated. Fungal species that has been isolated include Aspergillus sp, Rhizopus sp, Trichophyton sp and Mucor
[1]
. While fermentation process is ongoing by lactic acid bacteria, other bacteria such as Listeria monocytogenes, Salmonella paratyphi, Brucella melitensis, Clostridium botulinum and Escherichia coli may create possible threat as they produce toxins that cause intoxication of the products
[4]
. The coliform count may be less in some studies
[22, 10]
but their presence is an indication of fecal contamination and this can pose a health risk to the consumers. Enhancements in approaches (culture dependent and culture independent) used to identify microorganisms has greatly improved our knowledge on the micro biota of fresh milk
[16, 6]
.
2. Materials and Method
2.1. Study Area
Onitsha Main Market, located in Anambra State, Nigeria, is one of the largest and busiest markets in West Africa. It spans a vast area and features thousands of stalls specializing in a wide range of goods, from electronics to textiles. The market is a major economic hub, providing employment for thousands and generating significant revenue for the state. Despite its bustling activity, the market faces challenges such as congestion, poor infrastructure, and security issues. Nonetheless, it remains a crucial center for commerce and trade, with ongoing plans for modernization and expansion. It has a geographic coordinate of Approximately 6.1454° N latitude and 6.7850° E longitude, the market is close to the famous River Niger Bridge, which connects Onitsha to Asaba in Delta State. These landmarks and the strategic location of the Onitsha Main Market contribute significantly to its status as a major commercial hub in the region.
2.2. Sample Collection and Processing
Samples (Nono, Soybean and Yoghurt) were collected from three difference vendors, hawkers and retail outlets in Onitsha main market, Anambra State. The samples were collected under aseptic conditions into sterile airtight sampling jars. They were further conveyed to Microbiology Department laboratory of Legacy University Okija, in an insulated icebox immediately for bacteria analyses.
2.3. Enumeration and Isolation of Microorganisms from Yoghurt, Soybean Milk and Nono
The microbial enumeration was done by determining the total viable counts (TVC) and total coliform count (TCC) using the techniques of
[15]
.
2.4. Total Bacterial Count: Total Viable Counts and Total Coliform Counts
Ten mililitre (10 mL) of each sample was added aseptically to sterilized beaker tube containing 10 mL of distilled water and mixed thoroughly to achieve a stock solution (sample) for 10-folds serial dilution. The aforementioned dilution was used to make subsequent dilutions. About 0.1 mL of the sample was inoculated into well-labeled plate of Nutrient, MaConkey and Salmonella/Shigella agar using a pipette from the 10-2, 10-4 and 10-6 dilution tubes (diluents). The inoculated plates were incubated at 37°C for 24 hours after they were well labelled. The colonies on the plates were then counted and recorded as (cfu/mL) using a colony counter.
2.5. Purification of the Isolates
Colonies of various colours, shapes and sizes were picked from various plates and sub-cultured by streaking repetitively to acquire pure isolated. The pure isolates were stored on agar slant for further characterization and identification.
2.6. Identification of Isolates from Yoghurt, Soybean Milk and Nono
Bacterial isolates were identified by cultural, morphological and biochemical methods as described by
[13, 15]
. Bacterial identification was done using the appearance of colonies on culture media, Gram’s reaction, biochemical tests (coagulase, indole, catalase, methyl red, Vogues-proskauer, citrate, carbohydrate fermentation test). Cultural identification was done by observing the appearance of isolates on the culture media and taking notes of the colour, sizes and shapes of colonies
[13, 15]
.
3. Results
The results obtained after 24 hrs of incubation are as represented in the table.
Table 1. Results of Total heterotrophic and coliform count.

Nutrient Agar (cfu/ml)

MacConkey Agar (cfu/ml)

Salmonella/ Shigella Agar (cfu/ml)

10-2

10-4

10-6

10-2

10-4

10-6

10-2

10-4

10-6

NONO

60

58

56

20

15

11

6

3

1

SOYBEAN

40

35

30

12

10

9

1

NG

NG

YOGHURT

50

47

40

15

12

10

NG

NG

NG
Table 2. Results of Total heterotrophic and coliform count (standard representation).

Nutrient Agar (cfu/ml)

MacConkey Agar (cfu/ml)

Salmonella/ Shigella Agar (cfu/ml)

10 -2

10-4

10-6

10 -2

10-4

10-6

10 -2

10-4

10-6

NONO

6.0 x 10-4

5.8 x 10-6

5.6 x 10-8

2.0 x 10-4

1.5 x 10-6

1.1 x 10-8

6 x 10-3

3 x 10-5

1 x 10-7

SOYBEAN

4.0 x 10-4

3.5 x 10-6

3.0 x 10-8

1.2 x 10-4

1.0 x 10-6

0.9 x 10-7

1 x 10-3

NG

NG

YOGHURT

5.0 x 10-4

4.7 x 10-6

4.0 x 10-8

1.5 x 10-4

1.2 x 10-6

1.0 x 10-7

NG

NG

NG
Cfu/ml: coliform forming unit, NG: No growth
Table 3. Morphological, cultural and BIOCHEMICAL, characteristics of bacterial isolates during the study.

GR

CA

OX

CT

IN

MR

COA

M

Possible Organism

-R

+

-

+

-

-

-

Kebsiella spp

+R

+

-

+

-

-

+

Bacillus subtilis

+

-

+

-

+

-

+

Salmonella spp

-R

+

-

-

+

+

-

+

Escherichia coli

-R

+

-

-

-

+

+

Citrobacter spp

+C

+

-

+

-

+

+

-

Staphylococcus spp

+R

-

-

-

-

-

-

Lactobacillus sp

+C

-

-

-

Streptococcus sp

-R

-

+

-

-

-

-

Vibrio sp

+C

-

-

+

+

+

+

Lctococcus sp

-R

+

-

-

-

+

-

-

Shigella sp

-R

+

-

+

-

-

+

Proteus sp
Legend:
NG: No growth, GR = Gram reaction, CA = Catalase test OX = Oxidase test, CT = citrate test, IN = Indole test, MR = Methylred test, COA = Cozigulase test, M= motility test.
Figure 1. Colony count for NONO on the three media.
Figure 2. Colony count for Soybean on the three media.
Figure 3. Colony count for Yoghurt on the three media.
Figure 4. Place where the Nono was purchased.
Figure 5. Colonies on some of the plate viewed on magnifying glass.
Figure 6. Media used and other materials.
Figure 7. Agar slant, Slide for Gram staining and oxidase test.
4. Discussion
From the results as shown in both the tables and charts revealed that all the locally sourced products are contaminated. Of the three locally sourced samples, Nono has high bacterial load followed by the soybean. There are many risk factors associated with contamination of fermented milk food
[12, 19]
. The source of the contamination may not be unconnected with poor handling, preparation and storage condition. The soybean may be contaminated right from the farm. Another way of contamination may be through the processing machine. From Table 1, NONO revealed that there was a growth on the Salmonella/Shigella agar. Because of the nutrient content of Nono, any bacterium that finds its way into it by any means will grow well
 [11]
. There are several ways the Nono may have been contaminated. This could be from the water used in its final preparation. There is also the possibility of fecal contamination of the water used. At stage 1 during collection of samples directly from the udder, the presence of coliform in this stage may be due to contamination by fecal matter attached to the teats of the animal or the hand of the milking man.
5. Results of Further Investigation on the Source of the Samples
Research team visited the place where these samples were prepared and processed. The following were observed.
1. The entrance/ route to the place where the NONO was prepared was waterlogged.
2. The water used by the locals to mix the powders in the preparation showed clear evidence of fecal contamination which was evidenced in the presence of growth on the SS agar.
3. The youth used in the mass production of the NONO do not observe routine hand washing.
4. Their dressing and outfit are not clean.
5. As shown in the photo, they use pit latrine.
6. Those preparing the NONO do not wear hand glove.
7. The milk for NONO production are not pretreated (pasteurized or sterilized) used use.
8. There is no sterilization technique adopted for the raw material or finished products.
9. The machine used in blending the soybean was not cleaned thoroughly before using it for another.
10. It was also observed that the farmers that bring the soybean do not was the seed very well.
11. There is a well proven case of carriage container contamination of the soybean.
12. The hawkers of NONO move around leaving the prepared NONO open while on their head.
6. Recommendation by the Research Team
At the end of the research and visitation, the research team recommend as follows.
1. Clean water should be provided for them.
2. Access to the production site should be made clean.
3. The water should be checked for fecal contamination before use.
4. The NONO should be kept in the fridge and preparation to avoid contamination.
Abbreviations

SSA

Salmonell/Shigella Agar

CFU

Colony Forming Unit
Author Contributions
Osuji Malachy Ikeokwu: Conceptualization, Funding acquisition, Supervision
Conflicting of Interest
The authors declare no conflicts of interest.
References
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    Ikeokwu, O. M., Celestine, N. R. (2025). Some Locally Fermented Milk Products Sold in Onitsha Main Market and Their Bacteriological Analysis. American Journal of BioScience, 13(4), 102-107. https://doi.org/10.11648/j.ajbio.20251304.12

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    ACS Style

    Ikeokwu, O. M.; Celestine, N. R. Some Locally Fermented Milk Products Sold in Onitsha Main Market and Their Bacteriological Analysis. Am. J. BioScience 2025, 13(4), 102-107. doi: 10.11648/j.ajbio.20251304.12

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    AMA Style

    Ikeokwu OM, Celestine NR. Some Locally Fermented Milk Products Sold in Onitsha Main Market and Their Bacteriological Analysis. Am J BioScience. 2025;13(4):102-107. doi: 10.11648/j.ajbio.20251304.12

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  • @article{10.11648/j.ajbio.20251304.12,
  author = {Osuji Malachy Ikeokwu and Nkwonta Rosemary Celestine},
  title = {Some Locally Fermented Milk Products Sold in Onitsha Main Market and Their Bacteriological Analysis
},
  journal = {American Journal of BioScience},
  volume = {13},
  number = {4},
  pages = {102-107},
  doi = {10.11648/j.ajbio.20251304.12},
  url = {https://doi.org/10.11648/j.ajbio.20251304.12},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajbio.20251304.12},
  abstract = {This research was done to unveil the bacteria associated with three locally sourced fermented milk product sold at Onitsha main market in Anambra state in the South-East of Nigeria. The three locally sourced samples include; Nono, Soybean and Yoghurt. They were obtained aseptically from three different hawkers one for each. Ten-fold serial dilution was done while spread plate technique was adopted. After the inoculation of the second, forth and six diluents followed by 24 hours incubation. The some of the results obtained include; 6.0 x 104, 2.0 x 104 and 1.0 104 for NONO corresponding to nutrient agar, MaConkey and SS agar plate. For the Soybean sample, 4.0 x 104, 1.2 x 104 and 1.0 x 104 corresponding to nutrient agar, MaConkey and SS agar plate. Finally, 5.0 x 104, 1, 5 x 104 corresponding to nutrient agar, MaConkey agar plate. No growth was observed for SS agar. Streaking method was used for purification and isolates were stored in agar slants. Biochemical identification tests done revealed possible organisms like; Kebsiella spp, Bacillus spp, Salmonella spp, Escherichia coli, Shigella sp, Lactobacillussp, Vibrio sp. Further visit to the production and processing place of NONO by the research team revealed why it was more contaminated than other samples. Government should assist the locals to maintain good sanitary condition of the place or outright closure.},
 year = {2025}
}

    Copy | Download 

  • TY  - JOUR
T1  - Some Locally Fermented Milk Products Sold in Onitsha Main Market and Their Bacteriological Analysis

AU  - Osuji Malachy Ikeokwu
AU  - Nkwonta Rosemary Celestine
Y1  - 2025/08/27
PY  - 2025
N1  - https://doi.org/10.11648/j.ajbio.20251304.12
DO  - 10.11648/j.ajbio.20251304.12
T2  - American Journal of BioScience
JF  - American Journal of BioScience
JO  - American Journal of BioScience
SP  - 102
EP  - 107
PB  - Science Publishing Group
SN  - 2330-0167
UR  - https://doi.org/10.11648/j.ajbio.20251304.12
AB  - This research was done to unveil the bacteria associated with three locally sourced fermented milk product sold at Onitsha main market in Anambra state in the South-East of Nigeria. The three locally sourced samples include; Nono, Soybean and Yoghurt. They were obtained aseptically from three different hawkers one for each. Ten-fold serial dilution was done while spread plate technique was adopted. After the inoculation of the second, forth and six diluents followed by 24 hours incubation. The some of the results obtained include; 6.0 x 104, 2.0 x 104 and 1.0 104 for NONO corresponding to nutrient agar, MaConkey and SS agar plate. For the Soybean sample, 4.0 x 104, 1.2 x 104 and 1.0 x 104 corresponding to nutrient agar, MaConkey and SS agar plate. Finally, 5.0 x 104, 1, 5 x 104 corresponding to nutrient agar, MaConkey agar plate. No growth was observed for SS agar. Streaking method was used for purification and isolates were stored in agar slants. Biochemical identification tests done revealed possible organisms like; Kebsiella spp, Bacillus spp, Salmonella spp, Escherichia coli, Shigella sp, Lactobacillussp, Vibrio sp. Further visit to the production and processing place of NONO by the research team revealed why it was more contaminated than other samples. Government should assist the locals to maintain good sanitary condition of the place or outright closure.
VL  - 13
IS  - 4
ER  -

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