# Isolation, Characterization and Development of Starter Culture from Traditional Djibna baida Cheese

Adam Ahmat Moussa, Boureima Kagambèga, Abdoullahi Hissein Ousman, Moussa Idriss Mahamat, Al-Lamadine Mahamat, Ngandolo Bongo Nare Richard, Abdelsalam Tidjani

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## To cite this version:

Adam Ahmat Moussa, Boureima Kagambèga, Abdoullahi Hissein Ousman, Moussa Idriss Mahamat, Al-Lamadine Mahamat, et al.. Isolation, Characterization and Development of Starter Culture from Traditional Djibna baida Cheese. Current Journal of Applied Science and Technology, 2025, 44, pp.54 - 70. 10.9734/cjast/2025/v44i104619. hal-05321591

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# Isolation, Characterization and Development of Starter Culture from Traditional Djibna baida Cheese

Adam Ahmat Moussa<sup>a</sup>, Boureima Kagambèga<sup>b*</sup>, Abdoullahi Hissein Ousman<sup>a</sup>, Moussa Idriss Mahamat<sup>c</sup>, Al-lamadine Mahamat<sup>a</sup>, Ngandolo Bongo Nare Richard<sup>c</sup> and Abdelsalam Tidjani<sup>a</sup>

<sup>a</sup> Food Science and Nutrition Research Laboratory (LARSAN), Faculty of Human Health Sciences, University of N'Djamena, PO Box 1117, Chad.

<sup>b</sup> Applied Biochemistry and Immunology Laboratory (LaBIA), Joseph KIZERBO University, PO Box 7021 Ouagadougou, Burkina Faso.

<sup>c</sup> Livestock Research Institute for Development (IRED), PO Box 433, Chad.

## Authors' contributions

This work was carried out in collaboration among all authors. Authors AAM, BK, AHO, MIM, A-IM, NBNR and AT participated in the finalization of this article. From data collection to analysis, each made a significant contribution. The writing and editing of this manuscript were the result of a perfect collaboration. The submission was validated by all authors. All authors read and approved the final manuscript.

## Article Information

DOI: https://doi.org/10.9734/cjast/2025/v44i104619

### Open Peer Review History:

This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/145604

### Original Research Article

Received: 20/08/2025

Published: 18/10/2025

<footnote>*Corresponding author: E-mail: kagambou@gmail.com;</footnote>
ABSTRACT

**Aims:** To characterize the technological capabilities of lactic acid bacteria isolated from traditional cheeses coagulated by Jibben with a view to optimizing their use as starters in the manufacture of local cheeses.

**Study Design:** Descriptive study based on analyses, property tests and interpretations of results.

**Place and Duration of Study:** The study was carried out in the city of N'Djamena at the food microbiology laboratory of the Institute for Research in Livestock for Development (IRED), between January, 2023 to June, 2024.

**Methodology:** Lactic acid bacteria were isolated from 9 samples of artisanal cheeses produced from cow's milk coagulated by Jibben (*Solanum dubium*). The identification of lactic acid bacteria isolates were carried out using classical microbiological techniques, such as evaluating their morphological, physiological and biochemical characteristics. Technological capabilities and interactions studies were carried out, followed by fermentation profiling.

**Results:** The results showed that the microbial population of selected cheese is dominated by *Lactococcus* (32.06%), mesophilic *Lactobacillus* (26.77%), and *Leuconostoc* (16.03%) bacteria. The study revealed the ability of some of the selected strains to rapidly produce lactic acid and cause a drop in pH. The interaction studies between the previously identified strains reflect to compose two mesophilic ferments FI and FII. The FI ferment is composed of four strains of the genus *Lactococcus* (Lc7, Lc8, Lc11 and Lc12) and one belonging to that of *Leuconostoc* (Leuc4). The second FII ferment contains two strains of *Lactococcus* (Lc15 and Lc16) and two others belonging respectively to the genus *Leuconostoc* (Leuc6) and mesophilic *Lactobacillus* (Lbl1). The interaction between the strains was positive through the technological evaluation. The acidification and proteolysis rates as well as the lipolysis and texturizing power also gave interesting results.

**Conclusion:** The study highlighted the satisfactory technological characteristics of ferment I consisting of lactic acid bacteria isolates. These strains, after further characterization and investigation, could be used as starter cultures to improve the traditional cheese-making process.

**Keywords:** Traditional cheese; lactic bacteria; technological properties; ferments; starter; Jibben; Chad.

1. INTRODUCTION

Dairy products are known for their high nutritional value and contribute to the dietary balance of many populations (Stobiecka et al., 2022); Agbo et al., 2023). Milk contains essential nutrients with a high-water content recommended as a food or beverage for all age groups (Gondimo et al., 2024). In Chad, the dairy sector occupies an important place, bringing together the processing of fermented and powdered milk, butters and traditional cheese from local raw cow or goat milk (Beral et al., 2023; Niang et al., 2023). The high water content of milk favors the development of several germs including lactic acid bacteria (Cogan et al., 1997). These lactic acid bacteria are a group of heterogeneous cells classified according to their ability to produce lactic acid as the end product of their fermentative metabolism. Their roles in the traditional fermentation of foods of plant and animal origin have been highlighted by several authors (Zhou et al., 2025; Fall et al., 2019). The use of these bacteria in the fermentation of foods, particularly dairy products,

is closely linked to human history (Gasbarrini et al., 2016; Cherel, 2022). Their metabolic capacities not only contribute to the development of desirable sensory characteristics of food products, but also help to maintain or even improve the nutritional value of the raw material (Cao et al., 2022; Lutin et al., 2024). In addition, certain species of lactic acid bacteria have probiotic properties that are very beneficial to consumers (Hussain et al., 2025). In Chad, local bacteria present in milk contribute to the acidification of milk and the production of a traditional fresh cheese called *Djibna baida*. This cheese is obtained using a traditional and empirical technology based on mixed coagulation obtained by the combined action of an extract of Jibben fruit (*Solanum dibium*) and lactic acid bacteria (Cogan et al., 1997; Adnan & Tan, 2007). It is an activity that mainly employs women and micro-enterprises operating in a sector where the transformation process is almost traditional. The application of this technology, without specific precautions of good hygiene practices, increases the health risk and
affects the quality of the processed milk, particularly the texture, taste and organoleptic qualities (Beral et al., 2023). The introduction of imported commercial ferments can contribute to the standardization of this product and the reduction of risks of development of pathogenic germs and alteration (Demeulenaere & Lagrola, 2021). But with its global uses, artisanal cheeses tend to lose their originality. In order to preserve the richness and typicality of the organoleptic profiles of artisanal cheeses, it seems wise to consider using sourdoughs directly from the local production environment. The objective of this study was to characterize the strains of lactic acid bacteria in traditional cheese (Djibna baida) coagulated by Jibben in order to optimize their reuse as starter cultures in the manufacturing process.

## 2. MATERIAL AND METHODS

### 2.1 Study Location and Sampling

#### 2.1.1 Study location

The study was carried out in the city of N'Djamena, between January 17, 2023 and June 13, 2024.

#### 2.1.2 Sampling

Lactic acid bacteria were isolated from nine samples of artisanal cheeses produced from cow's milk coagulated by Jibben (Solanum dibium). These cheeses were collected from urban and peri-urban areas of the city of N'Djamena.

<figure><img src="image_4.png" /><figcaption>Fig. 1. Location of the study site</figcaption></figure>
<table><caption>Table 1. Culture media for the isolation of lactic acid bacteria</caption><thead><tr><th>Microorganisms</th><th>Culture media</th><th>T °C</th><th>Duration (H)</th><th>Incubation</th></tr></thead><tbody><tr><td>Entérococcus</td><td>M17 pH (6.5)</td><td>45</td><td>72</td><td>Aerobiosis</td></tr><tr><td>Lactococcus</td><td>M17 pH (6.5)</td><td>30</td><td>72</td><td>Aerobiosis</td></tr><tr><td>Leuconostoc</td><td>Hypersaline medium (6.5 %) ; pH (9.6)</td><td>30</td><td>72 to 96</td><td>Aerobiosis</td></tr><tr><td>Pediococcus</td><td>M17 pH (6.5)</td><td>30</td><td>72</td><td>Aerobiosis</td></tr><tr><td>Mesophilic</td><td>MRS pH (6 and 5.5)</td><td>30</td><td>72</td><td>Aerobiosis to Anaerobiosis</td></tr><tr><td>Lactobacillus</td><td>MRS pH (6 and 5.5)</td><td>45</td><td>72</td><td>Anaerobiosis</td></tr><tr><td>Thermophilic</td><td>MRS pH (6 and 5.5)</td><td>45</td><td>72</td><td>Anaerobiosis</td></tr><tr><td>Lactobacillus</td><td>MRS pH (6 and 5.5)</td><td>45</td><td>72</td><td>Anaerobiosis</td></tr></tbody></table>

Source: Dahou et al. (2015)

## 2.2 Isolation, Purification and the cell shape, size, association mode, and the presence or absence of spores.

### 2.2.1 Isolation and purification

Man-Rogosa-Sharpe (MRS) and M17 medium were used for isolation followed by successive subcultures to obtain pure (distinct and homogeneous) colonies. Table 1 summarizes the strain isolation conditions.

### 2.2.2 Strains conservation

Short-term conservation of pure strains was carried out on solid slant media. After growth at optimal temperature, cultures were maintained at 4°C and strain renewal was performed by successive subcultures every four (4) weeks. Long-term conservation of purified isolates were carried out in a medium containing 70% skim milk (enriched with 0.05% yeast extract and 0.05% glucose) and 30% glycerol and stored at -20°C (El Ahmadi et al., 2025).

## 2.3 Identification of Isolates

The identification of lactic acid bacteria isolates was carried out in two stages. The first consisted of testing all isolates using Gram staining (Albuquerque et al., 2025), catalase production, and oxidase detection (Mercy et al., 2025). Gram-positive, oxidase-negative, and catalase-negative bacteria were selected for further analysis. The second stage was based on the morphological, physiological, and biochemical characteristics of the strains.

### 2.3.1 Determination of morphological characteristics

Macroscopic observation of pure and isolated colonies highlights the colony shape, size, color, and appearance. Microscopic study can confirm

the cell shape, size, association mode, and the presence or absence of spores.

### 2.3.2 Determination of biochemical and physiological characteristics

#### 2.3.2.1 Thermoresistance

This test consisted of inoculating the bacterial strains into tubes containing MRS broth, then incubating them at 63.5°C for 30 minutes in a water bath to differentiate between mesophilic and thermophilic lactic acid strains (Bennani et al., 2017). The absence of turbidity indicates a negative result, which defines mesophilic strains, while turbidity results from the growth of thermoresistant strains (Badis et al., 2004; Joffin & Leyral, 2006).

#### 2.3.2.2 Growth at pH 9.6

MRS medium at pH 9.6 was inoculated with fresh cultures and incubated at 30 and 37°C for 24 hours, which is generally hostile to lactic acid bacteria, which cannot grow in alkaline media (Mathara et al., 2004).

#### 2.3.2.3 Growth in Hypersaline Broth

This study was conducted by inoculating MRS medium supplemented with 6.5% NaCl with a young culture. Incubation was carried out at 30 and 37°C for 24 to 48 hours. Unlike enterococci, lactic acid bacteria are sensitive to hypersaline media (Mathara et al., 2004).

#### 2.3.2.4 Fermentation type test

This test determines the nature of sugar fermentation (homofermentative or heterofermentative). The test strain was incubated in liquid MRS medium containing the Durham bell jar at 30 and 37°C for 24 hours. The
development of heterofermentative bacteria is manifested by the appearance of gas in the Durham bell jar, which is absent in homofermentative bacteria (Goa et al., 2022).

### 2.3.2.5 CO₂ Production from citrate

This test was performed using the method described by MacWilliams (2009). The strain was inoculated into a tube containing 10.5 ml of sterile 10% skimmed milk, supplemented with 0.5 ml of sterile sodium citrate solution (10%) and 4 ml of sterilized white agar (1.5%), and then incubated at 30°C. Gas production in the bulk of the medium reflects the decomposition of the citrate.

## 2.3.3 Study of the technological aptitudes of isolated lactic acid bacteria

### 2.3.3.1 Acidifying and coagulating power

Acidifying activity was assessed using the method of Larpent-Gourgaud et al. (1997). This involved monitoring the pH of the different cultures over time and simultaneously measuring total acidity using sodium hydroxide. To test the cultures, 100 ml of sterile skimmed milk was inoculated with 3 ml of culture. After incubation at 37°C for 24 hours, the volume of exuded whey was measured and the appearance of the resulting coagulum was noted. The pH change and lactic acid content were determined.

### 2.3.3.2 Proteolytic and lipolytic activities

The proteolytic activity of the strains was determined with method used by Dahou et al. (2015) modified. Sterile Wattman paper discs were placed on the surface of MRS agar supplemented with 10% skimmed milk. Each disc received a 20 µL volume of a young culture. After incubation at 37°C for 24 hours, proteolysis was revealed by clear zones around the discs. Lipolytic activity was demonstrated on triglyceride agar. Sterile Wattman paper discs were placed on the surface of this agar, and then each disc received 10 µL of a young culture. After incubation at 37°C for two days, lipolysis was revealed by a clearing zone surrounded by a deposit around the discs (Guiraud, 2003).

### 2.3.3.3 Texturizing and flavoring power

The strains to be tested were streaked onto hypersucrose agar (supplemented with 10% sucrose). Dextran-producing strains are characterized by the formation of large, viscous,

and sticky colonies (Zarour et al., 2017, Benhouna et al., 2019). The ability of mixed cultures to produce flavoring compounds during fermentation was demonstrated on skim milk. To do this, each tube containing sterile skim milk was inoculated with one of the cultures. After incubation for 24 hours and coagulation of the milk, the Vogues-Proskaeur reagents VPI and VPII were added and allowed to stand. The presence of flavor is revealed by the appearance of a red ring (Colombo et al., 2020).

### 2.3.3.4 Interaction study

The selected strains were spot-inoculated onto the previously poured and solidified MRS agar. The mixture was allowed to dry at room temperature, and at the same time, 7 ml of the MRS agar, already melted and cooled to 39°C, was inoculated with 0.5 ml of the indicator strain. The mixture was then poured onto the surface of the spot-inoculated MRS agar. Symbiosis is revealed by the absence of inhibition zones, while antagonism is indicated by their presence after incubation at 37°C for 24 hours (Fleming et al., 1975; Moraes et al., 2010). Strains exhibiting symbiosis with each other were chosen for the reconstitution of our cultures.

## 2.4 Formation of a Mesophilic Starter

### 2.4.1 Selection of strains

The selection of strains were based on the results of technological aptitudes and the study of strain interactions. Strains with technological power and that expressed no inhibition between them were selected to constitute our starter cultures.

### 2.4.2 Preparation of starters

The starter cultures were prepared using 10% skimmed milk. The milk was pasteurized at a temperature of 72°C for 30 minutes, followed by cooling to 50°C and heating to 95°C for 15 seconds (Tyndallization technique). The milk was then cooled to room temperature and then inoculated at a rate of 3% with the selected strains, then incubated at 37°C for 16 hours.

## 2.5 Data Processing

Data entry was performed using Office Excel 2016 to create figures (graphs, histograms) and tables, enhanced with Paint Net software.
# 3. RESULTS AND DISCUSSION

## 3.1 Results

### 3.1.1 Isolation and Identification of Lactic Acid Bacteria Strains

Macroscopic observation allowed us to identify whitish, sometimes transparent, round or lenticular colonies of varying size from one colony to another. Microscopic examination revealed 131 isolates that were Gram positive, oxidase-negative, and catalase-negative. 54 isolates in bacillary form and 77 isolates in cocci form. The results of biochemical and physiological characterization allowed to classify the isolates according to the type of fermentation (homofermentative or heterofermentative), the optimal growth temperature (mesophilic or thermophilic), the capacity to produce gas from citrate, the capacity to grow at different concentrations of NaCl (4% and 6.5%), growth in alkaline medium (pH 4.5, 6.5 and 9.5) and development on Sherman milk (0.1% and 0.3%). Thus, six (06) groups of lactic acid bacteria strains were identified: *Lactococcus* (42 isolates or 32.06%), mesophilic *Lactobacillus* (39 isolates corresponding to 26.77%), *Leuconostoc* (21 isolates representing 16.03%), thermophilic

*Lactobacillus* (15 isolates or 11.45%), *Pediococcus* (9 isolates or 6.87%) and *Enterococcus* (5 isolates equivalent to 3.81%). The presumed *Enterococcus* isolates were all thermophilic homofermentative, capable of growing in the presence of 4% and 6.5% NaCl, of growing in alkaline medium pH 6.9 and 9.6 and reducing 0.1% methylene blue. The isolates identified as *Lactococcus* were all homofermentative, capable of growing in the presence of 4% NaCl, 0.1% and 0.3% methylene blue and at pH 4.6. The isolates identified as *Leuconostoc* were heterofermentative strains, capable of producing CO2, growing in an alkaline medium at pH 6.5, and 4.5% and at a concentration of 6.5% NaCl. The presumed *Pediococcus* isolates were all homofermentative, able to grow at pH 6.9 but not at 9.6 or 4.5 and did not tolerate the 6.5% NaCl concentration. The isolates identified as mesophilic *Lactobacillus* were all homofermentative, able to grow at pH 6.5. Some strains showed growth capacity at pH 4.5, unable to produce CO2 and they did not grow at 45° C. The presumed thermophilic *Lactobacillus* isolates were all homofermentative bacteria capable of growth at 45° C, at pH 4.5 and 6.5 but not at 9.6. Table 2 illustrates the biochemical and physiological characteristics of the isolated strains.

<table><caption>Table 2. Morphology, biochemical and physiological characteristics of the strains</caption><thead><tr><th>Characteristics</th><th>Group 1</th><th>Group 2</th><th>Group 3</th><th>Group 4</th><th>Group 5</th><th>Group 6</th></tr></thead><tbody><tr><td>Form</td><td>Cocci</td><td>Cocci</td><td>Cocci</td><td>Cocci</td><td>Bacilli</td><td>Bacilli</td></tr><tr><td>Coloration Gram</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Catalase</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Oxidase</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Type of fermentation</td><td>Hom</td><td>Hom</td><td>Het</td><td>Hom</td><td>Hom</td><td>Hom</td></tr><tr><td>Growth at 30°C</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Growth at 45°C</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td></tr><tr><td>CO2 production</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Growth in a salty medium (NaCl)</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>4%</td><td>+</td><td>+</td><td>+</td><td></td><td></td><td></td></tr><tr><td>6.5%</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Growth in pH</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>4.5</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+/-</td><td>+</td></tr><tr><td>6.5</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>9.6</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Sherman's Milk Test</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0.1</td><td>+</td><td>+</td><td></td><td></td><td></td><td></td></tr><tr><td>0.3</td><td>-</td><td>+</td><td></td><td></td><td></td><td></td></tr><tr><td>Identification</td><td>Ec</td><td>Lc</td><td>Leuc</td><td>Pc</td><td>Lbl</td><td>Lbll</td></tr><tr><td>Number of isolates</td><td>05</td><td>42</td><td>21</td><td>09</td><td>39</td><td>15</td></tr><tr><td>Percentages %</td><td>3.81%</td><td>32.06%</td><td>16.03%</td><td>6.87%</td><td>26.77%</td><td>11.45%</td></tr></tbody></table>

Ec: Enterococcus; Lc: Lactococcus; Leuc: Leuconostoc; Pc: Pediococcus; Lbl: Mesophilic Lactobacillus; Lbll: Thermophilic Lactobacillus; Hom: Homofermentative; Het: heterofermentative
<table><caption>Table 3. Variation of pH and appearance of curd in the middle of skimmed milk in 18 hours of incubation</caption><thead><tr><th rowspan="2">Strains</th><th colspan="5">Incubation in skimmed milk</th></tr><tr><th>pH at 0H</th><th>pH after 2 H</th><th>pH after 6 H</th><th>pH after 18 H</th><th>Coagulation after 6 H</th></tr></thead><tbody><tr><td>Lc1</td><td>6.5</td><td>6.1</td><td>6.1</td><td>5.6</td><td>-</td></tr><tr><td> Labels</td><td>6.5</td><td>5.7</td><td>5.3</td><td>4.9</td><td>+++</td></tr><tr><td>Lc2</td><td>6.5</td><td>6.1</td><td>5.8</td><td>5.6</td><td>-</td></tr><tr><td>Lc3</td><td>6.5</td><td>6.1</td><td>5.7</td><td>5.4</td><td>-</td></tr><tr><td> Labels</td><td>6.5</td><td>6.3</td><td>6.1</td><td>5.4</td><td>-</td></tr><tr><td>Lc4</td><td>6.5</td><td>6.3</td><td>5.6</td><td>5.3</td><td>-</td></tr><tr><td> Leuc1</td><td>6.5</td><td>6.4</td><td>5.8</td><td>5.4</td><td>-</td></tr><tr><td>Lc5</td><td>6.5</td><td>6.3</td><td>5.6</td><td>5.4</td><td>-</td></tr><tr><td>Lc6</td><td>6.5</td><td>6.2</td><td>5.5</td><td>5.1</td><td>-</td></tr><tr><td>Lc7</td><td>6.5</td><td>6.1</td><td>5.7</td><td>4.9</td><td>+++</td></tr><tr><td>Lc8</td><td>6.5</td><td>5.9</td><td>5.3</td><td>4.2</td><td>+++</td></tr><tr><td>Lc11</td><td>6.5</td><td>6.3</td><td>5.9</td><td>5.6</td><td>+/-</td></tr><tr><td>Lc9</td><td>6.5</td><td>6.3</td><td>5.8</td><td>5.6</td><td>-</td></tr><tr><td>Lc12</td><td>6.5</td><td>6.1</td><td>5.6</td><td>5.3</td><td>+++</td></tr><tr><td> Leuc2</td><td>6.5</td><td>6.1</td><td>5.5</td><td>5.1</td><td>-</td></tr><tr><td>Lc10</td><td>6.5</td><td>6.3</td><td>5.6</td><td>5.1</td><td>-</td></tr><tr><td> Leuc3</td><td>6.5</td><td>6.2</td><td>5.5</td><td>5.2</td><td>-</td></tr><tr><td> Leuc4</td><td>6.5</td><td>6.1</td><td>5.3</td><td>4.6</td><td>+++</td></tr><tr><td> Labels</td><td>6.5</td><td>6.3</td><td>5.8</td><td>5.2</td><td>-</td></tr><tr><td>Lc15</td><td>6.5</td><td>6.3</td><td>5.7</td><td>5.2</td><td>-</td></tr><tr><td>Lc16</td><td>6.5</td><td>5.8</td><td>5.7</td><td>4.8</td><td>+++</td></tr><tr><td> Leuc5</td><td>6.5</td><td>5.8</td><td>4.5</td><td>3.7</td><td>+++</td></tr><tr><td> Leuc6</td><td>6.5</td><td>6</td><td>5.9</td><td>5.1</td><td>+/-</td></tr><tr><td> Leuc7</td><td>6.5</td><td>6.1</td><td>5.8</td><td>5.4</td><td>-</td></tr><tr><td>Lc17</td><td>6.5</td><td>6.4</td><td>5.3</td><td>5.1</td><td>-</td></tr></tbody></table>

Lc: Lactococcus; Leuc: Leuconostoc; Lbl: Mesophilic Lactobacillus, -: Absence of coagulation, +/-: Partial coagulation; +++: Total coagulation; H: Hour.

### 3.1.2 Technological aptitudes of isolated lactic acid bacteria

#### 3.1.2.1 Acidifying and/or coagulating activities

The majority of strains tested had the ability to lower pH values, with variability from one strain to another. We identified a total of 29 strains with the ability to lower pH to a unit ≥ 0.9 after 18 hours of incubation. Seventeen (17) strains of these isolates belong to the genus *Lactococcus*, seven isolates to the genus *Leuconostoc*, and five isolates to the mesophilic genus *Lactobacillus*. Table 3 illustrates the acidification kinetics of these strains. The fastest acidification recorded was 4.5, obtained by strain Leuc5 after 6 hours of incubation. Strains Lc8, Lc17, Leuc4, and Lbl1 have the ability to lower pH to 5.3 after 6 hours of incubation. The

pH value obtained during 18 hours of incubation by isolates belonging to the genus *Lactococcus* varies between 2.3 and 0.9. The highest value is obtained by strain Lc8 (2.3) and the lowest value was 0.9 obtained by strains Lc1, Lc2, Lc9 and Lc11 (Fig. 2). The acidifying potential of strains of the genus *Leuconostoc* is very important, their ΔpH after 18 hours of incubation varies between 1.1 and 2.8. The strain Leuc5 showed the highest acidifying potential; the pH lowering caused by this strain during 18 hours of incubation reached 3.7 with a ΔpH of 2.8. (Fig. 3). The mesophilic *Lactobacillus* strains Lbl1, Lbl2, Lbl3, Lbl4 and Lbl5 have a ΔpH that varies between 0.9 and 1.69 (Fig. 4). The most acidifying strain in this group was strain Lbl1. It decreased the pH to 4.9 during the 18-hour incubation.
<figure><img src="image_5.png" /><figcaption>**Fig. 2. Variation of pH with isolates of the genus Lactococcus**
*Lc: Lactococcus; ΔpH: Variation of pH*</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3. Variation of pH with isolates of the genus Leuconostoc**
*Leuc: Leuconostoc; ΔpH: Variation of pH*</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 4. Variation of pH with mesophilic Lactobacillus isolates**
* Labels: Lactobacillus; ΔpH: Variation of pH*</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Acidification kinetics of acidifying and/or coagulating strains
*Lc: Lactococcus*; *Leuc: Leuconostoc*; *Lbl: Lactobacillus*</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Amounts of lactic acid produced by acidifying and/or coagulating strains
*Lc: Lactococcus*; *Leuc: Leuconostoc*; *Lbl: Lactobacillus*</figcaption></figure>

The results showed that 5 isolates of the genus *Lactococcus* (Lc7, Lc8, Lc12, Lc15 and Lc16) caused total coagulation during 18 hours of incubation, the same result was observed in two (02) strains of *Leuconostoc* (Leuc4 and Leuc5) and one strain of mesophilic *Lactobacillus* (Lb11). Strains Lc11 and Leuc6, which belong to the genus *Lactococcus* and *Leuconostoc*, respectively, caused partial coagulation at the bottom of the tube. Strains Lc8, Leuc4, Leuc5, and Lb11 showed good acidifying and coagulating power. All of these strains showed the ability to decrease the pH to a value ≤ 5.3 during the 6 hours of incubation with complete coagulation after 18 hours of incubation. Strain L17 showed good acidifying power without having the coagulating potential. Fig. 5 presents the acidification kinetics of acidifying and/or coagulating strains during 24 hours of incubation. The acidity rate obtained by these strains during

24 hours of incubation varies between 52 and 78 D°. The highest acidity is obtained by the strain of the genus *Leuconostoc* Leuc5 with the production of 7.8 g/L of lactic acid after 24 hours of incubation (Fig. 6).

### 3.1.2.2 Proteolytic, lipolytic and texturizing activities

The proteolytic potential results for all strains tested showed lysis zone diameters ranging from 6 to 15 mm. Strains Lc11, Leuc4, and Lc15 exhibited the highest proteolytic activity with a diameter of 15 mm, followed by strain Lc7 with a lysis zone of 14 mm. Strains Lc3, Lc6, Leuc3, and Leuc7 recorded lower proteolytic activities (6 mm). Some of our strains exhibit lipolytic activity. This power is observed in four strains belonging to the genus *Lactococcus* (Lc7, Lc11 and Lc12), two strains of the mesophilic genus *Lactobacillus* (Lb11 and Lb15) and one strain of the genus
Leuconostoc (Leuc6). The exopolysaccharide production test was positive for ten (10) strains of the genus *Lactococcus* (Lc2, Lc5, Lc6, Lc7, Lc8, Lc11, Lc12, Lc14, Lc15 and Lc16), three strains belonging to the genus Leuconostoc (Leuc1, Leuc4 and Leuc6) and two strains of mesophilic *Lactobacillus* (Lb11 and Lb13). Table 4 illustrates the proteolytic, lipolytic and texturizing power of isolated strains (Table 4).

### 3.1.3 Sourdough composition

#### 3.1.3.1 Interstrain interactions

Most of the interactions between our strains appeared positive. However, all of our strains exhibited at least one antagonistic activity toward one of the strains studied. Strains Lb11 and Lc8 demonstrated remarkable antagonistic activity. The inhibition activity of Lb11 strain is observed against strains Leuc4, Lc12, Leuc5 e, Lc7 and Lc8 and that of Lc8 strain is observed against strains Lc15, Leuc6, Leuc5 and Lb11 (Table 5).

Based on this result, two mesophilic ferments were reconstituted F1 and FII. The F1 ferment is composed of four strains of the genus *Lactococcus* (Lc7, Lc8, Lc11 and Lc12) and one strain of *Leuconostoc* (Leuc4). The second ferment FII contains two strains of *Lactococcus* (Lc15 and Lc16) and two other strains belonging respectively to the genus *Leuconostoc* (Leuc6) and mesophilic *Lactobacillus* (Lb11).

#### 3.1.3.2 Technological capabilities of reconstituted mixed ferments

Both reconstituted ferments F1 and FII demonstrated significant acidifying potential, resulting in very rapid acidification. The pH values after 6 hours of fermentation were 4.2 and 4.3 for F1 and FII, respectively (Fig. 7). The pH variations after 18 hours of fermentation were 2.6 for F1 and 2.4 for FII. Each of the two ferments induced complete coagulation during 18 hours of incubation.

<table><caption>Table 4. Proteolytic, lipolytic and texturizing power of isolated strains</caption><thead><tr><th rowspan="2">Strains</th><th colspan="2">Proteolytic activity</th><th rowspan="2">Lipolytic activity</th><th rowspan="2">Texturizing power</th></tr><tr><th>Observation</th><th>Diameter (mm)</th></tr></thead><tbody><tr><td>Lc1</td><td>+</td><td>12</td><td>-</td><td>-</td></tr><tr><td>Lb11</td><td>+</td><td>13</td><td>+</td><td>+</td></tr><tr><td>Lb12</td><td>+</td><td>7</td><td>-</td><td>-</td></tr><tr><td>Lc2</td><td>+</td><td>9</td><td>-</td><td>+</td></tr><tr><td>Lc3</td><td>+</td><td>6</td><td>-</td><td>-</td></tr><tr><td>Lb13</td><td>+</td><td>9</td><td>-</td><td>+</td></tr><tr><td>Lc4</td><td>+</td><td>8</td><td>-</td><td>-</td></tr><tr><td>Leuc1</td><td>+</td><td>11</td><td>-</td><td>+</td></tr><tr><td>Lc5</td><td>+</td><td>13</td><td>-</td><td>+</td></tr><tr><td>Lc6</td><td>+</td><td>6</td><td>-</td><td>+</td></tr><tr><td>Lc7</td><td>+</td><td>14</td><td>+</td><td>+</td></tr><tr><td>Lc8</td><td>+</td><td>12</td><td>-</td><td>+</td></tr><tr><td>Lc11</td><td>+</td><td>15</td><td>+</td><td>+</td></tr><tr><td>Lc9</td><td>+</td><td>9</td><td>-</td><td>-</td></tr><tr><td>Lc12</td><td>+</td><td>9</td><td>+</td><td>+</td></tr><tr><td>Leuc2</td><td>+</td><td>13</td><td>-</td><td>-</td></tr><tr><td>Lc10</td><td>+</td><td>10</td><td>-</td><td>-</td></tr><tr><td>Leuc3</td><td>+</td><td>6</td><td>-</td><td>-</td></tr><tr><td>Leuc4</td><td>+</td><td>15</td><td>-</td><td>+</td></tr><tr><td>Lb14</td><td>+</td><td>12</td><td>-</td><td>-</td></tr><tr><td>Lb15</td><td>+</td><td>10</td><td>+</td><td>-</td></tr><tr><td>Lc13</td><td>+</td><td>11</td><td>-</td><td>-</td></tr><tr><td>Lc14</td><td>+</td><td>12</td><td>-</td><td>+</td></tr><tr><td>Lc15</td><td>+</td><td>15</td><td>+</td><td>+</td></tr><tr><td>Lc16</td><td>+</td><td>13</td><td>-</td><td>+</td></tr><tr><td>Leuc5</td><td>+</td><td>13</td><td>-</td><td>-</td></tr><tr><td>Leuc6</td><td>+</td><td>13</td><td>+</td><td>+</td></tr><tr><td>Leuc7</td><td>+</td><td>6</td><td>-</td><td>-</td></tr><tr><td>Lc17</td><td>+</td><td>11</td><td>-</td><td>-</td></tr></tbody></table>

Lc: *Lactococcus*; Leuc: Leuconostoc; Lb1: Mesophilic *Lactobacillus*, -: Absence of activities, +: Presence of activities; mm: millimeter
<table><caption>Table 5. Interaction between isolated strains</caption><thead><tr><th>Strains</th><th>Leuc4</th><th>Lc11</th><th>Lc8</th><th>Lc12</th><th>Lc15</th><th>Leuc6</th><th>Lc16</th><th>Leuc5</th><th> Label1</th><th>Lc7</th></tr></thead><tbody><tr><td>Leuc4</td><td></td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Lc11</td><td>+</td><td></td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Lc8</td><td>+</td><td>+</td><td></td><td>+</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td><td>+</td></tr><tr><td>Lc12</td><td>+</td><td>+</td><td>+</td><td></td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Lc15</td><td>-</td><td>+</td><td>-</td><td>+</td><td></td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Leuc6</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td></td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Lc16</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td><td>+</td><td>+</td><td>+</td></tr><tr><td>Leuc5</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td><td>-</td><td>+</td></tr><tr><td>Label1</td><td>-</td><td>+</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td></tr><tr><td>Lc7</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td></td></tr></tbody></table>

Lc: Lactococcus; Leuc: Leuconostoc; Lbl: Mesophilic Lactobacillus; -: Absence of Interaction, +: Presence of Interaction

<figure><img src="image_10.png" /><figcaption>Fig. 7. Acidification kinetics of reconstituted ferments
FI: Ferment I; FII: Ferment II</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Fig. 8. Production of lactic acid by ferments I and II
FI: Ferment I; FII: Ferment II</figcaption></figure>
<table><caption>Table 6. Technological potential of FI and FII ferments</caption><thead><tr><th rowspan="2">Ferments</th><th rowspan="2">Coagulation (18H)</th><th colspan="2">Proteolytic activity</th><th rowspan="2">Lipolytic activity</th><th rowspan="2">Texturizing power</th></tr><tr><th>Observation</th><th>Diameter (mm)</th></tr></thead><tbody><tr><td>FI</td><td>+++</td><td>+</td><td>16</td><td>+</td><td>+</td></tr><tr><td>FII</td><td>+++</td><td>+</td><td>21</td><td>+</td><td>+</td></tr></tbody></table>

FI: Ferment I; FII: Ferment II

The acidity obtained after 6 hours of fermentation was 51 °D for FI and 47 °D for FII. The acidity caused by the two ferments during 18 hours of incubation was respectively 81 o D and 77 °D with a production of 8.1 and 7.7 g/L of lactic acid (Fig. 8) (Kaddem, 1993)

The proteolytic power test was positive for both ferments with the presence of a lysis zone of 16 mm in diameter for FI and 21 mm for FII. Regarding the flavoring power, it appears that the two ferments did not have the same capacity to produce aromas. The FI ferment produced a significant quantity of aromas unlike the FII which was unable to produce these molecules (Table 6)

## 3.2 Discussion

According to the morphological and biochemical identification performed, the strains isolated from samples of traditional cheese coagulated by Jibben belong to the genera *Lactococcus*, *Lactobacillus*, *Leuconostoc*, *Pediococcus*, and *Enterococcus*. This microbial population is dominated by species of the genus *Lactococcus*, followed by mesophilic *Lactobacillus* and *Leuconostoc*. This result shows that species belonging to these genera are the main agents responsible for the transformation of milk and the production of fermented dairy products. The same result was reported by Lairini et al. (2014) in a study carried out on traditional Moroccan dairy products. Gonçalves et al. (2018) identified the microbial ecology of Portuguese Serpa cheese and the genera *Lactococcus*, *Leuconostoc*, *Lactobacillus* and *Enterococcus* were the main groups identified. The genus *Enterococcus* is particularly ubiquitous and survives in harsh conditions, such as those found in the digestive tract of humans and other animals. The probiotic properties of the latter are increasingly explored with reservations (Hussain et al., 2025). Doutoum et al. (2013) also demonstrated the dominance of species belonging to the genus *Lactococcus* in traditional Chadian curd (Araújo-Rodrigues et al., 2020).

Our study revealed the ability of some of the strains to rapidly produce lactic acid and cause a

decrease in pH. According to Durango et al. (2023), lactic acid bacteria are said to be acidifying when they are able to reduce the pH of milk from its initial value of approximately 6.6 to 5.3 over a period of six (6) hours. We identified strains belonging to the genera *Lactococcus*, *Leuconostoc* and *Lactobacillus* that possess this characteristic. Acidity is the most important property of strains used as lactic ferments. In addition to its crucial role in milk curdling and triggering the desired change reactions in cheesemaking technology, such as whey expulsion, acidity contributes to the development of texture, aroma, flavor, and antimicrobial activity (Endo & Dicks, 2014). The selection criterion for our strains was essentially based on their acidifying potential. Coagulant activity was observed in all acidifying strains except strain Lc17 of the genus *Lactococcus*. Despite the rapid acidification achieved by this strain during the 6-hour incubation period, the pH remained at 5 after 18 hours of incubation and coagulation did not occur. Proteolytic activity was positive for all strains tested. This potential is essential for the growth of lactic acid bacteria (Hussain et al., 2025). These microorganisms have a high need for free amino acids to develop. Gómez de Cadiñanos et al. (2019) demonstrated that *Lactococcus* have a high requirement for glutamic acid, glycine, leucine, isoleucine, histidine, methionine and valine. This proteolysis also plays an important role due to its impact on the texture and flavors of cheeses (Frau et al., 2016; Araújo-Rodrigues et al., 2021). The study of lipolytic activity allowed us to identify strains with this property. They belong to the genera *Lactococcus* (Lc5, Lc11 and Lc12), *Lactobacillus* (Lb11 and Lb15) and *Leuconostoc* (Leuc6). This lipolysis is essential for the strengthening of proteolytic activity in cheese as well as for the improvement of desired technological characteristics such as the development of aromas and flavors by promoting the increase in the production of small peptides and free amines in cheese (Nicosia et al., 2023; Hulak et al., 2016). The exopolysaccharide production test was positive for the majority of tested strains 14/29. These strains belong to the genera *Lactococcus*, *Leuconostoc* and *Lactobacillus*.
The production of exopolysaccharides is considered an important characteristic when selecting lactic acid bacteria as starter cultures for certain dairy products (Câmara et al., 2019).

The study of the interaction between the previously identified strains of interest allowed us to compose two ferments. Each of the two ferments contains several strains that do not exhibit any antagonistic activity. The symbiosis between the selected bacterial strains became clear during the technological evaluation of our ferments, which implies cell viability and coexistence between strains. The rate of acidification and proteolysis increased in both ferments tested. Lipolysis and texturizing power were positive. However, aroma production was observed only in the FI ferment. The studies of Pailin et al. (2001) and Canon et al. (2022) highlighted the importance of the proteolytic system in the positive interactions between *Lactobacillus bulgaricus* species (involved in exopolysaccharide and aroma production) and *Streptococcus thermophilus* (involved in rapid acidification and aroma production). Similar studies have shown viability above 90% for *Streptococcus thermophilus*, *Lactobacillus delbrueckii* and *Lactobacillus helveticus* strains used as ambient starters in Comté cheese technology despite microbial diversity (Lutin et al., 2022; Lutin et al., 2024).

The selection of high-performance strains to make cheese cultures will improve the quality of the finished product and increase production at the industrial level (Adnan & Tan, 2007; Zheng et al., 2021). This sector of cheese production in Chad is generally occupied by women from the collection of local raw milk to processing (Beral et al., 2023). An improvement in cheese production through the selection of appropriate cultures will promote scaled-up exploitation with the advantage of job creation for women and an increase in household income.

## 4. CONCLUSION

The study isolated and identified lactic acid bacteria strains from traditional cheeses. The selected strains produced two mesophilic starter cultures, FI and FII. These starter cultures consist of lactic acid bacteria strains belonging to the genera *Lactococcus*, *Leuconostoc*, and *Lactobacillus*. The combination of these bacteria has contributed to the improvement of several parameters sought in cheese technology. The inability of FII ferment to produce aromas will call into question its use and requires improvement.

However, the FI ferment has satisfactory technological characteristics and can be used as a starter culture in the production of traditional cheeses. In order to be able to exploit this work, other studies must be carried out, namely the molecular characterization of the identified strains of technological interest, the study of their probiotic effects and then the development and evaluation of a test cheese.

## DISCLAIMER (ARTIFICIAL INTELLIGENCE)

Author(s) hereby declare that NO generative AI technologies such as Large Language Models (ChatGPT, COPILOT, etc) and text-to-image generators have been used during writing or editing of this manuscript.

## ACKNOWLEDGEMENTS

This study was supported by the Laboratory of Food Science and Nutrition (LaRSAN), University of N'Djamena, the Livestock Research Institute for Development (IRED) and Laboratory of Biochemistry and Applied Immunology (LaBIA), University Joseph Ki-Zerbo. The authors also acknowledge these structures, the heads of the schools and the vendors who cooperated fully.

## COMPETING INTERESTS

Authors have declared that no competing interests exist.

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