Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online
Journal website www.isnff-jfb.com

Original Research

Volume 35, September 2026, pages 57-68

Probiotics characterization of fermented tiger nut-millet-blended beverage

Figures

Figure 1.
Figure 1. Percentage survival of LAB isolates at different acid levels after 3 hours of exposure. (n = 3). Data are presented as mean ± SEM (n = 3 independent replicates per isolate). Error bars denote ± SEM. For each isolate, survival was compared across the three pH levels using one-way ANOVA followed by pairwise t-tests with Bonferroni correction; survival differed significantly among all three pH levels for every isolate tested (p < 0.05 in all cases). Bars labeled a, b, and c within an isolate differ significantly from one another. Key: TMD (2, 3 and 5) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Date fruit; TMSP (2, 4, and 7) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Sweet potato; TMDSP (1, 3, 4 and 6) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Date fruit and Sweet potato.
Figure 2.
Figure 2. Percentage survival of LAB isolates at different bile salt concentrations after 3 hours of exposure (n = 3). Data are presented as mean ± SEM (n = 3). Error bars denote ± SEM. Survival at 0.3% versus 2% bile salt was compared for each isolate using Student’s t-test; survival differed significantly between the two concentrations for every isolate (p < 0.001). Key: TMD (2, 3 and 5) = Bacterial isolates from Tiger nut-Millet beverage sweetened with Date fruit; TMSP (2, 4, and 7) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Sweet potato; TMDSP (1, 3, 4 and 6) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Date fruit and Sweet potato.
Figure 3.
Figure 3. Percentage survival of LAB isolates at different phenol concentrations after 3 hours of exposure (n = 3). Data are presented as mean ± SEM (n = 3). Error bars denote ± SEM. Survival at 0.2% versus 0.3% phenol was compared for each isolate using Student’s t-test; survival differed significantly between the two concentrations for every isolate (p < 0.001). Key: TMD (2, 3 and 5) = Bacterial isolates from Tiger nut-Millet beverage sweetened with Date fruit; TMSP (2, 4, and 7) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Sweet potato; TMDSP (1, 3, 4 and 6) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Date fruit and Sweet potato.

Tables

Table 1. Lactic acid bacteria count (CFU/mL) from fermented spicy tiger nut-millet beverage
 
Sample CodeLog10 CFU/mL
0 hour6 hours12 hours18 hours24 hours30 hours36 hours
Values were presented as mean ± standard deviation (n = 3 for each sample). The letters across the column represent significant differences. Key: TMD F1-F6 = Tiger nut-Millet beverage sweetened with Date fruit; TMSP F1-F7 = Tiger nut-Millet beverage sweetened with Sweet potato; TMDSP F1-F7 = Tiger nut-Millet beverage sweetened with Date fruit and Sweet potato
TMD F13.45 ± 0.23a4.91 ± 0.27b4.97 ± 0.69b5.01 ± 0.60c6.07 ± 0.60d6.16 ± 0.80d6.30 ± 0.75d
TMD F23.92 ± 0.45a5.03 ± 0.72b5.10 ± 0.69b5.28 ± 0.68b6.03 ± 0.71c6.04 ± 0.73c6.20 ± 0.67c
TMD F34.01 ± 0.47a4.96 ± 0.80a5.08 ± 0.76b5.20 ± 0.77b6.04 ± 0.73c6.27 ± 0.75c6.31 ± 0.70c
TMD F44.23 ± 0.40a5.00 ± 0.90b5.11 ± 0.67b5.31 ± 0.60b6.01 ± 0.58c6.24 ± 0.79c6.31 ± 0.77c
TMD F52.45 ± 0.32a4.78 ± 0.84b5.17 ± 0.82b5.10 ± 0.67b5.92 ± 0.99c6.05 ± 0.88c6.16 ± 0.81c
TMD F63.73 ± 0.24a4.83 ± 0.81b5.05 ± 0.83c5.08 ± 0.67b6.04 ± 0.91c6.08 ± 0.90c6.18 ± 0.83c
TMSP F13.04 ± 0.35a4.87 ± 0.75b5.07 ± 0.71c5.10 ± 0.70c6.06 ± 0.65d6.04 ± 0.74d6.13 ± 0.77d
TMSP F23.86 ± 0.50a5.43 ± 0.70b5.70 ± 0.79b6.11 ± 1.04c6.03 ± 0.76c6.13 ± 0.74c6.26 ± 0.69
TMSP F33.64 ± 0.34a5.27 ± 0.68b6.16 ± 0.57c6.26 ± 0.76c6.30 ± 0.71c6.37 ± 0.74c6.40 ± 0.70
TMSP F43.30 ± 0.50a4.82 ± 0.35b5.31 ± 0.73c5.36 ± 0.73c6.11 ± 0.86d6.21 ± 0.83d6.26 ± 0.80d
TMSP F52.30 ± 0.40a4.91 ± 0.37b5.25 ± 0.53b6.28 ± 0.53c6.30 ± 0.56c6.34 ± 0.58c6.38 ± 0.64c
TMSP F63.23 ± 0.51a4.97 ± 0.47b5.11 ± 0.40c5.97 ± 0.86c6.04 ± 0.86c6.13 ± 0.85c6.22 ± 0.78c
TMSP F72.86 ± 0.34a4.57 ± 0.71b5.61 ± 0.70c5.69 ± 0.77c5.97 ± 0.85c6.09 ± 0.80c6.16 ± 0.82c
TMDSP F12.87 ± 0.25a4.81 ± 0.81b5.00 ± 0.83b5.14 ± 0.79b6.27 ± 0.79c6.31 ± 0.74c6.31 ± 0.77c
TMDSP F23.44 ± 0.30a4.64 ± 0.82b4.83 ± 0.62b4.93 ± 0.70b5.83 ± 0.88c5.98 ± 0.82c6.01 ± 0.82c
TMDSP F32.76 ± 0.51a4.70 ± 0.95b4.90 ± 0.83b4.94 ± 0.68b5.77 ± 0.88c5.95 ± 0.82c6.07 ± 0.78c
TMDSP F43.64 ± 0.42a4.91 ± 0.67b4.95 ± 0.70b5.00 ± 0.76b5.87 ± 0.67c6.00 ± 0.76c6.06 ± 0.81c
TMDSP F52.87 ± 0.23a4.73 ± 0.85b4.93 ± 0.81b4.98 ± 0.80b5.77 ± 0.53c5.92 ± 0.58c6.05 ± 0.65c
TMDSP F63.01 ± 0.22a4.74 ± 0.10b4.78 ± 0.98b4.95 ± 0.83b5.95 ± 0.59c6.05 ± 0.70c6.13 ± 0.76c
TMDSP F72.90 ± 0.35a4.89 ± 0.78b4.92 ± 0.79b4.95 ± 0.80b5.96 ± 0.63c5.98 ± 0.63c6.02 ± 0.64c
Control2.68 ± 0.42a4.87 ± 0.57b5.08 ± 0.48b5.59 ± 0.34b5.94 ± 0.71b6.02 ± 0.54c6.20 ± 0.60c

 

Table 2. Biochemical characterization and Physiological ability of LAB isolated from naturally sweetened spicy fermented tiger nut-Millet beverage
 
CodesCatalaseCitrateOxidaseH2S testSugar FermentationTemperature (oC)NaCl (%)
GlucoseLactoseSucrose154546.58
Key: TMD D1-D5 = Tiger nut-Millet beverage sweetened with Date fruit; TMSP P1-P7 = Tiger nut-Millet beverage sweetened with Sweet potato; TMDSP P1-P6 = Tiger nut-Millet beverage sweetened with Date fruit and Sweet potato
TMD1−−−−+ (gas)+ (gas)+ (gas)+++++
TMD2−−−−+ (gas)+ (gas)+ (gas)+++++
TMD3−−−−+ (gas)+ (gas)+ (gas)+++++
TMD4−−−−+ (gas)+ (gas)+ (gas)+−++−
TMD5−−−−+ (gas)+ (gas)+ (gas)+++++
TMSP1−−−−+ (gas)+ (gas)+ (gas)+++++
TMSP2−−−−+ (gas)+ (gas)+ (gas)+++++
TMSP3−−−−+ (gas)+ (gas)+ (gas)+−+++
TMSP4−−−−+ (gas)+ (gas)+ (gas)+++++
TMSP5−−−−+ (gas)+ (gas)+ (gas)+−++−
TMSP6−−−−+ (gas)+ (gas)+ (gas)+++++
TMSP7−−−−+ (gas)+ (gas)+ (gas)+−+++
TMDSP1−−−−+ (gas)+ (gas)+ (gas)+++++
TMDSP2−−−−+ (gas)+ (gas)+ (gas)+−++−
TMDSP3−−−−+ (gas)+ (gas)+ (gas)+++++
TMDSP4−−−−+ (gas)+ (gas)+ (gas)+++++
TMDSP5−−−−+ (gas)+ (gas)+ (gas)+−++−
TMDSP6−−−−+ (gas)+ (gas)+ (gas)+++++

 

Table 3. Identification of isolated LAB using the standard API 50 CHL system
 
IsolatesLAB Significant Taxa% accuracyRemarks
Key: TMD (2, 3 and 5) = Bacterial isolates from Tiger nut-Millet beverage sweetened with Date fruit; TMSP (2, 4, and 7) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Sweet potato; TMDSP (1, 3, 4 and 6) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Date fruit and Sweet potato
TMD2Lactobacillus plantarum 199.9Excellent identification
TMD3Lactobacillus plantarum 174.4Good identification
TMD5Lactobacillus plantarum 199.9Excellent identification
TMSP2Lactobacillus brevis 199.7Very good identification
TMSP4Lactobacillus plantarum 199.6Good identification
TMSP7Lactobacillus plantarum 199.9Excellent identification
TMDSP1Lactobacillus plantarum 199.9Excellent identification
TMDSP3Lactobacillus plantarum 190.4Good identification
TMDSP4Lactobacillus plantarum 198.5Very good identification
TMDSP6Lactobacillus pentosus69.5Doubtful profile

 

Table 4. Antibiotic susceptibility profile of selected probiotic LAB isolates
 
Antibiotics usedDisc conc. (µg)Zone of Inhibition (mm) (Interpretation)
TMD 2TMD 3TMD 5TMSP 2TMSP 4TMSP 7TMDSP 1TMDSP 3TMDSP 4TMDSP 6
Zone of inhibition (diameter in mm) for each antibiotic was measured and expressed as susceptible, S (≥21 mm), and resistant, R (≤15 mm). Key: TMD (2, 3 and 5) = Bacterial isolates from Tiger nut-Millet beverage sweetened with Date fruit; TMSP (2, 4, and 7) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Sweet potato; TMDSP (1, 3, 4 and 6) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Date fruit and Sweet potato.
Ampicillin (AMP)1024 (S)25 (S)25 (S)24 (S)27 (S)25 (S)26 (S)21 (S)30 (S)25 (S)
Cotrimoxazole (COT)2528 (S)28 (S)26 (S)28 (S)29 (S)27 (S)21 (S)25 (S)28 (S)22 (S)
Gentamicin (GEN)1026 (S)27 (S)26 (S)28 (S)31 (S)29 (S)28 (S)23 (S)27 (S)25 (S)
Erythromycin (ERY)524 (S)29 (S)21 (S)24 (S)27 (S)28 (S)34 (S)31 (S)29 (S)24 (S)
Tetracycline (TET)3029 (S)27 (S)26 (S)31 (S)29 (S)28 (S)25 (S)27 (S)29 (S)26 (S)
Vancomycin (VAN)3030 (S)22 (S)29 (S)27 (S)29 (S)21 (S)28 (S)24 (S)27 (S)23 (S)
Meropenem (MEM)106 (R)15 (R)7 (R)9 (R)10 (R)8 (R)11 (R)9 (R)13 (R)10 (R)
Augmentin (AUG)309 (R)11 (R)12 (R)14 (R)9 (R)10 (R)8 (R)11 (R)10 (R)8 (R)
Ceftazidime (CPZ)308 (R)9 (R)10 (R)11 (R)8 (R)8 (R)10 (R)9 (R)11 (R)10 (R)
Cefuroxime (CRX)109 (R)12 (R)10 (R)13 (R)12 (R)9 (R)10 (R)9 (R)12 (R)11 (R)
Ciprofloxacin (CIP)524 (S)27 (S)26 (S)29 (S)21 (S)20 (R)25 (S)21 (S)28 (S)29 (S)
Cephalexin (CP)1510 (R)9 (R)12 (R)10 (R)8 (R)10 (R)9 (R)11 (R)10 (R)12 (R)

 

Table 5. Antibacterial activities of selected probiotic LAB isolates
 
Test organismsZone of Inhibition (mm)
TMD 2TMD 3TMD 5TMSP 2TMSP 4TMSP 7TMDSP 1TMDSP 3TMDSP 4TMDSP 6
Values were presented as mean ± standard deviation (n = 3 for each sample). The letters across the column represent significant differences. Key: TMD (2, 3 and 5) = Bacterial isolates from Tiger nut-Millet beverage sweetened with Date fruit; TMSP (2, 4, and 7) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Sweet potato; TMDSP (1, 3, 4 and 6) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Date fruit and Sweet potato.
Bacillus cereus NCIB 634912.00 ± 0.00c11.00 ± 0.00b0.00 ± 0.00a15.00 ± 0.01d0.00 ± 0.00a15.00 ± 0.00d0.00 ± 0.00a11.00 ± 0.00b15.00 ± 0.0112.00 ± 0.00c
Staphylococcus aureus ATCC 259230.00 ± .00a0.00 ± 0.00a0.00 ± 0.00a20.00 ± 0.010.00 ± 0.00a11.00 ± 0.01b13.00 ± 0.01c0.00 ± 0.00a0.00 ± 0.00a17.00 ± 0.01d
Pseudomonas aeruginosa NCIB 34710.00 ± 0.00a19.00 ± 0.02e12.00 ± 0.01c11.00 ± 0.00b18.00 ± 0.010.00 ± 0.00a0.00 ± 0.00a11.00 ± 0.01b14.00 ± 0.02d20.00 ± 0.00f
Klebsiella pneumoniae20.00 ± 0.00e8.00 ± 0.00b23.00 ± 0.02g0.00 ± 0.00a20.00 ± 0.0114.00 ± 0.01c16.00 ± 0.01d21.00 ± 0.01f16.00 ± 0.01d0.00 ± 0.00a
Klebsiella edwardsii0.00 ± 0.00a19.00 ± 0.01e21.00 ± 0.01f14.00 ± 0.02d10.00 ± 0.00b0.00 ± 0.00a11.00 ± 0.00c10.00 ± 0.00b10.00 ± 0.00b0.00 ± 0.00a
Escherichia coli17.00 ± 0.01f15.00 ± 0.00d0.00 ± 0.00a20.00 ± 0.01h16.00 ± 0.02e15.00 ± 0.01d7.00 ± 0.01b0.00 ± 0.00a12.00 ± 0.01c16.00 ± 0.01e

 

Table 6. 16S rRNA gene sequence identification of isolated LAB strains
 
Isolate codeIsolate identitySimilarity (%)GenBank Accession number
Key: TMD (2 and 5) = Bacterial isolates from Tiger nut-Millet beverage sweetened with Date fruit; TMSP (2 and 4) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Sweet potato; TMDSP (1 and 6) = Bacterial Isolates from Tiger nut-Millet beverage sweetened with Date fruit and Sweet potato.
TMD2Lactiplantibacillus plantarum98.39NR_025447.1
TMD5Lactiplantibacillus plantarum96.89NR_117813.1
TMSP2Leuconostoc mesenteroides99.61NR_074957.1
TMSP4Lysinibacillus sphaericus90.43NR_042073.1
TMDSP1Lactiplantibacillus pentosus98.68NR_113338.1
TMDSP6Lactiplantibacillus pentosus99.73NR_029133.1