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IN VITRO SCREENING OF BACTERIAL ISOLATES FROM TRADITIONAL FERMENTED SORGHUM PRODUCTS FOR PROBIOTIC USE T.R. Duche 1 , L. I. Ezeogu 2  and T. N. Nwagu 2 1 Department of Microbiology, Federal University of Agriculture, Makurdi Benue State 2 Department of Microbiology, University of Nigeria Nsukka, Enugu State

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Potential probiotic bacteria from fermented sorghum products were screened

For  ability to survive gastro-intestinal acidity at different pH using simulated gastric juice

They were also tested for bile tolerance and antimicrobial inhibition on selected pathogens

The result provides a basis for further screening of isolates from fermented sorghum products from Africa to be considered for probiotic use.

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In African countries, particularly Nigeria, the most common fermented beverages are made from sorghum and they include  kunu ,  burukutu  and  pito  (Achi and  Ukwuru , 2015).  They have been part of the human diet since the dawn of human civilization, and have been used as a means of improving the shelf life, safety, digestibility, and nourishment for more than 6,000 years  ( Ayorinde   et al ., 2017),  These products have a high content of bacteria with a large bio-diversity ( Paludan - Muller  et al ., 2002;  Muyanja   et al ., 2003) most of which are lactic acid bacteria Nwachukwu  et al . (2010)  It’s on this basis that we decided to explore the microflora of these beverages focusing on  the  beneficial ones for possible probiotic traits

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Acid tolerance test Bile tolerance test Overlay method for antimicrobial activity Agar well diffusion for possible production of bacteriocin Coculture interference of potential probiotic bacteria and selected pathogens

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0.5h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN9 KN10 BK4 BK5 BK8 Pt1 Pt7 Pt8 298000 224000 298000 211000 285000 203000 216000 68000 37000 114000 1h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN9 KN10 BK4 BK5 BK8 Pt1 Pt7 Pt8 204000 2000 291000 200000 250000 48000 7000 28000 28000 2000 2h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN9 KN10 BK4 BK5 BK8 Pt1 Pt7 Pt8 90000 15000 260000 37000 211000 3000 3000 10000 12000 3000 3h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN9 KN10 BK4 BK5 BK8 Pt1 Pt7 Pt8 50000 3000 272000 222000 201000 0 1000 7000 0 0 4h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN9 KN10 BK4 BK5 BK8 Pt1 Pt7 Pt8 32000 0 58000 112000 199000 0 37000 5000 0 0 12h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN9 KN10 BK4 BK5 BK8 Pt1 Pt7 Pt8 0 0 6000 3000 104000 0 2000 0 0 0

0.5h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 Pt1 Pt4 Pt7 Pt8 226000 296000 820000 299000 230000 289000 196000 226000 101000 30000 67000 211000 1h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 Pt1 Pt4 Pt7 Pt8 206000 285000 150000 312000 212000 262000 192000 120000 105000 51000 58000 116000 2h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 Pt1 Pt4 Pt7 Pt8 117000 282000 89000 286000 217000 228000 143000 56000 44000 44000 34000 26000 3h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 Pt1 Pt4 Pt7 Pt8 216000 272000 75000 218000 248000 220000 127000 81000 27000 39000 34000 15000 4h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 Pt1 Pt4 Pt7 Pt8 240000 272000 136000 200000 1000 215000 122000 30000 73000 0 47000 16000 12h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 Pt1 Pt4 Pt7 Pt8 146000 27000 21000 129000 0 201000 21000 1000 31000 0 26000 6000

0.5h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK1 BK2 BK4 BK5 BK8 BK9 Pt1 Pt4 Pt7 Pt8 290000 274000 820000 271000 289000 156000 228000 284000 227000 290000 179000 135000 39000 38000 69000 1h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK1 BK2 BK4 BK5 BK8 BK9 Pt1 Pt4 Pt7 Pt8 284000 296000 150000 200000 278000 50000 1000 262000 217000 281000 132000 146000 64000 27000 213000 2h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK1 BK2 BK4 BK5 BK8 BK9 Pt1 Pt4 Pt7 Pt8 220000 304000 89000 186000 276000 17000 1000 251000 180000 237000 142000 80000 68000 317000 93000 3h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK1 BK2 BK4 BK5 BK8 BK9 Pt1 Pt4 Pt7 Pt8 230000 220000 75000 183000 231000 8000 1000 249000 226000 200000 870000 43000 39000 35000 98000 4h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK1 BK2 BK4 BK5 BK8 BK9 Pt1 Pt4 Pt7 Pt8 165000 261000 136000 192000 217000 50000 29000 212000 319000 195000 380000 106000 40000 37000 169000 12h 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ 1.5×10⁸ KN3 KN5 KN6 KN9 KN10 BK1 BK2 BK4 BK5 BK8 BK9 Pt1 Pt4 Pt7 Pt8 178000 188000 21000 278000 249000 40000 5000 1000 348000 215000 263000 270000 40000 28000 102000

Acid tolerance test of sorghum products at pH1 Acid tolerance test of sorghum products at pH2 Acid tolerance test of sorghum products at pH3

At pH1 only 3 isolates (KN 10, KN9 and BK4) survived up to 4 h point. At pH2, all the isolates could survive up to 12 h.  At pH3, growth kept increasing across all potential candidates, though isolate PT7 and PT4 showed the least counts.

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0h KN3 KN5 KN6 KN9 KN10 BK4  BK5 BK8 PT1 150000000 150000000 150000000 150000000 150000000 150000000 150000000 150000000 150000000 0.5h KN3 KN5 KN6 KN9 KN10 BK4  BK5 BK8 PT1 2980000 2930000 2620000 430000 420000 2200000 350000 390000 1900000 3h KN3 KN5 KN6 KN9 KN10 BK4  BK5 BK8 PT1 2920000 2760000 2700000 340000 370000 1270000 230000 620000 2010000 12h KN3 KN5 KN6 KN9 KN10 BK4  BK5 BK8 PT1 2900000 2520000 1210000 40000 200000 1480000 50000 600000 410000 24h KN3 KN5 KN6 KN9 KN10 BK4  BK5 BK8 PT1 1890000 2310000 1720000 1040000 260000 2420000 70000 560000 380000

0h KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 PT1 150000000 150000000 150000000 150000000 150000000 150000000 150000000 150000000 150000000 0.5h KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 PT1 2960000 2880000 2900000 400000 340000 2200000 250000 1190000 2110000 3h KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 PT1 2900000 2690000 2930000 140000 330000 2170000 240000 400000 2270000 12h KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 PT1 2860000 2330000 1560000 170000 130000 1300000 60000 630000 450000 24h KN3 KN5 KN6 KN9 KN10 BK4 BK5 BK8 PT1 1280000 2300000 2110000 260000 130000 1490000 430000 470000 360000

Fig. 4. Bile Tolerance test of sorghum products at 1%          Fig . 5. Bile Tolerance test of sorghum products at 2%

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Table 1 . Antagonistic activity of potential probiotic bacteria isolated from fermented sorghum products against selected pathogens by overlay method

Isolate Salmonella spp E.coli SH. Spp L.Spp   PT1     0.00±0.00 d   20.67±0.67 d   0.00±0.00 d   8.67±0.67 d KN3   22.00±1.16 c 24.67±2.03 cd 30.00±0.00 ab 40.67±1.76 b BK8   21.67±0.67 c 4.67±0.88 e 28.67±0.88 b 5.33±0.33 d KN10   0.00±0.00 d 0.00±0.00 e 14.67±1.45 c 0.00±0.00 e KN6   27.33±1.45 b 23.67±1.86 cd 11.00±1.00 c 21.33±0.67 c BK5   22.67±0.67 c 70.00±0.00 a 0.00±0.00 d 24.00±2.08 c KN5   23.33±2.03 c 70.00±0.00 a 33.00±1.73 a 39.67±2.03 b BK4   20.33±0.33 c 36.67±5.70 b 32.67±2.67 ab 23.00±1.00 c KN9   31.00±1.00 a 30.00±1.73 c 14.33±1.45 c 56.67±2.03 a

Isolate Salmonella spp E.coli SH. Spp L.Spp   PT1     10.33±0.88 a   9.33±0.33 cd   0.00±0.00    0.00±0.00 c KN3   6.00±0.00 bc 14.00±1.00 a 0.00±0.00 0.00±0.00 c KN5   3.33±0.88 c 0.00±0.00 f 0.00±0.00 0.00±0.00 c KN6   10.00±1.16 a 10.67±0.33 bc 0.00±0.00 3.33±0.88 b KN9   3.67±1.20 c 12.33±1.45 ab 0.00±0.00 0.00±0.00 c KN10   6.67±0.88 b 0.00±0.00 f 0.00±0.00 0.00±0.00 c BK4   12.33±1.45 a 5.00±0.00 e 0.00±0.00 0.00±0.00 c BK5   0.00±0.00 d 0.00±0.00 f 0.00±0.00 10.33±0.88 a BK8   12.00±0.00 a 7.67±0.88 d 0.00±0.00 4.33±0.67 b

Table 2 . Antimicrobial activity of potential probiotic bacteria isolated from fermented sorghum products against selected pathogens by agar well diffusion

a,b,c Means  with different superscript along the column are significant (p<0.05 )

a,b,c Means  with different superscript along the column are significant (p<0.05)

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Table 3. Coculture interference of potential probiotics isolated from fermented sorghum products with  Salmonella  spp

Isolate 0hr 4hr 8hr 12hr 16hr 20hr 24hr   PT1   2.84 a   2.51 b   2.41 c   2.55 b   2.62 cd   2.58 c   2.54 d   Salmonella 1.44 ef 1.25 b 1.25 g 0.87 f 0.69 g 0.60 f 0.74 f   KN3 2.09 cd 2.02 b 1.85 f 2.22 cd 2.68 c 2.60c 2.18 e   Salmonella 1.89 d 6.57 a 0.92 h 0.93 f 0.52 h 0.42 g 0.40 g   KN5 2.55 b 2.80 b 2.78 a 2.58 b 2.52 d 2.58 c 2.68 cd   Salmonella 1.87 d 0.89 b 0.60 i 0.73 g 0.12 i 0.02 h 0.001 h   KN6 2.84 a 2.65 b 2.62 b 2.61 b 2.80 b 2.78 b 2.81 abc   Salmonella 2.21 c 1.03 b 1.09 g 0.10 h 0.10 ij 0.001 h 0.001 h   KN9 2.18 c 2.13 b 1.82 f 1.87 e 1.90 f 1.91 e 2.22 e   Salmonella 1.09 g 0.84 b 0.22 j 0.19 h 0.17 i 0.06 h 0.00 h   KN10 2.91 a 2.20 b 2.11 de 2.26 c 2.96 a 2.98 a 2.97 a   Salmonella 1.26 fg 1.15 b 0.27 j 0.15 h 0.00 j 0.00 h 0.00 h   BK4 2.91 a 2.83 b 2.25 cd 2.20 cd 2.18 e 2.90 ab 2.91 ab   Salmonella 2.18 c 1.30 b 1.11 g 0.90 f 0.60 gh 0.00 h 0.00 h   BK5 2.89 a 2.56 b 2.30 c 2.86 a 2.81 b 2.74 bc 2.74 bc   Salmonella 1.27 fg 0.70 b 0.71 i 0.61 g 0.59 gh 0.55 fg 0.22 g   BK8 2.77 ab 2.30 b 2.07 e 2.11 d 2.09 e 2.20 d 2.30 e   Salmonella 1.60 e 0.71 b 0.25 j 0.10 h 0.70 ij 0.01 h 0.00 h

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Table 4. Coculture interference pf potential probiotics isolated from fermented sorghum products with  E. coli

Isolate pathogen 0hr 4hr 8hr 12hr 16hr 20hr 24hr   PT1   2.27 k   2.36 h   2.51 a   2.69 d   2.63 d   2.01 j   2.20 c   E.coli 2.72 hi 2.72 e 2.52 e 2.31 h 2.20 h 2.00 j 2.05 f   KN3 2,21 l 2.12 j 2.10 i 2.17 i 2.18 h 2.22 g 2.23 c   E.coli 2.97 a 2.93 a 2.86 a 2.83 b 2.80b 2.81 b 2.83 b   KN5 2.70 i 1.11 l 0.10 l 0.03 n 0.005 l 0.00 m 0.00 i   E.coli 2.78 ef 2.50 g 2.50 f 2.63 e 2.68 c 2.77 c 2.85 b   KN6 2.90 c 2.20 i 2.15 i 1.12 l 0.31 i 0.07 l 0.00 i   E.coli 2.81 e 2.74 e 2.30 h 2.19 h 2.19 g 2.14 i 2.14 de   KN9 2.52 j 2.51 g 2.40 g 2.45 g 2.46 g 2.46 f 2.18 cd   E.coli 2.96 ab 2.83 cd 2.71 d 2.51 f 2.78 b 2.18 h 2.04 f   KN10 2.86 d 2.80 d 2.31 g 1.36 j 0.07 j 0.003 m 0.00 i   E.coli 2.77 fg 2.83 d 2.74 cd 2.89 a 2.91 a 2.94 a 2.98 a   BK4 2.93 abc 2.89 abc 2.84 ab 2.70 d 2.45 g 2.51 e 1.87 g   E.coli 2.74 gh 2.65 f 2.55 de 2.30 h 2.59 e 2.73 d 2.22 c   BK5 2.74 fgh 1.09 l 0.68 k 0.43 m 0.03 kl 0.00 m 0.00 i   E.coli 2.93 bc 2.92 ab 2.81 b 2.74 c 2.53 f 2.23 g 2.12 e   BK8 2.04 c 2.86 bcd 2.77 c 2.63 e 2.55 f 1.80 k 0.15 h   E.coli 2.71 m 2.00 k 1.87 j 1.21 k 0.06 jk 0.04 l 0.03 i

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Plate. 1. showing activity of isolate 8BM9 on  S.  flexneri   by culture overlay

Plate. 2. agar well diffusion plate showing different zones of inhibition exhibited by LAB isolates on  S. typhimurium

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DISCUSSION OF RESULTS Twenty-nine percent of our isolates survived in simulated gastric and intestinal juices up to 12 h of incubation (Chen  et al ., 2019)  Results of agar well diffusion reported that 80 and 60% of the isolates showed inhibition by agar well diffusion on both  S. typhimurium  and enteropathogenic  E. coli An evidence that these isolates have the possibilities of eliciting  bacteriocinogenic  properties against pathogens implicated in food related infections . [ Kumar  et al . (2016); Kang  et   al . (2017);  Ahn   et al . (2018); Chen  et al . (2019) and Monteiro  et al . (2019) ] who also reported various degrees of antimicrobial activities of LAB on different relevant food related pathogens .

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Conclusion and Recommendation Our findings will serve as a basis for the promising role of probiotic bacteria from fermented sorghum beverages to be utilized in the control of food related infections and the food industry at large. Our limitation in this study was that we couldn’t demonstrate the latent survival of these potential probiotic cultures during gastrointestinal passage . Another limitation here was that we couldn’t screen the isolates for the quality and quantity of antimicrobial substances produced  We suggest further studies to step up from there.

IN VITRO SCREENING OF BACTERIAL ISOLATES FROM TRADITIONAL FERMENTED SORGHUM PRODUCTS FOR PROBIOTIC USE
T.R. Duche1, L. I. Ezeogu2 and T. N. Nwagu2
1Department of Microbiology, Federal University of Agriculture, Makurdi Benue State
2Department of Microbiol

IN VITRO SCREENING OF BACTERIAL ISOLATES FROM TRADITIONAL FERMENTED SORGHUM PRODUCTS FOR PROBIOTIC USE T.R. Duche1, L. I. Ezeogu2 and T. N. Nwagu2 1Department of Microbiology, Federal University of Agriculture, Makurdi Benue State 2Department of Microbiol

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