A survey of propionic acid bacteria and the optimisation of vitamin b12 in motoho, an African Beverage

Abstract

Cereal-based fermentation is an extensive, common and popular practice across the African continent. The variety of fermented food products and beverages produced by means of sorghum fermenting is testament to the practice. Motoho is a sour, porridge-like snack beverage made from brown or red sorghum. Motoho is produced by means of spontaneous fermentation by back slopping with tomoso (starter culture). Lactic acid bacteria (LAB) are reported to be the most dominant microorganisms in spontaneous cereal fermentation, followed by yeasts. Vitamin B12 (vit B12) deficiency is reported to have negative health effects, and therefore, fortification by means of cereal fermentation with Generally Recognised as Safe Organisms (GRAS) has been proposed as a method for addressing vitamin deficiencies. The vitamin content of motoho, especially vit B12 is unknown. Propionibacterium freudenreichii. P. freudenreichii biosynthesises vit B12 naturally and has GRAS status. The aim of this study was to isolate Propionibacterium spp. present in motoho, and optimise the production of vit B12 by means of co-inoculating tomoso with P. freudenreichii subsp shermanii (PAB-J17) at low and high concentrations. The traditional method (back-slopping with tomoso) being utilised as the control. Motoho was prepared using two methods: (i) the traditional method (TFM) (back-slopping with tomoso) and (ii) by co-inoculating tomoso with P. freudenreichii (PABJ-17) in a low (LPAB-J17) and high (HPAB-J17) cell concentrations, respectively. Fermentation was facilitated at 32 °C for 12 h with sampling performed every 3 h. The pH was monitored and the following organisms were enumerated: Total Bacterial Counts, coliforms, lactic acid bacteria (LAB), yeasts and moulds. LAB were the most dominant organisms during the whole fermentation process, with cell counts obtained for TFM being significantly higher (p ≤ 0.05) than LPAB-J17 and HPAB-J17. For total coliform counts, there was no significant difference (p ≥ 0.05) between LPAB-J17 and HPAB-J17. After 12 h of fermentation, there was no yeast growth for TFM. HPAB-J17 obtained the lowest final pH (3.6) after fermentation, with TFM and LPAB-J17 obtaining the same final pH of 4.75. Isolate MFS1 was isolated from MFS by pre-enrichment in Yel-broth (30 °C for 5 d) and sub-culturing on Yel-agar (30 °C for 7 d anaerobically). Isolate MFS1 was Gram-positive with an endospore. The NCBI BLAST resulted in 99.5 % similarity with seven strains. It had 99.5% similarity to the type species of Bacillus rugosus sp. nov. B. rugosus is an emerging organism of the Bacillus genus. MFS1 formed a relatively stable phylogeny at 68 %; the cluster was located in a clade that was 99 % stable. The optimisation of vit B12 was accomplished by co-inoculating tomoso, which was back-slopped with PAB-J17 in a low cell inoculum of 1 × 104 cfu.g-1 (LPAB-J17) and a high cell inoculum of 1 × 108 cfu.g-1 (HPAB-J17). The traditionally fermented motoho (TFM), which was the control obtained a significantly higher (p ≤ 0.05) vit B12 concentration than LPAB-J17; LPAB-J17 vit B12 concentration was significantly (p ≤ 0.05) lower than TFM and HPAB-J17. Inoculum optimisation was successful for HPAB-J17, obtaining vit B12 which was 2-fold higher than the control (TFM) and 5-fold higher than LPAB-J17.