ISOLATION AND CHARACTERIZATION OF LACTOBACILLUS AND WEISSELLA STRAINS FROM KIMERE TOWARDS DEVELOPMENT OF A NOVEL PROBIOTIC YOGHURT FOR AFLATOXIN B1 CONTROL

Abstract

In Kenya, maize is an important staple food. Unfortunately, household maize is generally spoilt by fungi whose mycotoxin metabolites have been found to cause adverse effect to animal and human health. The study had four objectives: i) to determine aflatoxin levels in maize and maize flours in three different selected regions in Kenya. ii) To isolate and characterize Lactobacillus and Weissella strains in Kimere and determine their individual strain capacity to sequester aflatoxins. iii) To assess growth of the isolated Lactobacillus and Weissella strains in milk so as to develop a novel yoghurt using the highest Lactobacillus and Weissella aflatoxins binding strains. iv) To determine the capacity to lower aflatoxin B1 (AFB1) metabolite (aflatoxin M1) in urine of children from Eastern Kenya by Lactobacillus and Weissella isolated strains. To achieve the objectives, 75 maize grains and 27 flour samples were collected from three parts of Kenya and analysed for aflatoxins by Enzyme linked immunosorbent assay (ELISA) plus spectrophotometric technique. Basic and advanced molecular microbiology techniques were employed to obtain Lactobacillus and Weissella isolates from Kimere. These included growing the Lactobacillus and Weissella in a selective media followed by Gram staining. To identify the species of the isolated Lactobacillus and Weissella, Polymerase Chain Reaction (PCR) was used to amplify the DNA extracts. DNA blocks were sequenced and identified using the Blast software process. A novel yoghurt was developed using an isolate of Lactobacillus rhamnosus and Weissella, along with starter culture Streptococcus thermophilus. The capacity of the isolates to sequester or bind AFB1, in vitro and in vivo was determined using ELISA. Where applicable High Performance Liquid Chromatography (HPLC) as well as Spectrophotometric techniques were used. Sequestration of AFB1 in vivo was examined indirectly by measuring aflatoxin M1 (AFM1) in urine of school children in Eastern Kenya whose diet was mainly maize or maize-based. Samples of urine from children were analyzed by ELISA, and Liquid chromatography-mass spectrophotometry combination (LC-MS). The results for objective one showed a significant difference between the AFB1 levels in Maize grains (P<0.05) from different regions. Maize samples from Eastern Kenya region had the highest contamination. Maize samples from Nairobi had the lowest concentration at 6.02±0.31145 ppb. There was no significant difference in the total aflatoxins in Maize flour samples from all regions (P>0.05). Moreover there was significant difference in AFB1 concentrations between stores for example from Western and Eastern regions (P<0.05). Total aflatoxin levels in maize flours were slightly above international standard of 5ppb but lower than the Kenyan standard which is 10 ppb. The results for objective two indicated that out of 300 isolates from Kimere that were analysed for capacity to lower the risk of aflatoxin exposure in vivo or in vitro, Weissella cibaria NN20 had highest but statistically insignificant survival in low acidic condition than probiotic Lactobacillus rhamnosus GR-1 and Escherichia coli GR12 (P>0.05). Weissella cibaria NN20 bound 43.7±2.3 % of total available AFB1 in modulated media. Objective three and four had results showing that lactobacillus isolated from Kimere had capacity to ferment milk same as yoghurt and be able to reduce aflatoxin M1 in urine in vivo from 6.3ppb to 2.6ppb. The results confirmed the hypothesis that consumers were at a risk of aflatoxin exposure and that they were indeed consuming the AFB1 contaminated maize and maize products. In conclusion, the findings confirmed relatively high concentrations of Aflatoxins in maize and maize flours despite government efforts to prevent this. These results call for further intervention at table level. The Lactobacillus and Weissella isolates reduced AFB1 both in vitro and in vivo, suggesting that probiotic yoghurt has the potential to prevent aflatoxicosis among consumers of maize and maize based diets.