Optimization of Solid Substrate Fermentation of Prosopis Africana Taub for Production of Okpeye, an African Seasoning Agent

Abstract

Okpeye is a traditional seasoning of West Africa made from the solid substrate fermentation of the seeds of Prosopis africana. The profiles of microorganisms associated with the traditional solid substrate fermentation were investigated. The organisms were characterized and identified using a combination of standard physiological tests and 16S rDNA gene sequencing with Basic Local Alignment Search Tool (BLAST) analysis. Potential toxigenicity of isolates was assayed by detection of genes encoding haemolysin BL (HblA, HblC, HblD) and non- haemolytic enterotoxin NHE (NheA, NheB, NheC). Physicochemical parameters including pH, free amino nitrogen, moisture content, reducing sugars and enzymatic activities related to fermentation were investigated using standard procedures. The predominant organisms from the traditional processes (Bacillus subtilis, B. velezensis, and B. amyloliquefaciens) were used as starter cultures, singly, in pairs and cumulatively to optimize the fermentation in controlled reactions. Products of the fermentations and commercial samples were evaluated for organoleptic attributes. Proximate, mineral composition and amino acid profiles of the different products were determined. Both the natural (traditional) and controlled (optimized) fermentation of P. africana seeds produced okpeye as clearly identified by its sensory attributes. Product quality consistently correlated with microbial activities and physicochemical parameters of the processes. A distinct group of bacteria were involved in the traditional fermentation of P. africana to okpeye. Diverse species and strains of Bacillus: viz B. subtilis (6) strains, B. velezensis (6), B. amyloliquefaciens (4), B. cereus (2), B. thuringiensis (2), B. anthracis (1) and B. licheniformis (1) were the predominant, consistent and most actively involved micro flora. Haemolysin BL genes (HblA, HblC, HblD) were not detected in any of the Bacillus isolates. However, NheA genes encoding non-haemolytic enterotoxin were detected in five isolates of B. subtilis, two of B. thuringiensis, and the only isolate of B. anthracis while NheB genes were detected in three isolates of B. subtilis, one of B. thuringiensis and the only isolate of B. anthracis. NheC gene was not detected in any of the Bacillus isolates. None of the toxic genes was detected in other species of Bacillus. Apart from Bacillus, other bacteria isolated include species of Enterobacter (3), Pseudomonas (3), Proteus (1), Staphylococcus (1) and Micrococcus (1). The population of Bacillus species, increased significantly (p < 0.05) (102 – 109 CFU/g) and persisted until the end of the primary fermentation (96 h) and into the secondary fermentation or maturation phase, while populations of the other organisms mostly dropped off by 48 h. The mean values of pH (6.22 – 8.21), temperature (30 – 45OC), reducing sugar (1.56 – 14.62 mg/g) and free amino nitrogen (0.21 – 33.06mg/g) increased significantly (p < 0.05) with time, peaking at 72 h. There were also significant (p < 0.05) increases in protease (1.15 – 46.19 units/ml) and amylase (2.33 – 5.61 units/ml) activities with peaks at 72 h while the moisture content (56.05 - 62.40%) and the lipase activity (0.14 – 2.86 units/ml) increased steadily throughout the duration of fermentation. Optimum temperature for solid substrate fermentation was (45OC), moisture content (56 - 58%) and inoculum size (105 -107 CFU/ml). Physicochemical changes observed for samples inoculated with single and mixed starter cultures were comparable. The mean sensory scores of the different products differed significantly (p < 0.05). Samples prepared with starter cultures were rated higher (mean score of 4.50) than samples prepared by the traditional process (3.83) and commercial sample (3.75). Products prepared with starter cultures contained higher amounts of crude protein (40.44 - 50.25%) than traditionally prepared (33.42%) and commercial (34.33%) samples. The crude fat, fibre, ash, total carbohydrate and moisture contents also varied considerably. The most abundant minerals were calcium, magnesium, iron and potassium. Naturally fermented and commercial products had lower amounts of potassium and manganese compared to products fermented with starter cultures. In the course of the primary fermentation and the maturation, marked increases in concentrations of amino acids including Threonine, Glycine, Alanine, Phenylalanine, and Asparagine were observed. The quantities of amino acids were higher in products fermented with starter cultures compared to naturally fermented and commercial samples. The study has established protocol for reproducible production of Okpeye, and suggests that room exist for modulation of organoleptic characteristics and quality of this important condiment through appropriate manipulation of microbes and culture condition.