Rev Chil Nutr Vol. 39, Nº4, Diciembre 2012

Studies on Akamu, a traditional fermented food

Estudios en Akamu, una comida tradicional de maiz fermentado

ABSTRACT Microbiological and some physicochemical properties of akamu, a fermented maize food were studied. Microbial population, pH, titratable acidity, protein, sugar and starch were measured during the fermentation. The initial microflora consisted of a heterogenous mixture of microorganisms namely Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus amylovorus, Pseudomonas aeruginosa, Pseudomonas alkaligenes, Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Candida utilis, Candida tropicalis, Saccharomyces cerevisiae, Aspergillus oryzae, Aspergillus niger, Penicillium citrinum, Rhizopus micros- porus and Rhizopus oligosporus. Within 24 h, lactobacilli and aerobic mesophilic bacteria accounted for a major portion of the total microflora. The lactobacilli numbers increased from 1.6 x107 cfu/g after 24 h to 7.1 x108 cfu/g after 72 h. Total aerobic mesophilic bacteria increased from 2.5 x109 cfu/g after 24 h to 4.2 x108 cfu/g Nwokoro, Ogbonnaya (1) after 72 h. Enterobacteriaceae count decreased from 6.3 x103 cfu/g Chukwu, Bernice Chidinma (2) after 24 h to 2.5x102 cfu/g after 72 h, although this level remained (1) Industrial Microbiology and Biotechnology Laboratory, significantly high for a finished food product. increased Department of Microbiology, University of , Nsukka, Nigeria. steadily and reached 6.8 x105 cfu/g after 72 h. Mould counts de- (2) Department of Biochemistry, creased from 6.3 x103 cfu/g after 24 h to 1.3 x102 cfu/g after 72 University of Nigeria, Nsukka, Nigeria. h. Moulds were largely responsible for amylolytic activity in pure culture. Fermentation caused a general decrease in pH from 6.6 Corresponding author: to 3.9 after 72 h and titratable acidity increased from 0.48 to 0.79 Nwokoro Ogbonnaya Industrial Microbiology and Biotechnology Laboratory after 72 h. Starch concentration decreased from 68 g/100 g to 37.4 Department of Microbiology g/100g. Protein and reducing sugar concentrations increased from University of Nigeria, 12.8 g/100 g to 18.5 g/100 g and from 5.3 g/100 g to 17.6 g/100 Nsukka, Nigeria Tel: +2348034402414 g, respectively. The types and numbers of microorganisms isolated E-mail: [email protected] from akamu could pose a health risk to consumers especially where this food product is used as a weaning product for infants. Key words: Akamu, microbial population levels, protein, reducing Este trabajo fue recibido el 24 de Mayo de 2012 sugar, starch. y aceptado para ser publicado el 28 de Octubre de 2012.

INTRODUCTION is sieved to smooth slurry which is allowed to settle and the Fermentation process serves as a mean of providing a supernatant decanted. The slurry is mixed with hot water with source of nourishment for large rural populations Fermentation stirring until it forms a get which serves as food. enhances the nutrient content of foods through the synthesis Fermented foods have become a major source of dia- of proteins, vitamin, and essential amino acids (1). rrhoeal diseases (5, 6). Moterjemi et al., (7) reported that Akamu is a porridge prepared from fermented maize. It maize porridge samples prepared for infants in Ghana were is a popular cereal and infant weaning food among contaminated with pathogenic bacteria including Aeromonas, the Igbo – speaking people of Nigeria. Akamu is similar to Bacillus cereus, Salmonella, Staphylococcus aureus and Vibrio ogi, a lactic acid fermented food made from maize, sorghum cholerae. This present investigation was aimed at assessing or millet which may the fortified with legumes (2-4). Akamu the microbiological and some physicochemical properties of is prepared by soaking clean maize grains in water for 2-3 akamu and the association of some pathogenic microorganisms days. The grains are washed and ground to a paste. The paste with the food.

180 Studies on Akamu, a traditional fermented maize food

MATERIALS AND METHODS bottle. An aliquot (4 mL) of this reagent was added to tubes Traditional preparation of Akamu: Five kilograms of maize containing 1mL of glucose solution and to distilled water (Zea mays) was cleaned and steeped in 6 L of tap water in a blanks. The tubes were placed in boiling water for 10 minutes plastic container for three days. The water was decanted and and cooled to room temperature. The solutions were read the grains were wet – milled before sieving with muslin cloth. in a Spectrum Lab 23A spectrophotometer at 540 nm. The The pomace was discarded and the starch suspension was readings were used to draw a standard curve for micrograms allowed to sediment during which fermentation occurred by glucose equivalents per mL against absorbance. Titratable the natural flora of the grains. acidity was determined using the standard method described by Amoa – Awua et al; (12). A 10 g sample was blended with Isolation of microorganisms 100 mL of distilled water and filtered through two successive At 0 h and then at 24 h intervals, 1 g sample was asep- Whatman No 1 filter papers. Then 25 mL of the filtrate was tically withdrawn and used for microbiological analysis. The titrated with 0.25M NaOH and 1% phenolphthalein as an sample was homogenized in 10 mL of sterile distilled water indicator. The titratable acidity was calculated as percent lactic using a sterile mortar and pestle. Then 1mL of the pulp was acid in the sample using the relationship given by Kimaryo et added into a test tube containing 9 mL 0.1% sterile peptone al., (13). Starch content was determined by anthrone method water diluents and mixed. Then 0.1mL was aseptically with- of Clegg (14). drawn with a sterile pipette and inoculated onto MRS agar (Oxoid Ltd., UK) for the isolation of lactic acid bacteria, Plate RESULTS AND DISCUSSION Count Agar (Difco) plates for total aerobic mesophilic bac- The microbial composition of akamu sample is shown in teria, Mac Conkey agar (Oxoid ) for Enterobacteriaceae and table 1. Within the first 48 h of fermentation, the organisms Sabouraud Dextrose agar containing 0.1% chloramphenicol isolated included Lactobacillus delbrueckii, Lactobacillus plan- for yeasts and mould counts. The plates were incubated at tarum, Lactobacillus fermentum, Lactobacillus amylovorus, 30 oC for 24-72 h and the number of colony forming units Pseudomonas aeruginosa, Pseudomonas alkaligenes, Bacillus per gram (CFU/g) was determined. The morphological, cereus, Bacillus licheniformis, Bacillus subtilis, Candida utilis, physiological and biochemical characterization of bacterial Candida tropicalis, Saccharomyces cerevisiae, Aspergillus isolates were carried out as described by Holt et al., (8). Car- oryzae, Aspergillus niger, Penicillium citrinum, Rhizopus mi- bohydrate fermentation patterns of the lactic acid bacterial crosporus and Rhizopus oligosporus. The presence of these (LAB) isolates were determined by using API 50 CHL test kit. organisms in significant numbers at this time indicated that On-line software of Bio Merieux (www.apiweb.biomerieux. they constituted part of the maize microflora. Mucor circi- com) was used to identify the isolates. Yeasts and fungi were nelloides and Rhodotorula glutinis were the only organisms identified based on the taxonomic schemes described by Pitt isolated beyond 48 h. and Hocking (9). Qualitative test for starch hydrolysis by the Bacillus subtilis, Bacillus cereus and Bacillus licheniformis isolates was done by surface – plating starch (Merck) agar showed saccharolytic activities and these organisms persisted plates with the isolates. The plates were flooded with 5 mL towards the end of the fermentation indicating that they iodine solution and visualized for zones of clearing around continued the saacharification of maize starch to release su- the colonies after 2 d incubation. gars. The majority of lactic acid bacteria isolated from akamu belonged to the genera Lactobacillus. These organisms arose Chemical analyses early in the fermentation, increasing rapidly from1.6 x107 Sample (100 g) was treated with 5% NaOH, 8% urea and cfu/g to 7.1 x 108 cfu/g after 72 h. The decrease in sugar 0.05 % sodium dodecyl sulphate and incubated at 32oC for 16 concentrations could be attributed largely to the activities h. After incubation, the sample was centrifuged at 2515xg and of these organisms which metabolized and converted sugars the supernatant was used for protein assay. Protein content into organic acids during maize fermentation (15). Giraud was determined by the method of Lowry et al., (10) using et al., (16) demonstrated amylase ability of Lactobacillus bovine serum albumin (Sigma-Aldrich) as a standard. The pH plantarum but all the lactic acid bacteria isolated from akamu was determined using a glass electrode pH meter (PYE Unicam, were unable to hydrolyze starch in pure culture. Lactobacilli England). Reducing sugar was assayed by a modification of the are the most important organisms that produced acidity in dinitrosalicylic acid (DNS) method of Miller (11): DNS (10 g) maize fermentation (17).These organisms have also been was dissolved in 200 mL of 0.2 M NaOH. Potassium sodium implicated as partly responsible for initiating acidification tartrate (300 g) was dissolved in 800 mL of distilled water. in fermenting maize dough (18). This work confirms this The two solutions were mixed and stored in an air tight dark suggestion because lactobacilli had the highest population

TABLE 1

Microbial population levels (cfu/g) during the fermentation of Akamu

Fermentation Lactic Total aerobic Enterobacteriaceae Yeasts Moulds Time (h) acid bacteria mesophilic bacteria

0 1.6x 107 2.5 x 109 6.3 x 103 2.0 x 104 6.3 x 103 24 3.2 x 107 4.0 x 109 1.6 x 103 8.0 x 104 7.9 x 102 48 4.0 x 107 7.9 x 108 3.8 x 102 1.6 x 105 2.5 x 102 72 7.1 x 108 4.2 x 108 2.5 x 102 6.8 x 105 1.3 x 102

181 Ogbonnaya N. y Bernice C. counts towards the end of the fermentation when pH was 3.9 riaceae isolated were identified as Escherichia coli, Proteus and titratable acidity was 0.79. This highly acidic condition sp, Serratia sp. and Enterobacter aerogenes. E coli and E. which developed at the latter part of the fermentation may aerogenes are generally considered as indicators of faecal con- be responsible for the dominance of lactobacilli towards the tamination. Their presence may be due to the contamination end of the fermentation. of water samples used to prepare the akamu sample. Enterobacteriaceae showed a slight decrease during the The , Saccharomyces proliferated rapidly during early stages of the fermentation (table 1). The Enterobacte- fermentation and then fell in counts disappearing towards the

FIGURE 1

Changes in pH during the fermentation of Akamu

FIGURE 2

Changes in titratable acidity during the fermentation of Akamu

182 Studies on Akamu, a traditional fermented maize food end of the fermentation at which time other yeasts namely of foods against food – borne pathogens. Fermentation is Candida spp. became predominant. The role of Saccharomyces protective against food-borne diseases and it is usually re- spp. is not easy to ascertain especially as ethanol, the man commended as a cheaper way of preparing foods especially product of its fermentation of carbohydrates has not been in the developing countries (26). However the occurrence of detected in fermented maize. However Saccharomyces rouxii bacterial pathogens in fermented foods suggests a need for (19) and Saccharomyces cerevisiae (20) have been implicated caution in the use of these foods for infant feeding. Challen- as being partly responsible for the organoleptic properties of ge tests have shown the possibility of pathogens to survive fermented maize. All the yeast strains isolated did not show and grow in fermented foods (27, 28). Akamu is a traditional amylolytic activity in pure culture. food for weaning infants who are fed at least once a day as a The moulds Aspergillus oryzae, Aspergillus niger, Rhi- supplement to breast milk. From this study, Akamu could pose zopus microsporus and Rhizopus oligosporus were able to a health risk to consumer based on the types and numbers of degrade starch in pure culture whereas Penicillium citrinum pathogenic microorganisms isolated. was unable to do so. These organisms decreased in population as the fermentation progressed and their population reduced RESUMEN significantly after 24 h (table 1). Ekundayo (21) reported that Se estudiaron las propiedades microbiológicas y fisi- Penicillium sp and Aspergillus sp. were responsible for the coquímicas del akamu, un alimento fermentado a base de conversion of starch to sugars during the steeping of maize for maíz. Se midió la población microbiológica, el pH, la acidez the production of pito. Rhizopus microsporus is mycotoxigenic por titulación, azúcar y almidón durante la fermentación. and was found in 18% of maize samples (9) La microflora inicial consistía en una mezcla heterógenea The results of pH development and titratable acidity of de microrganismos denominados Lactobacillus delbruecki, akamu during fermentation are shown in figures 1 and 2. The Lactobacillus plantarum, Lactobacillus fermentum, Lactoba- pH decreased from 6.6 at the start of the fermentation to 3.9 cillus amylovorus, Pseudonomas aeruginosa, Pseudomonas after 72 h. Titratable acidity increased from 0.48 at 0 h to 0.79 alkaligenes, Bacillus cereus, Bacillus licheniformis, Bacillus after 72 h. The acidity of ogi was attributed to the presence of subtilis, Candida utilis, Candida tropicvallis, Saccharomyces lactic acid, acetic acid and butyric acid (20). Banigo and Muller cerevisiae,Aspergillus oryzae, Aspergillus niger, Penicillium ci- (2) detected the presence formic acid in ogi. Production of trinum, Rhizopus microsporus y Rhizopus oligosporus. Al cabo acid during natural fermentation of maize led to a significant de 24 horas, las bacterias lactobacillus y mesofílicas aeróbicas reduction of pH which determined the shelf-life of the products formaron una parte importante de la microflora total. Las bac- as well as the destruction of enteric pathogens (22). terias lactobacilli aumentaron a 1.6 x107 cfu/g a las 24 horas Changes in starch, protein and total reducing sugar con- y al cabo de 72 horas a 7.1 x 10 8 cfu/g. El total de bacterias centrations of akamu are shown in table 2. Starch consisted mesofílicas aeróbicas aumentaron desde 2.5 x 109 en 24 horas 68.1% of unfermented maize but this decreased to 37.4 % y a 4.2 x 10 8 a las 72 horas. El recuento de enterobacterias after 72 h. The total reducing sugar increased from 5.3g/100 disminuyó de 6.3 x 103 en 24 horas a 2.5x 102 cfu/g a las 72 g to 17.6 g/100 g. The sugar production may be as a result horas, aunque este nivel permaneció significativamente alto of starch degradation by the fermenting microflora. The para un producto alimentario final. Las levaduras aumentaron protein content increased significantly (p≤0.01) from 12.8 en forma significativa y alcanzaron un rango de 6.8 x 105 a g/100 g to 18.5 g/100 g after 72 h. The increase in protein las 72 horas. Los recuentos de hongos disminuyeron desde may be due to microbial growth with a resultant increase in 6.3 x 103 cfu/g a las 24 horas a un rango de 1.3 x 102 cfu/g a microbial biomass during the fermentation process. Some las 72 horas. Los hongos fueron responsables de la actividad microbial strains have been found useful for protein enrich- amilolitica en el cultivo puro. La fermentación provocó una ment of foods (23,24). Since no external source of nitrogen disminución general en el pH desde 6.6 a 3.9 a las 72 horas y was applied in this study, this could be due to conversion of la acidez por titulación aumentó a 0.48 a 0.79 a las 72 horas. some of the plant proteins or other nitrogenous compounds La concentración de almidón disminuyó desde 68 g/100 g into microbial protein. a 37.4 g/100 g. Las concentraciones de proteínas y azúcar Several authors have highlighted the importance of ade- reducida aumentaron desde 12.8 g/100 g a 18.5 g/100 g y quate nutritional quality and hygiene during the preparation desde 5.3 g/100 g a 17.6 g/100 g respectivamente. Los tipos of foods and also the link between infection and nutrition (5). y cantidades de microrganismos aislados del akamu suponen Oyelana and Coker (25) reported the presence of pathogenic un riesgo sanitario para los consumidores especialmente dado bacterium such as Staphylococcus aureus and mycotoxigenic que este producto es utilizado como una formula láctea para fungi such as Aspergillus flavus and Fusarium oxysporium lactantes. from fermented maize samples. Fermented foods have many Palabras clave: Akamu, recuento bacteriano y hongos, advantages such as improving the nutritional value and safety proteina, azúcares reductoras.

TABLE 2

Changes in nutritional contents of Akamu

Component (g/100 g) Non fermented Fermented P (t-test)

Protein 12.8±0.57 18.5±0.42 < 0.01 Starch 68.1±12.73 37.4±2.83 <0.001 Reducing sugar 5.3±0.71 17.6±0.99 < 0.001

183 Ogbonnaya N. y Bernice C.

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