Microbiological assessment of maize ogi cofermented with pigeon pea

Abstract Maize was cofermented with pigeon pea for ogi production and evaluated for microbiological qualities. White maize and pigeon pea were mixed at ratios of 90:10, 80:20, 70:30, 60:40, and 50:50, respectively, with 100:0 serving as the control. Mixtures were cofermented for 96 h at 27 ± 2°C, and microbiological and sensory qualities analyzed were carried out using analysis of variance. Values were significant at p ≤ .05. Results showed that there was a gradual decrease in the pH and increase in total titratable acidity (TTA), respectively, during fermentation in all the samples. At the end of fermentation, pH ranged from 3.47 to 4.27 and TTA ranged from 0.47% to 0.54%, respectively. Total heterotrophic count (THPC) ranged from 5.76 to 5.90 log cfu/g; lactic acid bacteria (LAB) from 6.15 to 5.98 log cfu/g; and yeasts from 5.51 to 5.79 log cfu/g. Microorganisms isolated were lactic acid bacteria (Lactobacillus buchneri, L. casei, L. pentosus, Pediococcus pentosaceus), yeasts (Saccharomyces cerevisiae, Candida kefyr, C. krusei, C. tropicalis), molds (Aspergillus niger, A. flavus, Penicillium oxalicum, Mucor racemosus and Rhizopus stolonifer) and other aerobic bacteria (Klebsiella oxytoca, Enterobacter amnigenus, Staphylococcus xylosus, Bacillus subtilis, B. firmus, Corynebacterium kutscheri, C. striatum, and C. afermentans). In conclusion, the total heterotrophic plate count (THPC) in fortified maize: pigeon pea products was very high. This could constitute health hazards to infants as weaning foods. However, the microbial loads could be reduced through heat treatment as ogi is usually boiled or treated with boiled water before consumption and it can therefore be concluded that the fortified maize: pigeon pea products could be used as weaning foods.

The objective of this study is to evaluate the microbiological qualities of ogi developed from maize and pigeon pea.

| Sample collection
The pigeon pea (Cajanus cajan) and the maize grains (Zea mays) used in this study were purchased from Bariga market, Bariga, Lagos State, Nigeria.

| Cleaning and weighing of maize/pigeon pea samples
The grains/peas were sorted to separate them from stones, dirt, etc.
Six different proportions of maize: Pigeon peas were prepared as in Table 1.

| Fermentation of maize-pigeon pea blends
The grains/peas were washed thoroughly and steeped in tap water in the ratio of 1:2 (w/v) in properly labeled plastic buckets with lids.
Maize-pigeon pea ogi was prepared using a modified traditional fermentation method (Odunfa & Adeleye, 1985). The different maizepigeon pea blends were steeped for 48 h, washed, wet milled, and then sieved using muslin cloth to separate the pomace from the filtrate. The filtrates were allowed to settle and sour for another 48 h ( Figure 1). During the fermentation (steeping and souring) process, samples were taken at 24-h interval for microbiological, chemical, nutritional, and antinutritional analysis.

| Acidity changes during fermentation of the different maize-pigeon pea blends
The pH and total titratable acidity (TTA) of the different maize-pigeon pea blends were determined at 24-h interval during fermentation.

| pH
The pH of the fermenting maize-pigeon pea blends was determined at 24-h interval throughout the 96-h fermentation period by the method of Omemu (2011).
TA B L E 1 Materials used for the production of weaning food

| Isolation of microorganisms
Samples (1 g) of fermenting maize-pigeon pea ogi were collected aseptically from the different buckets at 24-h interval and homogenized in a mortar, previously cleaned with ethanol and passed over Bunsen flame. The homogenized samples were suspended in sterile 9 ml distilled water tubes and serially diluted (10-fold dilution).
Dilutions (0.1 ml) of 10 −3 -10 −5 were inoculated on sterile disposable petri dishes by pour plate method. The plates were previously labeled appropriately based on the different media used; deMann

| Preservation of isolates
The purified isolates were kept on slants of their respective growth agar as stock cultures under refrigeration temperature. The isolates were subcultured and transferred unto fresh agar slants at 2-month interval.

| Characterization and identification of isolates
The identification of isolates was carried out in four phases; cultural characterization, morphological characterization, biochemical characterization, and the use of Analytical Profile Index (API) kits.

| Cultural characterization
Grouping of pure cultures of bacteria and yeasts randomly picked from inoculated plates was carried out on the basis of their colonial characteristics such as colony elevation, color, size, shape, surface, edge, and pigmentation. Macroscopic identification of molds was carried out by observing and recording the colony form, surface color, and pigmentation on plates.

| Morphological characterization
This was done by examining the isolates microscopically for cellular morphology. Day-old cultures of the bacteria isolates were gramstained (section 3.6.2.1), and their color (purple or pink), shape (cocci or rods), and arrangement (singles, pairs, chains, or clusters) were observed and recorded. Day-old cultures of the yeast and mold isolates were stained with lactophenol cotton blue and observed microscopically for cell shape, size, and sporulation. The method of vegetative reproduction (fission, filaments or budding) of the yeast isolates was also observed.

| Gram staining
Using a sterile loop, a light suspension of organism in sterile distilled water was prepared on a clean microscope slide. The film was airdried and heat-fixed by passing the slide twice through a gas flame.
The slide was then allowed to cool. The slide was placed on a stain-  (Cheesbrough, 2006).

Oxidase test
This test was used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain.
It was also used to identify Candida spp. which are yeasts and are oxidase positive. A piece of filter paper in a Petri dish was moistened with 2-3 drops of Kovac's oxidase reagent (1% tetramethyl-p-phenylenediamine). Using a wire loop, a colony of the test organism was transferred to the filter paper and rubbed on the moistened area. Purple coloration within 30 sec indicated the production of cytochrome c oxidase (Cheesbrough, 2006).

Indole test
This test was carried out to determine the ability of bacteria to break down tryptophan to indole by the enzyme tryptophanase. The test organism was inoculated in a bijou bottle containing 3 ml of sterile tryptone water and incubated at 35-37°C for 48 h. Indole was tested for by adding 0.5 ml (5 drops) of Kovac's reagent (isoamyl alcohol; para-dimethyl aminobenzaldehyde; concentrated hydrochloric acid) and shaking gently. A red color in the surface layer within 10 min indicated a positive reaction while a yellow color indicated a negative reaction (Harrigan & McCance,1976).

Methyl red test
This test was carried out to identify enteric bacteria based on their pattern of glucose metabolism (mixed acid fermenters are positive to this test). The bacterium was inoculated into glucose phosphate broth, which contains glucose and a phosphate buffer and incubated at 37°C for 48 h. The pH of the medium was tested by the addition of five drops of methyl red reagent. The tube was gently rolled between the palms to disperse the methyl red reagent. Development of red color was taken as positive and yellow as negative (Somogyi, 1952).

Voges-Proskauer test
This test was carried out to detect acetoin in enteric broth culture with the aim of differentiating the enteric bacteria. The bacterium was inoculated into glucose phosphate broth and incubated for 48 h.
Alpha-naphthol solution (0.6 ml of 5%) in ethanol was added to the broth and shaken. The tube was allowed to stand for 15 min. Cherry red color was taken as positive while no color change indicated negative (Somogyi, 1952).

Citrate utilization test
This test was carried out to differentiate the enteric bacteria.
Bacterial colonies from fresh (18-to 24-h-old) plates were picked up with wire loop, inoculated onto a slope of Simmons citrate agar and incubated overnight at 37°C. A change of medium from green to blue indicated a positive reaction, that is, the organism has the ability to utilize citrate as sole source of carbon and energy (Kiiyukic, 2003).

Urease test
This test was carried out to differentiate the enteric bacteria.
Organisms positive to this test hydrolyze urea to produce ammonium ions with subsequent change in pH to alkaline (reaching 8.1) from an initial pH of 6.8. The test organism was inoculated heavily in a bijou bottle containing 3 ml sterile urea broth and incubated at 35°C for up to 7 days. Color change from yellow to rose pink was taken as positive (Harrigan & McCance, 1976).  (Payne, 1973).

Spore test
This test was carried out to identify the Bacillus spp. A film of the test organism was prepared on a clean microscope slide. The film was flooded with 10% aqueous malachite green solution, left to stand for 40-45 min, and washed under running tap water. It was again flooded with 0.5% aqueous safranin solution and left for 15 sec before rinsing under running tap water. The film was allowed to air-dry.

H 2 S production
This test was carried out to determine the ability of the bacteria and yeast isolates to reduce sulfur-containing compounds to sulfides during metabolism. The sulfide produces combines with iron compounds to produce ferrous sulfide (FeS), a black precipitate. Tubes of triple sugar iron (TSI) agar slopes were prepared, and using straight wire, the test organism was inoculated deep into butt of the medium and streaked up the slope. The tubes were incubated for 18-24 h at 37°C and examined for blackening of the medium (Somogyi, 1952).

Starch hydrolysis test
Starch agar (nutrient agar to which starch is added) plates were prepared and spot inoculated with test organisms. The plates were incubated at 35°C for 48 h. After incubation, the growth and surrounding areas was covered with Gram's iodine and the areas surrounding growth examined for clearing (Morello, Mizer, & Granato, 2003).

Casein hydrolysis test
Skim milk agar plates were inoculated with test bacteria and incubated at 35°C for 48 h. Zone of clearing around bacterial growth indicated the ability to secrete the proteolytic exoenzyme casesase that hydrolyze casein (Morello et al., 2003).

Sugar fermentation test
The tests were used to assess the sugar fermentation abilities of bacteria and yeasts isolated. Ten percent (10%) solutions of some test sugars (carbohydrates) such as glucose, fructose, lactose, galactose, mannitol, sucrose, raffinose, rhamnose, and melibiose were prepared and sterilized at 115°C for 10 min so as not to denature the sugars.
To 90 ml sterile peptone water, 10 ml of the sterile 10% sugar solution was added with 1-2 ml 0.01% phenol red indicator. The sugar solution (5 ml) was transferred aseptically into sterile test tubes with inverted Durham tubes to check for gas production. The tubes were incubated overnight to check for sterility, and then, the tubes were inoculated with pure culture of the test organisms and incubated at 37°C for 4 days. D Yellow coloration indicates acid production while gas production was indicated by displacement of the medium in the Durham tube (Fawole & Oso, 2004).

| Analytical profile index
Analytical profile index kits were used in confirming the iden-

| Analytical profile index (API 20C) for yeast identification
In the preparation of the inoculum, an ampoule of API suspension was opened and test colony was scooped with inoculating loop aseptically and transferred into the ampoule. This was mixed properly until the suspension became turbid. An ampoule of API C medium was opened, and approximately an aliquot of 100 l was transferred into the API C medium and gently homogenized. In the preparation of the API 20 C AUX strips, 5 ml of sterile distilled water was added into the honey-combed wells of the incubation tray before the strip was placed in the incubation box. The cupules of the strip (0-29) were filled with the yeast suspension, obtained in the ampoule of API C medium, to the indicated inoculum level. The lid was placed onto the box, and the preparation was incubated at 29°C for 48-72 h, after which the strip was read and recorded in the result sheet. Identification was by the API identification system (apiweb).

| Changes in pH of the fermenting maize-pigeon pea ogi blends
The changes in pH of the fermenting maize: pigeon pea blends during the 48-h steeping and 48-h souring process are presented in

| Changes in total titratable acidity of the fermenting maize-pigeon pea ogi blends
The changes in total titratable acidity (TTA) of the fermenting maize:

| Total heterotrophic plate count (THPC)
The total heterotrophic plate count (THPC) at the different phases of fermentation (steeping and souring) of all the maize-pigeon pea blends is shown in Table 3. At the 0-h steeping period, the THPC ranged from 5.70 to 6.04 log cfu/g. As fermentation progressed, there was an observed significant (p ≤ .05) increase in the microbial count till the end of the steeping process (48-h steeping). In contrast, at the 0-h souring period, the total plate count was seen to significantly (p ≥ .05) decrease gradually till the end of souring. At the end of the souring phase, the THPC ranged from 6.03 log cfu/g in the 50:50 maize: pigeon pea blend to 6.26 log cfu/g in the 100:0 maize: pigeon pea blend.

| Total lactic acid bacteria count
The total LAB count at the different phases of fermentation (steeping and souring) of all the maize-pigeon pea blends is shown in

| Total yeast count
The total yeast count at the different phases of fermentation (steeping and souring) of all the maize-pigeon pea blends is presented in Table 5. At the 0-h steeping period, no yeast was isolated but at 24-h steeping, and total yeast count ranged from 4.95 to 5.07 log cfu/g.
The yeasts increased significantly (p ≤ .05) in number in all the fermentation setups from this period till the end of the souring phase.
At the 48-h souring period which marked the end of the fermentation process, yeasts were seen ranging from 5.53 log cfu/g in the 100:0 maize: pigeon pea blend to 5.79 log cfu/g in the 50:50 maize: pigeon pea blend.

| Mold count
The mold count at the different phases of fermentation (steeping and souring) of all the maize-pigeon pea blends is presented in Figure 3. At the 0-h steeping period, molds encountered ranged from 2.81 log cfu/g in the 100:0 maize: pigeon pea blend to 2.91 log cfu/g in the 60:40 and 50:50 maize: pigeon pea blends. As fermentation progressed till the end of the fermentation process, there was a complete disappearance of molds.

| Coliform count
The coliform count at the different phases of fermentation (steeping and souring) of all the maize-pigeon pea blends is shown in

| Bacillus count
The Bacillus count at the different phases of fermentation (steeping and souring) of all the maize-pigeon pea blends is shown in Table 6.

| Cultural, morphological, and biochemical characteristics of microorganisms isolated from the different maize: pigeon pea mixtures
The cultural, morphological, and biochemical characteristics of bacteria, yeasts, and molds isolated from the fermenting maize:

| D ISCUSS I ON
There was a steady decrease in the pH during fermentation in all the ogi samples. This might be as a result of production of lactic acid by fermentative organisms (mainly lactic acid bacteria) responsible for the fermentation of ogi (Oluwafemi & Adetunji, 2011). A significant increase in the total titratable acidity (TTA) during fermentation in all the ogi samples was recorded in this study, and this could be as a result of production of lactic acid and other organic acids by organisms responsible for the fermentation, as is the case with the decrease in pH. This observation of decrease in pH and increase in total titratable acidity (TTA) agrees with most studies on ogi fermentation including those of Wakil and Kazeem (2012), Okwute and Olafiaji (2013), and Modu et al. (2013). The early rise in titratable acidity and reduced pH is important to avoid proliferation of undesirable organisms resulting in poor fermentation (Mbata, Ikenebomeh, & Alaneme, 2009).
This study revealed high total heterotrophic plate count (THPC) which may be attributable to availability of some easily metabolizable nutritional components of grains and legumes, which may be essential for the growth of microbes (Adebayo, Ogunsina, & Gbadamosi, 2013). A high total bacterial count has also been observed (Nsofor, Ume, & Uzor, 2014;Nwogwugwu, Ogbulie, Chinakwe, Nwachukwu, & Onyemekara, 2012) and reported to likely indicate a potential hazard to consumers. These counts are, however, lower than 1.0 × 10 8 cfu/ml staphylococcal count normally considered as potentially hazardous (Nsofor et al., 2014). The THPC ranged from 6.03 log cfu/g in the 50:50 maize: pigeon pea blend to 6.26 log cfu/g in the 100:0 maize: pigeon pea blend. These values were too high for foods for infants that could be immunecompromised people.
The increase in the lactic acid bacteria count with fermentation as observed in all the fermentation setups could be due to an increase in the acidity and the anaerobic condition of the fermenting medium, favouring the growth of only facultative anaerobes, and/or aciduric organisms. It may also have been as a result of the inhibitory effect of antimicrobial products from the lactic acid bacteria on the growth of unwanted harmful or spoilage organisms (Adebayo et al., 2013). Toxic substances such as hydrogen peroxide, diacetyl, carbon dioxide (CO 2 ), organic acid, and bacteriocins have been shown to be released by lactic acid bacteria into the fermenting medium during food fermentation (Mataragas, Melaxopoulous, & Drosinos, 2002;Nsofor et al., 2014). These substances are toxic to pathogenic organisms that may be present in the fermenting substrates.
The lactic acid bacteria isolated in this study include mostly the

Lactobacillus species (Lactobacillus buchnerii, Lactobacillus casei, and
Lactobacillus pentosus). The higher prevalence of rod-shaped lactic acid bacteria in this study corroborated the study of Nwokoro & Chukwu, 2012 who reported that the genus Lactobacillaceace commonly predominates during food fermentation. This is because they are the most aciduric of all lactic acid bacteria (Nsofor et al., 2014;Nwokoro & Chukwu, 2012).

Yeasts isolated in this study include Saccharomyces cerevisiae,
Candida kefyr, and Candida tropicalis. Previous workers have found several yeasts species in spontaneous lactic fermenting cereals including species of Saccharomyces and Candida (Jespersen, Halm, Kpodo, & Jakobsen, 1994). The appearance and increase in the yeast count after 24 h of fermentation is attributed to the decrease in the pH that creates conditions ideal for yeast growth. This is similar with the finding reported for other fermented beverages (Abegaz, Beyene, Langsrud, & Narvhus, 2002). Roles of yeasts have also been reported to include improving the organoleptic qualities by producing different flavors and aroma in different foods (Inyang & Idoko, 2006 (Mbata et al., 2009). The sources of these microorganisms could be human skin, cooking utensils, processing equipment, the environment and water (Ogbulie, 1991), or from the seeds. Jespersen et al. (1994) similarly reported the presence of molds such as Penicillium and Aspergillus in maize fermentation during kenkey production with drastic reduction in their numbers within 24 h of dough fermentation.
The subsequent disappearance of molds after 24 h observed in this study as well as previous studies (Mbata et al., 2009;Nsofor et al., 2014;Nwokoro & Chukwu, 2012) was probably due to the low oxygen tension in the fermenting matrix. It could also be due to the presence of organic acids especially lactic acid as it could be seen that the growth of lactic bacteria and yeasts increased gradually throughout fermentation while the numbers of molds decreased. Edema and Sanni (2008) also reported that the growth of lactic acid bacteria and yeasts can inhibit the growth of molds.
Enteric bacteria isolated in this study were Klebsiella oxytoca and Enterobacter amnigenus. It could be seen that as pH decreased and total titratable acidity increased, the presence of enteric bacteria decreased as they are acid intolerant, and at the 48 h of steeping, no enteric bacteria was isolated from all the fermentation setups. This was also corroborated by reports of Oluwafemi and Adetunji (2011) and Omemu and Faniran (2011).
The Bacillus species isolated in this study include Bacillus formis and Bacillus subtilis. Previous reports (Nwogwugwu et al., 2012;Nwokoro & Chukwu, 2012;Ogbulie, 1991) have also isolated Bacillus species in the fermentation of maize for ogi production, and they have been reported to show saccharolytic activities (Nwokoro & Chukwu, 2012). These organisms persisted toward the end of the fermentation indicating that they continued the saccharification of maize starch to release sugars. Corynebacteria (C. kutscheri,