Research Journal of Microbiology

Year: 2011 | Volume: 6 | Issue: 2 | Page No.: 105-114
DOI: 10.17311/jm.2011.105.114

Biochemical Evaluation of Combined State Fermentation of Canavalia ensiformis (L.) using Mixed Cultures

R.A.O. Gabriel-Ajobiewe

Abstract: This study was conducted to evaluate the effect of mixed cultures on the nutritional and antinutritional content of combined fermented jack beans. The combined role played by individually selected microorganisms (bacteria and fungi) involved in the natural fermentation of jack beans, under controlled environment was investigated. One hundred gramme of ground, samples were inoculated with the mixed cultures under aseptic condition using 1 mL of each inoculum and fermentation allowed to take place under controlled environment of temperature of 40°C for the liquid state fermentation (with bacteria being the inoculum) for 7 days. Thereafter the fungi were inoculated aseptically for the commencement of the solid state phase of the combined fermentation for another 7 days at the temperature of 30°C. The inocula range between 3-7 species in number. Physical sensory changes, fermentation parameters, proximate and antinutritional composition were determined during fermentation. The total titratable acidity values of the multiple starter culture fermenting substrates increased from 0.04-0.43%. While the pH values decreased from 6.5 to 4.3. The higher the number of species used as inoculums, the more acceptable the fermented samples (in terms of sensory properties). Irrespective of the number of species used in the fermentation, there was a significant increase in the crude protein from 26.20-39.82 g/100 g, while the fat (11.95-4.33 g/00 g) and ash content (3.50-2.23 g/100 g) shows a decrease in comparison with the control. The mineral composition showed a significant increase in magnesium (30.07 to 46.77 mg g^-1), sodium (18.51 to 34.34 mg g^-1), potassium (23.51 to 40.88 mg g^-1) and iron (0.00 to 0.08 mg g^-1) when compared with the control. Of all the antinutrient content analysed, only phytate (58.66 to 5.08 g/100 g) and canavanine (0.79 to 0.40 mg g^-1) has significant decrease in comparison with the control. Hence, it can be deduce from this work that the use of multiple starter culture in combined fermentation can be used to improve the nutritional content of Canavalia ensiformis L.

Keywords: bacterial, antinutritional composition, Nutritional, fungal isolates and fermentation

INTRODUCTION

Food legumes represent a diverse group of plants, which are found worldwide. These are an important component of animal and human diets. Legumes are consumed in one form or the other, serving as sources of both protein and energy, all over the world. The increasing demand for sources of protein in developing countries, coupled with the relatively high cost of imported proteins, has led to a search for alternatives, particularly unconvertional legumes indigenous to the tropics (Famurewa and Raji, 2005). Foods are fermented for many reasons including enhancement of nutritive value, improvement in sensory properties, digestibility. The increased nutritive value of fermented foods are due to the breakdown of complex components such as carbohydrates, proteins and lipids to more easily digested sugars, free fatty acids, amino acids as well as synthesis of certain vitamins (Steinkraus, 1997).

The popularity of legume based fermented food is due to desirable changes in the legumes that include texture, organoleptic characteristics (especially elimination of beany flavours and improvement in digestibility), enhancement in keeping quality of the product, improved safety-absence of toxins and partial and/or complete elimination of antinutritional factors, increase in nutrition and reduced cooking time. The organoleptic characteristics of fermented foods make them more attractive to the consumers than those of raw beans or legumes (Sahlin, 1999).

Underutilized legumes such as jack beans have been reported to be rich in protein and could serve as potential protein source and that it also has a high potential as protein replacer (Seena et al., 2006). In addition, FAO (1994) publication information has reportedly expressed the proximate composition of some underutilized tropical seeds and their usefulness as food for humans.

Commercial legume fermentations are still generally carried out by means of the natural microbial flora of the plant to be fermented. This substantially increases production costs, because the success of the fermentation may be random and the time of processing is usually long and is dependent on the initial flora and the progress of the microbial succession (Rombouts and Nout, 1995).

Canavalia ensiformis (commonly known as jack bean) is, considered to be the one with the highest potential as an economic crop of all the Canavalia genus, in view of it’s excellent agronomic characteristics (Udedibie and Nwaiwu, 1988) requiring no staking if planted on open land. Apart from jack bean’s ability to establish relatively with ease, many of it’s unusual growth attributes include, outstanding capability of well nodulated mycorrhizal colonized for continuous growth and harsh climatic conditions. To fulfill the growing demands of plant-based proteins for humans and livestock need, research on the possibilities of employing underutilized legumes as inexpensive and elegant source of protein than the conventional sources has to be pursued. Some underutilized wild legumes adapted to adverse conditions have been explored for their nutritional advantages (Bhagya et al., 2006; Sridha and Seena, 2006); but they possess some anti-nutritional factors which make their utilization very difficult, hence the need for their detoxification. Many means of detoxification has been explored but found to be expensive for the poor, fermentation as an affordable means of detoxification is being considered in this work. This study therefore utilized the ability of individual starter mould culture fermentation to improve the nutritional content of jack bean seeds.

MATERIALS AND METHODS

The jack bean seeds used for this study were obtained in June 2008, from International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. The clean healthy seeds were soaked with boiled distilled water for 3 h to remove the seed coat and rinsed in changes of distilled water. The dehulled seeds were dried at 60°C for 48 h in the drying cabinet and ground with the Marlex portable stainless grinder. The materials were stored in sterile transparent polythene bags, tightened and kept at -20°C for further usage.

Preparation of starter cultures: Cultures of Lactobacillus fermentum strain LF4801 Pediococcus pentosaceus strain PP4601 Bacillus polymyxa strain BP1801, Alcaligenes faecalis strain AF1803, Klebsiella ozaenae strain KO1802 and Rhizopus oryzae strain MF4801, Mucor mucedo strain MF4802, Aspergillus niger strain MF4803, Varicosporium elodea strain MF4804, Neurospora crassa strain MF4806, Brettanomyces bruxellensis strain YF2401, Schizosaccharomyces pombes strain YF2404, previously isolated from natural fermentation were used. The bacterial isolates with the exception of lactobacilli were cultivated by streaking on nutrient agar plates and incubated at 37°C for 24 h. One colony was picked and transferred to a tube containing 10 mL nutrient broth and incubated at 37°C for 24 h. 0.1 mL of this culture was used to inoculate10 mL nutrient broth and incubated at 37°C for 16 h. This culture was centrifuged (3000 rpm, 10 min), the pellet was washed in 10 mL sterile peptone physiological salt solution (1 g peptone, 8.5 g NaCl in 1000 mL distilled water, pH 7.2), centrifuged again and redistributed in peptone physiological salt solution. This procedure was repeated for the lactobacilli using MRS agar plates and MRS broth tubes. For the two categories, the procedure achieved an inocuum containing 10⁹ cfu mL^-1, checking as viable count in respective agar. The fungal isolates which comprises of both moulds and yeasts were cultivated by inoculating tubes containing 10 mL malt extract broth incubated at 30°C for 24 and 48 h (for both yeasts and moulds respectively). These cultures were centrifuged and washed as described previously. This procedure achieved inoculums containing 10⁷-10⁸ sfu mL^-1, as viable count in both malt extract and malt yeast glucose agar. The mixed stains of bacterial and fungal inocula comprised of:

Preparation of samples: A 100 g of ground jack beans each, were pressure cooked for 40 min before inoculation. Samples were inoculated with the mixed cultures under aseptic condition using 1 mL of each inoculum and fermentation allowed to take place under controlled environment of temperature for 14 days. This was done by placing the covered transparent plastic vessel used for pressure cooking in the water bath at optimal temperature of 40°C for the liquid state fermentation (with bacteria being the inoculum). The liquid state controlled fermentation was terminated after 7 days, under aseptic condition using a sterile mucein cloth to drained out excess water from the substrate. Thereafter the fungi were inoculated aseptically for the commencement of the solid state phase of the combined fermentation for another 7 days and the temperature readjusted to 30°C. Control was set-up by pressure cooking 1 kg oven dried, ground jack beans for 40 min and transferred aseptically into dry sterile 1 L beaker and covered with non-absorbent cotton wool and aluminium foil for 14 days (Njoku et al., 1990).

Physico-chemical analysis: At every 48 h interval for the whole 14 days samples were taken during the combined fermentation and analyzed for, titratable acidity and pH (AOAC, 2000).

pH determination: The pH of the samples was determined according to the method of AOAC (2000). Ten gram of sample was mixed in 100 mL of CO₂ - free distilled water. The mixture was allowed to stand for 15 min, shaken at 5 min interval and filtered with Whatman No. 14 filter paper. The pH of the filtrate was measured using a pH meter (Model HM-305, Tokyo, Japan).

Total titratable acidity (T.T.A): Ten milliliter aliquots (triplicates) were pipetted and titrated against O.1 M NaOH to phenolphthalein end-point and the acidity was calculated as g lactic acid/100.

Sensory evaluation: Sensory characteristics of the fermented were assessed by 10 trained members of the department of microbiology of the Fededral university of Technology, Akure, Nigeria. Physical states of the substrates were assessed for their changes in colour, odour (aroma), sliminess, texture and overall acceptability during the fermentation period (Steinkraus, 1997). The panelists were instructed to sip water before and after assessing each product. The judges recorded sensory characteristics of each sample using 8 - point hedonic scale as described by Ihekoronye and Ngoddy (1985), where:

Each treatment was evaluated three times by each panelist.

Proximate analyses: After fermentation, all the substrates were dried in the oven at 50°C, ground, sieved and kept at -20°C pending analysis. The following proximate parameters were determined; moisture content, crude protein, ash content, crude fibre, fat and carbohydrate (was estimated by difference) (AOAC, 2000).

Mineral composition: Preparation of aqueous solution of substrates for Atomic Absorption Spectrophotometer (AAS) analysis which was used for calcium, iron, magnesium, potassium, sodium and zinc; were carried out and 2 g of the seeds samples were ashed. Fifteen milliliter of 20% v/v of nitric acid solution was added to the crucible to break up the ash. This was boiled and filtered into a 100 mL volumetric flask and then diluted to 100 mL with glass distilled deionised water (AOAC, 2000).

Anti-nutritional factors: Tannin content was determined by the method of Makkar et al. (1993). Phytate was determined using the modified procedure of Young and Greaves according to Oboh (2006). Canavanine content was determined by HPLC using method for gradient condition (Acamovic and D’Mello, 1990). Oxalates were determined by standard method (AOAC, 2000).

Statistical analysis: Data were analyzed according to the analysis of variance (ANOVA) procedures (Gomez and Gomez, 1984).

RESULTS

There was a significant increase in the total titratable acidity of the substrates fermented with mixed cultures of Lf, Pp and Mm (0.05 to 0.27%) and those of Bp, Af, Ko, Ro, An and Nc (0.15 to 0.39%) while the substrates fermented with mixed cultures of Sp, Pp, Ve and Nc (0.22 to 0.18%) and those of Bb, Lf, Pp, Bp, An, Ve and Mm (0.33 to 0.24%) had a significant decrease. The highest total titratable acidity value was 0.39% for mixed fermented substrate of Bp, Af, Ko, Ro, An and Nc and the lowest value was 0.18% for mixed fermented substrate of Sp, Pp, Ve and Nc (Fig. 1). The pH value of all the mixed fermented substrates showed a steady decrease between 6.5 to 4.5, with the substrate fermented with Bp, Af, Ko, Ro, An and Nc (from 6.5 to 4.5 ) being the most reduced (Fig. 2).

The mixed fermented substrates with four and above cultures (2.9) showed significant colour changes while the sample with only three cultures did not show any changes when compared with the control at the end of fermentation (2.2). The degree of slilminess increased significantly for all the fermented substrates between 2.9 to 3.5, but those samples whose cultures include yeasts has higher degree of slimlness (3.5) when compared with the control (2.2). The changes in texture for all the fermented samples were the same (2.9), being significantly higher when compared with the control (2.2). Generally, there was a significant increase in the score for odour change from 3.5 to 5.5 when compared with the control (2.2). The sample fermented with the mixed cultures of Bb, Lf, Pp, Bp, An, Ve and Mm (5.5) had the highest score for odour change.

There was an increase in the overall acceptability scores of the fermented samples as the number of the mixed cultures increases from 4.0 for Lf, Pp, Mm to 5.6 for Bb, Lf, Pp, Bp, An, Ve, Mm (Table 1).

The crude protein content of all the combined mixed fermented samples showed significant increase between 34.14 to 39.82 g/100g when compared with the control of 26.29 g/100 g. There was a significant decrease in the fat content of all the mixed culture fermented substrates from 11.95 g/100 g of the control to 4.33 g/100 g for Bp, Af, Ko, Ro, An, Nc which is the lowest and also in that of the ash content from 3.50 for the control to 2.23 g/100 g in Sp, Pp, Ve, Nc. With the crude fibre and carbohydrate content of the mixed fermented samples showing significant difference when compared with the control (47.52 g/100 g) (Table 2).

Table 3 shows that the mineral content of all the mixed combined fermented samples increased significantly in magnesium (from 30.07 to 46.77 mg g^-1), sodium (from 18.51 to 34.34 mg g^-1), potassium (from 23.51 to 40.88 mg g^-1) and iron (from 0.00 to 0.08 mg g^-1) when compared to the control. With both calcium (from 3.55 to 1.35 mg g^-1) and zinc (from 0.14 to 0.00 mg g^-1) content having a significant decreased in all the samples when compared with the control.

Of all the antinutrient content analysed for in all the combined mixed fermented samples, it was only phytate (58.66 to 5.07 g/100 g) and canavanine from 0.79 to 0.40 mg g^-1 that showed significant decrease in comparison with the control (Table 4).

DISCUSSION

In principle the mixed cultures acted in a similar way independent of the plant material to be fermented as if in a spontaneous fermentation and the cultivation time determines the quantity and degree of end products, this was in line with the findings of Rombouts and Nout (1995), Mbajunwa et al. (1998), Amadi et al. (1999), Sherfi and Hamad (2001) and Egounlety (2003). In this study, the effect of mixed culture fermentation on the nutritional status of C. ensiformis, L was observed. Increase in the total titratable acidity of cultures comprising of Lf, Pp and Mm and Bp, Af, Ko, Ro, An and Nc showed that secretion of some organic acids might have occurred, this was in accordance with the report of Park et al. (1996) and Ojokoh (2005). While the decrease in the total titratable acidity in those of Sp, Pp, Ve and Nc and Bb, Lf, Pp, Bp, An, Ve and Mm is an indication that there was less of organic acid production but more of proteinase activity was what Nout (1994) detected. The general decrease of the pH of all the fermented substrates was an indication of reduced alkalinity in order to hydrolyse the available carbohydrate to acid or the break down of protein to low molecular weight amino acids which agreed with the reports of other workers (Pederson, 1991; Caplice and Fitzgerald, 1999; Sahlin, 1999). The significant activeness of mixed cultures in the fermentation of Jack beans for the desirable physical characteristics of colour, texture, odour and overall acceptability was in agreement with Holzapfel (1997) and Ibrahim et al. (2005). This work revealed an increase in crude protein which might be as a result of more active proteolytic involvement and microbial biomass according to the findings of Yigzaw et al. (2001) and Ibrahim et al. (2005). The general significant decrease in the fat content observed from all the combined mixed fermentations might be due to usage of the available lipids by the different microbial culture involved. The decrease in the ash content of the fermented substrates was an indication that most of these cultures might have been the usage of some elements in their metabolites. The carbohydrate content showing significant difference was an indication that, for Bp, Af, Ko, Ro, An and Nc; Sp, Pp, Ve and Nc; Bb, Lf, Pp, Bp, An, Ve and Mm mixed fermented substrates, there might have been production of metabolites which make use of carbohydrate while for Lf, Pp and Mm fermented substrates, there might have been some breakdown of components which was what was detected by Dakwa et al. (2005) and Idris et al. (2005). The significant increase (p≤0.05) in magnesium, sodium, potassium and iron, of all the fermented substrates was an indication that they could be a good source of minerals as released by these microorganisms, these findings was in line with the finding of Gabriel (2002). The fact that calcium and zinc decreased significantly in all the fermented substrates could be an indication that the mixed cultures might have utilised calcium and zinc for their metabolism, this conform with the result of Hassan et al. (2005). The significant decrease in canavanine and phytate in all the fermented substrates showed that these microorganisms had enzymes that are capable of hydrolyzing these antinutrients, which was supported by the findings of Gabriel et al. (2004).

REFERENCES