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 American Journal of  
Food Science and Technology (AJFST)

Microbial Lactic Acid Fermentation Improves Nutritional and Organoleptic 
Profile of  Non-Dairy Milk Made from Bambara Groundnut 

Chude C.O.1*, Nwagbo C.C.1, Dibua N.A.2, Okoye E.C.S.2, Okpalanma E.F.3, Okoyeuzu C.F.4

Volume 2 Issue 1, Year 2023
ISSN: 2834-0086 (Online)

DOI: https://doi.org/10.54536/ajfst.v2i1.1025
https://journals.e-palli.com/home/index.php/ajfst

Article Information ABSTRACT

Received: November 28, 2022

Accepted: March 02, 2023

Published: April 11, 2023

Non-dairy milk was produced from bioprocessed Bambara groundnut using submerged 
fermentation with bacterial strains of  Lactobacillus plantarum [NRRL B-4306] and 
Lactobacillus fermentum [NRRL B-1932] obtained from the United States Department of  
Agriculture (USDA). Bambara groundnut was submerged in sterile water, inoculated with 
the starter culture containing 106 CFU/mL, and allowed to stand for 3 days. Bambara milk 
was produced by wet-milling the nut and the resulting paste cooked on medium heat for 20 
min and strained with cheesecloth to remove the particles. Milk from non-inoculated 
Bambara nut and cow milk was used as positive and negative controls for sensory 
characteristics and nutritional comparison. The nutritional and sensory profile was compared 
to meet WHO recommended daily intake (RDA) with cow milk. Proximate composition of  
the samples ranged from 20.80 - 19.70, 57.20 - 52.25, 6.80 - 8.79% and 368.10 - 425.10 
Kcal/100g for protein, carbohydrate, fat and energy, respectively. Results show that protein 
content of  the fermented Bambara milk (19.70) was higher than cow milk (3.4) while fat 
content (8.79) was higher compared to cow milk (3.6 g/100g). Amino acids content ranged 
from 3.90 - 5.00, 9.00 - 14.20, 5.62 - 6.80, 0.6 – 0.92, 17.20 – 19.50, 3.35 – 3.80, 2.50 – 3.00, 
3.80 – 4.15, 7.00 – 8.00, 2.90 – 4.50, 2.80 – 3.20, 4.80 – 5.10, 3.80 – 4.50, 2.60 – 3.80, 2.60 
– 4.00, 3.50 – 3.90 and 4.10 – 4.85 for alanine, arginine, aspartic acid, cystine, glutamic acid, 
histidine, isoluecine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, 
tyrosine and valine, respectively.  In-vitro protein digestibility increased from 70.74 - 89.70%, 
while anti-nutritional factors decreased from 4.72 – 2.08, 870.30 – 383.70, 1470.15 – 1023.10 
and 1.85 – 0.55 mg/100g for tannin, polyphenol, phytate, oxalate and trypsin inhibition 
activity, respectively. Intensities of  the sensory attributes was assessed based on parameters 
of  appearance, aroma, mouth feel, consistency and overall acceptability using a nine-point 
Hedonic scale rating, and the results found to compare favorably with cow milk. 

Keywords

Probiotic Non-Dairy Products, 
Plant-Based Foods, Milk 
Alternatives, Vegan Foods, 
Sensory Profile, Recommended 
Daily Allowance

1 Department of  Food Science and Technology, Chukwuemeka Odumegwu Ojukwu University, Nigeria
2 Department of  Microbiology, Chukwuemeka Odumegwu Ojukwu University, Nigeria
3 Department of  Food Science and Technology, Madonna University, Nigeria
4 Department of  Food Science and Technology, University of  Nigeria, Nigeria
* Corresponding author’s e-mail: co.chude@coou.edu.ng 

INTRODUCTION
With the increasing demand for alternative protein 
sources and healthier less processed foods, the food 
process industry is constantly searching for nutritious 
and consumer acceptable plant-based foods to meet 
the nutritional needs of  various age groups. Bambara 
groundnut (Vigna subterranean (L)) is a legume crop in the 
sub-Saharan Africa grown mainly by subsistence farmers. 
It is indigenous to West and Central Africa and considered 
a highly nutritive but underutilized grain legume. Our 
recent study (Chude et al., 2021) noted that the nutritional 
composition of  Bambara grains varies with cultivar and 
growing locations but is considered a complete food 
because of  its high protein content (9.60–40.0%); and is 
also considered to have a good balance of  the essential 
amino acids. Previous studies noted that Bambara grains 
contains 54.5 - 69.3% carbohydrate, 17 - 24.6% protein, 
5.3 - 7.8% oil, and the gross energy value is greater than 
that of  other common pulses like pigeon pea (Cajanus 
cajan) and cowpea, lentil (Lens esculenta) (Azam-Ali et al., 
2001; Quaye & Kanda, 2004). Despite less interest in the 
commercial production of  the crop and the almost total 
scientific neglect to improve yield, Bambara groundnut 
greatly contributes to the dietary structure of  many parts 
of  Africa (National Research Council, 2006). In addition 

to its commendable nutritional composition, Bambara 
groundnut possesses outstanding traits for drought 
tolerance, nitrogen fixation, and ability to produce yields 
in marginal soils, among others. Improved Bambara 
groundnut cultivars do not exist (Linnemann & Azam-
Ali, 1993); it exists as landraces, which actually composed 
of  many genotypes that is reported to result in an ability 
to endure stresses under local agricultural systems (Zeven, 
1998). 
Lack of  adequate processing techniques is said to hinder 
the utilization of  this legume crop which has limited 
its production. Fermentation using lactic acid bacteria 
(LAB) provides a good measure to improve its hard-to-
cook phenomenon and optimize its utilization through 
improved palatability and nutrient availability (Chude et 
al., 2018a, b; Masood et al., 2011). LAB fermentation has 
been used to improve preservative and detoxifying effects 
on fermented food product (Day & Morawicki, 2018). 
The process of  LAB fermentation also provides a cheap 
processing technique that has the potential to improve 
the digestibility of  protein in grains and increase the 
protein content (Chude et al., 2021). Bambara groundnut 
like many underutilized crops in the African region has 
the potential to improve food security measures and also 
contribute to problems of  insufficient protein availability 

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which is a major problem in many developing countries 
due to the high cost of  protein from animal sources. 
Alternative measures to improve protein availability 
through other sources than animal protein becomes 
necessary to alleviate this problem.
Lots of  work has been done on the production and 
nutritional status of  non-dairy plant-based milks like 
soy milk, almond milk, coconut milk, rice milk or hemp 
milk. However, Bambara groundnut also has the potential 
of  a milk substitute that can be utilized by individuals 
intending to avoid products of  animal origin for ethical 
or health reasons, or simply for taste preferences. Most 
food producers market plant-based milk substitutes as 
being healthier than cow milk because they may be lower 
in saturated fat and, if  entirely free of  animal products, 
cholesterol-free. 
Cow milk related health problems such as lactose 
intolerance in persons deficient in the enzyme lactase 
which is responsible for breaking down the lactose 
in the intestine can be solved with non-diary milk 
alternatives. Health symptoms such as abdominal cramps, 
constipation, bloating or diarrhea may result in lactose 
intolerant individuals who consume a dairy product. 
Amongst infants and children, dairy milk is considered 
common allergens although many tend to outgrow it in 
later years. More so, dairy milk especially unpasteurized 
milk is considered to have food safety concerns and has 
been linked to outbreaks of  food pathogens around the 
world such as Salmonella and E. coli outbreaks. Owing to 
the outstanding qualities of  Bambara grains, there is need 
to research novel ways that can be used to effectively 
harness its use and potentials in various food product 
applications. Hence, the study aims to evaluate the 
nutritional and sensory properties of  non-diary milk 
produced from fermented Bambara grains. 

MATERIALS AND METHODS
Our previous studies (Chude et al., 2021; Chude et al., 
2018a, b) described the pre-handling operations of  the 
Bambara groundnut and LAB starter culture used in the 
fermentation process. Briefly, the nuts were carefully 
cleaned and all extraneous materials and damaged nuts 
removed prior to use. Washing of  the nuts was done twice 
using ordinary water while rinsing was done with distilled 
water prior to cooking to softness as a pretreatment 
measure and to eliminate existing microflora before 
starter cultures inoculation. Lactobacillus plantarum [NRRL 
B-4306] and Lactobacillus fermentum [NRRL B-1932] 
used in this experiment were obtained from USDA 
Agricultural Research Services Culture Collection as pure 
cultures of  freeze dried cells preserved in a dormant 
state.  Inoculation in 25 ml Nutrient Broth and incubated 
in CO2 enriched jars for 24 h was used to bring the freeze 
dried cells to active state; cell recovery was achieved by 
centrifugation at 3600-x g for 15 min. 
The recovered cells were rinsed using 10 ml sterile 
distilled water and the spine repeated twice. Afterwards, 
a suspension of  the cells in 9 ml sterile distilled water 

was made and serially diluted before plating on Plate 
Count Agar using pour plating method. The colonies on 
each plate of  the dilution factor were counted after 24 h 
incubation in CO2 enriched jars and only plates containing 
approximately 106 cfu/ml was utilized in the inoculation 
of  the fermentation process.

Production of  Bambara Milk 
The pre-treated Bambara groundnut used in this 
experiment for the production of  Bambara milk was 
submerged in 30 liters of  sterile distilled water and 
inoculated with 106 cfu/ml of  the Lactobacillus plantarum 
[NRRL B-4306] and Lactobacillus fermentum [NRRL 
B-1932] obtained from USDA. Fermentation was allowed 
for 3 days after which the LAB-fermented Bambara 
groundnut and control was wet-milled. The resulting 
paste was poured into a pot, cooked on medium heat for 
15 min, and stirred continuously so it does not stick to the 
bottom of  the pot. After 15 min, the foam was scooped 
off  and the samples allowed to cool down to about 
100C. Then cheesecloth was used to strain the samples 
to remove the particles, and it was pasteurized at low heat 
for 5 min, poured into a bottle, refrigerated and analyzed 
within 12 hours of  production. Milk from non-inoculated 
Bambara groundnut and Cow milk was used as control 
for sensory characteristics and nutritional comparison.

Determination of  Proximate Composition and Total 
Energy 
Standard procedures described by the Association 
of  Official Analytical Chemists (1990) were used to 
determine the nutrient composition of  the samples. 
Briefly, the Kjeldahl method was used for protein (N 
× 6.25) determination while soxhlet extraction with 
a known weight of  sample in petroleum ether (boiling 
point, 40 to 60°C) was used for crude fat determination. 
The carbohydrate content was determined by sample 
differences of  subtracting the total fat, crude protein, ash, 
and crude fiber from the total dry weight (100g) of  the 
sample. A ballistic bomb calorimeter and the caloric value 
estimation was used for the gross energy determination 
according to Antia et al. (2006) by summing the multiplied 
values for crude protein, oil, and carbohydrate by their 
respective factors.

Determination of  Mineral Content 
The dry ashing method originally described by Chapman 
and Pratt (1982) was used for minerals determination. 
Sample size of  2 g was acid-digested with diacid mixture 
(HNO3:HClO4, 5:1, v/v) in a digestion chamber and 
the digested samples dissolved in double-distilled water 
and filtered (Whatman No. 42). The filtrate was made 
to 50 ml with double-distilled water and used for the 
determination of  total calcium, phosphorus and iron. 
Calcium was determined by a titration method. Iron was 
determined by atomic absorption spectrophotometer. 
Phosphorus and other minerals were determined 
spectrophotometrically using molybdovanadate method.

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Determination of  amino acids 
The amino acids compositions of  the samples were 
measured on protein hydrolysates based on high 
performance liquid chromatography technique according 
to the method described by Chude & Nkama (2020). The 
fermented and non-fermented Bambara milk samples 
were weighed into conical flasks with 100 mL of  70 % 
methanol. The mixture was shaken for 5 min using a 
mechanical rotor and allowed to stand for 10 min. The 
resultant supernatant was filtered through Whatman #1 
filter papers and diluted with deionized water in a volume 
ratio of  1:3; and the pH adjusted to a range of  5-8 using 
HCl and NaOH as described by Jayaratne et al. (2020). 
The filtered samples will be utilized for HPLC following 
protocol outlined by the manufacture. A standard solution 
containing 1.25 μmol/mL of  each amino acid in 0.1N 
hydrochloric acid was created.
Derivatization of  amino acids were done using a standard 

solution (20 μL) pipetted into a 10 × 5-mm tube and 
dried in vacuo at 65°C. The residue was added 30 μL of  
methanol-water-Phenylisothiocianate (2:2:1 [v/v]) and 
then removed in vacuo at 65°C, after which 30 μL of  the 
derivatizing reagent methanol-water-Phenylisothiocianate 
(7:1:1:1 [v/v]) was added, and the tube was vortexed 
for 30 sec and allowed to stand for 20 min at ambient 
temperature. Aliquot of  150 μL of  the diluent containing 
5mM sodium phosphate with 5% acetonitrile was added 
to each tube before injection. Chromatographic analysis 
was obtained at 30°C using a gradient elution (Table 1). 
An aqueous buffer (Eluant A) was prepared using 0.5 
mL/L Triethylamine added to 0.14M sodium acetate and 
titrated to pH 6.20 with glacial acetic acid while eluant B 
was acetonitrile-water (60:40 [v/v]). Each amino acid was 
calculated based on the proportional molar concentration 
using the concentration of  standard amino acids and 
expressed as μ g amino acid/mg sample.

Table 1: Gradient program employed for the separation of  PTC-amino acids
Time Flow rate
(min) (mL/min) % Eluent A % Eluent B 
0 1.0 90 10 
12.0 1.0 70 30 
20.0 1.0 52 48 
22.0 1.0 0 100 
24.0 1.0 0 100 
30.0 1.5 0 100 
37.0 1.0 90 10 

Determination of  anti-nutrients Composition 
Determination of  Tannin Content
The modified vanillin-HCl method originally described 
by Price et al. (1978) was used for the quantitation of  
tannins in the test and control samples. Briefly, 200 mL of  
the samples were extracted for 20 min in capped rotating 
test tubes using 10 mL 1% (v/v) conc. HCl in methanol. 
Aliquot of  0.5% of  5 ml vanillin reagent was added to the 
extract (1 ml) and the absorbance of  the color determined 
at 30°C after 20 min and the absorbance read at 500 nm. 
An interference natural light pigment was corrected in 
the sample by subjecting the extract to same conditions 
of  the reaction without the vanillin reagent. The results 
were expressed as catechin equivalents using standard 
curve, i.e amount of  catechin (mg per ml) which gives 
a color intensity equivalent to that given by tannins after 
correcting for blank.

Determination of  Phytic acid Content 
Following methods originally described by Wheeler 
& Ferrel (1971), 3 mL of  the samples were used for 
extraction of  phytic acid using 3% trichloro-acetic acid 
kept on a vortex shaker at ambient temperature and 
subsequently centrifuged on high speed. Precipitation 
was used to obtain the phytic acid from the supernatant 
by estimating the ferric phytate and iron in the samples. 

Thus, a 4:6 iron: phosphorus molecular ratio was used 
to calculate the Phytate-phosphorus (phytate-P) from the 
iron determined. The phytic acid was then estimated by 
multiplying the amount of  phytate-phosphorus by the 
factor 3.55 based on the empirical formula C6P6O24H18. 

Determination of  Oxalate Content 
The AOAC (2010) method was utilized for oxalate 
determination. To this regard, 1 g each of  the samples was 
weighed into 100 ml conical flask and was added 75 ml 
of  3 M H2SO4. The mixture was intermittently stirred for 
about 1 hour with the aid of  a magnetic stirrer and then 
filtered using whatman No.1 filter paper. The filtrate was 
adjusted to 25 ml and titrated at 80 - 90°C using a solution 
of  0.1 N KMnO4 until a faint pink colour that persisted 
for at least 30 sec appeared. The oxalate concentration in 
each sample was obtained from the calculation: 1 ml 0.1 
permanganate = 0.006303 g oxalate.

Determination of  Polyphenol Content
The spectrophotometric method originally described 
by Price and Butler (1977) was utilized for polyphenols 
quantification using sample size of  50 ml mixed with 3 
ml of  methanol in a test tube and was manually shaken 
for 60 sec. The mixture was filtered using whatman No.1 
and the filtrate mixed with 50 ml of  water. Analysis of  

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the samples was done within an hour. For the analysis, 3 
ml aliquot of  0.1 M FeCl3 in 0.1 N HCl was added to 1.0 
ml of  the filtrate and timed addition of  3 ml of  0.008 M 
K3Fe(CN)6 was done. The absorbance was read at 720 
nm after 10 min using a spectrophotometer. A standard 
curve was prepared using tannic acid and following the 
above procedure.

Determination of  Trypsin Inhibition Activity 
The inhibitory action of  the bovine trypsin (EC 
3.4.21.4) on substrate benzoyl-DL-arginine-p-nitrianilide 
(BAPNA) hydrochloric was used to determine trypsin 
inhibition activity (Kakade et al., 1974). The samples (1 ml 
each) were extracted for 3 hours with 50 ml, 10 M NaOH 
using a mechanical shaker continuously at ambient 
temperature. The pH of  the resulting solution was 
adjusted to 9.4 - 9.6 with 1 M NaOH and the suspension 
was shaken and diluted with distilled water to produced 
trypsin inhibition of  40 - 60% at 37°C. The respective 
dilutions were noted. Consequently, TIA was calculated 
in terms of  mg pure trypsin (Sigma type lll, lot 20H0868). 

Where D is the dilution factor, A is the change in 
absorbance at 410 mm due to trypsin inhibition per cm3 
diluted sample extract and S is the weight of  the sample.

Determination of  in-vitro Protein Digestibility 
Measurement of  in-vitro protein digestibility of  the 
samples was determined according to the method 
originally described by Maliwal (1983). In triplicate, 
a known quantity of  the test and control samples was 
mixed with 16 mg nitrogen and digested in 15 ml of  
0.1 M HCl for 2 h at 37ºC using 1 mg pepsin. Aliquot 
of  15 ml of  10% trichloroacetic acid (TCA) was added 
to stop the reaction. Centrifugation of  the mixture was 
done at 630 rev. for 5 min and the resultant filtrate passed 
through Whatman No. 1 filter paper for quantification. 
Thus, the TCA soluble fraction was assayed for nitrogen 
using the micro-Kjeldahl method (AOAC, 2010). The 
following equation was used to obtain the digestibility of  
the samples: 

Determination of  Sensory Characteristics 
Sensory qualities of  the test and control products were 
conducted with the 10 member panelists trained to 
identify retronasal aroma and taste to determine sensory 
attributes. A consent form was given to the panelists and 
was also educated on their duties. A randomized, single-
blind manner was employed for the evaluations were 
panelists were blinded from the identity of  the specific 
product to be consumed. The product was labeled prior 

to the start of  experiment with a randomization code and 
the code list kept confidential from the panelist during the 
experiment. The test was conducted while the samples 
were still fresh and the panelists were required to observe 
the sample, taste and score; then rinse their mouth with 
water before tasting another sample/product. 
As described by Arteaga et al. (2021), 20 mL of  each 
sample presented in random order at ambient temperature 
in glass cups sensory analysis was done using. The sensory 
analysis was divided into two sessions for presentations 
of  fermented / unfermented Bambara milk sample and 
fermented / cow milk samples per session. For palate 
cleansing, water and plain crackers were provided. The 
panelists assessed the intensities of  the attributes based 
on parameters of  appearance, aroma, mouth feel, 
consistency and overall acceptability using a nine-point 
Hedonic scale rating of  9 = liked extremely down to 1 = 
disliked extremely.

Data Analysis 
Analysis of  the data generated from the experiment was 
conducted in triplicates and expressed as mean ± SD 
using Tukey–Kramer multiple comparisons test or two-
way analysis of  variance (ANOVA). Pair-wise statistical 
comparison and the relevance between samples was 
evaluated using the Mann-Whitney rank sum test in 
SigmaPlot (Systat Software, USA). Statistical difference 
between fermented and non-fermented Bambara milk 
was evaluated using the Student’s T-test. P-value of  
< 0.05 was considered statistically significant.

RESULTS AND DISCUSSION
Table 2 presents the proximate composition and total 
energy of  the test and control Bambara milk samples 
in comparison to recommended daily intake of  adults. 
The protein content of  non-inoculated Bambara milk 
was found to be 20.80% which was similar to that 
reported by Abdulsalami & Sheriff  (2010) but higher 
than that reported by Okonkwo & Opara (2010) for 
raw Bambara seed. The protein content of  the Bambara 
milk slightly decreased after fermentation (19.70%). 
The result indicates that bioprocessing of  the seeds had 
no significant effect on protein content. However, an 
increment in protein content after soaking was observed 
by Hassan et al. (2005) for lupin seeds and explained that 
the increment resulted due to quantitative reduction of  the 
antinutritional factors (tannin and phytic acid) and other 
water soluble constituents. In this study, the decrease in 
protein content after bioprocessing may have resulted 
from precooking the nut possibly due to solublization of  
protein by heating that lead to loss of  protein in the final 
product as explained by DeMan (1999). Similar reduction 
in protein after cooking was observed by Hamed et al. 
(2008) and Hainida et al. (2008) for pumpkin and roselle 
seeds, respectively. 

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Table 2: Proximate composition, mineral content and total energy of  samples
Paramet+1:10ers    NBM        IBM Cow Milk RDI*
Protein (g/100g)    20.80±0.8%a        19.70±0.2%a   3.4. ± 0.9      50g
Fat (g/100g)    6.80±0.1%a        8.79±0.5%b 3.6. ± 0.2      Less 70g
Carbohydrate (g/100g)     57.20±0.4%a        52.25±0.7%b 53.40 ± 0.2      Least 260g
Mn (mg/100 g)    3.00 c±0.60               1.90 d±0.40    11.7 ± 1.0                 2.0mg
P (mg/100 g)    265.80 a±0.40           248.40 b±0.60        93.5 ± 0.4                  1,000.00mg
Ca (mg/100 g)    220.30 a±0.80           198.80 b±0.2012 6.4 ± 0.5  1,000.00mg 
K (mg/100 g)    50.20 c±0.90        38.90 d±0.20 138.5 ± 0.1      3,500.00mg
Na (mg/100 g)    11.50 c±0.50        7.80 d±1.00 58.2 ± 1.0      2300mg
Energy (Kcal/100g)    368.10±1.0b        425.10±1.5a 252.58 ± 0.01      2,000Kcal

Values are means ± SD (n = 3). Values in the same row with different superscripts are significantly (p ˂ 0.05) different. NBM: Non-
inoculated Bambara Milk, IBM: Inoculated Bambara Milk*Recommended Daily Intake (RDI) of  Adults (WHO, 1996; NIH, 2020)

The fat content of  non-inoculated milk was 6.80% and 
was similar to that reported by Abdulsalami & Sheriff  
(2010) and higher than that of  Mune et al. (2007) for 
raw Bambara seeds. Bioprocess significantly increased 
fat content of  the Bambara milk (P˂0.05). Bradbury 
et al. (1984) did not observe any significant changes 
in the crude fat content of  sorghum after lactic acid 
fermentation for 4 days. Chavan (1988) found a slight 
increase in crude fat content of  sorghum and sorghum 
plus green gram blend during natural fermentation. More 
so, the carbohydrate content of  non-inoculated sample 
was found to be 57.20% and was similar to that reported 
by Okonkwo and Opara (2010) for raw Bambara 
seed. The carbohydrate content significantly (P˃0.05) 
decreased after fermentation. The changes observed are 
possibly due to leaching of  soluble components into 
cooking (Yagoub & Abdalla, 2007) and fermentation 
water; and possibly the breakdown and utilization of  the 
sugars by the fermenting organisms as a ready source 
of  energy. Carbohydrates particularly starch and soluble 
sugars are principal substrates for fermentation with 
lactics. Hence, significant degradation and a subsequent 
decrease in starch content are expected to occur during 
fermentation of  legumes. The gross energy content 
significantly (P˂0.05) increased with a maximum value 
of  425.10 kCal/100g. The calculated metabolizable 
energy values which ranged between 368.10 and 425.10 
kCal/100 g showed that Bambara groundnut have energy 
concentrations favorably comparable to cow milk.
Determination of  mineral content indicates that the 
non-inoculated Bambara milk was found to be rich in 
calcium. Calcium content of  flour from non-inoculated 
nuts was 220.30 mg/100 g which decreased to 198.80 
mg/100 g after fermentation. The results indicated that 
fermentation of  the nut, significantly (P˂0.05) reduced 
the calcium content of  Bambara groundnut. The loss 
of  calcium during the treatment may be attributed to its 
leaching out into the discarded water used for cooking 
and fermentation. The results are in close consistence 
with the results of  Duhan et al. (2002) who also reported 
a significant decline in the total calcium content on 
water soaking. All other major minerals followed a trend 

similar to that obtained for calcium (Table 2). The iron 
content of  non-inoculated sample was 5.90 mg/100 g; 
bioprocessing of  the seeds reduced iron content to 3.80 
mg/100 g. The reduction in iron content may also be due 
to loss of  iron in the fermentation medium. The results 
are in agreement with those of  Lestienne et al. (2005), who 
observed reduction in iron content of  the soaked grains 
as compared to raw ones. However, the mineral content 
of  the inoculated Bambara milk despite the decrease was 
found to compare favorably with cow milk and with the 
RDA of  WHO (1996). 
Figures 1 and 2 shows the chromatogram for amino 
acid analysis of  the test (fermented) and control 
(non-fermented) samples. The study for amino acid 
composition observed that glutamic acid, aspartic 
acid and leucine are the most abundant amino acids in 
Bambara milk. Amino acid contents slightly increased 
after fermentation. Similar observation has been reported 
by Olaofe & Akintayo (2000), and Adeyeye & Afolabi 
(2004) for soaking and cooking of  Bambara groundnut; 
glutamic acid was the most concentrated essential 
amino acid (17.00%). Other researchers have also 
reported similar observations on the increasing effects 
of  fermentation on amino acids content. According to 
Sarkar et al. (1997), Bacillus fermented soybean led to an 
increase in free amino acids and ammonia by 60- and 40-
fold, respectively. Baumann & Bisping (1995) found that 
certain Rhizopus strains with high proteolytic activity 
were able to release nearly 5 times more amino acids. Song 
et al. (2008) reported that fermentation of  soybean with 
L. plantarum and B. lactis caused increase in amino acids 
content while fermentation with S. cerevisae showed the 
opposite results. Ferial & Esmat (2011) observed 21.8% 
increases in essential amino acids in fermented chickpea 
with Rhizopus at 48 hours of  fermentation. Thus, when 
comparing the essential amino acids in Bambara milk 
with the recommended FAO/WHO provisional pattern, 
it was superior with respect to aspartic acid, threonine, 
methionine, leucine, tyrosine, phenylalanine, histidine and 
arginine and adequate in valine and isoleucine (Table 3), 
and it was only for lysine that supplementation might be 
required.

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Figure 1: HPLC Chromatogram of  LAB-fermented Bambara groundnut

Figure 2: HPLC Chromatogram of  non-fermented Bambara groundnut

Table 3: Amino acid composition of  samples
Amino Acids NBM IBM Cow Milk* Reference Daily
Alanine 3.9 5.0 4.15
Arginine 5.0 6.2 2.9 – 4.2 2.0
Aspartic Acid 5.62 6.8 6.2 – 7.8 4.0
Cystine 0.6 0.92 0.65
Glutamic Acid 17.2 19.5 15.8 – 23.2
Glycine 3.35 3.8 0.8 – 2.1
Histidine 2.5 3.0 3.0 2.4
Isoleucine 3.8 4.15 4.1 – 6.2 4.2
Leucine 7.0 8.0 3.2 – 8.3 4.8
Lysine 2.9 4.5 8.1 4.2
Methionine 2.8 3.2 3.2 2.2
Phenylalanine 4.8 5.1 5.4 2.8
Proline 3.8 4.5 10.1 – 11.8
Serine 2.6 3.8 6.6
Threonine 2.6 4.0 5.8 2.6
Tyrosine 3.5 3.9 5.8 1.4
Valine 4.1 4.85 7.5 4.2
Values are mean of  triplicates. NBM: Non-inoculated Bambara Milk. IBM: Inoculated Bambara Milk.
* Saima et al. (2016)* *WHO (1996)

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The antinutritional factors of  the samples are shown in 
Table 4. Tannin content of  the non-inoculated Bambara 
milk (4.72 mg/100g) was higher than that reported by 
Abiodun & Adepeju (2011) for Bambara groundnut. 
Fermentation significantly (P ˃ 0.05) decreased tannin 
content to 2.08 mg/100g. Similar trends was observed by 
Mubarak (2005), Hassan et al. (2005) and Abedel-Hady et 
al. (2005) for soaking and cooking of  mug bean, lubin, 
maize and lentil seeds, respectively. Polyphenol content 
of  non-inoculated sample was 870.30 mg/100g, and 
significantly (p ˃ 0.05) decreased after fermentation to 
383.70 mg/100g. These results were in agreement with 
the findings of  Yagoup et al. (2004) for roselle seeds. 
The phytate content also had a similar trend of  decrease. 
Bambara groundnut is rich in protein; therefore they had 
high phytate levels. In legumes, phytates are associated 
with protein bodies (Sulieman et al., 2007) and, therefore, 
phytate levels should increase with increasing protein 
content. 
These results revealed that fermentation could lower 
the level of  these antinutrients. The loss in phytates 
during fermentation of  Bambara groundnut may be 
due to leaching of  phytate ions into the fermentation 
water under the influence of  a concentration gradient 
(difference in chemical potential) which governs the rate 
of  diffusion. Similar results for reduction in phytic acid 
in the soaked bean have been earlier reported (Bishnoi 
et al., 1994). Fermentation also significantly (p ˃ 0.05) 

decreased oxalate content to 0.55 mg/100g; while trypsin 
inhibition activity has a content of  8.40 mg/100g for 
non-inoculated sample against 3.30 mg/100g observed 
after fermentation. 
Table 4 also presents the in-vitro protein digestibility of  
the test and control samples. The increment in protein 
digestibility after fermentation is likely due to reduction in 
antinutrients as a result fermentation. Effective reduction 
of  antinutrients has been reported to improve the protein 
digestibility in legumes (Babiker and ElTinay, 1993). 
Legume consumption has been associated to deleterious 
effects such as growth retardation (Martinez et al., 1995), 
lowered digestibility and absorption of  dietary nutrients 
(Pusztai et al., 1995) and physiological, metabolic and 
immunological disturbances (Hajobs et al., 1995). It is 
evident from the study that antinutrient concentration in 
legumes can be eliminated or reduced to tolerable level 
through lactic acid bioprocess. 
Sensory attributes were determined to monitor the 
potentials of  the product and ascertain the consumer 
perception of  the fermented Bambara milk in comparison 
to the non-fermented sample and cow milk, and the data 
presented in Table 5. Evaluation for color, consistency, 
mouth feel, taste and overall acceptability showed that 
Bambara milk can compete favorably with cow milk in 
consumer acceptability. If  a new product or formulation 
fails to appeal to consumers, the new product is considered 
as an unsuccessful development. 

Table 4: Antinutritional factors and in-vitro protein digestibility of  samples
Antinutrients NBM         IBM Daily Reference Intake*
Tannin (mg/100 g) 4.72±1.0a 2.08±0.2b 1.5–2.5 g
Polyphenols (mg/100 g) 870.30±0.3a 383.70±0.5b 500mg to 1500mg
Phytic acid (mg/100 g) 1470.15±0.5a 1023.10±0.1b 100-400 mg
Oxalate (mg/100 gm) 1.85±0.8a 0.55±0.5b 50 – 100 mg
Trypsin Inhibitor (mg/100 g) 8.40±0.2a 3.30±0.4b -----
IVPD (%) 70.74±1.0b 89.70±0.6a -----

Values are means ± SD (n = 3). Values on the same row with different superscripts are significantly (p ˂ 0.05) different. NBM: 
Non-inoculated Bambara Milk, IBM: Inoculated Bambara Milk. *(Finkielstein and Goldfarb, 2006; Taguchi et al. 2015; Raj et 
al. 2015; Sharma et al. 2019)

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Figure 1: Box plot illustrating sensory evaluation of  fermented Bambara milk and control

The result for color showed that fermented Bambara-
milk had a mean score of  8.3, which was extremely 
liked. Color is considered an important property of  
food product and could affect consumer acceptance of  
the product. The Bambara-milk had a pale yellow color 
and contained no artificial color additive. The mean 
value for consistence was shown to be 7.7 and had no 
significant (p ˃ 0.05) difference, when compared to 
values of  7.4 and 7.8 obtained for non-inoculated milk 
and cow milk respectively. Consistency is an important 
attribute for beverages and in commercial beverage 
products; emulsifiers and stabilizers, such as guar gum, 
κ-carrageenan and xanthan gum, may be added to improve 
the quality of  the product by promoting its thickness. 
Hence, the result obtained for consistency of  the product 
indicates that this will not be needed in commercializing 
Bambara-milk as the product was liked without artificial 
emulsifiers and stabilizers. 
Bioprocessed Bambara milk had a mean score of  7.8 
for taste and no significant (P ˃ 0.05) difference with 
the control sample. It was observed that the fermented 
sample had no beany flavor ordinarily observed in 
products made from Bambara groundnut. This was 
attributed to the actions of  the lactic acid bacteria used 
in the fermentation process. It could also be possible 
that the fermented Bambara nuts used in the production 
was lipoxygenase free through fermentation. According 
to Yuan and Chang (2007), the beany flavor components 
are mainly the oxidation products of  unsaturated lipids 
catalyzed by lipoxygenases. When no lipoxygenase exists 
in legumes, polyunsaturated lipids will not be catalyzed 
to produce the undesirable flavor. Mouth feel was used 
as an indication of  texture for the Bambara milk and 
characterized by degree of  hardness or softness of  the 
product. The sensorial data obtained for mouth feel had a 
mean score of  6.7 and no significant (P ˃ 0.05) difference 
with cow milk which was preferred. Hence, mouth feel 
is an important parameter to consider in developing new 
beverage products, as this will also influence consumer 
perception and acceptability.

CONCLUSION 
Bambara groundnut (Vigna subterranean (L)), is a seed 
whose origin is of  West and Central Africa where research 
and development is ineffective, however, the crop has been 
identified to have the potential to improve malnutrition 
and boost food availability. This study has shown that 
milk from this fermented legume can compare favorably 
with cow milk with regards to consumer acceptability 
as well as the recommended FAO/WHO nutritional 
provisional pattern. This will provide a good alternative 
for consumers who wish to avoid intake of  animal 
products due to cholesterol content or personal belief. 
The study also showed that microbial fermentation may 
offer the simplest and economic means to improve the 
safety, quality and functionality of  Bambara groundnut, 
hence, optimize its utilization as plant-based milk. In 
developing regions where population is constantly on 
the increase, food security becomes paramount and 
there is no better means of  ensuring food security than 
harnessing the potentials of  indigenous crops. 
Considering the qualities mentioned for Bambara 
groundnut, it has great opportunities towards food 
security, sustainability, income generation, product 
development, and dietary diversification. Although the 
crop is limited by the content of  anti-nutritional factors, 
microbial fermentation provides an effective and safe 
means to biodegrade these anti-nutrients. Owing to the 
outstanding potentials of  Bambara groundnut in helping 
to curb food security issues, it should not only be seen 
and cultivated as subsistence crop; rather it should be 
seen as a crop that is relevant to food security.
However, there is need to further investigate the scope 
for improvement in fermentation of  Bambara groundnut, 
since most fermented foods are produced at household 
level in a majority of  African countries where this 
legume is indigenous. Upgrading the production process 
for fermented Bambara groundnut will necessitate 
several critical steps in the commercialization of  plant-
based milk from this important legume crop. Identified 
microorganisms, which have the ability to effect beneficial 

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changes in the fermentation process of  this legume 
should be selected and subjected to genetic improvement 
geared towards maximizing desirable quality attributes 
and limiting any undesirable attributes such as anti-
nutrients and beany-off  flavour.

Acknowledgement 
The authors wish to acknowledge the Agricultural 
Research Services Culture Collection, Bacterial 
Foodborne Pathogens and Mycology Research Unit; 
National Center for Agricultural Utilization Research 
of  the United States Department of  Agriculture, Peoria 
Illinois USA for providing the bacterial culture used for 
this study. 

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