Food Measure DOI 10.1007/s11694-017-9541-2

ORIGINAL PAPER

Production, nutritional and biological value of bambara groundnut (Vigna subterranea) milk and yoghurt Majeste Mbiada Pahane1 · Leopold Ngoune Tatsadjieu2 · Clemence Bernard3 · Nicolas Yanou Njintang4   

Received: 3 February 2017 / Accepted: 8 May 2017 © Springer Science+Business Media New York 2017

Abstract  The purpose of this study was to assess the nutritional properties of Bambara milk and yoghurt. The milk was produced by aqueous extraction of flour obtained by dehulling the seeds followed by parboiling. It was then fermented using a mix culture of L. Bulgaricus, S. thermophilus and L. plantarum, followed by the evaluation of the protein biological value on rats. During the process of flour production, from the whole seeds to the flour, a significant drop in total polyphenol content (1.00 ± 0.10– 0.41 ± 0.01  mg/100  g) and phytates (1.18 ± 0.03– 0.32 ± 0.01 mg/100 g) was observed while the protein content increased (19.7 ± 1.2–25.47 ± 2.28 g/100 g). During the fermentation of the milk into yogurt, a significant decrease in phytate content (0.29 ± 0.01–0.03 ± 0.01  g/100  g), an increase in the protein content (1.8 ± 0.1–2.6 ± 0.1 g/100 g) and the protein digestibility (91.5–96%) were equally observed. Red blood cell, glycaemia, the ASAT and ALAT contents of rat bloods fed Bambara milk or yoghurt were * Nicolas Yanou Njintang [email protected] Leopold Ngoune Tatsadjieu [email protected] Clemence Bernard cl.bernard@ebi‑edu.com 1

National School of Agro‑Industrial Sciences (ENSAI), University of Ngaoundere, P.O. Box 455, Ngaoundere, Cameroon

2

University Institute of Technology (IUT), University of Ngaoundere, P.O. Box 455, Ngaoundere, Cameroon

3

Laboratoire EBInnov, Ecole de Biologie Industrielle, 32 boulevard du port, 95094 Cergy Pontoise Cedex, France

4

Faculty of Sciences (FS), University of Ngaoundere, P.O. Box 454, Ngaoundere, Cameroon





not significantly different to rats fed casein as protein reference. In conclusion Bambara groundnut is a source of protein which the quality may be enhanced through processing of high value yogurt. Keywords  Bambara groundnut · Milk production · Fermentation · Nutrients · Antinutrients · In vivo protein digestibility

Introduction Vegetal milks are aqueous extracts of plant materials generally made from pulse and oil legumes. Production of vegetal milks has been the subject of many studies in view to alleviate the protein energy malnutrition in developing countries and to benefit from its health promoting properties. In this respect vegetal milk is proposed as alternatives to animal milk [1] which is not only expensive and scarce, but it also induced in some cases allergy and metabolic disorders such as hypercholesterolemia and galactosemia [1]. The most important legume seed that has given more scientific attention in vegetal milk production is soya bean and more research is still being undertaken nowadays to improve the quality and commercialization of soymilk [2]. Several studies associated consumption of soya products with health benefits such as low risk of cancers, cardiovascular-associated diseases, hypercholesterolemia, diabetes, bone and kidney diseases [3]. The technology of vegetable milk production include soaking for variable times 1–4 days, dehulling or not before being ground to a slurry which is diluted in water (ratio volume to mass varying from 3 to 5 v/g), filtered and pasteurized [4, 5]. In some conditions the milk is fermented before being consumed [6]. In Cameroon the unfermented product is

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commercialized under the brand name “SOY RIVER” and has become an important food staple or beverage for not only the infants, but also for adults. In households, the soy milk is usually produced and consumed after fermentation using yogurt Streptococcus thermophilus and Lactobacillus bulgaricus bacteria starter. In order to increase the market and offer new product with high nutritional and commercial value, the Cameroon industry in cooperation with our laboratory is investigating the use of other leguminous such as Bambara groundnut, mucuna, cowpea as raw material in the production of vegetal milk [5, 7]. The condition under which this is feasible needs to be investigated and is the subject of this piece of work. Bambara groundnut is a pulse legume with high nutritional value. The carbohydrate (50–61.3%), protein (16–21%) and lipid (6.0%) contents are relatively equilibrated and give Bambara groundnut a gross energy value greater than those of common pulses such as cowpea, lentil and pigeon pea [8]. Bambara groundnut has advantage over soybean and many others because of its resistance to drought and its ability to grow on poor soils. In addition, bambara groundnut can be consumed without treatment suggesting it contains less antinutrients and toxicants. The annual world production of Bambara groundnut was estimated to 330.000 tonnes in 2012 with 45–50% produced in West Africa [9]. The production in Cameroon was estimated in 2010 to 23 882 tonnes [10] while it ranks third in terms of food leguminous production after groundnuts and cowpea. In a functionality view point, investigations on Bambara groundnut flour reported relatively high water absorption capacity (149–159  g/100  g), and oil absorption capacity (OAC 0.82–0.91  mL/g flour) but lower than OAC from soy flour (1.94  mL/g), [11]. The Bambara groundnut flour also exhibits high emulsion capacity (42–66  mL/100  L) and stability (38–62  mL/100  mL), foaming capacity (38–43  mL/100  mL) and stability (53–65  mL/100  mL) and high gelling power (LGC  =  13–14  g/100  mL) as compared to soya flour (LGC > 18  g/100 mL) [11]. Though studies on Bambara groundnut are fragmentary, it appears that the crop has a great potential which needs to be investigated. Some authors investigated the physicochemical, sensory and microbiological properties of Bambara groundnut milk and yoghurt. Poulter and Caygill [12] assessed the potential of Bambara groundnut in vegetable milk production in Ghana to alleviate the intermittent use of existing dairy facilities in lesser developed countries. Obizoba and Egbuna [13] and later, Brough et  al. [14] reported that Bambara groundnut milk gives much accepted flavor compared to the cowpea, pigeon pea and soy milk. Falade et al. [6] recently reported higher yield (52%) of soy milk, compared to Bambara groundnut milk (yield 47%). Very few studies were reported on Bambara

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groundnut yoghurt. Murevanhema [15] evaluated the optimal conditions of fermentation of Bambara groundnut milk using lactic acid bacteria. Falade et  al. [6] recently reported the production of Bambara groundnut yoghurt using starter cultures of Lactobacillus delbruieckii subspp. bulgaricus and Streptococcus salivarus subspp. thermophilus. They found that the total solid of Bambara groundnut yoghurt (9.7%) was lower than that of soya yoghurt (10.4%). In addition, the viscosity of Bambara groundnut yoghurt (698  Cp) was higher but decreased rapidly during storage than that of soya milk (530  Cp). The sensory aroma, taste, colour and general acceptability of Bambara groundnut yoghurt were significantly higher than that of soy yogurt. All these results further highlighted the high potential of Bambara groundnut in producing yogurt. However the nutritional characteristics of Bambara groundnut milk as well as yoghurt are still to be investigated. In addition, very few or no data exists on the change in nutrient and antinutrient compositions during milk and yoghurt production. In other words what are the nutrients, antinutrients and biological values of the flour, milk and yoghurt made from Bambara groundnuts? The objective of the work was to evaluate the nutrient and antinutrient compositions of whole, dehulled and blanched Bambara groundnut flour as well as the nutritional value of the Bambara groundnut milk and yoghurt produced.

Materials and methods Mature and dried seeds of Bambara groundnuts were obtained from the Agricultural and development research institute (IRAD) Center of Ngaoundere-Cameroun. The animal material used were male born albino rats Rattus norvegicus, Wistar strain. Production of Bambara groundnut flour Bambara groundnut flour was produced as described by Kaptso et al. [16] with minor modifications as presented in Fig. 1. The seeds of similar sizes were selected, washed and soaked in distilled water in the ratio 1/5 (w/v) for 24  h at ambient temperature. The seeds were then drained, manually dehulled and blanched in hot water at 100 °C for 20 s. They were then dried for 48 h in a ventilated electric oven (CKA 2000, Ngaoundere, Cameroon) set at 40 ± 5 °C till they attained a moisture level of about 10%. The dried seeds were ground in a hammer milling machine to pass through a 500  µm mesh sieve, packaged in polyethylene bags and stored at 4 °C for further analysis.

Production, nutritional and biological value of bambara groundnut (Vigna subterranea)… Bambara groundnuts (dry seeds)

Sampling 1

Water

Production of Bambara groundnut yoghurt

Soaking in water (1:5 g/mL; 24h, 25°C) Manual Dehulling

Hulls Sampling 2

Dehulled beans

Blanching in water (20s, 100°C) Drying oven (40°C, 48h)

Water

Dehulled Blanched dried beans flour

Whole raw beans flour

Sampling 3

Distilled water (1/5 g/mL) Agitation (130 rpm/min, 3 hours) Filtration (muslin cloth) Bottom Recycling, same conditions

Filtrate 1

Filtrate 2

Bambara milk

Sampling 4

Pasteurisation (90°C, 10min) Seeding -2/3 Streptococcus thermophilus and Lactobacilllus bulgaricus -1/3 Lactobacillus plantatum

Pasteurised, cooled Bambara milk Fermentation (40°C, 5 hours) Bambara yoghurt

Bambara groundnut yoghurt was produced according to Yaou et al. [17] with few modifications. The milk was preheated in a steam cooker at 95 °C for 5 min, then cooled to 42 °C and inoculated with a culture made of 3 lactic acid bacteria 2/3 Lb. bulgaricus and S. thermophilus and 1/3 Lb. plantarum A6. The different preparations were incubated at 42 °C for 5 h and then cooled to 4 °C in a refrigerator. Animal experimentation

Hammer grinding, (500µm mesh) Dehulled, unblanched dried beans flour

glass vessels and stored at 4 °C in the refrigerator for analysis within a maximum of 6 h.

Sampling 5

Fig. 1  Flow diagram for the production of Bambara groundnut yoghurt

Production of Bambara groundnut milk The extraction of Bambara groundnut milk was made from flours as recently reported with some modifications [7]. 500 g of flour were dissolved in 2500 mL of distilled water and stirred using an electric homogeniser (TECHNICON stirrer motor, England) for 3 h at 130 rpm in a water batch set at 60 °C. The mixture was then filtrated with muslin cloth (double-layer) and the sediment resubmitted to a second extraction under the same conditions. The first and the second filtrate were combined and the Bambara groundnut milk so obtained was packaged in 2500  mL volumetric

Authorization for the animal experimentation was obtained from the National ethic committee, region of Ngaoundere. The animals were young male albino rats of the Wistar strain, aged between 21 and 31 days and weighing 45–75 g. The rats were placed in metabolic cages randomly parted into four groups of six rats each. The diets were formulated as shown in Table  1. The test groups were fed diets containing 10% protein while a negative control group was fed diet without protein. The three groups of test rats were different with the sources of protein which were fermented milk, unfermented milk and casein (positive control). At the end of the 28  days experiment, rats were fasted for 14  h, anaesthetized by inhalation of diethyl ether impregnated on a cotton wool, and blood was collected via cardiac puncture. The liver, kidneys, brain, testicles and heart were removed. One part of the blood was collected on EDTA tubes for hematological analysis while another part of blood was submitted to centrifugation at 3500  rpm for 15  min to obtain serum. Some biochemical and haematological parameters were determined with organ biometry. Kidneys and liver of different groups were removed and fixed in formaldehyde 10%. Physicochemical analyses In order to evaluate the effect of pre-treatment on the physicochemical properties of Bambara groundnut flour, sampling was done at the different steps of flour processing (Fig. 1): the whole seeds, the dehulled seeds and blanched seeds. All the seeds samples were converted into flour as described above. The flours, milk and yoghurt were analysed for their physicochemical properties according to standard methods. The water and ash contents were determined according to AOAC [18]. The crude protein content was determined after mineralisation using the spectrophotometric nitrogen assay [19] and the conventional protein conversion factor of 6.25. The crude lipid content was determined using the hot extraction soxhlet method with

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Table 1  Composition of the diets (100 g) used in the experiments with rats

Ingredients

Mineral mix (g) Vitamin mix (g) Cellulose (g) Tournesol oil (g) Casein (g) Bambara groundnut milk (g equivalent dry matter) Bambara yoghurt (g equivalent dry matter) Cassava starch (g) Total (%)

Diets Protein free diet

Casein

Bambara milk

Bambara yoghurt

1 4 5 10 – –

1 4 5 10 10 –

1 4 – 10 – 43.88

1 4 – 10 – –

–

–

–

38.07

80 100

70 100

41.12 100

46.93 100

hexane as solvent [20]. The total sugar content [21], the phytate content [22], the total polyphenols and the tannin (PVPP) contents [23] using Folin–Ciocalteu reagent were equally determined.

Organ to body weight ratio determination

Determination of protein digestibility

Biochemical evaluation

The biological value of the milk and yoghurt was determined as recently reported [7]. During animal experiment, the individual rat body weight, feed intake and feed waste were measured every 2  days, and used in calculating day weight gain or loss and protein intake. The indices of protein digestibility were determined following formula given below as recently reported [7]: Protein Efficiency Ratio (PER), Net Protein efficiency Ratio (NPER), true (TD) and apparent (AD) digestibility.

The serum samples of rats were used to quantify some biochemical parameters as reported recently [7]. Creatinine, alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) were assessed using kits (RANDOX kits). Total cholesterol (TC), triglycerides (TG) and high density lipoprotein cholesterol (HDL-C) were evaluated using enzymatic kits (HUMAN kits). Low density lipoprotein cholesterol (LDL-C) was then calculated according to Friedewal et al.’s [24] formula.

PER =

Gain in body weight (g) Protein consumed (g)

NPER =

Gain in weight (g) + Loss in body weight of protein free diet Protein consumed (g)

TD =

AD =

Ni − (NF1 − NF2) × 100 Ni Ni − NF1 × 100 Ni

In the equations, Ni is the Nitrogen intake of animals fed the test diet; NF1 is the Nitrogen excreted in feces of animals fed the test diet; NF2 is the Nitrogen excreted in feces of animals fed the protein-free diet.

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The organs liver, kidneys and heart of each rat sample were weighted (balance precision 0.01 g) and the ratio of organ to body weight calculated.

Hematological analyses Hematological analyses were performed on blood samples using an automated hematology analyzer (Humacount; Human, Weisbaden, Germany). The recorded parameters were white blood cells, lymphocytes, granulocytes, red blood cells, hemoglobin, hematocrit and platelets. Histopathological examination Histopathological examinations were done on kidneys and livers samples. In the procedure, organs fixed in formaldehyde (10%) were subsequently dehydrated in upgraded concentrations of ethanol (10–90%), cleaned in xylene, impregnated and embedded in paraffin. Sections of 5  µm were cut using a microtome, stained with hematoxylin and

Production, nutritional and biological value of bambara groundnut (Vigna subterranea)…

eosin stains. Light microscopic examination of multiple tissue sections from each organ in all groups was performed and image representatives of the typical histological profile were examined. Statistical analyses The results obtained were expressed as mean ± SD. The analysis of variance (ANOVA) and LSD multiple comparison test (Least significant difference) for significant differences were carried out using Statgraphics 5.0 software. Statistical significance was defined at p < 0.05.

Results and discussion Change in nutrient and antinutrient contents during processing of Bambara flour The chemical composition of Bambara groundnut at different stages of flour production is presented in Table  2. The dry seeds and Bambara flour were very low in moisture which not only contributes to secure the grain during storage but also concentrates the other biomolecules. In this respect Bambara groundnuts are good source of proteins and ash (thus minerals) with level increasing during flour production from 19.7  g/100  g  dw and 3.9  g/100  g  dw in the dry seeds to 25.6 g/100 g dw and 4.8 g/100 g dw in the flour, respectively. In reverse the lipids and sugars content diminished from 6.5  g/100  g  dw to 5.10  g/100  g  dw and 62.0–60.1 g/100 g dw, respectively. On a comparative basis, the proximate composition of whole Bambara groundnut seeds fall within the range reported by other authors [13, 16, 25, 26]. These data showed that soaking/dehulling of the seeds led to 14% increase in proteins and 13% increase in ash, while blanching dehulled seeds and milling into flour induced 14 and 8% increase in protein and ash, for respectively overall increase of 29 and 22%. The overall Table 2  Some nutrient and antinutrient contents of Bambara groundnut at different steps of flour production

decreases of crude lipids and total sugars were 22 and 3% respectively. The step which shown a large decrease in lipid content was soaking/dehulling with 14%, while blanching induced 8% decrease. The variation in seeds composition during flour production suggests the prominent role of seed coat. Kaptso et  al. [16] reported that Bambara groundnut seed coat represents 14–16 g/100 g weight of the dry seeds. Since these constituents are differently distributed in the coat and the cotyledon, it is likely that the dehulling during flour production induced change in their level. The legume seeds coats are generally poor in proteins and sugars, and their removal was expected to increase their respective level in the flour. In this respect, Olaleye et al. [27] reported increase in protein (18.4–22.9 g/100 g) and total carbohydrate contents (60.8–61.9  g/100  g) following dehulling of Bambara groundnut. In addition, Mang et  al. [5] reported increase in total protein and sugars after dehulling of mucuna flour. Worth to mention are the opposite actions of blanching and soaking: blanching theoretically contributes to the leaching of soluble molecules, hence the expected reduction of protein and carbohydrate contents of the flour. In this respect Andriamamonjy [28] reported increase in protein content during blanching of Bambara groundnut, and suggested this could be a consequence of leaching of soluble components such as low molecular weight antinutrients and soluble sugars, with a subsequent concentration of proteins. In contrast while many authors report decrease in ash content with dehulling and blanching [5, 27], we observed no significant increase. The most interesting decrease during the dehulling and blanching operations was observed on phytate and polyphenols. During the flour production, the phytate and total phenol contents decreased by 73 and 59% respectively. Similar important losses in polyphenols and phytic acid during mucuna flour production were recently reported as they are generally less concentrated in the seeds cotyledon [5]. Another account for the loss of polyphenols is the leaching into water during soaking [13]. Globally our observations on the nutrient and

Properties

Whole Bambara groundnuts

Dehulled Bambara groundnuts

Dehulled-blanched Bambara groundnut flour

Moisture (g/100 g) Ash (g/100 g) Crude proteins (g/100 g) Crude lipids (g/100 g) Total carbohydrates (g/100 g) Total phenols (g/100 g) Total phytates (g/100 g) Total tannins (g/100 g)

9.7 ± 0.4b 3.9 ± 0.3a 19.7 ± 1.2a 6.5 ± 0.1c 62.0 ± 0.1a 1.00 ± 0.10a 1.18 ± 0.03a –

8.5 ± 0.4ab 4.4 ± 0.3a 22.4 ± 1.6b 5.5 ± 0.1b 62.0 ± 0.1a 0.82 ± 0.01b 0.92 ± 0.03b –

8.1 ± 0.4a 4.8 ± 0.3a 25.5 ± 2.3c 5.1 ± 0.1a 60.1 ± 0.1b 0.41 ± 0.01c 0.32 ± 0.01c –

Data are expressed as mean ± standard deviation; n = 3; Figures on the same row with different letters are significantly different at p < 0.05

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antinutrient contents meet the objective of the work, which was to keep higher the protein content and lower the antinutrient content, the latter’s incidence on nutrient depreciation having been established. Nutrient and antinutrient composition of Bambara groundnut milk and yogurt Table  3 presents the nutrient and some nutrient composition of Bambara groundnut milk and yogurt. In a comparative view point, the Bambara groundnut yogurt is higher in dry matter, ash and protein contents, but lower in total sugar, phenols and phytate contents than Bambara groundnut milk. Compared to reported data, our yogurt dry solid, 9.9%, was quite similar to 9.7 and 10.4% reported for Bambara groundnut and Soy yogurt, respectively [6]. The protein content is lower, and ash higher than reported values for soymilk, 3.62 and 0.50% respectively [29]. In addition Poulter and Caugill, [12] reported higher protein content for Bambara groundnut milk (2.04  g/100  mL) and soymilk (3.47  g/100  mL). The difference in the protein content of legume milks may depend not only to the variety, but also and to a large extend to the extraction conditions. In this respect we recently reported protein content of 14.8 g/100 mL for mucuna milk produced at optimised extraction conditions of temperature, time and solid to water ratio [30]. Investigations on optimization of the extraction conditions are needed to improve the protein content of Bambara groundnut milk. An important aspect to mention is the effect of fermentation on the reduction of phenols and phytates. The

reduction of phytates during fermentation is well established as a consequence of action of phytase produced by microorganisms from which Lactobacilli species are competent [31]. The drop in the phenolic compound content may be attributed as suggested earlier to the action of microbial polyphenol oxidase, peroxidase or laccase [32, 33]. While the phytate and polyphenol contents decreased, a 39% increase in the protein content was observed during yogurt production probably as a consequence of the increasing microorganism biomass. In addition the resulting drop in the free sugar content can be accounted for by their utilisation as carbon substrate for fermentation. In overall the Bambara groundnut milk and yogurt produced in this work as in other reported studies are poor in solid matter. Fermentation improved the protein and ash contents of the milk, as well as reduced the antinutrient phytates. Investigations are needed to improve the solid matter of the Bambara groundnut milk and yogurt. Effect of fermentation on nutritional properties of Bambara groundnut milk Table 4 shows the food intake of rats during the experimentation. The food intake and growing rate varied with the protein source, with casein based diet having the highest value (Fig.  2.). Two major factors have been attributed to food appetence of rats: presence of antinutrient factors [34] and protein essential—non essential amino acid balance [35]. We found that our milk and yoghurt samples were pour in antinutrients, thus suggesting that the low food

Table 3  Effect of fermentation on the nutrient and antinutrient content of Bambara groundnut milk Bambara sample

Moisture (g/100 g)

Ash (g/100 g)

Crude protein (g/100 g)

Total carbohydrates (g /100 g)

Total Phenol (mg /100 g)

Phytates (mg/100 g)

Bambara milk Bambara yogurt

92.1 ± 0.1b 90.1 ± 0.1a

1.5 ± 0.1a 2.4 ± 0.1b

1.8 ± 0.1a 2.6 ± 0.1b

4.2 ± 0.1b 3.1 ± 0.1a

0.42 ± 0.01a 0.02 ± 0.01b

0.29 ± 0.00a 0.03 ± 0.01b

Data are expressed as mean ± standard deviation; n = 6 ; Figures on the same row with different letters are significantly different at p < 0.05 Table 4  Effect of fermentation on the nutritional properties of Bambara groundnut milk

Biological parameters

Food intake Weight gain Food intake index Protein efficiency ratio Apparent digestibility True digestibility

Diet formulations Protein free diet

Bambara milk diet

Bambara yoghurt diet

Casein diet

20.4 ± 0.5a −22.3 ± 2.2a −1.0 ± 0.1a

27.7 ± 1.5b 19.8 ± 2.2b 1.5 ± 0.2b 3.2 ± 0.2a 71.0 ± 0.7b 91.5 ± 0.7b

29.7 ± 0.6c 25.8 ± 2.2bc 1.3 ± 0.1b 4.2 ± 0.2b 76.0 ± 0.7c 96.0 ± 0.7c

37.3 ± 0.4d 29.0 ± 2.2c 1.3 ± 0.4b 4.5 ± 0.2b 78.0 ± 0.7c 98.0 ± 0.7c

Data are expressed as mean ± standard deviation; n = 3; Figures on the same row with different letters are significantly different at p < 0.05

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Production, nutritional and biological value of bambara groundnut (Vigna subterranea)…

1.5 and 2, and high quality for PER > 2. In addition Mang et al. [7] recently reported PER range 1.5–3.8 as good for growth. The PER value reported for Bambara protein milk and yogurt are similar to that reported by Friedman [36] for soy protein (2.74), but significantly lower than that of Colza protein (4.59). In addition TD reflects ability of the protein to digestion in the intestine, with lower values indicating increased faecal nitrogen. True protein digestibility of Bambara milk and yoghurt fall within the range reported for vegetable protein (75–90%) and significantly lower than that of animal protein which are close to 98% [37].

Protein free diet Bambara milk diet Bambara yoghurt diet casein diet

40

Mean body weight of rats (g)

30 20 10 0

-10

Effect of Bambara groundnut milk and yogurt on the blood biochemical and hematological parameters

-20 -30

0

5

10

15

20

Feeding time (day)

25

30

35

Fig. 2  Body weight of rat groups fed with diets formulated with different proteins sources. Casein diet, Bambara milk diet, Bambara yoghurt diet, protein free diet

intake of test sample may be attributed to the imbalance of the amino acids of our samples. The digestibility parameters shown in Table 4 evidence the relatively high quality of Bambara groundnut milk protein as compared to casein, in particular the digestibility of Bambara groundnut yoghurt protein which was improved due to fermentation. The protein efficiency ratio (PER), apparent digestibility (AD), and the True digestibility (TD) of the protein varied respectively from 3.2, 71 and 91.5% for Bambara groundnut milk protein to 4.5, 78 and 98% for casein. Generally not only the digestibility of Bambara yoghurt protein was significantly higher than that of Bambara protein milk, it was also significantly (p < 0.05) different from that of casein. PER referred as the contribution of protein to growth is an index of digestibility directly link to the growth indicator. In this respect Friedman [36] classified proteins according to PER as low quality for PER < 1.5, intermediate quality for PER in between Table 5  Some blood biochemical parameters of rats fed according to the diet formulation

Parameters Creatinin (mg/dL) Urea (mg/dL) ALAT (UI/L) ASAT (UI/L) Cholesterol-HDL (mg/dL) Triglycerides (mg/dL) Cholesterol- LDL (mg/dL) Glycemia (g/L)

The level in some blood biochemical parameters are presented in Table  5. Generally the ALAT, ASAT, urea, glucose and creatinin concentration in blood did not changed significantly with the diet, suggesting no apparent acute toxicity of the Bambara groundnut milk and yogurt on the rats’ organs. However significant differences were observed on the lipid profile. In this respect the HDL cholesterol concentrations were lower and the LDL cholesterol were higher in the blood of rats fed protein free diet compared to those fed casein and Bambara groundnut yogurt. Generally the behavior of HDL and triglyceride profile was similar to that of protein digestibility suggesting the dependence of the lipid profile to protein quality. We can then clearly see that fermentation not only increased the HDL cholesterol, but also decreased the LDL cholesterol and triglyceride levels in rat blood. The hematological parameters also varied amongst diet groups according to their protein quality (Table  6). As recently reported [7], the reference protein (casein) diet had the highest white blood cells  (WBC), red blood cells  (RBC), lymphocytes, granulocytes, hematocrit and platelets, while the protein free diet had the lowest. Generally the hematological parameters of groups fed Bambara groundnut yogurt were not significantly different to

Protein free diet b

8.0 ± 0.5 53 ± 4b 125 ± 10a 130 ± 12a 45 ± 6a 162 ± 5a 133 ± 4a 0.86 ± 0.08a

Bambara milk diet a

6.7 ± 0.5 38 ± 4a 119 ± 12a 119 ± 12a 63 ± 7b 132 ± 8b 123 ± 6a 0.81 ± 0.08a

Bambara yoghurt diet a

6.2 ± 0.4 34 ± 4a 101 ± 15a 101 ± 12a 86 ± 6c 113 ± 8c 107 ± 5b 0.80 ± 0.08a

Casein diet 6.9 ± 0.6a 33 ± 4a 94 ± 15a 98 ± 11a 101 ± 6c 111 ± 8c 102 ± 4b 0.66 ± 0.08a

Data are expressed as mean ± standard deviation; n = 8; Figures on the same row with different letters are significantly different at p < 0.05

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Table 6  Hematological parameters of rats according to the diet formulation Parameters

Proteins free diet

Bambara milk diet

Bambara yoghurt diet

Casein diet

White blood cell count (×109/L) Red blood cells (×1012/L) Lymphocyte (%) Granulocyte (%) Hemoglobin (g/dl) Hematocrit (%) Platelets (×109/l)

5.3 ± 0.4a 4.6 ± 1.4a 68.9 ± 2.1a 11.9 ± 1.8a 9.2 ± 1.4a 23.5 ± 3.7a 451 ± 38a

6.0 ± 0.4a 4.8 ± 1.4ab 70.2 ± 2.1ab 11.9 ± 1.8a 9.6 ± 1.4a 34.7 ± 3.7ab 427 ± 38a

7.4 ± 0.4b 5.3 ± 1.4ab 70.8 ± 2.1ab 15.2 ± 1.8b 12.0 ± 1.4a 31.8 ± 3.7ab 498 ± 38a

8.1 ± 0.4b 10.0 ± 1.4b 76.0 ± 2.1b 17.0 ± 1.8b 13.0 ± 1.4a 38.9 ± 3.7b 542 ± 38a

Data are expressed as mean ± standard deviation; n = 6; Figures on the same row with different letters are significantly different at p < 0.05

that of rat fed casein based diet but significantly (p < 0.05) higher than that of the group fed Bambara groundnut milk diet. The hematopoietic system represents a significant marker of the state of human and animals. The reduction of RBC rate, hemoglobin, and hematocrit in diet without proteins would indicate the presence of anemia and could be explained by their reduction in the erythropoiesis, the reduction of hemoglobin synthesis and the hemolysis of RBC [38]. Organ to body weight ratio and organ histology Table 7 shows the organ-to-body weight ratio of rats from different diet regimes. It was observed that the rats fed free protein diet had high values while rats fed casein diet possessed low values. The rat groups fed Bambara groundnut products had organ to body weight ratio in between both free protein and casein diet regimes, with Bambara milk exhibiting higher value. The increase in organ-tobody ratio was correlated to the increase protein quality of the diet in the order free diet < Bambara milk < Bambara yogurt < casein. The hypertrophy or kidney and liver enlargement of rats fed low quality protein diet may result from the impairment of homeostasis and consequently hyperactivity of liver and kidney. Consequently the cells increase in volume in order to increase their activity and achieve their goals. Ngatchic et  al. [39] observed similar increase in organ to body ratio when casein was substituted with mucuna protein and attributed this to natural occurring toxicant present in the mucuna product. Although this might be right, the hypertrophy of rats group fed free Table 7  Effect of diet formulation on organ-to-body weight ratio of rats

protein diet reinforces rather the idea of hyperactivity than toxicity. The histological consequences of the consumption of Bambara groundnut milk and yoghurt (Fig. 3) showed several structural changes in the kidney and liver. As shown in Fig.  3a the kidneys of control group (casein diet “D”) showed normal histological structure while, when the protein free diet was given to the rat, it caused loss of kidney (A) function characterized by interstitial infiltrates, fibrosis and inflammatory hypertrophy. The findings are in keeping with Mugendi et al. [40]. Concerning the liver, Fig. 3b represents liver tissues collected from rats according to diet formulation. The histological section of liver of rats fed on protein free diet (A’) relieved liver infiltrates, vacuolar degeneration, venous congestion and necrosis of liver cells indicating a partial loss of liver function. These organ lesions are mainly due to oxidative stress resulting in excessive generation of free radicals due to protein deficiency. This consequently causes oxidative damage to biomolecules such as membrane lipids and a variety of transport proteins which results in cell death and organ-dysfunction [41].

Conclusion This study shows that the pre-treatment applied to the grains during the processing of milk reduces by more than 50% their phytate and phenolic compound contents, but increase by more than 30% the protein content, blanching significantly affecting the process when compared to

Organs

Proteins free diet

Bambara milk diet

Bambara yoghurt diet

Casein diet

Heart (g/100 g) Liver (g/100 g) Kidney (g/100 g)

0.41 ± 0.03b 4.85 ± 0.28b 0.82 ± 0.05c

0.37 ± 0.02b 4.53 ± 0.25b 0.69 ± 0.05b

0.22 ± 0.02a 3.57 ± 0.25ab 0.44 ± 0.05a

0.23 ± 0.02a 3.17 ± 0.25a 0.59 ± 0.05ab

Data are expressed as mean ± standard deviation; n = 6; Figures on the same row with different letters are significantly different at p < 0.05

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Production, nutritional and biological value of bambara groundnut (Vigna subterranea)…

Fig. 3  Section of the kidney of rats according to the diet formulation a Hematological section through kidney of rats submitted to different protein-source diet A protein free diet, B Bambara milk, C Bambara yoghurt diet, D casein diet, control showing normal group of kidney. Magnification: 400×: G glomerulus, P Podocytes, U Urinary space b

Hematological section of liver of rats submitted to different proteinsource diet D’ casein diet showing a normal liver histology, A’ protein free diet, B’ Bambara milk, C’ Bambara yoghurt. Magnification: 400×; a central vein, b hepatic cord, c liver sinusoid

dehulling. This variation is further more important when the milk produced from these flours is submitted to fermentation with a greater than 30% increase in protein content and a reduction of more than 95% of phytates and phenolic compounds content. Besides, fermentation increases the digestibility of the proteins and utilisation, conferring to Bambara protein a quality close to that of casein. Consumption of Bambara groundnut yogurt had no detrimental effect on the liver, kidney, and heart. These properties offer Bambara groundnut yogurt and to a lesser extend Bambara groundnut milk a better perspective pertaining to the role it can play in reducing protein-energy malnutrition.

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Acknowledgements  The authors greatly thank the Agence Universitaire de la Francophonie (AUF) for work supervision through the College Doctoral BIOVEG-AGRO program.

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