Nutritional, antioxidant, carbohydrate hydrolyzing enzyme inhibitory activities, and glyceamic index of wheat bread as influence by bambara groundnut substitution

The research was designed to ascertain the potential of bambara groundnut inclusion in wheat bread to improve antioxidant activity, modulate carbohydrate hydrolyzing enzyme activities, and lower glyceamic index/ load. Protein (g/100 g) (11.2—11.73) and energy value (kcal/100 g) (421.5—435.5) of the bread were significantly higher than commercial wheat flour bread (CWF—10.45; 388.7). However, developed experimental bread samples exhibited higher growth performance in rats, free radical scavenging potentials, inhibitory activities against carbohydrate hydrolyzing enzymes and low glycemic index than other bread samples. Nevertheless, experimental bread samples were rated lower compared with the controls samples as regards organoleptic properties. The study authenticates that WBO3—25% wheat, and 75% bamabara groundnut WBO3 exhibits higher potentials as regards nutritional composition, growth indices, free radical scavenging potentials, ability to modulate carbohydrate hydrolyzing enzyme and lower glycemic index/ load. Hence, WBO3 may be recommended as functional bread for hyperglycemia prevention/ management.


Background
Consumers attitude are tilted toward consumption of foods/ food products known as functional foods which are capable of providing health benefits beyond basic nutritional needs due to the presences of bioactive compounds [1]. Li et al. [2] further established that these foods possess the ability to reduce cardiovascular diseases, and maintain human wellness. Evidence has shown that there is a strong correlation between regular intakes of healthy diets and prevention of several degenerative diseases such as diabetes, hypertension etc. [3]. This has recently necessitated production of varieties of wheat-based functional foods like biscuits and bread enriched with indigenous agricultural crops to boost the functionality, nutritional content and antioxidative compounds.
Bread is a wheat-based baked product, rich in calories and widely consumed across all ages globally [4,5]. However, it is limiting in protein and micronutrients [6]. Recent times, efforts have been shifted towards production of nutritious and healthy bread by supplementing with legumes [7,8]. According to Adewale et al. [9] these plantbased food materials contain large amount of bioactive phytochemicals, protein and minerals, and possess medicinal properties. Studies have also shown that to reduce the glyceamic index of wheat-based bread it involves incorporation of dietary fiber either from the whole grains or through the inclusion of other plant-based food materials such as bambara groundnut into the formulation [10,11]. Dietary fibres helps in reduction of hyperglyceamia and exhibites good therapeutical potential as regards insulin resistance syndrome and lipid oxidation [12,13]. Hence, help in weight control and diabetic treatment [14].
Bambara groundnut (Vigna subterrenea, L.) is a known plant-based protein source, cultivated for the purpose of animal feeding [15,16], and production of various local diets, beverage ("akara, moin-moin, okpa", milk, "kunnu, tuwo"), and for management of diseases [17][18][19]. Recently, there is a shift towards production and consumption of functional foods in order to prevent oxidative stress and associated degenerative diseases like diabetes, hypertension, etc. [20]. In view of this, studies have formulated verities of functional foods from wheat enriched with local food materials [21][22][23]. However, there is scanty information on enriching wheat bread with inclusion of bambara groundnut. Bambara groundnut is included in the present study in bread production to boast the protein and bioactive compounds in experimental bread samples. Therefore, present research aimed at evaluating the potential of bambara groundnut inclusion in wheat bread to improve antioxidant activity, modulate carbohydrate hydrolyzing enzyme activities, and lower glyceamic index/ load.

Sources of food samples
Wheat, bambara groundnut and orange fruits were sourced from Kings Market, Akure, Ondo state, while Albino Wistar rats were obtained from Colony unit, Animal House, University of Ibadan, Ibadan, Nigeria.

Processing of wheat and bambara groundnut flour samples
The raw wheat and bambara groundnut were processed into flour in which the food materials were thoroughly cleaned, dried at 55 °C in an automated electrical oven (Sunshine scientific equipment, 07AFHPN3371D1ZL, New Delhi, India) for 48 h, milled using Waring Commercial Laboratory Blender (Model WF2211210; Chicago, USA) and sieved through no 200 wire mesh sieve (British standard). The flour samples were then store at room temperature (27 °C) until further use for analysis.

Preparation of aqueous extracts of wheat-bamabara groundnut bread
The bread samples flour (500 g) was extracted exhaustively via maceration for 48

Determination of amino acid composition of wheat-bambara groundnut bread samples
Amino acid profile of the bread samples was evaluated scientific method of AOAC [25]. And results were calculated in gram per 100 g of crude protein.

Determination of anti-nutritional factor of wheat-bambara groundnut bread samples
Tannin content was determined according to the method of Fagbemi et al. [28]. Flavonoid was evaluated using the method described by Boham and Kocipai [29]. Oxalate was determined as described by the modified method Adeniyi et al. [30]. Total alkaloids were evaluated using the method of Harborne [31]. Cardiac glycoside content in the sample was evaluated using Buljet's reagent as described by El-Olemy et al. [32]. The phenol in the food samples was determined as described by the method of Georgé et al. [33] with minor modifications. The amount of phenolic compounds was expressed as mg of gallic acid per g of extract (mgGAE/g). Steroid content was determined with the method described by Okeke and Elekwa [34].

Molar ratio of oxalate and phytate to minerals
The moles of oxalate/phytate and minerals were determined using the methods earlier stated by Gemede [35].

Determination of antioxidative potential of wheat-bambara groundnut bread samples
The free radicals scavenging activity of aqueous extract of the bread samples on 2, 2-Diphenyl-1-picryhydrazyl (DPPH) was determined as described by Girgih et al. [36]. The free radical scavenging activity of the bread samples against 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was determined using modified method of Re et al. [37]. The metal chelating activity of bread aqueous extract samples were determined according to the method of Girgih et al. [36]. The hydroxyl radical scavenging activity of the bread aqueous extract samples was determined as described by Girgih et al. [36]. The Ferric-reducing antioxidant power (FRAP) activity of the bread aqueous extract samples was determined using method of Mau et al. [38].

Carbohydrate hydrolyzing enzymes inhibitory ability of wheat-bambara groundnut bread samples
In vitro α -amylase and α -glucosidase inhibition assay of bread samples was determined using spectrophotometric method described by Worthington [39] and Oboh et al. [40] respectively.

Determination of glycemic index and glycemic load of flour blends:
University of Ibadan, Ibadan, Nigeria were grouped (4 rats/group), and housed individually in metabolic cages with free access to feed and water ad libitum. The rats were acclimatized under standard laboratory conditions (22 °C ± 3 °C; 12 h light and dark periods, respectively and humidity-40-45%) [42] for 7 days. After 7 days acclimatization, the experimental food samples (WBO 1 , WBO 2 and WBO 3 ), control samples (CWF and LWF) and glucose in a portion that was calculated to contain 2.0 g of available carbohydrate were dissolved in warm distilled water (40 °C, 5 mL) and administered to the rats through oral gavage. Immediately after the oral feeding, the initial blood glucose concentration of the rat was measured via the tip tail, while the subsequent readings were taken at the interval of 30 min for 120 min. using an automatic glucose analyzer ('Accu-Chek Active' Diabetes monitoring kit; Roche Diagnostic, Indianapolis, USA). The glycaemic Index (GI) (%) for each food sample was calculated as described by Wolever et al. [43]. The Glycemic Load (GL) for each of the food samples was determined as described by Salmerón et al. [44] and categorized as follows: Low-GI = < 55%, Medium-GI = 56-69%, and High-GI = > 70% [45]. Low-GL = < 10, Medium-GL = 11-19 and High-GL = > 20 [45].

Growth performance and biochemical activity in rats of wheat-bambara groundnut bread samples
Nutritional status of rats fed on Wheat-Bambara groundnut bread samples: The anthropometric measurements, i.e., weight and length, of the rats were measured at three days' interval for 28 days. Length-For-Age (Stunting) and Weight-For-Age (Underweight) of Albino Wistar rats fed on experimental bread and control samples for 28 days were measured. Biochemical activity of rats fed on wheat-bambara groundnut bread samples: On 28 day of the experimental period, all the rats were starved for about 3 h and weighed. Each rat was anaesthetized with chloroform before been euthanized. Blood was collected into Bijour bottles containing a speck of dried tetracetic ethylenediamine acid powder. The biochemical parameters were analysed using methods described by Jasper et al. [46]. After the experiment, the animals' carcass was hygienically buried below the soil level as detailed by the study ethical protocol committee. The blood sample was first centrifuged at 1,500 × g for 10 min at ambient temperature. The serum was then separated and used for liver function assessment employing measurements of the enzymes aspartate aminotransferase (AST ), alanine aminotransferase (ALT) and Alkaline Phosphate (ALP). Renal function was evaluated using serum concentrations of urea and creatinine. These tests were performed using disposable kits obtained from Labtest Diagnostica S.A. (Lagoa Santa, Minas Gerais, Brazil).

Determination of sensory attributes of wheat-bambara groundnut bread samples
Sensory attributes of developed bread samples were carried out under standard sensory conditions with respect to lighting and environmental odour using 30 semi-trained personnel. Bread samples were coded using three digits and randomly distributed to personnel for assessment based on product aroma, appearance, texture, and overall acceptability. Product ranking was done using 9-point hedonic scale ranging from 1 = dislike extremely and 9 = like extremely [47].

Statistical analysis
Data were obtained in triplicate and subjected to analysis using statistical package for social sciences (SPSS) (version 21), expressed as mean ± standard error of mean (SEM) using New Duncan Multiple Range Test (NDMRT) and Graphs were plotted using GraphPad Prism 8. Results were considered to be significant at p ≤ 0.05.

Amino acid composition of wheat-bambara groundnut bread
The amino acid profiles of wheat-bambara groundnut bread samples are presented in Table 2. Glutamic acid (18.48-31.03 mg/100 g protein) was present in abundant concentration, while tryptophan (1.2-1.29 mg/100 g protein) had the lowest concentration in the experimental bread samples, and these values were comparable to that of CWF (32.17 and 1.37 mg/100 g protein) and LWF (30.98 and 1.78 mg/100 g protein). Likewise, a large increase in the content of lysine was observed, which certainly affects the color of the bread and the level of the non-enzymatic browning reaction compounds.

In vitro antioxidant activities of wheat-bambara groundnut bread samples
The in vitro antioxidant activities of wheat-bambara groundnut bread samples are shown in Table 4

Glycaemic index and glycaemic load of wheat-bambara groundnut bread samples
The glycaemic index (GI) and glycaemic load (GL) of the experimental bread samples are presented in Fig. 2. Glycaemic index of the formulated bread samples varied from 35.0 in WBO 3 to 55% in WBO 1, and the values were significantly (p < 0.05) lower than in the control samples, that is, CWF (60.8%) and LWF (57.9%), respectively. For the GL, the values varied from 17% in WBO 3 to 27% in WBO 1 , and were significantly lower than that of CWF (28.8%) and LWF (28.3%), respectively.

Growth performance and biochemical activities of rats fed on wheat-bambara groundnut bread samples
Growth performance: The effect of formulated bread samples on the growth performance of rats is presented in Fig. 3. The rats fed on WBO 3 sample had highest growth performance when compared with the rats fed on WBO 2 , WBO 1 and control samples (CWF and LWF). Biochemical properties: The effects of formulated bread samples on the biochemical activities of the liver and kidney with reference to aspartate transaminase (AST), alanine

Sensory attributes of wheat-bambara groundnut bread samples
The sensory attributes of bread formulated from wheatbambara groundnut flour blends and control samples are presented in Table 6. The results of aroma, appearance and taste ranged from 4.35-7.8, 4.3-8.0 and 2.95-8.1, respectively, while that of texture and overall acceptability were 2.65-7.9 and 3.9-8.0, respectively.

Discussion
The moisture content of bread produced in this present study was in line with the wheat-leafy vegetable-based bread [23], wheat-Bambara Groundnut-based bread [7] and wheat-bambara groundnut-yellow cassava-based bread [48]. Food products with high moisture content tends to have rapid reproduction rate of spoilage microorganism, hence reduced storage life. Experimental bread moisture content in this study indicates that the food products can only be stored for a short time.
The ash content indicates that WBO 3 might have higher mineral content when compare to other experimental bread samples including controls (CWF, 0.85 g/100 g; LWF, 1.03 g/100 g). The ash contents of wheat-bambara groundnut-orange peel-based bread in this study agreed with values reported for wheat-legume branbased bread (3.2 -5.4 g/100 g) [21]; but, higher than what reported for wheat-bambara groundnut-based bread (1.71-2.44 g/100 g) [7] and that of wheat-leafy vegetablebased bread (1.1 -2.4 g/100 g) [23]. The variation in ash contents of these products mighty be attributed to differences in food materials, climatic condition and processing techniques.
The protein content and energy value of the bread samples were observed to be increasing as the level of bambara groundnut added increased. This finding could be as a result of inclusion effect between protein, fat and carbohydrate content of the wheat and Bambara groundnut. For instance, study has shown that bambara groundnut contains between 18-24% of protein, while    [7] and that of Odunlade et al. [23]. The high protein and energy of the bread samples in this study mighty be useful to prevent protein-energy malnutrition in all age groups, particularly among low-income people who cannot afford expensive quality diets [49]. The findings observed in minerals contents of experimental bread samples may be associated with inclusion effects of mineral composition of the blended raw materials, i.e., wheat and bambara groundnut [50]. One major Fig. 3 Growth performance of rats fed on wheat-bambara groundnut breads for 28 days. [75% wheat, 25% and bambara groundnut (WBO 1 ); 50% wheat, and 50% bambara groundnut (WBO 2 ); 25% wheat, and 75% bambara groundnut (WBO 3 ); Commercial wheat flour (CWF) and Laboratory wheat flour (LWF)] Table 5 Biochemical parameters of wheat-bambara groundnut bread samples Means (± SD) with different alphabetical superscripts in the same row are significantly different at P < 0.05 [75% wheat, and 25% bambara groundnut (WBO 1 ); 50% wheat, and 50% bambara groundnut (WBO 2 ); 25% wheat, and 75% bambara groundnut (WBO 3 ); Commercial wheat flour (CWF) and Laboratory wheat flour (LWF)] Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Alkaline Phosphate (ALP) *R: Giannini et al. [89] and Diana [90] Samples nutrients required for growth in children and healthy well-being in adults are minerals. Hence, these formulated bread samples in this study are rich in minerals that could promote such good health for both children and adults [51]. The mineral ratios are nutritional indices, which provides information on the interrelationships of dietary minerals regarding their status and disease states [27]. Na:K ratio of the experimental bread samples was less than recommended value (< 1) [27]. Nutritionally, it could be deduced that the bread samples contained higher amount of potassium relatively to low sodium, hence, regular consumption of these bread samples may be suitable for management of hypertension [52,53]. Ca:P of experimental bread were less than recommended values (< 0.5 poor; > 1 good) [52]. This indicates that the formulated bread samples were low in phosphorous relatively to calcium. Higher calcium-phosphorous content is recommended in diets for effective calcium metabolism and utilization in the body [54], likewise, diet containing protein and phosphorus may enhance calcium wastage via urine [55]. The low Ca/P obtained in experimental bread samples has implications nutritionally, as regards the growing-aged infants and adults, in need of high intake of calcium and phosphorus for strong bone and teeth formation and prevention of osteoporosis. Hence, consumer of experimental bread samples may need to source for calcium supplements to avoid deformation of bones.
The glutamic acid concentration in the bread samples was observed to be statistically (P < 0.05) higher compared with other amino acids evaluated, and this observation is similar to Ndungu et al. [56] and Wiedemair et al. [57], reports that glutamic acid is abundant in plant-based food products. The aromatic, hydrophobic, branched chain, essential amino acids and Arginine/lysine ratios of the formulated bread samples ranged from 9.32-10.31, 39.09-42.93, 14.79-15.77, 31.11-40.53 mg/100 g protein and 0.95-1.46, respectively, and these values were statistically (P < 0.05) higher in the formulated bread samples compared with control samples, except for Arginine/lysine ratio. Constant consumption of these amino acids may prevent chronic diseases like type-2 diabetes and high blood pressure [58,59]. For instance, arginine intakes help in secretion of nitric oxide, which relax arteries for easy transfer of blood, hence prevent the occurrence of hypertension [58]. Comparatively, total essential amino acids in experimental bread samples were higher compared with wheat-based bread enriched with Oyster Mushroom reported by Ndungu et al. [56]. This may be associated to high essential amino acids in legume (bambara groundnut) than Oyster Mushroom.
The antinutrients in experimental bread samples were either lower or higher than in control samples. However, the contents of antinutrients in experimental bread samples are within the tolerable levels. Antinutrients are generally toxic at high concentration, but have some health benefits at lower concentration. For instance, phytate has ability to chelate with divalent cations resulting into formation of insoluble complexes with proteins at elevated pH [60,61], thereby impair digestibility and bioavailability of these vital nutrients in humans and/or animals. It is well established that some of these antinutrients have antioxidant activities against free radicals' formation in humans [62]; they help scavenge free radicals and prevent oxidative stress hence, preventing chronic diseases like cancer, hypertension and diabetes [63].
The phytate and divalent minerals molar ratios (an index of mineral bioavailability) of the formulated bread samples were calculated.
The phyatate/ mineral molar ratios of the experimental bread samples were significantly lower than LWF, but higher than in CWF, except for Phytate/Calcium and Phytate/iron molar ratios. However, these values were comparably lower than the critical molar ratio values (Phyt/Ca, < 0.24; Phyt/Zn, < 10; Phyt/Fe, > 0.15; Phytate*Ca/ Zn, 0.5 and Oxalate/Calcium, < 1), indicating high absorption and bioavailability of calcium, zinc and iron in the formulated bread samples [64][65][66][67]. The Oxalate/Calcium molar ratio was less than one in this study; this is of significance since the amount of oxalate in the bread sample may not interfere with the absorption of calcium. Oxalate in foods may possess negative effects on human health, by reducing the rate of calcium metabolism as well as facilitating kidney stones occurrence [64]. Antioxidants, are chemical substance that acts as oxidation reaction inhibitors and as such prevents the production of free radicals, and plays major roles in preventing oxidative stress and associated chronic diseases like hypertension, diabetes, obesity and cancers [68,69]. The antioxidant activity of these formulated bread samples indicates that regular intakes of the food products could inhibit free radicals' formation and thereby preventing oxidative stress occurrence as well as hypertension, diabetes etc.
The enzyme inhibitory activities of the formulated bread samples were observed to be increasing with inclusion of bambara groundnut, which may be attributed to the additive effects of bioactive components in Bambara groundnut, wheat and orange peel. Comparatively, the enzyme inhibitory power of experimental bread samples was statistically (p < 0.05) higher compared with CWF (50.7% and 51.7%) and LWF (71.5% and 49.9%). The difference between the enzyme inhibitory activities of the formulated bread samples and the control could be due to inhibitory activities of bambara groundnut flour included in the production of experimental bread samples which is absent in control samples. The carbohydrate hydrolyzing enzyme inhibitory activities of the formulated bread samples may be of health benefits, particularly to control overweight/obesity and high blood glucose in diabetes. It is evident that α-amylase-glucosidase enzyme inhibition from hydrolyzing carbohydrate are the main strategy to prevent obesity and diabetes [70]. α-amylase is the enzyme that hydrolyse α-(1,4)-D-glycosidic linkages of starch to disaccharides and oligosaccharides, while α-glucosidase hydrolyse oligosaccharides to monosaccharides (glucose) [71]. Inhibition of these enzymes usually leads to prevention of starch from breakdown and thereby lowering blood glucose levels [70,71].
In this study, it was recorded that the GI of experimental bread samples reduce as the proportion of bambara groundnut inclusion increase. This finding may be associated to difference in carbohydrate properties of the blending food materials. It is well established that mixing two or more carbohydrates such as cereals (wheat) of higher GI value and legumes (bambara groundnut) of lower GI value may resulted into modification of the physical property and chemical composition of the resulting foods [72]. Hence, reducing the GI of the food blends, which may be of nutritional benefits to disorders like diabetes. The GI values of experimental bread samples in this study were lower than what obtained for whole grain based multigrain Indian bread samples (Rotis) (63.2-66.2%) reported by Nagaraju et al. [73], and also, lower than recommended value for low GI foods (< 55%). This indicates that the formulated bread samples, particularly WBO 3 , may be recommended as functional bread for the prevention of weight gain and hyperglycaemia in diabetes. Foods with GI values in the range of < 55, 55-69, and > 70 are classified as low, medium, and high GI foods, respectively [73]. Low GI and GL foods play vital roles in the management of diabetes [74] and overweight/obese [75].
The high growth performance of rats that were fed on WBO 3 could be attributed to additive effects of protein, essential amino acids and energy value of the bread products. It is well established that a food that deficient in quality protein, energy value and micronutrient may lead to development of malnutrition, hence, hindering growth and development in humans or animals. Epidemiological studies have implicated deficient of protein and energy in foods as a major contributory factor to high prevalence of protein-energy malnutrition in developing countries [76][77][78]. Nutritionally, the growth performance of rats fed on the formulated bread samples, particularly WBO 3 , is an indication that the bread products are of nutritional quality, and that regular intakes of the bread products could prevent protein related diseases in children and adults.
The values of bilirubin, creatinine, urea and urea/creatinine enzymes in this study are either lower or within the normal recommended values. Evidences have shown that values above the normal reference of ALP, AST, ALT, urea and creatinine indicate haemoglobin, liver and kidney breakdown and dysfunction, respectively [79][80][81]. It is well established increased serum AST and ALT are associated with internal tissue damaged. Hence, AST/ALT ratio (> 1.5) are termed injured viral hepatitis [82]. Creatinine are kidney waste products in which elevated serum concentration is associated with poor kidney efficiency. The low concentration of these enzymes in experimental animal fed on the form with experimental bread samples implies safety for consumption.
The scoring of sensory attributes of experimental bread samples with respects to assessed parameters decrease from WBO 1 to WBO 3, and this observation could be associated to inclusion of bambara groundnut in bread samples. Sensory attributes of experimental bread samples were statically (p < 0.05) rated lower compared to LWF and CWF, respectively. This finding could be attributed to the familiarity of the panelists to the commercial bread (i.e., wheat-based bread), bread composition or processing techniques. Rating WBO 1 by the panelists above other formulated bread samples in sensory attributes indicates that inclusion of 25% Bambara groundnut in wheat bread samples could be acceptable. This finding agreed with the report of wheat-based biscuit and bread enriched with Bambara groundnut-orange peel and waxy rice-wheat respectively [83,84]. However, the texture of the experimental bread samples was poorly rated lower compared to

Conclusion
The study developed and evaluated nutritional quality, antioxidant, carbohydrate enzyme inhibitory activity and glycaemic index of bread products from wheat, and bambara groundnut flour blends. The finding established that the formulated bread samples, particularly WBO 3 (75% wheat, and 25% bambara groundnut) exhibited high nutritional quality, free radical scavenging potentials, carbohydrate enzyme inhibitory activity and low glyceamic index. Hence, this bread sample may be suitable to prevent hyperglycaemia and oxidative stress. However, there is a need for clinical study to substantiate the therapeutical potentials of the bread products.
Ethical approval and consent to participate Ethical Committee of School of Agriculture and Agricultural Technology, Federal University of Technology Akure, Nigeria approved the study protocol with approval number FUTA/SAAT/2019/011.

Consent for publication Not Applicable.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.