Nutritional, organoleptic, and physical properties of biscuits made with cassava flour: effects of eggs substitution with kidney bean milk (Phaseolus vulgaris L.)

ABSTRACT Common bean forms a significant part of the diet in Africa and hence plays a critical role in human nutrition. In order to promote it, this study was designed to investigate the effects of fully substituting eggs with bean milk on the physical, nutritional and organoleptic properties of biscuits made with cassava flour. Replacement of egg by bean milk increased the biscuits’ fat, carbohydrates, crude protein, and energy content. On the other hand, there were no significant differences in mineral contents between the cassava biscuits with eggs which served as the control and cassava biscuits with bean milk following substitution by bean milk. There was no significant difference (p < .05) in the Saponin and Phytate contents regarding anti-nutrients contents between bean milk and cassava bean milk biscuits. In contrast, Tannin contents were significantly higher in biscuits than in bean milk. Biscuit made with eggs, was rated as “very good,” while the test biscuits were rated as “good.” Substitution of egg by bean milk in cassava biscuits increased the biscuits’ protein, the fat, and carbohydrates contents with an appreciable taste. These biscuits made with bean milk can be used as a food supplement to help fight protein malnutrition in vulnerable groups.


Introduction
Common bean forms a significant part of the diet in Africa and hence plays a critical role in human nutrition, providing as much as 45% or more of the total proteins consumed. It provides a critical source of protein to the diet, complementing staple carbohydrate sources . [1] In addition to being an essential source of protein and carbohydrates, it also supplies essential vitamins and micronutrients such as Zn and Fe, an integral component to the health and wellbeing of women and children . [2] The vitamins and minerals in the crop lower cholesterol levels and have preventive and curative faculties to terminal diseases such as cancer . [3] In Cameroon, peanuts (Arachis hypogea), common beans (Phaseolus vulgaris L.), cowpea (Vigna unguiculata), soybean (Glycine max), and voandzou (Voandziea subterranea) are the main cultivated legumes . [4] Common bean (Phaseolus vulgaris L.) is arguably the most important legume for human consumption, [1] particularly for smallholder farmers across tropical regions. Cameroon is ranked the seventh in haricot bean production in Africa and first in Central Africa, with production estimated at 402,054 metric tons .According to FAO statistics, common bean is the second most frequently cultivated crop after maize in Cameroon . [5] Most farmers who grow common beans dry them in the sun to conserve them and increase their shelf-life, making them almost permanently available in than those of wheat gluten (only ∼5 m 2 of oil covered/g of isolate and 9 min of emulsifying stability, and foaming activity and stability indexes of 29% and 40%). The emulsifying stability and other interfacial properties of beans can also be evaluated as ingredients for biscuit-based egg substitution.
The biscuit-based cassava flour (TMS I070593) and milk bean combination can add vitamin A which contributes to better vision, build the immune system, and improve reproduction. However, so far, minimal research has been done on the use of bean milk as an egg replacer in the production of biscuits.This study was designed to investigate the effects of fully substituting eggs with bean milk on the physical, nutritional and organoleptic properties of biscuits made with cassava flour. It also aimed to provide a biscuit high in proteins, iron, calcium, and Vitamin A to vulnerable populations at an affordable cost.

Materials and methods
Common beans (Phaseolus vulgaris L.) MAC 33, a biofortified (high in iron and zinc) red bean variety, were obtained from the Institute of Agricultural Research for Development (IRAD), Foumbot in the West Region. The cassava roots of cultivar TMS I070593 were purchased at the International Institute of Tropical Agriculture (IITA), Nkolbisson. The other ingredients, such as baking powder, eggs, salt, sugar, butter, and vanilla essence, were procured from the local markets in Yaoundé, Cameroon.

Preparation of bean milk
The common bean milk preparation process was modified from the method previously described by Calvince et al.,, [7] as shown in Figure 1. Briefly, common bean (1000 g) was thoroughly cleaned, rinsed, and soaked in 3000 ml of tap water for 18 hours at room temperature (27°C). The soaked common bean seed was drained, rinsed, dehulled by hands, and ground in a commercial blender. Water was added to make a common bean slurry in a ratio of 1:3 on a weight basis. The resulting slurry was passed through 2 layers of muslin cloth to filter the water-soluble common bean milk material from other insoluble matter. The strained milk was heated in a heavy bottom pan to 100°C, and this temperature was held for 20 min, stirring frequently to prevent sticking. The heated bean milk was placed at room temperature (≈ 25°C) and left to cool for 6 hours, and after that, one part was stored until further use at 4°-C. A hundred grams (100 g) of cooled bean milk were distributed on five (5) petri dishes, and the filled petri dishes were introduced into an oven (Panasonic MOV-212) set at 50°C and dehydrated for 20-25 min. This dried bean milk was stored at 20°C for chemical analysis.

Production of cassava flour
Edible cassava flour (TMS I070593) was prepared from the raw roots by washing, peeling, and slicing into 0.5 cm chips, followed by oven drying (55°C during 48 hours) and powdering. The flour was sieved (30-mesh size) to obtain a fine grade sample. The flour sample was kept in sealed plastics and stored at 4°C for future use.

Production process of biscuits
Two types of biscuits were made. These were biscuits made with 100 % cassava with eggs which served as the control (Control), and cassava biscuits with bean milk (CBMB), with measurements presented in Table 1. The biscuits were produced following the method described by . [22] For biscuit production, butter was put in a clean bowl and creamed thoroughly with a stainless-steel spatula till it became soft and smooth. Powdered sugar was added and creamed to give a homogenous mixture. Cassava flour was mixed together with baking powder and salt. This flour-baking powder-salt mixture was added to the butter-sugar mixture and homogenized with the spatula to form a paste. Egg or bean milk and liquid flavor were then added, and the mixture stirred to obtain the final biscuit dough. The dough was then put into the biscuit mold, and the desired biscuit shape formed on a tray lightly greased with butter. The filled trays were introduced into an oven (Panasonic MOV-212) set at 160 °C and baked for 20-25 min. The light brown baked biscuits were removed and put in a large tray to cool at room temperature before packaging. They were packaged in high-density polyethylene, labeled, and stored at ambient temperature for various analyses. Schematic presentation of the bean milk production process adopted from [10] .

Nutritional analysis
Chemical analysis was done on bean-derived products and biscuit samples to measure moisture content and total fat using standard Association of Official Analytical Chemists methods . [23] The total nitrogen was determined by the Kjeldahl method using 6.25 as the nitrogen conversion factor to total protein. The total carbohydrates were determined by the Anthrone method . [24] The mineral content (Ca, Mg, Fe, Zn) was determined by atomic absorption spectrophotometry (Varian Vista, Victoria, Australia). The caloric value was determined by multiplying the protein and carbohydrate values by a factor of four and lipid values by a factor of nine described by Paul and Southgate . [25]

Anti-nutritional factors analysis
Phytate content was determined by titration with iron III solutions after acid digestion [26] . Saponin content was determined by weight difference after extraction in solvent . [27] Tannin content was evaluated following the method described by Ndhlala et al., . [28]

Physical characteristics of biscuits
The color value of different cookies was estimated using a Hunter's Lab color analyzer (Hunter lab scan XE, Reston, VA, USA). In the Hunter colorimeter, the color of a sample is designated by the three dimensions, L *, a *, and b *. This color was measured by placing the aperture of the equipment on the sample with white paper as the reference. L*, a*, and b* indicates lightness, redness (+)/greenness (−), yellowish (+)/blueness (−) of the sample, respectively. The color of the samples was measured after placing the samples in front of the tiniest opening . [29] In order to obtain data reflecting the color of samples, different points were taken into consideration for each sample. All data were collected with three replications.

Sensory analysis
The sensory test was carried out in the Food and Technology Laboratory of IRAD. A total of ten trained (10) panelists (6 women and 4 men) were invited to perform the sensory analysis. The bean biscuits were coded and presented to the panelists. The parameters were the intensity of aroma, basic taste (sweet, salty, acidic, bitter), texture (granular, melty, greasy, crumbly, crunchy), bean flavor, and overall taste quality on 6-point hedonic scales. Parameters were scored using a scale from 0 (absence) to 5 (very pronounced) for basic taste and texture and the overall acceptability from 0 (very disagreeable) to 5 (very pleasant) as given in Table 5.
Drinking water was prepared for panelists, and they were asked to rinse their mouths after tasting each biscuit sample. The sensory analysis was performed three times.

Cost evaluation of cassava-based biscuits
Cost evaluation of bean milk, control sample, and cassava biscuit fortified with bean milk was done based on direct costs (DC) (using the prices of the ingredients used for the food process of each of the products) and indirect costs (including Electricity, Miscellaneous fees (2.5 % DC) and packaging materials).

Statistical analysis
All data were subjected to one-way analysis of variance (ANOVA) on the triplicate data using XLSTAT version 2020.1.2 with a significance level of 5 %. Graphs and Radar charts were generated in Excel 2018 software (Office 365, Microsoft Corp.) from the color and sensory analysis results to determine differences in L*, a*, and b* color variables and attributes between the formulated biscuits.

Nutritional analysis
The proximate composition of bean milk and cassava-based biscuits fortified with bean milk is shown in Table 2. There was a significant difference (p < .05) in the crude fat, carbohydrates, crude protein, and energy content of the biscuits. Vitamin A is a fat-soluble vitamin essential in the growth, development of epithelial tissue, reproduction, and functioning of the visual cycle . [30] Carotenoids are important micronutrients for human health. They function as a precursor of vitamin A. For the total carotenoid content, there was no significant difference (p < .05) between cassava -egg biscuits (1.5 ± 0.05) µg/ g and cassava -bean milk biscuits (1.7 ± 0.08) µg/g. The carotenoid contents of the formulated biscuits were lower than values for biscuits prepared from pro-vitamin reported by Sogo et al., [31] but higher than values in yellow staining roots of cassava recorded by Carvalho et al., . [32] The present study values are within the carotenoid content range (1.47 to 19.18 μg/g) of fresh weight reported by Da Silva et al., . [33] The moisture content plays a vital role in determining the shelf life of the product. The water content of bean milk was higher (6.16 ± 0.55) % than cassava-based biscuits fortified with bean milk (3.62 ± 0.12) % and control biscuits (2.92 ± 0.01) %. The low water content may increase the quality and stability of biscuits fortified with bean milk, as previously reported for other foods . [33] The crude fat content of the bean milk was (2.10 ± 0.06) %, which was within the range of 1.8 to 2.7% previously reported for some common beans . [7] The fat content of CBBM was significantly higher (25.70 ± 0.91) % than that of CEB (19.10 ± 0.56) %; this shows that the substitution of eggs with bean milk significantly changed the lipid content of those biscuits.
Carbohydrates were significantly higher (p < .05) in cassava-based biscuits fortified with bean milk (47.08 ± 0.06) % than that of bean milk and control biscuits (36.25 ± 0.96) %. This was expected as the bean is richer in carbohydrates than eggs. The protein content increased as eggs were replaced by bean milk. This could be attributed to higher protein content in bean milk relative to hens' eggs. Other authors observed similar results [34] ; with red kidney bean flour. The results suggest that the substitution of eggs by bean milk in whole cassava biscuits may be useful as a food supplement for alleviating protein malnutrition in vulnerable groups. Energy content is a function of the total protein, fat, and carbohydrates present in the food. In this study, sample CBMB with the highest fat, carbohydrates, and protein value also recorded the highest energy value (438.3 ± 4.12) kcal/g while the least energy value was for CEB (324.1 ± 3.50) kcal/g. The energy content of the CBMB samples was within the range (397-457) kcal/g reported by Abiodun and Ehimen . [35] Table 3 shows the variation in mineral content of cassava-based biscuits and bean milk. There were significant differences (p < .05) in the calcium, magnesium, zinc, and iron contents in bean milk compared with cassava-based biscuits CEB (control) and CBMB. This finding agrees with several studies, [36] which reported that kidney beans are rich in minerals essential for human development and growth. On the other hand, there were no significant differences in mineral contents between the control biscuits and cassava-based biscuits fortified with bean milk (CBMB). Calcium concentration was increased from bean milk (33.00 ± 1.61) mg/100 g to CBMB (54.00 ± 2.60) mg/100 g while magnesium, zinc and iron contents were decreased (from 146.00 ± 7.00; 3.72 ± 0.51; 12.82 ± 1.51) mg/100 g to (59.00 ± 0.19; 0.97 ± 0.09; 1.300 ± 0.01) mg/100 g respectively. Magnesium is the most abundant mineral in bean milk (146.00 ± 7.00) g/100 g, followed by calcium, iron, and zinc being the least. This result corroborates with that found for other common beans by . [7] CBM biscuits contained the highest concentration of Ca (54.00 ± 2.6) mg/100 g. Calcium, the most abundant mineral in the body, makes up much of the structure of bones and teeth. The calcium recorded in biscuits produced in this study was far higher than values recorded for biscuits fortified with safou . [22] This result was in the range of calcium values (41.5 ± 0.9 to 62.3 ± 4.1) mg/100 g in dry beans obtained by . [7] The amounts of calcium contained in CBMB are lower than the average daily intake of calcium from foods and beverages (from 842 to 1,083 mg) independently of age and sex . [37] However, they contribute to calcium intake, even though they contain small amounts of calcium because people consume them frequently. The observed higher magnesium in cassava bean milk biscuits agrees with the reports from other researchers . [35,38] It is worth noting that magnesium is a cofactor in many enzyme systems that regulate diverse biochemical reactions in the body . [39] Biscuits with a high iron content can be used as an alternative snack to overcome iron deficiency or anemia experienced mostly by children, the elderly, pregnant women, and nursing mothers . [40] Iron concentrations in bean milk (12.82 ± 1.51) mg/100 g were comparable to those reported for different common beans in the range of 6.0 mg/100 g to 23.8 mg/100 g . [7,32] The amounts of iron contained in CBMB were higher than the range (31.7 to 36.7 ppm) recorded for other biscuits . [22,40] The required daily intake (RDI) is age-and sex-dependent except for infants younger than 6 months whose recommended intake is 0.27 mg/day; the recommended intake is generally between 7 and 18 mg/day . [37] Zinc is found in cells and is needed during pregnancy, infancy, and childhood. The body needs zinc to grow and develop properly . [41,42] The RDI is also age-and sex-dependent, 2-8 mg/day for infants and children and 11-13 mg/day for adolescents and adults . [37] Although there are low levels of zinc compared to other minerals in the biscuits, consuming at least 100 g of cassava-based biscuits per day would be a good source of zinc.  Table 4 shows the composition of anti-nutrients in bean milk and cassava-based biscuits fortified with bean milk. Legumes and some tubers contain some natural toxicants. Some of these substances can interfere with the absorption of proteins, carbohydrates, and certain minerals . [43,44] Generally, there was no significant difference (p < .05) in the Saponin and Phytate contents between bean milk and cassava -bean milk biscuits, meaning that the only source of these elements in biscuits is bean milk. Furthermore, the phytate contents (3.25 to 3.22 %) were lower than previously reported values for common beans by . [45] Tannin contents were significantly higher in biscuits than in bean milk, which is likely related to the additional source from cassava. The tannin contents of bean milk (0.048 %) and CBMB (0.092 %) products were very low when compared with the usual tannin content of cassava products (0.4-0.5 %) . [46] Regarding the bean milk, its tannin contents is far lower than previously reported values for bean milk . [10] The products could also be considered safe regarding tannin poisoning since the levels found in this study are below the critical value of 0.7-0.9 % . [47]

Physical analysis of cassava-based biscuits
Color is an important sensory attribute of any food because it influences acceptability. From Figure 2, the a* values of the cookie bars were not different between formulas. There was an increase of the brightness (L) from 41 to 45 and a decrease in yellowish (b*) from 26 to 11 as the eggs were replaced by bean milk in biscuits. This signifies that the color of biscuits becomes a light golden color with bean milk, most likely because of the naturally milky pigmentation of bean milk. In addition, the luminosity (L*) was influenced by non-enzymatic browning reactions  that occurred during baking. When heated, the Maillard reaction occurs between reducing sugars and free amino acids (especially lysine) and peptides . [21] Since CBMB had higher protein levels than control biscuits, this CEB was also expected to have more non-enzymatic browning, thus lowering luminosity values. However, the results obtained in the present work do not agree with those of, [48] who reported that pasta with more bean flour was darker.

Sensory evaluation of biscuits
The sensory evaluation of the biscuits was presented in Figure 3. Biscuit made with eggs (CEB), which served as the control, had the highest rating for crunchy (2.8), crumbly (2.5), and overall acceptability (4.1) compared to Cassava -Bean Milk Biscuits which recorded the highest score for sweet (3.7), crispy (4.2), milky (4.0) and bean flavor (3.0). However, according to the panelists, there was no significant difference in the variation of color between them. The higher rating observed for the control sample may be because panel members are familiar with bakery products made with eggs, which could have influenced their rating for the control sample. The added bean milk changed the sensory properties of the biscuit, as evident in the rating recorded by the panelists. Panelists revealed that they detected the bean aroma in the final biscuits. The overall acceptability obtained for the CEB was 4.15, while CBMB obtained 3.2. Crispy is the noise and strength when the sample of biscuit breaks or cracks when chewed on the first and second . [49] The results showed that supplementation with bean milk increased crispiness. Therefore, the substitution of egg with bean milk affected the crispiness of the biscuits. These results are not in line with those of Fieben et al.,, [50] who reported that the crispiness of the biscuits decreased as haricot bean flour was added. This difference could be explained by the fact that we used bean milk instead of flour in this current study. Acidity and bitterness were not felt, recording a score of zero in both the control and CBM biscuits.

Production cost analysis
The production cost of control and cassava-based biscuits is shown in Table 6. This table shows that replacing eggs with bean milk in the cassava biscuits can decrease production by 28.6 % per biscuit produced (Cost of CEB = 13.14 FCFA/Biscuit while Cost of CMBM = 9.38 FCFA/Biscuit). Thus, substituting of eggs by bean milk is most economically achievable for the maximum quantity of biscuits (Table 6).

Conclusion
This study was designed to investigate the effects of fully substituting eggs with bean milk on the physical, nutritional and organoleptic properties of biscuits made with cassava flour. It was found CBMB is affordable and sustainable for different entrepreneurs, making it easy to engage private sector partners to produce these nutritious products for different target groups. Good option to reduce micronutrient deficiency in the country. Is it a great market opportunity to meet the demands of vegans by providing them with an alternative to powdered cow milk. Our study provides valuable information about product attributes of processed CBMB likely to influence consumers and processors producing and buying intentions. Further studies can be undertaken for rheology and in vivo assessment purposes

Acknowledgments
The authors acknowledge the Institute of Agricultural Research for Development (IRAD) Nkolbisson, Yaoundé-Cameroon, for providing the facilities to carry out this research work and the Pan African Bean Research Alliance for funding (PABRA) 2 through the Alliance of Biodiversity International and the International Center for Tropical Agriculture for financial support from Global Affairs Canada. Bill and Melinda Gates Foundation funded the publication of this work under the Tropical Legume III (TLIII) and Accelerated varietal improvement and seed delivery of legumes and cereals in Africa (AVISA) projects.

Disclosure statement
No potential conflict of interest was reported by the author(s).