Antioxidative properties and sensory evaluation of white bread containing pre-treated cowpea (Vigna unguiculata L) flour

Cowpea (Vigna unguiculata L.) has limited characteristics since it has a high content of antinutritional properties. The pretreatment process by a combination of germination and roasting of cowpeas, called pretreated cowpea was done to prepare the flour made from cowpeas. The purpose of the study was to investigate how the pretreatment of cowpea flour affected the antioxidant content and other characteristics of white bread. This study used 0-10.4% (w/w) of the flour as a wheat flour substitute for making white bread. The dough was baked at 175°C for 40 minutes. The results showed that substituting pre-treated cowpea flour affected the bread colour, volume expansion, total phenolic content as well as antioxidant capacity. Pre-treated cowpea flour enhanced the total phenolic compound, antioxidant activity, moisture content, and darker colour, whereas the expansion rate was reduced. The best formula was found by using 5.2% of pretreated cowpea flour. It has good characteristics at colour properties, volume expansion by 12.14%; moisture content was 27.62%, total phenol was 86.24 mg GAE/100 g, antioxidant activity was 29.67 %RSA, and sensory score was 3.75 (preferred). Therefore, the pre-treated cowpea flour can be utilized as a natural antioxidant source in white bread recipes.


Introduction
White bread consumption grew gradually between 2014 and 2017, going from 0.062 packs/cap/year to 0.37 packs/cap/year [1].The primary ingredient in white bread is wheat flour.National wheat flour consumption in 2019 reached 8 million metric tons (mt).Indonesians use wheat flour as a necessary food component to make a variety of culinary products.To promote the consumption of locally produced goods, agricultural product diversification is crucial.Cowpeas (Vigna unguiculata L) are a type of legume which is a source of vegetable protein.These beans contain the second highest protein after soybeans [2].These legumes contain polyphenols, peptides, dietary fibre, resistant starch, vitamins, and minerals [3]. 100 g of cowpeas consist of 24.4 g of protein, 56.6 g of carbs, 1.9 g of fat, 481 mg of calcium, 399 mg of phosphorus, and 2.68 g of phytic acid.Compared to other legumes, cowpeas are a type of multifunctional legume that have several benefits, including low fat (1% db), high protein content (23-32% db), feasible mineral and folic acid sources, and a high concentration of nutraceuticals such dietary fibre and antioxidants [4].Based on research [5], the phenolic content of cowpea flour ranges from 34.6 to 376.6 mg/100 g of flour..The addition of cowpeas to composite flour with sorghum and cassava can be used to enhance the nutritional aspects of bread products [6].In IOP Publishing doi:10.1088/1755-1315/1338/1/012035 2 some studies, cowpea flour has been used to replace 20% of the flour in crackers when preparing various food products, which produces products that are acceptable to panellists [7].Other research has also been carried out regarding adding 25% cowpea flour, which can increase the rising power of French bread [8].Adding 50% cowpea flour produced biscuits with the best characteristics [9], and high levels of antioxidant compounds in cereals [10].
However, cowpeas also have weaknesses, such as containing several anti-nutritional substances, namely trypsin inhibitors (13.70 mg/gram), phytic acid (12.80 mg/gram), and tannins (9.70 mg/gram) [2].The unpleasant smell causes the product to become less accepted by the consumer.The higher the phytic acid can lower the amount of iron absorption.The phytic acid compounds can inhibit the bioavailability of iron in food because it binds with minerals and protein [11].The pretreatment of the legume was required to minimize the anti-nutrient compounds and increase nutrient digestibility in cowpeas.The quality of the bread can be impacted by the yellow peas (Pisum sativum) germination and toasting.Toasted peas can increase dough stability and the technological value of pea flour [12].Germinating cowpeas can increase protein and starch digestibility, increasing the concentration of niacin, thiamin, and riboflavin.Soaking followed by germinating cowpeas can increase water content, crude protein, fibre, ascorbic acid, and ash [13].In addition, mineral concentrations of sodium, potassium, copper, calcium, zinc, manganese, magnesium and iron also increased.[14].Therefore, pretreatment of cowpeas before making flour can improve their functional characteristics.
In this research, the preliminary treatment of cowpeas was germination followed by roasting before making flour.Once obtained, the flour is used to make bread as an alternative to wheat flour.The intended outcome of this procedure was to improve the produced bread's properties and antioxidant activity.The objective of this research was to investigate the effect of pretreatment cowpea flour on the antioxidant content and white bread characteristics.

Materials
Cowpea from the local market in Sleman, Yogyakarta.The materials for making bread were wheat flour, yeast, skim milk, bread improver, salt, sugar, margarine, and egg were also obtained from the local market in Yogyakarta.The chemical reagents were obtained from Merck (Germany).

Methods
2.2.1.Making the flour from cowpeas A combination of germination and toasting pretreated the cowpeas.These pretreatments refer to [15].Three litres of water were used to soak one kilogram of cowpea seeds at a 1:3 (w/v) ratio for a whole day.The seeds are then rinsed thoroughly using running water and drained for 5 minutes.Cowpea sprouts that have been dried using a cabinet dryer for 8 hours at a temperature of 50ºC, then spread evenly on a tray and roasted at a temperature of 140ºC for 40 minutes with even stirring, cooled, ground using, sifted to a size of 80 mesh, packed in high-density polyethylene bags, and stored in the refrigerator at 4ºC until use.

Bread formulation
Making plain bread containing cowpea flour refers to [16] with slight modifications.The addition of cowpea flour is 0, 5.2, 7.8, and 10.4% (w/w).The formula is shown in Table 1.The dough was baked at 175°C for 40 minutes.The bread samples were then tested for colour characteristics, swelling volume, water content, total phenolic, antioxidants, and sensory activity.The color of the loaf was expressed using the L*, a*, and b* color scales and quantified using a colorimeter.Three readings of the data were obtained.b.Moisture content The bread samples' moisture content was determined using the method of [17].c.Phenolic total The bread samples' determined phenolic content corresponds to [18] d.Antioxidant activity To analyze the antioxidant activity in the bread samples, the DPPH radical scavenging activity method was used [19] e. Expansion volume The expansion volume of the bread was measured according to [20] f.Hedonic test The color, flavor, texture, smell, and general acceptance of the bread samples were assessed using sensory evaluation; the methodology was modified from [21].After being cut into uniformly sized pieces, the samples were shown beside the water.On a 5-point rating system, panelists rated the bread sample as follows: 1 for really disliked, 2 for disliked, 3 for liked, 4 for preferred, and 5 for extremely liked.

Statistical analysis
The samples have undergone triplicate analysis.Using SPSS version 22, an analysis of variance (ANOVA) was used on the color determination (p<0.05), and statistical significance was considered.On the other hand, Microsoft Excel 2019 was used to do regression analysis on the remaining data.

Colour characteristics
The addition of pretreated cowpea flour provides significant alteration in colour of L, a, b, C, and H (Table 2).Using 5.2% of this flour can provide a significant difference in each colour parameter (p<0.05).The white bread samples' L (lightness), b, C, and H values can be decreased by increasing the amount of cowpea flour used.This result showed that adding cowpea flour has a darker colour effect on the resulting white bread.The characteristics of cowpea flour itself are also darker in color when compared to wheat flour.These color characteristics are influenced by preliminary treatment, namely germination and roasting of cowpeas.The roasting process in cowpeas can increase the Maillard reaction, which results in browning.The same results were also presented by [12], that adding roasted pea flour can reduce the L* value in a bread crust.In a* value parameter, which shows a reddish color, the addition of cowpea flour provides a notable rise in a* value in contrast to the control group (p<0.05).Additionally, b, C, and H significantly decreased once the flour was added.The values b, C, and H indicate the bluish color, color intensity, and hue, respectively.This result aligns with the L value parameter, which decreases as the number of cowpeas increases.The darker color of the bread, along with the increase in the number of cowpeas, led to the formation of this trend.The addition of ingredients other than wheat flour in making bread changes the color of the product.For example, [16] added purple yam flour to white bread, resulting in a color change pattern.

Water content
Table 3 demonstrates that the addition of cowpea flour up to 7.8% (w/w) did not significantly change the parameters determining the moisture content.Adding the flour by 20% can increase the water content in bread significantly (p<0.05).This outcome demonstrated that the flour's increased viscoelasticity can enhance the dough's rheological qualities [22].

Expansion Volume
The ratio of the bread's volume difference before and after baking can be used to define the bread expansion volume.The capacity of the dough to generate and hold on to gasses created during fermentation allows the bread to rise.In wheat flour, the gluten component affects how much bread expands in volume.The bread's hollow interior shape causes the gluten to coagulate during baking, firming it and preventing the bread's volume from dropping [16].The impact of pretreatment cowpea flour on the bread sample's expansion volume can be seen in Figure 1.

Figure 1. The impact of pretreatment cowpea flour on the bread sample's expansion volume
This flour's addition is also known to lessen the bread's volume growth.The decrease in bread expansion volume was 0.178% for each percentage of cowpea flour addition (R 2 = 0.9402).Adding 10.4% cowpea flour caused the smallest sample volume expansion of 10%.The decrease in bread development volume aligns with the reduced amount of gluten formed.The higher the portion of cowpea flour added, the amount of gluten also decreases, this may have an impact on the capacity of the dough to hold produced throughout the process for making bread.The amount of bread development decreased due to the of purple yam flour [16].

Antioxidant activity and phenolic total
A graph of the increase in antioxidant activity in white bread is presented in Figure 2, and Figure 3 shows the impact of pre-treatment cowpea flour on the bread's phenolic total.It is known that as the addition of cowpea flour increases, it has a positive influence on the increase in antioxidant activity in white bread samples (p<0.05).In bread samples used as a control, antioxidant activity was 15%, then increased to 35% when 20% cowpea flour was added.The rate of increase in antioxidant activity reaches 1.9% for every 1% increase in flour.This result was supported by the potential of cowpeas as carriers of natural antioxidants.Numerous beneficial substances, including epicatechin (0.48-8.67 mg/100 g), gallic acid (45.4 -93.4 mg/100 g), ferulic acid (11.1 -32.07 mg/100 g), catechin (2.07 -6.48 mg/100 g), and chlorogenic acid (0.59 -3.08 mg/100 g) are reported to be present in cowpeas [22].Another study discovered that cheese bread containing cowpeas had higher levels of condensed tannins, flavonoids, and total phenolic content.These results also align with the increase in antioxidant activity, which reached 497.5 and 731 µmol TEAC/100 gr using the DPPH and ABTS free radical scavenging methods [23]  In addition, the components of the Maillard process in the crust increase the activity of bread.The antioxidant qualities of bread products can be impacted by phenolic compounds found in dietary components and intermediate intermediates resulting from the heat breakdown of phenolic compounds, such as polyphenol-protein/starch compound complexes [23].In line with the results of antioxidant activity analysis, total phenolic in bread samples also increased along with adding cowpea flour through the equation y = 5.3588x + 61.029 (R 2 = 0.9342).The increase in total phenolic reached 5.36% for every 1% addition of this flour.This result is also supported by the potential of cowpeas as carriers of bioactive compounds, which contribute to a significant increase in phenolic total in bread samples (p<0.05).

Sensory evaluation
Samples of white bread were analyzed visually based on colour, flavor, texture, aroma, and acceptability as a whole.Ratings range from 1 to 5, with the options being highly hated, disliked, liked, preferred, and very liked.Table 2 displays the findings of the study of the sensory evaluation.The addition of cowpeas to white bread had a substantial effect on every measurement that was studied.(p<0.05).Participants expressed a decreasing preference for the bread sample's colour with increasing cowpea additions.The panellists' opinion of acceptability is impacted by the white bread's darker colour due to the addition of cowpea flour.The bread samples with 10.4% and the control samples showed significantly different panellist preferences for sample colour (p<0.05).The colour was more advantageous in the control sample, namely 4.25, whereas when adding 10.4% cowpea flour, it became 2.50.This result was in line with the previous results of testing colour characteristics in Table 1 explaining that the more cowpeas added, the brightness of the sample decreases, and the intensity of the dark colour becomes higher.This result showed that panellists prefer bread products that have brighter colours.Table 4 shows the sensory evaluation result Results for the parameters of taste, texture, and odor were similar.The amount of prepared cowpea flour added to the white bread samples results in a decrease in their sensory value.But based on the overall results, adding 5.2% the flour gave the best treatment because there was not a significant distinction between it and the control group (p>0.05).Overall, bread-making effectiveness using 5.2% cowpea flour was successful without losing sensory quality.The Maillard reaction causes the characteristic darker colour of flour.The effects resulting from the Maillard reaction also influence the aroma and flavor, limiting the amount of flour used.

4.
Conclusion According to the study, using pre-treated cowpea flour instead of regular flour had an impact on the bread's color, antioxidant activity, volume expansion, and total phenolic compound.Pre-treated cowpea flour the amount of antioxidant activity and total of phenolic compound, moisture content, and darker color, whereas the expansion rate was reduced.The best formula was found by using 5.2% of pretreated cowpea flour.It has good characteristics at color properties, volume expansion by 12.14%; moisture content was 27.62%, total phenol was 86.24 mg GAE/100 g, antioxidant activity was 29.67 %RSA, and sensory score was 3.75 (preferred).Therefore, the pretreated cowpea flour can be utilized as a natural antioxidant source in white bread recipes.

Figure 2 .Figure 3 .
Figure 2. The impact of pretreatment cowpea flour on the bread sample's antioxidant activity

Table 1 .
The bread formula containing pretreated cowpea flour

Table 2 .
The color properties of the bread examples with different amounts of pretreatment cowpea

Table 3 .
The water content of the bread samples containing various pretreated cowpea flour content