The Effect of Red Bean Juice and Butterfly Pea Extract on Flavonoid Content, Antioxidant Activity, and Sensory Quality of Stirred Yoghurt

This study aimed to determine the effect of adding red bean juice and butterfly pea extract to stirred yogurt on antioxidant activity, total flavonoids, and sensory properties. Preliminary research on total plate count was conducted, followed by antioxidant activity, total flavonoids, and sensory properties. Data analysis used one-way ANOVA, Tukey HSD, and the Pearson correlation test. The addition of 10% butterfly pea extract (A3), 10% red bean juice and 10% butterfly pea extract (A4) significantly increased the antioxidant activity. All treatments had a significant effect on the increase of total flavonoids, but significantly decreased the preference of colour. The addition of 10% butterfly pea extract (A3), 10% red bean juice and 10% butterfly pea extract (A4) significantly decreased overall preference. The addition of 10% red bean juice and 10% butterfly pea extract (A4) significantly decreased the preference of consistency as compared to the control.


Introduction
A free radical is reactive with the surrounding cells as they do not have an electron pair.Electrons that were taken from the body cells will turn into free radicals, resulting in oxidative stress.Humans produce antioxidants, which take part in blocking free radicals by donating electrons, [1].Nevertheless, whenever the number of free radicals increased by exposing continually in the long term; antioxidants will no longer be sufficient, hence, need more antioxidants from external sources to protect from free radical attack.Red beans (Phaseolus vulgaris L.) and butterfly pea (Clitoria ternatea) contain natural antioxidants and could increase antioxidant activity.These two ingredients are rich in antioxidants, could be combined with other products, such as yogurt, a popular processed dairy product that is favorite by Indonesians.This combination aims to maximize its utilization and give added value to the yogurt product by improving the functionality such as antioxidant activities, total flavonoids, and sensory properties.
Taken together, the extract of butterfly pea contains anthocyanin which is sensitive to pH.Stirred Yogurt has a low pH, so even though the antioxidant activities increased, the anthocyanin pigments in yogurt turned into purplish blue colour, and less acceptance to the panelist.Furthermore, the addition of red bean juice is expected to increase the antioxidant activities and total flavonoids of stirred yogurt, but the beany flavor may affect the sensory properties.Therefore, the purpose of this research is to formulate 1324 (2024) 012116 IOP Publishing doi:10.1088/1755-1315/1324/1/012116 2 stirred yogurt with red bean and butterfly pea extracts and to assess their effect on antioxidant activity, total flavonoids, and sensory properties of stirred yogurt, supporting sustainable development goals, especially good health, and well-being.In this study, quantitative methods were used in analyzing antioxidant activity and total flavonoids, as well as the home-run test method for sensory evaluation.Panelists were asked to fill out an online sensory survey via Google Form.

Research methodology
The flowchart of the research process carried out in the study is shown in Figure 1.

Figure 1. Research Flow Chart
There were 4 group of stirred yogurt formulations: 100% stirred yogurt control (A1), 10% addition of red bean juice (A2), 10% addition of butterfly pea extract without red bean juice (A3), and 10% addition of red bean juice and 10% butterfly pea extract (A4).The process of making stirred yogurt products started by preparing red bean juice and butterfly pea extract using the aquadest maceration method, [2], then producing of stirred yogurt formulation accordingly, followed by incubation at 37°C for 12 hours, [3].
Preliminary study was conducted to enumerate the viability of lactic acid bacteria by serial dilution and plate counting on Man, Rogosa and Sharpe Agar (MRS) (Merck, Darmstadt, Germany) and expressed in CFU/g, [4].All formulated stirred yogurt were assessed for antioxidant activity by using the DPPH method, [5] and expressed by the IC50 value obtained from the linear regression equation between inhibition percentage at five different concentrations in each stirred yogurt sample.The IC50 value is a number that indicates the concentration of the extract which enables it to effectively inhibit DPPH (free radical) activity by 50%, [6].
Total flavonoids were determined by the UV-Vis spectrophotometry method.This analysis consists of two stages; the first step was preparing a standard curve solution of quercetin so that a standard curve is obtained.After measuring the absorbance from the newly made sample solution, the second step was to plot the absorbances into the linear equation of the quercetin standard curve in mgQE/g, [7].The third step was to do the sensory evaluation using the home-run test method, with a 5-point hedonic scale from 100 untrained panelists at the Jakarta-Tangerang location, representing the degree of liking that was categorized into 5 points, starting from point 1 = 'disliked very much', point 2 = 'disliked', point 3 = 'neutral', point 4 = 'liked', and point 5 = 'liked very much', [14].The sensory attributes were evaluated for taste, aroma, texture, colour, and overall acceptance of stirred yogurt.The panelists tested the sample and filled out the prepared Google form and then statistical analysis was conducted using one-way analysis of variance (ANOVA) in the 25 th version of the IBM SPSS Statistics application (Armonk, New York), followed by a post-hoc test for significant findings with the Tukey HSD test, [8].

Total viable counts of lactic acid bacteria in stirred yogurt
Enumeration of total viable lactic acid bacteria in yogurt products is important to meet the requirements of yogurt quality based on Standard National Indonesia.SNI and in line with the regulation of Badan Pengawas Obat dan Makanan Number 1 of 2022 especially for claims on processed food labels and advertisements [9].The results of enumerating viable lactic acid bacteria showed the addition of 10% red bean juice and 10% butterfly pea extract did not inhibit the growth of yogurt starter cultures and met the criteria for the viable counts for starter culture, [10,11].The results are shown in Table 1.  1 shows that total viable counts of lactic acid bacteria in four stirred yogurt formulations were in a range of 8.06 ± 0.043 to 8.67 ± 0.104 and meet the SNI standard quality requirements for total lactic acid bacteria which is higher than 10 7 CFU/mL or 7 log CFU/mL.

Antioxidant activity and total flavonoid of stirred yogurt
The Antioxidant activity were expressed in IC50 value.It was obtained from the linear regression equation between the inhibition percentages at five different concentrations in each stirred yoghurt sample.The IC50 value is a number that indicates the concentration of the extract that can effectively inhibit DPPH (free radical) activity by 50%, [7].The IC50 value of all stirred yogurt formulations are shown in Figure 2. Figure 2 shows that stirred yogurt control (A1) had the highest IC50 value, followed by stirred yogurt with the addition of 10% red bean juice (A2), the addition of 10% butterfly pea extract (A3), and the lowest IC50 value was shown in the addition of 10% red bean and 10% butterfly pea extract (A4).The IC50 value is inversely proportional to the antioxidant activity; the lower the IC50 value, the higher the antioxidant activity, [12].Hence, addition of red bean juice and butterfly pea extract significantly increased antioxidant activity and the highest among other formulations of stirred yogurt.This result was expressed using One-Way ANOVA analysis that showed that the addition of red bean juice and butterfly pea extract significantly reduced the IC50 value (P = 0.002).Tukey's HSD analysis revealed that the addition of 10% butterfly pea extract (A3) (P = 0.006) as well as the addition of 10% red bean juice and 10% butterfly pea extract (A4) (P= 0.001) significantly increased antioxidant activity significantly compared to the control stirred yogurt (A1).This significant increase could be due to the content of various bioactive compounds in red bean juice and butterfly pea extract which play role in increasing antioxidant activity.
Total flavonoids were assessed by UV-Vis spectrophotometry method.Figure 3 shows that the lowest total flavonoids found in stirred yogurt control (A1), followed by the addition of 10% red bean juice (A2), 10% butterfly pea extract (A3), and the highest was stirred yogurt with the addition of 10% red bean juice and 10% butterfly pea extract (A4).The addition of red bean juice and butterfly pea extract significantly increased total flavonoids (P= 0.000), Tukey HSD analysis demonstrated significant increase of total flavonoids in the formulated stirred yogurt with addition of 10% butterfly pea extract (A3) (P = 0.008), 10% red bean juice and 10% butterfly pea extract (A4) (P = 0.000) significantly increased the total flavonoids compared to the stirred yogurt control (A1), hence, flavonoids present in red bean and butterfly pea extracts.2007) reported that kidney beans contain a total of 3.39 mg CE/g of flavonoids.Butterfly pea also contain flavonoids of 20.07 mmol/mg, it contains flavonols, which are compounds that belong to the flavonoid group, in the form of kaempferol, quercetin, and myricetin glycosides, [15].Correlation between total flavonoids and antioxidant activity was also analyzed (Figure 4), using Pearson correlation test ( = 0.05) and revealed that the total flavonoids had negative correlation with the IC50 value, with Pearson correlation coefficient value of -0.95**, a very strong correlation.Total flavonoids and IC50 values showed a significant correlation (P = 0.000).

Sensory evaluation of stirred yogurt formulations
A total of 100 untrained panels was involved in sensory evaluation of stirred yogurt formulation.The control stirred yoghurt sample had the most preferred for all attributes, such as colour, aroma, texture, taste, and overall acceptance.followed by stirred yogurt with the addition of 10% red bean juice (A2) and stirred yogurt with the addition of 10% butterfly pea extract (A3).The stirred yogurt with the addition of 10% red bean juice and 10% butterfly pea extract (A4) was the least preferred.

3.3.1.
Colour preference of stirred yogurt formulations The most preferred was control stirred yogurt (A1) in a range of 'liked' to 'liked very much' category, followed by formulated with 10% red bean juice (A2) in the neutral category, then 10% butterfly pea extract (A3), and the lowest value was the addition of 10% red bean juice and 10% butterfly pea extract (A4), where both formulations were in a range of 'neutral' to 'liked' category (Figure 5).

Figure 5. Colour Preference of Stirred Yogurt Formulations
One-Way ANOVA revealed significant decreased in preference of colour of formulated stirred yogurt in the presence of as compared to the stirred yogurt control (P = 0.000), hence, followed by the Tukey HSD post-hoc statistical analysis.The addition of 10% red bean juice (A2), 10% butterfly pea extract (A3), and the addition of both red bean juice and butterfly pea extract at 10% each (A4) in stirred yogurt significantly decreased colour preference of formulated stirred yogurt as compared to the control (P = 0.01).The colour of the stirred yogurt on the addition of 10% red bean juice (A2) became cloudy, the colour on the addition of 10% butterfly pea extract (A3) became bluish white, the addition of 10% red bean juice and 10% butterfly pea extract (A4) was also slightly cloudier compared to the control.The bluish colour could be due to anthocyanin content, which is sensitive to low pH because of fermentation so that it turned to bluish purple, [13].The cloudy colour of stirred yogurt was caused by the red colour from anthocyanin content in red beans.

3.3.2.
Aroma preference of stirred yogurt formulations Figure 6 shows no significant difference (P = 0.361) in aroma preference of stirred yogurt formulations.All treatments are in range of 'neutral' to 'liked'.Consistency preference of stirred yogurt formulations Figure 7 shows that the control (A1) has the highest preference of consistency, followed by the addition of 10% red bean juice (A2).Both are in 'liked' to 'liked very much' category.No significant difference among control (A1), the addition of 10% red bean juice (A2), and addition of 10% butterfly pea extract (A3).The addition of addition of 10% red bean juice and 10% butterfly pea extract (A4) significantly lowering the preference of consistency (P = 0.035) as compared to other formulated stirred yogurt, in a range of 'neutral' to 'liked' category.

Figure 7. Consistency Preference of Stirred Yogurt Formulations
The Preference of consistency for stirred yogurt with the addition of 10% red bean juice and 10% butterfly pea extract (A4) showed slightly more condensed and could be due to higher viable lactic acid bacteria, as compared to the control-stirred yogurt with smoother texture, The viability of lactic acid bacteria tended to be higher as compared to control as shown in Table 1, 8.67 ± 0.104 log CFU/mL, and 8.38 ± 0.039 log CFU/mL, respectively.

Taste preference of stirred yogurt formulations
Figure 8 shows no significant difference (P = 0.141) among all stirred yogurt formulations.The control (A1) and the addition of 10% red bean juice (A2) are in 'liked' to 'liked very much' category.The addition of 10% butterfly pea extract (A3) and the addition of 10% red bean juice and 10% butterfly pea extract (A4) are in 'neutral' to 'liked' category.

Overall preference of stirred yogurt formulations
Figure 9 shows the highest average score for control stirred yogurt (A1), in a range of 'liked' to 'liked very much', followed by the addition of 10% red bean juice (A2), in a range of 'liked' to 'liked very much', the addition of 10% butterfly pea extract (A3) in a range of 'neutral' to 'liked', and the lowest score was the addition of 10% red bean juice and 10% butterfly pea extract (A4) in a range of the 'neutral' to 'liked'.

Figure 9. Overall Preference of Stirred Yogurt Formulations
The stirred yogurt formulation with the addition of 10% butterfly pea extract (A3) and 10% red bean juice, and 10% butterfly pea extract (A4) significantly less preferred for the overall preference, compared to control (A1), (P =0.049), and (P = 0.03), respectively, from 'liked' to 'liked very much' into 'neutral' to 'liked'.This is due to colour turned into bluish white in the stirred yogurt added with the butterfly pea extract and red bean juice (A3) and slightly cloudy in 10% red bean juice and 10% butterfly pea extract (A4).Moreover, the consistency of stirred yogurt with the addition of 10% red bean juice and 10% butterfly pea extract (A4) was thicker compared to the control (A1) which related to relatively higher amount of viable lactic acid bacteria (Table 1).

Conclusion and further research
Stirred yogurt with the addition of 10% butterfly pea extract (A3), 10% red bean juice and 10% butterfly pea extract (A4) significantly increased total flavonoids and antioxidant activity compared to control stirred yogurt (A1).The addition of 10% red bean juice (A2) significantly increased the total flavonoids but significantly decreased the level of colour preference compared to the control.The addition of 10% red bean juice and 10% butterfly pea extract (A4) showed a significant decreased in consistency preference and the overall acceptance compared to control (A1).The addition of 10% butterfly pea extract (A3) significantly decreased the overall acceptance.
The best formulation is the addition of 10% red bean juice and 10% butterfly pea extract (A4), both for antioxidant activity, expressed as IC50 and total flavonoids, at 7.87 ± 0.751 mg/mL, and 9.13 ± 0.044 mgQE/g, respectively.However, it is less acceptable from sensory properties, so it has the potential to be reformulated by adding bright coloured fruit extracts to improve colour preference, addition of CMC stabilizer to produce homogeneous yogurt product and improve its consistency.

Figure 4 .
Figure 4. Correlation between Total Flavonoid and IC50 Value of Stirred Yogurt

Figure 8 .
Figure 8. Taste Preference of Stirred Yogurt Formulations

Table 1 .
Total viable lactic acid bacteria in stirred yogurt