Nutritional Profile, Antioxidant Activities and Organoleptic Properties of Tempeh Fermented with Additional Butterfly Pea Flower Petals

Tempeh is a superfood rich in benefits and nutrients, and it contains several amino acids that make protein beneficial for immunity and more efficiently absorbed by the body than other proteins. Although tempeh is rich in antioxidant content, its antioxidant is weaker than vitamin C and anthocyanins. This study aimed to determine the formulation of tempeh with added butterfly pea that consumers most favour, has the best nutritional content, and has the highest antioxidant activity. The variation of butterfly pea concentration added was 0%, 0,7%, 1,4%, and 2%. The results showed that 0,7% concentration of butterfly pea was the most preferred formulation with an overall score of 6.08 (somewhat like) and a moisture content of 61.88(%wb), ash content 0.04(%wb), protein content 16.40(%wb), fat content 9.66(%wb), carbohydrate content 12.11(%wb), antioxidant activity of 82.94%.


Introduction
Tempeh is a traditional Indonesian food originating from the ancestral cultural heritage of the Indonesian people which is a product of soybean fermentation by Rhizopus moulds such as Rhizopus oryzae, Rhizopus arrhizus, Rhizopus oligosprorus, Rhizopus stolonifera [1].Tempeh consumption per year can reach 7 kilograms per capita.Tempeh is a superfood that is rich in benefits and nutrients, and it contains several amino acids that makeup proteins that are beneficial for immunity and are more easily absorbed by the body compared to other proteins.The content of bioactive compounds in it, such as isoflavones, sterols, and fiber from the oligosaccharide group, can have a positive impact on health and prevent the onset of degenerative diseases.Tempeh also contains vitamin B12, probiotic bacteria, and a source of antioxidants derived from isoflavone compounds and Superoxide dismutase (SOD) [2].
Although tempeh is rich in antioxidant content, its antioxidant content is less strong or weaker when compared to vitamin C and anthocyanins [3].Based on research antioxidant activity of tempeh taken from 10 tempeh producers with the same method of making and type of soybeans, the value of antioxidant activity ranged from 52 -70% and had an average of 58%.In comparison, vitamin C has antioxidant activity with IC50 of 9.05 ppm, which is included in the powerful group [4].Anthocyanins have antioxidant activity with an IC50 of 87.86 ppm, which is classified as a strong antioxidant.Efforts that can be made to increase antioxidant activity in tempeh can be made by adding other food ingredients such as butterfly pea flower [5].Consuming tempeh butterfly pea can ensure healthy lives and promote 1324 (2024) 012118 IOP Publishing doi:10.1088/1755-1315/1324/1/012118 2 improved well-being because tempeh butterfly pea contains antioxidants that can prevent degenerative diseases [23].
The addition of vegetable materials to increase the antioxidant activity of tempeh has already done many times before, such as the addition of tempeh with red chilli powder with a concentration of 1.5% can reduce the IC50 value from 6798.53 ppm to 422 ppm [6].Adding butterfly pea flowers with a concentration of 2% can reduce the IC50 value from 5340 ppm to 2398 ppm [7].Adding angkak with a concentration of 1.5% can reduce the IC50 value from 6803.43 ppm to 4532.01 ppm [8].
The addition of butterfly pea flowers to food and beverages has been widely done.However, the addition of butterfly pea flowers in making tempeh is still rare, as evidenced by the need for more research on this matter.In previous studies, the nutritional content of butterfly pea flower tempeh, such as moisture, ash, protein, fat, and carbohydrate content, has not been tested.Nutritional content is very important to know because it determines whether the quality of tempeh has met the standards owned by the government.If it does not meet the standards, tempeh cannot be circulated or traded.In addition, in previous studies, antioxidant analysis was carried out through a drying process using a rotary evaporator, which made the antioxidant content of the sample damaged due to exposure to hot temperatures [7].
This study will analyze the nutritional content of proximate tempeh butterfly pea to determine whether the nutrition of tempeh butterfly peas has met SNI standards or not.To maximize antioxidant analysis without damaging the antioxidant, that can be done by taking butterfly pea tempeh filtrate using a blender.Tempeh butterfly pea will be blended and then filtered using filter paper, and then the filtrate will be taken for analysis.Using this method it can minimize the damage of antioxidants because it does not go through the drying or heating process at all [9].
The addition of butterfly pea flowers to tempeh also has its advantages because of its abundant production and easy to obtain [10].Therefore, further research is needed in the formulation of food products by combining tempeh with other food ingredients, such as butterfly pea flower.

Sample Preparation
Analytical balance weighs the ingredients of tempeh making.After that, wash the soybeans in running water until clean and then boil for 30 minutes.After cooking, soybeans are soaked for 24 hours to produce acidic conditions.Then, rinse the soaked soybeans with water and peel the skin.The soybeans are then irrigated again, then boiled for 40 minutes.The cooked soybeans are spread on a thin container and wait until it's cool and the water drips out for ± 40 minutes.After that, in 50 g of soybeans, add 0.1 gram tempeh yeast and mix evenly, followed by the addition of butterfly pea powder with a concentration of F1 (0%) as much as 0 gram, F2 (0.7%) as much as 0.35 gram, F3 (1.4%) as much as 0.7 gram, and F4 (2%) as much as 1 gram, then stirred until evenly distributed, then put into a Ziploc plastic and give the plastic a hole using a toothpick as air circulation, after that incubated at room temperature for 30 hour [11]

Water Content Analysis
In this research, analysis of moisture content using oven method began with put an empty aluminium cup into the oven at (100 ± 5) ℃ for 1 hour, then set it into a desiccator to cool for 20-30 minutes, and then weight the cup.Put 2 g samples into the aluminium cup and weighed.Fill the cup with the sample and put it into the oven at a temperature of (95 -100)℃ with a pressure of ≤ 100 mmHg (13.3 kPA) for 5 hours.Then, put the sample into a desiccator to cool for 20 -30 minutes and weigh.Then heat again for 1 hour.Obtain the moisture content value through the following formula [12] : Water content (% Wb) = " !"($"%) !# × 100 Description : X = Sample Weight Y = Weight of sample cup after drying A = Weight of empty cup

Ash Content Analysis
Put an empty porcelain cup into the oven at 105 ℃ for 15 minutes.After that, put the porcelain cup into a desiccator for 10 minutes until it cooled down and weigh it.Weigh Samples as much as 2 -3 g and put them into a porcelain cup whose weight was already known.Then, place the porcelain cup in a series of three-foot burners, Bunsen burners, and porcelain triangles that are ignited above the flame of the Bunsen burner until there is no more smoke.After that, the sample was put into an electric furnace at 550 ℃ for 4 -6 hours until it formed a white ash.Then, put the sample into a desiccator for 10 minutes to cool and weight it (13).Obtain the ash content value through the following formula: Ash content (% wb) =

Protein Content Analysis
Analysis of protein content using the Kjeldahl method, which began by weighing a sample of 0.5 -1 gram, then putting it into a 300 ml Kjeldahl tube and adding a mixture of selenium and concentrated H2SO4, then preheating the KjelDigester tool.Store the 300 ml Kjeldahl tube containing the sample in the KjelDigester tool, turn on the scrubber unit and the sample is deconstructed for 1 hour.After that, stop the KjelDigester tool, lift the tube rack and allow it to cool to room temperature.Then, insert the tube containing the deconstructed sample into the distillation unit tool and distilled water and 40% NaOH in excess.Carry out the addition using the distillation unit tool.Place a reservoir in the distillation unit, a 250 ml Erlenmeyer filled with 4% H3BO3.Carry out the distillation process until the volume of distillate reaches 3x the initial container.After that, titrate the distillate with 0.2 N HCl.Carry out every cycle of destruction with blank work.Obtain the value of protein content through the following formula:

Fat Content Analysis
Analysis of fat content using the gravimetric method, which begins with weighing a sample of 2 -3 grams, then adding 25% HCl, distilled water, and several boiling stones, after which the glass cup is closed and placed on a hot plate, in a hot state filter the residue using grey filter paper.Wash the residue with hot distilled water.After that, dry the residue in an oven and put it into a filter paper sleeve (hulls); put hulls into a Soxhlet device that connects to a 300 ml volumetric flask containing boiling stones that have been dried and known weight.Then hexane was added, paired with a condenser and water bath, and extracted.Distillate the hexane and dry the fat residue in an oven.After that, cool it in a desiccator to room temperature and weigh it.Drying in the oven was repeated until the weight was constant.Obtain the value of fat content through the following formula: Fat content (%) =

Carbohydrate Content Analysis
The analysis of carbohydrate content using difference obtained by subtracting the percentage of 100% from the percentage of components contained in the sample, namely water, ash, protein, and fat [14] .Obtain the value of carbohydrate content through the calculation of the following formula: Carbohydrate content (% wb) = 100% -(Water + Ash + Protein + Fat)

Antioxidant Analysis
Antioxidant analysis begins with extracting the sample by mixing raw tempeh with 95% ethanol in a ratio of 1:3 (w/v).After that blend, filter the resulting slurry using filter paper to separate the pulp and filtrate.The resulting filtrate can be stored in the freezer (-20℃) for further analysis [9].Put 1 ml of filtrate into a test tube coated with aluminium foil, then add 2 ml of 0.2 mM DPPH.They were then incubated in a dark room at room temperature for 30 minutes.After that, measure the absorbance using a spectrophotometer at a wavelength of 517 nm.Calculate the antioxidant activity value using the following formula [3] : Antioxidant activity (%) = " %78.9:;<=:>"?78.8?@A>B %78.9:;<=:> # × 100

Organoleptic
Organoleptic analysis of tempeh was conducted using a hedonic rating test.Tempeh was served fried without the addition of any seasoning.60 untrained panellists conducted hedonic testing.Panelists will be presented 5 kinds of samples and asked to rate each sample using a hedonic scale of 1 -9, namely 1 = very dislike, 2 = very dislike, 3 = dislike, 4 = somewhat dislike, 5 = neutral, 6 = somewhat like, 7 = like, 8 = very like, and 9 = very like.Parameters assessed for tempeh included liking for color, scent, taste, texture, and overall.

Data Analysis
The proximate analysis results, antioxidant activity, and organoleptic were processed statistically with IBM SPSS Statistics version 23.Carry out data processing using the analysis of variance technique or One-way ANOVA with a confidence level of 95%.If there is a significant difference in the treatment, it is continued with the Tukey further test to determine the treatment that causes the difference.

Proximate Analysis
Variations in the concentration of butterfly pea flowers added to soybeans are F1 (0%), F2 (0.7%), F3 (1.4%), and F4 (2%).The results of proximate analysis can be seen in table 1.The water content of the four treatments ranged from 59.26 -63.34%wb.The lowest percentage value of water content was in the F4 treatment, while the F1, F2, and F3 treatments were not significantly different.The ANOVA test results obtained a significant value of 0.003 (P < 0.05), meaning there is a significant difference between F1, F2, F3, and F4 treatments on the water content of soybean tempeh with the addition of butterfly pea flower powder.Based on SNI 3144: 2015, the percentage of water content of tempeh with the addition of butterfly pea flowers has met the quality requirements of SNI tempeh, which is a maximum of 65%.The decreased water content can be caused by butterfly pea flower powder because more total solids in the product will reduce the water proportion [15] .
The ash content of the four treatments ranged from 0.03 -0.04 %wb; the ANOVA test results obtained a significant value of 0.843 (P > 0.05), which means it is not significantly different, which indicates that the addition of butterfly pea flower powder does not affect the ash content of tempeh.Butterfly pea flowers have an ash content of 0.45% and have mineral content in the form of boron, calcium, cobalt, chromium, copper, iron, potassium, magnesium, manganese, molybdenum, sodium, selenium, and zinc [16].Ash content in butterfly pea flower tempeh produces an insignificant increase because butterfly pea concentration is too small to affect the ash content of the product [17] .
The protein content of the four treatments ranged from 15.60 -17.08%wb; the ANOVA test results obtained a significant value of 0.057 (P > 0.05), which means not significantly different, which indicates that the addition of butterfly pea flower powder does not affect the protein content of tempeh.Based on SNI 3144: 2015, the percentage of protein content of tempeh with the addition of butterfly pea flowers has met the quality requirements of SNI tempeh, which is at least 15%.Butterfly pea flowers have a protein content that is not too high, which is 0.32%.Protein levels have insignificant results due to the small concentration of butterfly pea flower powder and the low protein content in butterfly pea flowers [16] .
The fat content of the four treatments ranged from 9.29 -11.80 %wb.The highest percentage of fat content was in treatment F4, but F1, F2, and F3 were not significantly different.The ANOVA test results obtained a significant value of 0.007 (P < 0.05), meaning significantly different, indicating that adding butterfly pea flower powder affects the fat content of tempeh.Based on SNI 3144: 2015, the percentage of the fat content of tempeh with the addition of butterfly pea flowers has met the quality requirements of SNI tempeh, which is at least 7%.Bunga butterfly pea has a fat content of 2.5%, and its addition to tempeh should not show significant results.However, the results are significant because the butterfly pea powder has a high fat content, as proven by the butterfly pea flower powder that smells rancid [18].
The carbohydrate content of the four treatments ranged from 11.26 -12.94%wb.The lowest percentage of carbohydrate content was in the F1 treatment, while the highest was in the F4 treatment.The ANOVA test results obtained a significant value of 0.039 (P < 0.05), which means significantly different, indicating that adding butterfly pea flower powder affects the carbohydrate content of tempeh.Butterfly pea flowers have a carbohydrate content of 2.23%.The addition of butterfly pea flower powder should give insignificant results because the carbohydrate content is relatively low; the results are significant because the butterfly pea powder has carbohydrate content that makes its addition have a significant effect [16] .
It concluded whether or not the concentration of butterfly pea flowers can influence the nutritional content of tempeh increases and the type of butterfly pea flowers used.Each type of butterfly pea flower has a different nutritional content, influenced by the place of growth and temperature.In the lowlands, 6 plants tend to have a higher nutritional content when compared to the highlands because the lower the area, the temperature, the amount of CO2 concentration, and the increasing sunlight.The need for sunlight to photosynthesize in the lowlands is more fulfilled when compared to the highlands, which makes the highlands have cooler temperatures when compared to the lowlands [19].In addition, the nutritional content of butterfly pea flower powder can increase due to food additives such as dressing materials because butterfly pea flowers have anthocyanins, which are unstable dyes.If the nutritional content of butterfly pea flowers is high, adding a small amount will give significant results.However, if the nutritional content is low, more additions or higher concentrations are needed for significant results.

Antioxidant Analysis
Antioxidant activity analysis was conducted using the a,a-diphenyl-b-pricryl-hydrazyl (DPPH) radical scavenging method.DPPH is a stable free radical that can react with other radicals to form stable compounds; DPPH can also react with hydrogen atoms in antioxidants that will produce reduced DPPH.This DPPH method does not specifically test antioxidants to measure food ingredients' overall total antioxidant activity.The smaller the absorbance value produced, the higher the antioxidant activity, indicating that the DPPH solution, which acts as a free radical, can be dampened by the presence of antioxidants in the test sample [20].The results of antioxidant activity analysis can be seen in Table 2.The antioxidant activity of the four treatments ranged from 61.92 -91.54%.The lowest percentage of antioxidant activity was in the F1 treatment, while the highest was in the F4 treatment.The ANOVA test results obtained a significant value of 0.000 (P < 0.05), which means there is a significant difference in treatment (F1, F2, F3, and F4) on the antioxidant activity of soybean tempeh with the addition of butterfly pea flower powder.
The increase in antioxidant activity in tempeh is due to the increased concentration of butterfly pea flower powder in each treatment.Butterfly pea flowers have high antioxidant activity with an IC50 of 53.61 ppm but will experience a decrease after being applied to food products; this can be caused by the fermentation process, which makes changes in food properties due to the breakdown of complex compounds that turn into simpler ones [21].The more the addition of butterfly pea flowers, the higher the concentration can increase antioxidant levels in the product [7].The addition of butterfly pea flowers has a significant inhibitor [22].
The increase in antioxidants is also evidenced by changes in the color of DPPH, which was initially purple; after adding the filtrate, the color changes to bright yellow.In addition, it is also characterized by the absorbance value, which decreases over time.The increase in antioxidant activity in tempeh occurs due to the content of saponins, tannins, steroids, alkaloids, flavonoids, and quinones contained in butterfly pea flowers [7].The content of butterfly pea flower compounds acts as a donor that will release Hydrogen atoms and bind to DPPH and then form new compounds in the form of diphenyl picrylhydrazine, which are more stable and harmless [23].

Organoleptic Analysis
Tempeh with different concentrations of butterfly pea flower was tested organoleptically using the hedonic rating method with parameters including color, scent, flavour, texture, and overall.Tempeh samples will be fried and consumed without the addition of flavour.The results of tempeh before and after frying can be seen in Figure 1 and Figure 2. The results of the organoleptic analysis can be seen in Table 3.The average value of the results of organoleptic analysis of tempeh color parameters ranged from 4.43 -7.23.The most preferred color was tempeh in the F1 treatment at 7.23 (like), while the least preferred tempeh color was F4 treatment at 4.43 (somewhat dislike).The ANOVA test results obtained a significant value of 0.000 (P < 0.05), which means there is a significant difference in treatment (F1, F2, F3, and F4) on the color parameters of tempeh with the addition of butterfly pea flower powder.The addition of butterfly pea flowers reduces the level of liking for color because the higher concentration of butterfly pea flowers will make tempeh have a dark blue color.The color is obtained through the crown of the butterfly pea flower, which contains flavonoid compounds in the form of anthocyanins [7].Anthocyanins are pigment compounds that can dissolve in water and are a source of antioxidants.In addition, fried tempeh will produce a brownish color caused by the Maillard reaction.This reaction can occur because the protein contained in tempeh can react with reducing sugar groups.This brownish color is what makes the blue color of tempeh darker and almost blackish [24] .
The average value of the results of the organoleptic analysis of tempeh scent parameters ranged from 6.17 -6.93.The preferred scent is tempeh in the F1 treatment at 6.93 (somewhat like), while the least preferred tempeh scent is in the F3 treatment at 6.17 (somewhat like).The ANOVA test results obtained a significant value of 0.010 (P < 0.05), which means there is a significant difference in treatment (F1, F2, F3, and F4) on tempeh scent parameters with the addition of butterfly pea flower powder.In addition to the scent of frying results, there is also the scent of butterfly pea flowers that smells rancid, which makes the scent value decrease.Because each panellist has a different olfactory sensitivity, some panellists realize the smell of butterfly pea flowers, and some feel that there is no difference in scent at all [7].
The average value of the results of the organoleptic analysis of tempeh flavour parameters ranged from 5.23 -6.37.The preferred flavour was tempeh in treatment F1 at 6.37 (Somewhat like), while the least preferred tempeh flavour was in treatment F4 at 5.23 (neutral).The ANOVA test results obtained a significant value of 0.000 (P < 0.05), which means there is a real difference so that the addition of butterfly pea flower powder affects the taste of tempeh.The higher concentration of butterfly pea flowers makes the tempeh taste less favourable, which can be caused by the appearance of tartness caused by the content of active compounds in butterfly pea flowers [25].The tannin content in butterfly pea flowers makes it have a bitter taste, so butterfly pea flowers and their processed products are still difficult to accept by the public [26] .
The average value of the results of organoleptic analysis of tempeh texture parameters ranged from 6.00 -6.10.The preferred texture was tempeh in the F1 treatment at 6.10 (somewhat like), while the least preferred tempeh texture was in the F2 treatment at 6.00 (somewhat like).The ANOVA test results obtained a significant value of 0.988 (P > 0.05), which means there is no significant difference, so the addition of butterfly pea flower powder does not affect the texture of tempeh.The tempeh served had a crispy texture and was also soft.Each sample treatment was fried using low heat for 4 minutes.Because there is no difference in the way tempeh is fried, the resulting texture will be the same and not much different.
The average value of the results of organoleptic analysis of overall parameters tempeh ranged from 5.47 -6.68.The overall preferred tempeh is tempeh in the F1 treatment at 6.68 (Somewhat like), while the least preferred overall tempeh is in the F4 treatment at 5.47 (neutral).The ANOVA test results obtained a significant value of 0.000 (P < 0.05), which means the addition of butterfly pea flowers affects the overall acceptance of tempeh.The overall acceptance value is influenced by the color, scent, flavour, and texture attributes found in tempeh.Based on the overall value, the tempeh formulation with the addition of butterfly pea flowers most preferred by panellists is F2 because the blue color produced is still bright, the rancid odour possessed by butterfly pea flowers is still not smelled because the scent value is still the same as the control or not significantly different, the taste is not much different from tempeh in general because the value is not significantly different.

Conclusion
From the research that has been done, it can be concluded that the tempeh of butterfly pea flowers that has the best formulation is F2, namely tempeh with a concentration of 0.7% of butterfly pea flowers.Formulation panellists prefer F2 because the color produced is still bright blue; the smell of rancid and bitter taste of butterfly pea flowers still needs to be smelled and felt because the addition is in small quantities.In addition, F2 has a water content of 61.88% wb, ash content of 0.04% wb, protein content of 16.40% wb, fat content of 9.66% wb, carbohydrate content of 12.11% wb and antioxidant activity of 82.94%.Although the highest antioxidant activity and nutritional content is owned by treatment F4, F2 already has significant results in antioxidant activity compared to F1 by 61.92%, and nutritional content such as water content, ash, protein, fat, and carbohydrates are not significantly different so that the content is still the same as F1 treatment.
of the cup and sample after ignition B = Weight of empty cup C = Weight of sample before ignition of HCl required for sample titration (ml) Vb = Volume of HCl required for the blank titration (ml) N = Normality of HCl solution Fk = Protein conversion factor W = Weight of sample (g) IOP Publishing doi:10.1088/1755-1315/1324/1/0121184 of empty fat flash (g) B = Sample weight (g) C = Constant weight of fat flask + sample after heating (g)

Table 1 .
Proximate Analysis Result

Table 2 .
Antioxidant Analysis Result

Table 3 .
Organoleptic Analysis Results