Application of hesperidin crude extract from Siamese orange peel as an alternative natural antioxidant in the butter making process

Butter is a form of fat product that is quickly rancid. Reactions that cause rancidity can be prevented by adding synthetic antioxidant compounds. Behind that, the use of synthetic antioxidants triggers various side effects. Based on these problems, this study aimed to determine the effect of adding hesperidin crude extract from Siamese orange peel on the quality of the resulting butter product. In general, this research was divided into two parts: the isolation of the crude extract of hesperidin and the preparation and characterization of the final quality of butter. The crude extract of hesperidin showed positive for flavonoids and polyphenols. In general, the addition of hesperidin crude extract showed an effect by increasing the value of antioxidant activity and decreasing the peroxide value. The conclusion that can be obtained is the shelf life and the addition of hesperidin crude extract showed an effect on the final quality of butter. The best formulation in this study was the addition of 1.5% hesperidin crude extract. The formulation proved to have the highest value for antioxidant activity and the lowest value for peroxide value.


Introduction
Butter is a form of oil and fat emulsion product or water in oil commonly consumed by the public as processed food.Butter consists of milk fat, water, and salt (optional).Butter can be made by separating the liquid and solid phases of the milk fat.The solid phase will be given additional ingredients and then cured [1].Based on the regulations of SNI 3744:2018, SNI 01-3744-1995, and SNI 3741-2013, good quality butter must have a distinct buttery aroma (no rancid odour) and a pale-yellow colour.Compositionally, butter must have a maximum water content of 16%, a minimum of 80% milk fat, and a maximum of 2% non-fat milk solids.In terms of quality, butter must have a Polenske number between 1.6 -3.5, a Reichert Meissl number between 23-32, a maximum content of free fatty acids (butyric acid) of 0.5%, and a maximum peroxide value of 10 meq O2/kg.
Butter products have a long shelf life.However, butter is still susceptible to damage when exposed to air, water, and placed in less-than-ideal storage conditions.Butter spoilage is expected due to rancidity [2].Rancidity can arise due to the occurrence of oxidation reactions in unsaturated fatty acids [3].This oxidation reaction occurs through an autoxidation process through initiation, propagation, and termination stages.The initiation stage is where oxygen breaks down unsaturated fatty acids into radical fatty acids and hydrogen.In the propagation stage, oxygen reacts with fatty acid radicals and forms peroxide radicals.In the termination stage, free radicals react with active peroxide radicals to form compounds that cause rancidity, namely aldehydes and ketones [4].The method commonly used to overcome rancidity in butter is by using antioxidant compounds.Antioxidant compounds are compounds that have a phenol group.The phenol group will donate hydrogen atoms to free radicals, resulting in a slowdown in forming compounds that cause rancidity.Antioxidant compounds commonly used in the food industry are synthetic antioxidant compounds, such as propyl gallate, tertiary butylhydroquinone, and butylated hydroxyanisole [5].The advantages of synthetic antioxidants are good consistency of the final product, high efficiency, heat resistance, low cost, and easy to buy.However, the use of synthetic antioxidants in large quantities and continuously can cause side effects for the body [6].In addition, most of the synthetic antioxidants used by the food industry in Indonesia are mostly imported from abroad.
Alternative efforts that can be made to reduce the negative effects of using synthetic antioxidants is to use natural antioxidants made from local sources, such as orange fruits.Orange fruit has potential as a source of natural antioxidants because of its very abundant availability in Indonesia, which is ranked fourth in fruit with the highest productivity every year.Most of the compounds that have a role as antioxidants found in orange fruits are phenolic and flavonoid groups [7].The highest flavonoid content is found in the skin.One of the flavonoid components found in orange peel is hesperidin.The hesperidin content is higher than quercetin or naringenin.One type of orange that has the highest flavonoid content compared to all citrus variants is the Siamese orange.Addition of hesperidin component has the potential to be used as a natural antioxidant in fat products, because of their ability to donate hydrogen atoms so the fatty acid oxidation chain reaction becomes slower.[8].Based on the problems that have been described, this study aims to determine the effect of adding hesperidin crude extract from Siamese orange peel on the quality of the resulting butter product.

Hesperidin Crude Extract Isolation
The process of isolating the crude extract of hesperidin carried out using the soxhlet method.This method began with the selected of Siamese oranges (unripe oranges with a dark green to yellowish green colour, not rotten or physically deformed) [9].Siamese oranges that have been cleaned were crushed and squeezed, while the skin was dried in an oven (50-60°C, 2 hours).Dried orange peel were crushed to form a powder [10].Orange peel powder (15 g) was added to the cellulose thimble and placed in Soxhlet [11].Petroleum ether (110 mL) was added in the round flask, and the fat removal was extracted for 1 hour [12].The petroleum ether was exchanged with 110 ml of methanol for the crude hesperidin extraction process.Orange peel extraction with methanol was carried out for 2 hours.The crude extract obtained was concentrated for 20 minutes and precipitated using a 6% acetic acid solution.The crude extract was stored for 48 hours in the refrigerator.The precipitated hesperidin crude extract was filtered and rinsed with pure methanol.The crude extract left on the filter paper were dried using a dehydrator (40-50℃, 6-8 hours).The dried hesperidin crude extract were weighed and used for further analysis [11].

Qualitative Test for Flavonoid and Polyphenol Content
Hesperidin crude extract testing was carried out qualitatively on the flavonoid and polyphenol content.Qualitative testing of flavonoids was carried out with the Shinoda test.Isolated hesperidin crude extract (1 mg) was dissolved in 2 mL of methanol.Samples (1-2 drops) were mixed with Mg powder.The sample was dripped with pure HCl until a colour change forms in the sample.The sample was proven positive if there was a colour change from yellow to orange to reddish [11].
Meanwhile, qualitative testing of polyphenol content was carried out by dissolving 1 mg of the isolated crude extract of hesperidin in 2 mL of methanol.On a drop plate, 1-2 drops of sample were mixed with a few drops of 1% FeCl3 solution.The sample was proven positive if there was a colour change from yellow to blue, green, or black [11].

Hesperidin Crude Extract Concentration Test in Butter
Concentration testing began with preparing a standard hesperidin standard curve with a concentration series of 8, 12, 16, 20, 24, and 28 ppm using a spectrophotometer (λ=284 nm).The regresion equation obtained was utilized to calculate the concentration of hesperidin crude extract inside the sample.The concentration of hesperidin crude extract was tested in the sample by mixing 100 mg of butter sample and 2.4 mL of ethyl acetate.The sample was homogenized, and 1 mL of the solution was transferred to a 10 mL volumetric flask and measured.The samples were then analyzed using a spectrophotometer (λ=284 nm) [15].

Peroxide Value Test
Butter sample (5 g) was added with 30 mL of acetic acid and chloroform (18 mL:12 mL), then homogenized.The sample was added with 0.5 mL of saturated KI and incubated for 1 minute.The sample was added with 30 mL of aquadest and 0.5 mL of 1% starch indicator.The sample was incubated for 30 minutes before being titrated using 0.01 N Na2S2O3 solution until the purple colour in the sample disappeared [16].The peroxide number in the sample was calculated using Equation 1.

Antioxidant Activity Testing
Butter sample (0.2 mL) was added with 3.8 mL of ethyl acetate & 1 mL of DPPH (6.34 × 10 -5 mol/L).Samples were incubated for 10 minutes and analyzed using a spectrophotometer (λ=520 nm).The blank for this study was mixture of 1 mL of DPPH and 4 mL of ethyl acetate [10].Antioxidant activity in the sample was calculated using the formula Equation 2.

Statistical Analysis
This study determined the significance of the values between samples using the Two Way Analysis of Variance statistical method (variables were hesperidin crude extract addition concentration and shelf life) followed by Duncan method if there are significant differences between samples (p-value <0.05).Statistical analysis were performed using the IBM SPSS Statistics Version 25 application.

Hesperidin Crude Extract Isolation
The isolated hesperidin using the Soxhlet method can be seen in Figure 1.The isolated hesperidin has a powder form with a light brown color.The crude extract will then be tested for the content of flavonoids and polyphenols.

Flavonoid Qualitative Testing
The hesperidin crude extract tested result for flavonoid groups can be seen in Figure 2. In the Shinoda test, it can be seen that mixing the sample with HCl and mg powder causes a change in the color of the sample from yellow to pink.These results indicate that the isolated sample contains flavonoids.The color change from yellow to pink in this test is relatively consistent with previous literature [11].The addition of Mg powder and HCl was carried out to ensure the presence of hydroxyl groups (due to the structure of most flavonoids filled with hydroxyl groups) in the ring structure A & B in the isolated sample.Addition of Mg powder to the hesperidin crude extract is intended as a reducing agent in a redox reaction.Reducing agent is expected to reducing flavonoids to form an orange-to-reddish complex (flavilium salt) [17].The addition of HCl to the hesperidin crude extract is intended to help the process of Mg and flavonoids redox reaction, also HCl play a role in hydrolyzing flavonoids into its aglycones structure [18].Changes in the color of the sample to pink can be caused by differences in the flavonoids groups (differences in the chemical structure of each flavonoid).The hesperidin crude extract gives a pink colour because the compound belongs to the flavanol group [19].

Polyphenol Qualitative Testing
The hesperidin crude extract tested result for polyphenol groups can be seen in Figure 2. In the polyphenol content test, it can be seen that mixing the sample with 1% FeCl3 solution resulted in a change in the colour of the sample from yellow to blackish brown.These results indicate that the isolated sample contains polyphenols.The colour change from yellowish to blackish brown in this test is relatively consistent with previous literature [11].The addition of FeCl3 solution was carried out to ensure the presence of hydroxyl groups in the isolated crude extract.Before reacting with the sample, FeCl3 will first split into Fe 3+ and 3Cl -ions.Fe 3+ ions will react with H ions from the hydroxyl groups, which previously formed phenoxy ions.This phenoxy ion will react with Fe 3+ ions to form green, blue, to black complexes, namely iron (III) hexafolate [20].

Production and Characterization of Butter
The butter samples formulated using hesperidin crude extract can be seen in Figure 3.It can be seen that there is a difference in colour between the samples, which were given a lot, a little, or no hesperidin crude extract added.The sample will be tested for its final quality.The quality parameters to be tested were the total concentration of hesperidin crude extract, the peroxide value, and the antioxidant activity value of the sample.Furthermore, the quality test results will be further processed using statistical tests to see significant differences between samples (the effect of shelf life and the amount of hesperidin crude extract added to the final quality of butter).This test was carried out using a Two-way ANOVA with Post hoc of Duncan at the 5% significance level.

Hesperidin Crude Extract
Before testing the hesperidin crude extract concentration, regression data will be collected between several series of standard hesperidin solutions (8-28 ppm with four ppm intervals) with their absorbance values.The graph gives the regression equation y = 0.0037x -0.0264 (R 2 = 0.9694).From the regression equation that has been obtained, the hesperidin crude extract concentration will be calculated.The graph can be seen in Figure 4.The results of testing the concentration of hesperidin in butter during 28 days of storage can be seen in Figure 5.During storage process, the concentration of hesperidin tended to increase from day 7 to day 14, decreased on day 21, and increased again on day 28.Based on statistical tests one way ANOVA and Duncan's post hoc test (α = 5%) can be seen if the concentration of hesperidin contained in the samples gives significantly different (p-value <0.05) over the storage period.In general, hesperidin concentrations will decrease during storage.This is caused by exposure to air, light, and storage temperature, which have the potential to accelerate the process of oxidation of fatty acids in butter.The oxidation process can be caused by free radicals that use hesperidin hydrogen atoms as oxidation targets to replace free fatty acids, thereby forming stable products.Continuous use of hydrogen atoms will decrease the concentration of the hesperidin crude extract during storage [21].
Except for the oxidation process of fatty acids in butter, the crude extract of hesperidin can precipitate.This precipitation is caused by the difference in solubility between the hesperidin crude extract and the butter.The hesperidin crude extract has hydrophobic groups (hesperetin group) and hydrophilic (rutinose sugar group).The existence of hydrophilic groups or water (polar) is quite contrary to the solubility of butter (non-polar) [22].The difference in solubility between the two materials can cause the hydrophilic groups to become insoluble and precipitate at the bottom of the sample.This precipitation could reduce the concentration of the crude extract of hesperidin during sample analysis because only the supernatant is used in the analysis.That way, there is a possibility that the hesperidin that is precipitated at the bottom of the sample is not counted [23].

Peroxide Value Testing
The results of testing the peroxide value in butter during 28 days of storage can be seen in Figure 7.During the storage process, the peroxide value of the samples tended to increase.Based on the oneway ANOVA statistical test and Duncan's post hoc test (α = 5%), it can be seen that the peroxide value of the samples gives significantly different values (p-value <0.05) each time of sampling.The highest peroxide value of all samples can be found on day 28, and the lowest on day 0. In Figure 8. it can be seen that the addition of hesperidin and the shelf life had a significant effect (p-value <0.05) on the peroxide value of the samples.The initial peroxide value of the butter sample ranged from 0-0.99 meq/kg, the final peroxide value ranged from 2.18-10.23 meq/kg, and the average peroxide value ranged from 1.04-3.62meq/ kg.Samples with the highest peroxide value were commercial; the lowest were 1.5% and 2% formulations.The increase in peroxide value during storage is due to continuous oxidation.This oxidation process will not stop if the air is still in contact with the butter and all unsaturated fatty acids have not been oxidized [4].Peroxide number is commonly used as a determinant of the appropriateness of consumption of fat products.The peroxide number describes how far the fat has been oxidized.The principle of the peroxide number test is to determine the amount of sodium thiosulphate that reacts with free iodine.The presence of free iodine can be caused by a reaction between peroxide and saturated KI in acidic conditions.The amount of free iodine contained in the sample will be calculated as the amount of peroxide in fat [24].Therefore, the calculation of the sample peroxide number was carried out using the iodometric titration method.This method will react saturated potassium iodide with sodium thiosulfate, resulting in an oxidation process [25].An increase in the value of the peroxide number shows if there is a new peroxide formed during the storage process [10].In Table 1. it can be seen that the addition of hesperidin crude extract added affects the value of the peroxide number of the sample.This effect can be seen in the Pearson correlation test (-0.174).This value shows the correlation between tests is very weak and has a negative value (the more hesperidin crude extracts added, the peroxide number value will decrease).Then, the p-value of the sample was 0.135 (p-val>0.05),which showed that the correlation between the addition of hesperidin and the peroxide number was not significant.
The addition of hesperidin crude extract can cause a negative correlation between the two tests.The more hesperidin crude extract added, the more agents are available to donate hydrogen atoms to free radicals so that the process of fatty acid oxidation will be hampered and decrease the rate of formation of new peroxides.The low rate of peroxide formation will cause the value of the peroxide number obtained to be lower [26].
The discrepancy between theory and this research is thought to be caused by impurities (residues of chemicals/other components) or errors in reading the concentration of hesperidin crude extract in the samples.These impurities can change the characteristics of hesperidin crude extract, causing the antioxidant properties of hesperidin crude extract to change or become inactive.In effect, the hydrogen donor process or the role of hesperidin crude extract as an antioxidant agent will be disrupted, affecting the formation of peroxides in the sample [27].In addition, errors in reading the concentration of hesperidin in the sample can occur due to the hydrophilic structure of hesperidin crude extract (routine group) and hydrophobic (hesperetin group), causing precipitation in the butter sample due to differences in solubility [22].This precipitation indicates that hesperidin crude extract is not completely dissolved and during analysis the concentration is not calculated [23].

Antioxidant Activity Testing
The results of testing the antioxidant activity of butter during 28 days of storage can be seen in Figure 9.During the storage process, the antioxidant activity of the samples tended to decrease.Based on the one-way ANOVA statistical test and Duncan's post hoc test (α = 5%), it can be seen that the antioxidant activity of the 0% -1.5% formulation samples gave significantly different values (p-value <0.05) and the commercial samples, the 2% gives a value that is not significantly different (p-value > 0.05) at each sampling time.The highest antioxidant activity can be found on days 0 (0% and 1% formulation) and 7 (0.5% and 1.5% formulation).Meanwhile, the lowest antioxidant activity was found on day 28 (0% -1.5% formulation).In Figure 10. it can be seen that the addition of hesperidin crude extract and the shelf life had a significant effect (p-value <0.05) on the value of the sample's antioxidant activity.The value of the initial antioxidant activity of the butter samples ranged from 31.75-45.87%,the final antioxidant activity ranged from 11.09-26.88%,and the average antioxidant activity ranged from 23.62-30.80%.Samples with the highest antioxidant activity were 1%, 1.5%, and 2% formulations, and the lowest were commercial formulations, 0% and 0.5%.
Antioxidant activity in this study was carried out using the DPPH method.DPPH is one of the chemicals with reactive free radical properties.The principle of this method is to measure the ability of hesperidin crude extract antioxidant agent to reduce DPPH.The reduction process is done by donating a hydrogen atom to a free radical group.The hydrogen atom donor process will cause the stability of free radicals.Physically, the reaction between antioxidants and DPPH will cause a colour change from deep purple to yellow.The decolourization of DPPH caused the colour change during the reduction process.The more the amount of DPPH that is reduced, the more intense the yellow colour will be.The colour change will be measured by the colour absorption using a spectrophotometer [28].The addition of antioxidants to butter products is highly supported, considering that antioxidants can prevent rancidity caused by their ability to donate hydrogen atoms so that the fatty acid oxidation chain reaction becomes slower [29].
In Table 1. it can be seen that the addition of hesperidin crude extract affects the value of the antioxidant activity of the sample.This effect can be seen in the Pearson correlation test (0.188).This value shows the correlation between tests is very weak and has a positive value (the higher the addition of hesperidin, the increase the value of the antioxidant activity).Then, the p-value of the sample was 0.106 (p-val>0.05),which showed that the correlation between the addition of hesperidin crude extract and antioxidant activity was not significant.
The addition of hesperidin crude extract can cause a positive correlation between the two tests.The more hesperidin crude extract is added, the more hydrogen atoms are available for free radical reduction.The antioxidant activity of the sample will increase because the antioxidant activity is determined by the ability of the components to donate hydrogen [26].
The discrepancy between theory and this study is thought to be caused by an error in reading the hesperidin concentration in the sample and the presence of pigment in the commercial sample (results equivalent to 0.5%, 1%, and 2% samples even though hesperidin was not added).Antioxidant activity in commercial butter samples is obtained from carotene pigments.Carotene pigment has the ability as an antioxidant agent in butter [30].This pigment is obtained from the primary raw material for butter, milk (derived from the feed consumed by dairy cattle) [31].
In Table 1. it can be seen that the antioxidant activity affects the value of the peroxide number of the sample.This effect can be seen in the Pearson correlation test (-0.51).This value shows the correlation between tests is quite strong and has a negative value (the higher the value of the antioxidant activity, the value of the peroxide number will decrease).Then, the p-value of the sample was 0 (p-val <0.05), which showed a significant correlation between antioxidant activity and peroxide number.The negative correlation between the two tests can be caused by the higher ability of hydrogen atoms to donate atoms.The higher the value of antioxidant activity, the more hydrogen atoms are available to slow down the performance of the oxidizing agent [26].In effect, the process of fatty acid oxidation will be inhibited and cause the formation rate of new peroxides to be delayed.This will cause a lower peroxide value in the sample [10].The discrepancy between theory and research is thought to be caused by the presence of impurities in hesperidin which causes changes in the ability of hesperidin as an antioxidant agent and causes inhibition of free radical reduction reactions [27].

Conclusions and Recommendations
The treatment with the addition of hesperidin crude extract and the shelf life affects the final quality of the butter product.The addition of hesperidin crude extract showed positive results on butter's peroxide value and antioxidant activity.The best addition formulation from this study was the addition of 1.5% hesperidin crude extract.This formulation showed the best stability during 28 days of storage, with the highest antioxidant value (30.8%) and the lowest peroxide value (1.05 meq/kg) as tested statistically using Two-way ANOVA and Post hoc Duncan.For further research, it is possible to purify the hesperidin crude extract and characterise it.Purification is recommended to be carried out, considering that impurities in hesperidin crude extract caused some errors in research.In addition, it is also possible to add an emulsifier to the hesperidin crude extract, so it can be completely dissolved in non-polar products such as fat.With the addition of an emulsifier to the hesperidin crude extract, the results given in the test will be better and provide higher extract effectiveness.

Figure 1 .
Figure 1.Hesperidin Crude Extract Result of Isolation

Figure 2 .
Figure 2. Flavonoid and Polyphenol Group Qualitative Testing

Figure 3 .
Figure 3. Physical Appearance of (a) Commercial butter, Butter with 0% and 0.5% Addition of Hesperidin Crude Extract (b) Butter with 1-2% Addition of Hesperidin Crude Extract

Figure 6 .
A two-way ANOVA statistical test and Duncan's post hoc test (α = 5%) were performed.It can be seen that the addition of hesperidin and the shelf life had a significant effect (p-value <0.05) on the hesperidin concentration in the sample.Initial concentration values of butter samples ranged from 109.48-154.43 ppm, final concentrations ranged from 98.76-130.38 ppm, and average concentrations ranged from 123.42-166.81ppm.The sample with the highest hesperidin concentration was the 0.5% formulation and the lowest was the 0% formulation.

Figure 7 .Figure 8 .
Figure 7. Peroxide Value of Butter Sample During Storage Figure 8. Statistical Analysis of Peroxide Value and Sample Formulation (Different superscripts indicate significantly different in Duncan's statistical test at α = 5%)

Figure 9 .Figure 10 .
Figure 9. Antioxidant Activity of Butter Sample During Storage Figure 10.Statistical Analysis of Antioxidant Activity and Sample Formulation (Different superscripts indicate significantly different in Duncan's statistical test at α = 5%)

Table 1 .
Pearson Correlation Test