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Effect of Stir-Frying on Calcium Inside Terrestrial Water Spinach (Ipomoea Reptans Poir) with Water Spinach (Ipomoea Aquatica Forks) with Complexometric Titration Method

Water spinach is a variant of vegetables containing a lot of nutrients, including calsium. Inappropriate cooking will affect nutrients inside the foods. This research aimed to determine the effect of stir-frying on calcium inside terrestrial water spinach and water spinach using complexometric titration.This research usedterrestrial water spinach (Ipomoea Reptans Poir)and water spinach (Ipomoea Aquatica Forks). Researchers chose stir-frying as a cooking process as commonly popular and known to affecting calsium inside foods. The research used laboratory experiments. Results on qualitative research showed that samples positively contained calcium, marked with a white sediment. On quantitative, levels of calcium inside terrestrial water spinach before stir-frying was 0,0540% w/v while 0,0715% w/v after the cooking process. Meanwhile, levels of calcium on water spinach before stir-frying was 0,0549% w/v while 0,0746% w/v after the cooking process. The research had concluded that there were differences on the levels of calcium, before and after stir-frying, p = 0,000 (terrestrial water spinach) and 0,018<0,05(water spinach). In contrast, there were no differences on the effect of stir-frying on calcium levels on the terrestrial water spinach and water spinach, with independent t-test p = 0,310 > 0,05.


Introduction
Vegetables are a source of vitamins and minerals for the body and are needed by human as an intake of healthy and fresh foods. One vegetable that has a beneficial nutrients is water spinach. There are two types of water spinach available in Indonesia, namely terrestrial water spinach and water spinach 13. . The differences on environment where the plants grow affect nutrients inside both water spinach types 16 .
Water spinaches have complete nutrients, one of them is calcium. Calcium is a mineral needed by human body in large enough amounts. It plays an important role for the body to regulating cell function as well as a growth factor. It is formed from 1.5% to 2% of adult bodyweight or about 1 kg and 99% found in bones 2 The levels of calcium can either be reduced or increased after undergoing cooking processes. Calcium will reduce when using cooking media, such as oil and water, and food surfaces in contact with air 19 . Stir-frying will cause the outer skin to constrict due to dehydrating process. Water inside foods will evaporate because of the heat from oils so the foods get constricted 9 . Empty spaces previously filled with water and other components will be filled mostly by the oils during the cooking process. It is thought to cause calcium inside vegetables gets decreased 15 . This mineral has a boiling Researchers used complexometric titration methods with EDTA reagents as they are faster, cheaper with a high precision and accuracy to form a calcium complex 17 . The reagents are also easily soluble in water, its stability in forming chelates is very constant so the reaction goes perfectly and can react quickly with calcium metal 12 .
Research by Aisyah 1 confirmed that heating process can affect antioxidants inside vegetables, while the results of study by Setyopratiwi 14 showed that foods high in calcium are generally not resistant to heating process. The results of the study are supported by a research conducted by Burhanuddin 5 which showed that there were significant differences on the interaction between processing and reheating variations in the calcium inside Moringa leaves. In contrast, a research by Hidayat 7 showed that there was no difference before and after boiling with high temperatures.
Researchers, based on all descriptions above, were interested to conduct a scientific research on the effect of stir-frying to calcium inside terrestrial water spinach leaves and water spinach. Researchers chose both terrestrial water spinach and water spinach as there were differences in the environments where the plants grow that can affect calcium. This research was intended to find out whether cooking process can affect calcium or not, so people can still meet their daily calcium needs after processing, especially in the stir-frying process.
Materials used in this research included leaves of terrestrial water spinach and water spinach, 0.01 M EDTA reagents, CaCO3 solution, 1% EBT indicator, HCl, Methyl Red, 1 M NaOH solution, ammonium oxalate solution, and aquadest.

Methods
The method of this research was laboratory experimental, while the design was One Group Pre-test Post-test. In this study, samples were treated with a stir-fry cooking process 10 . Populations in this study were terrestrial water spinach taken from a vegetable garden at Tegalsari while water spinach taken from a paddy pond at Tegalsari, Kemudo, Prambanan, Klaten. It was because people in the locations cultivate the plants.
Samples in this study were water spinach leaves taken when still fresh, colored green or dark green, and having long-form with pointed leaves for terrestrial water spinach while rounded or having widerlooking for water spinach. All samples were taken as many as one kilogram.
After collection, identification of calcium inside water spinach was immediately conducted using ammonium oxalate to form a white precipitate.
Quantitative test for samples before stir-frying was conducted by titrating samples namely the leaves of fresh terrestrial water spinach and water spinach. Titration was added with Eriochrom Black T indicator and titrated with 0.01 M EDTA solution until the red color turned into blue 3 . Samples were then given the effect of processing in the form of stir frying by heating leaves of terrestrial water spinach and water spinach in a pan filled with a little cooking oil (30 ml), which had been heated with a temperature of 80-100 o C, then sautéed for 5 minutes on medium heat, while stir-frying for several times. Determination of calcium levels after stir-frying was also done by complexometric titration method using 3-time replication 11 .
Data analysis used descriptive analysis of percentages with mean and SD and then proceeded with Kolmogorov-Smirnov analysis, Paired T-Test and Independent T-Test 8 .

Plant Determination
Samples were taken in Tegalsari, Kemudo, Prambanan, Klaten. Terrestrial water spinaches were obtained from the cultivation of vegetable garden, while water spinach came from ponds in rice fields. They were then determined in the Biology Laboratory, Faculty of Mathematics and Natural Sciences, Gadjah Mada University, Yogyakarta. The result of the determination showed that samples used in this study were actually terrestrial water spinach and water spinach with species name known as Ipomoea aquatica Forssk.

Qualitative Calcium Test
Qualitative tests using ammonium oxalate showed that samples positively contained calcium characterized by the formation of white sediments.

Determination of Calcium Inside Samples
Calcium inside samples was analyzed using the complexometric titration method with 3-times replication or repetition. Based on the tables above, calcium obtained from terrestrial water spinach prior to stir-frying was averagely 0.0540% w/v ± 0.0015, while after stir-frying was 0.0715% w/v ± 0.0018. Calcium obtained from water spinach before stir-frying was 0.0549% w/v ± 0.0012, while after stir-frying was 0.0746% w/v ± 0.0042.

Analyze Data of Sample Testing Results
Calcium inside samples that were stir-fried and not stir-fried was analyzed using statistical analysis paired t-test. Based on table 6, the significance value (p) of terrestrial water spinach and water spinach samples after stir-frying was 0.310, both of which have p> 0.05 so that Ho is accepted, then Ha is accepted. It means that there is no difference in the effect of stir-frying on calcium inside the leaves of terrestrial water spinach and water spinach.

Discussion
This study aimed to analyze the effect of stir-frying on differences in calcium levels inside the leaves of terrestrial water spinach and water spinach. Water spinaches were chosen because they are one of the high calcium vegetables. People generally consume water spinach on the leaves. In this study, water spinach samples have been proven through determination in the Plant Systematic Laboratory, Faculty of Biology, Gadjah Mada University, Yogyakarta. The results of the determination showed that samples used in this study were actually terrestrial water spinach and water spinach with species name known as Ipomoea aquatica Forssk.
The study was conducted with two tests, namely qualitative and quantitative test. The qualitative test was conducted at the Analysis Laboratory of STIKES Muhammadiyah Klaten. Based on tests using ammonium oxalate, it was showed that the sample solutions contained calcium, known from the formation of white deposits which are calcium oxalate deposits (CaC2O4) 12 . Reaction between calcium and ammonium oxalate was as follow: Ca 2+ + (NH4)2C2O4 CaC2O4 + 2 NH4 + (1) Standardization of EDTA solutions was replicated 3 times. The aim of this process was to standardize because the sodium salt from EDTA did not meet the requirements as the main standard solution. Test of the titration results was obtained molarity of 0.02 M. It is in accordance with the literature and the desired molarity so that the solution is considered standard and can be used in complexometric titration.
After standardization, the EDTA solution can already be used to titrate the solution of terrestrial water spinach and water spinach with the complexometric titration method. In the titration, it is necessary to add 1.0 M NaOH which acts as a buffer to prevent pH changes in the sample solution 12 . Of such titration, a calcium-EDTA complex compound was produced in the form of a solution. The reaction of the formation of complex compounds is commonly referred to as the Lewis Acid Base reaction 12 .
Based on the results of quantitative tests, calcium inside terrestrial water spinach before stir-frying was 0.0540% w/v ± 0.0015 and 0.0715% w/v ± 0.0018 after stir-frying; meanwhile calcium inside water spinach before stir-frying was 0.0549% w/v ± 0.0012 and 0.0746% w/v ± 0.0042 after stirfrying. From these results, it was known that there are differences in calcium levels before and after stir-frying. In comparison of calcium levels in the terrestrial water spinach and water spinach, the results were calcium levels get higher after stir-frying.
Based on the literature, minerals inside foods can be lost during processing or can be influenced by cooking utensils used to process foods and differences in temperature exposure can affect food quality, one of which is in terms of calcium inside foods 5 .
The influence of heating also causes calcium to react with water to form a base solution of calcium hydroxide. Calcium hydroxide dissolves easily in water. If calcium hydroxide is made continuously, it will rise the concentration of the solution and a calcium hydroxide suspension will be formed thus causing high levels of calcium. In stir-frying, the oil used is coconut oil which contains calcium so that the oil will be hydrolyzed into water spinach thus causing increased calcium levels after stir-frying. Lipase enzymes in oil can hydrolyze triglycerides to produce free fatty acids and glycerol 6 .
From the results of research by Burhanuddin 5 which showed the same results, there are differences between before and after heating. It is different from a research conducted by Hidayat 7 where there was no difference in calcium before and after boiling. Based on a quantitative test after normality test using Kolmogorov-Smirnov One Sample test, it was found that the data were normally distributed. Statistical analysis of Paired T-Test showed that there were significant differences in calcium before and after stir-frying for each type of water spinach. Differences in calcium before and after stir-frying due to high temperature food processing can cause the evaporation of water in these foods 18 .
Since the digestibility of human body is limited, it is recommended to consume water spinach after processing, namely by stir-frying because the results of the study show there is no difference in the effect of stir-frying on calcium inside terrestrial water spinach and water spinach. Based on statistical analysis, Independent T-Test, it was found that there was no difference in the effect of stir-frying on calcium inside both terrestrial water spinach and water spinach. It is caused by the duration of the heating process, type of cooking utensils, method of processing and same temperature.

Conclusion
 Calcium levels of terrestrial water spinach before stir-frying was 0.0540% w/v ± 0.0015 and after stir-frying was 0.0715% w/v ± 0.0018; meanwhile water spinach contains 0.0549% w/v ± 0.0012 before stir-frying and 0.0746% w / v ± 0.0042 after stir-frying.  There are significant differences in calcium levels before and after stir-frying in both terrestrial water spinach and water spinach with significant values p = 0,000 <0.05 for terrestrial water spinach and 0.018 <0.05 for water spinach.  There is no difference in the effect of stir-frying on calcium levels of terrestrial water spinach and water spinach, independent t-test p = 0.310> 0.05.