, Hypolipidemic Effects of Modified Edamame Tempeh Flour on Lipid Profile Levels in Dyslipidemia Rats Efek Hipolipidemia Tepung Tempe Edamame Modifikasi terhadap Profil Lipid Tikus Model Dislipidemia

Background: Dyslipidemia is a lipid metabolism disorder that causes an increase or decrease in plasma lipid concentrations. Comprehensive management is an attempt to prevent and reduce dyslipidemia, one of which is nutritional therapy. Edamame contains nutrients such as protein, fat, fiber


INTRODUCTION
Dyslipidemia is an abnormal lipid metabolism that causes an increase or decrease in plasma lipid concentrations characterized by increased levels of total cholesterol, LDL-C, triglycerides, and decreased levels of HDL-C 1 . Dyslipidemia contributes to endothelial dysfunction which is a major factor in the pathogenesis of hypertension, thrombosis, and atherosclerosis. 2 Atherosclerosis causes blood flow to the heart muscle to be blocked and results in cardiovascular disease 1,3 .
One of the management of dyslipidemia therapy is through food regulation. Nutritional therapy is an important supporting factor in the management of dyslipidemia. Nutrition therapy has shown effectiveness in efforts to reduce dyslipidemia including proper energy intake to maintain a normal weight, limiting saturated fat intake, limiting simple carbohydrate intake, consuming high fiber foods, limiting alcohol consumption, and implementing the consumption of functional food sources in the daily diet [4][5][6] . One of the functional foods that are closely related to antidyslipidemia is tempeh 7 . Tempeh has high nutritional values such as a source of protein, vitamin B12, and bioactive compounds. The nutritional value of tempeh is recognized to support the improvement of dyslipidemia 8 . The main protein content in soybeans is globulin 7S (β-conglycinin) and globulin 11S (glycinin) which play a role in increasing bile salt secretion and inhibiting cholesterol absorption 9 .
In general, tempeh is made from yellow soybeans or mature soybeans, but edamame can also be an alternative material for tempeh production 10 . Edamame or vegetable soybeans are rich in protein, vitamins, minerals, dietary fiber, calcium, iron, and bioactive compounds such as omega-3 fatty acids, sterols, isoflavones, and saponins, and high in essential amino acids [11][12][13] . Another advantage is the low content of anti-nutritional substances such as phytic acid, antitrypsin, phenolics, and tannins 12 . Edamame tempeh is a processed food from soybean derivatives as the attempts to increase the nutritional content and digestibility of the product 10 . The presence of enzymatic activity from the fungus Rhizopus oligosporus can reduce antinutrient compounds in beans and convert them from macromolecular substrates into simple forms such as amino acids, fatty acids, isoflavone aglycones, and other bioactive compounds so that they are more easily utilized by the body [14][15][16][17] .
In addition, during the soybean fermentation process, other microorganisms can grow, such as the yeast Saccharomyces cerevisiae. Yeast S. cerevisiae is involved in the breakdown of glycosides into aglycones increasing the availability of isoflavone aglycones 18,19 . S. cerevisiae is one of the microorganisms that produce βglucan 20 . β-glucan is effective in preventing an increase in blood cholesterol levels 21 . Therefore, tempeh produced with the addition of S. cerevisiae has the advantage of increasing the quality of tempeh with high β-glucan content 22 .
Previous research has studied the health benefits of tempeh, while in this research the main material used is edamame. In addition, edamame tempeh in this research was modified by adding yeast S. cerevisiae. This modification process produces edamame tempeh with higher nutritional value. Based on the background, this research aimed to analyze the effect of edamame tempeh flour (ET) and modified edamame tempeh flour (MET) on lipid profile levels of dyslipidemic rats.

Research Sample
The sample used was a white rat (Rattus norvegicus) Sprague Dawley strain with inclusion criteria including male rats, body weight ranging from 150-200 g, 8 weeks old, and normal behavior. Samples that were not included in the research if they had the exclusion criteria, the rats experienced behavioral changes and diarrhea which was indicated by watery stools, and the dropout criteria were that the rats died during the research.
This research used 36 rats divided into six treatment groups. The calculation of the sample size for each group referred to the provisions of the World Health Organization in 2000, namely in each group a minimum of five rats and an estimated dropout of 20% were added, so that six rats were obtained in each treatment group. The determination of each group was conducted by randomization.
Before the intervention, the rats were adapted for seven days. Rats were placed in groups of 6 rats. Each group of cages has an individual cage, and each cage is equipped with a small hole. The cage is placed at an ambient temperature of 22-25°C and humidity of 30-70%. A light reception is divided into the light cycle and dark cycle, each cycle for 12 hours. During the adaptation, rats were given standard comfeed of 20g/200gBW/day, drinking water ad libitum, and induction of a high-fat diet (HFD) for 14 days. The composition of the HFD induction consisted of 2 ml/200gBW pork oil and 1 ml/200gBW duck egg yolk administered through a gastric probe 25  The treatment group consisted of negative control (K-), namely dyslipidemic rats that were not given the intervention, a positive control (K+) that was given the simvastatin drug, KP1 was the group that was given the intervention of ET flour at a dose of 2.7 g/200gBW, KP2 was the group that was given the intervention of ET at a dose of 5.4/200gBW, KP3 is the group that was given the intervention of MET flour at a dose of 2.7 g/200gBW, and KP4 is the group that was given the MET flour intervention at a dose of 5.4/200gBW.

Dosage Determination
The dose of simvastatin for dyslipidemic adults is 10 mg/day converted according to Laurence and Bacharach's table for Rattus norvegicus so that the dose of simvastatin is given to rats is 0.18 mg/200gBW 27 . The determination of dose was determined based on the consumption of tempeh per day according to the vegetable protein exchange unit equivalent to 150 g of tempeh 28 . The amount was converted so that the dose of ET flour was 2.7 g/200gBW (normal dose) and 5.4 g/200gBW (high dose) and MET flour 2.7 g/200gBB (normal dose) and 5.4 g/200gBB (high dose). The intervention was given once per day in the morning for 28 days 28 .

Data Collection
Parameters observed in this research included levels of lipid profile (total cholesterol, LDL-C, HDL-C, and triglycerides). The sample used was rat' blood serum taken through the vein retro-orbital. Examination of total cholesterol, LDL-C, and HDL-C levels was obtained from the results of the enzymatic photometric Cholesterol Oxidase-Peroxidase Aminoantipyrine Phenol (CHOP-PAP) test and triglyceride levels using the Glycerol-3-Phosphate-Peroxidase Aminoantipyrine Phenol (GPO-PAP) method. Then it was analyzed using a measure the spectrophotometer. Lipid profile levels in rats were measured three times after adaptation (before HFD induction), before intervention (after HFD induction), and after intervention.

Data Analysis
The data obtained were tested for normality using the Shapiro-Wilk test. Next, the data that were normally distributed were followed by a homogeneity test using Levene's Test. Data that were normally distributed and homogeneous can be continued with the One-Way ANOVA test. A One-Way ANOVA test was used to analyze the differences between each treatment group. Next, it was followed by the Post Hoc Tukey High Significant Difference (HSD) test, the data were normally distributed but not homogeneous. The next test was the Games Howell test. The data were not normally distributed but homogeneous so using the Kruskal Wallis non-parametric statistical test followed by the Mann-Whitney test. All statistical tests used a 95% significance level.

Total Cholesterol
Based on Table 2, the average total cholesterol levels in all treatment groups decreased after being given the intervention. The results of statistical tests indicated significant differences in the four groups compared to before the intervention (p<0.05). The highest decrease occurred in the positive control group who were given simvastatin as much as 52.55% and the lowest decrease in cholesterol was in KP1 as much as 30.28%. The intervention group that experienced a decrease in cholesterol levels was closed to the positive control group that is the KP4 group (50.17%).  Table 3 is a Mann-Whitney test to determine the difference in the difference in total cholesterol levels in each treatment group before and after being given the intervention. The results of the analysis showed the difference in total cholesterol levels was significantly different between the treatment groups after being given the intervention for 28 days (p<0.05), but in KP2 and KP3 there was no significant difference (p>0.05). This showed that the administration of ET flour at a dose of 5.4 g/200gBW and MET flour at a dose of 2.7 g/200gBW has almost the same effect on lowering total cholesterol levels. Soybeans and their processed products were closely related to foods high in protein, essential fatty acids, as well as vitamins, and minerals. This had a direct effect on the body's metabolic processes 28 . Based on the results of statistical analysis, total cholesterol levels decreased significantly in the intervention group with ET flour and MET flour. The hypocholesterolemic effect on MET flour occurred because soy protein was a fundamental component in soybeans. After all, it had a beneficial effect on lipid metabolism 29 . The main protein content in soybeans are 7S globulin (β-conglycinin) and 11S globulin (glycinin) 9 31 . In addition, the low content of methionine in soybeans is associated with hypocholesterol activity which has positive homocysteine levels and can induce hypercholesterolemia by increasing cholesterol synthesis in the liver, so that in an increase in plasma cholesterol levels 32 . The recommendation for soy protein intake in improving lipid profiles was 25 g/day 33 .
In addition, the fiber content in soybeans played a role in reducing cholesterol levels caused by the ability of fiber to modulate intestinal microbiota to reduce cholesterol synthesis due to the formation of propionate which inhibits the action of the HMG-CoA reductase enzyme in decreasing the diffusion of bile salts 34, .The recommended daily fiber intake for adults was 25-38 g 21 . Research by Huang et al., (2018) proved that hyperglycemic rats induced by HFD experienced a decrease in cholesterol levels after being given tempeh supplementation which was inoculated with Lactobacillus plantarum. Based on the results of research related to cholesterol levels, it was found that the MET flour group was more effective than ET flour due to the compounds contained in the two products. Edamame tempeh added S. cerevisiae contains β-glucan. S. cerevisiae was a source of β-glucan 20 . It was known that β-glucan could reduce bile salts and increase bile salt secretion 37,38 . These mechanisms caused cholesterol to decrease and prevented reabsorption back to the liver 38 . Higher isoflavone aglycones in MET flour compared to ET flour because S. cerevisiae helped in increasing isoflavone aglycones 19,23 .
Soy isoflavones had an anticholesterol effect because they were able to bind to estrogen receptors on body cells, thereby affecting the function of estrogen in binding DNA sequences and inducing DNA transcription. Through this mechanism, isoflavones became ligands for lipid regulating proteins such as PPARs (Peroxisome Proliferator-Activated Receptors), liver X receptors, and farnesoid X receptors that carried out a leading role in lipid metabolism. Therefore, this mechanism could reduce lipid synthesis in the liver, bile salt synthesis, and cholesterol reabsorption 39 . Isoflavones in tempeh were isoflavone aglycones that were more easily absorbed by the body. During the fermentation process, microorganisms helped activate isoflavones from glycone forms (daidzin and genistin) to aglycones (daidzein and genistein) 1 . 7

Low-Density Lipoprotein Cholesterol (LDL-C)
Based on table 4, it can be seen that the average change in LDL-C levels in the rats group after the intervention decreased. Statistical analysis showed a significant difference between LDL-C levels before and after the intervention (p<0.05). The treatment group that experienced the highest decrease in LDL-C levels was the positive control group (simvastatin), which was 60.51%. The effect of giving the intervention of MET flour at a dose of 5.4 g/200gBW (KP4) could reduce LDL-C levels (57.27%) close to the positive control group and the lowest reduction in LDL-C levels is in KP1 of 33.82%. LDL-C was the main lipoprotein that had atherogenic properties so it become a target for improving the lipid profile 40 . Edamame had nutritional components and bioactive compounds in improving LDL-C levels including protein, unsaturated fatty acids, fiber, isoflavones, and vitamins 4 . Amino acids, fiber, and high isoflavones content in soybeans had the potential as cardioprotective in lowering LDL-C levels 41 .
The results of the research related to LDL-C levels indicated that each treatment group that was given the intervention of ET flour and MET flour had a significant decrease. Based on the results of the analysis, it was recognized that the reduction in LDL-C levels was more effective in the group with MET flour compared to ET flour. This might happened because the main protein components in soybeans were globulin 7S (βconglycinin) and globulin 11S (glycinin). This peptide had a physiological effect in reducing LDL-C levels by degrading LDL-C in the liver, resulting in a decrease in serum LDL-C levels 4,32 . In addition, the peptides obtained from soybean glycinin are IAVPGEVA, IAVPTGVA, and LPYP. These three peptides could increase LDL-C receptor activity and work in vitro as inhibitors of HMG-CoA Reductase activity to regulate cholesterol biosynthesis. This would decrease the synthesis of cholesterol in the intracellular which led to the activation of SREBP-2 and increased the absorption of LDL-C by Hep2 cells. Another peptide derived from βconglycinin, namely FVVNATSN could increase the transcription of LDL-C receptors in hepatocytes and the small subunit of albumin 2S protein (lunasin) plays a role in improving LDL-C levels through the mechanism of decreasing HMG-CoA reductase expression and increasing LDL-C receptor expression 31 .
The fiber content and unsaturated fatty acids in soy such as omega-3 and omega-9 fatty acids have been shown to reduce LDL-C levels in the blood 42 . The fat contained in soybeans was mostly unsaturated in the form of arachidonic fatty acids, linoleic fatty acid, and oleic fatty acid, and contained phospholipids such as lipopositol, sepalin, and lecithin 43 . This is in line with the research of Huang et al. (2018) reported that supplementation of tempeh co-inoculated with Lactobacillus Plantarum in induced hyperglycemic rats HFD significantly lowers LDL-C levels.
The decrease in LDL-C levels was more common in the group with MET flour administration also due to the presence of β-glucan compounds which played a role in increasing the number of LDL-C receptors in the liver, thereby eliminating LDL-C particles in plasma 44 . This was evidenced by the research of Grundy et al. (2018) stated that there was a decrease in LDL-C levels in hypercholesterolemic rats fed a diet containing β-glucan. Table 6 shows that HDL-C levels in the rats treatment group increased with the administration of ET flour and MET flour interventions for 28 days and statistically the average HDL-C levels before and after the intervention were significantly different (p<0,05). The KP4 group was known to have the best HDL levels among the other intervention groups with a value of 71,55 mg/dl or an increase of 204.21% compared to HDL-C levels before the intervention. KP4 is the intervention group that is closest to the positive control HDL-C levels, which was 70.71 mg/dl (209.45%).   Table 7 showed that there was a significant difference between the treatment groups (p<0.05), but the positive control was not significantly different from KP3 and KP4, KP2 and KP3, KP3 and KP4 (p>0.05). Based on the results of the analysis, showed that MET flour was able to increase HDL-C levels close to positive control compared to ET flour. The group given ET flour at a dose of 5.4 g/200gBW had the effect of increasing HDL-C levels which was almost the same as the MET flour at a dose of 2.7 g/200gBW, while the group given TEM flour at a dose of 2.7 g/200gBW was able to increase HDL-C levels in rats not much different from the dose of 5  HDL-C was considered good cholesterol because it acted as a cleanser of excess cholesterol in the arteries by transporting LDL back to the liver and excreted by the body 46 . Table 6 shows that the HDL-C of rats with the effect of administration of the intervention of ET flour and MET flour a significant increase. This research was in line with the research of Astawan et al. (2015) which stated that rats fed tempeh rations experienced an increase in HDL-C levels compared to boiled soybean or casein rations. This was influenced by the presence of oleic, linoleic, and linolenic acids in tempeh which had the potential to increase HDL-C levels 48 .

Post-Hoc Tukey HSD test in
In addition, the results obtained showed that MET flour increased higher HDL-C levels in rats compared to ET flour. This was because the high content of isoflavone aglycones in MET flour has the potential to increase HDL-C levels 23,49 . The higher isoflavone content in the product had a greater effect on increasing HDL-C levels 49 . Soy isoflavone aglycones consisting of genistein, daidzein, and glycitein exhibit estrogenic effects which acted as a cardioprotective effect to maintain and increase HDL-C levels 50 .
Edamame contained about 60% unsaturated fatty acids 42 . High levels of HDL-C in the blood could reduce the occurrence of deposition and plaque formation in the circulatory system 48 . Based on the results of HDL-C analysis obtained, tempeh could be used as a functional food to help reduce the risk of cardiovascular disease through various mechanisms such as removing cholesterol from macrophages, improving endothelial function, and having antiinflammatory properties 51 .

Triglycerides
The results of the analysis showed that the intervention of giving ET flour and MET flour for 28 days was able to reduce triglyceride levels in all treatment groups. This was evidenced by statistical tests showing a significant difference compared to before the intervention (p<0.05). The treatment group after being given the intervention had the highest triglyceride levels in the KP1 group (104.11 mg/dl) and the lowest was a positive control (81.50 mg/dl) followed by KP4 (84.02 mg/dl). The results of the research related to triglyceride parameters showed that the intervention treatment group with ET flour and MET flour decreased significantly. This is related to soy protein including βconglycinin which had been shown to help in lipid metabolism and inhibit lipid synthesis in the liver and cause a decrease in serum triglycerides in rats 31,52 . Triglyceride levels in the intervention group with MET flour had a better lowering effect than ET flour with the same dose.
Besides, MET flour had a higher content of isoflavone aglycones so there was a greater decrease than the intervention group of ET flour 23 . Isoflavones found in soybeans significantly reduce triglyceride levels 53 . Soy isoflavones were able to modulate lipoproteins resulting in the transformation of several important enzymes regulating lipid metabolisms such as lipoprotein lipase (LPL), and hepatic lipase (HL), or hepatic triglyceride lipase (HTGL), and 7 alphahydroxylase 54 . According to the results of a metaanalysis by Nachvak et al. (2019) revealed that the concentration of triglycerides decreased significantly when individuals consumed soy protein containing soy isoflavones.

CONCLUSIONS
The administration of ET flour and MET flour gave significant results in reducing total cholesterol, LDL, and triglyceride levels as well as increasing HDL-C levels. Based on the results of the analysis, it was found that MET flour showed better effectiveness as a hypolipidemic functional food compared to ET flour, especially when given MET flour at a dose of 5.4 g/200gBW. This was indicated by the difference in the decrease in total cholesterol, LDL-C, and triglyceride levels as well as an increase in HDL-C levels approaching the positive control group.