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Research Article

Increased Inhibition of Angiotensin Converting Enzyme (ACE) Obtained from Indonesian Buffalo Meat Protein Using SEP-PAK Plus C18

Rio Olympias Sujarwanta, Jamhari , Edi Suryanto, Rusman , Setiyono , Endy Triyannanto, Yuny Erwanto and Ragil Yuliatmo
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Objective: This study aimed to investigate angiotensin converting enzyme (ACE) inhibitory activity derived from Indonesian native buffalo meat and its purified protein using SEP-PAK Plus C18. Methodology: Buffalo meat was hydrolyzed with pepsin, trypsin and a combination of pepsin and trypsin. These hydrolysates were assessed for dissolved protein, the molecular weight of proteins and ACE inhibitory activity. The hydrolysate with the highest ACE inhibitory activity was subsequently purified using SEP-PAK Plus C18. The molecular weight of the proteins was analyzed using descriptive statistics and the dissolved protein content and ACE inhibitory activity were evaluated in a complete randomized design. Results: Buffalo meat contained protein (22.66±0.2%) and dissolved protein (3.67±0.59 mg mL–1). Enzymatic hydrolysis significantly elevated the level of dissolved protein and decreased the molecular weight of the proteins. The dissolved protein content following hydrolysis by pepsin, trypsin and a combination of pepsin and trypsin was 10.91±0.4, 8.09±0.8 and 10.90±0.4 mg mL–1, respectively. The molecular weight of proteins in the buffalo meat homogenate ranged from 40-70 kDa, whereas, the hydrolysates were generally less than 40 kDa. The ACE inhibitory activity of the purified hydrolysate (IC50 = 75.8±20 μg mL–1) was approximately twice that of the hydrolysate (IC50 = 133±18 μg mL–1). Purification using SEP-PAK Plus C18 increased ACE inhibitory activity. Conclusion: Buffalo meat hydrolyzed by pepsin had the highest ACE inhibitory activity compared with other hydrolysates. Purification using SEP-PAK Plus C18 almost doubled the ACE inhibitory activity.

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Rio Olympias Sujarwanta, Jamhari , Edi Suryanto, Rusman , Setiyono , Endy Triyannanto, Yuny Erwanto and Ragil Yuliatmo, 2018. Increased Inhibition of Angiotensin Converting Enzyme (ACE) Obtained from Indonesian Buffalo Meat Protein Using SEP-PAK Plus C18. Pakistan Journal of Nutrition, 17: 434-440.

DOI: 10.3923/pjn.2018.434.440

Received: December 22, 2017; Accepted: March 28, 2018; Published: August 15, 2018

Copyright: © 2018. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.


Hypertension is a major health problem and is a major risk factor for the development of arteriosclerosis, cardiovascular disease, stroke and myocardial infarction1. According to Forouzanfar et al.2, the number of adults in the world with hypertension is ~3.5 billion. In 2015, China, India, Russia, Indonesia and the United States accounted for more than half of the global Disability-Adjusted Life-Years (DALYs) related to a systolic blood pressure (SBP) of at least 110-115 mm Hg2. Stressful, fiercely competitive lifestyles and food habits have compounded this problem3.

The treatment of hypertension involves chronic control of blood pressure under normal conditions. Angiotensin-I converting enzyme (ACE) inhibitors are one of several classes of pharmacologic agents that have been widely used in the treatment of hypertension. Natural peptides with ACE inhibitory activity were first found in snake venom4 and that discovery encouraged the development of synthetic ACE inhibitors. A strong ACE inhibitor was developed in the 1980s and is currently in use5. The ACE inhibitors are effective in lowering the mean, systolic and diastolic pressures in hypertensive patients as well as in salt-depleted normotensive subjects3.

Recently, many bioactive peptides derived from food proteins, including ACE inhibitors, have attracted attention due to their ability to prevent hypertension6. Some peptides from food, including porcine skeletal muscle proteins7-11, salmon12, fermented egg albumen13, chicken legs14 and goat milk casein15, have been shown to have antihypertensive effects. The ACE inhibitory peptides, such as those reported in the studies above16, are obtained from enzyme-hydrolyzed proteins that are produced using enzymes, such as trypsin or pepsin. Thus, peptides can be produced by the hydrolysis of food proteins during processing and digestion in the gastrointestinal tract17. To purify hydrolysates, Jamhari et al.18 and Katayama et al.6 used SEP-PAK Plus C18 and Yuliatmo et al.14 also reported that using this technique led to an increase in ACE inhibitory activity.

Buffalo meat has several properties that differentiate it from beef, such as less intermuscular fat and cholesterol, fewer calories, more essential amino acids and higher iron content, it also has significant potential for the production of bioactive peptides19. In Indonesia, buffalo meat has become an important source of protein20 because it contains as much protein as beef. However, no studies have been conducted on the peptides that can be derived from buffalo meat. This study aimed to investigate the potential of buffalo meat as a source of bioactive peptides that increase the angiotensin-converting enzyme inhibitory activity. Additionally, given the findings of Yuliatmo et al.14, the use of SEP-PAK Plus C18 to purify protein was investigated to determine whether this technique increases bioactivity.


Sample collection and preparation: Buffalo meat was obtained from a buffalo loin (Longissimus dorsi) from the Central Java area in Indonesia. Pepsin (porcine stomach mucosa) and trypsin were purchased from the Wako Pure Chemical Industry Ltd., Tokyo, Japan. Angiotensin-converting enzyme (ACE) from rabbit lung was obtained from the Sigma Chemical Co., St. Louis, USA and hippuryl-L-histidyl-leucine (HHL) free base was purchased from Nacalai Tesque, Kyoto, Japan.

The meat was homogenized following the method of Jamhari et al.18. Water (100 mL) was added to a 50 g sample of meat and blended using a Philips HR 2116 food processor (Philips, Indonesia) for 5 min. The meat extract was then homogenized using a Silverson L4R homogenizer (Silverson, England) for 10 min. The meat homogenates were incubated in a Memmert water bath (Memmert, Schwabach, Germany) for 30 min at 70°C and then cooled with ice. The homogenates were made and used immediately prior to analysis.

Chemical composition analysis: The moisture content, crude fat and crude protein of the buffalo meat were determined using standard methods of the Association of Official Analytical Chemists (AOAC)21.

Preparation of hydrolysates: Buffalo meat homogenates were separated into three treatments, hydrolyzed by pepsin (BMP), trypsin (BMT) or a combination of pepsin and trypsin (BMPT). The hydrolyzation by pepsin was adjusted to pH 2, the hydrolyzation by trypsin was adjusted to pH 7.0 and the third treatment was hydrolyzed first by pepsin then hydrolysis was continued with trypsin. Prior to incubation, 0.01 g of enzyme was added to the homogenate. After 2 h of incubation at 37°C, the pH of the hydrolysate was adjusted to 7.0 and hydrolysis was terminated by heating to 95°C for 10 min followed by cooling in ice. The hydrolysates obtained were assayed to determine the concentration of dissolved protein, the molecular weight of the proteins and the ACE inhibitory activity.

Determination of dissolved protein: The concentration of dissolved protein in the hydrolysates was determined with the Biuret method using a spectrophotometer (Shimadzu, Tokyo, Japan) at a wavelength of 750 nm22. The protein concentration was determined by comparing the absorbance of the sample with the absorbance of a bovine serum albumin (Sigma, St. Louis, USA) standard.

Molecular weight of proteins using SDS PAGE: To determine the molecular weight of the proteins, the extracts were subjected to SDS PAGE according to the method of Laemmli23 using 12% resolving gel and 5% stacking gel at a constant voltage of 140 V for 3 h. Protein bands were visualized by staining with Coomassie Brilliant Blue R-250 (Sigma, St. Louis, USA). The molecular weights of the proteins were estimated by comparison with molecular weight standards.

Inhibition of angiotensin I-converting enzyme activity: The ACE inhibition assays were performed using the method of Cushman and Cheung24 after modification. Briefly, 30 μL of sample was added to 250 μL of 0.1 M borate buffer (pH 8.5) that included 7.6 mM hippuryl-L-histidyl-L-leucine (HHL) (Nacalai Tesque Inc., Kyoto, Japan) as a substrate and 0.608 M sodium chloride. The reaction was started by the addition of 100 μL of 60 mU mL–1 rabbit lung ACE in 0.25 M borate buffer (pH 8.5). To terminate the reaction, 544 μL of 1 M hydrochloric acid (Merck, Darmstadt, Germany) was added. To extract the hippuric acid liberated from the HHL by the ACE, 1.5 mL of ethyl acetate (Merck, Darmstadt, Germany) was added the tubes, which were vigorously shaken and then centrifuged for 10 min at 3,600 rpm. About 1 mL of the ethyl acetate layer was collected into another tube and dried for 10 min at 100°C. The residue obtained was dissolved in 1 mL of 1 M sodium chloride (Merck, Darmstadt, Germany) and the absorbance of the solution was determined at 228 nm. The ACE inhibitory activity was calculated as follows:

Image for - Increased Inhibition of Angiotensin Converting Enzyme (ACE) Obtained from Indonesian Buffalo Meat Protein Using SEP-PAK Plus C18

where, S is the absorbance of the sample (inhibitor) plus ACE (enzyme) and HHL (substrate), C is absorbance of the water (positive control) plus ACE (enzyme) and HHL (substrate) and B is the absorbance of water (negative control) plus ACE (enzyme), HHL (substrate) and hydrochloric acid (added prior to the reaction). The inhibitory activity was expressed as the IC50 (the concentration needed to inhibit 50% of the original ACE activity) of the sample. A decrease in the IC50 indicates an increase in ACE inhibitory activity. The inhibitory activity of the hydrolysate fraction was expressed as a percentage of ACE inhibition.

Purification of hydrolyzed peptides using a SEP-PAK Plus C18 cartridge: The hydrolysates with the highest ACE inhibitory activity were purified using a SEP-PAK Plus C18 cartridge (Waters Co., Milford, MA, USA) and eluted with 2% acetonitrile (Merck, Darmstadt, Germany) and 0.1% trifluoroacetic acid (Merck, Darmstadt, Germany) in water followed by 65% acetonitrile (Merck, Darmstadt, Germany) and 0.1% trifluoroacetic acid in water. The purpose of this step was to remove the non-protein components in each hydrolysate. The filtrates were dried using a freeze dryer and were then diluted with water14.

Statistical analysis: The data on chemical composition and the molecular weight of proteins were analyzed using descriptive statistics, whereas, the data on dissolved protein concentration and ACE inhibitory activities were analyzed using a one-way ANOVA in SPSS v15.0. The results are expressed as the mean±standard deviation. Differences were considered significant at p<0.05. When significant differences were detected, the analysis was followed with a Duncan’s new multiple range test25.


Chemical composition of buffalo meat: The average chemical composition of the buffalo loin consisted of 72.16±0.24% water, 2.44±0.03% fat, 1.46±0.18% collagen and 22.67±0.20% protein and is shown in Table 1.

Dissolved protein content of buffalo meat hydrolysate: The dissolved protein of the hydrolysates of buffalo meat using trypsin (8.09±0.8 mg mL–1) showed a significant increase (p<0.05) compared with the homogenate without hydrolysis (3.67±0.59 mg mL–1), the highest levels of dissolved protein occurred in samples buffalo meat hydrolyzed by pepsin (BMP) (10.91±0.4 mg mL–1). These results are shown in Table 2.

Table 1:Chemical composition of buffalo meat
Image for - Increased Inhibition of Angiotensin Converting Enzyme (ACE) Obtained from Indonesian Buffalo Meat Protein Using SEP-PAK Plus C18

Image for - Increased Inhibition of Angiotensin Converting Enzyme (ACE) Obtained from Indonesian Buffalo Meat Protein Using SEP-PAK Plus C18
Fig. 1:Results of SDS PAGE
  M: Marker, 1: Homogenate, 2: BMP, 3: BMT, 4: BMPT

Image for - Increased Inhibition of Angiotensin Converting Enzyme (ACE) Obtained from Indonesian Buffalo Meat Protein Using SEP-PAK Plus C18
Fig. 2:Determination of the IC50 values
  BMP: Buffalo meat hydrolyzed by pepsin, BMT: Buffalo meat hydrolyzed by trypsin, BMPT: Buffalo meat hydrolyzed by pepsin+trypsin, BMP-P: Purified protein of BMP

Table 2:Dissolved protein contents after hydrolysis of buffalo meat
Image for - Increased Inhibition of Angiotensin Converting Enzyme (ACE) Obtained from Indonesian Buffalo Meat Protein Using SEP-PAK Plus C18
*Different superscripts in the same column indicate significant differences (p<0.05)

Analysis of molecular weights of proteins using SDS PAGE: The homogenate of the non-hydrolyzed samples produced three bands of molecular weights between 40-70 kDa (Fig. 1), whereas, the hydrolysates produced many bands all with a molecular weight below ~40 kDa.

Inhibition of ACE activity: Buffalo homogenate did not inhibit the ACE. The average IC50 of the sample BMP and BMT were 133±18 and 353±48 μg mL–1, respectively, whereas, the sample BMPT was 333±45 μg mL–1 (Fig. 2). However, the purified hydrolysates showed higher ACE inhibitory activities (IC50 = 75.8±20 μg mL–1). The IC50 of the purified hydrolysates was significantly decreased (p<0.05) compared with the non-purified hydrolysates, indicating greater ACE inhibitory activity as a result of this treatment.


The protein content of the buffalo meat was high (22.66%) and the concentration of the protein differed slightly from the value reported by Kandeepan et al.26, who found that frozen buffalo meat contained 20.41% protein. The result suggested that its protein content was higher than other meats such as beef27 (18.48%) and pork28 (20.57%). This high nutritional content makes food products from buffalo meat functionally important for purposes such as antihypertension.

Hydrolysis significantly increased the content of soluble proteins (p<0.05). This increase was due to the hydrolytic process, which is able to break peptide bonds and convert complex peptides to simpler peptides. As a result, the number of peptides increased and the concentration of dissolved proteins also increased. As a result, the hydrolysates from protein hydrolyzed by digestive enzymes showed activities up to 3 times higher compared with the treatment without the enzyme.

The results of the SDS PAGE showed differences among the samples. Homogenized protein had bands at the top of the lane (approximately 70 kDa) and typically had only 3 bands. One of these bands corresponded to homogenate protein as described by Darewicz et al.12, also called the myosin heavy chain (MHC) fragment. These types of peptides are commonly found in muscle or meat. According to Wells et al.29, MHC is a motor protein composed of thick muscle filaments. Most organisms produce many MHC isoforms with regular temporal and spatial expression patterns.

Protein samples that were hydrolyzed by pepsin and trypsin, such as BMP, BMT and BMPT, had more bands of lower molecular weight: Approximately 40 kDa and below. The decrease in the molecular weight of these bands may be due to the process of enzymatic hydrolysis. The hydrolytic process results in changes in peptide structure producing shorter peptides. The shorter the peptide, the more peptides are contained in the solution and the easier the material passes through the pores of the acrylamide gel, thus appearing on the lower part of the gel. The protein band with a molecular weight of 45 kDa was identified as an actin fragment and the three bands of lower molecular weight may correspond to the myosin light chain (MLC) and a tropomyosin fragment. These results are consistent with the results of Martinez et al.30 and Lin et al.31. The electrophoresis results from the BMPT treatment were unclear and barely visible. A similar result was observed by Katayama et al.10. This may have occurred because the concentration of hydrolysate in the lane was too low to either be stained by Commasie Brilliant Blue (CBB) or to remain in the gel.

The IC50 of buffalo meat could not be determined in this experiment. This is because the protein in the buffalo meat retains its complex protein structure and therefore, it has no inhibitory activity as do the bioactive peptides. The ACE-inhibitor is a protein that exists in the food in an inactive form and it therefore requires the enzymatic process to break down the proteins12. The hydrolysates had high ACE inhibitory activity (IC50 = 133±18 μg mL–1), which is consistent with the results of Saiga et al.32 (IC50 = 130 μg mL–1). Moreover, this similarity was paralleled by the similarity in molecular weight of 30 kDa reported by Saiga et al.32, which is comparable to the molecular weight of approximately 40 kDa documented in this study.

The ACE inhibitory activity of BMT (IC50 = 353 μg mL–1) and BMPT (IC50 = 333 μg mL–1) hydrolysates were lower than that of BMP. The ACE inhibitory activity can be affected by differences in the enzyme used for hydrolysis, which will have different effects on the structure of the protein and the products of the hydrolytic process such as the amino acid sequences or oligopeptides. This hypothesis is supported by Katayama et al.9, who stated that different enzymes could produce different amino acid sequences and therefore, the activity of the resulting peptide could be different. Iwaniak et al.33 further argued that the smaller peptides produced by digestion/hydrolysis resulted in stronger ACE inhibition than the larger peptides because they were more suitable for the ACE active sites and therefore, changed activities. Studies by Akillioglu and Karakaya34 and Park et al.35 stated that ACE inhibitors were proteins or peptides that were presented in foods but were generally not present in an active state. Therefore, enzymatic or fermentation processes are required to break down the proteins.

The purified hydrolysate had a higher ACE inhibitory activity (IC50 = 75.8±20 μg mL–1) than the hydrolysate without purification. Yuliatmo et al.14 also revealed that protein purification using SEP-PAK Plus C18 could increase ACE inhibitory activity. Thus, this research confirms the prior discovery that increased ACE inhibitory activity can be achieved using SEP-PAK Plus C18.


Buffalo meat, with its high percentage of crude protein (22.66%), is good source of protein for enzymatic digestion. Hydrolysates from BMP have potential as ACE inhibitors with an IC50 of 133 μg mL–1. The inhibitory activity of ACE was enhanced in purified hydrolysates (IC50 75.8±20 μg mL–1). Protein purification using SEP-PAK Plus C18 increased ACE inhibitory activity.


This study demonstrates that hydrolysates containing peptides can inhibit the activity of ACE and therefore may be a beneficial antihypertensive drug ingredient. Previous studies have already shown that these peptides can be purified from foods, including porcine skeletal muscle proteins, salmon and chicken legs, but the study of buffalo meat as a source of hydrolysates and as a food that can function as an antihypertensive agent has not previously been performed. Current study also reported that buffalo meat is able to produce bioactive peptides and that SEP-PAK Plus C18 can be used to increase ACE inhibitory activity. Researchers can use this study as an initial reference for developing buffalo meat as a functional food, this study also confirms the utilization of SEP-PAK Plus C18 for the purification of hydrolysates to increase the activity of bioactive peptides.


Authors are thankful to the Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, for the Research Grant 2017 that was provided to implement the experiment. The authors also gratefully thank the Ministry of Research, Technology and Higher Education (KEMENRISTEK DIKTI) of the Republic of Indonesia for support through the World Class Professor Program (No. 168.A10/D2/KP/2017).


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