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Effect of Solvent Type and Germination Time on The Level of Free Amino Acid and Peptides of Germinated Pigeon Pea
(Cajanus cajan (L.) Millsp) Extract
Ketut Ratnayani1*, Ni Wayan Wisaniyasa2, Ni Putu Wiwik Oktayuni1
1Departement of Chemistry, Faculty of Mathematics & Natural Sciences, Udayana University, UNUD Campus Street, Jimbaran Bali, 80361, Indonesia.
2Departement of Food Science & Technology, Faculty of Agricultural Technology, Udayana University, UNUD Campus Street, Jimbaran Bali, 80361, Indonesia.
*Corresponding email: ketut_ratnayani@unud.ac.id Email author 1: ketut_ratnayani@unud.ac.id Email author 2: wisaniyasa_2007@yahoo.com Email author 3: wiwikoktayuni241099@gmail.com
Received 25 January 2022; Accepted 28 July 2022
ABSTRACT
Germination can activate the degradation of storage protein in legumes releasing peptides and free amino acids for seed growth. These compounds have many benefits in many fields, especially in food and health. This study aimed to determine the effect of solvent type and germination time on the level of free amino acids (FAA) and the level of peptides (MW < 10 kDa) of germinated pigeon pea extract. The extraction of free amino acids and the dissolved protein from germinated pigeon pea flour was carried out using two kinds of solvents, namely water, and 0.1 N HCl. The variation of germination times of pigeon pea seeds was carried out at 12, 24, 36, 48, 60, 72, 84, and 96 hours. The level of FAA was determined spectrophotometrically using the ninhydrin method after the deproteination step, while the level of peptides was determined spectrophotometrically using the biuret method. The results showed that using 0.1 N HCl as a solvent produced a higher level of FAA and peptides in the extract than using water. The increase of germination time can increase the level of FAA with the peak achieved in 36 hours. The increase of germination time can increase the level of peptides with the peak of peptides level achieved on 84 hours of germination. These results showed that seed germination is a potential method for producing free amino acids and peptides which accumulate at a specific time.
Keywords: free amino acid, small peptide, germinated pigeon pea
INTRODUCTION
Pigeon pea (“kacang gude” in Indonesian) is one of the underutilized legumes that have the potential to be developed as a substitute for soybeans because it contains complete nutrients that are not inferior to soybeans and is resistant to dry land. Protein levels in pigeon pea is in the range of 15.5-28 % [1]. Like other legumes, globulins are the main protein storage (50-72
%) which is mainly concentrated in the cotyledons of pigeon pea seed.
Various studies have proven that germination has been able to increase the nutritional value of legumes. During the germination process, reactions occur which include hydrolysis, oxidation and synthesis, and mobilization of protein in seeds. At the beginning of germination,
there is an increase in the activity of endogenous protease enzymes which can hydrolyze storage proteins with large molecular weights and produce peptides and free amino acids for seed growth. The germination of pigeon pea has been shown to be able to increase the soluble protein levels in pigeon pea [2]. Yu et al (2020) have analyzed and compared the proteomic and peptide profiles of mung bean seeds and sprouts, and found that both the types and concentration of the peptides in mung bean sprouts were higher than those in seeds [3].
However, there is very limited data on the profile of free amino acid and peptides levels during the germination of pigeon pea. Based on the background, it is necessary to determine the germination time resulting in the accumulation of free amino acid and peptide components during the germination of pigeon pea.
Amino acids and peptides have special importance among the other metabolites since they are not only components of tissues, but also reactive organic compounds which are important regulators of metabolism. Amino acids play vital roles in the central metabolism of seeds. They are primarily utilized for the synthesis of seed storage proteins, but also serve as precursors for the biosynthesis of secondary metabolites and as a source of energy [4]. Free amino acids and small peptides have many benefits, including in the pharmaceutical and medical fields, one of the benefits of amino acids as drug delivery agents and reducing the toxic effects of drugs [5]. Free amino acid supplementation is also beneficial especially for sports athletes and as a component of skin care [6-7]. Small peptides released in the germination process also potential to be used as a source of bioactive peptides which are useful in the food and health fields [8]. Thus, the extraction process of free amino acids and small peptides from germinated pigeon pea flour needs to be optimized. The effectiveness of the extraction of a compound by a solvent is highly dependent on the solubility of the compound in the solvent.
The pH of an aqueous solution can affect the solubility of the solute. By changing the pH of the solution, we can change the charge state of the solute.
This study aimed to determine a good solvent for extracting the free amino acids and small peptides from pigeon pea germinated flour and also to determine the effect of germination time on the level of free amino acids (FAA) and the level of peptides of germinated pigeon pea extract.
EXPERIMENT
Material and instrumentation
Mature and dried pigeon pea seeds were supplied from the local market, Hydrochloric Acid (Merck), Sodium Hydroxide (Merck), Ninhydrin (Sigma), Ethanol (Merck), CuSO4 (Merck), NaKTartrat (Merck), Sodium Carbonate (Merck), Leucine (Sigma-Aldrich), Amicon Ultrafiltration Membrane Filter-MWCO 10 kDa (Sigma-Aldrich). Spectrophotometer (Shimadzu UV 2600), Centrifuge TOMY MX-105.
Procedure
Germination of Pigeon Pea
A total of 1 kg of pigeon pea were soaked for 30 minutes with 0.07 % sodium hypochlorite with a ratio of 1:5. The next stage is the imbibition process by adding warm water at 50 oC with a ratio of 1:3 and leaving it submerged at room temperature for 5 hours. The seeds were then germinated in a box at room temperature with 99 % humidity and sprayed with distilled water every 12 hours. The variation of germination time carried out were 12, 24, 36, 48, 60, 72, 84, and 96 hours. The germinated pigeon pea were stored at -20 oC for at least 24 hours to stop the germination process. The germinated pigeon pea was then oven-dried at 50
oC for 24 hours, peeled to get the cotyledon, ground into flour, and sieved through a 60 mesh sieve. So that the germinated pigeon pea cotyledon flour is obtained.
Extraction of Free Amino Acids and Peptide from Germinated Pigeon Pea Fluor
Solvent selection on the extraction of free amino acid components and peptides was carried out using two types of solvents, namely water solvents and 0.1 N HCl solvent.
Extraction was carried out by mixing germinated pigeon pea flour with a solvent in a ratio of 1:10. The mixture was then homogenized by shaking at 40 oC for 15 minutes. This mixture was then centrifuged at 5000 rpm for 15 minutes at 4 oC. The supernatant obtained was then stored at -20 oC and called germinated pigeon pea extract (GPPE).
Determination of FAA Level of GPPE
Before determining the FAA level of the extract, the deproteination step was carried out using TCA precipitation methods (the soluble protein was precipitated using TCA 24 %, followed by centrifugation and the filtrate pH was set to 5-6) [9]. A total of 0.2 mL of ninhydrin reagent was added to 1 mL of the GPPE sample. The tube was tightly closed with parafilm, then heated at a temperature of 80-100 oC for 4 - 7 minutes until a purple color was formed.
The FAA level was determined by spectrophotometer at a wavelength of 570 nm using leucine as an amino acid standard. The treatment was repeated 3 times.
Determination of Peptide Level of GPPE
Before determining the peptide level of the extract, the large protein content (MW<10 kDa) was eliminated by passing the extract into a 10 kDa ultrafiltration membrane. The filtrate obtained was then measured for its peptide level by spectrophotometric methods using a biuret reagent and tryptone as a peptides standard. As much as 4 mL of Biuret reagent was added to 1 mL of the sample solution in a test tube. The test tube was then shaken and allowed to stand for 30 minutes at room temperature, then read the absorbance at 540 nm. The treatment was repeated three times [10].
RESULT AND DISCUSSION
The Effect of Solvent Type on The Level of FAA and Peptide of GPPE
Table 1. The Level of FAA and Peptide Using Two Kinds of Solvent of GPPE
Data is expressed as means ± standard deviation
In this solvent selection stage, two types of solvents were used to extract free amino acids and peptides from germinated pigeon pea flour, namely water and 0.1 N HCl, using the same germination time of 3 days (72 hours). Based on Table 1, showed that the levels of free amino acids and peptides (MW<10 kDa) in the 0.1 N HCl extract were higher than in the aqueous extract. These data showed that the use of 0.1 N HCl solvent can increase the solubility of free amino acids and peptides so they can easier be extracted from the germinated pigeon pea flour. Based on Table 1, it also showed that the use of 0.1 N HCl as a solvent resulted in a 7 times higher level of free amino acid than water solvent, while the levels of small peptides Parameters Water Extract (mg/mL) 0.1 N HCl Extract (mg/mL) Free Amino Acids Level 3.1643 ± 0.0360 21.7256 ± 0.0412 Peptide Level (MW<10 kDa) 9.3080 ± 0.3395 10.898 ± 0.1694
only increased slightly. This data showed that the use of HCl as a solvent is more effective in increasing the solubility of free amino acids compared to the solubility of peptides. This is due to the solubility of amino acids in an aqueous solution being affected by the pH of the solution because amino acids dissociate into different ionic forms in aqueous solution: zwitterions, cationic and anionic species. These are not only different forms in chemical equilibrium with each other but also with hydrogen ions in an aqueous solution [11]. The use of 0.1 N HCl solvent with a pH of 1.1 can cause an increase in the solubility of amino acids, considering that this pH value is quite far from the isoelectric point range of the amino acids contained in pigeon pea which is 3.2-10.8. The closer the pH of the solution to the isoelectric point of a compound (amino acid/peptide/protein), the lower the solubility of the compound. At low pH (acidic atmosphere), amino acids will be positively charged while at high pH (alkaline atmosphere) will be negatively charged. Amino acids can be dissolved if they are given excessive acid treatment, this happens because of positive ions in HCl which causes amino acids that were originally neutral or zero to become positively charged which causes increased solubility. One type of proteogenic amino acid that cannot form a purple complex with ninhydrin reagent is proline, so the presence of proline in the extract cannot be quantified in the analysis of free amino acid levels carried out in this study. Proline contains no primary amino group, which is not involve producing the colored ninhydrin chromophore called Ruhemann’s purple (λ max 570 nm) [12].
The solubility of a peptide in an aqueous solution is affected by the amount and type of ionic charge in the peptide molecule. This is influenced by the number and composition of the amino acids composing of peptide. Some peptides easily dissolve in aqueous solutions, but a common problem encountered is very low solubility or even insolubility, especially peptides with long sequences of hydrophobic amino acids. The use of 0.1 N HCl solvent with a pH of 1.1 was able to increase the solubility of peptides compared to water solvents, possibly due to excess acid conditions causing the –NH2 group to be protonated so that it would be positively charged. In addition, the pH value of 1.1 is probably the pH value of the solution which is far from the isoelectric point of the peptides component contained in the germinated pigeon pea flour. Ghodsvali et al. (2005) reported that the isoelectric point of the rapeseed proteins was in the range of pH 4.5 to 5.5 [13]. Antioxidant peptides of flaxseed have a broad range of isoelectric points (3.7 – 10.8) [14].
The Effect of Germination Time on Free Amino Acid Level of the GPPE
According to Figure 1., it can be seen that the increase in germination time was able to increase the free amino acid level in the extract with fluctuation and reached its peak at 36 hours of germination. This is because at the beginning of germination there is an intensive degradation of storage protein to meet the need for free amino acids as building blocks to build new plant proteins. This is reinforced by the study of Ramakrishna and Rao (2005) which stated that there was an increase in protease activity in Indian beans (Dolichos lablab L. var. lignosus) at the early of germination (reached a peak on the 4th day) and decrease later [15].
Figure 1. Germination Time vs Free Amino Acid Levels of GPPE
It can also be noted a significant increase in free amino acid levels between the 1st day and 2nd day (36 hours) of germination. The reason for the peak accumulation of the FFA on 36 hours germination maybe due to it was the time when the activity of exopeptidase reach its peak in degradation of peptide and produced free amino acid. These results were similar to the profile of the total free amino acid levels in the germination of Cedrela fissilis Vellozo (Meliaceae) seeds which increased at the beginning of germination and there was a significant increase in levels at germination on the 2nd day [16].
The Effect of Germination Time on The Peptides Level of the GPPE
According to data in Figure 2. it can be seen that the increase in germination time was able to increase the peptide level in the extract starting from 24 hours, which reached its peak at 84 hours of germination. This is due to there was increasing protease activity at the early of germination and then it was decreased, such as the study found in the study of protease enzyme activity in Indian bean germination. The presence of disulfide bonds in storage protein will slow down the protein degradation process and thereby prevent uncontrolled loss of nitrogen reserves. The cleavage of a specific peptide bond on the storage protein substrate that occurs early in germination produce a high molecular peptides [17].
Figure 2. Germination Time vs Peptide Levels of GPPE
0 1 2 3 4 5
0 20 40 60 80 100 120
FFA Level (mg/mL)
Germination Times (hours)
0 0.2 0.4 0.6 0.8 1 1.2 1.4
0 20 40 60 80 100 120
PEPTIDES LEVEL (MG/ML)
GERMINATION TIMES (HOURS)
A similar pattern was also found in soybean germination, it was found that there was an increase in peptide levels starting on the 2nd day and decreasing on the 6th day of germination [16]. The profile of peptide levels obtained in this study is also in accordance with similar studies that have been carried out previously, Ratnayani et al (2019) have investigated the effect of germination time on peptide levels of GPPE using 24 hours of time interval and found that there was an increase in peptide levels starting on the 2nd day and reached its peak on the 3rd day of germination (72 hours) [18]. Considering this study used a germination time interval of 12 hours, and found a peak in peptide levels at 84 hours (between 3rd and 4th day). In this study, it was found that 12 hours after the 3rd day (72 hours) there was still an increase in peptide levels and decreased after 96 hours (the 4th day). Thus the profile of peptide levels obtained in this study is more detailed than the previous study.
CONCLUSION
The type of solvent which is better in extracting free amino acids and small peptides (size
> 10 kDa) from germinated pigeon pea flour was 0.1 N HCl solvent because it produces a higher amino acid level than water solvent in the extract. The increase in germination time was able to increase the free amino acid level in the extract which reached its peak at 36 hours of germination. The increase in germination time was able to increase the peptide level in the extract starting from 24 hours and reached its peak at 84 hours of germination. This result showed that germination of pigeon pea seed is a potential process in producing free amino acid and peptides naturally, and this potency can be more optimized later in the germination step and extraction step.
ACKNOWLEDGMENT
The present work was financially supported by the PUU-PNBP UNUD Grant 2021 (Based on the Research Implementation Agreement Letter, Number: B/99-57/UN14.4.A/PT.01.05/2021, May-03- 2021).
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