In the present context, the objective of this study was to synthesize and analyze the content of AA of macadamia protein and the impact of hydrogen ion concentration (pH) on AA composition. The determination of AA mainly by cation-exchange chromatography was also investigated. Reproducible and reliable techniques for quantification and identification of AA usually require derivatization. However, techniques such as AA analyzer are composed of cation-exchange chromatography and other components can sideline the derivatization with significant accuracy. The present analysis revealed a higher concentration of essential amino acids especially acidic AA, Glu and Asp and basic AA, Arg than other AA in macadamia protein. The study constitutes first report of use of bubble chart for evaluation of AA and explaination of AAS. The results may elaborate that the degradation of AA of macadamia protein for extraction of pH 11 is caused by the impact of pH. Moreover, the nutritional values of AA present in macadamia protein could change for the better by adjusting pH of extraction.
Macadamia nuts as a natural healthy and nutritious food contain no cholesterol and are a good source of protein [
Amino acids are the building blocks (monomers) of proteins, and 20 different amino acids are used to synthesize proteins. Those requirements in humans were even emphasized on the metabolic availability of amino acids investigated by Rajavel et al. (2009) [
Many methods and approaches have been used for determination of amino acids, such as gas chromatography-mass spectroscopy [
In the present context, the content of amino acids of macadamia protein and the
impact of hydrogen ion concentration (pH) on amino acids composition were investigated. The determination of amino acids was mainly relied upon cation-exchange chromatography.
The residue of Macadamia after extracting oil were obtained from Key Laboratory of Tropical Crop Products Processing Ministry of Agriculture in China (our own laboratory). Hydrochloric acid (HCl) and sodium hydroxide (NaOH) were procured from Guangdong Guanghua Chemical Factory Co., Ltd. (China). HH-W600 electric heated water bath, 752N spectrophotometer, electronic balance, PHS-25 acidity meter, circulating water vacuum pump and low-speed desktop centrifuge were used in this experiment. All other chemicals used were analytical grade for the experiments and analysis.
Box-Behnken Design (BBD) was used to estimate and optimize the experiment for improving the yield of protein. The optimum parameters were materials to water ratios 1: 91, extraction time 2.5 h, extraction temperature 55˚C and pH 9.0. Under the optimized conditions, macadamia protein was extracted and centrifuged at 5000 rpm for 10 min. Then the supernatant was collected and and pH was adjusted to 4.6 to precipitate protein. After incubation at room temperature for one hour, the content was centrifuged again at 5000 rpm for 10 min and the supernatant was discarded. The protein left in the centrifuge tubes was freezing dried. Parallel experiments were conducted with extraction at pH 11. Extracted proteins before and after freezing dried could be found in
The analysis was carried out in a column packed with 4.6 mm ID * 60 mm Hitachi custom
ion exchange resin and connected with EZChrom Elite software. Analysis time: 30 minutes approximately; Reproducibility of peak retention time: CV 0.3% (Arg), 0.5% (Ala); Reproducibility of peak area: CV 1.0% (Gly, His); Detection limit: 3 pmol (S/N = 2, Asp); Spectrophotometer: Aplanatic concave diffraction grating with 570 nm and 440 nm Analyzer; CPU: 32 bits OS, Windows xp; Operating temperature range: 15˚C to 35˚C; Power supply: 100 - 115 V AC/220 - 240 V AC, 800 VA and over, w/in 50/60 Hz ± 0.5 Hz; N2 gas source must be prepared. Amino Acid Analyzer is composed of these parameter and components.
All experiments were performed in triplicate, and statistical analysis of the biological replicates was conducted using Excel or OriginPro 8.5 (www.originlab.com).
The content of amino acids was explained by the following formula:
where A1, A0 represent the peak area of amino acid in sample and standard, respectively; C0 is the concentration of amino acids in sample (nmol/20μl); N: dilution multiple of sample; M: molecular weight of amino acid; W: mass of sample.
The amino acids scores was explained according to the following formula:
where A: content of essential amino acid in sample; B: content of essential amino acid in the FAO/WHO pattern (1973) [
There was no significant difference in the amino acids analysis of the first and second protein samples from extraction at pH 9 or pH 11. The content of amino acids of macadamia protein and the impact of pH on amino acids composition were investigated in
Amino acid | WMa | RTd ± SDb | Name | Height ± SDb | Area ± SDb | nmol/20ul (ESTDc) | Content (%) |
---|---|---|---|---|---|---|---|
Asparagine | 133.1 | 6.350 ± 0.052 | Asp | 1,128,458 ± 30,123 | 15,716,467 ± 479,053 | 15.824 ± 0.483 | 9.672 ± 0.241 |
Threonine | 119.1 | 6.903 ± 0.042 | Thr* | 383,097 ± 12,474 | 5,226,508 ± 161,985 | 5.551 ± 0.172 | 3.036 ± 0.086 |
Serine | 105.1 | 7.530 ± 0.042 | Ser | 795,034 ± 33,284 | 11,503,966 ± 363,067 | 10.726 ± 0.339 | 5.177 ± 0.169 |
Glutamic acid | 147.1 | 8.470 ± 0.033 | Glu | 1,777,937 ± 65,392 | 28,731,041 ± 842,915 | 24.330 ± 0.713 | 16.435 ± 0.357 |
Glycine | 115.1 | 11.990 ± 0.024 | Gly | 455,711 ± 18,037 | 11,834,126 ± 275,591 | 12.712 ± 0.296 | 6.719 ± 0.148 |
Alanine | 89.1 | 12.794 ± 0.009 | Ala | 794,397 ± 26,423 | 10,637,708 ± 282,414 | 8.597 ± 0.228 | 3.518 ± 0.114 |
Cysteine | 240.3 | 13.267 | Cys | 121,031 ± 4392 | 1,578,146 ± 38,772 | 1.181 ± 0.029 | 1.303 ± 0.014 |
Valine | 117.1 | 13.964 ± 0.005 | Val* | 549,259 ± 12,794 | 8,612,192 ± 225,252 | 6.907 ± 0.181 | 3.714 ± 0.091 |
Methionine | 149.2 | 15.357 ± 0.005 | Met* | 101,196 ± 2499 | 2,171,777 ± 56,909 | 0.921 ± 0.024 | 0.631 ± 0.012 |
Isoleucine | 131.1 | 17.624 ± 0.005 | Ile* | 202,741 ± 3784 | 5,842,291 ± 126,612 | 4.916 ± 0.106 | 2.960 ± 0.053 |
Leucine | 131.1 | 18.680 | Leu* | 425,150 ± 12,428 | 9,997,132 ± 294,910 | 10.770 ± 0.317 | 6.484 ± 0.159 |
Tyrosine | 181.2 | 19.353 | Tyr | 351,554 ± 10,670 | 5,687,614 ± 164,320 | 5.161 ± 0.149 | 4.294 ± 0.075 |
Phenylalanine | 165.2 | 20.213 | Phe* | 236,647 ± 5652 | 5,707,902 ± 117,455 | 3.733 ± 0.077 | 2.832 ± 0.039 |
Lysine | 146.2 | 22.453 | Lys* | 416,752 ± 13,561 | 5,881,191 ± 171,025 | 4.179 ± 0.122 | 2.806 ± 0.061 |
Histidine | 155.2 | 24.670 ± 0.004 | His | 122,402 ± 2143 | 2,194,588 ± 8543 | 2.406 ± 0.009 | 1.714 ± 0.005 |
Arginine | 174.2 | 28.597 ± 0.005 | Arg | 497,336 ± 12,041 | 14,813,470 ± 199,486 | 14.722 ± 0.198 | 11.777 ± 0.099 |
Proline | 115.1 | 9.084 ± 0.033 | Pro | 101,328 ± 5146 | 1,726,726 ± 84,991 | 6.034 ± 0.297 | 3.189 ± 0.148 |
Tryptophan | 204.2 | ND | Trp | ND | ND | ND | ND |
Totals | 8,460,027 | 1,478,628,415 | 138.644 | 86.259 |
aWM: Molecular weight of amino acid; bSD: Standard deviation; cESTD: External standard method; dRT: Retention Time; *: Essential amino acids; ND: Not detectable; *P-value < 0.05. Data represent mean ± SD (3 biological replicates).
0.05.
The cation-exchange chromatography analysis of the protein samples of pH 11 revealed the content of 18 AA quantified by cation-exchange chromatography ranged between 4.38 and 117.18 mg/g ,and the highest content was of Glu 117.18 mg/g followed by Arg 78.72 mg/g, Asp 63.58 mg/g and Gly 45.47 mg/g (
Conformationally, constrained amino acids are a useful way of tailoring the rigidity of peptides [
Amino acid | WMa | RTd ± SDb | Name | Height ± SDb | Area ± SDb | nmol/20ul (ESTDc) | Content (%) |
---|---|---|---|---|---|---|---|
Asparagine | 133.1 | 6.347 ± 0.057 | Asp | 698,892 ± 32,892 | 9,748,698 ± 414,652 | 9.815 ± 0.417 | 6.358 ± 0.417 |
Threonine | 119.1 | 6.907 ± 0.057 | Thr* | 260,327 ± 13,711 | 3,623,733 ± 165,286 | 3.849 ± 0.176 | 2.231 ± 0.176 |
Serine | 105.1 | 7.527 ± 0.047 | Ser | 479,449 ± 30,358 | 6,945,418 ± 292,983 | 6.476 ± 0.274 | 3.312 ± 0.274 |
Glutamic acid | 147.1 | 8.467 ± 0.037 | Glu | 1,196,464 ± 68,461 | 19,329,519 ± 730,629 | 16.369 ± 0.619 | 11.718 ± 0.619 |
Glycine | 115.1 | 11.990 ± 0.033 | Gly | 294,886 ± 16,445 | 7,557,786 ± 231,760 | 8.118 ± 0.249 | 4.547 ± 0.249 |
Alanine | 89.1 | 12.797 ± 0.005 | Ala | 567,213 ± 27,605 | 7,605,155 ± 231,748 | 6.147 ± 0.187 | 2.665 ± 0.187 |
Cysteine | 240.3 | 13.273 | Cys | 84,715 ± 2190 | 1,137,223 ± 16,612 | 0.851 ± 0.012 | 0.995 ± 0.012 |
Valine | 117.1 | 13.970 ± 0.004 | Val* | 361,025 ± 15,981 | 5,544,967 ± 190,768 | 4.447 ± 0.153 | 2.534 ± 0.152 |
Methionine | 149.2 | 15.367 | Met* | 67,361 ± 3190 | 1,422,654 ± 59,294 | 0.604 ± 0.025 | 0.438 ± 0.025 |
Isoleucine | 131.1 | 17.644 ± 0.005 | Ile* | 132,890 ± 5524 | 3,808,508 ± 141,624 | 3.205 ± 0.120 | 2.045 ± 0.120 |
Leucine | 131.1 | 18.710 ± 0.004 | Leu* | 284,259 ± 11,044 | 6,467,194 ± 265,396 | 6.967 ± 0.286 | 4.445 ± 0.286 |
Tyrosine | 181.2 | 19.360 ± 0.010 | Tyr | 234,290 ± 9142 | 3,774,921 ± 139,195 | 3.425 ± 0.126 | 3.020 ± 0.126 |
Phenylalanine | 165.2 | 20.220 ± 0.010 | Phe* | 168,127 ± 6912 | 4,090,728 ± 129,761 | 2.675 ± 0.085 | 2.151 ± 0.085 |
Lysine | 146.2 | 22.450 ± 0.014 | Lys* | 276,786 ± 12224 | 3,882,139 ± 151,657 | 2.759 ± 0.108 | 1.963 ± 0.108 |
Histidine | 155.2 | 24.677 ± 0.014 | His | 74,571 ± 1203 | 1,233,249 ± 9257 | 1.352 ± 0.011 | 1.021 ± 0.011 |
Arginine | 174.2 | 28.620 ± 0.010 | Arg | 322,733 ± 6858 | 9,344,176 ± 83,756 | 9.286 ± 0.083 | 7.872 ± 0.083 |
Proline | 115.1 | 9.083 ± 0.042 | Pro | 66,692 ± 5478 | 1,143,911 ± 83,046 | 3.997 ± 0.290 | 2.239 ± 0.290 |
Tryptophan | 204.2 | ND | Trp | ND | ND | ND | ND |
Totals | 5,570,675 | 96,659,974 | 90.3375 | 59.55341 |
aWM: Molecular weight of amino acid; bSD: Standard deviation; cESTD: External standard method; dRT: Retention Time; *: Essential amino acids; ND: Not detectable; *P-value < 0.05. Data represent mean ± SD (3 biological replicates).
on α-amino acid to accelerate hard segment degradation [
In order to evaluate the degradation of AA of macadamia protein caused by the impact of pH, we introduced this new bubble chart for explaination of AAS. The absence of some essential AA in protein causes other AA difficult to be fully utilized and the overall protein digestibility was reduced. Therefore, nutritional value of protein in food depends on species, quantity and proportion of essential AA. The amount of essential AA, nonessential AA and total AA was also shown in the
11 was only 19.882%, 39.672% and 59.554%, the ratio of amount of essential AA and nonessential AA, total AA was 0.50 and 0.33. The latter was more close to the FAO/ WHO protein pattern of reference.
The objective of this study was to synthesize and analyze the content of AA of macadamia protein and the impact of hydrogen ion concentration (pH) on AA composition. The determination of AA and its content mainly by cation-exchange chromatography was also investigated. The E, N and T from extraction at pH 9 was 22.463%, 63.796% and 86.259%, respectively. And E/N, E/T was 0.35 and 0.26, respectively. Compared with AA from extraction at pH 9, the E, N and T from extraction at pH 11 was only 19.882%, 39.672% and 59.554%. However, E/N, E/T was 0.50 and 0.33. The latter was more close to the FAO/WHO protein pattern of reference. Meanwhile, the AAS of Met + Cys, Ile, Thr, Leu, Val + Tyr and total AA was 55.3, 74, 75.9, 92.6, 160.1, 64.2 and 40.9, 51.1, 55.8, 63.5, 111.1, 56.6 respectively. The results may elaborate that the degradation of AA of macadamia protein for extraction of pH 11 is caused by the impact of pH. Moreover, the nutritional value of AA present in macadamia protein could change for the better by adjusting pH of extraction.
Ye, J.Z., Han, Z.P. and Tan, W. (2016) Impact of Hydrogen Ion Concentration on Amino Acids Composition of Macadamia Protein: Approached Us- ing Cation-Exchange Chromatography. Jour- nal of Biosciences and Medicines, 4, 6-14. http://dx.doi.org/10.4236/jbm.2016.410002
AA Amino acids
pH Hydrogen ion concentration
AAS Amino acids scores
ID Inside diameter
WM Molecular weight
CV Coefficient of variation
AC Alternating current
WTO World Trade Organization
FAO Food and Agricultural Organization
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