STUDY AND COMPARISON OF AMINO ACIDS IN RED AND GREEN APPLES

STUDY AND COMPARISON OF AMINO ACIDS IN RED AND GREEN APPLES

Maha Abdalla Alnuwaiser

Department of Chemistry, Faculty of Science, Princess Nora Bint Abdul Raman University, P.O. Box 28445, Riyadh, 11437, Saudi Arabia.

ABSTRACT: Apples are rich of vital components to the human health, such as the amino acids, but their presence differ according to the type and colour of the apples. In this study we compared the concentrations of amino acids in the peels and pulps of red and green apples, by using the HLPC for analysis. 16 amino acids were found with different concentrations according to the locality in which they exist in the red and green apples cultivars. The content of amino acids in the pulps was found higher than in the peels in both the red and green apples cultivars. The content of amino acids in the green apples recorded higher value than in the red apples. Higher concentrations of Aspartic acid were found in the peels and pulps of the red and green apples while the content of Methionine acid decreased, which was considered as the lowest amino acid found in the two apples cultivars and Histidine amino acid was not detected in both red and green apples cultivars.

HLPC, Aspartic acid, Glutamic acid, Serine, Methionine

Introduction: The general perception that apples are good for human health, together with the consumer’s increasing demand for functional foods, has encouraged researchers to study in depth the polyphenolic profiles and antioxidant properties of many apple cultivars. It is well known that apples are one of the most important natural sources of polyphenols, exhibiting antioxidant activity, which can potentially prevent chronic diseases. The popularity of apples is not only a result of their taste, but also of their high nutritional value and healthful properties. It is a well-known fact that apples are a rich source of polyphenols ¹.

What is more, the concentration of free phenolics in apples is the highest amongst all fruits and for that reason these Apple juice composition compounds may be assimilated in higher amounts from apples. Phenolics, which are naturally existing antioxidants, demonstrate a variety of biological activities that may protect against the development of some chronic diseases, such as cardiovascular disease, obesity, diabetes, cancer and asthma; they may also have a beneficial influence on preventing premature ageing.

Apples refill the reserves of Vitamins, minerals, phenolic compounds, amino acids, dietary fiber and trace elements in the human body ² therefore; they have been a highly popular snack for a long time throughout the world. The color, soluble sugars and organic acids are important components of fruit taste, and together with the aroma, they have a strong impact on the overall organoleptic quality of fruits.

Currently, red-fleshed apples are receiving increased attention because of their remarkable anthocyanin contents, as it has been reported that anthocyanin contributes more to hydrogen peroxide scavenging than other phenolic compounds in the apple peel ³, and they are very important natural flavonoid compounds for the apple colour.

Anthocyanins are the main pigments responsible for the colour of apples, where in the previous studies; cy3-gal was reported to be the most abundant anthocyanin inthe red apples cultivars ⁴. Moreover, soluble sugars and organic acids also play an important role in fruit coloration. Organic acids could serve to stabilize anthocyanins ⁵. Colour is the most important indicator of maturity and quality in many fruit species. Anthocyanins represent a group of natural flavonoid compounds in plants and are responsible for coloration. Development of red colour of fruits is influenced by genetic factors and various environmental factors.

Amino acids in apple juice are main nitrogen source for yeasts. Many amino acids are the intermediates or precursors of some volatile compounds, especially the production of higher alcohols. Nitrogen compounds can be the limiting factor for yeast growth and activity ⁶. So far, several researchers have studied cider production from dessert apples ⁷.

However, none of these studies have attempted to investigate the change profile of chemical compositions, especially volatile compounds and amino acids during the process. Apples are among the main vegetal food sources rich in phenolic compounds, and the schools health programs indicates that the nutrition of school-age children should be supplemented with vegetal products as apples. Malic acid is the main acid in apple fruit and has an important influence on the sour taste of apples. In cultivars with low amounts of malic acid, the sweet taste becomes predominant.

“Starkrimson” is a black red apple cultivar. It was developed a long time ago but is still widely planted in some regions. In the arid area of the northern Wei River of China, “Starkrimson” is a very popular apple cultivar due to its deep red colour, large fruit, appropriate sugar: acid ratio and well-balanced aroma, which all have a strong impact on the overall organoleptic quality of the fruit.

Where the apples fruits are vital nutrients, as they contain several amino acids, the contents of amino acids in the peels and pulps of the red and green apples were examined by HLPC, and then the concentrations of these acids and their presence were compared between peels and pulps. The red and green apples were also compared in terms of their amino acids contents.

Materialsand Methods:

Sampling Method: Tow cultivars of imported apples were obtained including the red and green apples, as the study aimed to compare amino acids found in the peels and pulps of both red and green apples.

Sample Preparation: The exterior peel of both samples was separated and seeds were removed, so we have two samples of each cultivar for analysis which are the peel and pulps. The samples were crushed using electric blender, and each type of samples was homogenized separately.

Experimental:

• Proportion of water was determined in each sample so as calculate the results of analysis in a form of a dry matter.
• Analysis of amino acids in the four samples using the standard methods AOAC-9772.
• Materials: All the chemicals used in the analysis are of high purity and solid materials were obtained from Sigma Corporation.
• HPLC apparatus was used in determination of amino acids with the following specifications and operating conditions:
• Waters e2695 Separation Module (alliance).
• Waters 2475 FLR detector (Excitation wavelength 250nm and Emission wave-length 395nm).
• Column: AccQ Tag 3.9mm for hydrolyzed amino acids analysis.
• Mobile Phase: Eluent A: Aqueous buffer, Waters AccQ. Tag, Eluent B: HLPC grade acetonitrile, Eluent C: Milli-Q water.
• Preparation of the Samples: According to AccQ Tag Method.

Identification of the chromatographic peaks of different amino acids in the 4 samples was achieved by comparing the retention times and spectral characteristics UV-Vis of them with the peaks of the reference compounds of amino acids. With regard to the quantitative analysis, a calibration curve of amino acids was obtained by the injection of known concentrations of different standard materials of amino acids. The concentrations of amino acids identified in the four samples of apples were within the limits of the calibration curves. 17 amino acids represented in the four samples of the peels and pulps of red and green apples were examined and including: Glutamic acid (Glu), Serine (SER) Aspartic acid (Asp), Proline (Pro), Alanine (Ala), Threonine (THR), Arginine (ARG), Histidine (HIS), Glycine (GLY), Leucine (LEU), Lysine (LYS), Methionine (MET), Valine (VAL), Tyrosine (TYR), Cysteine (CYS), Isoleucine (IEU), Phenylalanine (PHE), as shown in Table 1, Fig. 1.

FIG. 1: HLPC CHROMATOGRAMS OF STANDARD AMINO ACIDS USING THE COLUMN; 3.9×105mm FOR HYDROLYZED AMINO ACIDS ANALYSIS AND USING DETECTOR (EXCITATION WAVELENGTH 250nm and EMISSION WAVELENGTH 395nm)

TABLE 1: CONCENTRATION AND RETENTION TIME OF STANDARD AMINO ACIDS

Results and Discussion: Chemical analyses of amino acids in peels and pulps of the red apples were done by HLPC system. Chromatograms of amino acids in peels and pulps of red apples were obtained, where 16 peaks of amino acids were detected as shown in Fig. 2 in red apples peels and Fig. 3 in red apples pulps, these amino acids include: Glutamic acid (GLU), Serine (SER) Aspartic acid (ASP), Proline (PRO), Alanine (ALA), Threonine (THR), Arginine (ARG), Glycine (GLY), Leucine (LEU), Lysine (LYS), Methionine (MET), Valine (VAL), Tyrosine (TYR), Cysteine (CYS), Isoleucine (IEU), Phenylalanine (PHE). Table 2 shows amino acids content in the red apples peels and Table 3 shows the concentration of amino acids in red apples pulps.

FIG. 2: HLPC CHROMATOGRAMS OF AMINO ACIDS IN THE PEELS OF RED APPLESUSING THE COLUMN; 3.9×105mm FOR HYDROLYZED AMINO ACIDS ANALYSIS AND USING DETECTOR (EXCITATION WAVE-LENGTH 250nm AND EMISSION WAVELENGTH 395nm)

TABLE 2: CONCENTRATION OF AMINO ACIDS IN THE PEELS OF RED APPLES

Fig. 3: HLPC Chromatograms of amino acids in the pulps of red applesusing the column; 3.9×105mm for hydrolyzed amino acids analysis and using detector (excitation wavelength 250nm and emission wavelength 395nm)

Table 3: Concentration of amino acids in the pulps of red apples

Fig. 4: HLPC Chromatograms of amino acids in the peels of green apples using the column; 3.9×105mm for hydrolyzed amino acids analysis and using detector (excitation wavelength 250nm and emission wavelength 395nm)

 TABLE 4: CONCENTRATION OF AMINO ACIDS IN THE PEELS OF GREEN APPLES

Fig. 5: HLPC Chromatograms of amino acids in the pulps of green apples using the column; 3.9×105mm for hydrolyzed amino acids analysis and using detector (excitation wavelength 250nm and emission wavelength 395nm)

TABLE 5: CONCENTRATION OF AMINO ACIDS IN THE PULPS OF GREEN APPLES

From Table 2 it became obvious that Aspartic acid was the predominant amino acid in red apples peels with the concentration of 37.896 ppm, followed by Glutamic acid with the concentration of 28.185 ppm, then Glycine, Serine and Lysine. Methionine was the minority amino acid with the concentration of 2.003 ppm, which is considered as the lowest content in the red apples peels. Histidine was not detected in red apples peels as it was shown in Table 2. The total concentration of amino acids in red apples peels was approximately 204.844 ppm. When examining the amino acids in red apples pulps, high contents were found, and aspartic acid was the predominant amino acid in red apples pulps with the concentration of 76.323 ppm, followed by Glutamic acid with the concentration of 30.050 ppm, then Serine and Glycine. Methionine was the minority amino acid with the concentration of 1.476 ppm, which is considered as the lowest content in the red apples pulps. Histidine amino acid was not detected in red apples pulps as it was shown in Table 3. The total concentration of amino acids in red apples pulps was 226.445 ppm.

When the concentrations of amino acids were compared between the red apples peels, Table 2 and red apples pulps, Table 3 the prominent result was that, the red apples pulps contained Aspartic acid more than twice its content in the peels of the same apples cultivar. Small differences were observed when other amino acids contents in red apples peels and pulps were compared. No statistically significant differences were observed in concentration difference of the remaining amino acids in the red apples peels and pulps. The higher total content of amino acids was found in the red apples pulps more than in the peels.

Moreover, amino acids in green apples peels and pulps were analyzed using HLPC, as it was shown in Fig. 4, 5. The chromatographic profile was complex to the green apples peels and pulps in terms of the numbers of compounds and peak areas. 16 amino acids were extracted with different concentrations as it was shown in Fig. 4 which represents the HPLC chromatogram of amino acids present in the green apples peels, and Fig. 5 which represents the HPLC chromatogram of amino acids present in the green apples pulps.

When amino acids contents in green apples peels were investigated it became obvious from Table 4, which represent the changes in theamino acids concentrations in green apples peels, a significant increase in the concentration of Aspartic acid has occurred which is considered as the main amino acid in green apples peels with the concentration of (108.934 ppm) followed by a Glutamic acid which was present with the concentration of (39.397ppm) then Lysine and Glycine followed by Leucine and Serine acids with nearly similar concentrations. Methionine was the minority amino acid in green apples peels with the concentration of2.089 ppm. Histidine amino acid was not detected in green apples peels. The total concentration of amino acids in green apples peels recorded 318.702 ppm.

When examining the concentration of amino acids in green apples pulps as indicated in Table 4, Aspartic acid was the predominant amino acid in green apples pulps with the concentration of 172.296 ppm, followed by glutamic acid with the concentration of 49.787 ppm, then Glycine followed by Proline and Serine acids with nearly similar concentrations, then Lysine. Methionine was the minority amino acid in green apples pulps with the concentration of1.888 ppm. Histidine amino acid was not detected in green apples pulps. The total concentration of amino acids in green apples pulps recorded 431.59 ppm. When comparing the changes of amino acids concentrations between the green apples peels, Table 4 and green apples pulps, Table 5 the higher content of amino acids was found in the green apples as it was significantly concentrated in the pulps than in peels, where the concentrations of all amino acids in pulps were much higher than in peels and Aspartic acid was the predominant amino acid which represent a higher content in green apples peels and pulps. When comparing the total contents of all amino acids concentrations in the green apples pulps (431.59 ppm), they were found significantly exceeding amino acids concentrations in the green apples peels which recorded (318.702 ppm).

When comparing the red and green apples cultivars, it was found that the green apples outmatched the red apples as they recorded higher content of amino acids in both the peels and pulps, The total concentration of amino acids in green apples reached nearly twice its content in red apples as it recorded 750.292 ppm while the total concentration of amino acids in red apples recorded 431.289 ppm, which revealed to us the importance of this apples cultivar as a valuable source of amino acids. The total amino acids concentration was found higher in pulps than in peels of both red and green apples, which proved the importance of the apples pulps in obtainment of amino acids in both cultivars.

Significance of aspartic acid became evident, as it was considered the predominant amino acid in the peels and pulps of red and green apples, and which was found with higher concentrations especially in the green apples pulps as it recorded 172.296 ppm compared to the concentrations of other acids. Moreover, Glutamic acid showed a relatively high content in both apples cultivars, but a significant decline was observed in Methionine acid compared to the contents of other acids in both apples cultivars. Interestingly, no Histidine acid was found in the peels and pulps of red and green apples. A comparison was carried out between the ratios of amino acids in the peels and pulps of both red and green apples before and after drying as shown in Table 6, Fig. 6. The ratio of amino acids contents in the samples recorded significant increase after drying, where the total ratio of amino acids in the green apples peels recorded 2.41% after drying compared to 0.47% before drying. The total ratio of amino acids in the green apples pulps amounted to 3.06% compared to 0.40% before drying. Further-more, we observed increase in the total ratio of amino acids in the peels and pulps of red apples after drying compared to their ratio before drying as indicated in Table 6, Fig. 6.

Fig. 6: Ratio ofamino acids in peels and pulps of the red and green apples before and after drying

TABLE 6: RATIO OF AMINO ACIDS IN PEELS AND PULPS OF THE RED AND GREEN APPLES BEFORE AND AFTER DRYING

Conclusion: In this study the variation in amino acids content in green and red apples pulps and peels were determined by HPLC system. The results showed the distribution patterns of amino acids in both apples cultivars, as 16 amino acids were present. For both cultivars, the qualitative and quantitative distribution of amino acids varied between the peels and pulps. The high total amino acids content was found in apples pulps. Moreover, amino acids content was by far greater in green apples than in red apples, where this type of apples is considered as incomparable variety and a valuable source of amino acids. Aspartic acids had the higher concentration in both cultivars while Methionine was the minority amino acid in both apples cultivars.

ACKNOWLEDGMENT: The author would like to thank Department of Chemistry, Faculty of Science, Princess Nora bint Abdul Raman University for their cooperation.

CONFLICTS OF INTEREST: Nil.

References:

1. Juranovic CI, Zeiner M, Kröppl M and Stingeder G: Comparison of sample preparation methods for the ICP-AES determination of major and minor elements in clarified apple juices. Microchemical Journal 2011; 99: 364–
2. Wang XQ, Li CY, Liang D, Zou YJ, Li PM and Ma FW: Phenolic compounds and antioxidant activity in red-fleshed apples. Journal of Functional Foods 2016; 18: 1086–1094.
3. Bi XL, Zhang JL, Chen CS, Zhang D, Li PM and Ma FW: Anthocyanin contributes more to hydrogen peroxide scavenging than other phenolics in apple peel. Food Chemistry 2014; 152: 205–209.
4. Solovchenko AE, Merzlyak MN and Pogosyan SI: Light-induced decrease of reflectance provides an insight in the photoprotective mechanisms of ripening apple fruit. Plant Science. 178: 281–288.
5. Pérez-Ramírez IF, Castaño-Tostado E, Ramírez-de León JA, Rocha-Guzmán NE and Reynoso-Camacho R: Effect of stevia and citric acid on the stability of phenolic compounds and in vitro antioxidant and antidiabetic capacity of a roselle (biscus sabdariffa) beverage. Food Chemistry 2015; 172: 885–892.
6. Kelkar S and Dolan K: Modeling the effects of initial nitrogen content and temperature on fermentation kinetics of hard cider. Journal of Food Engineering 2012; 109: 588–596.
7. Alberti A, Vieira RG, Drilleau JF, Wosiacki G and Nogueira A: Apple wine processing with different nitrogen contents. Brazilian Archives of Biology and Technology 2011; 54: 551–558.
   

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   How to cite this article:

   Alnuwaiser MA: Study and comparison of amino acids in red and green apples. Int J Pharm Sci Res 2017; 8(10): 4097-05.doi: 10.13040/ IJPSR.0975-8232.8(10).4097-05.

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