Amino Acids Composition of V. Doniana and S. Comorensis Fruits

doi:10.21203/rs.3.rs-4157856/v1

Download PDF

Research Article

Amino Acids Composition of V. Doniana and S. Comorensis Fruits

https://doi.org/10.21203/rs.3.rs-4157856/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

The amino acids composition of S. comorensis and V. doniana fruits were investigated in this study. The analysis was done using Liquid Chromatography - Mass Spectrometry (LC-MS). It was found that the various essential amino acids in S. comorensis fruits ranged from 0.01 ± 0.18 mg/kg to 55.01 ± 2.15 mg/kg and V. doniana fruits ranged from 0.023 ± 0.00 mg/kg to 88.64 ± 0.87 mg/kg. For each of the plant fruits there were no significant differences in terms of amino acids amounts between the two regions (P > 0.05) except for phenylalanine in V. doniana. The phenylalanine content in V. doniana ranged from 51.36 mg/kg to 53.14 mg/kg for the fruit samples from Pwani and 39.61 mg/kg to 39.87 mg/kg for the fruit samples from Tanga with a significant difference between the two regions {P < 0.05). Leucine was the most abundant essential amino acid in S. comorensis fruits with a range between 44.78 mg/kg and 51.42 mg/kg for the fruit samples from Tanga region and 52.86 mg/kg to 57.16 mg/kg for the fruit’s samples from Pwani region with no significant difference (P > 0.05) in content between two regions. It was notable that lysine, histidine and threonine ware not detected in any of the samples from the two species. The study shows that these fruits are potential essential amino acids food sources and can complement with other sources to make a balanced diet for a healthy human body.

Nutritive values

Essential Amino Acid (EAA)

Wild fruits

Food

Health

Wild fruits contribute to food security, health and income generation in some local communities in Tanzania ¹. However, the nutritional value of wild fruits in terms of amino acids is poorly documented and not recognized as important in human nutrition and health. Amino acids are generally the most abundant macromolecules in biological systems ² that serve as a precursor of many enzymes, hormones, neurotransmitters, nucleic acid as well as molecules of life ². These essential amino acids such as histidine, valine, methionine, threonine, leucine, lysine and tryptophan, phenylalanine and isoleucine presented in figure 3:1 can only be obtained from various dietary sources including fruits ³. Some of the essential amino acids such as histidine, methionine, lysine and tryptophan act as antioxidants ⁴. The deficiency of amino acids in human body can cause cell destruction, slowdown of development and growth in children and sometimes causes degenerative diseases ⁴^,⁵. Almost all the amino acids consumed by man are initially synthesized in the plants ⁶ which are needed by the body ⁷. Some amino acids from plants are lost during food preparation either through cooking or industrial processes. It is therefore recommended to supplement the loss by consuming a variety of wild fruits ⁸.

The nutritional value of dietary protein sources may not be predicted from a determination of its essential amino acids content alone, as there are other factors that can affect the utilization of the protein. A mixture of plant essential amino acids can adequately meet human nutritional requirements and therefore consumers do not need to be concerned about amino acid imbalances when plant food proteins are part of the diets ⁹. This study is reporting the amino acids profile in S. comorensis and V. doniana wild fruits from the coastal forests of Tanzania.

3.2.1 Wild Fruit Sample Collection and Processing

The samples of S. comorensis and V. doniana fruits were taken randomly from different coastal forests of Pwani and Tanga regions in Tanzania. Total of 240 Fruit samples of S. comorensis were collected randomly from four areas that included Kibiti (60 fruits), Ngomboloni (60 fruits), Mkuranga (60 fruits) and Nyakikai (60 fruits) in the coastal forests. From Tanga region a total of 180 fruits samples were taken randomly from different local markets that obtained them from nearby coastal forests. V. doniana fruits were also collected from four areas that included Kibiti, Ngomboloni, Mkuranga and Nyakikai in the coastal forests. From Tanga region the fruits were taken randomly from different local markets that obtained them from nearby coastal forests.

The pulps and seeds were removed by using a metal spoon. The pulp together with seeds were put in a dish and blended manually to separate the pulp and seeds. The seeds with residual pulp were dried in an oven at 60 ^oC for 5 hours to completely separate seeds from residue pulp. The seeds were separated by peeling off the pulp residue and the edible parts were homogenized using mortar and pestle. The edible part of the fruits (mesocarp) was dried in shades for two weeks then taken to the oven overnight 65 ^oC. After drying, the sample was ground to a powder using a grinding machine found at zoology department, university of Dar es salaam.

3.2.2 Amino Acid Analysis

The amino acid composition in fruit samples were determined by the method described by Spackman et al. (1958) with modification. Fifty grams of the sample fruit pulp was oven dried at 105 ^oC. Five gram of dried sample fruit was defatted with chloroform/methanol (2:1) mixture for 15 hours, using Soxlet extractor (Association of Official Analytical Chemists 1995). One gram of the defatted sample was weighed into a quickfit test tube and 7 mL of 6 M HCl added. In order to prevent possible oxidation of some amino acids during hydrolysis, oxygen was expelled by passing nitrogen into the quickfit test tube. The quickfit test tube was then closed and dried at 105 ± 5 ^oC for 22 hours. The test tube was opened after cooling, and the content was filtered. The filtrate was evaporated to dryness at 40 ^oC under a vacuum rotary evaporator and residues were dissolved in 5 mL of acetate buffer (pH 2.0) and put in the freezer at 20 ^oC. The Liquid Chromatography Mass Spectrometry (LCMS) present at Government Chemist Laboratory Authority (GCLA) was used for determination of essential amino acids (valine, histidine, leucine, isoleucine, threonine, phenylalanine, methionine, tryptophane and lysine) in fruit samples of S. comorensis and V. doniana.

3.2.3 Preparation of Standard

The nine essential amino acids (leucine, lysine, methionine, phenlyalalanine, threonine, histidine, isoleucine, tryptophan and valine) were dissolved into 0.1 M HCl to make final concentration of 1, 2, 3, 4 and 8 g/mL for each amino acids (Appendix 3), this was done to prepare calibration standards. Individual solutions of each of the essential amino acids were prepared from weighed standards at a concentration of 5 g/mL in 0.1 M HCl for optimization of instrument parameters (Le et al. 2014).

3.2.4 Instrument Settings and Sample Analysis

Thermo Fisher LC-MS/MS UltiMate 3000 was used for amino acids analyses in fruits samples. The column used was Hypersil Gold aQ with column length (150 mm), internal diameter (4.6 mm), particle size (1.9 m). The column temperature was 40 ^oC, flow rate 0.35 mL/min, and nebulizer pressure of 60 psi. The mass spectrometer was Q-exactive with collision energy of 45 eV and scanning range of 50–750 m/z. The chromatographic condition was developed as gradient elution and used for amino acids analysis as summarized in Table 3.1

Table 3

1: Chromatographic Condition for Amino Acid Analysis
Time (mn)	Phase A (%)	Phase B (%)
00.00	97.7	2.3
3.00	97.6	2.4
6.00	95.0	5.0
8.00	10.0	90.0
10.00	10.0	90.0
10.50	95.0	5.0
15.00	92.0	2.0

After suitable chromatographic conditions were set, the test samples (5 µL each) were introduced into the LC-MS/MS by serial analysis starting with the blank (0.1 M HCl) followed by all essential amino acid’s standards and then samples. The mobile phase used were 1 mM Tridecafluoroheptanoic acid in water as phase A and Tridecafluoroheptanoic acid (TDFHA) in acetonitrile as phase B (Le et al. 2014).

3.2.5 Data analysis

Data was analyzed using t-test available in statistical software Paleontological Statistics (PAST) version 2.17. The significant different was considered significant when P > 0.05.

The LCMS chromatogram of standard mixture of amino acid and standard profile with retention time is shown in Table 3.2. The identification and quantification of the essential amino acids were done based on the comparison in the retention time between individual fruit sample and amino acid standard. Data on the amino acid profile in the two wild fruits is summarized in Table 3.3 and Table 3.4. A total of six essential amino acids (valine, leucine, isoleucine, phenylalanine, methionine and tryptophan) were identified in S. comorensis and V. doniana fruits while three essential amino acids (lysine, histidine and threonine) were not detected.

The amounts of essential amino acids in S. comorensis fruits samples from Pwani and Tanga were not significantly different (P > 0.05) based on two sample t-test. The most abundant essential amino acids in S. comorensis fruits were leucine which ranged between 44.78 mg/kg and 51.42 mg/kg for the fruit samples from Tanga and 52.86 mg/kg and 57.16 mg/kg for the fruit’s samples from Pwani. Similarly, the differences in leucine content in S. comorensis fruits between the two regions was not significant (t = 2.4994, df = 1, P > 0.05). Tryptophan (Tanga 0.023 ± 0.00 mg/kg, Pwani 0.024 ± 0.00 mg/kg) was the lowest detected in S. comorensis. Except for valine, the other essential amino acid identified in S. comorensis fruit samples from Tanga; 48.1 ± 3.32 mg/kg, 3.18 ± 0.13 mg/kg, 21.23 ± 1.26 mg/kg, 21.32 ± 0.52 mg/kg and 0.023 ± 0.00 mg/kg for leucine, methionine, phenylalanine, tryptophan and isoleucine respectively (Appendix 4 and 5) were comparatively lower than those from Pwani. Conversely, the content of valine was comparatively higher in samples from Tanga (24.62 ± 0.77 mg/kg) than samples from Pwani (0.01 ± 0.18 mg/kg). These differences are however not statistically significant based on two sample t- tests (P > 0.05).

Samples of V. doniana fruits from Pwani had higher amino acids content than those from Tanga region. The differences were, however, not significant (t= -1.3082, df = 1, P > 0.05) except for phenylalanine content which was significant (t = 19.581, df = 1, P < 0.05). The lowest detected amino acid in V. doniana from both regions was tryptophan (0.023 ± 0.00 mg/kg from Pwani and 0.025 ± 0.00 mg/kg from Tanga). The highest detected amino acid in V. doniana from both regions was leucine (88.64 ± 0.87 mg/kg from Pwani and 74.94 ± 1.31 mg/kg from Tanga).

Leucine, methionine, phenylalanine and isoleucine were the highest detected essential amino acids in all fruit samples. On the other hand, tryptophan was the least detected in all samples.

Table 3

2: Amino Acid Profile of Essential Amino Acids Standards.
AMINO ACID	RETENTION TIMES	CONCENTRATION (ppm)
Histidine	3.07	8
Isoleucine	2.77	8
Leucine	3.47	8
Lysine	2.38	8
Methionine	3.84	8
Phenylalanine	6.07	8
Threonine	2.4	8
Tryptophane	11.35	8
Valine	2.70	8

Table 3

3: Amino Acid Profile of Essential Amino Acid in *S. comorensis* fruits.
Essential Amino Acids	S. comorensis from Tanga.	S. comorensis from Pwani	t-value	p-value	Conclusion
	mg/kg
Valine	24.62 ± 0.77	0.01 ± 0.18	1.4261	P > 0.05	Not significant
Leucine	48.1 ± 3.32	55.01 ± 2.15	2.4994	P > 0.05	Not significant
Lysine	ND	ND	-	-	-
Methionine	3.18 ± 0.13	4.3 ± 0.06	3.8865	P > 0.05	Not significant
Phenylalanine	21.23 ± 1.26	24.87 ± 0.21	3.5874	P > 0.05	Not significant
Histidine	ND	ND	-	-	-
Isoleucine	21.32 ± 0.52	21.64 ± 0.28	0.7765	P > 0.05	Not significant
Tryptophan	0.023 ± 0.00	0.024 ± 0.00	1.3885	P > 0.05	Not significant
Threonine	ND	ND	-	-	-
t(0.05) (2) 1 = 12.706 ND = Not Detected

Table 3

4: Amino Acid Profile of Essential Amino Acid in *V. doniana* fruits.
Essential Amino Acids	V. doniana from Pwani.	V. doniana from Tanga.	t- value	P- value	Conclusion
	mg/kg
Valine	45.42 ± 0.81	38.45 ± 1.61	5.474	P > 0.05	Not significant
Leucine	88.64 ± 0.87	74.94 ± 1.31	12.297	P > 0.05	Not significant
Lysine	ND	ND	-	-	-
Methionine	18.11 ± 0.49	11.24 ± 4.08	2.3618	P > 0.05	Not significant
Phenylalanine	52.25 ± 0.89	39.74 ± 0.13	19.581	P < 0.05	Significant
Histidine	ND	ND	-	-	-
Isoleucine	31.83 ± 0.56	33.2 ± 1.22	1.4424	P > 0.05	Not significant
Tryptophan	0.023 ± 0.00	0.025 ± 0.00	1.3082	P > 0.05	Not significant
Threonine	ND	ND	-	-	-
t(0.05) (2) 1 = 12.706 ND = Not Detected

A number of studies have been done on the amino acids profile of wild fruits and revealed their nutritional richness which in some cases surpass cultivated exotic fruits ¹⁰. This study has shown that S. comorensis and V. doniana fruit are good sources of essential amino acids notably, leucine, methionine, phenylalanine and isoleucine. The other amino acids are present in moderate amounts and some (histidine, lysine and tryptophan) were not detected. Essential amino acids have crucial roles in metabolism.

Methionine, which is one of the essential amino acids present in the two fruits investigated and acts as an antioxidant which helps to scavenge free radicals in the body ⁴. Valine in S. comorensis was higher than those reported for the exotic fruit Mangifera indica which are 5.8 g/100g ¹¹ and 3.8 g/100g ¹². Other wild fruits have also higher valine content ⁵ in Edestus giganteus (17.85%), Solanum melongena (27.05%), Tetrapleura tetraptera (15.85%) and Phoenix dactylifera fruits (1.92 g/100g) ¹³. This confirms the superiority of these fruits over exotic ones. Valine is an important amino acid since it is involved in the regulation of the immune system.

The amount of isoleucine in S. comorensis was also higher compared to some exotic fruits such as Carica papaya (2.60 g/kg) ¹⁴ and M. indica (4.4 g/100g) ¹¹. Also, other wild fruits such as Aframomum danielii (4.83 g/100g) ⁵ reveal wild fruits to have higher amount of isoleucine. Deficiency of isoleucine can cause physical and mental disorder to human ⁵. Leucine, isoleucine and valine play a role in promoting muscle function, bones and skin ^5,7.

Phenylalanine is used in the brain for producing norepinephrine that transmits signals between nerve cells. Phenylalanine regulates human mood and is used for treatments of various diseases such as Parkinson’s diseases, depression and obesity ^5,15.

S. comorensis fruits from Pwani were found to have higher amounts of essential amino acids (except for valine) compared to fruits from Tanga. These differences can be attributed to environmental factors like soil fertility and geographic location. Statistically, however, there were no significant differences between the two regions (P > 0.05).

Similar to S. comorensis, the valine content of V. doniana fruits, were higher than some popular exotic fruit ¹⁶ in M. indica (5 g/100g) and Umesh (2017) in C. papaya which had 3.05 g/kg. The valine content in V. doniana are also comparable to other wild fruits such as Azanza garckeana (6.00g/100g) ^7,17.

Leucine was the most abundant essential amino acid in V. doniana fruits samples and was found to be higher than the exotic Opuntial ficus-indica. It has also been reported in higher amounts in other wild fruits such as A. garckeana fruits (12.01 g/100g) and its leaves (12.97 g/100g) ¹⁷.

Isoleucine contents in V. doniana fruits from both Pwani and Tanga regions were higher than exotic fruits such as Mangifera indica (4.4 g/100g) ¹¹, 4.0 g/100g ¹⁶ and 3.6 g/100g ¹². Other wild fruits such as Aframomum danielii (4.83 g/100g) ⁵, 4.98 g/100g in A. garckeana fruits and 5.00 g/100g in A. garckeana leaves ¹⁷.

In general, the concentration of amino acid in S. comorensis and V. doniana detected in these two wild fruits are higher than in some popular domesticated exotic fruits as also reported by others. For example, ⁶ observed lowest amount of methionine (1.255 ± 1.3 µmol/100g) and the highest amount of leucine (59.80 ± 17.73 µmol/100g) in Musa acuminate. Citrullus lanatus recorded lowest amount of methionine (2.524 ± 0.141 µmol/100g) and the highest composition of leucine (13.18 ± 0.176 µmol/100g), Fragaria ananassa recorded lowest amount of isoleucine (2.356 ± 1.764 µmol/100g) but highest in amount of leucine (15.34 ± 1.230 µmol/100g). These above mentioned domesticated exotic fruits have lower composition of essential amino acids compared to wild fruits.

Through both S. comorensis and V. doniana are excellent source of essential amino acid V. doniana fruits have higher quantities than S. comorensis fruits. This may be due to the nature of the plants on the uptake and absorption of nutrients from the soil in which the plants were grown.

The analysis of amino acids contents in S. comorensis and V. doniana indicates that the fruits are good sources of proteins and essential amino acids. In some cases, they are even superior to the commonly used exotic fruits. It is hence important to encourage effective consumption these fruits. It can be concluded that, when these wild fruits are taken regularly and/ or in combination with other fruits oo other protein sources, may contribute or complement to a better diet beneficial to human body health.

Author Contribution

Name of the author with designation Email Contribution Dr. Clarence Mgina [email protected] Editor, Field Supervisor, data analysisMr. Dominic Charles [email protected] Writing, collecting data, analysis and Interpretation. All authors reviewed the manuscript.

Bharucha. Z, Pretty. J. 2010. The roles and values of wild foods in agricultural systems. Philosophical Transactions of Royal Sociaty. B Biological. Science. 365, 2913–2926.
Talole, B. 2013. A c a d e m i c S c i e n c e s. International Journal of Pharmaceutical Science 5, 3–6.
Chakravorty, J., Ghosh, S., Megu, K., Jung, C. & Meyer-Rochow, V. B. 2016. Nutritional and anti-nutritional composition of Oecophylla smaragdina (Hymenoptera: Formicidae) and Odontotermes sp. (Isoptera: Termitidae): Two preferred edible insects of Arunachal Pradesh, India. Journal Asia Pacific Entomology. 19, 711–720.
Okada, Y. & Okada, M. 1998. Scavenging Effect of Water Soluble Proteins in Broad Beans on Free Radicals and Active Oxygen Species. Journal of Agriculture and Food Chemistry. 46, 401–406.
Bouba, A. A. 2016. Amino Acid and Fatty Acid Profile of Twenty Wild Plants Used as Spices in Cameroon . Am. J. Food Science and Technology 2016, Vol. 4, No. 2, 29-37 4, 29–37.
Tabuti, J. R. S., Dhillion, S. S. & Lye, K. A. 2004. The status of wild food plants in Bulamogi County, Uganda. International Journal of Food Science and Nutrition. 55, 485–498.
Mohanty, B. 2014. Amino Acid Compositions of 27 Food Fishes and Their Importance in Clinical Nutrition. Journal of Amino Acids 2014, 1–7.
Gerloff, E. 1965. Amino Acid Composition of Leaf Protein Concentrates. 139–143.
Young, V. R. & Pellett, P. L. 1994. Plant proteins in relation to human protein and amino acid nutrition. American Journal of Clinical and Nutrition. 59.
Song, X.. 2021. Analysis, occurrence, and potential sensory significance of tropical fruit aroma thiols, 3-mercaptohexanol and 4-methyl-4-mercapto-2-pentanone, in Chinese Baijiu. Food Chemistry. 363, 130232.
Abdalla, A. A. 2017. Survey on the Moisture and Ash Contents in Agricultural Commodities in Al-Rass Governorate , Saudi Arabia. 2017, 55–62 (2017).
Elegbede, J. A. T 1995. Composition of Mango ( Mangnzfem Indica ) Seed Kernel From Nigeria. Journal of Food Biochemistry. 19, 391–398.
E, Y. S. 2015. Nutritional and anti-nutritional composition of date palm (Phoenix dactylifera L.) fruits sold in major markets of Minna Niger State, Nigeria. African Journal of Pure Applied Chemistry. 9, 167–174.
Umesh, M., Priyanka, K., Thazeem, B. & Preethi, K. 2017. Production of Single Cell Protein and Polyhydroxyalkanoate from Carica papaya Waste. Arabic Journal of Science and Engineering. 42, 2361–2369.
Fernstrom, J. D., Fernstrom, M. H. 2007. Tyrosine, phenylalanine, and catecholamine synthesis and function in the brain. Journal of Nutrition. 137, 1539–1547.
Arogba, S. S. 1997. Physical, chemical and functional properties of Nigerian mango (Mangifera indica) kernel and its processed flour. Journal of Science and Food Agriculture. 73, 321–328.
Nkafamiya, I., Ardo, B., Osemeahon, S. & Akinterinwa, A. 2016. Evaluation of Nutritional, Non-nutritional, Elemental Content and Amino Acid Profile of Azanza garckeana (Goron Tula). British Journal of Applied Science and Technology 12, 1–10.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Amino Acids Composition of V. Doniana and S. Comorensis Fruits

Status:

Version 1

Abstract

Figures

Introduction

Material and Methods

3.2.1 Wild Fruit Sample Collection and Processing

3.2.2 Amino Acid Analysis

3.2.3 Preparation of Standard

3.2.4 Instrument Settings and Sample Analysis

3.2.5 Data analysis

Results

Discussion

Conclusions

Declarations

Author Contribution

References

Additional Declarations

Status:

Version 1