Determination and Analysis of Amino Acid Content in Maca from Different Habitats and Varieties

Yuting LI, Ziyi LI, Lin JIANG, Deli XIAO,3*

1. Department of Pharmacology, Yongzhou Vocational Technical College, Yongzhou 425000, China; 2. Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, China; 3. Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing 210009, China

Abstract [Objectives] To determine and analyze amino acid content in Maca from different habitats and varieties. [Methods] Maca from two habitats and three colors were selected and a kind of turnip commonly used was to fake Maca. After optimizing the hydrolysis conditions, 100 mg Maca samples were hydrolyzed in the concentration of 6 mol/L hydrochloric acid at 110 ℃ for 24 h. The content range of amino acids in each sample was detected by HPLC. [Results] The average total content of amino acids in Maca was 9.52 g/100 g. Yunnan yellow Maca contained 16 kinds of amino acids, including 7 kinds of human essential amino acids, accounting for 17.2% of the total amino acids while Yunnan purple Maca held 16 kinds of amino acids, counting 7 kinds of human essential amino acids, accounting for 16.5% of the total amino acids. The amino acid ratio coefficient method also showed that the two kinds of Maca had higher nutritional value than the others.15 kinds of amino acids were detected in Yunnan black Maca, including 6 kinds of essential amino acids, accounting for 9.60% of the total amino acids. Among amino acids in Yunnan Maca, the highest content was Arg. A total of 12 kinds of amino acids, together with 6 essential human amino acids, were determined in Peru black Maca, accounting for 11.6% of the total amino acids. In Peru black Maca the highest concentration of amino acid was Pro. It was noteworthy that the amino acid species were comprehensive in Maca, and high level of amino acids resulted in high nutritional value. Its specific amino acid composition ratio was obviously different from Turnip, the counterfeit goods to deceive customers. [Conclusions] The method of automatic amino acid analyzer is simple and accurate, and can be used for the identification and content determination of amino acids in Maca as amino acid is an essential nutrient in Maca.

Key words Maca, Habitats, Varieties, Amino acid content

1 Introduction

Maca is a rare alpine and edible plant native to the Andes of Peru. It has been cultivated for at least 2 000 years at altitudes of up to 3 500-4 500 m, which brings it robust tolerance to extreme environmental stresses like rocky formations, intense sunlight, strong winds, and other extreme weather conditions[1-3]. Since 1992, Maca has been widely promoted by the Food and Agriculture Organization (FAO) of the United Nations throughout the world. The study of Maca is progressively popular in the pharmaceutical field. In recent years, as functional foods and natural health products, Maca has a broad application prospect, and can be seen in worldwide markets and attracts increasing attention.

Reputed as "South American Ginseng" throughout the world, Maca has a high nutritional value, not only being rich in nutrients, such as proteins, essential amino acids, various minerals, and numerous vitamins, but also comprehending unsaturated fatty acid, for instance, linoleic acid and linolenic acid, and secondary metabolism active composition like alkaloids (macamides), sterols, macaenes, glucosinolates, and polyphenols[4-12]. These nutrients warrant its development as an important economic crop and functional food, and it has also well recorded that Maca has the medicinal efficacy of improving fertility, increasing sperm number, improving sexual function, regulating endocrine, enhancing immunity, anti-aging, antioxidation, anti-fatigue, anti-cancer, anti-osteoporosis, preventing prostatic hypertrophy, improving the exercise capacity[13-21], has the potential benefit in glucose homeostasis and diabetes, and has a good effect on rheumatism, gastrointestinal motility, respiratory diseases, depression, anaemia,etc.

Health issues have attracted increasing concerns as the citizens’ life quality is continuously improving, thus health-care foods have gradually become daily necessities. However, in recent years, the safety issues of health foods have become prominent and become the highlight of public concern throughout the country and even the world. Driven by huge profits, some law-breakers exaggerate the "treatment" effect and even add illegal chemical composition to healthy foods in order to extract huge profits. Furthermore, some merchants use defective products to substitute the qualified ones and take advantage of the lack of public perceptions and regulatory loopholes to disturb the normal market order and threaten consumers’ health.

Unfortunately, compared with high concern about Maca, there are few reports of the content of nutrients, let alone the amino acid, the chief nutrient component in Maca, whose systematic research is even rarer. Researchers have analyzed the composition of Maca dry powder and compared it with the components in potatoes. The results shows that the protein content of Maca in Peru is 4-5 times that of potatoes, and the content of essential amino acids is also very rich[25]. In order to establish a detailed and complete amino acid data analysis method, and protect consumers’ rights and expand health products markets, the identification of various Maca and the determination of its nutritional components have important theoretical significance and practical application value. Thus it is urgent to establish a scientific and effective Maca quality assessment method. In this work, we collected Maca samples of different producing areas and colors randomly and utilized automatic amino acid analyzer to determine the amino acid content and analyze its composition characteristics, so as to provide a theoretical basis for establishing a scientific analysis system of Maca and have a deeper understanding of the representative nutritional component (amino acids) in Maca.

2 Materials and methods

2.1 Instruments and materialsA300 full-automatic amino acid analyzer (Membrapure GmbH, Germany); DCY-12G nitrogen evaporator (Haike Chemical Group, Shandong, China); BS224 analytical balance (Sartorius, Germany); DZF vacuum drying oven (Yiheng Scientific Instrument Corporation, Shanghai, China).

The Maca was purchased from Peru and Yunnan as one is the origin and the other is an important economic production area. This sample set provided major Maca varieties which were commercially grown, processed, packed and marketed reflecting the general consumption situation in the Chinese market. After washing and drying under natural sunlight, we pulverized Maca samples to 60 mesh, dried at a constant temperature of 70 ℃ to constant weight, cut it into the same weight, and kept for later use.

Amino acid mixed standard solution (Membrapure GmbH, Germany, containing 18 kinds of amino acids); acetic acid, sodium acetate, citric acid, sodium citrate, sodium chloride, and hydrochloric acid (all reagents were of analytical grade and came from Sinopharm Group).

2.2 Chromatographic conditionsHigh performance liquid chromatography (HPLC) analysis was performed using cation separation column (4.6 mm×150 mm, 7 μm). The reaction column temperature was 70 ℃ and the reactor temperature was 110 ℃. The flow rate of elution pump was 0.25 mL/min and the flow rate of the derivative pump was 0.125 mL/min. The gradient elution program: 0-23.5 min, A, R; 23.5-31.5 min, B, R; 31.5-37.5 min, C, R; 37.5-58.0 min, D, R; 58.0-65.0 min, F, W. The column required being equilibrated 10 min after each test. A, B, C, D, F were sodium ion eluents with different concentrations and pH, respectively, R was 2.0% NaOH regeneration liquid, W was ninhydrin derivative agent. The injection volume was 20 μL and the column effluent was monitored at 570 and 440 nm.

2.3 Preparation of sample solutionThe experiment process referred to theNationalFoodSafetyStandard,DeterminationofAminoAcidsinFood(China national standard GB 5009.124-2016). The Maca example was entirely ground and mixed. Then, 0.1 g Maca powder was put into a hydrolysis tube and dissolved with 10 mL of 6 mol/L hydrochloric acid solution, then added 3 to 4 drops of phenol to it. After that, the hydrolysis tube was put into the cryogen and was frozen for 3-5 min. The hydrolysis tube was vacuumized and filled with nitrogen, then the process of vacuumizing and filling nitrogen was repeated 3 times and the tube was sealed under nitrogen atmosphere. The sealed hydrolysis tube was placed in an electric-heating blast thermostat or a hydrolysis oven where proteins were hydrolyzed at (110± 1) ℃ for 24 h. Subsequently, the hydrolysis tube was removed and cooled to room temperature. The hydrolysate was filtered into a 50 mL volumetric flask and the hydrolysis tube was rinsed several times with a little water. The washing solution was transferred to the same 50 mL volumetric flask, and the volumetric flask was diluted to the mark with water and mixed well. Then 1.0 mL of filtrate was transferred into a 25 mL test tube precisely and was dried under decompression (heat to 40-50 ℃) through a test tube concentrator or parallel evaporator. After drying, the residue was dissolved with 1.0-2.0 mL water and dried under decompression once again. Afterward, the ultimate residue was dissolved with 1.0-2.0 mL sodium citrate buffer solution of pH 2.2, and then shook and mixed well. Finally, the solution was filtered through a 0.22 μm filter membrane into the autosampler vials.

2.4 Optimization of experimental conditionsWe used Yunnan yellow Maca as an illustration and treated it according to conditions in Section2.3. Kept the other parameters unchanged and explored the following experimental conditions to seek an optimum design proposal.

2.4.1Investigation of the concentration of hydrochloric acid. The concentration of hydrochloric acid was set at 2, 4, 6, 8 and 10 mol/L, respectively. The amino acid determination experiments were performed 5 times simultaneously at each concentration.

2.4.2Investigation of hydrolysis temperature. The hydrolysis temperature was set at 90, 100, 110, 120 and 150 ℃, respectively. The determination experiments were accomplished 5 times at each hydrolysis temperature at the same time.

2.4.3Investigation of hydrolysis time. The hydrolysis time was set at 8, 16, 24, 32 and 48 h, respectively. The amino acid determination experiments were conducted 5 times concurrently at each hydrolysis time.

2.4.4Investigation of the degree of hydrolysis completeness. The weight of the Maca was set at 100, 120, 200, 500 and 1 000 mg. The experiments were performed 5 times simultaneously at each weight of the Maca.

2.5 Determination of amino acidThe optimal hydrolysis conditions of protein in Maca were: 6 mol/L hydrochloric acid, hydrolysis at 110 ℃ for 24 h, and 100 mg Maca. We utilized the optimal hydrolysis conditions to examine amino acid content among a variety of Maca.

2.6 Amino acid ratio coefficient methodThe amino acid ratio coefficient method was proposed by WHO/FAO in 1973 for evaluating the protein quality. We compared the amino acid content of Yunnan yellow Maca, Yunnan purple Maca, Yunnan black Maca and Peru black Maca with the assistance of this approach when processing data. The ratio of amino acids (RAA), the ratio of coefficient of amino acids (RC), and the score of RC (SRC) analysis of the essential amino acids in the 4 kinds of Maca were calculated according to formulas (1)-(3).

(1)

(2)

SRC=100-CV×100 (CVis the variation coefficient ofRC,

(3)

3 Results and analysis

3.1 Optimization of hydrolysis conditions

3.1.1Optimal concentration of hydrochloric acid. The content of amino acids obtained by hydrolysis gradually developed with the increase in the concentration of hydrochloric acid. When the concentration of hydrochloric acid was 6-8 mol/L, the content of amino acids was the highest and the hydrolysis degree was most comprehensive. Keeping increasing the concentration of hydrochloric acid, it was observed that amino acid content had no obvious improvement. Therefore, we chose a 6 mol/L of hydrochloric acid for the subsequent researches.

3.1.2Optimal hydrolysis temperature. The amino acid content gradually increased when hydrolysis temperature rising. It was easily concluded that at 110-120 ℃, the content of amino acid was the maximum and the kinds were the most comprehensive. Keeping raising temperature, it had no significant promotion on amino acid hydrolysis. In consequence, we selected a hydrolysis temperature of 110 ℃ in this experiment.

3.1.3Optimal hydrolysis time. When the hydrolysis time was 24 h, the content of amino acid was the utmost and the kinds of amino acid were the most. When prolonging reaction time, the content of amino acid was diminished on the contrary. Accordingly, the hydrolysis time was set at 24 h in the following experiment.

3.1.4Optimal weight of Maca. When the weight of the Maca sample was 100-120 mg, the amino acid content was the maximum and the hydrolysis degree was the greatest. When the weight of Maca sample was 500 mg and 1 g, the agglomeration phenomenon possibly would occur after hydrolysis, indicating that the amount of Maca sample was too large to hydrolyze completely as a consequence the results on the two units could not be calculated. Eventually, we used a Maca sample of 100 mg in this investigation.

In brief, comprehensively considering the results of optimizing experiments, and the environmental protection principle of saving raw materials, time and energy consumption, the experiments utilized the optimization conditions: 6 mol/L hydrochloric acid, 110 ℃ hydrolysis for 24 h, and 100 mg Maca sample.

3.2 Content of amino acids in Maca from different origins

The 11 kinds of amino acids were detected in Peru black Maca, including Asp, Thr, Ser, Glu, Ile, Leu, Phe, His, Lys, Arg, and Pro. In Yunnan black Maca we had identified 15 kinds of amino acids, counting Asn, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr, Phe, His, Lys, Arg, and Pro (Fig.1). For the reason that the method of hydrochloric acid hydrolysis had been adopted in the operation, Trp was frequently destroyed during acid hydrolysis, Cys was partially hydrolyzed, Gln and Asn released ammonia and transformed into Glu or Asp during this process. Therefore, it could not be ruled out that it was possible to involve Trp, Cys, Gln, and Asn in the Maca example. Horizontally comparing the amino acid content, it was considered that the content of Pro was highest in Peru black Maca, accounting for 46.3% of total amino acids. In Yunnan black Maca the uppermost content was Arg, making up 33.0% of the whole. It was reported that Arg had obvious effect on improving fertility: not only because Arg was the main amino acid in sperm, but also because Arg could improve motility of sperm cells[17-18]. Therefore, Yunnan black Maca might have a more significant effect on improving the fertility than Peru black Maca. Moreover, the content of Asp (7.7%) and Arg (18.9%) was relatively more in Peru black Maca, compared with the other amino acids with content less than 0.35 g/100 g. And the second and third highest content were Pro (20.0%) and Glu (19.0%) in Yunnan black Maca. Their content was much higher than other amino acids with content less than 0.68 g/100 g. Peru black Maca lacked the essential amino acids Val and Cys while Yunnan black Maca lacked Cys merely, thus the kinds of essential amino acids were more comprehensive in Yunnan black Maca. In summary, from both the perspective of maintaining amino acid balance and the content of nutrient intake, Yunnan Maca should be a superior nutritious food (Table 1).

3.3 Content of amino acids in Maca of different colorsThe 16 kinds of amino acids were detected in Yunnan yellow Maca and Yunnan purple Maca respectively, both including Asp, Thr, Ser, Gln, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, His, Lys, Arg, and Pro, together with 7 essential amino acids among them. 15 kinds of amino acids, including 6 kinds of essential amino acids, were identified in Yunnan black Maca (Fig. 2). We assumed that there was no Met in Yunnan black Maca or it could not be distinguished due to its extremely low content in Yunnan black Maca. In these three kinds of Maca, the contents of Arg, Pro, and Glu were enormously high. On average, they separately made up 30.3%, 17.6% and 17.3% of overall amino acid. The content of Met in all three kinds of Maca was lowest in accordance with the outcome in Yunnan black Maca. Horizontally comparing the amino acid content, it was discovered that yellow Maca and purple Maca comprised abundant proteins. The overall amino acid content of yellow Maca and purple Maca was slightly higher than the black Maca. The content of essential amino acids in yellow Maca accounted for 17.2% of total amino acid content, which was higher than that of the purple Maca (16.5%) and black Maca (9.60%). Besides, branched chain amino acids such as Val, Leu and Ile had the best anti-fatigue effect among all the amino acids. So it could be learned that the effect of Yunnan black Maca on resisting fatigue was slightly worse than that of Yunnan purple Maca and Yunnan yellow Maca. In summary, the amino acid kinds and content of yellow Maca was more comprehensive and plentiful in these three diverse colors of Maca. Hence Yunnan yellow Maca was a healthier amino acid product (Table 2).

Fig.2 Differences of amino acid content among different colors of Maca

Table 2 Content of Amino acid of Yunnan black Maca, Yunnan purple Maca and Yunnan yellow Maca (g/100 g)

In addition, we summarized the effects of different colors of Maca on the human body in detail so that people can be more targeted and specific while choosing the Maca they need. Yunnan Yellow Maca had the ability to increase sperm count, but the ability is weaker than that of Yunnan Black Maca. Yunnan Purple Maca could inhibit prostatic hyperplasia, protect the bone structure, enhance antioxidant enzyme activity, and resist strong ultraviolet radiation. Yunnan Black Maca had strong fertility and learning and memory ability, and it also could protect bone structure. Besides, all the kinds of Maca helped prevent depression, increase energy, develop immunity from disease, and improve attention[13-25].

3.4 Authenticity identification of MacaTurnip’s shape was approximate to yellow Maca, so turnip was substituted for Maca in the market frequently. Thus in succeeding procedures, we compared the amino acid content of Yunnan yellow Maca with turnip sample as Yunnan yellow Maca was the most common and representative kind in the Chinese market nowadays. The comparison results were exhibited in Fig.3 and Table 3. It could be known from Table 3 that the total content of amino acids in Yunnan yellow Maca (11.1 g/100 g) was further more than that in turnip (6.41 g/100 g). Turnip encompassed 12 kinds of amino acids, in which the content of Glu and Leu was particularly high, whereas the maximum content in Yunnan yellow Maca was Arg. The varieties and amino acid content in turnip were fairly different from those of Maca, which contributed to distinguishing turnip and Maca as a significant basis.

Fig. 3 Significant difference of amino acid content between Yunnan yellow Maca and turnip

Table 3 Content of amino acid in t and Yunnan yellow Maca (g/100 g)

3.5 Amino acid evaluation methodAccording to the formulas (1)-(3), the results of protein quality among the four kinds of Maca were exposed as Table 4. It was clearly known that the utmost RC values were Thr and the minimum RC values were Met altogether. Therefore, Thr was bountiful in the four kinds of Maca, and Met was the first limiting amino acid of the four kinds of Maca. The SRC value was a common index designed for estimating the nutritional value of proteins. The taller SRC value was, the more balanced amino acid composition in the protein was, and the better protein utilization was. The consequences demonstrated that the SRC values of the four kinds of Maca contrasted significantly. The SRC values ranking from largest to smallest were: Yunnan purple Maca (54.8)>Yunnan yellow Maca (54.3)>Yunnan black Maca (41.1)>Peru black Maca (17.6). Yunnan purple Maca and Yunnan yellow Maca held tremendously high SRC values comparing with the other two kinds. Furthermore, Peru black Maca embraced a smallest SRC value. Hence the amino acid composition of Yunnan purple Maca and Yunnan yellow Maca were the most balanced among these four kinds of Maca, and their nutritional value was relatively high.

Table 4 Parameters of amino acid in different Maca obtained by amino acid ratio coefficient method

4 Conclusions and discussion

In this study, we determined the composition and content of amino acid in two habitats and three colors of Maca and compared Maca with the common counterfeit (turnip). Maca has much higher nutritional value than turnip, and there is no considerable distinction in amino acid composition of two producing areas and in the content among three colors of Maca. In addition, the amino acid ratio coefficient method indicates that the amino acids in the Yunnan purple and Yunnan yellow Maca are better utilized in the human body and have considerably higher nutritional value. In summary, we used automatic amino acid analyzer to explore the characteristics of amino acid content and composition in Maca. The detection method meets the needs of the market and is conducive to strengthening the supervision of Maca. In the future, the mechanism of action of maca active ingredients will be the focus of research, which is expected to promote the rational development and deep utilization of Maca resources.