Nutritional compositions of Indian Moringa oleifera seed and antioxidant activity of its polypeptides

Abstract To study the nutritional composition of Indian Moringa oleifera seed and the antioxidant activity of M. oleifera seed polypeptide, Indian M. oleifera seed was used as raw material for composition analysis and content determination. After extraction of the seed protein, enzymatic hydrolysis with flavourzyme, dispase, papain, pepsin, and alcalase was conducted for different time, and the optimal enzymatic hydrolysis conditions was determined with DPPH scavenging capacity as an indicator. The seed polypeptides obtained by enzymatic hydrolysis were ultrafiltered, and the active peptide fragments were tracked with DPPH, HO (•OH), ABTS and superoxide anion (O2•−) free radical scavenging ability and lipid oxidation inhibition rate as indicators. The results showed that the protein content in Indian M. oleifera seed was high to 40.34%, containing seven essential amino acids. The content of macroelements such as potassium, sodium, and magnesium is high, with the potassium content as high as 2,357.71 mg/kg, among the microelements, the iron content as high as 36.2 mg/kg. The optimum enzymatic hydrolysis conditions were as follows: enzymatic hydrolysis with flavourzyme (50°C, pH 6.7) for 300 min, and DPPH scavenging capacity was 84.76%. Activity tracing found that the polypeptide fragment with molecular weight <3.5 kDa had the strongest antioxidant capacity, and the EC50 values of DPPH, •OH, ABTS, and O2•− free radical scavenging rates were 4.0, 4.2, 5.3, and 4.3 mg/ml, respectively. The above results show that Indian M. oleifera seed not only has high nutritional value, but its protease enzymatic hydrolyzate also has significant antioxidant activity, which can be further developed into nutrition products, healthcare products, functional foods, beauty and skin care products, liver protection drugs, etc.

as India and Myanmar. M. oleifera seed is rich in oils, proteins, and minerals. It can be used in food, medicine, cosmetics, and water purification, and has good research prospects. It has significant effects in reducing blood lipids, blood pressure, slimming, regulating the stomach, protecting the liver from alcohol, and enhancing the body's immunity (Du, Sun, Yan, Luo, & Dai, 2017;González et al., 2017;Pereira, Oliverira, & Oliverira, 2011). In addition, studies have confirmed that M. oleifera protein is soluble in aqueous solution and can be completely digested (Fan, Shao, Ye, & Yang, 2016). Lin found that M. oleifera seed polypeptide has protective effect on the erythrocytes which suffered oxidative damage (Lin, Zhu, & Zhao, 2018), and Zhao found the extract of M. oleifera leaves can strongly scavenge DPPH and superoxide anion free radical and absorb oxidative free radical (Zhao, Li, Lin, & Yang, 2017). M. oleifera root protein has anti-inflammatory and analgesic functions. At present, there are few studies on M. oleifera seed protein. The only articles are the research provided by Aderinola and his coworkers. Their study showed M. oleifera seed protein has antioxidant properties (Aderinola, Fagbemi, Enujiugha, Alashi, & Aluko, 2018), especially after hydrolysis by trypsin or alcalase, the hydrolyzate shows in vitro antihypertensive and antioxidative properties (Aderinola, Fagbemi, Enujiugha, Alashi, & Aluko, 2019a, 2019b. In this article, we analyze the Indian M. oleifera seed nutrient composition and the antioxidant activity of its peptides hydrolyzed by flavourzyme, dispase, papain, pepsin, and alcalase, expected to provide data support for the in-depth research and development of M. oleifera seed polypeptide.

| Materials and reagents
Moringa oleifera seeds were collected in the southern Himalayas of

Determination of general nutrients
Determination of crude protein content refers to GB/T5009.5-2016 "Determination of protein in food," using Kjeldahl method; determination of ash content refers to GB 5009.4-2016 "Determination of ash in food," using muffle furnace burning method; determination of crude fat refers to GB 5009.6-20163 "Determination of fat in food," using Soxhlet extraction method; determination of mineral elements refers to GB5009.268-2016 "Determination of multi-elements in food"; determination of moisture refers to GB5009.3-2016 "Determination of moisture in food," using direct drying method; determination of nucleic acid refers to GB5009.87-2016 "Determination of phosphorus in food," using Fiske-Subbarow method; and determination of carbohydrate acid refers to NY/T 2332-2013.

Determination of amino acid composition and content
After the M. oleifera seeds were dried and pulverized, 0.5 g was weighed and was digested with 6 mol/L hydrochloric acid at a constant temperature of 110°C for 22 hr, and then the amino acid content was determined by an L-8900 amino acid analyzer (Hwee & Chee, 2013;Zhang, 2016).

| Determination of the antioxidant activity of M. oleifera seed protein, Vc as positive control
Determination of the scavenging ability of M. oleifera seed protein to DPPH free radicals Two milliliters of samples consisting of distilled water and different concentrations of the analytes were placed in cuvettes, and 500 μl of an ethanolic solution of DPPH (0.02%) and 1.0 ml of ethanol were added. A control sample containing the DPPH solution without the sample was also prepared. In the blank, the DPPH solution was substituted with ethanol. The antioxidant activity of the sample was evaluated using the inhibition percentage of the DPPH radical with the following equation: A c : Absorbance value of the control group; A o : Absorbance value of the blank group; A s : Absorbance value of the sample group (Pan, Zhao, Hu, & Wang, 2016).
Determination of the scavenging ability of M. oleifera seed protein to hydroxyl radicals 1.0 ml of a 1.87 mM 1,10-phenanthroline solution and 2.0 ml of the sample were added to a screw-capped tube and mixed. Then, 1.0 ml of a FeSO 4 ·7H 2 O solution (1.87 mM) was added to the mixture. The reaction was initiated by adding 1.0 ml of H 2 O 2 (0.03%, v/v). After incubating at 37°C for 60 min in a water bath, the absorbance of the reaction mixture was measured at 536 nm against a reagent blank. The reaction mixture without any antioxidant was used as the negative control, and a mixture without H 2 O 2 was used as the blank. The hydroxyl radical scavenging activity (HRSA) was calculated using the following formula:

Enzymatic hydrolysis
The protein solution with concentration of 0.01 g/ml and the enzyme with concentration of 0.005 g/ml were extracted from M. oleifera 2015) was conducted, respectively, and DPPH scavenging capacity was used as an indicator to select the optimal enzymatic hydrolysis conditions. The DPPH scavenging capacity is determined as above.

Ultrafiltration
Peptide fragments with different molecular sizes of >5 kDa, 3.5-5 kDa, and <3.5 kDa were obtained after ultrafiltration using a 5 kDa, 3.5 kDa ultrafiltration membrane. The three sections of the solution were separately collected for lyophilization, and the dried peptide was stored at −20°C.

Screening for active components
The ability of the peptide fragments with three different molecular sizes to scavenge DPPH, •OH, ABTS, O 2 • − free radicals and the EC 50 values of lipid antioxidants were studied to screen for more active components. The DPPH•, •OH, ABTS•, O 2 • − scavenging power and lipid antioxidants were determined as above.

| Proximate composition of M. oleifera seed
As can be seen from and 21.06%) (Liu & Pan, 2016;Zheng, Jin, Geng, & Yu, 2015), the crude protein content of Indian M. oleifera seed is equivalent to soybean, higher than peanut; its crude lipid content is much higher than soybeans, slightly lower than peanuts. It is reported that M. oleifera seed protein has the effect of water purification and anticoagulation (Aline et al., 2017;Luciana et al., 2013;Marichamy & Ramasamy, 2015), and M. oleifera oil can accelerate the healing of wounds (Duan et al., 2011). It shows that the Indian M. oleifera seed is not only rich in crude protein and lipid, but also has various effects. We can deeply develop its plant protein and vegetable oil, such as functional foods, feed, water purification, cosmetic raw materials, plant growth promoters, and fungicides.

| Content of mineral elements in M. oleifera seed
As can be seen from  (Juhaimi, Ghafoor, Babiker, Matthaus, & Ozcan, 2017). M. oleifera seeds also contain four heavy metal elements of arsenic, cadmium, lead, and tin, which are 0.01, 0.018, 0.015, and 0.007 mg/kg, respectively. These are low in content and are in line with GB2762-2017 "The national food safety standard, food contaminants limit" (arsenic is limited to 0.5 mg/kg in grains and their products; cadmium and lead are limited to 0.5 and 0.2 mg/kg, respectively; and in nuts and seeds, tin is limited to 250 mg/kg in food).

| Amino acid composition and content in M. oleifera seeds
As can be seen from Table 3, M. oleifera seed contains seven essential amino acids, namely threonine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine, with the total content of 0.824 g/100g, accounting for 25.65% of the total amino acid content. This result is close to the 28.8% of essential amino acids in Liaoning peanuts reported by Shi (Shi, Yu, & Han, 2017). The M. oleifera seeds contain seven kinds of hydrophobic amino acids: alanine, valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine, and the total content is 0.834 g/100g. The antioxidant activity of the protein is closely related to its amino acid composition. Peptides containing hydrophobic amino acids can increase their solubility at the water-lipid interface and better interact with free radicals (Zheng, Si, Baseer, Li, & Zhang, 2018).

| Protein antioxidant activity
As shown in Figure 1, when the M. oleifera seed protein was 10 mg/ ml, the DPPH•, ABTS•, •OH, and O 2 • − scavenging rates were 63.25%, 52.45%, 55.25%, and 59.32%, respectively. And the lipid oxidation inhibition rate was 43.25%. It shows that M. oleifera seed protein extraction has good antioxidant activity against different antioxidant models.
As shown in Figure 2, the DPPH scavenging rate of the five enzymatic hydrolyzates increased gradually with the increase in When the flavourzyme was used for the enzymatic hydrolysis of the M. oleifera seed protein for 300 min at 50°C and pH 6.7, the DPPH scavenging rate was up to 85%. Therefore, this condition was selected as the optimum enzymatic hydrolysis process for M. oleifera seed protein for subsequent studies.

| Antioxidant activity of different peptide fragments
After ultrafiltration, the enzymatic peptide was divided into three parts, the molecular weight of the peptide was <3.5 kDa, 3.5-5 kDa, >5 kDa, and the EC 50 values of DPPH, •OH, ABTS, O 2 • − free radical scavenging rate and the lipid antioxidant inhibition rate are shown in Figure 3.
As shown in Figure 3, for the peptide fragment with molecular

KD-5 KD
and magnesium is much higher than other metals, and the K content is 2,537.71 mg/kg. Indian M. oleifera seeds contain seven essential amino acids and seven hydrophobic amino acids, and the hydrophobic amino acids contribute to the antioxidant activity of M. oleifera seeds. The optimum enzymatic conditions were flavourzyme hydrolysis of M. oleifera seed protein for 300 min at 50°C and pH 6.7.
<3.5 kDa polypeptide fragment has higher EC 50 values for DPPH•, •OH, ABTS•, and O 2 • − scavenging rates than fragments 3.5-5 kDa and >5 kDa, and only the lipid oxidation inhibition rate of <3.5 kDa polypeptide fragment is lower than >5 kDa. Therefore, the <3.5 kDa polypeptide fragment is the strongest antioxidant. It has the potential to be further developed into nutritional products, health products, functional foods, beauty and skin care products, and liver protection drugs.