Composition and tocopherol, fatty acid, and phytosterol contents in micro-endosperm ultra-high oil corn

Q. Li^a, X. Tang^a, S. Lu^b and J. Wu^b,*

^aLight Industry and Food Engineering College, Guangxi University, Nanning, 530004, China.

^bGuangxi Forestry Research Institute, Nanning 530002, Guangxi, China.

*Corresponding author: wujianwen627@163.com

1. INTRODUCTIONTOP

Oils are among the most important nutrients for humans. According to the United States Department of Agriculture statistics, from 2013 to 2017, the average annual production of vegetables oils was 178.81 million metric tons, while the average annual consumption was 175.20 million metric tons (USDA Foreign Agricultural Service Oilseeds, 2018). However, China, a populous country, still needs to import a large amount of edible oils every year to fill the gap in the availability of edible oils. In 2016, the self-sufficiency rate of edible oils in China was only 32.3%, with an external dependence of 67.7%. Therefore, the current status of the Chinese vegetable oil supply is not encouraging. The development of new oil species is needed to meet the demands of global population growth and bio-energy development. HJ-1, the first micro-endosperm ultra-high oil corn without transgenics, was approved by the Chinese government in 2011 (approval no. Guishenyu 2011017). HJ-1 is a new type of oilseed crop cultivated by Guangxi University and Nanning Guifuyuan Agriculture Co. Since 1994, Professor Wu Zikai of Guangxi University and his team have made great breakthroughs in the breeding of Micro-endosperm ultra-high oil corn (Qin et al., 2005; Wu et al., 2006; Dai et al., 2009). The typical characteristics of HJ-1 are a seed oil content exceeding 20% at corn seed maturity, a small endosperm, and an embryo weight ratio ≥ 40%, which means that HJ-1 can be used directly for oil extraction without the need to extract the corn embryo.

HJ-1 is universally suitable for planting, with a yield of 4200 kg/ha and a dry seed oil content of 27%, which would make it suitable for large-scale use. Regarding oil extraction, the embryo-to-weight ratio of HJ-1 can reach more than 53%, which not only allows the complicated process of embryo lifting to be avoided, but also improves the utilization rate of the raw materials. Therefore, as a new oil seed, analyzing the nutrient content of HJ-1 oil is important.

Tocopherol and phytosterol contents are important indices for measuring the nutritional value of corn oil. Natural tocopherols comprise four different structures, namely α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, of which α-tocopherol exhibits the highest biological activity (Shi et al., 2014). Tocopherols can reduce the speed of oil oxidation and remove free radicals. Phytosterols are important components of plant cell membranes, and mainly comprise β-sitosterol, stigmasterol, and campesterol. Furthermore, phytosterols have comprehensive biological activities, such as antioxidation, anti-inflammation, and anticancer activities. Related studies have shown that phytosterols can inhibit the absorption of low-density lipoprotein cholesterol, resulting in a reduction in total cholesterol in the blood (de Jong et al., 2003). Phytosterols cannot be synthesized by the body so are obtained solely through the diet.

This study aims to analyze the composition and contents in tocopherols, fatty acids, and phytosterols in HJ-1 oil, compare them with those of seven common vegetable oils, and explore the nutritional value of HJ-1 oil.

2. MATERIALS AND METHODSTOP

2.1. Materials TOP

HJ-1 oil, obtained by drying HJ-1 seeds to a moisture content of about 8% then pressing the whole seeds using a screw press, was kindly provided by Nanning Guifuyuan Agriculture Co. (Model YZYX130, Guangxin Cereals and Oils Machinery Manufacturing Co., Mianyang, China). The HJ-1 corn came from the Nanning, Wuming and Tanluo regions of China. Three samples were randomly selected from these three different regions for pressing the whole corn kernels to produce HJ-1 oil. The pressed HJ-1 corn oil was then directly tested without any processing.

2.2. HPLC analysis of tocopherolsTOP

Samples (1.00 g) were mixed with BHT (0.1 g) in a 25-mL brown volumetric flask, with a mobile phase (10 mL; 90% hexane + 10% BHA/MTBE–THF–methanol (20:1:0.1), v/v) which was freshly prepared before each use. After dissolving the sample by vortexing, the solution was made up to 25 mL with the mobile phase and then shaken. The solution was then filtered through a 0.22-μm membrane filter into brown autosampler vials before injecting into the HPLC system, following the ISO 9936 method (ISO, 2016).

An HPLC system (Model 1200, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an amino column (150 mm × 3 mm × 1.7 μm) and a fluorescence detector was used for tocopherol analysis. Chromatographic analysis was performed using the following conditions: column temperature, 30 °C; mobile phase, 90% hexane + 10% BHA/MTBE–THF–methanol (20:1:0.1), v/v; flow rate, 0.8 mL/min; excitation wavelength, 294 nm; emission wavelength, 328 nm; and injection volume, 10 μL.

2.3. GC analysis of fatty acidsTOP

Samples (0.2 g) were added to a 2% NaOH–methanol solution (8 mL) and refluxed over an 80 ± 1 °C water bath until the oil droplets disappeared. Boron trifluoride–methanol (7 mL, 15%) was then added into the top of the reflux condenser and reflux was continued for 2 min. The reflux condenser was rinsed with a little water and the reaction flask was removed and rapidly cooled to room temperature. After adding n-heptane (20 mL), the mixture was shaken for 2 min and saturated aqueous NaCl was added. After allowing the layers to separate, 5 mL of the n-heptane extract (upper layer) were transferred to a 25-mL test tube and anhydrous Na₂SO₄ (approx. 4 g) was added, followed by shaking for 1 min and allowing to stand for 5 min. The dried extract was then transferred to a sample vial for determination according to the ISO 12966-2 method (ISO, 2017).

Gas chromatography (GC; 7890B, Agilent Technologies Inc.) was performed using a capillary column (100 m × 0.25 mm × 0.2 μm) with high polarity polypolar cyanopropyl siloxane as the stationary phase. GC was performed under the following conditions: sampler temperature, 270 °C; detector temperature, 280 °C; carrier gas, nitrogen; split ratio, 100:1; and injection volume, 1.0 μL. The GC temperature program was as follows: initial temperature, 100 °C for 13 min; increased to 180 °C at 10 °C/min and held at 180 °C for 6 min; increased to 200 °C at 1 °C/min for 20 min; and finally, increased to 230 °C at 4 °C/min and held at 230 °C for 10.5 min.

2.4. HPLC analysis of phytosterolsTOP

Samples (2 g) were added to a NaOH–ethanol solution (20 mL, 2%) and refluxed for 1 h. Distilled water (100 mL) was then added into the top of the condenser and the reaction mixture was shaken. This saponification mixture was then transferred into a separatory funnel, extracted several times with petroleum ether (100 mL), and the solvent was evaporated. The resultant unsaponifiable matter was then dissolved with ethanol to a volume of 10 mL and filtered through a 0.45-μm filter for determination (Ito et al., 2017).

Phytosterol analysis was performed using an HPLC system (Agilent Technologies Inc.) equipped with a UV detector using an IODS column (250 mm × 4.6 mm × 5 μm, Agilent Technologies Inc., Palo Alto, CA, USA). HPLC analysis was performed under the following conditions: mobile phase, methanol; volume temperature, 25 °C; flow rate, 1.0 mL/min; injection volume, 10 μL; and detection wavelength, 210 nm.

2.5. Data treatmentTOP

All measurements were performed in triplicate and results are shown as means ± standard deviation.

3. RESULTS and DISCUSSIONTOP

3.1. Tocopherol composition and contentTOP

Vitamin E is an essential human nutrient because of its important physiological functions and it is obtained solely through foods and supplements (Burton et al., 1983). Vegetable oils, which contain 700–1900 mg of tocopherols per kg of oil, are the main source of tocopherols in the human diet (Gliszczyńska-Świgło and Sikorska, 2004). Unlike other vitamins, vitamin E contains tocopherols and tocotrienols, which are closely related fat-soluble components (Azzi and Stocker, 2000). Tocopherols are mainly found in corn, soybean, and olive oils, while palm, rice bran, and barley oils are rich in tocotrienols. Therefore, this study determined the levels of four tocopherols in HJ-1 oil and compared them with those found in seven other selected edible vegetable oils (Table 1).

Tocopherols in HJ-1 oil were dominated by α-tocopherol and γ-tocopherol, as also observed in corn, rapeseed, and soybean oils. Furthermore, HJ-1 oil contained 38.41 ± 0.005 mg/kg of δ-tocopherol, which was not detected in corn oil. δ-Tocopherol was also detected in soybean, rapeseed, palm, and camellia oils. Jung et al., (2016) have previously shown that the presence of δ-tocopherol is beneficial for maintaining the stability of other tocopherols with a certain moisture content. Therefore, the nutrients in HJ-1 might be more stable than those in corn oil. Table 1 also shows that the main tocopherol in olive and corn oils was α-tocopherol, but that the contents of all four tocopherols in olive oil were lower than those in HJ-1. However, the contents of the four tocopherols in soybean oil were higher than those in HJ-1 oil.

3.2. Fatty acid composition TOP

Edible vegetable oil plays a significant role in the human diet, providing energy for growth and metabolism. Furthermore, many essential fatty acids, such as linoleic and linolenic acids, cannot be synthesized in the human body. Therefore, data on fatty acid compositions and contents are essential for evaluating oil quality. In the present study, the fatty acid composition of HJ-1 oil was determined and compared with those of seven other edible vegetable oils (Table 2).

Seven kinds of fatty acids were detected in HJ-1 oil, of which palmitic (C16:0), oleic (C18:1-9c), and linoleic (C18:2-9c12c) acids were the main components, as also observed in normal corn oil. The oleic acid (C18:1-9c) content in HJ-1 oil was higher than that of linoleic acid (C18:2-9c12c), while the opposite was observed for normal corn oil. Furthermore, the unsaturated fatty acid content in HJ-1 oil was approximately 83.56%, which was higher than that of corn oil (79.85%).

Olive oil is rich in unsaturated fatty acids, which has played a key role in its global recognition as a superior oil. Table 2 shows that the fatty acid composition of camellia oil is similar to that of olive oil. Compared with the other selected oils, the most outstanding feature of HJ-1 oil was an oleic acid to linoleic acid ratio close to 1:1. Among saturated fatty acids, long-chain saturated fatty acids (such as palmitic acid 16:0) are prone to raising serum cholesterol. Therefore, people tend to reduce the intake of saturated fatty acids in the diet. Many scholars have proposed reasonable ingestion ratios for saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids. For example, the Ministry of Health and Welfare of Japan advocates a 1:1.5:1 ratio of saturated fatty acids/monounsaturated fatty acids/polyunsaturated fatty acids, with the World Health Organization and the Food and Agriculture Organization of the United Nations proposing a fatty acid ratio of 1:1:1. Therefore, HJ-1 oil could be suitable for use either alone or as a raw material in blended oils.

3.3. Phytosterol compositionTOP

Phytosterols, such as β-sitosterol, campesterol, and stigmasterol, are natural ingredients found in plant cell membranes and have high contents in vegetable oils, nuts, seeds, and cereals. Phytosterols are mainly found as free sterols or sterol esters of fatty acids in vegetable oils. Many studies have confirmed the ability of phytosterols to reduce blood cholesterol levels (Yuan et al., 2017). The present study measured the contents of three phytosterols in HJ-1 oil and compared them with those in seven other selected edible vegetable oils (Table 3).

The composition and content of phytosterols among different types of vegetable oils varied greatly. Table 3 shows that β-sitosterol was the dominant phytosterol in HJ-1 oil, as also observed in the other seven oils, accounting for more than 50% of the total phytosterols. The β-sitosterol and campesterol contents in corn oil and rapeseed oil were higher than those in HJ-1 oil, especially the β-sitosterol content in corn oil, which was about 2.6 times that in HJ-1 oil. The stigmasterol content in HJ-1 oil was the highest among the oils analyzed, at approximately 105.7 mg/100 g, which was about twice that in corn oil. Unlike other vegetable oils, the contents of the three phytosterols in HJ-1 oil were very similar, indicating a relatively balanced content compared with the other seven oils. According to Roche et al., (2010), sunflower seeds are a major source of natural phytosterols. The β-sitosterol content in HJ-1 oil was equivalent to that in sunflower oil, while the contents of campesterol and stigmasterol were much higher than those in sunflower oil. Therefore, HJ-1 oil shows great potential for industrial applications.

4. CONCLUSIONSTOP

HJ-1 oil is derived from whole corn kernels, without the traditional embryo extraction process, which effectively reduces processing costs. The contents of four vitamins E in HJ-1 oil were much higher than those in olive oil. No δ-tocopherol was detected in traditional corn oil but the contents of α-tocopherol and β-tocopherol in HJ-1 oil were lower than in traditional corn oil. The content of unsaturated fatty acids in HJ-1 oil was higher than that in traditional corn oil. Unlike traditional corn oil, the content of oleic acid in Huajian I was higher than its linoleic acid content. Like other vegetable oils, the phytosterols in HJ-1 were mainly β-sitosterol. Although the total amount of the three phytosterols in HJ-1 was not as high as in traditional corn oil, the content of stigmasterol in HJ-1 was twice as high.

ABBREVIATIONSTOP

HJ-1, Huajian No. 1; HPLC, High performance liquid chromatography, GC; Gas chromatography; BHT, 2,6-Di-tert-butyl-4 methylphenol; BHA, Butylated hydroxyanisole; MTBE, Methyl tert-butyl ether; THF, Tetrahydrofuran

ACKNOWLEDGMENTSTOP

Financial support was provided by the Science and Technology Planning Project of Guangxi Province, China (grant number: Gui Ke AB16380107). We thank Simon Partridge, PhD and Philip Creed, PhD from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript, and GUANGXI Ultra High Oil and High Protein Corn Processing and Engineering Technology Research Center.

REFERENCESTOP