Effects of Lanthanum and Cerium on Seeds Fatty Acid Composition in Soybean

Ren Hong-yu, Zhuang Zhong, Liu Yan-yu, Li Xue-cong, Zhu Hua-li, Su Xiao-lei, Zhang Shu-ying, Liu Wenshu, and Zhang Xing-wen

1 College of Resources and Environmental Science, Northeast Agricultural University, Harbin 150030, China

2 College of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China

Abstract: Soybean Dongnong 42 from northeast of China was used as an experimental material.The samples were tested by spraying different concentrations of LaCl3, CeCl3 and LaCl3+CeCl3 solutions on the leaves at the seedling stage and the early flowering stage of the soybeans.Gas chromatography was used to determine the fatty acid content and ratio of the soybean seeds at the maturity stage.The fatty acid ratio of soybean seeds were evaluated by calculating saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA), unsaturated fatty acids (UFA), UFA/SFA, index unsaturated fatty acid (IUFA) and double bond index (DBI).(MUFA+PUFA)/SFA was used to study the effects of rare earths on the quality of soybean seeds.The results showed that spraying the rare earth solution could significantly optimize the fatty acid distribution ratio of soybean seeds.Compared with the control group (CK), stearic acid was reduced by 5% and linolenic acid was reduced by 3.72%.Except for La10, the oleic acid content increased significantly by 2.22%.Under the treatment of 30 mg·L-1 CeCl3 , UFA, UFA/SFA and DBI were 83.56, 5.08 and 9.47, respectively, which were the highest in each group.Under the treatment of 150 mg·L-1 LaCl3, (MUFA+PUFA)/SFA was 4.89, the closest meal to the recommended ratio.The above results showed that spraying appropriate concentrations of rare earth at the seedling stage and the early flowering stage could optimize the fatty acid ratio of soybean seeds and improve dietary fatty acid ratios.

Key words: lanthanum, cerium, soybean, fatty acid

Introduction

Rare earth is a general term of 17 elements in the periodic table, including lanthanum (La) and cerium (Ce), together with their families of scandium (Sc) and yttrium (Y).As a major rare earth resource and agricultural country, China has used rare earth in agricultural production from as early as the 1970s (Yanget al., 2009).Lanthanum and cerium are light rare earth elements that are currently being encouraged by the state and are also the most commonly used agricultural rare earth elements.Its promotional effect on crop growth and development has been widely recognized, and its safety and reliability as a fertilizer in a low dose range have also been proven (Zhang, 2012).Therefore, rare earth agriculture has a wide range of scientific research and application prospects.

As an important oil crop, soybeans contain about 20% oil, where 90% of them are fatty acids (Nanetal., 2015).This includes approximately 16% saturated fatty acids (palmitic acid [C16:0] and stearic acids [C18:0]), 24% of monounsaturated fatty acids with one unsaturated bond (oleic acid[C18:1]), and 60% of polyunsaturated fatty acids with more than two unsaturated bonds (linoleic acid [C18:2] and linolenic acid [C18:3]).Among them, n-6 linoleic acid and n-3 linolenic acid have important physiological effects.The two cannot be synthesized in the body and must be provided by food, so they are called essential fatty acids (Hamurcuet al., 2019).With the improvement of living standards, people's requirements for oil and fat not only stay on the content, however, more importantly, on the proportion of each component of fatty acids.Therefore, the optimization of the distribution ratio of soybean fatty acid has become the main goal of improving soybean quality.Domestic and foreign studies have confirmed that the composition and proportion of fatty acids in soybeans can change with the changes in the ecological environment.The contents of different fatty acids have a significant correlation with factors, such as geographic latitude, average temperature and precipitation during the growing season (Ning and Yang, 2003; Ghassemi-Golezani and Farhangi-Abriz, 2018).Our research group has also confirmed in many years of experiments that rare earth has a significant impact on soybean yield, stress resistance, chlorophyll content, nutrient accumulation protein accumulation and quality (Renet al., 2019).

In order to further clarify the effect of rare earth on the fatty acid composition of soybean seeds, Dongnong 42 was used as the experimental material in this experiment.The light rare earth elements lanthanum and cerium, which were currently encouraged to be developed and applied by the state, were sprayed at the seedling stage and the early flowering stage.Taking soybean grain fatty acid composition as the measurement index, the effects of rare earth on the combination of fatty acid quality and the appropriate diet ratio were studied with the aim to provide data supports for clarifying the mechanism of the effect of rare earth on soybean qualities.

Materials and Methods

Plant materials and field experiment

The experiment was conducted at Northeast Agricultural University in 2018.The experimental soybean variety Dongnong 42, provided by the Soybean Research Institute of Northeast Agricultural University, was planted in garden pots with a height of 30 cm, a diameter of 35 cm at the top and 25 cm at the bottom.Each pot contained 17.5 kg of black soil, where eight full and uniformly-sized seeds were sown in each one.Four seedlings were kept when the seedlings grew to 5-8 cm.The experiment soil was typical black soil.The chemical properties of the soil were organic matter 27.40 g·kg-1, pH 7.1, alkaline hydrolyzed nitrogen 1 137.62 mg·kg-1, fast-acting phosphorus 54.55 mg·kg-1and fast-acting potassium 194.00 mg·kg-1.Each pot was administered with 0.44 g of urea, 1.22 g of diammonium phosphate and 1.25 g of potassium sulfate.

The experiment referred to the results of the previous experiment of this research group.During the soybean seedling stage (June 26th, 2018) and the early flowering stage (July 6th, 2018).CeCl3solution with a concentration of 30, 60 and 90 mg·L-1(which were represented by Ce3, Ce6 and Ce9, respectively) and LaCl3solution with a concentration of 100, 150 and 200 mg·L-1(which were represented by La10, La15 and La20, respectively) as well as with the groups of 40,60 and 70 mg·L-1LaCl3+CeCl3, according to a 1 : 1 ratio and volume mixing solution (which were represented by LC4, LC6 and LC7, respectively) was sprayed evenly on the front and back of the soybean leaves.They were sprayed with about 150 mL per pot in the seedling stage and about 300 mL per pot in the early flowering stage.The control group (CK) was sprayed with an equal amount of distilled water.Samples were taken after maturation and each sample had three replications.

Determination of fatty acids

With reference to the gas chromatography method described by Fanet al(2015).The 30 mg of soybean powder was crushed through a 100 mesh sieve and accurately weighed into a 2 mL centrifuge tube, to which was added 1.00 mL of n-hexane for 20 min.Afterwards, added 1 mL of 0.5 mol·L-1sodium methoxide solution and shaken well for 10 min.The supernatant was placed in a sample bottle of chromatography after holding and stored in a refrigerator at 4℃ for the experiment.

The equipment used was a Shimadzu GC-14C gas chromatograph and a 30 m×0.25 mm×0.25 μm FFAP elastic quartz capillary.The chromatographic conditions were an injection volume of 1 μL; a split injection, split ratio of 40: 1; an inlet temperature of 250℃; carrier gas nitrogen of 54 mL·min-1; hydrogen that was 40 mL·min-1and air that was 400 mL·min-1.It was kept at 180℃ for 1.5 min and raised to 210 ℃ at 10℃·min-1, where it was kept for 2 min before being raised to 220℃ at 5℃·min-1and kept there for 5 min.The flame ion detector (FID) temperature was 300℃.Each sample was experimental for 20 min.and the interval between sample detections was 2 min.The area normalization method was used to calculate the percentage of the fatty acid content.

Statistical analyses were carried out using Microsoft-excel software 2007.Statistical analyses were performed using one-way analysis of variance (ANOVA) and the group means were compared by duncan experiment using SPSS software 19.0.

Saturated fatty acid (SFA)=palmitic acid+stearic acid;

Monounsaturated fatty acid (MUFA) =oleic acid;

Polyunsaturated fatty acid (PUFA) =linoleic acid+linolenic acid;

Unsaturated fatty acid (UFA) =oleic acid+linoleic acid+linolenic acid;

Index unsaturated fatty acid (IUFA) =oleic acid×1+linoleic acid×2+linolenic acid×3;

Double bond index(DBI)= [Σ (relative content of unsaturated fatty acids × number of double bonds) ]/ (Σ relative content of saturated fatty acids).

Results

Table 1 showed that from the perspective of the changes in the content of each component, rare earth had different effects on different fatty acid components.Compared with CK, the content of palmitic acid in La20 decreased significantly by 0.97%, and the content of palmitic acid in LC7 increased significantly by 2.35%.In addition, the content of palmitic acid in Ce9, La15 and LC6 increased significantly.Compared with CK, Ce3, Ce6, Ce9 and LC4 stearic acid contents were significantly reduced, of which Ce3 treatment content reached the lowest with a decrease of 5%.Except for La10, the content of oleic acid in each group increased significantly and the content of La20 treatment reached the highest, increasing by 2.22%.The contents of linoleic acid in La15 and LC7 groups were significantly lower than those in CK, among which the content in LC7 group was the lowest which a decrease of 1.04%.The other treatment effects were not significant.Except for the La10, LC4 and LC7 treatments, the content of linolenic acid was significantly lower than that of CK, and the content of La20 treatment was the lowest with a decrease of 3.72%.

Table 2 showed the order of unsaturated fatty acid content in soybean seeds as the following: PUFA> MUFA> SFA.Compared with CK, Ce3 and LC4 SFA decreased significantly, of which Ce3 group contents was the lowest with a decrease of 1.67%.La15 and LC7 content increased significantly.Compared with CK, MUFA content showed an increasing trend.The content of PUFA showed a general downward trend compared with CK, among which the content of LC7 was the lowest with a decrease of 0.98%.The total UFA content in Ce3 group increased significantly by 0.34%.The change trend of UFA/SFA accorded with the change trend of saturated fatty acid and unsaturated fatty acid content.When compared with CK, Ce3 increased significantly by 2%.In summary, Ce3 had the best effect in increasing the content of unsaturated fatty acids in soybean seeds.

Table 1 Fatty acid components in soybean seeds under each rare earth treatment

Table 2 Fatty acid components in soybean seeds under each rare earth treatment

Compared with CK, IUFA showed a downward trend, and the Ce9, La15, La20, LC6 and LC7 groups decreased significantly form 0.35% to 0.58%.The results showed that spraying rare earth could reduce the fatty acid IUFA of soybean seeds, and the degree of decrease had a tendency to increase with the increase of rare earth concentration.Ce3 and LC4 DBI increased significantly, while La15 and LC7 decreased significantly.

Fig.1 showed that the range of (MUFA+PUFA)/SFA in the experiment was form 4.90 to 5.08.Compared with CK, the ratio of Ce3, Ce6 and LC4 groups increased significantly.Ce3 was the highest, with an increase of 2.06%.The ratio of the La15 and LC7 groups decreased significantly, of which La15 was the lowest with a decrease of 1.81%.It showed that RE had a regulating effected on (MUFA+PUFA)/SFA.The range of n-6/n-3 ranged from 6.64 to 6.92.Compared with CK, the ratios of Ce3, Ce6, Ce9, La20 and LC6 increased significantly from 2.10% to 3.60%, of which La20 was the highest.The processing ratio of LC7 decreased, but the decrease was not obvious.

Fig.1 Effect of rare earth treatment on dietary fat ratio of soybean seeds

Discussion

Effects of rare earth treatments on content of fatty acid components in soybean seeds

The results of the relative content of fatty acids in each group of the experiment were consistent with the results of previous studies (Lu, 2018).This showed that the regulation of soy fatty acid components by the rare earth only fluctuated in a small range and could not cause a wide range of its proportion.The effect of rare earth on palmitic acid had both promotional and inhibitive effects, consistent with the results believed by most scholars that there was "two-way regulation" of rare earth (Renet al., 2018).It showed that the types and concentrations of rare earth spraying could be reasonably selected according to different production demand goals.Significant effects on stearic acid content was made by cerium, indicating that compared with lanthanum, stearic acid was more significantly regulated by cerium.Compared with other fatty acid components, oleic acid had the most obvious response to rare earth regulation.Studies had shown that palmitic acid (16:0) produced stearic acid (18:0) through carbon chain extension, stearic acid forms oleic acid (18:1) through desaturation oleic acid forms linoleic acid (18:2), and linoleic acid desaturated to form linolenic acid (18 : 3) (Zhaoet al., 2013).Therefore, the above results indicated that the rare earth might adjust the content of fatty acid components in soybean seeds by increasing the rate of stearic acid desaturation and decreasing the rate of oleic acid and linoleic acid.

In terms of fatty acid composition optimization, most scholars had believed that a suitable amount of stearic acid could improve human health.However, as a saturated fatty acid, it was not conducive to human absorption and utilization.Unsaturated fatty acids, as essential fatty acids in the human body, had the functions of preventing the deposition of cholesterol in serum, preventing cardiovascular and cerebrovascular diseases, and maintaining the stability of biofilms (Chen, 2018; Haet al., 2010).However, because linolenic acid contained three unsaturated bonds, it was more easily oxidized by lipoxygenase, which produced a fishy smell and reduced the storage quality of soybean oil (Zhaoet al., 2014).Therefore, reducing the ratio of stearic acid and linolenic acid, while improving its antioxidant properties, was more conducive to the optimization of soybean fatty acid components.However, oleic acid showed significant negative correlation with linoleic acid and linolenic acid content, and linoleic acid showed significant positive correlation with linoleic acid content (Liuet al., 2009).In summary, Ce3 had a more prominent effect in reducing stearic acid while optimizing the components.La20 had the best optimization effect in increasing oleic acid and reducing the content of linolenic acid.In addition, La10 did no significant effect on the fatty acid content of each component, which might be due to the low concentration of rare earth, which did not played a regulatory role.

Effects of rare earth treatments on dietary fat ratio of soybean seeds

In the experiment results, Ce3 and LC4 groups (MUFA+PUFA)/SFA increased, because SFA content was significantly reduced.Ce3 treated SFA and MUFA+PUFA were the highest among the treatment groups, so the ratio of Ce3 was the maximum value.At the same time, the minimum La15 was the maximum SFA content group, consistent with the previous conclusion.It showed that rare earth mainly affected (MUFA+PUFA)/SFA value by adjusting SFA content.Su and Guo (2003) believed that the total fat intake from the diet and SFA/MUFA/PUFA ratio in the diet were generally not scientific eough to say whether a fatty acid was good or bad.When PUFA was excessively increased, it was susceptible to free radical attack, and its carbon chain was oxidatively cleaved into aldehydes, ketones and some epoxides, resulting in changes in the cell membrane and lipoproteins containing PUFA.These were considered to be one of the mechanisms by which tumors and atherosclerosis were formed (Su and Guo, 2003).Therefore, the ratio of (MUFA+PUFA)/SFA should not be greater than two.Although the rare earth adjustment did not adjust this ratio to the optimal range, the experimental results showed that rare earth had the effect of optimizing the ratio of (MUFA+PUFA)/SFA, and La15 was the best.

The reason for the increase of n-6/n-3 was that the effect of rare earth on the reduction of linolenic acid content was obvious.Studies had shown that the ratio of n-6/n-3 would affect the immune function of the body.Since n-6 and n-3 polyunsaturated fatty acids could form eicosanoic acid with different molecular structures in the body, eicosanoic acid derived from n-6 arachidonic acid might have higher immune activity than the source eicosanoic acid at n-3 (Su and Guo, 2003).Therefore, considering the effect of PUFA on immune function, an appropriate n-6/n-3 ratio was also necessary.The Chinese Nutrition Society put forward the appropriate ratio of polyunsaturated fatty acids n-6/n-3 in the "Reference intake of dietary nutrients for Chinese residents" as (4-6):1.The results showed that the n-6/n-3 ratio in soybean seeds was slightly higher than the recommended ratio.LC7 reduced the ratio, but it was not significant.This showed that high-concentration rare earth might reduce the n-6/n-3 ratio of soybean and make it closer to the optimal ratio trend.The rare earth concentration could be appropriately increased in the future experiments.

Conclusions

Under the conditions of this experiment, foliar spraying of lanthanum and cerium could adjust the content and ratio of fatty acid components in soybean seeds, affect its saturation, and make the fatty acid dietary ratio closer to the recommended ratio.There were differences in the regulation of soybean fatty acid content by different types and concentrations of rare earth.The results of this experiment indicated that the method of regulating rare earth could be used to optimize the fatty acid ratio of soybean seeds and improve the dietary fatty acid ratio.