INTRODUCTION

Efforts to increase soybean production in Indonesia have been intensively performed through planting of high yielding varieties, improved cultivation practices, and extending the harvested area, particularly to outside of Java. However, the agro ecological conditions were marginal, primarily due to acid soil. This exists in Kalimantan, Sumatera, Maluku, Papua, Bali, and Nusa Tenggara that accounts for 69.4% of the total dry land area (108.8 million ha) in Indonesia (Mulayani et al., 2004). Spesific characteristics of acid soil, such as low pH (< 5,5), high Al and Mn, high P fixation, low organic nutrients (N, P, K, Ca, Mg, Mo), and ion exchange capacity (Samac & Tesfaye, 2003) and low population of beneficial micro-organisms (Rhizobium spp.) (Kresović et al., 2010) may limit the soybean productivity. Therefore, soybean varieties tolerant to acid soil are essential as improvement of soil acidity by the addition of lime (amelioration) is yet costly for marginal farmers (Uguru et al., 2012).

A number of soybean promising lines tolerant to acid soil are currently available through conventional breeding (Kuswantoro et al., 2014 and Kuswantoro, 2016) and genemutation (Giono et al., 2014). Supportive data on the nutritional value, particularly protein and fat is needed as normally they are affected by genetic and environmental conditions (Rotundo & Westgate, 2009 and Whent et al., 2009). Soybeans also play an important role in the Indonesian diet as a protein source, primarily as tempe and tofu with the consumption level of 6.96 kg and 7.07 kg/capita/ year, respectively (Data and Information Centre of Agriculture, 2015). High protein content (≥ 40% dw) of soybeans is tailored for tofu ingredient, resulting in high yield recovery (Ginting et al., 2009). Similarly, the seed protein content which would dictate the final protein in tempe, tofu, soymilk, and soy sauce (Ginting et al., 2009) is essential as all national standard qualities for soybean products have established the minimal requirement levels for protein.

Soybeans contain approximately 20% of fat (dw) (Clemente & Cahoon, 2009), which consist of 85% unsaturated fatty acids (UFA), namely oleic, linoleic, and linolenic and 15% saturated fatty acids, such as palmitic and stearic acids (Lee et al., 2007). This is an important health issue as linoleic and linolenic belong to essential fatty acids and a diet containing fat high in UFA may lower the risks for high blood cholesterol and coronary heart disease (Lee et al., 2007). Increased intake of linoleic acid (ω-6) and linolenic acid (ω-3) in soybean foods is desirable as it may reduce the cardiovascular disease and improve the cognitive function. However, they are susceptible to oxidation, thus more amounts of oleic acid (ω-9) is desirable in soybean oil (Pham et al., 2012).

Isoflavones, the bioactive components in soybeans are beneficial to health due to their biological activities as phytoestrogens and antioxidant capacities that protect cells from oxidative damage (premature aging), prevent osteoporosis, coronary heart disease, cancer, and health problems associated with menopause (Luthria et al., 2007). Naturally occurring isoflavones are present as conjugation with sugars (glycones) and as free compounds (aglycones). About 12 isoflavones have been identified in soybeans, which the majority belong to genistin, daidzin, and glycitin glycones and genistein, daidzein, and glycitein aglycones (Luthria et al., 2007 and Ming et al., 2011). The total isoflavones and their profiles are highly dependent on soybean cultivar, planting season, cultivation practices, environmental conditions, and postharvest handling (Lee et al., 2003, Riedi et al., 2007, and Murphy et al., 2009). Therefore, a wide range of total isoflavones in soybeans has been reported, c.a. 682.4 μg to 4,778.1 μg/g (Kim et al., 2012) and 551 μg up to 7,584 μg/g (Zhang et al. 2014). This study was performed to quantitatively identify the protein, fat, fatty acids, and isoflavones in soybean promising lines tolerant to acid soil in terms of supporting their release as new varieties and enhancing the utilization for foods.

MATERIALS AND METHODS

The study was performed at the Laboratory of Plant Physiology, National Institute of Crop Science, Rural Development Administration (RDA), Miryang, South Korea. The materials used were twenty soybean promising lines tolerant to acid soil as a result of cross-breeding of Tanggamus variety (acid soil tolerant and medium-seeded) with Anjasmoro variety (large-seeded). They were grown at Tegineneng Experimental Field in Lampung, Indonesia during the rainy season up to beginning of the dry season (Februari-May) with a plot size of 2.4 m × 4.5 m, planting distance of 40 cm × 15 cm and two plants per hill. The average of daily temperature and relative humidity was 27.6oC and 82.8% and the monthly rainfall average was 89 mm. The soil was fairly acid with a pH of 4.7 Fertilizer application included 33.75 kg of N, 45 kg of P2O5, 37.5 kg of K2O per ha, while soil tillage, drainage, weed, pest and disease controls were optimally performed as referred to Kuswantoro et al. (2017a). Prior to analysis, working samples were prepared through grinding 5 g of seeds for each soybean line using a Hi-speed Vibrating Sample Mill TI- 100 (CMT Co. Ltd. Japan) to reach the refinement level of 150 mesh.

Protein Analysis

Dumas method was used for protein analysis (Jung et al., 2003). About 50 mg of ground sample was weighed on the oil paper, then subsequently pressed into a pellet form and inserted into the sample hole of combustion analyzer (Rapid N Cube Analyzer Type CR-100, Elementar, Germany). The protein content is calculated by multiplying the % N obtained from the analysis with a particular conversion factor for soybean (5.75).

Fat and Fatty Acid Analysis

Direct extraction method was applied for fat analysis according to Ha et al. (2009). About 2 g of ground sample was extracted in 200 ml of hexane using Fat Extraction System B-811 (Büchi, Switzerland) and distilling unit (Eyela CCA-110, Tokyo Rikakikai Co, Ltd Japan), then oven-dried, cooled in a desiccator and weighed. For fatty acid analysis, derivatization into fatty acid methyl esters (FAMEs) as described by Ha et al. (2009) was performed and slightly modified. The extracted fat was mixed with a solvent containing sulphuric acid, methanol, and toluene (1:20:10 v/v) and heated in a boiling water at 100-120oC for 1 h. After reaching room temperature, water was added and shaked. Water at the top was removed and the bottom oil layer (FAMEs) was collected into a 2-mL vial containing anhydrous Na2SO4 to absorb the excess water and capped with a 11-mm silver aluminum cap. Using a split mode (split ratio 1:50), about 1 μL of the analyte was injected onto the GC system (Agilent 7890A series, Boeblingen, Germany) that was equipped with a HP-FFAP capillary column (25 m × 0.32 μm i.d., 0.5 μm film thickness, Agilent Technologies), an auto sampler (Agilent 7683B), and a flame ionization detector (FID). The injector temperature was 250oC and oven temperature was programmed from 150o for 1 min, then 150oC to 230oC at 2.5oC/min, then held for 2 min. Flame ionization was detected at 260oC and N2 was used as a carrier gas at a flow rate of 1 mL/min. Standard mixtures of palmitic, stearic, oleic, linoleic and linolenic acid methyl esters (Sigma, Aldrich, St. Louis, MO, USA) were used for determination of fatty acids.

Isoflavones Analysis

Extraction of isoflavones was referred to Ha et al. (2009) with a minor modification. About 1 g of ground sample was extracted with 20 mL of 50% methanol and shaked for 24 h at room temperature, then filterred through a Whatmann paper No. 42. One mL of the filtrate was taken using a sterile hypodermic syringe (Korea Vaccine Co, Ltd) and filtered through a microfilter (0.45 μm, 33 mm i.d., SLHV033RS/Millex, Millipore, Merck). Separation and detection of the extract was performed using a HPLC (Agilent 1100 series, Boeblingen, Germany), which was equipped with a quaternary pump (Agilent G1311A,), a degasser (Agilent G1322A), an auto sampler and DAD detector. Twenty μL of the extract was injected onto a reversed-phase Lichrospher 100-RP-18e column (250 mm × 4 mm i.d., 5 μm, LichroCART, Merck KGaA). The isoflavones were separated through the gradient elution of acetonitrile containing 0.1% acetic acid (A) and water containing acetic acid 0.1% (B) as follows: 0 min 10% B, 20 min 20% B, 30 min 25% B, 40 min 35% B, 50 min 40% B and decreased back to 10% of B after 10 min. The flow rate was 1.0 mL/min and the analyte was monitored at a wavelength of 260 nm. Retention time and calibration curve of 12 isoflavones standards (Sigma, Aldrich, St. Louis, MO, USA) were used for identification and quantification of isoflavones in the sample extract. Analysis of protein, fat, fatty acids, and isoflavones was done in duplicate, respectively and data was presented as a mean and standar deviation of duplicate analysis. Analysis of variance (ANOVA) was applied for statistical analysis of total isoflavones, followed by DMRT for analysis of mean differences between soybean lines at a probability level of 0.05.

RESULTS AND DISCUSSION

Protein

The protein content of 20 soybean lines ranged from 39.64% up to 46,76% dw (Table 1). Three lines contained considerably high protein (≥ 45% dw), which were slightly higher than that of Tanggamus, a released variety tolerant to acid soil (44.5% dw) (Iletri, 2016). Genetic variation may contribute to such differences as protein content is quantitatively inherited traits (Hwang et al., 2014). Soybean cultivars also have different tolerance levels to Al toxicity in acid soils (Foy et al., 1992 and Kuswantoro, 2017b), resulting in different nutrients uptake (Kuswantoro, 2014) and seed chemical composition (Medic et al., 2014). Acidic soil conditions cause inhibition of cell elongation and division, leading to stunting of the primary roots. This results in low uptake of water and macronutrients in plants (Samac & Tesfaye, 2003 and Hanum et al., 2009). Kang et al. (2011) noted the negative correlations between Al content and K (r = 0.890), Ca (r = 0.913), and P (r =0.925) in wild soybean grown in acidic soil of Thailand. In addition, reduced nitrogen fixation may also occur in soybean due to sensitivity of the symbiotic rhizobia to soil acidity (Kresović et al., 2010). Thus, such limited uptake of nutrients may affect the seed chemical composition, particularly protein. Grobogan, Tanggamus, Anjasmoro, and Wilis varieties were reported to contain 3-5% lower of protein when grown in acidic dry land of Lampung (Ginting et al., 2012) relative to normal conditions (Iletri, 2016). Under non-irrigated conditions, Bellaloui & Mengistu (2008) also noted that Dwight variety gave lower protein and higher fat content compared to full season and reproductive stage irrigation treatments.

The protein contents of Tgm/Anj-888, Tgm/Anj-862 and Tgm/Anj-858 lines were approximately similar to those of Detam 1 and Detam 2, which contain the highest protein among Indonesian improved soybean varieties, c.a. 45.4% and 45.6% dw, respectively (Ginting et al., 2009). However, Saedanbaek, a Korean soybean variety (Shin et al., 2012) showed higher protein content (48.2% dw) relative to such three lines. Environmental growing conditions may also contribute to differences in protein content of soybean. Kumar et al. (2006) reported a variation of protein content from 36.8% to 42.1% dw of Pb 1 variety over four locations in India. The application of nitrogen fertilizer at <100 kg/ha increased the soybean protein content about 2% and up to 14% when >200 kg N/ha was applied. However, water stress and elevated temperature decreased the protein content by 16% and 9%, respectively (Rotundo & Westgate, 2009). Protein content is an essential quality trait in soybean as it positively correlated with the yield recovery and hardness of tofu produced. Also, soy sauce, soymilk, soybean flour, and protein isolate need high protein soybeans as ingredients (Ginting et al., 2009). Therefore, acid tolerant soybean lines with high protein content are promising in terms of releasing new variety as a supportive character to their desired agronomic characters, such as high yield, early maturity, and tolerant to major pests and diseases.

Fat and Fatty Acids

The fat contents ranged from 16.85% to 21.20% dw (Table 1) considerably due to genetic variation (Shi et al., 2010 and Hwang et al., 2014) and tolerance level to acidic soil conditions. In terms of low uptake of water and nutrients in acid soil, similar phenomenon in protein may also occur in fat content of soybean. However, Grobogan, Tanggamus, Anjasmoro, and Wilis varieties showed 1.5- 2% higher in fat contents when grown in acid soil (Ginting et al., 2012) relative to optimal conditions (Iletri, 2016). This may due to a decrease in protein content of these varieties as discussed previously since fat normally has negative correlation with protein (Lee et al., 2007).

In addition to genotype, soybean growing conditions significantly contribute to fat content (Clemente & Cahoon, 2009). Kumar et al. (2006) revealed that the fat content of Pb1 variety varied from 16.9% to 18.6% dw over four locations in India. Water stress and elevated temperature markedly increase the fat content (Wolf et al., 1982 and Bellaloui et al., 2013) as well as low altitude (Shin et al., 2009), while it remains unchanged when nitrogen supply increased (Rotundo & Westgate, 2009).

Similar values of fat obtained in this study were reported in 10 soybean varieties/lines (16.6-21.7% dw) (Antarlina et al., 2002) and in most of Indonesian improved varieties (Iletri, 2016). Tgm/Anj-847, Tgm/Anj-857, and Tgm/Anj- 832 lines contained considerably high fat (≥ 20% dw), approaching the fat content of imported soybean c.a. 21.4- 21.7% dw (Ginting et al., 2009). High fat content of soybean is desirable for oil extraction purposes as in the USA, hence imported soybean normally has lower protein content and higher fat content compared to domestic varieties. It seems difficult to develop soybean variety with high protein (≥ 45% dw) and fat contents (Lee et al., 2007) as both traits are likely controlled by the same genes (Hwang et al., 2014) and therefore gave negative correlation as seen in Fig. 1.

The profile of fatty acids varied between soybean lines with the major differences in oleic and linoleic acids (Table 1) that was primarily dictated by genetic variation (Lee et al., 2007 and Estiasih et al., 2011). On average, oleic acid showed the major proportion of soybean fat (43.93%), followed by linoleic (38.53%), and palmitic acids (9.42%) with total UFA of 87.81% (Fig. 2). However, Estiasih et al. (2011) noted that linoleic was the predominant fatty acid in Anjasmoro and Burangrang varieties (48.1% and 52.5%, respectively), followed by oleic (32.3% and 24.7%) and palmitic acids (11.5% and 11.7%). Another study from Indonesia also showed relatively higher values of linoleic (53.86%) and linolenic acids (7.15%) in soybean seed (Isa, 2011). This suggests that environmental growing conditions such as acid soil may also contribute to higher oleic acid obtained at present study. On average, the ratio of oleic/linoleic acids was 1.21 which varied from 0.45 to 2.52 and both of them negatively correlated (r =-0.98). Shin et al. (2012) also reported a significant negative correlation between oleic and linoleic acids in 18 soybean cultivars (r =-0.94) and in 721 soybean lines (Bachlava et al., 2008). Oleic acid was reported to increase along with water stress and elevated temperature, while linoleic and linolenic acids decreased (Jung et al., 2012 and Bellaloui et al., 2013).

Fat profile with low in saturated fatty acids (palmitic and stearic) and high in polyunsaturated fatty acids (linoleic and linolenic) is desirable with regard to health benefits in reducing coronary heart disease and improvement of brain function (Griffith & Morse, 2006). Therefore, they are suitable for direct food consumption and food supplements (Lee et al., 2007 and Estiasih et al., 2011). Tgm/Anj-832 and Tgm/Anj-862 lines with > 50% of linoleic and 6% of linolenic acids (Table 1) are tailored for such purposes.

However for food industry applications, soybean oil with more oleic acid (monounsaturated fatty acid) is preferred as it is less susceptible to oxidation (rancidity) and may reduce the trans-fat accumulation due to hydrogenation process for solid fat production, such as margarine and shortening (Kumar et al., 2006 and Pham et al., 2012). Soybean oil in the USA normally consists of 10-12% palmitic acid, 3-5% stearic acid, 23-24% oleic acid, 53-54% linoleic acid, and 8% linolenic acid with total UFA of 85% (Lee et al., 2007 and Bellaloui et al., 2013). Therefore, breeding of soybean is focused to increase oleic acid up to > 55% (Lee et al., 2007), even as high as 80% using mutant genes (Fehr, 2007, Pham et al., 2011, and Pham et al., 2012) and decrease linoleic acid to as low as 3%. Two lines, namely Tgm/Anj-889 and Tgm/Anj-857 could achieve such level with oleic acid of 55.54-60.35% (Table 1), thus suitable for oil-based food industry purposes.

Isoflavones

At present study, isoflavones were identified in their natural forms (mostly as glycones), thus no hydrolysis was performed during analysis. Among 12 isoflavones, only 10 of them were obtained in this study, while acetyldaidzin and acetylglycitin were not detected that might be present in trace amounts. Kim et al. (2014) also noted small amounts of both isoflavones in 44 soybean cultivars. On average, malonylgenistin was the major component (29.49%) of isoflavones in 20 soybean lines, followed by malonyldaidzin (19.04%) and glycitin (16.70%), while daidzein gave the lowest value (Fig. 3). One line, namely Tgm/Anj-832 contained almost similar amounts of malonylgenistin (446.0 μg/g) and malonyldaidzin (436.3 μg/g) (Table 2). As grown at the same location, variation in isoflavone profiles was primarily due to genotype responses to acidic soil conditions. Liang et al. (2007) reported that maternal genetic effects were predominant on isoflavone trait relative to the environment interaction effects.

The amounts of glycone isoflavones were much greater than those of the aglycones with in order of malonyl glucosides > glucosides > aglycones > acetyl glucoside (Table 2). Sakthivelu et al. (2008) also reported a similar order in 11 soybean cultivars with malonylgenistin and malonyldaidzin at the first and second rank, however genistin was found at the third rank and glycitein was the lowest. Malonyl isoflavones that normally account for 68-93% significantly correlate with total isoflavones (Sakthivelu et al., 2008 and Júnior & Ida, 2015). Prior to gastrointestinal absorption in human body, glycone isoflavones are hydrolized to their responding aglycones. Aglycone isoflavones show higher antioxidant activities relative to the glycones and genistein exhibits the highest activity. Also, individual isoflavone possesses different biological activity and bioavailability as well as stability during processing (Shao et al., 2011). Therefore, profiling soybean isoflavones is essential in terms of producing soy foods with greater health benefits. Due to their antioxidant capacities and biological activities as phytoestrogens, isoflavones may prevent the incidence of degenerative diseases (Sakthivelu et al., 2008) and menopause-associated health problems (Luthria et al., 2007 and Boucher et al., 2013).

The total isoflavones were significantly different between soybean lines that ranged from 835.9 μg/g dw in Tgm/Anj-858 up to 2,130.2 μg/g dw in Tgm/Anj-995 (Table 2). These values were within the range of total isoflavones reported in soybean, c.a. 1,200 to 2,400 μg/g (Luthria et al., 2007), 558.2 to 1,084.6 μg/g in Indian cultivars and 627.9 to 1,716.9 μg/g in Bulgarian cultivars (Sakthivelu et al., 2008), and 275.8 μg to 1,709.2 μg/g in Korean, Japan and China cultivars (Kim et al., 2014). However, Daweon and Seoritae (Korean varieties) showed much higher total isoflavones c.a. 2,581.6 μg/g (Kim et al., 2005) and 6,780 μg/g (Kim, 2007), respectively as well as soybean varieties grown in Vietnam, c.a. 1,153 to 5,653 μg/g (Cong et al., 2011) and China, c.a. 945.01 to 4,207.57 μg/g and 551 to 7,584 μg/g (Ming et al., 2011 and Zhang et al., 2014). Such differences may occur due to genotype, tissue type, growing conditions (location, season/year, temperature, soil nutrition, irrigation, biotic and abiotic elicitors, organic farming) and storage durations (Sakthivelu et al., 2008, Boué et al., 2008, Whent et al., 2009, Jung et al., 2012, Balisteiro et al., 2013, and Teekachunhatean et al., 2013). Secondary metabolites, including isoflavones also tend to increase under both biotic and abiotic stresses (Boué et al., 2009). Tgm/Anj-995 line, which had the highest value in this study was approaching a highisoflavone variety, namely Devon 1, which was released in 2016 with total isoflavones of 2,219.7 μg/g (Iletri, 2016). This suggests that Tgm/Anj-995 is promising to be released as a high-isoflavone variety tolerant to acid soil in addition to other agronomic characters.

CONCLUSIONS

This study suggests that selected soybean lines tolerant to acid soil are promising with respect to their nutritional aspects. Three lines had high protein content (≥45% dw), namely Tgm/Anj-888, Tgm/Anj-862 and Tgm/Anj-858. Tgm/Anj-889 and Tgm/Anj-857 lines with oleic acid > 55% are tailored for oil-based food industry purposes. In terms of total isoflavones, Tgm/Anj-995 line showed the highest value (2,130.2 μg/g dw), followed by Tgm/Anj- 784 and Tgm/Anj-832. This information needs to be taken into account by the breeder in releasing new soybean varieties tolerant to acid soil to support their excellent agronomic characters.

적 요

산성토양의 한계지역은 인도네시아에 많이 분포해 있다. 하 지만, 산성토양과 관련된 낮은 pH와 영양소는 콩 생산성의 제 한요인이 된다. 따라서 콩의 식품 활용성을 위해 양분소와 생 리활성성분에 대한 정보가 산성토양에 내성인 콩 품종과 함께 필요하다. 본 연구는 산성토양 내성 콩 유망계통 20개의 단백 질, 지방, 지방산, 이소플라본 함량을 구명하기 위해 수행하였 다. 그 결과, Tgm/Anj-888, Tgm/Anj-862 및 Tgm/Anj-858 등 3개의 계통에서 단백질함량이 높은 것으로 나타났다(≥ ≥45% dry weight=dw). 지방함량은 16.85~21.20% dw의 범 위를 보였으며, 단백질함량과는 부의 상관관계를 보였다(r=- 0.64). 콩 지방은 평균적으로 팔미트산 9.42%(C16:0), 스테아릭 산 2.77%(C18:0), 올레산 43.93%(C18:1), 리놀레산 38.53% (C18:2) 및 리놀렌산 5.35%(C18:3)으로 구성되었다. 총 10종 류의 이소플라본이 분류되었으며, 주요성분은 말로닐-제니스틴 (29.49%)으로 그 뒤를 이어 말로닐-다이드진(19.04%), 글리시 틴(16.70%)으로 밝혀졌다. 이소플라본 함량은 Tgm/Anj-995계 통(2,130.2μg/g dw)에서 가장 높았으며, 그 뒤를 이어 Tgm/ Anj-784계통과 Tgm/Anj-832계통(각각 1,719.9 μg과 1,710.0 μg/g dw)인 것으로 나타났다. 본 영양소 정보는 육종가들이 산성토양에 내성인 새로운 콩 품종을 보급하는데 있어 작물 특성의 보완적인 자료로서 유용할 것이다.