Components of soybean meal
Soybean has been cultivated all over the world since ancient times for its high protein and lipid content. It is one of the most important agricultural products in the world and its global production is more than 220 million tons per year . Vegetable oil production from soybean is the highest among plant oils (30%) .
Soybean is used directly as food in Japan and several Asian countries. Recently, soybean protein was recognized as both healthy and tasty and is used in food such as Tofu and soy sauce. Soybean-meal, which remains after extraction of the vegetable oil, contains about 50% protein with well balanced amino acids. Therefore, soybean-meal is often re-utilized as animal foodstuff.
Soybean waste was utilized as an organic fertilizer prior to the 1940s [3-6]. However, a chemical fertilizer took the place of the organic fertilizer because it produced faster results. Organic fertilizers are now gradually being used again for increased food production safety and the protection of the environment.
Soybean cultivation is well known for improving soil fertility [3, 7, 8]. Root-nodules are formed by the soybean plant, and atmospheric N2 is fixed by the nitrogen fixing bacteria in the root-nodule . N2 is converted to NH4+ by nitrogenase from these nitrogen fixing bacteria, and this NH4+ is supplied to the soil environment.
Recently, investigations into the utilization of proteins from soybean waste have been carried out for the development of high quality foods. Protein fractions, such as soy protein isolates and whey protein are industrially produced, and these fractions are used as additives for the improvement of food nutrition . Moreover, several soybean proteins and peptides have been purified and utilized as medicines for hypotension, rheumatism, and cholesterol control [11-13]. The bioactive peptides of soybean protein have also been investigated [5, 6].
This chapter explains how soybean cultivation and soybean protein are nitrogen suppliers and describes the production of novel bioactive peptides from soybean and legumes.
2. Nitrogen supply by soybean cultivation
2.1. Nitrogen fixing bacteria
N2 is fixed by nitrogen fixing bacteria in the soil environment [14-17]. These bacteria convert N2 to NH4+. The biological reduction of atmospheric N2 to NH4+ (nitrogen fixation) provides about 65% of the biosphere's available nitrogen .
As long ago as 1890, a nitrogen fixing bacteria was isolated from a root nodule and identified as
Nitrogenase (EC 18.104.22.168) from nitrogen fixing bacteria catalyzes N2 to NH4+ (N2 + 8H2 + 8e- + 16ATP + 16H2O → 2NH3 + H2 + 16ADP + 16Pi). NH4+ is further converted to NO2- and NO3- by ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB).
2.2. Relationship between nitrogen fixing bacteria and soybean cultivation
The roots of soybean secrete flavonoids and enhance the growth of nitrogen fixing bacteria around the root . The nitrogen fixing bacteria infect the soybean root, and the root-nodule is formed. Bacteroids in the root-nodule fix and provide nitrogen from the air .
The change in soil microbial diversity after soybean cultivation has been analyzed by PCR-DGGE. Root-nodules were shown to be formed and specific bacteria were increased during cultivation (Figures 1 and 2) but not the total number of bacteria in the soil. Soybean cultivation caused nitrogen accumulation in the soil environment.
3. Enhancement of nitrogen circulation by soybean cultivation and soybean protein
3.1. Evaluation of nitrogen circulation in soil environment
The nitrogen cycle is illustrated in Figure 3. Organic forms of nitrogen such as protein are degraded to peptides and amino acids by soil microorganisms, and these peptides and amino acids are then converted to NH4+. Subsequently, NH4+ is further converted to NO2- and NO3- (nitrification). NO2- is denitrified to N2 by denitrifying bacteria and this N2 is converted to NH4+ by the nitrogen fixing bacteria, and NH4+ is accumulated in the soil environment again.
The nitrification process is the rate limiting step in the nitrogen cycle . To further investigate the soil nitrogen cycle, a new method for the evaluation of nitrogen circulation activity was constructed based on bacterial number, ammonium oxidizing activity (AOA), and nitrite oxidizing activity (NOA) (Figure 4) . These three indices were used to construct a radar chart of nitrogen circulation in the soil. The area of the radar chart was calculated, and then the value was treated as a nitrogen circulation activity (0–100 points).
3.2. Enhancement of nitrogen circulation
A database of nitrogen circulation activity was constructed using 155 agricultural soils (Figure 5). The nitrogen circulation activity of agricultural soil ranges from 0 to 99.6 points with an average of 26 points.
Soybean cultivation leads to nitrogen accumulation in the soil environment, and therefore nitrogen circulation activity should be enhanced by soybean cultivation. This enhancement was further analyzed (Figure 6) and activity was shown to be enhanced 26 to 95 points after soybean cultivation.
Soybean waste is also rich in nitrogen (Table 1), and is often used as an organic fertilizer. Soil nitrogen is increased by using soybean waste as fertilizer, and consequently nitrogen circulation is increased. Soybean waste is also rich in carbon (C/N values; 5.1), and therefore soil bacteria and bacterial activity may also be increased by the addition of soybean waste.
|Total carbon||450,000 mg/kg|
|Total nitrogen||87,500 mg/kg|
|Total phosphorous||6,100 mg/kg|
|Total potassium||18,900 mg/kg|
4. Bioactive peptides from soybean protein
4.1. Plant growth promoting peptides from soybean waste
For efficient use of soybean waste, it is treated with an alkaline protease from
4.2. Root hair promoting peptide in DSP
The number of root hairs in
The molecular mass of RHPP was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) . The molecular weight of the bioactive peptide was 1,198.2 Da (Figure 9), and the molecular weight of the amino acid sequence in KTI was searched. Positions 27–38 in KTI (Gly-Gly-Ile-Arg-Ala-Ala-Pro-Thr-Gly-Asn-Glu-Arg) were identical to this molecular weight, and this peptide was thus designated as the RHPP (Figure 9). The RHPP that was chemically synthesized was also shown to have root hair promoting activity (data not shown).
5. Novel plant bioactive peptides from other legume
Many other legumes form root-nodules with nitrogen fixing bacteria. The nitrogen fixing bacteria related to legume cultivation are classified into 13 genera (
Legumes such as
In order to find novel bioactive peptides, attempts to degrade protein biomass from
Soybean supplies nitrogen into the soil environment by forming root nodules and accumulating protein in its seed. Soybean cultivation has been shown to enhance nitrogen circulation by about 3.6 times accompanied with increases in nitrogen fixing bacteria.
DSP has been shown to increase the fresh weight of plants, and a peptide from DSP promoted root hair formation in