Indian Journal of Chemical Technology Vol. 10, September 2003, pp. 470-476
Articles
Improvement in the yield and quality of soybean oil through an integrated biotech approach
R P Patil, V V Gite#, V L Maheshwari* & R M Kothari
School of Life Sciences,# School of Chemical Sciences, North Maharashtra University, Jalgaon 425 001, India
Received 24 May 2002; revised received 3 April 2003; accepted I I June 2003
-~-;;~:.cultivation of-soybean o~ vast scale, desery~;:S ~onsi~eraqon~ priillari~y .. by· virtue :o.f its nutritional contentS and yield irnprqy~ment.to, beo,a viable o_ilseed- ~n. gl9bal1 benchmark. Usii:t~IPNM.. aP.proach; comprising of soil conditioner;:3. bio'fefti,lizers· anp planr gro\vth reguJators, .fa£tOfial ·'randomized block d(;!Sign ··)Has eoneeived for, experimental purpOSe tt1 deter~ 'ihffie th.e combination of inputs. that afforded i-iiai_i.~um output. ,Th~ perfoimance of experupental design was monitored by ~s):im~tirig the yield ·ofsoybeari seeds, its oil and_protein content;· as :also f~der and fuejr value addition for putrit;ion. By e~:ll"i>i!rameter,)t has been demonstrated that IPNM approach ~¥ i~heteQt' strejigth to improve the yield of. soyhean, fqdder as·well as quality of oil. _ · · · ~-, ' : · ·
Soybean (Glycine max (L) Merrill), a multi-faceted crop belonging to the family Papillionaceae, ranked eighth in the world's crop production'. Biochemically, it has (i) about 40% protein, (ii) complement of all essential amino acids, (iii) 24% carbohydrates, (iv) approximately 20% oil, supporting health by virtue of a complement of essential unsaturated fatty acids (EUFA, 87%), especially linolenic acid (6.5%), (v) minerals (4.5%), fibers (3.7%), (vi) a complement of several vitamjns (71 0 IU vitamjn A, 2.4 mg niacin, 0.73 mg vitamin B 1, 0.39 mg vitamin B2, traces of vitamin C, D, E and K periOO g) and (vii) as many as 7 among 14 phyto-chemicals as rejuvenators2
•3
.
Presently, India's edible oil consumption is put at 80 mmt and domestic availability at 70 mmt; India bridges this gap through import4
. The consumption of oil is likely to grow at a rate of 10% in near future due to awareness about merits of polyunsaturated fatty acids (PUFA) content in it. Soybean oil has a balanced ratio of MUFA to PUFA, both needed to control blood cholesterol level. No wonder, therefore, it is fast becoming an accepted cooking medium in common Indian households. It is estimated that per hectare, soybean has the highest protein yield5 (500 kg), compared to sunflower (375 kg), cottonseed (250 kg), com (180 kg), rice (175 kg), milk (60 kg), poultry (50 kg) and beef (25 kg). Yet, Soybean remained neglected as a source of oilseeds/pulses in India until
*For correspondence (E-mail: vlmaheshwari @hotmail.com; Fax: 0257-2252183)
1986. Failure of several oilseed and pulse crops, in several geo-climatic zones of India, under a short spell of monsoon in recent years, brought soybean into focus as the most sustainable Kharif and Rabi crop, as a builder of soil fertility as judged from its remunerative history, year after year and in varied agro-climatic conditions, without compromising with satisfactory returns. While an average world production was 1900 kg/ha, in India, productivity remained 900 kg/ha. Thus, in totality (productivity per hectare, protein-rich nature, complement of amino acids, profiles of fatty acids, etc), soybean appears to be the best pulse/oilseed/source of food (feed)/industrial raw material. This article deals with efforts made towards improving the yield and quality of soybean oil through integrated plant nutrition management (IPNM) strategy.
Experimental Procedure
Experimental site The field experiments were carried out on the re
search farm of the North Maharashtra University, during Kharif season 1998 and 1999. The farm site was properly leveled, uniform, silty clay in texture. A composite soil sample before sowing and after harvesting, withdrawn from 0-30 em depth, by usual soil sampling technique, was analyzed for its physicochemical and microbial properties. It was virgin prior to experimental trials conducted during Kharif 1998 and repeated in Kharif 1999.
Patil et al.: Improvement in the yield and quality of soybean oil through an integrated biotech approach Articles
Soybean seeds Certified seeds of soybean MACS-124 variety,
yellow in colour, bold in size, characterized for (a) dwarfness, (b) short duration growth cycle, (c) pest/disease resistance, (d) high yielding characteristics and (e) recommended for Maharashtra State, were used for sowing.
Biotech inputs Organic carbon rich soil conditioner (SC), 3 types
of microbes (biofertilizers, BFs) and amino acids based plant growth regulators (PGR) were added either individually or in combinations. Their preparation, properties, rate of application, agricultural practices followed etc. are dealt within detail by Pati16
.
Experimental set-up The trials were conducted twice, during Kharif
1998 and 1999. The experimental set-up had factorial randomized block design (RBD), with three replica-
tions of each treatment. Total, 24 treatment permutations and combinations (formulated from 8 SC/BF/CF treatments and 3 PGR treatments), as summarized in Table 1 were examined for their effect on growth and yield. For each treatment, the gross plot size was 6.0 x 2.4 m, while the net plot size was 5.4 x 1.8 m. Anallocation of the representative plot to any particular treatment was made at random.
Chemical/microbial analysis of soil Do the experimental treatments make a qualitative
difference in the composition of soil and quality of soybean seeds (digestibility, profiles of essential fatty acids), assumedly by increasing/decreasing the presence of endogenous inhibitor(s) lipase was assayed.
Soil sampling Representative soil samples from individual plots
were collected before sowing soybean seeds/after harvesting the crop, air dried in shade, pulverized, sieved
Table !-Profiles of treatments in the experimental set-up
Sr No Treatment particulars
2
3
4
5
6
7
8
9
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
Control (No inputs)
Chemical fertilizers +No plant growth regulator
Soil conditioner+ No plant growth regulator
Biofertilizers + No plant growth regulator
Soil conditioner+ Chemical fertilizers+ No plant growth regulator
Soil conditioner+ Biofertilizers + No plant growth regulator
Chemical fertilizers+ Biofertilizers + No plant growth regulator
Soil conditioner+ Biofertilizers +Chemical fertilizers+ No plant growth regulator
Plant growth regulator (Aminos) only
Chemical fertilizers+ Ami nos
Soil conditioner+ Ami nos
Biofertilizers + Aminos
Soil conditioner+ Chemical fertilizers+ Ami nos
Soil conditioner+ Biofertilizers + Aminos
Chemical fertilizers+ Biofertilizers + Ami nos
Soil conditioner+ Biofertilizers +Chemical fertilizers+ Ami nos
Plant growth regulator (Growmore) only
Chemical fertilizers + Growmore
Soil conditioner+ Growmore
Biofertilizers + Growmore
Soil conditioner + Chemical fertilizers + Grow more
Soil conditioner+ Biofertilizers + Growmore
Chemical fertilizers + Biofertilizers + Growmore
Soil conditioner+ Biofertilizers + Chemical fertilizers+ Growmore
Po= No PGR; P1 = Aminos; P2 = Growmore
Symbols
SC0 + BF0 + CF0 + Po
CF+P0
SC+Po
BF+P0
SC + CF +Po
SC+ BF+ Po
CF+ BF+Po
SC + BF + CF + P0
SC0 + BF0 + CF0 +P1
CF+P1
SC+P1
BF+ PI
sc + CF+ PI
sc + BF+ PI
CF+BF+P1
SC + BF+CF+ PI
SCo + BFo + CFo + P2
CF+ P2
SC+P2
BF+ P2
SC + CF+ P2
SC + BF + P2
CF + BF + P2
SC + BF + CF + P2
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Articles Indian J. Chern. Techno!., September 2003
Table 2-Profiles of mean yield of seeds and mean yield of stover as a function of treatments
Treatment
SC+ BF+CF
CFoSCoBFo
CF
sc BF
SC+CF
SC+BF
CF+BF
SC+CF+ BF
'F' test
SE±
COatS%
PGR Po
PI
p2
'F' test
SE±
COatS%
SC+BF+CFx P
'F' test
SE±
COatS%
General mean
Sigt: Significant, NS: Non-significant
Kharif, 1998 Seed yield
(kg/ha)
944
12 13
1337
1064
1397
1237
1254
1614
Sigt
26
73
1209
1292
1271
Sigt
16
45
Sigt
45
127
1257
through 2 mm sieve, packed in clean cloth bags, duly labelled and stored for chemical/microbial analysis.
Microbial analysis of soil Individual colony forming units (CFU) of 3 biofer
tilizers and total CFU per g of soil were counted by standard microbiological methods to examine their presence before sowing and survivability at harvesting as a function of treatments 7•
Chemical analysis of soil N, P, K and organic carbon content of soil, before
sowing and at harvest, were determined by microKjeldahl method8
, Olsen's method9, flame photome
try10 and Walkey-Black method 11, respectively.
Estimation of oil Total oil content of air-dried and finely pulverized
soybean seeds was determined by extraction in n-
472
Kharif, 1999 Seed yield (kg/ha)
956
1266
1451
1183
1428
1389
1322
1648
Sigt
20
57
1290
1362
1339
Sigt
12
35
Sigt
35
99
1330
Kharif, 1998 Stover yield
(kg/ha)
782
838
1053
777
905
824
873
849
NS
103
733
888
967
Sigt
63
179
Sigt
178
506
863
Kharif, 1999 Stover yield (kglha)
875
948
1148
885
1014
929
971
953
NS
101
836
995
1065
Sigt
62
176
Sigt
175
498
965
hexane using Soxhlet method. Its percentage was calculated by difference between an initial weight and weight after extraction and extrapolated for I 00 g.
Estimation of digestibility of oil For estimating the digestibility of lipids, inhibitors
were extracted. For this purpose, 1 g of an air-dried, finely pulverized soybean seed flour was incubated with 25 mL Df 0.9% sodium chloride solution at 37°C for 4 h, mimicking the extraction of inhibitor(s) in digestive tract. The incubate was centrifuged (5000 rpm, 5 min) and the supernatant analyzed for an inhibitor activity by examining the digestibility.
Digestibility of oil by pancreatic lipase was judged by (i) vortexing 2 mL olive oil + 0.1 mL bile salts (0.1%) + 3 mL PV A (2% ), (ii) adding pancreatic lipase (0.1 mL, 100 IU) in phosphate buffer (0.05 M, pH 7.0), (iii) fortifying the reaction mixture with 20
Patil et al.: Improvement in the yield and quality of soybean oil through an integrated biotech approach Articles
Table 3- Analytical profiles of research farm soil at NMU
SrNo Soil characteristics Before sowing Status After harvest- After harvest-
Silt(%)
2 Clay(%)
3 Fine sand(%)
4 Coarse sand (%)
5 Textural class
6 Ph
7 Total soil salinity (mmho/cm)
8 Water holding capacity(%)
9 Soil density (g/cm3)
10 Organic carbon (%)
II Available N (kg/ha)
12 Available P20 5 (kg/ha)
13 Available K20 (kg!ha)
14 Exchangeable Ca (meq/100 g soil)
15 Exchangeable Mg (meq/100 g soi l)
16 Exchangeable Na (meq/100 g soil)
17 Mn (l!g/g)
18 Cu (l!g/g)
19 Fe (l!g/g)
20 Zn (l!g/g)
21 Total microbial count (cfu/g)
mM CaC}z, (iv) incubating the whole reaction mixture at 37°C for 15 min and (v) determining the digestibility of oil in the presence and absence of an inhibitor by titrating the liberated free fatty acids against 0.01 N NaOH solution and a few drops of 0.5% phenolphthalein as an indicator. An amount of 0.01 N NaOH requirement was taken as a measure of digestibility of oil. In this way, digestibility of fats in soybean seeds as a function of 24 treatments was examined.
Gas chromatography It was carried out as per Danials et al. 12 on a gas
chromatograph (Nucon, Model 5765, New Delhi) equipped with (a) SP-2300 capillary column having 30 m length and 0.25 mm diameter, (b) flame ionization detector (FID) and (c) recording integrator. A 3 f..lL aliquot of a given sample was injected into the column at 185°C with injector and detector at 250°C. Ethyl acetate served as a solvent, N2 flow rate was 30 mUmin, attenuator at 64 and chart speed 1 em/min.
Results and Discussion The main objective of the present studies was to
ing, 98 ing, 99
46.8 46.8 46.8
33.5 33.5 33.6
12.4 12.4 12.3
7.5 7.4 7.4
Silty clay Silty clay Silty clay
7.8 Normal 7.6 7.5
0.46 Normal 0.43 0.41
52.0 Moderate 72.0 75.0
1.30 Normal 1.20 1.18
0.13 Very low 0.50 0.60
150 Low 210 230
18.0 Low 28.0 30.5
670.0 Very high 650.0 645.0
62.0 60.0 59.0
11.0 10.5 10.4
50.0 47.0 46.5
37.0 Enough 35 .0 35.0
11.0 -do- 10.0 10.0
3.0 -do- 2.8 2.8
2. 1 -do- 2.0 2.0
0.3x107 Low 2.8xl07 4.0x107
investigate if IPNM approach succeeded in increasing the yield. This is summarized in Table 2.
From the data of productivity of soybean seeds and stover (fodder) and its statistical analysis 13
, it is clear that IPNM succeeded in achieving the objective of improving the yield of seeds and fodder. Another objective here was to explore, if the soil after harvest was poor in fertility or soybean crop contributed to fertility . Data pertaining to these aspects are summarized in Table 3.
The soil before crop was high in available potasssium (670 kglha), low in available nitrogen (150 kg/ha), lower in available phosphorus (18 kglha) and alkaline in nature (pH 7 .8). After the crop, it improved in available nitrogen (230 kg/ha) and available phosphorous (30.5 kglha). Presumably, this was a contribution of 3 bioferti lizer (N2 fixer, P solubilizer and S metabolizer) . If this was so, it should reflect in total CFU. Microbial count recorded 10 times increase, which is a reflection of improved soil fertility. Thus, to address the problems associated with soybean cultivation reviewed from literature 14
, a cost-
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Articles Indian J. Chern. Techno!., September 2003
Table 4--Profil es of oil content in soybean seeds and digestibility of oil in soybean seeds as a function of treatments
Oil (g%) Digestibili ty No Treatment Kharif
98
I SCo + BFo + CFo + Po 11.3
2 CF+ P0 13.5
3 SC + P0 13.7
4 BF + P0 14.0
5 SC + CF +Po 14.5
6 SC + BF + P0 16.0
7 CF + BF + P0 12.3
8 SC + BF + CF + P0 18.1
9 SC0 + BF0 + CFo +P 1 10.7
10 CF+ P1 10.8
II SC + P 1 12.3
12 BF+ P 1 13.1
13 SC + CF+ P1 13.2
14 SC + BF+ P1 14.2
15 CF+ BF+ P1 15.8
16 SC +BF+CF+PI 17.9
17 SCo + BFo + CFo + P2 11.0
18 CF+ P2 11.6
19 SC+ P2 11.9
20 BF + P2 13.4
21 SC + CF + P2 13.5
22 SC + BF + P2 13.8
23 CF+ BF + P2 13.4
24 SC + BF + CF + P2 16.7
General mean 14.3
effective, eco-friendly and integrated plant nutrition management (JPNM) strategy was devised and implemented on farm scale trials during Kharif 1998 and 1999 seasons . Observations during this period, made for morphological characters of plants, pods and seeds, biochemical parameters like photosynthetic efficiency, chemical composition of seeds and overall yield of soybean were corroborative6
.
Since soybean is being seen as a major oil seed crop globally, the data pertaining to oil content in seeds (g%) as a function of various treatments are summarized in Table 4.
From Table 4, it is clear that during Kharif 98 and 99, (i) mean oil content in soybean seeds was 14.3 and 14.1 g%, respectively, (ii) no specific trend could be discerned in oil content in seeds which corroborated with biotech inputs, (iii) barring an isolated in-
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Kharif Kharif Kharif 99 98 99
10.1 0.16 0.15
10.4 0.17 0.16
13.9 0.18 0.18
14.2 0.13 0.14
14.5 0.14 0.15
14.6 0.15 0.16
15 .1 0.16 0.17
15.6 0.18 0.19
10.9 0.15 0.14
10.9 0.17 0.16
11.1 0.18 0.16
11.3 0.22 0.19
11.2 0.21 0.18
11.2 0.20 0.20
12.3 0.18 0.19
15.0 0.22 0.21
10.8 0.17 0.16
11.8 0.16 0.15
14.9 0.20 0.18
15.7 0.20 0.20
15.8 0.15 0.17
15.8 0.16 0.18
17.3 0.21 0.20
18.3 0.27 0.28
14.1 0.18 0. 18
stance, most of the inputs showed oil content around 14.3 ± 2%, (iv) composite treatments (8, 16 and 24) gave marginally higher mean oil content ( 17.6 and 16.3%) over the rest of the treatments and (v) minimum oil content (11.3 and 10.1 %) was observed in control.
Digestibility of oil
A profile of digestibility of soybean oil by pancreatic lipase as a function of various treatments is given in Table 4.
The results indicated that digestibility of oil in soybean seeds is (i) poor as a result of individual treatments 1-7 and (ii) marginally improved by virtue of composite treatment, as also general mean (see treatments at serial No 8, 16 and 24 versus 1). It gives to our knowledge for the first time an indication that
Patil eta/.: Improvement in the yield and quality of soybean oil through an integrated biotech approach Articles
Table 5-Percentage profiles of fatty acids
Treatment Fatt acids(%) MPI MP2 MP3 MP4 MP5 MP6
I 18.0 27.2 54.7 2 12.3 24.3 54.9 3.0 5.5 3 16.4 28.8 54.9 4 14.4 25.2 57.8 2.5 5 14.6 23.8 52.9 3.9 4.8 6 12.8 23.0 57.2 1.5 5.4 7 11.9 21.9 54.7 2.2 5.6 3.5 8 13.9 22.5 57.5 3.3 2.9 9 14.1 22.1 60.2 1.6 1.9 10 15.4 14.7 69.8 II 14.3 24.8 58.8 2.1 12 14.7 23.3 60.4 1.6 13 12.1 21.8 58.3 1.7 7.0 14 12.8 23.3 60.2 1.2 2.5 15 12.5 24.6 57.8 1.3 4.0 16 13.9 21.6 60.9 3.5 17 13.1 21.1 61.4 0.9 2.9 18 13.9 22.4 61.6 2.1 19 15.9 26.6 51.3 8.7 3.5 20 14.5 23.2 56.6 2.9 2.8 21 20.2 23.1 49.7 3.5 3.4 22 14.9 23.5 59.6 1.2 0.7 23 23.1 19.6 46.4 2.7 2.2 24 15.7 21.1 52.1 5.1 5.3
Mean 14.8 ± 2.5 23.1 ± 2.6 57 .1±4.6 2.7 ±1.8 3.6 ± 1.5
MP =Major peak/Minor peak; MPI-MP3 =Major peaks; MP4-MP6 =Minor peaks
IPNM suppresses the synthesis of lipase inhibitor(s), like that of proteases (data not shown) and thereby improves their digestibility and bio-availability of metabolites for human beings to conserve limited. resources.
Generally, nutrition/energy requirement of soil is (i) less for a starchy (such as potato) crop, (ii) more for a proteinaceous (such as legume) crop, (iii) yet more for an oil-rich (such as oilseeds) crop and (iv) maximum for an oil and protein-rich crop, as in the case of soybean. Therefore, it is creditable that IPNM treatment has imparted higher oil content (Table 4), even though marginally so. While there was no increase in protein content per se, increase in the yield contributed to increase in the proteins too. This observation indicated that while molecular biology approach may take some time to enhance protein/oil content, IPNM approach can be adopted for achieving the same objective, albeit partially.
Gas chromatographic profiles of fatty acids While oil content appeared to be on marginally
higher side and digestibility too seemed improved marginally, it was essential to find out if soybean oil
as a function of these treatments contained higher percentage of essential unsaturated fatty acids (EUFA). For this purpose, oil from seeds derived by different 24 treatments was methyl esterified and GC profiles were carried out as per Daniels et al. 12
. The fatty acid peaks (MP1-MP6) were identified from their retention time on the basis of peaks of standard mixture of fatty acids, run simultaneously. Percentage of individual fatty acids obtained from GC profiles is summarized in Table 5.
From Table 5, it is clear that (i) qualitatively fatty acid profiles contain minimum 3 and maximum 6 peaks, with 18 samples showing 5 peaks (some containing MP4, some containing MP5 and some both) and 2 samples showing 6 peaks, (ii) quantitatively 3 major peaks account for 95.01 ± 9.66%, (iii) among the major peaks, MP3 accounted for 57.1 ± 4.67%, MP2 for 23.1 ± 2.67% and MPI for 14.8 ± 2.54%, (iv) corresponding peaks in literature belong to linoleic acid (51 %), oleic acid (29%), palmitic acid (23%), linolenic acid (6.5%) and stearic acid (2.1%) and (v) among the minor peaks, MP4 accounted for 2.7 ± 1.8%, MP5 for 3.6 ± 1.52%, while only sample
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7 and 23 contained MP6. The minor peaks could not be identified for want of standards. Therefore, IPNM derived soybean seeds exhibited, by and large, the same percentage of EUFA, perhaps its constituents varying non-significantly. Thus, through IPNM strategy a concerted mission for enhancing the yield of soybean and thereby ushering into yellow (soybean) r ~volution is feasible .
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