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-

474

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 .

J ~eferences 1 Patil R H. Effect of organic farmin g on the yield of soybean

and soil fertility under cotton-sorghum-soybean rotation, MSc Dissertation. Panjabrao Krishi Yidyapeeth, Akola, In­dia, 1999

2 Chhabra N, Soybean high protein, wonder grain, Ku­rukshetra , XLIII, 1994, 18

3 Gopalan C, Rama Sastri , B V & Balasubramanian S C, Nu­tritive Value of Indian Foods, Natl In st of Nutrition (ICMR, Hyderabad, Indi a) , 1997

4 Mutha P C, Soybean Industry Scenario in India, All India Conf Soybean Development, Indore, 1996, 4

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Indian J. Chern. Techno!. , September 2003

5 Wiedermann L H, ICFOST, New Delhi, 1985 6 Patil R P, An integrated biotech approach for enhancing the

yield and quality of Soybean, Ph D dissertation, North Ma­harashtra University, Jalgaon, India, 2001

7 Krieg N R In : Bergey's Manual of Systematic Bacteriology, 9'11 Edn (Williams and Wilkins, Baltimore, USA), 1984

8 AOAC Official methods of analysis of Association of Agri­cultural Chemists, 9'h Edn (Washington, DC, USA), 1955

9 Olsen S R, Cole C G, Watanabe F S & Dean FA, Estimation of available 'P' in soil by extraction with NaHC01 USDA Cir, 1964,919

10 Hanway J & Heidal N, Soil Analysis Methods as Used in Iowa State College Soil Testing Laboratory, Iowa Agri, 57: 1967, I

II Ramamurthy V, Sharma R K & Kothari R M, Walkley-Black Method of Organic Carbon Estimation In: Soil Conditioner, TCRDC Publ, Patiala, 1993

12 Daniels D H, Warner C R & Fazio T, J Assoc Off Anal Chern, 65 (1982) 588

13 Panse V G & Sukhatme P V, Statistical Methodf for Agri­cultural Workers, ICAR, New Delhi, India, 1995

14 Patil R P, Mendki P S, Chaudhari A 8, Maheshwari V L & Kothari R M, J Physiol Mol Boil Plants, 8 (2002) 221