utilization of jatropha curcas seed cake as a plant growth stimulant

Original Research Paper

Utilization of Jatropha curcas seed cake as a plant growth stimulant

Onuma Selanon, Donlaporn Saetae, Worapot Suntornsuk n

Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand

a r t i c l e i n f o

Article history:Received 7 September 2013Received in revised form11 August 2014Accepted 11 August 2014

Keywords:Plant growth promotionJatropha curcas seed cakeProtein hydrolysateEnzymatic hydrolysis

a b s t r a c t

Jatropha curcas seed cake containing high protein has served as a source of protein hydrolysate appliedfor plant growth stimulation. Effects of enzymatic and acid protein hydrolysates on the growth of HuaReau chili were investigated. Toxic compounds, phorbol esters in the seed cake, were removed first. Theprotein isolate prepared from the phorbol ester-free seed cake was digested by Neutrase, papain, trypsin,pepsin and HCl using various hydrolysis times. Degree of hydrolysis of the protein varied, ranging from3.8% to 97.3% depending on hydrolysis methods and times. The growth of Hua Reau chili wasconsiderably stimulated by the protein hydrolysate obtained from Neutrase digestion at 2 h and wassignificantly higher than that of all controls. In addition, the protein hydrolysate of 10 μg/ml obtainedfrom Neutrase digestion at 2 h gave the maximum stem height and diameter of Chinese kale plants.Therefore, the protein hydrolysate of the phorbol ester-free seed cake of J. curcas would be an alternativeand novel source of natural plant growth stimulant.

& 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Jatropha curcas (Physic Nut), a member of the Euphorbiaceaefamily, is a drought resistant plant often found in Central andSouth America, South-east Asia, India and Africa. It has beenconsidered as an important oilseed crop with various industrialand medicinal uses. After oil is removed from the seeds, a seedcake is generated and becomes a by-product with minimallyvaluable applications. The seed cake, however, contains 50–62%protein with high levels of essential amino acids (Makkar et al.,2008). Seed proteins are generally complex mixtures of variousproteins which are different in structure, size, charge, shape,amino acid composition, solubility and other physico-chemical,functional and nutritional properties. J. curcas protein has beenreported to consist of albumins, globulins, prolamins and glutelins(10.8%, 27.4%, 0.6% and 56.9%, respectively) (Selje-Assmann et al.,2007). Although the seed cake is rich in protein, use for directfeeding is limited because several anti-nutritional factors and toxicphorbol esters are presented. Phorbol esters in the seed cake weresuccessfully removed by using ethanol extraction (Saetae andSuntornsuk, 2010, 2011), a combination of enzymatic treatmentand ethanol (Xiao et al., 2011), alkaline treatment (Rakshit et al.,2008), bleaching (Haas and Mittelbach, 2000), and microbialfermentations (Phengnuam and Suntornsuk, 2013). Protein fromthe phorbol ester-free seed cake could be applied to human foodsor animal feed as a protein source.

Protein wastes and protein by-products from animals andplants treated by enzyme and acid hydrolyzes were reported tobecome peptides with beneficial biological activities. The bioactivepeptides found in protein hydrolysates could play their roles asantioxidant, antimicrobial agent, ACE-inhibitory agent, antityrosi-nase and plant growth promoters (Dziuba et al., 2004). Proteinsand peptides found in different parts of J. curcas also havebiological activities (Devappa et al., 2010). In addition, J. curcasflour and protein isolate served as a good source for the produc-tion of protein hydrolysates, providing biological benefits(Marrufo-Estrada et al., 2013).

Plant growth promoting activity is an important function ofprotein hydrolysates applied to agriculture. The hydrolysate couldbe derived from hydrolysis of plant and animal proteins such assoybean and fish. Kubo et al. (1994) reported that peptides, aminoacids, and amides produced from soybean lees degraded bymicrobial proteases could be an effective growth stimulant forBrassica campestris. The phytosulfokine-α has been reported to bea peptidyl plant growth factor stimulating the cell division insingle-cell cultures of asparagus (Matsubayashi and Sakagami,1996) and rice (Matsubayashi et al., 1997). It was also found thatthe phytosulfokine-α could stimulate the somatic embryogenesisin carrot (Kobayashi et al., 1999) and Japanese larch (Umeharaet al., 2005). Systemin was found to be a peptide hormoneregulating systematic wound response in tomato (Sun et al.,2011). Matsumiya et al. (2007) found that a peptide obtained fromdegraded soybean protein by an alkaline protease from Bacilluscirculans HA12 was a bioactive peptide promoting the differentia-tion of root hairs of Brassica rapa. Another study reported thatpeptides included in degraded bluegrill products promoted the

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Biocatalysis and Agricultural Biotechnology

http://dx.doi.org/10.1016/j.bcab.2014.08.0011878-8181/& 2014 Elsevier Ltd. All rights reserved.

n Corresponding author. Tel.: þ66 2470 8890; fax: þ66 2470 8891.E-mail address: [email protected] (W. Suntornsuk).

Please cite this article as: Selanon, O., et al., Utilization of Jatropha curcas seed cake as a plant growth stimulant. Biocatal. Agric.Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.08.001i

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root hair number and adventitious root formation of Brassica rapa(Sanpa et al., 2006).

A plant growth stimulant produced from natural materials hassignificantly become a research focus because of environmentalconcerns and the detrimental effects on soil due to overuse ofchemical fertilizers and chemical plant growth promoters. There-fore, this work was aimed at producing protein hydrolysates fromphorbol ester-free proteins obtained from J. curcas seed cake byacid and enzymatic hydrolyzes and studying their effects on thegrowth of Hua Reau chili (Capsicum annuum L.) and Chinese kale(Brassica alboglabra L.) plants.

2. Materials and methods

2.1. Preparation of J. curcas seed cake

J. curcas seed cake, obtained after oil extraction by a screwpress, was kindly provided from Ladda Company (Bangkok, Thai-land). It was ground with a blender and dried by a vacuum oven(OV-12, Jeio Tech, Seoul, Korea) at 55 1C until its weight wasconstant. The dry seed cake was stored in a desiccator prior to use.

2.2. Preparation of phorbol ester-free J. curcas seed cake

Phorbol esters were removed from the seed cake according tothe modified method of Saetae and Suntornsuk (2011). The seedcake was weighed and extracted with 90% (v/v) ethanol (analyticalgrade; Fisher Scientific, Loughborough, UK) at a ratio of 1:3. Themixture was shaken at 150 rpm for 5 min and filtered through afilter paper. The seed cake was collected and repeatedly extractedwith 90% (v/v) ethanol for 3 times as described earlier. Then, theseed cake was dried by a vacuum oven at 55 1C until its weight wasconstant and was stored in a desiccator until used. The dry seedcake was referred to as the phorbol ester-free seed cake.

2.3. Preparation of protein isolate

Protein of the phorbol ester-free seed cake was precipitatedand isolated according to the modified method of Saetae andSuntornsuk (2011). The precipitated protein referred to as thephorbol ester-free protein isolate was collected and dried by afreeze dryer (Scanvac Coolsafe 110-4, Labogene, Lynge, Denmark).

2.4. Determination of protein and phorbol esters

Protein contents of the J. curcas seed cake, phorbol ester-freeseed cake and phorbol ester-free protein isolate were determinedby the Kjeldahl method according to standard procedures (AOAC,1995). Phorbol ester contents of the samples were extracted andanalyzed by HPLC following the method of Saetae and Suntornsuk(2010) with triplicate determinations.

2.5. Enzymatic hydrolysis of phorbol ester-free protein isolate

Enzymatic hydrolysis of phorbol ester-free protein isolate wascarried out using the commercial proteolytic enzymes: Neutrase,trypsin, papain and pepsin. Neutrase (Z0.8 U/g, from Bacillusamyloliquefaciens) and trypsin (10,100 U/mg, from bovine pan-creas) were purchased from Sigma-Aldrich (Copenhagen, Den-mark). Pepsin (0.8–2.5 U/g, from porcine gastric mucosa) andpapain (30,000 U/mg, from Carica papaya) were purchased fromMerck KGaA (Darmstadt, Germany). Neutrase solution (1%, w/v),trypsin solution (1%, w/v), and papain solution (1%, w/v) wereprepared in 0.1 M phosphate buffer pH 7, while the pepsin wasdissolved in 0.1 M citrate buffer pH 2.5 to obtain the concentration

of 1% (w/v). The protein isolate solution for Neutrase, trypsin andpapain hydrolyzes was prepared by mixing the protein isolate with0.1 M phosphate buffer pH 7 (1%, w/v), while the protein isolatesuspended in 0.1 M citrate buffer pH 2.5 (1%, w/v) was preparedfor pepsin hydrolysis. The enzymatic hydrolysis was conducted byadding an enzyme solution to the protein solution at the ratio of1:100 (v/v). Hydrolysis by Neutrase and papain was maintained at50 1C, while the reactions of trypsin and pepsin were incubated at37 1C. The suspensions were taken at 0, 1, 2, 4, 6, 8, 10 and 12 hfrom each reaction and immediately heated at 90 1C for 10 min toinactivate enzyme activity. The mixtures were then centrifuged at12,000g for 15 min. The supernatants were collected and stored at�20 1C before use. They were referred to as the enzymatic proteinhydrolysates.

2.6. Acid hydrolysis of phorbol ester-free protein isolate

The protein isolate was mixed with 6 M HCl at a ratio of 1% (w/v). The mixture was incubated at 95 1C and was collected at 0, 1, 2,4, 6 and 12 h. The sample was kept at room temperature to cooldown. The mixture was neutralized to pH 6.5–7.0 by 5 M NaOH. Itwas centrifuged at 12,000g for 15 min and the supernatant wascollected and then stored at –20 1C prior to use. The collectedsupernatant was referred to as the acid protein hydrolysate.

2.7. Determination of α-amino acid and degree of hydrolysis

Content of α-amino acid in the protein hydrolysate sampleswas analyzed by the TNBS method (Adler-Nissen, 1979) based onthe reaction of 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Sigma-Aldrich, St. Louis, MO, USA) with primary amino groups underalkaline conditions. The α-amino acid content was expressed interms of L-leucine (Sigma-Aldrich, St. Louis, MO, USA) at theabsorbance of 420 nm.

Degree of hydrolysis (DH) was determined according to themethod of Adler-Nissen (1979). The DH (%) was calculated as[(AN2–AN1)/Npb]�100. The AN1 and AN2 (mg/g protein) are theamino nitrogen content of the sample before and after hydrolysis,respectively. The Npb (mg/g protein) is the nitrogen content of thepeptide bonds in the sample.

2.8. SDS-PAGE analysis

Molecular weights of the protein hydrolysates were deter-mined by the sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) according to Laemmli's method (Laemmli,1970). They were compared to those of the protein isolate beforehydrolysis and commercial protein hydrolysate (Bio Life M80,Suboneyo Chemicals Pharmaceuticals, Maharashtra, India). Thestandard protein markers used in this study were myosin(209.0 kDa), β-galactosidase (124.0 kDa), bovin serum albumin(80.0 kDa), ovalbumin (49.1 kDa), carbonic anhydrase (34.8 kDa),soybean trypsin inhibitor (28.9 kDa), lysozyme (20.6 kDa), andaprotinin (7.1 kDa) (Bio-Rad Laboratories, Hercules, CA, USA).Electrophoresis was carried out at a constant voltage of 100 V.Protein bands on the gel were finally made visible by staining withCoomassie brilliant blue G-250.

2.9. Effect of phorbol ester-free protein hydrolysates on plant growth

2.9.1. Hua Reau chilliPlant growth promoting effect of the phorbol ester-free protein

hydrolysates was tested on Hua Reau chili (Capsicum annuum L.)seeds provided by the Division of Postharvest Technology, theSchool of Bioresources and Technology, King Mongkut's Universityof Technology Thonburi.

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The protein hydrolysates hydrolyzed by Neutrase, trypsin,papain, pepsin and hydrochloric acid at 0, 1, 2, 4 and 12 h with aconcentration of 30 μg amino acids/ml were tested for plantgrowth promotion. Water, phorbol ester-free protein before diges-tion (30 μg amino acids/ml), urea (30 mg nitrogen/ml) and com-mercial protein hydrolysate (Bio Life M80) (30 μg amino acids/ml)were used as controls. The experiments were designed as Com-pletely Randomized Design (CRD) and were done in 4 replicationswith 100 seeds/replication.

One hundred seeds were cultivated on the top of blotter paperin a 14.5�20.5�7 cm3 plastic box. The seeds were treated with30 ml of samples and controls on the first day. The seeds wereincubated at room temperature for 14 days with adding 5 ml ofwater to each box on a daily basis. Germination percentage, radicleemergence percentage, seedling growth rate and germinationindex of the chili seeds were recorded following the method ofthe International Seed Testing Association (ISTA) (1999).

2.9.1.1. Germination percentage. Germination percentage wascalculated as an equation below

Germination percentage ð%Þ

¼Number of germinated seeds at day 14Total seeds

� 100

2.9.1.2. Radicle emergence percentage. Radicle emergence percentagewas expressed as

Radicle emergence percentage ð%Þ

¼Number of seeds with a radicle at day 14Total seeds

� 100

2.9.1.3. Seedling growth rate. The seeds were cultivated followingthe method discussed in Section 2.9.1 for 6 days and then kept intoa dark box until 14 days. After that, their cotyledons wereremoved. Their remaining part was dried for 24 h at 80 1C andweighed. The seedling growth rate (mg/plant) was calculated asthe ratio of dry weight of the remaining part to number ofgerminated seeds.

2.9.1.4. Germination index. Germination index was calculated by afollowing formula:

Germination index¼∑Number of germinated seeds

Day of final count

� �

2.9.2. Chinese kaleThe growth of Chinese kale (Brassica alboglabra L.) seeds

supplied by Ladda company (Thailand) was tested using theprotein hydrolysates obtained by Neutrase hydrolysis at 2 h con-taining amino acid of 10, 20, 30, 40, and 50 mg/ml. Chinese kaleseeds were cultivated in 14.5�20.5�7 cm3 plastic boxes for5 days and the Chinese kale sprouts were transplanted to smallpots with 2 sprouts/pot. The experiments were done in 4 replica-tions with 4 pots/replication. Each replication was watered daily inthe morning with 500 ml tap water for 40 days and it was sprayedweekly by using 500 ml of the protein hydrolysate. The controls ofthis experiment were also carried out by water, commercialprotein hydrolysate (Bio Life M80) (15 μg amino acid/ml and30 μg amino acid/ml) and the protein before digestion (30 μgamino acid/ml). After 40 days, the Chinese kale plants were taken.Their stem heights and diameters were recorded.

2.10. Statistical analysis

Statistical data was analyzed by the Analysis of Variance(ANOVA). Means of each parameter were compared by Duncan'sNew Multiple Range Test (DMRT) using the SPSS software version17 (SPSS, Chicago, Illinois, USA). Differences were consideredstatically significant at po0.05.

3. Results and discussion

3.1. Protein and phorbol ester contents

Protein contents in the J. curcas seed cake, phorbol ester-freeseed cake and phorbol ester-free protein isolate are shown inTable 1. J. curcas seed cake contained 17.5% protein, while thephorbol ester-free seed cake had 14.7% protein. The results wereslightly different from those reported by Makkar et al. (2008) andSaetae and Suntornsuk (2011) who found that the protein contentin screw-pressed seed cake was approximately 23–24%, whileprotein content in the phorbol ester-free seed cake was 17.9% asreported by Saetae and Suntornsuk (2012). However, proteinmainly found in the phorbol ester-free protein isolate wasapproximately 74.3% which was similar to that reported byMakkar et al. (2008).

Phorbol esters in J. curcas seed cake were detected in a smallamount of 0.08 mg/g as shown in Table 1. It was lower than thatfound in the seed kernel of a non-toxic J. curcas variety (0.11 mg/g)(Makkar et al., 1998) and was also similar to a rat tolerable level(0.09 mg/g) (Aregheore et al., 2003). However, the phorbol esterswere not detected in the phorbol ester-free seed cake and theirproteins isolate (Table 1). The result confirms previous studies thatphorbol esters in the seed cake were effectively removed byethanol extraction and the protein isolate was safe to be appliedto animal feed or other agricultural products.

3.2. Free α-amino acid content

Amino acids released from the protein isolate by enzymatic andacid hydrolyzes at various hydrolysis times are illustrated graphi-cally in Fig. 1. The treatments of protein isolate with hydrochloricacid and Neutrase showed a dramatic increase in amino acidsduring the first 4 h of hydrolysis. The acid and Neutrase providedthe highest amount of amino acids released with approximately1.6 and 1.1 mg/ml within 4 and 10 h, respectively. The amino acidreleased by papain moderately increased during hydrolysis, whilepepsin and trypsin gave slightly small amounts of amino acids.Different protein digestibility may result from the enzyme speci-ficity to peptide bonds at different amino acid sequences in theJatropha proteins.

Table 1Protein and phorbol ester contents of J. curcas seed cake, phorbol ester-free seedcake and phorbol ester-free protein isolate (based on dry weight).

Samples Protein (%) Phorbol estersa

(mg/g dry sample)

J. curcas seed cake 17.570.1 0.0870.01Phorbol ester-free seed cake 14.770.3 NDb

Phorbol ester-free protein isolate 74.373.2 NDb

All values are means of triplicate determinations7standard deviations.a Equivalent to phorbol 12-myristate,13-acetate.b Not detected.

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3.3. Degree of hydrolysis (DH)

Extent of protein hydrolysis can be quantified by the degree ofhydrolysis (DH) which refers to the percentage of peptide bondscleaved. The effect of enzymatic and acid hydrolyzes on proteindigestion in the protein isolate is shown in Fig. 2. Enzymatichydrolysis profiles in this study were substantially differentdepending on hydrolysis time and enzyme type. At the initialhydrolysis, the rate of hydrolysis increased rapidly and then it wasquite constant after 4 h of the reaction except for papain. Thismight be due to a decrease in specific peptide bonds available forenzyme reaction, product inhibition, and enzyme inactivation. Atthe same hydrolysis time, the highest DH was attained in thepresence of Neutrase among the protease hydrolysis. The Neutraseshowed a rapid increase in the DH during initial 1 h of thereaction. Kim et al. (1997) also found that the hydrolysis reactionof Neutrase occurred rapidly during the first 30 min of the reactionon soybean protein. Extensive hydrolysis of Neutrase may bebecause the enzyme had broader specificities to cleave variouspeptide bonds than other proteases. At 12 h of hydrolysis, Neutraseexhibited the highest DH with 97%, while papain, pepsin, andtrypsin showed much lower DH with 65%, 29% and 18%, respec-tively (Fig. 2). It is observed that trypsin hydrolysis gave the lowestDH possibly because of the presence of trypsin inhibitors in theJatropha proteins which inhibited trypsin to the substrate. Inaddition, the resistance of a protein for enzymatic hydrolysismight depend on the structure and conformation of the proteinand the specificity of the enzyme used. However, in general, DH isaffected by enzyme specificity, physical state and chemical char-acteristics of intact protein, and hydrolysis conditions (Karamaćand Rybarczyk, 2008).

Acid hydrolysis also provided the high DH as well as Neutrase.Its DH remarkably increased with hydrolysis time up to 2 h and

slightly increased until it reached constant at 4 h with a maximumvalue of 84%. This agreed well with the report of Kaewka et al.(2009) that the rice bran protein hydrolysate showed higher DHwith increasing acid hydrolysis time. The DH rate was high at theinitial period of hydrolysis since a large number of peptide bondsin protein molecules were cleaved, resulting in increased solublepeptides in the reaction mixture. The released soluble peptidesmight be substrate competitors to compete with undigested orpartially digested proteins, leading to the steady or lower DH atthe end of hydrolysis time.

3.4. Effect of phorbol ester-free protein hydrolysates on the growth ofHua Reau chili

Plants require amino acids in essential quantities for theirgrowth. Several plants can absorb amino acids which are betternitrogen sources than ammonia or nitrate. Several reportsrevealed that peptides or amino acids were necessary for plantsto support their growths, developments and metabolisms includ-ing decreased plant stress. Lee and Lian (2006) found thatbioactive peptides and free amino acids generated by the proteo-lytic hydrolysis of squid processing by-products can be used as anorganic fertilizer. Enzymatic extract of carob germ containingpeptides, free amino acids with a high content of glutamine andarginine, and a small amount of phytohormones were used as abiofertilizer to promote the growth, flowering and fruiting oftomato plants (Parrado et al., 2008). A Plant model, Arabidopsisthaliana, and heathland species, Lobelia anceps, treated by peptidesshowed increased growth rates (Soper et al., 2011).

3.4.1. Germination percentageSeed germination is an important stage for plant growth gen-

erally affected by seedling development, survival, and populationdynamics. It can be determined in a term of germination percentagewhich is an estimate of the seed viability. Germination percentagesof chili seeds after 14 days of cultivation are shown in Fig. 3. Proteinhydrolysate digested by Neutrase for 2 h gave the highest germina-tion percentage of 75% and provided a significant difference ofgermination percentage with other treatments and all controls(po0.05) (Fig. 3). The chili germination percentages of all treat-ments were higher than those of water, while all hydrolysates at 2,4 and 12 h of hydrolysis time showed higher percentages than allcontrols. These results suggested that hydrolysates would beeffective stimulants to promote the chili seed germination.

3.4.2. Radicle emergence percentageRadicle emergence is considered as the completion of germination.

Radicle emergence percentages of chili seeds after 14 days of cultiva-tion are presented in Fig. 4. The hydrolysate obtained by Neutrase for

Hydrolysis time (h)

Am

ino

acid

con

cent

ratio

n (m

g/m

l)

Fig. 1. Effect of enzymatic and acid hydrolysis time on α-amino acids released fromphorbol ester-free protein isolate.

Hydrolysis time (h)

Deg

ree

of h

ydro

lysi

s (%

)

Fig. 2. Effect of enzymatic and acid hydrolysis time on degree of hydrolysis ofphorbol ester-free protein isolate.

Neutrase Papain Trypsin Pepsin HClControl

Ger

min

atio

n (%

)

Water

Urea

Bio Life M80

Protein

0 h

1 h

2 h

12 h

4 h

Fig. 3. Effect of phorbol ester-free protein isolate hydrolyzed by acid and enzymeson germination percentage of chili seeds.






2 h showed the highest radicle emergence with 96%. It also provided asignificant difference on radicle emergence percentage compared toother treatments and all controls (po0.05). In contrast, the hydro-lysates from other enzymes and hydrochloric acid at various hydrolysistimes had a lower radicle emergence of chili seeds in a range of 75–85%, which was similar to all controls.

3.4.3. Seedling growth rateSeedling growth is the developmental period of young plant

starting from its completed germination until it has enough foodthrough photosynthesis to sustain its growth. Nitrogen is the mostimportant nutrient affecting plant growth. Seedling growth rate iscontrolled by nitrogen concentration which is influenced by theuptake rate of plant. Amino acids, peptides and proteins seemed tobe well absorbed by the root hair for plant nutrition as a nitrogensource (Kielland et al., 2006; Paungfoo-Lonhienne et al., 2008).

Seedling growth rates of chili affected by protein hydrolysatesobtained from acid and enzymatic hydrolyzes are shown in Fig. 5. Theseedling growth rates of hydrolysate by Neutrase at 2, 4 and 12 h ofhydrolysis time were much higher than those of other hydrolysatesand the controls. The highest seedling growth rate of 4.5 mg/plant wasgiven by protein hydrolysate of Neutrase digestion at 2 h, while thehydrolysate from trypsin showed the lowest seedling growth rateamong all hydrolysis treatments. The treatment at 2 h provided asignificant difference on seedling growth rate compared to othertreatments and all controls (po0.05) (Fig. 5).

3.4.4. Germination indexGermination index is based on a combination of seed germination

and root elongation data obtained. The germination indices of thehydrolysates from acid and enzymes at various times are illustrated in

Fig. 6. The germination index of the hydrolysate obtained by Neutrasefor 2 h was the highest at 7.8, which was a significant difference fromthat of other treatments and all controls (po0.05). Therefore, thehydrolysate obtained by Neutrase for 2 h was the best stimulant topromote the chili seed germination and chilli growth.

3.5. Effect of phorbol ester-free protein hydrolysates on the growth ofChinese kale

The hydrolysates obtained by Neutrase digestion at 2 h con-siderably affected the growth of Chinese kale as shown in Fig. 7.The hydrolysate at 10 μg amino acid/ml gave the greatest height ofChinese kale (approximately 28 cm/plant) which was significantlydifferent from all controls (po0.05). It was also provided thelargest diameter of Chinese kale (0.43 cm/plant). However, itsvarious concentrations (10–50 μg amino acid/ml) did not make asignificant difference on the plant diameter. This demonstratedthat an amount of 10 μg amino acid/ml in the hydrolysate isrequired for Chinese kale growth promotion.

3.6. Molecular weight of protein hydrolysates

Revealed by SDS-PAGE, Jatropha proteins without digestion andprotein hydrolysate obtained by Neutrase hydrolysis at 0 h showed

Rad

icle

em

erge

nce

(%)

WaterUrea

Bio Life M80

Protein

1 h

0 h

12 h

2 h

4 h

Control Neutrase Papain Trypsin Pepsin HCl

Fig. 4. Effect of phorbol ester-free protein isolate hydrolyzed by acid and enzymeson radical emergence percentage of chili seeds.

Water

Urea

Bio Life M80

Protein

1 h

0 h

2 h

12 h

4 h

Control Neutrase Papain Trypsin Pepsin HCl

Seed

ling

grow

th ra

te (m

g/pl

ant)

Fig. 5. Effect of phorbol ester-free protein isolate hydrolyzed by acid and enzymeson seedling growth rate of chili seeds.

Ger

min

atio

n in

dex

Water

Urea

Bio Life M80

Protein

0 h

1 h

2 h

4 h

12 h

Control PapainNeutrase Trypsin Pepsin HCl

Fig. 6. Effect of phorbol ester-free protein isolate hydrolyzed by acid and enzymeson germination index of chili seeds.

Treatments1 2 4 6 8 9

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0

5

10

15

20

25

30

35DiameterHeight

753

Dia

met

er (c

m/p

lant

)

Hei

ght (

cm/p

lant

)

Fig. 7. Stem heights and diameters of Chinese kale tested by protein hydrolysatesobtained from Neutrase hydrolysis of the seed cake protein isolated at 2 h with theircontrols (1¼ water; 2¼commercial protein hydrolysate (Bio Life M80, 30 μg aminoacid/ml); 3¼commercial protein hydrolysate (Bio Life M80, 15μg amino acid/ml);4 ¼protein without enzymatic digestion (30 μg amino acid/ml); 5¼protein hydro-lysate at 10 μg amino acid/ml; 6¼protein hydrolysate at 20 μg amino acid/ml;7¼protein hydrolysate at 30 μg amino acid/ml; 8¼protein hydrolysate at 40 μgamino acid/ml; 9¼protein hydrolysate at 50 μg amino acid/ml).






the same protein molecular weight profile of 21–32 kDa, 79.5 kDaand 85 kDa. These proteins were albumins, globulins, prolamins,and glutelins which are generally found in cereals (Selje-Assmannet al., 2007), and a mixture of proteases from Neutrase. Whileprotein hydrolyzed by Neutrase at 2 h contained the proteinmolecular weights of 12–22 kDa, 39.8 kDa, and 79.5 kDa, thisresult indicated that the protein molecules were cleaved byprotease actions resulting in the lower protein molecular weightsthan original Jatropha proteins. In addition, the commercial pro-tein hydrolysate obtained from soybean digested by a protease(Bio Life M80) used as a control showed protein molecular weightsranging from 12 to 22 kDa which provided higher plant growthindices of chili seeds than other controls but much lower plantgrowth indices than the protein hydrolysate obtained by Neutrasehydrolysis at 2 h. It could be concluded that the protein fractionsin protein hydrolysates with the molecular weights of 12–22 kDaand 39.8 kDa may have contributed to plant growth-promotingactivities leading to significantly higher plant growth indices,compared to other protein hydrolysates and better growth ofChinese kale. Thus, the protein hydrolysate obtained from Neu-trase hydrolysis at 2 h would provide the effective peptides whichcould play a role as a plant growth promoter. However, thepurification and identification of the plant growth-promotingpeptides in the protein hydrolysates need to be done. As reportedby Matsumiya and Kubo (2011) the plant growth-promotingactivity of the degraded soybean meal products on Brassica rapawere resulted from bioactive peptides present in the products.Bioactive peptides were previously reported to be a ligand thatwould bind to its specific receptor, sending the signal to promoteplant growth, or could have similar properties of phytohormones(Sanpa et al., 2006) or would increase root hair numbers in plants(Hasegawa et al., 2002) or promote root hair differentiation inplants (Matsumiya et al., 2007) or enhance the proliferation ofembryogenic cells and plant cell divisions (Kobayashi et al., 1999).However, the mechanism of the proteins for plant growth promo-tion in this study is still unknown. Further experiments to revealtheir mechanism of plant growth promotion are in progress.

4. Conclusions

Protein isolate obtained from the phorbol ester-free J. curcasseed cake was an excellent source to become a protein hydrolysatethat acted as a plant growth stimulant. The degree of hydrolysisand plant growth-promoting activities of the protein hydrolysateswere influenced by protease type, reaction condition and reactiontime. The protein hydrolyzed by Neutrase at 12 h provided thehighest degree of hydrolysis (97%), while the hydrolysate at 2 hgave the maximum growth indices for Hua Reau chili and thehydrolysate of 10 μg amino acid/ml gave the maximum height anddiameter of Chinese kale plants. The Jatropha protein hydrolysatecould be used as a natural plant growth stimulant instead ofchemical fertilizers. It, therefore, can benefit agricultural produc-tion. In addition, further study of plant growth promotion underactual farming conditions is necessary to confirm the practicalvalue of the Jatropha protein hydrolysate.

Acknowledgments

The authors are thankful to the Thailand Research Fund –

Master Research Grants for a scholarship of Onuma Selanon andThe Thailand Research Fund (IUG5180003) for a research budget.We also thank Ladda Company for financial and material supports.We acknowledge Asst. Prof. Pongphen Jitareerat for her valuable

discussion in plant growth data and analysis. Finally, we wouldlike to thank Lynne Crow for her English proof-reading.

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utilization of jatropha curcas seed cake as a plant growth stimulant

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