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Bioresource Technology 99 (2008) 2312–2318

Production of a carob enzymatic extract: Potential use as a biofertilizer

a,*
J. Parrado , J. Bautista a, E.J. Romero a, A.M. Garcı́a-Martı́nez a, V. Friaza a, M. Tejada b

a
Departamento de Bioquı́mica, Bromatologı́a y Toxicologı́a, Facultad de Farmacia, Universidad de Sevilla,
C/ Profesor Garcı́a González sn, 41012 Sevilla, Spain
b
Departamento de Cristalografı́a, Mineralogı́a y Quı́mica Agrı́cola, E.U.I.T.A. Universidad de Sevilla, Crta de Utrera km. 1, 41013 Sevilla, Spain

Received 17 April 2006; received in revised form 7 May 2007; accepted 7 May 2007
Available online 29 June 2007

Abstract

In this paper, we describe a biological process that converts carob germ (CG), a proteinic vegetable by-product, into a water-soluble
enzymatic hydrolyzate extract (CGHE). The chemical and physical properties are also described. The conversion is done using a prote-
olytic enzyme mixture. The main component of CGHE extracted by the enzymatic process is protein (68%), in the form of peptides and
free amino acids, having a high content of glutamine and arginine, and a minor component of phytohormones, which are also extracted
and solubilized from the CG. We have also compared its potential fertilizer/biostimulant capacity on growth, flowering, and fruiting of
tomato plants (Licopericon pimpinellifolium cv. Momotaro) with that of an animal enzymatic protein hydrolyzate. CGHE had a signif-
icantly beneficial impact, most notably regarding the greater plant height, number of flowers per plant, and number of fruits per plant.
This could be due primarily to its phytohormonal action.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Carob; Enzymatic extract; Protease; Phytohormones

1. Introduction the photosynthetic activity as they are directly absorbed

The application of organic wastes, such as animal man-
ure (Haynes and Naidu, 1998), sewage sludge (Albiach
et al., 2001), city refuse (Eriksen et al., 1999), compost (Sik-
ora and Enkiri, 1999; Tejada and Gonzalez, 2003a), crop
residues (De Neve and Hofman, 2000; Trinsoutrot et al.,
2000), or by-products with high organic matter content
(Tejada and Gonzalez, 2003b, 2004a), to soil is a current
environmental and agricultural practice for maintaining
soil organic matter, reclaiming degraded soils, and supply-
ing plant nutrients.
The new products are biofertilizers or biostimulants,
they are organic products composed of peptides, amino
acids, polysaccharides, peptides, humic acids, and/or phy-
tohormones, etc. for immediate uptake and availability
within the plant. Their absorption does not depend on
*
Corresponding author. Tel.: +34 954556113; fax: +34 954233765.
E-mail address: parrado@us.es (J. Parrado).
by the plant, resulting in lower energy consumption. The
aim of these products is not to supply nutrition, but rather
to favour and stimulate the metabolism of the plant,
decrease plant stress, etc.
They are also claimed to enhance crop growth and yield
through a series of widely varying mechanisms including
activation of soil microbial activity, and promotion or aug-
mentation of the activities of critical soil enzymes or plant
growth hormones.
They decrease the need for soil-applied fertilizer,
decrease leaching and run-off of nutrients, decrease the
impact on the environment of fertilizer salts, supply the
nutritional requirements of the plant in the early stages
of development, and significantly increase the crop yield
(Ordoñez et al., 2001; Tejada and Gonzalez, 2004b).
Carob fruit – the fruit of the carob tree – comprises pulp
(90%) and seeds (10%). The pulp is sweet, and can be either
milled for used in confectionery or extracted to make syrup
(Marakis, 1996). Carob germ – the embryo of the seeds – is
a by-product and is processed for animal feedstuffs

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.05.029
 J. Parrado et al. / Bioresource Technology 99 (2008) 2312–2318
(Drouliscos and Malekafi, 1980). It has a high proportion
of very water-insoluble protein (Wang et al., 2001) but a
high content of arginine and glutamine (Boza et al.,
2000). It makes a good substrate for conversion into fertil-
izer/biostimulants for foliar fertilization and/or
fertirrigation.
The development of new fertilizers/biostimulants using
natural materials has become the focus of much research
interest. For this reason, the first aim of this paper was
to describe the production by an enzymatic process of a
vegetable extract from carob germ rich in amino acids
and peptides. The second aim was to determine the effects
of this by-product vs. other amino-acid- and peptide-rich
by-products of animal origin on morphological parameters
in a tomato crop (Licopericon pimpinellifolium cv.
Momotaro).
2. Methods
2.1. Determinations in CGHE
Ash was analyzed according to standard AOAC meth-
ods (AOAC, 1990). The protein content was determined
using the Kjeldahl procedure. Crude fat was determined
gravimetrically after CGHE extraction with hexane for
12 h in a Soxhlet extractor.
Total soluble carbohydrates were determined after
extraction with a mixture of ethanol/water (2/3) for 2 h.
After centrifugation at 4000g, the supernatant was filtered
through no.1 Whatman paper, and total soluble sugars
were estimated colorimetrically by the phenol–sulphuric
acid method, using a standard curve of glucose (Dubois
et al., 1956). Organic matter was determined by the dry
combustion method (MAPA, 1986).
Macro- and micronutrients in the sample were analyzed
by inductively coupled plasma atomic emission spectrome-
try (ICP-AES) using a Fisons-ARL 3410 sequential multi-
element instrument equipped with a data acquisition and
control system. The standard operational conditions of this
instrument are summarized as follows: the carrier gas, cool-
ant gas, and plasma gas is argon at 80 psi of pressure, the
carrier gas flow rate is 0.8 L min 1, the coolant gas flow
rate is 7.5 L min 1, the plasma gas flow rate is 0.8 L min 1,
and the integration time is 1 s. One mini-torch consumes
argon gas at a radio-frequency power of 650 W.
2.1.1. Protein characterization
Molecular mass distribution of peptides in CGHE and
Siapton was determined by size-exclusion chromatography
on a Superdex Peptide 10/300GL column (Amershambio-
tech) using an ÄKTA purifier (Amershambiotech) accord-
ing to the procedure described by Bautista et al. (1996).
Because of their high molecular weight, a Superdex 75 col-
umn (Amershambiotech) was used to analyze the soluble
proteins of CG.
The column was equilibrated and eluted with 0.25 M
Tris–HCl buffer (pH 7.0) in isocratic mode, at a flow-rate
2313
of 0.5 mL min 1, and peptides were detected at 215 and
280 nm. A protein standard mixture was used to cover
the range of 100 Da to 7 kDa.
Amino acid composition was determined by reversed-
phase HPLC analysis of 6-aminoquinolyl-N-hydroxysuccin-
imidyl carbamate (AQC) derivatives, with c-aminobutyric
acid as internal standard. Briefly, samples were hydro-
lyzed using 6 N HCl/1% (w/v) phenol vapour at 110 C for
24 h in vacuo. The amino acids were treated with AQC to
form AQC-derivatives, which were then analyzed using a
Waters HPLC system (Millipore Ltd.) fitted with a
reversed-phase C18 column. For cysteine estimation, ali-
quots were first oxidized with performic acid and then ana-
lyzed as above.
Protein solubility was determined using the method
described by Adler-Nissen (1977): briefly, 5 mL of 2.4 M
trichloroacetic acid was added to 10 mL of the sample
(1% w/v), the precipitate was removed by centrifugation
(8000g, 10 min), and the nitrogen concentration of the
supernatant was determined.
2.1.2. Phytohormone quantification
Auxins were determined as described by Schmelz et al.
(2003), and analyzed by GC–MS. For gibberellin quantifi-
cation, we extracted with EtOAc and, after some fraction-
ation steps, the fractions were trimethylsilylated with
N-methyl-N-(trimethylsilyl)-trifluoroacetamide, and ana-
lyzed by GC–MS, using a DB-1 capillary column (30 m ·
0.25 mm · 0.25 mm film thickness).
Cytokinin quantification: The sample was made up to
5 mL with 10 mM triethylammonium acetate (TEAA),
pH 7.0, and then part-purified by passage through a C18-
Sep Pak cartridge which had previously been primed with
10 mL of methanol followed by washing with 10 mL of
aqueous TEAA. Cytokinins were eluted in 10 mL of 50%
(v/v) aqueous methanol, and the methanol was removed
in vacuo at 35 C. The cytokinins in the exudate were sep-
arated by reversed-phase HPLC, and were detected at
268 nm.
2.2. Enzymatic hydrolysis
An enzymatic process to extract and reduce the protein
size of original carob germ (CG) protein was performed.
The substrate was modified by enzymatic hydrolysis, using
an endoprotease mixture as hydrolytic agent, and the pro-
teases were obtained by liquid fermentation with Bacillis
lichiniformis ATCC 21415. The fermentation broth, with
a proteolytic activity of 1000 U/mL, was used as hydrolytic
tool, using the pH-stat method (Adler-Nissen, 1986).
Briefly, it was diluted 1/20 in a bioreactor with controlled
temperature (60 C) and pH (pH 8) at a CG concentration
of 15% w/v. The processing of this hydrolyzed product fol-
lows different steps, including centrifugation, filtration, and
concentration, as has been described for similar products
(Parrado et al., 1991).
2314 J. Parrado et al. / Bioresource Technology 99 (2008) 2312–2318

2.3. Experimental layout and nutritional treatments
The study was conducted in a greenhouse under con-
trolled conditions: temperature of 20 C and relative
humidity of 60%. Tomato plants (Licopericon pimpinellifo-
lium cv. Momotaro) were chosen as the test crop, at a rate
of 8 plants per container. Tomato seeds were germinated
on well-washed and sterilized sand. Seedlings were trans-
ferred to a container with a commercial peat. The experi-
mental layout was in a randomized block of nine
containers. Three treatments were used (three replicates
per treatment): (1) treatment A0, containers fertilized with-
out by-product and treated with deionized water; (2) treat-
ment A1, containers fertilized with Siapton (a trademark of
Isagro), a protein hydrolyzate of animal origin, used as
plant biostimulant – at a dose of 1 cm3 500 mL 1; and
(3) treatment A2, containers fertilized with CGHE at a dose
of 1 cm3 500 mL 1.
The crop time was 18 weeks, during which plant height,
number of flowers per plant, and number of fruits per plant
were determined.
2.4. Statistical analysis
The results obtained were analyzed by ANOVA, using
the Statgraphics v. 5.0 software package (Statistical Graph-
ics Corporation, 1991), with the treatment as independent
variable. The means were separated by Tukey’s test, con-
sidering a significance level of p < 0.05 throughout the
study.
3. Results
The proteins of CG are efficiently hydrolyzed: 20%
hydrolysis is reached after 40 min of reaction. The final
vegetable by-product (CGHE) is a brown syrup completely
soluble in water. Table 1 shows the main chemical charac-
teristics of CGHE.
The enzymatic process increased the protein concentra-
tion in CGHE (64.2%). Due to the use of proteases, which
solubilize and hydrolyze the initial insoluble proteins, there
is a specific increase of soluble proteins, peptides, and free
amino acids. Their physical properties are somewhat differ-
ent to those of CG proteins; the proteins of CG are mainly
insoluble (see Fig. 2) and only the molecular weight of the
soluble protein fraction has been analyzed by size-exclusion
chromatography. To perform this analysis, we have to use
a column that can differentiate globular proteins (see Sec-
tion 2). The size percentage has also been included in
Fig. 1, and comprises mainly proteins of higher molecular
weight.
In CGHE, the proteins are soluble, and mainly in the
form of peptides and free amino acids. Fig. 1 shows the
molecular-weight distribution of the proteinaceous mate-
rial present in CGHE. The data show that it comprises
mainly peptides (<10 kDa) and free amino acids, almost
80% being under 1 kDa.

Table 1
Chemical characterization of CG, CGHE and Siapton
CG CGHE Syapton
Protein (g kg 1, w/w) 465 ± 12.0 642 ± 32.0 660 ± 23.0
Free amino acids (g kg 1, w/w) Nd 80 ± 3.2 85 ± 2.1
Fat (g kg 1, w/w) 65 ± 2.0 30 ± 1.5 20 ± 0.2
Carbohydrates (g kg 1, w/w) 380 ± 16.0 260 ± 8.0 Nd
Ash (g kg 1, w/w) 57 ± 0.4 65 ± 0.3 72 ± 0.4
Organic matter (g kg 1, w/w) 492 ± 20.0 494 ± 21.0 500 ± 23.0
P (g kg 1, w/w) 3.5 ± 0.2 6 7.7 ± 0.6 6.9 ± 0.5
K (g kg 1, w/w) 5.6 ± 0.3 22.8 ± 1.2 12.6 ± 1.0
Ca (g kg 1, w/w) 5.6 ± 0.4 3.1 ± 0.2 2.1 ± 0.1
Mg (g kg 1, w/w) 2.8 ± 0.1 3.5 ± 0.2 0,2 ± 0.0
S (g kg 1, w/w) 2.5 ± 0.1 4,3 ± 0.1 4,8 ± 0.1
Na (g kg 1, w/w) 1.2 ± 0.1 0.9 ± 0.0 11 ± 0.5
Fe (mg kg 1, w/w) 67 ± 3.1 65 ± 3.3 63 ± 3.5
Cu (mg kg 1, w/w) 25 ± 1.2 17 ± 1.1 –
Zn (mg kg 1, w/w) 36 ± 0.1 24 ± 1.2 3 ± 0.1
B (g kg 1, w/w) – 34 ± 2.1 40 ± 3.1
Mo (g kg 1, w/w) – 14 ± 0.8 –
Co (g kg 1, w/w) – 8 ± 0.5 –
Indoleacetic acid (IAA) (mg kg 1, w/w) Nd 108.5 ± 8.3 –
Naphtoxy acetic acid (NAA) (g kg 1, w/w) Nd 7.2 ± 0.5 –
Indolebutyric acid (IBA) (g kg 1, w/w) Nd 25.3 ± 0.16 –
Gibberellic acid (g kg 1, w/w) Nd 198.4 ± 11.0 –
Gibberellin A4+A7 (g kg 1, w/w) Nd 444.3 ± 29.0 –
Kinetin (g kg 1, w/w) Nd 23.1 ± 1.5 –
The values are on dry-weight basis.
Nd: non determined.
Data are the means of three samples.
 J. Parrado et al. / Bioresource Technology 99 (2008) 2312–2318 2315

1750 Table 2
Amino acid composition of CG and CGHE
1550 CG CGHE Siapton
Ala 3.95 ± 0.15 3.58 ± 0.12 15.06 ± 0.52
1350 Asp 8.86 ± 0.24 7.14 ± 0.21 3.96 ± 0.12
Cys 0.96 ± 0.01 0.20 ± 0.01 0.83 ± 0.02
Absorbancia (mAu)

1150 Glu 9.86 ± 0.25 9.80 ± 0.21 2.91 ± 0.08

Gln 18.23 ± 0.51 18.72 ± 0.61 0.90 ± 0.01
950 Gly 4.55 ± 0.12 3.57 ± 0.09 22.06 ± 0.10
CGHE
Pro 3.73 ± 0.15 3.75 ± 0.14 30.72 ± 0.15
750 Ser 4.98 ± 0.13 4.23 ± 0.16 0.97 ± 0.01
Tyr 3.62 ± 0.12 3.85 ± 0.13 0.77 ± 0.01
550 Siapton Arg 11.37 ± 0.31 13.82 ± 0.29 5.78 ± 0.20
His 2.93 ± 0.06 2.91 ± 0.07 2.30 ± 0.06
350
Ile 3.42 ± 0.12 3.64 ± 0.160 0.26 ± 0.01
Leu 5.34 ± 0.29 7.07 ± 0.32 4.70 ± 0.18
Val 4.06 ± 0.15 4.18 ± 0.14 1.11 ± 0.02
150
Lys 6.40 ± 0.29 5.49 ± 0.25 2.02 ± 0.05
Met 0.71 ± 0.02 1.22 ± 0.03 3.01 ± 0.11
-50 Phe 3.15 ± 0.10 3.28 ± 0.11 1.23 ± 0.05
0 5 10 15 20 25 30
Thr 3.95 ± 0.13 3.54 ± 0.12 1.01 ± 0.04
Ve (ml)
Results are expressed as grams per 100 g of proteins.

Molecular weight CHE Siapton CG*

(Daltons)
> 10.000 6.09 1.85 95 However, for element content, trends are different: Mg,
10.000-5.000 4.83 13.84 5 P, and K contents increased, and Ca, Na, Zn, and Cu
5.000-1.000 9.14 10.24 decreased.
1.000-300 20.80 23.34 An important component of CGHE is the water-soluble
< 300 59.11 50.73
phytohormonal content. We have evaluated all the classical
Fig. 1. Size-exclusion chromatography and molecular weight distribution phytohormones (auxins, gibberellins, and cytokinins) (see
(percent of protein Nitrogen) of CGHE and Siapton on Superdex Peptide Table 1). This phytohormonal content is extracted from
10/300GL high performance column. *To analyse the soluble proteins of the starting material (carob germ) by the hydrolytic event.
CG a Superdex 75 column (Amersham biotech) was used.
Table 3 shows the analyzed parameters in tomato crop
for the different treatments. The statistical analysis indi-
This change in the protein structure drastically modifies
cates significant differences of these parameters depending
the water solubility. The protein components of CGHE are
on the fertilizer treatment used. The greatest plant height,
totally soluble, independently of the pH, whereas CG pro-
number of flowers per plant, and number of fruits per plant
tein is limited in solubility, being practically insoluble at
was in the A3 treatment, while the least was in the A0 treat-
very acidic pH (Fig. 2).
ment (in which organic matter was not applied). We used
With regard to the amino acid composition, CGHE had
as a control of organic protein supply Siapton, a commer-
increased arginine, isoleucine, and leucine contents, and
cial product, composed of peptides and free amino acids
decreased asparagine, cysteine, glycine, and lysine contents,
with a molecular distribution similar to that of CGHE
while other organic components, such as carbohydrate,
(see Fig. 1); the chemical composition is also similar (Table
decreased in content (46%).
1).
The main difference was in the organic wastes to hydro-
CHGE
100
lyze: CG is of vegetable origin and leads to a product
(CGHE) with phytohormones. Flowering and fruiting
occurred earlier when CGHE was applied than when Siap-
Soluble Nitrogen %

80
ton was applied (A2 treatment) to the tomato plants.
CG
60
4. Discussion
40
The by-product (CGHE) obtained after the enzymatic
process presents important characteristics for its possible
20
agricultural use, in particular some important contents in
organic matter. Several works indicate the positive effect
0
3 4 5 6 7 8 9 10 11
of humic substances on the uptake of nutrients such as N
(Gamiz et al., 1998) and micronutrients (Gamiz et al.,
pH
1998; Mackowiak et al., 2001). Humic substances also have
Fig. 2. Nitrogen solubility of CG and CGHE at different pH values. a positive effect on the content of photosynthetic pigments
2316 J. Parrado et al. / Bioresource Technology 99 (2008) 2312–2318

Table 3
Parameters of tomato crop
Crop time Plant height (cm) Number of flowers per plant Number of fruits per plant
(weeks)
A0 A1 A2 A0 A1 A2 A0 A1 A2
treatment treatment treatment treatment treatment treatment treatment treatment treatment
8 Nd Nd Nd 1 – 1 – – –
10 Nd Nd Nd 3 – 4 – – –
12 Nd Nd Nd 8 – 7 1 - 1
14 Nd Nd Nd 9 – 15 1 – 1
16 Nd Nd Nd 9 2a 32a,b 2 – 4
18 53.7 69.5a 83.8a,b 9 16 33a,b 3 1 15a,b
Nd: not determined.
a
Statistically different when compared with A0 treatment (p < 0.05).
b
Statistically different when compared with A1 treatment (p < 0.05).

such as chlorophyll A and B and carotenoids in plants
(Asenjo et al., 2000), grain protein and grain starch concen-
trations (Durante et al., 1992), and crop production (Tej-
ada and Gonzalez, 2003a,b; Tejada and Gonzalez, 2003a,
2004a,b).
By solubilizing the organic components of vegetable
matter – such as its carbohydrates, protein, and fat – in
liquid, a nutritional product is obtained. The protein, char-
acterized by long molecular chains, is mostly divided into
its smaller components, such as peptides and, ultimately,
amino acids. The purpose of breaking down the organic
material is to make the amino acids and protein available
for easy absorption by plants. CGHE amino acids and pro-
tein supply nitrogen, phosphorous, potassium, and many
trace elements, micronutrients, and minerals at a steadier
rate than do synthetic products.
The protein in CGHE is completely soluble – the molec-
ular size being drastically reduced by the enzymatic attack,
converting the insoluble and aggregated proteins into pep-
tides and free amino acids – so that the organic N is easily
transported.
These changes in molecular weight also lead to an
increase in the nutritional functionality. The better bioab-
sorption of CGHE protein is due to two parameters: solu-
bility, which is higher than that of the original CG protein,
and molecular weight, comprising mainly short peptides
and free amino acids.
In relation to this, the transport of peptides might be a
more efficient means of nitrogen distribution than the
transport of individual amino acids (Higgins and Payne,
1980), especially for long-distance transport during the
bulk movement of protein-degradation products (e.g. in
leaf senescence and seed germination). Peptide transport
might also protect amino acids from catabolism by
enzymes known to be present in the phloem during trans-
port within the plant (Higgins and Payne, 1982).
The process is characterized by the ability of cells to
transport peptides across membranes in an energy-depen-
dent manner. Internalized peptides are rapidly hydrolyzed
by peptidases, and the resulting amino acids are used for
protein synthesis or as alternative sources of nitrogen and
carbon (Perry et al., 1994; Steiner et al., 1995).
The number and types of amino acids and their mix
determine the nutritional profile of the product. The main
chemical difference between CGHE and Siapton regarding
nitrogen nutritional support is the amino acid profile, as
shown in Table 2.
The amino acid profile of Siapton is included in Table 2.
The CGHE and Siapton amino acid profiles are somewhat
different, mainly due to their different origin: Siapton, from
an animal source (skin, hair wastes), has more than 50% in
only two amino acids (glycine + proline); by contrast,
CGHE – with a vegetable origin – has a greater spread in
distribution of the other amino acids – the most abundant
being glutamine (18%).
The amino acid profile of CGHE is of high quality due
to the high content of glutamine–glutamine-treated plants
accumulate amides (glutamine and asparagine) in their
roots. Glutamine can be used as a substrate in a number
of transamidation reactions, e.g. glutamate synthase
(GOGAT), asparagine synthetase (AS), and carbamoyl-
phosphate synthetase (CPS). Glutamine is also transami-
nated or used in the synthesis of proteins and in the export
of carbon and nitrogen to other parts of the plant (Sechley
et al., 1992). Thus, maximum growth rates were found in
plants supplied with glutamine, suggesting that this amino
acid may be an important natural source of nitrogen for
this plant species (Majerowicz et al., 2000).
The content of free amino acids in CGHE is an impor-
tant parameter for its agronomic evaluation; chemical
hydrolysis using a high temperature and acid concentra-
tions produced substantial racemization of the free amino
acids (Barret, 1985). Various negative or toxic effects of
D-amino acids on living organisms have been reported
(Friedman, 1999). Therefore, the presence of D-amino acids
may be considered a negative indicator of fertilizer quality.
However, in our product, the level of free amino acids is
8% of the total, and by using the enzymatic process we
have avoided the presence of D-amino acids.
To cope with nutrient deficiencies, higher plants have a
range of responses to both their internal nutritional status
and the external availability of nutrients. Thus, there are
transporters that supply essential nutrients to the plant
throughout development.
 J. Parrado et al. / Bioresource Technology 99 (2008) 2312–2318
The concentration of the macronutrients P and K
increased, an aspect of great interest due to their impor-
tance in plant mineral nutrition and in crop production.
Na concentration, which can negatively affect soil physical,
chemical, and biological properties, and thereby crop pro-
duction and quality (Haynes and Naidu, 1998), decreased.
Another aspect regarding CGHE is the hormone concen-
tration. Since Siapton is a by-product of animal origin, it
does not present any concentration of the analyzed hor-
mones. The presence of these hormones in CGHE is of
great importance, because of their fundamental role in crop
growth.
In this respect, auxins stimulate stem elongation and cell
elongation, induce root growth and apical dominance,
stimulate fruit development, delay fruit ripening, stimulate
growth of flowering parts, and are involved in absorption
of vital minerals and autumnal colour, etc. (Zerony and
Hall, 1980; Cohen and Bandurski, 1982). Gibberellins, like
auxins, promote cell elongation, act as chemical messengers
to stimulate the synthesis of enzymes such as a-amylase
and other hydrolytic enzymes important during germina-
tion of seedlings in order to ensure release of stored nutri-
ents, promote leaf growth, and stimulate flowering and
fruit development, etc. (Zerony and Hall, 1980; Philipson,
1985; Longman et al., 1986). Cytokinins stimulate cell divi-
sion and growth, seed germination, flowering, and fruit
development, and retard senescence, etc. (Hall, 1974; Zer-
ony and Hall, 1980).
Our experiment, carried out in tomato plants, demon-
strates the importance of the organic matter applied. The
best results were obtained in the treatment A2, indicating
that its chemical composition (phytohormone, aminoacid
content, etc.) in the by-product impacts positively on plant
height, number of flowers per plant, and number of fruits
per plant. These results suggest the advantage of applying
by-products of vegetable, rather than animal, origin and
their positive incidence in crop growth and development.
Acknowledgements
We thank the Ministerio de Medio Ambiente of Spain
for financial support (Gant No. 262/2006/2-1.2).
References
Adler-Nissen, J., 1977. Enzymatic hydrolysis of food proteins. Process
Biochem. 12, 18–23.
Adler-Nissen, J., 1986. Enzymic Hydrolysis of Food Proteins. Elsevier
Applied Science Publisher, London, New York.
Albiach, R., Canet, R., Pomares, F., Ingelmo, F., 2001. Organic matter
components, aggregate stability and biological activity in a horticul-
tural soil fertilized with different rates of two sewage sludges during ten
years. Biores. Technol. 77, 109–114.
AOAC., 1990. Official Methods of Analysis, 14th ed. Washington, DC.
Asenjo, M.C., Gonzalez, J.L., Maldonado, J.M., 2000. Influence of humic
extracts on germination and growth of ryegrass. Comm. Soil Sci. Plant
Anal. 31, 101–114.
Barret, G.C., 1985. Chemistry and Biochemistry of Amino Acids.
Chapman and Hall, London, p. 339.
2317
Bautista, J., Hernandez-Pinzón, I., Alaiz, M., Parrado, J., Millán, F.,
1996. Low molecular weight sunflower protein hydrolysate with low
concentration in aromatic acids. J. Agric. Food Chem. 44, 967–971.
Boza, J.J., Maire, J.C., Bovetto, L., Ballevre, O., 2000. Plasma glutamine
response to enteral administration of glutamine in human volunteers.
Nutrition 16, 1037–1042.
Cohen, J.D., Bandurski, R.S., 1982. Chemistry and physiology of bound
auxins. Ann. Rev. Plant Physiol. 33, 403–430.
De Neve, S., Hofman, G., 2000. Influence of soil compaction on carbon
and nitrogen mineralization of soil organic matter and crop residues.
Biol. Fertil. Soils 30, 544–549.
Drouliscos, N.J., Malekafi, V., 1980. Nutritional evaluation of the germ
meal and its protein isolate obtained from the carob seed (Ceratonia
siliqua) in the rat. Br. J. Nutr. 43, 115–123.
Dubois, M., Gilles, K., Hamilton, J., Rebers, P., Smith, F., 1956.
Colorimetric method for determination of sugars and related sub-
stances. Anal. Chem. 28, 350–353.
Durante, M., Attina, E., Nardi, S., Cacco, G., 1992. Changes induced by
humic substances and nitrate in the genomic structure of wheat roots.
In: Senesi, N., Miano, T.M. (Eds.), 6th International Meeting of IHSS,
Monopoli (Bari), p. 149.
Eriksen, G.N., Coale, F.J., Bollero, G.A., 1999. Soil nitrogen and maize
production in municipal solid waste amended soil. Agron. J. 91, 1009–
1016.
Friedman, M.J., 1999. Chemistry, nutrition, and microbiology of D-amino
acids. J Agric. Food Chem. 47, 3457–3479.
Gamiz, R., Espejo, J.A., Tejada, M., Dobao, M.M., Gonzalez, J.L., 1998.
Evolución de los contenidos de clorofilas en plantas de espárrago verde
(Asparagus officinalis, L.) tras la adición de aminoácidos y ácidos
húmicos. VII Simposio Nacional-III Ibérico sobre Nutrición Mineral
de las Plantas 1, 173–178.
Hall, R., 1974. Cytokinins as a probe of development processes. Ann. Rev.
Plant Physiol. 24, 415–444.
Haynes, R.J., Naidu, R., 1998. Influence of lime, fertilizer and manure
applications on soil organic matter content and soil physical condi-
tions: a review. Nutr. Cycl. Agroecosyst. 51, 123–137.
Higgins, C.F., Payne, J.W., 1980. Transport and utilization of amino acids
and peptides by higher plants. In: Payne, J.W. (Ed.), Microorganisms
and Nitrogen Sources. Wiley, pp. 609–637.
Higgins, C.F., Payne, J.W., 1982. Plant peptides. In: Boulder, D., Parthier,
B. (Eds.), Encyclopedia of Plant Physiology, vol. 14A. Springer, pp.
438–458.
Longman, K.A., Dick, J.McP., Mugglestone, M., Smith, R.I., 1986.
Effects of gibberellin A4/7 and bark-ringing on cone initiation in
mature Picea sitchensis grafts. Tree Physiol. 1, 101–113.
Mackowiak, C.L., Grossl, P.R., Bugbee, B.G., 2001. Beneficial effects of
humic acid on micronutrient availability to wheat. Soil Sci. Soc. Am. J.
65, 1744–1750.
Majerowicz, N., Kerbauy, G.B., Nievola, C.C., Suzuki, R.M., 2000.
Growth and nitrogen metabolism of Catasetum fimbriatum (orchida-
ceae) grown with different nitrogen sources. Environ. Exp. Bot. 44,
195–206.
MAPA, 1986. Métodos oficiales de análisis. Ministerio de Agricultura,
Pesca y Alimentación 1, 221–285.
Marakis, S., 1996. Carob bean in food and feed: current status and future
potentials – a critical appraisal. Food Sci. Technol. 33, 365–383.
Ordoñez, C., Asenjo, M.G., Benitez, C., Gonzalez, J.L., 2001. Obtaining a
protein concentrate from integral defatted sunflower flour. Biores.
Technol. 78, 187–190.
Parrado, J., Bautista, J., Machado, A., 1991. Production of soluble
enzymatic protein hydrolyzate from industrially deffated undehulled
sunflower meal. J. Agric. Food Chem. 39, 447–450.
Perry, J.R., Basrai, M.A., Steiner, H.Y., Naider, F., Becker, J.M., 1994.
Isolation and characterization of a Saccharomyces cerevisiae peptide
transport gene. Mol. Cell. Biol. 14, 104–115.
Philipson, J.J., 1985. The promotion of flowering in large field-grown
Sitka spruce by girdling and stern injections of gibberellin A4/7. Can. J.
Forest Res. 15, 166–170.
2318 J. Parrado et al. / Bioresource Technology 99 (2008) 2312–2318
Sechley, K.A., Yamaha, T., Oaks, A., 1992. Compartmentation of
nitrogen assimilation in higher plants. Int. Rev. Cytol. 134, 85–163.
Sikora, L.J., Enkiri, N.K., 1999. Growth of tall fescue in compost/
fertilizer blends. Soil Sci. 56, 125–137.
Schmelz, E.A., Engelberth, J., Alborn, H.T., O’Donnell, P., Sammons,
M., Toshima, H., Tumlinson, J.H., 2003. Simultaneous analysis of
phytohormones, phytotoxins, and volatile organic compounds in
plants. PNAS. 18, 10552–10557.
Statistical Graphics Corporation, 1991. Statgraphics 5.0. Statistical
Graphics System. Educational Institution Edition, USA, p. 105.
Steiner, H.Y., Naider, F., Becker, J.M., 1995. The PTR family: a new
group of peptide transporters. Mol. Microbiol. 16, 825–834.
Tejada, M., Gonzalez, J.L., 2003a. Effects of the application of a compost
originating from crushed cotton gin residues on wheat yield under
dryland conditions. Eur. J. Agron. 19, 357–368.
Tejada, M., Gonzalez, J.L., 2003b. Application of a byproduct of the two-
step olive oil mill process on rice yield. Agrochimica 47, 94–102.
Tejada, M., Gonzalez, J.L., 2004a. Effects of application of a byproduct of
the two-step olive oil mill process on maize yield. Agron. J. 96, 692–
699.
Tejada, M., Gonzalez, J.L., 2004b. Effects of foliar application of a
byproduct of the two-step olive oil mill process on rice yield. Eur. J.
Agron. 21, 31–40.
Trinsoutrot, J., Nicolardot, B., Justes, E., Recous, S., 2000. Decomposi-
tion in the field of residues of oilseed rape grown at two levels of
nitrogen fertilization. Effects on the dynamics of soil mineral nitrogen
between successive crops. Nutr. Cycl. Agroecosyst. 56, 125–137.
Wang, Y., Belton, P.S., Bridon, H., Garanger, E., Wellner, N., Parker,
M.L., Grant, A., Feillet, P., Noel, T.R., 2001. Physicochemical studies
of caroubin: a gluten-like protein. J. Agric. Food Chem. 49, 3414–
3419.
Zerony, M., Hall, M.A., 1980. Molecular effects of hormone treatment on
tissue. In: MacMillan, I.J. (Ed.), Hormonal Regulation of Develop-
ment. Springer-Verlag, Berlin.