Production of A Carob Enzymatic Extract
Production of A Carob Enzymatic Extract
com
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.
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 2313
(Drouliscos and Malekafi, 1980). It has a high proportion of 0.5 mL min 1, and peptides were detected at 215 and
of very water-insoluble protein (Wang et al., 2001) but a 280 nm. A protein standard mixture was used to cover
high content of arginine and glutamine (Boza et al., the range of 100 Da to 7 kDa.
2000). It makes a good substrate for conversion into fertil- Amino acid composition was determined by reversed-
izer/biostimulants for foliar fertilization and/or phase HPLC analysis of 6-aminoquinolyl-N-hydroxysuccin-
fertirrigation. imidyl carbamate (AQC) derivatives, with c-aminobutyric
The development of new fertilizers/biostimulants using acid as internal standard. Briefly, samples were hydro-
natural materials has become the focus of much research lyzed using 6 N HCl/1% (w/v) phenol vapour at 110 C for
interest. For this reason, the first aim of this paper was 24 h in vacuo. The amino acids were treated with AQC to
to describe the production by an enzymatic process of a form AQC-derivatives, which were then analyzed using a
vegetable extract from carob germ rich in amino acids Waters HPLC system (Millipore Ltd.) fitted with a
and peptides. The second aim was to determine the effects reversed-phase C18 column. For cysteine estimation, ali-
of this by-product vs. other amino-acid- and peptide-rich quots were first oxidized with performic acid and then ana-
by-products of animal origin on morphological parameters lyzed as above.
in a tomato crop (Licopericon pimpinellifolium cv. Protein solubility was determined using the method
Momotaro). described by Adler-Nissen (1977): briefly, 5 mL of 2.4 M
trichloroacetic acid was added to 10 mL of the sample
2. Methods (1% w/v), the precipitate was removed by centrifugation
(8000g, 10 min), and the nitrogen concentration of the
2.1. Determinations in CGHE supernatant was determined.
2.3. Experimental layout and nutritional treatments sidering a significance level of p < 0.05 throughout the
study.
The study was conducted in a greenhouse under con-
trolled conditions: temperature of 20 C and relative 3. Results
humidity of 60%. Tomato plants (Licopericon pimpinellifo-
lium cv. Momotaro) were chosen as the test crop, at a rate The proteins of CG are efficiently hydrolyzed: 20%
of 8 plants per container. Tomato seeds were germinated hydrolysis is reached after 40 min of reaction. The final
on well-washed and sterilized sand. Seedlings were trans- vegetable by-product (CGHE) is a brown syrup completely
ferred to a container with a commercial peat. The experi- soluble in water. Table 1 shows the main chemical charac-
mental layout was in a randomized block of nine teristics of CGHE.
containers. Three treatments were used (three replicates The enzymatic process increased the protein concentra-
per treatment): (1) treatment A0, containers fertilized with- tion in CGHE (64.2%). Due to the use of proteases, which
out by-product and treated with deionized water; (2) treat- solubilize and hydrolyze the initial insoluble proteins, there
ment A1, containers fertilized with Siapton (a trademark of is a specific increase of soluble proteins, peptides, and free
Isagro), a protein hydrolyzate of animal origin, used as amino acids. Their physical properties are somewhat differ-
plant biostimulant – at a dose of 1 cm3 500 mL 1; and ent to those of CG proteins; the proteins of CG are mainly
(3) treatment A2, containers fertilized with CGHE at a dose insoluble (see Fig. 2) and only the molecular weight of the
of 1 cm3 500 mL 1. soluble protein fraction has been analyzed by size-exclusion
The crop time was 18 weeks, during which plant height, chromatography. To perform this analysis, we have to use
number of flowers per plant, and number of fruits per plant a column that can differentiate globular proteins (see Sec-
were determined. tion 2). The size percentage has also been included in
Fig. 1, and comprises mainly proteins of higher molecular
weight.
2.4. Statistical analysis In CGHE, the proteins are soluble, and mainly in the
form of peptides and free amino acids. Fig. 1 shows the
The results obtained were analyzed by ANOVA, using molecular-weight distribution of the proteinaceous mate-
the Statgraphics v. 5.0 software package (Statistical Graph- rial present in CGHE. The data show that it comprises
ics Corporation, 1991), with the treatment as independent mainly peptides (<10 kDa) and free amino acids, almost
variable. The means were separated by Tukey’s test, con- 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)
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 The number and types of amino acids and their mix
(Asenjo et al., 2000), grain protein and grain starch concen- determine the nutritional profile of the product. The main
trations (Durante et al., 1992), and crop production (Tej- chemical difference between CGHE and Siapton regarding
ada and Gonzalez, 2003a,b; Tejada and Gonzalez, 2003a, nitrogen nutritional support is the amino acid profile, as
2004a,b). shown in Table 2.
By solubilizing the organic components of vegetable The amino acid profile of Siapton is included in Table 2.
matter – such as its carbohydrates, protein, and fat – in The CGHE and Siapton amino acid profiles are somewhat
liquid, a nutritional product is obtained. The protein, char- different, mainly due to their different origin: Siapton, from
acterized by long molecular chains, is mostly divided into an animal source (skin, hair wastes), has more than 50% in
its smaller components, such as peptides and, ultimately, only two amino acids (glycine + proline); by contrast,
amino acids. The purpose of breaking down the organic CGHE – with a vegetable origin – has a greater spread in
material is to make the amino acids and protein available distribution of the other amino acids – the most abundant
for easy absorption by plants. CGHE amino acids and pro- being glutamine (18%).
tein supply nitrogen, phosphorous, potassium, and many The amino acid profile of CGHE is of high quality due
trace elements, micronutrients, and minerals at a steadier to the high content of glutamine–glutamine-treated plants
rate than do synthetic products. accumulate amides (glutamine and asparagine) in their
The protein in CGHE is completely soluble – the molec- roots. Glutamine can be used as a substrate in a number
ular size being drastically reduced by the enzymatic attack, of transamidation reactions, e.g. glutamate synthase
converting the insoluble and aggregated proteins into pep- (GOGAT), asparagine synthetase (AS), and carbamoyl-
tides and free amino acids – so that the organic N is easily phosphate synthetase (CPS). Glutamine is also transami-
transported. nated or used in the synthesis of proteins and in the export
These changes in molecular weight also lead to an of carbon and nitrogen to other parts of the plant (Sechley
increase in the nutritional functionality. The better bioab- et al., 1992). Thus, maximum growth rates were found in
sorption of CGHE protein is due to two parameters: solu- plants supplied with glutamine, suggesting that this amino
bility, which is higher than that of the original CG protein, acid may be an important natural source of nitrogen for
and molecular weight, comprising mainly short peptides this plant species (Majerowicz et al., 2000).
and free amino acids. The content of free amino acids in CGHE is an impor-
In relation to this, the transport of peptides might be a tant parameter for its agronomic evaluation; chemical
more efficient means of nitrogen distribution than the hydrolysis using a high temperature and acid concentra-
transport of individual amino acids (Higgins and Payne, tions produced substantial racemization of the free amino
1980), especially for long-distance transport during the acids (Barret, 1985). Various negative or toxic effects of
bulk movement of protein-degradation products (e.g. in D-amino acids on living organisms have been reported
leaf senescence and seed germination). Peptide transport (Friedman, 1999). Therefore, the presence of D-amino acids
might also protect amino acids from catabolism by may be considered a negative indicator of fertilizer quality.
enzymes known to be present in the phloem during trans- However, in our product, the level of free amino acids is
port within the plant (Higgins and Payne, 1982). 8% of the total, and by using the enzymatic process we
The process is characterized by the ability of cells to have avoided the presence of D-amino acids.
transport peptides across membranes in an energy-depen- To cope with nutrient deficiencies, higher plants have a
dent manner. Internalized peptides are rapidly hydrolyzed range of responses to both their internal nutritional status
by peptidases, and the resulting amino acids are used for and the external availability of nutrients. Thus, there are
protein synthesis or as alternative sources of nitrogen and transporters that supply essential nutrients to the plant
carbon (Perry et al., 1994; Steiner et al., 1995). throughout development.
J. Parrado et al. / Bioresource Technology 99 (2008) 2312–2318 2317
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We thank the Ministerio de Medio Ambiente of Spain Majerowicz, N., Kerbauy, G.B., Nievola, C.C., Suzuki, R.M., 2000.
for financial support (Gant No. 262/2006/2-1.2). Growth and nitrogen metabolism of Catasetum fimbriatum (orchida-
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