Available online at www.sciencedirect.com
Bioresource Technology 99 (2008) 2312–2318
Production of a carob enzymatic extract: Potential use as a biofertilizer J. Parrado
a,*
, J. Bautista a, E.J. Romero a, A.M. Garcı´a-Martı´nez a, V. Friaza a, M. Tejada
b
a
b
Departamento de Bioquı´mica, Bromatologı´a y Toxicologı´a, Facultad de Farmacia, Universidad de Sevilla, C/ Profesor Garcı´a Gonza´lez sn, 41012 Sevilla, Spain Departamento de Cristalografı´a, Mineralogı´a y Quı´mica Agrı´cola, 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 proteolytic 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 significantly 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 application of organic wastes, such as animal manure (Haynes and Naidu, 1998), sewage sludge (Albiach et al., 2001), city refuse (Eriksen et al., 1999), compost (Sikora 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 supplying plant nutrients. The new products are biofertilizers or biostimulants, they are organic products composed of peptides, amino acids, polysaccharides, peptides, humic acids, and/or phytohormones, 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:
[email protected] (J. Parrado).
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.05.029
the photosynthetic activity as they are directly absorbed 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 augmentation 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 (Ordon˜ez 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
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(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 fertilizer/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 methods (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 spectrometry (ICP-AES) using a Fisons-ARL 3410 sequential multielement instrument equipped with a data acquisition and control system. The standard operational conditions of this instrument are summarized as follows: the carrier gas, coolant 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¨ KTA purifier (Amershambiotech) accordtech) using an A ing to the procedure described by Bautista et al. (1996). Because of their high molecular weight, a Superdex 75 column (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
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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 reversedphase HPLC analysis of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives, with c-aminobutyric acid as internal standard. Briefly, samples were hydrolyzed 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, aliquots were first oxidized with performic acid and then analyzed 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 quantification, we extracted with EtOAc and, after some fractionation steps, the fractions were trimethylsilylated with N-methyl-N-(trimethylsilyl)-trifluoroacetamide, and analyzed 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 C18Sep 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 separated 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 proteases 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 follows different steps, including centrifugation, filtration, and concentration, as has been described for similar products (Parrado et al., 1991).
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sidering a significance level of p < 0.05 throughout the study.
2.3. Experimental layout and nutritional treatments The study was conducted in a greenhouse under controlled conditions: temperature of 20 C and relative humidity of 60%. Tomato plants (Licopericon pimpinellifolium 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 transferred to a container with a commercial peat. The experimental layout was in a randomized block of nine containers. Three treatments were used (three replicates per treatment): (1) treatment A0, containers fertilized without by-product and treated with deionized water; (2) treatment 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 Graphics Corporation, 1991), with the treatment as independent variable. The means were separated by Tukey’s test, con-
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 characteristics of CGHE. The enzymatic process increased the protein concentration 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 different 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 Section 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 material present in CGHE. The data show that it comprises mainly peptides ( 10.000 10.000-5.000 5.000-1.000 1.000-300 < 300
Ala Asp Cys Glu Gln Gly Pro Ser Tyr Arg His Ile Leu Val Lys Met Phe Thr
CG
CGHE
Siapton
3.95 ± 0.15 8.86 ± 0.24 0.96 ± 0.01 9.86 ± 0.25 18.23 ± 0.51 4.55 ± 0.12 3.73 ± 0.15 4.98 ± 0.13 3.62 ± 0.12 11.37 ± 0.31 2.93 ± 0.06 3.42 ± 0.12 5.34 ± 0.29 4.06 ± 0.15 6.40 ± 0.29 0.71 ± 0.02 3.15 ± 0.10 3.95 ± 0.13
3.58 ± 0.12 7.14 ± 0.21 0.20 ± 0.01 9.80 ± 0.21 18.72 ± 0.61 3.57 ± 0.09 3.75 ± 0.14 4.23 ± 0.16 3.85 ± 0.13 13.82 ± 0.29 2.91 ± 0.07 3.64 ± 0.160 7.07 ± 0.32 4.18 ± 0.14 5.49 ± 0.25 1.22 ± 0.03 3.28 ± 0.11 3.54 ± 0.12
15.06 ± 0.52 3.96 ± 0.12 0.83 ± 0.02 2.91 ± 0.08 0.90 ± 0.01 22.06 ± 0.10 30.72 ± 0.15 0.97 ± 0.01 0.77 ± 0.01 5.78 ± 0.20 2.30 ± 0.06 0.26 ± 0.01 4.70 ± 0.18 1.11 ± 0.02 2.02 ± 0.05 3.01 ± 0.11 1.23 ± 0.05 1.01 ± 0.04
Results are expressed as grams per 100 g of proteins.
CHE
Siapton
CG*
6.09 4.83 9.14 20.80 59.11
1.85 13.84 10.24 23.34 50.73
95 5
(Daltons)
Fig. 1. Size-exclusion chromatography and molecular weight distribution (percent of protein Nitrogen) of CGHE and Siapton on Superdex Peptide 10/300GL high performance column. *To analyse the soluble proteins of CG a Superdex 75 column (Amersham biotech) was used.
This change in the protein structure drastically modifies the water solubility. The protein components of CGHE are totally soluble, independently of the pH, whereas CG protein is limited in solubility, being practically insoluble at very acidic pH (Fig. 2). With regard to the amino acid composition, CGHE had increased arginine, isoleucine, and leucine contents, and decreased asparagine, cysteine, glycine, and lysine contents, while other organic components, such as carbohydrate, decreased in content (46%). CHGE 100
Soluble Nitrogen %
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80
However, for element content, trends are different: Mg, P, and K contents increased, and Ca, Na, Zn, and Cu decreased. An important component of CGHE is the water-soluble phytohormonal content. We have evaluated all the classical phytohormones (auxins, gibberellins, and cytokinins) (see Table 1). This phytohormonal content is extracted from the starting material (carob germ) by the hydrolytic event. Table 3 shows the analyzed parameters in tomato crop for the different treatments. The statistical analysis indicates significant differences of these parameters depending on the fertilizer treatment used. The greatest plant height, number of flowers per plant, and number of fruits per plant was in the A3 treatment, while the least was in the A0 treatment (in which organic matter was not applied). We used as a control of organic protein supply Siapton, a commercial product, composed of peptides and free amino acids with a molecular distribution similar to that of CGHE (see Fig. 1); the chemical composition is also similar (Table 1). The main difference was in the organic wastes to hydrolyze: CG is of vegetable origin and leads to a product (CGHE) with phytohormones. Flowering and fruiting occurred earlier when CGHE was applied than when Siapton was applied (A2 treatment) to the tomato plants.
CG
60
4. Discussion
40 20 0 3
4
5
6
7
8
9
10
11
pH Fig. 2. Nitrogen solubility of CG and CGHE at different pH values.
The by-product (CGHE) obtained after the enzymatic process presents important characteristics for its possible agricultural use, in particular some important contents in organic matter. Several works indicate the positive effect of humic substances on the uptake of nutrients such as N (Gamiz et al., 1998) and micronutrients (Gamiz et al., 1998; Mackowiak et al., 2001). Humic substances also have a positive effect on the content of photosynthetic pigments
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Table 3 Parameters of tomato crop Crop time (weeks)
8 10 12 14 16 18
Plant height (cm)
Number of flowers per plant
Number of fruits per plant
A0 treatment
A1 treatment
A2 treatment
A0 treatment
A1 treatment
A2 treatment
A0 treatment
A1 treatment
A2 treatment
Nd Nd Nd Nd Nd 53.7
Nd Nd Nd Nd Nd 69.5a
Nd Nd Nd Nd Nd 83.8a,b
1 3 8 9 9 9
– – – – 2a 16
1 4 7 15 32a,b 33a,b
– – 1 1 2 3
– – – – 1
– – 1 1 4 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 concentrations (Durante et al., 1992), and crop production (Tejada 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, characterized 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 protein 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 molecular size being drastically reduced by the enzymatic attack, converting the insoluble and aggregated proteins into peptides 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 bioabsorption of CGHE protein is due to two parameters: solubility, 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 transport within the plant (Higgins and Payne, 1982). The process is characterized by the ability of cells to transport peptides across membranes in an energy-dependent 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 carbamoylphosphate synthetase (CPS). Glutamine is also transaminated 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 important parameter for its agronomic evaluation; chemical hydrolysis using a high temperature and acid concentrations 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.
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The concentration of the macronutrients P and K increased, an aspect of great interest due to their importance in plant mineral nutrition and in crop production. Na concentration, which can negatively affect soil physical, chemical, and biological properties, and thereby crop production and quality (Haynes and Naidu, 1998), decreased. Another aspect regarding CGHE is the hormone concentration. Since Siapton is a by-product of animal origin, it does not present any concentration of the analyzed hormones. 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 germination of seedlings in order to ensure release of stored nutrients, promote leaf growth, and stimulate flowering and fruit development, etc. (Zerony and Hall, 1980; Philipson, 1985; Longman et al., 1986). Cytokinins stimulate cell division and growth, seed germination, flowering, and fruit development, and retard senescence, etc. (Hall, 1974; Zerony and Hall, 1980). Our experiment, carried out in tomato plants, demonstrates 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 horticultural 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.
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