Enrichment of peat with a protein hydrolysate-based biostimulant obtained from wine lees: effect on pepper plants

Cogent Food & Agriculture ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/oafa20 Enrichment of peat with a protein hydrolysate-based biostimulant obtained from wine lees: effect on pepper plants Manuel Tejada , Jesús López-Rodríguez , Isidoro Gómez , Patricia Paneque , Salvadora Navarro-Torre , José M. Orts & Juan Parrado To cite this article: Manuel Tejada , Jesús López-Rodríguez , Isidoro Gómez , Patricia Paneque , Salvadora Navarro-Torre , José M. Orts & Juan Parrado (2025) Enrichment of peat with a protein hydrolysate-based biostimulant obtained from wine lees: effect on pepper plants, Cogent Food & Agriculture, 11:1, 2486516, DOI: 10.1080/23311932.2025.2486516 To link to this article: https://doi.org/10.1080/23311932.2025.2486516 © 2025 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 08 Apr 2025. Submit your article to this journal Article views: 160 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=oafa20 Soil & Crop SCienCeS | reSearCh artiCle Cogent Food & AgriCulture 2025, Vol. 11, no. 1, 2486516 Enrichment of peat with a protein hydrolysate-based biostimulant obtained from wine lees: effect on pepper plants Manuel tejadaa, Jesús lópez-rodríguezb, isidoro Gómeza, patricia panequea, Salvadora navarro-torrec, José M. ortsb and Juan parradob aenvironmental Soil Science research group, department of Crystallography, Mineralogy and Agricultural Chemistry, e.t.S.i.A. university of Seville, Seville, Spain; bdepartment of Biochemistry and Molecular Biology, university of Seville, Seville, Spain; cdepartment of Microbiology and Parasitology, university of Seville, Seville, Spain ABSTRACT Currently, the use of biostimulants is considered an environmentally friendly alternative to synthetic fertilizers. in this work, we investigated the use of a biostimulant obtained from winemaking sludge by enzymatic hydrolysis processes in peat enrichment. the aim was to observe how this biostimulant affects the growth and development of green pepper (Capsicum annuum l. cv. italian) seedlings. the biostimulant was applied in three doses (1, 2, and 3 g/l) for a total of four applications. Seedlings were harvested at a height of 8–16 cm and with 5–7 leaves. plant height, weight of aboveground and belowground parts, macroand micronutrient contents, photosynthetic pigments in leaves, and enzymes related to plant stress were determined. the results obtained indicated that plant height, weight of the aboveground and belowground parts, and macroand micronutrient content in the aboveground and root parts were higher in plants treated with the highest dose of biostimulant. Compared with the control treatment (without amendments), the contents of chlorophyll a, chlorophyll b, and total carotenoids increased by 57%, 51.9%, and 53.1%, respectively, in plants treated with the highest dose of biostimulant. these results suggest that peat enrichment with this biostimulant could be useful for improving bell pepper quality. 1. Introduction the use of organic substrates in seedbeds for seed germination and seedling growth is a very common practice in modern horticulture (Massa et al., 2018). peat is currently, one of the most commonly used organic substrates is peat, as it represents an optimal growing medium given its physicochemical properties (Kitir et al., 2018; Massa etal., 2018). thus, it has been demonstrated that the use of peat for horticultural plants provides balanced aeration to the roots, low bulk density and optimal water retention, thus allowing these roots to develop optimally in terms of oxygenation and water absorption (Kitir et al., 2018). as regards chemical properties, peat is characterized by a high cation exchange capacity and nutrient supply after mineralization (Kitir et al., 2018; Massa et al., 2018). in addition, peat is considered a clean organic substrate, as it is free from weeds and pathogens (Kitir et al., 2018). however, the organic compounds that form part of peat need some time to mineralize and provide nutrients to the plant in its initial vegetative stages. this is of great importance since after seed germination, the mineral nutrition of the plant and therefore its growth will depend on the mineral elements obtained after said mineralization (tejada & Benítez, 2015). the use of protein-hydrolysate-based biostimulants in horticultural crops is currently of great interest, since it has been shown that these compounds improve plant mineral nutrition, and therefore, crop productivity and quality (Ávila-pozo etal., 2022, 2023; Shahrajabian et al., 2021). this is because these biostimulants generally consist of low molecular weight peptides, amino acids, polysaccharides, and macroand micronutrients, all of which can be assimilated by the plant. the enrichment of peat with these protein-hydrolysatebased biostimulants could be very of particular interest, since they could supply the seedling with a series of nutrients needed during its first vegetative stages, while mineralization of the organic © 2025 the Author(s). Published by informa uK limited, trading as taylor & Francis group CONTACT Manuel tejada [email protected] environmental Soil Science research group, department of Crystallography, Mineralogy and Agricultural Chemistry, e.t.S.i.A. university of Seville, Crta de utrera km. 1, Seville, Spain https://doi.org/10.1080/23311932.2025.2486516 this is an open Access article distributed under the terms of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. ARTICLE HISTORY received 22 october 2024 revised 14 March 2025 accepted 24 March 2025 KEYWORDS peat; biostimulant; wine lees; pepper nutrition; photosynthetic pigments SUBJECTS agriculture & environmental Sciences; Soil Sciences; Botany 2M. teJaDa etal. compounds that make up the peat give rise to these nutrients for the mora mature plant. one of the most common residues originating from wine production is wine lees, which are obtained after fermentation, storage and winemaking, and include filtration and centrifugation residues (yeasts and mineral residues) (De iseppi et al., 2020; Scarponi et al., 2024). it is estimated that 5 l of wine lees are produced for every hectoliter of wine (Da ros et al., 2017). Various authors consider that wine lees present a series of very diverse chemical constituents such as macro and micronutrients, vitamins (B1 and C), polyols and phenyl alcohols (for example, 2,3-butanediol, phenylethyl alcohol) (ioannidou et al., 2022; nagai et al., 2019). as a consequence, one strategy to be developed in the valorization of this residue is its use in the mineral nutrition of plants. Use of this by-product of the wine industry to develop protein-hydrolysate-based biostimulants could be of great interest since this waste has few applications in the circular economy (pérez-Bibbins etal., 2015) and it could be a safe and effective alternative to address sustainability challenges in agriculture (Ávila-pozo et al., 2022, 2023). the fruit of the green pepper (Capsicum anuum, l.) is considered to be a valuable source of elements that are very beneficial for health, such as phenolic compounds, vitamin C, vitamin e, carotenoids, capsaicinoids and minerals such as iron, phosphorus, copper and zinc (Seğmen & Ünlü, 2023). these compounds are of great importance for human health as they are related to the prevention of different diseases (Ávila-pozo etal., 2023). Since the use of biostimulants can enhance this range of chemical compounds (Ávila-pozo et al., 2023), the use of biostimulants obtained from wine lees in green pepper crops could be considered to be important in improving human health by enhancing these beneficial substances. We hypothesize that application of the biostimulant obtained from red wine lees during the growth of pepper seedlings in a seedbed could provide essential nutrients for growth until the peat is sufficiently mineralized to provide nutrients for the seedling. this should positively influence the growth and development of said seedling. as such, the objective of this study is to investigate the effect of enriching peat used as a substrate with a biostimulant obtained from red wine lees on the growth and development of pepper seedlings. this study represents a novel contribution since, to the best of our knowledge, there are no studies of the use of this type of biostimulant in agriculture and there is also no information about the enrichment of peat in seedbeds through the use of biostimulants. 2. Material and methods 2.1. Biostimulant characteristics the biostimulant was obtained from wine lees from vinification of the red grape varieties Syrah and tempranillo provided by the winery ‘nuestra Señora del Socorro’ located in rociana del Condado (huelva, Spain). the two varieties were vinified together in a 50:50 ratio in the 2023 vintage in accordance with the usual protocol followed in this winery. Specifically, the lees used for production of the biostimulant were those from the first and second fermentation, that is, those formed during alcoholic fermentation and malolactic fermentation, and they were mixed in a single sample. to obtain the biostimulant, the lees were subjected to enzymatic hydrolysis using the enzyme subtilisin (eC 3.4.21.62) as hydrolyzing agent in a bioreactor with controlled temperature (55 °C) and ph (ph = 9), using the ph-stat method (parrado et al., 2006). Figure 1 shows how the biostimulant was obtained. tables 1 and 2 show the chemical composition of the biostimulant. the methodology used to determine macro and micronutrients as well as the peptide profile of the biostimulant has been described previously (rodríguez-Morgado et al., 2014). the free amino acid content was determined using the methodology described by Kıvrak et al. (2014). For this purpose, a water/methanol mixture (Meoh/h2o, 80:20) was used for extraction. after sonication and centrifugation, the sample was filtered through a pVDF membrane filter with a pore size of 0.22 µm. Quantification was performed using a high-resolution UplC aCQUitY i-ClaSS chromatographic system equipped with a luna omega polar C18 column (2.1 x 50 mm) with a particle size of 1.6 µm, a flow rate of 0.4 ml min−1, an injection volume of 3 µl, two mobile phases (h2o + 0.5% hCooh and Me/oh/h2o (50:50 v/v) + 0.5% hCooh) and a column temperature of 40 °C. the phytohormone contents was determined following the methodology proposed by Müller and Munné-Bosch (2011). the extraction was similar to that described for free amino acids, and indoleacetic acid, 6-benzyladenine, abscisic acid and jasmonic acid were quantified using a high-resolution UplC chromatographic system coupled to a QtoF-MS mass CoGent FooD & aGriCUltUre 3 spectrometer, using a luna omega polar C18 column (2.1 x 50 mm) and 1.7 µm particle size, a flow rate of 0.4 ml min-1, an injection volume of 3 µl, two mobile phases (h2o + 0.05 hCooh) and a column temperature of 25 0C. 2.2. Experimental scheme the experiment was carried out in a greenhouse under controlled conditions of humidity (80 ± 1.4%) and temperature 25 ± 1.3 °C). only natural light was used during the experiment with no additional light being provided. as such, the natural light cycle corresponded to the hours of light in the study area. the peat used in the experiment (Floraska type 2) is characterized by being a blond peat with a ph (CaCl2) of 5.4–5.9, an electrical conductivity 400–500 µS cm−1 and n content of 190–250 mg l−1, a p2o5 content of 130–170 mg l−1 and a K2o content of 210–290 mg l−1. Green pepper seeds (Capsicum annuum l. cv italian) were acquired from a commercial nursery in the study area. the pepper seeds were planted manually on april 18 in seedbeds containing the experimental peat (one seed per compartment of the seedbed). three doses of the biostimulant (1, 2 and 3 g l−1 on dry matter) were added to the peat during the experimental period. these doses were completely random. table 3 shows the biostimulat treatments carried out in the experiment. each experimental treatment was performed in triplicate. the biostimulant was applied at a 14-day interval, such that it was applied on May 24, June 7, June 21 and July 5. this 14-day interval was chosen according to the criteria described by tejada et al. (2016), who observed that the persistence of biostimulants obtained by enzymatic hydrolysis in soil was very short due to the rapid absorption of its constituents by microorganisms. Consequently, the total doses of Table 1. Chemical composition and protein molecular weight distribution (mean ± standard error, n = 3) of biostimulant. dry matter (%) 9.2 ± 0.7 pH 7.8 ± 0.3 organic matter (g kg-1)431 ± 20 n (g kg-1)6.9 ± 1.2 P (g kg-1)10.4 ± 2.5 K (g kg-1)16.6 ± 2.9 S (g kg-1)4.5 ± 0.9 Ca (g kg-1)3.3 ± 0.4 Mg (g kg-1)1.2 ± 0.2 Fe (g kg-1)77.8 ± 8.3 Cu (mg kg-1)68.1 ± 8.6 Mn (mg kg-1)13.6 ± 1.1 Zn (mg kg-1)8.7 ± 0.2 indolacetic acid 66.1 ± 4.2 6-benzyladenine 192 ± 6.3 Abscisic acid 381 ± 23 Jasmonic acid 15.4 ± 1.96 Protein molecular weight distribution (da) >10,000 38.8 ± 2.1 10,000–5000 6.5 ± 2.2 5000–3000 4.3 ± 1.4 3000–1000 8.7 ± 2.0 1000–300 8.8 ± 1.6 <300 32.8 ± 2.3 Table 2. Biostimulant free aminoacid composition (mean ± standard error, n = 3). results are expressed as µg 100 g−1 of proteins. Ala 2130 ± 145 Asn nd Cys nd glu 928 ± 46.2 gln 23.1 ± 2.8 gly 721 ± 26.6 Pro 22 ± 3.2 Ser 481 ± 27.5 tyr 334 ± 16.9 Arg 58.2 ± 13.4 His 115 ± 26 ile 2487 ± 116 leu 3644 ± 197 lys 116 ± 19 Met 31.1 ± 1.6 Phe 470 ± 27.5 thr 679 ± 39.8 Val 679 ± 55.2 Asp 411 ± 27.3 nd: not detected. Figure 1. diagram of the enzymatic hydrolysis process used in vinification less to obtain the biostimulant. 4M. teJaDa etal. biostimulant applied throughout the experiment were 4, 8 and 12 g l−1 on dry matter. Considering that the microorganisms that may be part of the peat could use the biostimulant as a source of energy, said biostimulant was applied 4 times in the 14-day interval described so that the plant could assimilate the chemical constituents of said experimental biostimulant for a longer time. During the experimental period, the pepper seedlings were watered every 5–6 days depending on the moisture content of the peat. Drainage was prevented during each irrigation, and the acceptable irrigation volume was estimated using a control seedbed. pepper seedlings were harvested on July 13, when the plants had 5–7 leaves and were 8–16 cm tall. 2.3. Plant analysis For each seedbed and for each replicate of each biostimulant treatment, the height of the plant, the weight of the root and the weight of the aerial part were determined. to evaluate the nutritional status of the plant, 20 seedlings were collected from each seedbed and biostimulant treatment and the macro and micronutrient content of the underground (root) and aerial parts (stems + leaves) was analyzed. For each of these plant organs, the plant material was washed with distilled water, oven dried at 70 °C and crushed according to the procedure reported by Madejón et al. (2014). Kjeldahl-n was determined using the Mapa (1986) method for fresh material. For this purpose, a 0.5 g sample of plant material was digested with concentrated h2So4, using na2So4 and Se as catalysts. after digestion, the sample was distilled after adding 1 ml of 40% naoh solution. Finally, the sample was titrated with a standard solution of 0.02 n h2So4 to the end point and the n concentration was calculated. regarding the determination of other macro and micronutrients (p, K, S, Ca, Mg, Fe, Cu, Mn and Zn), after grinding the plant material it was digested with concentrated hno3 (65%, trace analysis grade) under pressure in a microwave oven. in the extract obtained these elements were determined by iCp-oeS. to determine the photosynthetic pigments, these were extracted with acetone using 0.25 g of the plant material. after 10 min of centrifugation, the content of chlorophyll a (at a wavelength of 662 nm), chlorophyll b (at a wavelength of 646 nm) and total carotenoids (at a wavelength of 470 nm) was determined. the concentration of these photosynthetic pigments was calculated according to the formulas described by lichtenthaler and Wellburn (1983). to observe how the enrichment of peat with the biostimulant affected plant stress, various antioxidant enzymes such as ascorbate peroxidase (apx), superoxide dismutase (SoD), guaicol peroxidase (Gpx) and catalase (Cat) were determined following the method described by Duarte et al. (2015). to that end, a plant extract was extracted (500 mg of leaves) with 50 mM sodium phosphate buffer (ph = 7.6). Cat was then determined at 240 nm in a solution containing 50 mM sodium phosphate buffer (ph = 7) and 100 mM h2o2. apx was determined using a solution of 12 mM h2o2 and 0.25 mM l-ascorbate and measured at 290 nm. SoD was determined by monitoring the oxidation of pyrogallol at 325 nm. Gpx was measured at 470 nm in a mixture with the assay buffer, 2 nm h2o2 and 20 mM guaiacol. the total protein content in the enzyme extracts was determined according to Bradford (1976). 2.4. Statistical analysis the statistical analysis was performed with the r software (r Core team, 2024), using different packages depending on the analysis required. to check whether a parametric test could be applied to analyze whether there were statistically significant differences between treatments, the Shapiro-Wilk normality test and the Bartlett test of homogeneity of variances included in the base r package were first carried out. to check whether the different doses of the biostimulant have a significant effect on the measured variables, an anoVa was performed with the aov fusion of the r base package (r Core team, 2024). When the result of the analysis showed that there were statistically significant differences, a tukey hSD test was performed using the agricolae package (by Mendiburu, 2023). to explore the data set and see how the measured variables are grouped and determine which ones have the most weight in explaining the Table 3. Biostimulant treatments were carried out in the experiment. l0 treatment: Control. no biostimulant was added to the peat l1 treatment: the peat was enriched with the biostimulant at a rate of 1 g l-1 (on dry matter) l2 treatment: the peat was enriched with the biostimulant at a rate of 2 g l-1 (on dry matter) l3 treatment: the peat was enriched with the biostimulant at a rate of 3 g l-1 (on dry matter) CoGent FooD & aGriCUltUre 5 variance of the system, a principal Component analysis was performed. For this, the FactoMiner (lê et al., 2008) and factoextra (Kassambara & Mundt, 2020) r packages were used. 3. Results tables 4 and 5 show the results of the Shapiro-Wilk test. as can be seen, since p > 0.05 the null hypothesis was not rejected and therefore it was verified that the data followed a normal distribution. on the other hand, table 6 shows the results of the Bartlett test for homogeneity of variances. in this case, it can also be verified that p > 0.05, so the null hypothesis was not rejected either and the equality of variances could be assumed. taking these results into account, the anoVa test was applied to check whether the addition of the different doses of the experimental biostimulant significantly affected the variables studied. enrichment of the peat with the biostimulant significantly increased (p < 0.05) the height of the plant, as well as the weight of the aerial and underground parts (table 7). this increase in these parameters was greater when the dose of biostimulant used increased. table 8 shows the content of macroand micronutrients in the aerial part (stems + leaves) and root, expressed in dry matter, for all biostimulat treatments. the enrichment of peat with the biostimulant obtained from wine less increased the contents of these nutrients in both the aerial and underground parts of the plant in comparison with the control treatments. Moreover, this increase was greater as the dose of biostimulant applied to the peat increased. thus, with regard to the macronutrients analyzed in the aerial part, treatment l3 caused a significant increase (p < 0.05) of 42.3% in n, 22.2% in p, 25.7% in K, 21% in Ca and 22.6% in Mg compared to the control treatment. no significant differences were observed with respect to the values obtained for S. Similarly, for the micronutrients analyzed, treatment l3 resulted in a significant increase (p < 0.05) of 16.7% in Fe, 18.3% in Cu, 58.8% in Mn and 37.1% in Zn compared to the control treatment. With regard the underground part of the plant, the macroand micronutrient contents increased significantly (p < 0.05) in plants for treatment l3, compared with the control treatment. Table 4. Shapiro-Wilk’s normality test of plant height, weight of aerial part, root weight, chlorophyll a, chlorophyll b, total carotenoids, ascorbate peroxidase, guaiacol peroxidase, superoxide dismutase and catalase. treatment Plant Height Weight aerial part root Weight Chlorophyll a Chlorophyll b total Carotenoids Ascorbate peroxidase guaiacol peroxidase Superoxide dismutase Catalase l0 W 0.9619 0.9720 0.9824 0.9429 0.9243 0.9733 0.9807 0.9054 0.9500 0.9320 p-value 0.5839 0.7969 0.9612 0.2723 0.1202 0.8231 0.9431 0.0547 0.3674 0.1680 l1 W 0.9622 0.9553 0.9703 0.9691 0.9557 0.9411 0.9465 0.9076 0.9654 0.9400 p-value 0.6514 0.4605 0.7603 0.7362 0.4631 0.2514 0.3182 0.0574 0.6569 0.2437 l2 W 0.9702 0.9649 0.9644 0.9320 0.9779 0.9846 0.9488 0.9890 0.9269 0.9500 p-value 0.7596 0.6456 0.6354 0.1689 0.9052 0.9794 0.3496 0.9968 0.1348 0.3730 l3 W 0.9546 0.9642 0.9607 0.9441 0.9866 0.9540 0.9624 0.9230 0.9599 0.9780 p-value 0.4437 0.6310 0.5595 0.2866 0.9897 0.4320 0.5939 0.1080 0.5429 0.9100 Table 5. Shapiro-Wilk normality test of macro and micronturients in aerial part and root. treatment n P K S Ca Mg Fe Cu Mn Zn Aerial part l0 W 0.9458 0.9362 0.9436 0.9706 0.9677 0.9625 0.9672 0.9574 0.9249 0.9658 p-value 0.3082 0.2033 0.2799 0.7677 0.7059 0.5965 0.6955 0.4932 0.1230 0.6647 l1 W 0.9124 0.9380 0.9585 0.9447 0.9017 0.9673 0.9591 0.97469 0.9835 0.9649 p-value 0.0709 0.2199 0.5148 0.2941 0.0443 0.6969 0.5266 0.8490 0.9711 0.6459 l2 W 0.9821 0.9450 0.9755 0.9651 0.9266 0.9605 0.9468 0.9778 0.9416 0.9654 p-value 0.9586 0.8146 0.864 0.6492 0.1329 0.5538 0.3212 0.9022 0.257 0.6558 l3 W 0.9587 0.9793 0.9717 0.9538 0.9780 0.9749 0.9461 0.97811 0.9694 0.9351 p-value 0.5176 0.9249 0.7910 0.4288 0.9059 0.8531 0.3123 0.9074 0.7428 0.1936 root l0 W 0.9609 0.9380 0.9766 0.9654 0.9403 0.9847 0.9650 0.9702 0.9680 0.9076 p-value 0.5627 0.8162 0.8831 0.6560 0.2427 0.9795 0.6482 0.7598 0.7129 0.0575 l1 W 0.9059 0.9351 0.9573 0.9738 0.9178 0.9322 0.9626 0.97615 0.9670 0.9528 p-value 0.0534 0.1943 0.4932 0.8331 0.0902 0.1702 0.5976 0.8753 0.6915 0.4122 l2 W 0.9517 0.9144 0.9471 0.9676 0.9739 0.9705 0.9593 0.9666 0.9603 0.9679 p-value 0.3935 0.0775 0.3256 0.7050 0.8345 0.7663 0.5305 0.6821 0.5503 0.7109 l3 W 0.9707 0.9531 0.9605 0.9595 0.9642 0.9208 0.9745 0.9729 0.9781 0.9731 p-value 0.7697 0.418 0.5533 0.5346 0.6317 0.1026 0.8447 0.8145 0.9079 0.8191 6M. teJaDa etal. the chlorophyll a, chlorophyll b and total carotenoid contents determined in the leaf followed a very similar trend to that reported for the macroand micronutrient contents (table 9). in addition, these photosynthetic pigment content increased as the dose of biostimulant applied increased. thus, for treatment l3, chlorophyll a content increased by 57%, chlorophyll b content increased by 51.9% and total carotenoid content increased by 53.1% compared to treatment l0. With regard to the enzymes determined, the results obtained showed that only apx increased significantly (p < 0.05) as the biostimulant dose increased (table 10). this, in comparison with treatment l0, apx increased by 98.2% for treatment l3. in contrast the other enzymes determined did not show significant changes with respect to the control treatment. regarding the principal component analysis (pCa) performed, Figure 2 shows the graph of variables used in the pCa. of all the variables measured, the sulfur content in the aerial part and the enzymes guaiacol peroxidase (GpX), superoxide dismutase (SoD) and catalase (Cat) have been eliminated, because they do not present statistically significant differences between groups. the graph shows that most of the variables analyzed have a high influence on the first component, which explains 53.5% of the variance of the system. these variables include most of the macro and microelements analyzed in the aerial part and in the root of the pepper plants, as well as the pigments analyzed (chlorophylls a and B and carotenes) and the enzyme ascorbate peroxidase. Factor 2, which explains only 5.1% of the variance, includes some of the macro and micronutrients, both from the aerial part and the root. in this graph, the length of the vector and the angle it forms with the axes indicate the weight of the variables in a given component. in this way, it can be seen that in component 1, the variables with the greatest weight are the contents of n, K, Ca, Zn and Mn in the root, the contents of Mn and Zn in the aerial part, the contents of chlorophyll a and B and carotenes, the enzyme ascorbate peroxidase, the height of the plant and the weights of the aerial part and the root. Figure 3 represents the measurements of the variables used in the pCa. First, it can be observed that the treatment has a strong influence on these variables, so that four groups are clearly separated, which correspond to the four treatments. treatments l0 and l1 are separated from treatments l2 and l3 by component 1. this indicates that treatment l1 has values closer to the control (l0). in addition, it can be observed that the distance between l2 and l3 is greater than that between the other groups. therefore, l3 is the treatment that produces the greatest differentiation in the measured parameters. Based on what has been stated in the previous paragraph, it can be said that the pCa detects relevant patterns in the data that confirm, on the one hand, the existence of a difference in the effect of the biostimulant with respect to the control and, on the other, that this effect is dose-dependent. Table 6. Bartlett test of homogeneity of variances of plant height, weight of aerial part, root weight, enzymes, pigments and macro and micronutrients in aerial part and root. Plant Height Weight aerial part root Weight Chlorophyll a Chlorophyll b total Carotenoids Ascorbate peroxidase guaiacol peroxidase Superoxide dismutase Catalase K-squared 2.527 2.4195 6.2322 7.3967 4.9278 0.3639 6.4587 1.356 1.834 0.1961 df 3 3 3 3 3 3 3 3 3 3 p-value 0.4703 0.4900 0.1008 0.0603 0.1772 0.9476 0.091 0.716 0.6071 4.688 n P K S Ca Mg Fe Cu Mn Zn Aerial part K-squared 6.1113 4.8606 4.1192 3.3461 3.956 0.8327 3.0508 1.084 1.795 7.3801 df 3 3 3 3 3 3 3 3 3 p-value 0.1063 0.1823 0.2489 0.3413 0.3624 0.8416 0.3839 0.7808 0.6160 0.061 root K-squared 6.736 6.226 2.555 5.897 5.952 7.387 0.9589 5.4900 5.643 3.6544 df 3 3 3 3 3 3 3 3 3 3 p-value 0.0808 0.1011 0.4653 0.1167 0.1139 0.0605 0.8112 0.1392 0.1304 0.3013 Table 7. Plant height, weight of aerial part and root weight (mean ± standard error) for each fertilizer treatment. rows followed by the same letter(s) are not significantly different (p < 0.05). treatment Plant Height (cm) l0 8.5a ± 0.4 l1 10.7b ± 1.0 l2 11.2b ± 1.2 l3 15.7c ± 1.3 Weight of aerial part (g) l0 0.76a ± 0.03 l1 0.99b ± 0.05 l2 1.4c ± 0.2 l3 1.9d ± 0.2 root weight (g) l0 0.17a ± 0.002 l1 0.41b ± 0.05 l2 0.50b ± 0.03 l3 0.63c ± 0.04 CoGent FooD & aGriCUltUre 7 4. Discussion application of the new biostimulant obtained from red wine lees by way of enzymatic hydrolysis processes stimulates both the growth of pepper seedlings as well as their mineral nutrition. these results are in line with those obtained by other authors after application of various types of biostimulants obtained by enzymatic hydrolysis, with high contents of amino acids, low molecular weight peptides and organic matter to various types of crops, thereby stimulating the growth of these crops. thus, Ávila-pozo et al. (2022, 2023) observed an improvement in the morphological properties of tomato and green bell pepper plants, mineral nutrition, photosynthetic pigments and productivity after the application of a biostimulant obtained from slaughterhouse sludge. Similarly, Jain and Badve (2022) observed a higher germination percentage and growth of wheat plants (shoot and root length and dry weight) after application of a biostimulant obtained by enzymatic hydrolysis processes of soybean meal. after the application of a biostimulant based on defatted seed meal rich in free amino acids and low molecular weight peptides to mung bean (Vigna radiata) cuttings, Ugolini etal. (2023) observed a positive effect on the root development of the plant, as well as a high nitrogen content. this biostimulant effect on plant growth is a consequence of the chemical constituents present in the biostimulant (Ávila-pozo etal. 2022, 2023). thus, several authors have indicated that the biostimulant effect of these compounds, which contain amino acids, low molecular weight peptides, organic matter and phytohormones is responsible for the physiological improvement of crops, which will cause greater plant growth and development (Francesca et al., 2021; rouphael et al., 2021). in the opinion of some authors, the biostimulant effect of a single chemical constituent cannot be isolated from that resulting from the rest of the constituents of the biostimulant (Bulgari et al. (2019). as such, the combined action of all the chemical compounds that make up the biostimulant is responsible for the physiological improvement of the plant. in our study, application of the biostimulant to the organic substrate improved the root development of pepper seedlings. these results are in Table 8. Macro and micronutrient contents in roots and aerial parts (stems + leaves) in pepper planta for each fertilizer treatment. rows followed by the same letter(s) are not significantly different (p < 0.05). treatment n† (%) P (%) K (%) S (%) Ca (%) Mg (%) Fe (mg kg-1) Cu (mg kg-1) Mn (mg kg-1) Zn (mg kg-1) Aerial part l0 1.5a ± 0.2 0.28a ± 0.05 2.6a ± 0.1 0.32a ± 0.08 1.5a ± 0.3 0.24a ± 0.07 110a ± 18 5.8a ± 1.1 24.3a ± 3.7 38.9a ± 3.9 l1 1.8ab ± 0.4 0.32b ± 0.08 3.0b ± 0.3 0.31a ± 0.08 1.6a ± 0.2 0.28ab ± 0.05 118ab ± 14 6.1ab ± 1.4 33.1b ± 2.8 48.4b ± 2.1 l2 2.2b ± 0.4 0.33b ± 0.06 3.2b ± 0.3 0.33a ± 0.06 1.6a ± 0.3 0.29b ± 0.05 129b ± 21 6.9b ± 1.3 43.2c ± 3.0 52.0bc ± 3.7 l3 2.6c ± 0.5 0.36b ± 0.06 3.5c ± 0.4 0.34a ± 0.09 1.9b ± 0.4 0.31b ± 0.06 132b ± 17 7.1b ± 1.5 59.0d ± 3.6 61.8c ± 3.8 root l0 1.1a ± 0.1 0.15a ± 0.04 0.63a ± 0.08 0.11a ± 0.05 1.1a ± 0.2 0.14a ± 0.03 104a ± 18 3.1a ± 0.9 17.5a ± 1.8 13.9a ± 1.4 l1 1.4ab ± 0.3 0.19a ± 0.04 0.85b ± 0.1 0.13a ± 0.03 1.2a ± 0.1 0.17ab ± 0.03 106a ± 16 4.3ab ± 0.7 22.1ab ± 2.0 18.6b ± 2.1 l2 1.7b ± 0.2 0.21ab ± 0.06 0.95c ± 0.07 0.13a ± 0.05 1.3a ± 0.2 0.17ab ± 0.02 123b ± 20 4.1ab ± 0.8 28.2b ± 1.4 20.2bc ± 1.9 l3 2.3c ± 0.4 0.24b ± 0.05 1.1c ± 0.1 0.15a ± 0.04 1.4b ± 0.3 0.19b ± 0.04 137c ± 21 5.2b ± 1.1 34.9c ± 1.8 21.4c ± 2.1 †Fresh weight. Table 9. Photosynthetic pigment contents in pepper leaves for each fertilizer treatments. Columns followed by the same letter(s) are not significantly different (p < 0.05). treatment Chlorophyll a (g kg-1, FW) Chlorophyll b (g kg-1, FW) total Carotenoids (g kg-1, FW) l0 0.86a ± 0.11 0.38a ± 0.08 0.23a ± 0.07 l1 1.1b ± 0.2 0.59b ± 0.10 0.31b ± 0.08 l2 1.7c ± 0.3 0.63bc ± 0.09 0.40bc ± 0.08 l3 2.0c ± 0.3 0.79c ± 0.17 0.49c ± 0.1 FW: fresh weight. Table 10. effect of biostimulant on antioxidant enzymatic activities in pepper leaves. Columns followed by the same letter(s) are not significantly different (p < 0.05). treatment Ascorbate peroxidase (u min-1 µg-1 protein) guaiacol peroxidase (u min-1 µg-1 protein) Superoxide dismutase (u min-1 µg-1 protein) Catalase (u min-1 µg-1 protein) l0 0.0096a ± 0.0051 0.0003a ± 0.0001 3.0a ± 0.5 0.008a ± 0.002 l1 0.02b ± 0.007 0.0003a ± 0.0001 2.5a ± 0.5 0.007a ± 0.001 l2 0.39c ± 0.01 0.0004a ± 0.0001 2.8a ± 0.4 0.01a ± 0.003 l3 0.56d ± 0.012 0.0004a ± 0.0001 2.7a ± 0.4 0.009a ± 0.02 8M. teJaDa etal. agreement with those obtained by De pascale et al. (2018), pylak etal. (2019) and Ávila-pozo etal. (2022, 2023), who observed better growth and development of the roots of different plants when applying biostimulants rich in amino acids, low molecular weight peptides and organic matter. Several authors have suggested that plant-derived biostimulants can have auxin-like effects, there by promoting crop yield (Colla et al., 2014; Malécange et al., 2023; rouphael et al., 2017). these authors suggest that the constituent peptides of these biostimulants play an important role in such auxin-like activities. auxin is one of the most important hormones responsible for promoting plant root growth and development (Ubeda-tomás et al., 2012). on the other hand, the chemical composition of the experimental biostimulant contains indoleacetic acid. the application of this auxin to the plant could also be responsible for the increased root development seen in the experimental plants. this increased root development after application of the biostimulant is of great importance, as it leads to increased root expansion and therefore leads to increased nutrient uptake by the plant. it is very likely that this is one of the reasons why plants with greater root development show better mineral nutrition. in this sense, Colla et al. (2015) suggest that one of the reasons for the higher nutrient uptake in plants treated with biostimulants is a consequence of the modifications that occur in the root architecture of crops, particularly in root length, density and number of lateral roots. the amino acids and peptide content in the biostimulant are also a consequence of improved mineral nutrition of plants. in this regard, there are studies indicating that biostimulants could promote plant growth by stimulating metabolism and nitrogen uptake and assimilation (ertani etal., 2017; Khan et al., 2019). Similarly, several studies also that indicate that biostimulants promote the absorption of other macronutrients. With regard to the rest of the macronutrients analyzed, the p, K, Ca and Mg contents increased in plants to which biostimulants were applied. these Figure 2. graph of variables used in the principal component analysis.