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Article

Evaluation of the Biostimulatory Potential of Waste Protein Hydrolysates in Coriander (Coriandrum sativum L.)

by
Monika Skwarek-Fadecka
1,*,
Paulina Pipiak
2,
Katarzyna Sieczyńska
2,
Małgorzata Krępska
2 and
Małgorzata M. Posmyk
1
1
Department of Plant Ecophysiology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237 Łódź, Poland
2
Łukasiewicz Research Network—Lodz Institute of Technology, Marii Skłodowskiej-Curie 19/27, 90-570 Łódź, Poland
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2026, 27(5), 2188; https://doi.org/10.3390/ijms27052188
Submission received: 23 January 2026 / Revised: 19 February 2026 / Accepted: 24 February 2026 / Published: 26 February 2026
(This article belongs to the Section Molecular Biology)
Browse Figures
Versions Notes

Abstract

This study evaluated protein hydrolysates from fish collagen (Col) and sheep keratin (Ker) as potential biostimulants in the hydro-priming of coriander (Coriandrum sativum L.) seeds. Seeds treated with low, non-nutritional doses of Col (0.5%) and Ker (1%) were compared with non-primed (C) and water-primed (H) controls under optimal conditions and after high-temperature stress (35 °C, 9 days). After stress removal, H-Col and H-Ker seeds achieved ~90% germination, whereas H and C reached 78% and 60%, respectively, confirming improved seed quality and post-stress recovery. Seedlings from Col- and Ker-treated seeds showed enhanced growth, higher biomass, and increased chlorophyll and precursor content. High-temperature stress also acted as a priming factor, modifying elemental profiles and stimulating carotenoid antioxidant synthesis. ATR–FTIR analyses indicated changes in cell wall composition and protein structure, particularly in the H-Ker variant. The results demonstrate that collagen and keratin hydrolysates, as industrial by-products, possess strong phytobiostimulatory potential and can be applied in sustainable strategies to improve seed quality and plant stress resilience.

1. Introduction

The continuous growth of the human population and negative climate change pose serious challenges to agriculture and are problems for scientists to solve [1]. Food security is a major concern currently. Plants, including crop plants, can suffer severe damage due to environmental stresses such as heat, drought, salinity, and others [2]. The need to maximise crop production and meet the increasing demand for nutritious and functional foods is driving the development and implementation of environmentally safe innovations in crop cultivation [3,4]. It would be extremely valuable to combine a modern approach to the circular economy with the secondary use of its non-toxic waste for crop cultivation [5]. Industrial and food waste could be used to produce fertilisers and biostimulants that support plant growth and yield, particularly in suboptimal conditions. The bioactive compounds in biostimulants depend on the waste/substrate used to produce them. Biostimulants derived from animal or plant protein waste can be categorised as either peptide–amino acid hydrolysates or amino acid mixtures [6]. When applied appropriately, their main advantage is that they provide plants with ready-made organic nitrogen compounds [7].
It is well known that the effective biostimulation of plant growth and increased resistance, particularly during the early stages of development when plants are susceptible to environmental stress, has a significant impact on the final yield [7]. This relates to the ability to initiate vegetation earlier, even at suboptimal or supraoptimal temperatures, and consequently extend this period—in particular, under climate changes. It is also important to stimulate secondary metabolite biosynthesis because this can increase a plant’s tolerance to environmental stresses. For instance, it can reduce the likelihood of secondary oxidative stress occurring [8] or counteract pests [9]. It should be remembered that plants richer in secondary metabolites are more valuable due to their aromatic and flavour qualities, as well as their medicinal properties resulting from their antimicrobial and antioxidant properties.
Based on the available data, it can be concluded that the effectiveness of biostimulants depends on several factors being met [10]. Firstly, they have to be effectively delivered to the plant—via seeds, shoots, or roots—dependent of species and developmental stage. The method and timing of application depend on the preparation’s form and composition, the crop type, and its agronomic requirements; however, they are primarily determined by the expected outcome. Basically, they are assumed to have a preventive effect, i.e., increasing resistance, preparing the plant for stress, or an interventional effect, such as supporting its fight against negative environmental pressure and eliminating the effects of stress, or supporting regeneration [10]. The dose is also important, and the optimum of which should always be determined experimentally.
Coriander (Coriandrum sativum L.) is an annual herbaceous plant from the celery family (Apiaceae), native to the Middle East and the Mediterranean region. Historically, it has been widely used for its nutritional and medicinal properties, and today, it is one of the most important spices, honey plants, and medicinal plants cultivated commercially around the world, including in Poland [11]. Coriander, also known as cilantro or Chinese parsley, is an herb whose taste is perceived very differently by those who enjoy it and those who dislike it. It is intriguing to note that certain individuals portray cilantro leaves as ‘fresh’ or ‘fragrant’, while others employ terms such as ‘soapy’ or ‘mouldy’ to describe them [12]. The plant has a single stem with many branches, and it grows to be 50 cm tall. Its small flowers are pink or white and grow in a round shape [13].
In this work, we tried to improve the quality of coriander seeds and the vigour of the seedlings growing from them by adding the following before sowing via hydroconditioning: collagen (Col) and keratin (Ker)—food industry waste products. Hydro-priming involves introducing natural or synthetic bioactive compounds into seeds prior to germination. This technique is based on controlled seed hydration, which induces a specific physiological state in plants, i.e., the initial stages of germination in the strict sense, but without embryo growth [14]. The seeds are then subjected to secondary desiccation to initial moisture, which allows for their storage and/or transport to the time and place of sowing. In this technique, seeds can be treated with water or water solutions of active substances. Seeds treated in this way increase their germination capacity, expand their temperature range, and improve the speed and uniformity of emergence of the resulting seedlings. The success of crop production is dependent on the use of high-quality seeds. The components of seed quality that are of critical importance are germination potential, seed health, and genetic and physical purity [15]. In view of the fact that florae are perpetually subjected to a variety of extreme ecological pressures, such as elevated temperatures, which are regarded as among the most significant abiotic pressures impacting the structure and distribution of natural environments, as well as the productivity of agriculturally significant plants on a worldwide scale [16,17], we investigated whether seed priming with collagen and keratin could biostimulate seedling growth and development, particularly under high-temperature stress conditions. This is important because the impact of seed priming on improving crop resilience to heat stress has not yet been widely studied.
The research hypothesis assumes that the appropriate application of the aforementioned hydrolysates—applied pre-sowing via hydro-priming into seeds—will positively affect the growth and development of plants previously subjected to supraoptimal temperature stress. This will be achieved by improving the supply of assimilable organic compounds and strengthening the plant’s resistance to stress, for example, by intensifying secondary metabolism. The future synergy effect of the research is based on two aspects: (1) ecological waste management to limit environmental pollution and (2) intensifying green biomass production, even in suboptimal conditions. This increases the quantity and quality of the yield and generates more intensive CO2 binding during the vegetation period of biostimulated crops. This study aimed to verify whether administering protein hydrolysate (an industrial waste product) to seeds would have a biostimulating effect on coriander seedlings, promoting their germination, growth, and development and influencing their elemental composition and photosynthetic pigments.

2. Results

2.1. Seed Germination and Growth Parametres

In optimal conditions (20 °C), coriander seeds achieved 47%, 39%, 39%, and 15.5% germination on the fourth day, and 92%, 91%, 91%, and 87% on the last (14 d) day of the experiment for C, H, H-Col, and H-Ker, respectively (Figure 1A). At the optimal conditions, the positive effects of hydro-priming were not visible, as all the different types of seeds germinated very well (Figure 1A). During the 9-day incubation at 35 °C, neither variant germinated, which confirms its stressful nature. However, after moving the seeds to 20 °C, a recovery effect was analysed. Interestingly, primed seeds germinated more effectively than at the optimal temperature, reaching 45%, 52%, 59%, and 70% on day 4, and 62%, 78%, 91%, and 88% on the final day for C, H, H-Col, and H-Ker, respectively (Figure 1B). For the H-Col and H-Ker variants, a maximum germination rate was obtained on days 9 and 8, respectively. The experimental scheme for seedling growth conditions was created based on the presented germination tests for coriander seeds.
Table 1 presents photos, and Table 2 presents measurable parameters for representative 24-day-old seedlings grown from the tested seed variants (C, H, H-Col, and H-Ker) germinated under optimal conditions or after an initial 9-day stress of 35 °C (post-stress recovery). The photos (Table 1) show the slight positive effect of seed conditioning on seedling growth at optimal conditions, but there are no clear differences between plants grown under optimal conditions and those showing post-stress recovery. However, statistically significant differences are indicated by measurable seedling growth parameters (Table 2).
In seedlings grown under optimal conditions from H-Col and H-Ker seeds, a statistically significant increase in shoot fresh weight and root fresh weight was observed (Table 2). Given that the dry mass of these organs did not differ among all seedling variants, this indicates improved water accumulation in the shoots and roots of H-Col and H-Ker seedlings. The post-stress recovery significantly altered almost all seedling growth parameters (except for the number of branches, root length, and root dry mass). Seedlings grown from seeds previously subjected to high-temperature stress exhibited slightly longer shoots, a higher number of leaves (particularly in the H-Ker variants), greater shoot fresh mass (especially in the H-Col and H-Ker variants), greater shoot dry mass (especially in the H, H-Col, and H-Ker variants), and greater root fresh mass (especially in the H, H-Col, and H-Ker variants) (Table 2).

2.2. Changes in Elemental Composition

The following tables present the compositions of macro and microelements in shoots (Table 3) and roots (Table 4) of 24-day-old coriander seedlings grown from all seed variants (C, H, H-Col, H-Ker) under optimal or post-stress conditions.
In terms of element composition in the shoots (Table 3) under optimal conditions, there were no changes between seedling variants. Only the level of K decreased significantly in the H-Col and H-Ker plants compared to the control ones, while in the shoots of seedlings from primed seeds, the Mo level increased significantly. There were also no significant changes between seedling variants during post-stress recovery except for the levels of Na and Mn, which decreased in the primed seed variants, and an increase in Mo levels, which was again observed in the shoots of seedlings from primed seeds.
The elemental composition of the shoots was clearly influenced by the cultivation conditions. In post-stress regeneration conditions, an increase in Ca, Mg, Na, Cu, Mn, and Zn levels was observed in all seedling variants compared to optimal conditions. However, high-temperature stress caused a decrease in K and Mo levels in the shoots of all seedling variants during regeneration.
In terms of element composition in the roots (Table 4) under optimal conditions, there were no big changes between seedling variants. Only the levels of Na and Mn increased significantly in the H, H-Col, and H-Ker plants compared to the control ones. Statistically significant differences were also observed in the Mo levels in roots between seedling variants.
Slightly more differences in root element composition between seedling variants under post-stress recovery conditions were observed. The differences were found in Mg, Na, and Zn, the highest concentrations of which were determined in the roots of H-Ker seedlings. Fe was highest in the roots of seedlings grown from seeds primed in the opposite manner, compared to Cu, which was highest in the roots of control seedlings. The roots of H-Col and H-Ker plants also had significantly lower Mn concentrations under post-stress conditions.
As in the case of shoots, the elemental composition of the roots was clearly influenced by the cultivation conditions. Post-stress conditions decreased K, Na, P, and Mo levels in all seedlings while increasing Ca and Cu levels in the roots. Unexpectedly, Mg levels in the roots increased in C and H plants, while in H-Col and H-Ker, they decreased. However, the Fe level in post-stress conditions was significantly higher in the roots of plants grown from primed seeds compared to the control ones.

2.3. Changes in Nitrogen Concentration

The determination of total nitrogen in 24-day-old coriander seedlings showed that the shoots contained significantly more nitrogen (2.5–3 times more) (Figure 2A) compared to the roots (Figure 2B).
Seed hydroconditioning treatments (H, H-Col, H-Ker) resulted in a slight increase in nitrogen content only in the shoots of seedlings grown under optimal conditions (Figure 2A). However, this increase was at the threshold of statistical significance compared with the control seedlings (C).
The factor that clearly contributed to the increase in total nitrogen content in the seedlings was high-temperature stress experienced by the seeds prior to germination. All seedling variants in the post-stress recovery showed increased nitrogen content in both the roots and shoots (Figure 2A,B). The most pronounced increase was recorded in the H-Col variant relative to the nitrogen level under optimal conditions.

2.4. Changes in Nitrate and Nitrite Reductase Activities

The potential activity of two key enzymes involved in plant nitrogen assimilation was determined in the green shoots (where nitrogen assimilation occurs) of 24-day-old coriander seedlings: nitrate reductase (NR) and nitrite reductase (NiR) (Figure 3A,B).
NR activity was very low (up to 0.1 µM mg−1 protein) (Figure 3A). However, statistically significant changes in the activity of this enzyme were observed only in H seedlings—a significant reduction in activity under post-stress conditions—and in H-Col, which had exceptionally high activity in these seedlings grown under optimal conditions. A general trend also appears to be a decrease in NR activity under post-stress recovery conditions (except for variant C).
NiR activity was significantly higher (up to 1–2 µM mg−1 protein) and did not differ between seedling variants or change under different cultivation conditions (Figure 3B).

2.5. Changes in Photosynthetic Pigments and Their Precursors

The results showed no significant changes in carotenoids or in the ratio of chlorophyll a + b to carotenoids (Figure 4B,C). However, there was a significant increase in chlorophyll a + b content in seedling variants H-Col and H-Ker during post-stress recovery compared to optimal conditions (Figure 4A). The values for all other variants were similar for chlorophyll a + b, carotenoids, and the ratio of chlorophyll a + b to carotenoids.
The changes in chlorophyll content described above were confirmed by the profile of its precursors studied. Although the analyses did not reveal significant changes in porphyrin and protochlorophyllide content (Figure 5A,B) between seedling variants grown under optimal conditions, the greatest increase in protoporphyrin content was observed in the H-Col seedling variant and a significant increase in H-Ker seedlings during recovery from stress compared to optimal conditions.

2.6. Changes in Lutein, Zeaxanthin, and β-Carotene

Although the total pool of carotenoids (carotenes and xanthophylls) did not change under the experimental conditions (Figure 4B), analyses of its selected elements showed statistically significant changes.
Basically, no differences were found in the content of lutein, zeaxanthin, and β-carotene in seedlings grown under optimal conditions (Figure 6A–C), except for a slight decrease in lutein in H-Col seedlings (Figure 6A).
Compared to optimal conditions, a significant increase in the content of lutein, zeaxanthin, and β-carotene was observed in all variants following the recovery period after stress (Figure 6A–C).

2.7. ATR–FTIR Analysis Differences

In this study, the aim was to compare the spectra obtained for coriander leaf samples in order to identify significant spectral differences that may indicate changes in their biochemical composition. To facilitate further analysis of the recorded ATR–FTIR spectra for the test samples, transformations of the raw spectra were carried out. Normalisation of the resulting spectra was conducted for both the test sample and control sample using the peak 2850 cm−1 as a reference. This allowed for a more detailed analysis of the changes observed in the spectra of the test samples compared to the control sample. Two spectral variants were recorded: one for samples under optimal conditions and one for samples experiencing the recovery effect after stress. These are shown in Figure 7 and Figure 8, respectively. The recorded spectra exhibited bands corresponding to chemical components typical of coriander leaves, including proteins, carbohydrates (notably cellulose), and fats, as well as numerous bioactive compounds such as flavonoids, phenolic acids, and carotenoids [18]. Characteristic absorption bands for plant material were observed in all samples. The band from 1730 to 1740 cm–1 is attributed to the C=O stretching vibrations typical of esters such as pectins and hemicelluloses [19]. The band observed at 1600 cm−1 corresponds to the amide II region, which is characteristic of proteins, and may also reflect the presence of carboxylate groups (COO−) in polyphenols [20]. The 1370 cm−1 band is associated with deformation vibrations of CH3 and CH2 groups found in lipids, structural proteins, and cellulose [21]. Absorption features around 1200–1260 cm−1 are commonly attributed to C–O and C–C vibrations in polysaccharides and lignin [22]. In contrast, absorption bands in the 1100–1150 cm−1 and 1010–1020 cm−1 regions correspond to C–O–C and C–O stretching vibrations typical of cellulose and hemicellulose, respectively [23]. The ATR–FTIR spectra showed noticeable variations in band position and shape within the 1300–1400 cm−1 range, which may indicate differences in the composition or structure of the chemical compounds present in the analysed samples.

3. Discussion

In line with the search for innovative and sustainable methods of plant cultivation while simultaneously promoting the concept of a circular economy, this present study tested the possibility of using non-toxic industrial waste as potential plant biostimulants. To improve the quality of coriander seeds and the vigour of the resulting seedlings, non-toxic waste products—protein hydrolysates derived from fish collagen (Col) and sheep keratin (Ker)—were applied.
The importance of amino acids in promoting plant growth and development has been demonstrated in numerous studies [24,25]. For instance, glutamic acid, arginine, alanine, glycine, and proline are particularly valuable for plant growth [26]. Glutamic acid, proline, serine, and valine mitigate the effects of high temperatures, while glycine, alanine, glutamic acid, and arginine improve chlorophyll production. Proline mitigates salinity stress due to its chelating effect [26].
Germination and seedling development are critical stages of plant ontogenesis [27]. Various cereals, legumes, oilseeds, and horticultural crops, such as wheat, rice, beans, mustard, and tomatoes, show reductions in germination-related parameters—including germination percentage, seedling vigour, root and coleoptile growth, and seedling dry mass—mainly because of heat stress [28]. A study by Tamindžić et al. 2023 [29] demonstrated that the use of different priming techniques improved seed germination, seedling development, and physiological traits of garden pea cultivars under both optimal and adverse conditions, alleviating the negative effects of heat stress. Hassanein et al. 2022 [30] investigated the beneficial effects of seed priming under salt stress by evaluating the use of potassium silicate, humic acid, and gamma irradiation. The results showed that the pre-treatment of coriander seeds with potassium silicate or humic acid, followed by short gamma irradiation, mitigated the effects of salt stress and improved key metabolic processes, and thus growth parameters and yield.
Therefore, in this present study, coriander seeds were pre-sown and hydro-primed with the addition of the above-mentioned protein hydrolysates at doses excluding their nutritional significance, i.e., 0.5% Col and 1% Ker solutions were used. As control variants, non-primed seeds and seedlings (C) and seeds hydro-primed with water only (H) were tested. The experiments were conducted at the germination stage and during early seedling development.
The optimal temperature range for coriander seed germination is 15–20 °C [31]. Temperatures above 25 °C cause a delay or inhibition of germination and/or a reduction in germination percentage (pilot tests) and therefore generate stress in coriander seeds. In the conducted experiments, not a single seed germinated in any coriander seed variant at 35 °C (seeds imbibed, but no radicle emergence was observed). Therefore, the conditions of 9 days of incubation in darkness, on a moist substrate, at 35 °C were adopted as a model of high-temperature stress during seed germination and potential seedling emergence.
Positive effects of seed priming—especially in seeds of good initial quality—are rarely visible in tests conducted under optimal conditions [10], which was confirmed by coriander germination tests at 20 °C. All seed variants started to germinate on day 3 and reached about 90% germination by day 14. A slightly different course of germination (curve shape) was observed only in the H-Ker variant, but this is acceptable because the final test outcomes were comparable with the other variants. As expected, the positive effect of protein hydrolysate application became evident after transferring the seeds from high-temperature stress (9 d/35 °C) to optimal conditions (20 °C). Although, in all seeds germinating after stress, the rate of germination increased (Figure 1A,B: see the first growth phase of the germination curves, i.e., days 2–6) because imbibition had already begun during the 9-day incubation period at 35 °C, and the total number of germinated seeds was markedly reduced. Under these conditions, control seeds (C) germinated at about 60%, which clearly confirms the negative effect of high temperature. Slightly higher germination was observed in H seeds (78%), as even hydro-priming with water alone has a positive effect on seed physiology under stress conditions [10]. In contrast, the germination of H-Col and H-Ker seeds remained at a level comparable to optimal conditions, i.e., about 90%, which indicates a positive biostimulatory effect of the applied protein hydrolysates on seed physiology during post-stress recovery. Thus, the aim of improving seed quality was achieved. The next stage of the study was to monitor the development of seedlings derived from all seed variants under optimal and post-stress recovery conditions.
It is known that the positive effects of seed conditioning are often reflected in better growth and development of the seedlings growing from them (especially in the earliest, critical period of emergence) [10,32]. Growth tests showed that seed-applied Col and Ker improved water accumulation in the shoots and roots of H-Col and H-Ker seedlings under optimal conditions. Moreover, post-stress recovery significantly altered almost all seedling growth parameters, but not in a negative way. As mentioned above, seedlings grown from seeds previously subjected to high-temperature stress exhibited slightly longer shoots, a higher number of leaves (particularly in the H-Ker variants), greater shoot fresh mass (especially in the H-Col and H-Ker variants), greater shoot dry mass (especially in the H, H-Col, and H-Ker variants), and greater root fresh mass (especially in the H, H-Col, and H-Ker variants) (Table 2). This phenomenon results from the fact that the high-temperature stress applied at the seed germination stage caused a preselection of the strongest individuals for further experiments (in the C variant from 60%, in H from 78%, and in H-Col and H-Ker from 90% of germinated plants). Moreover, in plant ontogeny, seeds are the most stress-resistant developmental stage—they have evolved to allow the plant to survive periods unfavourable for vegetation. Therefore, 35 °C stress in seeds that survived it must have activated defence strategies, the positive effects of which were observed during post-stress recovery, including enhanced seedling growth. The applied stress turned out to be, for the strongest individuals of the C and H variants and for all individuals of the H-Col and H-Ker variants, an additional priming factor—this was also confirmed by further tests, including those related to the biosynthesis of antioxidants (lutein, zeaxanthin, and β-carotene).
The concept of plant ionomics assumes that maintaining mineral balance in plants is a complex process involving many mechanisms. A change in the amount of one or several elements may affect the status of the others [33]. Protein hydrolysates stimulate carbon and nitrogen metabolism and regulate nitrogen uptake. Moreover, data from the literature indicate that peptides and amino acids derived from protein hydrolysates can form complexes and chelates with soil micronutrients such as Cu, Fe, Mn, and Zn. This contributes to better availability and uptake of nutrients by the root system and to increased root density [34]. Micronutrients chelated by amino acids form very small, electrically neutral molecules, which accelerates their absorption and transport in the plant [35]. Popko et al. 2018 [35] demonstrated the biostimulatory potential of keratin hydrolysate-based preparations obtained from feathers and applied as foliar sprays. Their studies showed that amino acid preparations had a positive effect on winter wheat cultivation, increasing the content of nutrients in grain, especially macronutrients such as Na and Ca and micronutrients such as Cu and Mo. Both the availability of mineral nutrients and the development of an efficient root system are crucial for plant growth and development. Several factors play a key role in regulating how plants acquire nutrients. These include rhizospheric traits, root morphology, architecture, and kinetics [36,37]. Plants require a total of 17 mineral elements for growth and development [37]. Macro and micronutrients play a vital role in stress responses. Heat stress leads to a decline in nutrient content. This is due to reduced root growth and biomass [38]. It is also due to limited water and nutrient uptake. The decrease in nutrient uptake during periods of heat stress can be explained by several factors, including a reduction in root mass and surface area, as well as decreased nutrient uptake per unit of root [37,39,40]. Ca improves the plant’s response to stress by regulating physiological processes and acting as a secondary messenger that develops stress resistance in plants [41]. Leaves with a high level of Mg can help keep the water content during drought [42]. The presence of Zn results in the increased stability of antioxidant properties, such as superoxide dismutase (SOD) activity, under heat stress conditions [42]. Cu is a transition metal capable of undergoing redox reactions. It plays a significant role in vital cellular processes, including photosynthesis, carbon and nitrogen metabolism, respiration, and protection against oxidative stress [43].
The application of protein hydrolysates at the proposed doses to seeds did not significantly affect the composition of macro and microelement profiles in either the roots (except for an increase in Na and a decrease in Mo) or in shoots (except for a decrease in K and an increase in Mo) of 24-day-old coriander seedlings. It was the stress treatment that significantly modified these profiles in a similar manner for all studied seedling variants during post-stress recovery. Modifications in nutrient uptake, accumulation, and distribution represent a series of adaptive responses that facilitate the maintenance of cellular homeostasis and ensure the survival of plants under adverse conditions [44]. The ability to sense water availability in soil is primarily down to the roots. They then relay the necessary hormonal and electrical signals to the shoots, enabling a coordinated response to stress [44,45]. Nevertheless, drought-induced upheavals are compounded by a decrease in transpiration rates, which are vital for transporting nutrients from the roots to the shoots [46]. The differences in the content of macro and microelements between roots and shoots are due to various factors [47]. These include the different physiological functions of these organs, the mobility of elements in the plant, selective transport mechanisms and the influence of environmental conditions and the developmental stage [48].
Changes in the total nitrogen pool showed a slightly different pattern. All seed hydroconditioning treatments (especially H-Col and H-Ker) resulted in a slight increase in nitrogen content only in the shoots of seedlings grown under optimal conditions (Figure 2A). However, this increase was at the threshold of statistical significance compared with control seedlings (C). This confirms that the applied protein hydrolysates—0.5% from fish collagen and 1% from sheep keratin—had no trophic significance; they were not fertilisers. Their effects—those that we observed in this work—were biostimulatory in nature. Once again, the factor that induced an increase in the total nitrogen pool in both the roots and shoots of 24-day-old coriander seedlings was high-temperature stress. All seedling variants in post-stress recovery showed increased nitrogen content in both roots and shoots (Figure 2A,B). The most pronounced increase was recorded in the H-Col variant relative to the nitrogen level under optimal conditions.
Nitrogen availability is a key factor in plant growth and crop productivity. Plants use various forms of N in natural soils; inorganic forms include nitrate, nitrite, and ammonium [49]. In most well-aerated soils, nitrate predominates, whereas in some acidic and/or anaerobic environments, ammonium may dominate [50]. Nitrate is reduced to ammonium by nitrate reductase (NR), whereas nitrite reductase (NiR) requires eight moles of electrons per mole of nitrate. Therefore, the use of ammonium significantly reduces the energy consumption required for the synthesis of organic nitrogen compounds [51]. The Mo cofactor (Moco) of nitrate reductase contains molybdenum, which is therefore essential in nitrate metabolism [52]. In the study by Nieves-Silva et al. 2024 [53], organic fertilisers increased NR activity in the vegetative phase, probably due to the higher availability of nitrogen derived from organic matter, which is rapidly used at this stage [54,55]. In our study, under optimal conditions, the highest NR activity was found in seedlings grown from seeds treated with collagen, while after stress, the activity of this enzyme slightly decreased in all seedling variants. According to Onwueme et al. 1971 [56], heat stress leads to the inactivation of NR. The same conclusion was reached by other researchers. For example, Gautam et al. 2020 [57] demonstrated that NR activity decreased in two Oryza sativa cultivars under high-temperature stress. Khan et al. 2013 [58] obtained similar results when studying NR activity, N content, and photosynthetic NUE (the allocation of N to Rubisco) in wheat under high-temperature stress (40 °C). In contrast, Majeed et al. 2020 [59] reported a significant increase in NR activity in sodium nitroprusside (SNP)-treated maize plants under drought stress.
Scientific reports indicate that nitrogen level may affect chlorophyll a and b content, i.e., an increase in nitrogen content correlates with an increase in chlorophyll concentration [60]. In our study, such a correlation was confirmed in 24-day-old coriander seedlings of the H-Col variant during post-stress recovery; however, it was not confirmed in the H-Ker variant, where an increase in chlorophyll content after stress was observed despite low nitrogen levels in tissues. This is difficult to explain because organic nitrogen from protein hydrolysates was assumed to be applied to seeds at non-nutritional doses—it should not affect the total nitrogen pool. However, it seems that the application of a twofold higher concentration of organic nitrogen from Ker (1%) compared with Col (0.5%) was sufficient to significantly stimulate chlorophyll biosynthesis (Figure 4A) and that of its precursors, protoporphyrins (Figure 5A) and protochlorophyllide (Figure 5B), in 24-day-old seedlings grown from seeds conditioned with both protein hydrolysates (H-Col and H-Ker). Chamle and Raut, in 2021 [61], reported, for example, that sorghum plants with higher chlorophyll b content have an advantage, as this chlorophyll is associated with the expression of chlorophyllide a oxygenase, which leads to a greater number of proteins in the electron transport chain and better light harvesting. Increasing photosynthetic efficiency—the main process that, in the light phase, converts light into metabolically useful energy (ATP) and reduces power (NADPH), and, in the dark phase, provides carbon skeletons (precursors) and high-energy compounds (sugars)—is the basic strategy enabling rapid and effective recovery after stress [62]. Thus, all factors that support the structure and function of the photosynthetic apparatus, especially under stress conditions and after its cessation, are undoubtedly biostimulants.
Another anti-stress strategy is the protection of cellular redox balance, i.e., counteracting or mitigating secondary oxidative stress [63]. Although the determination of the total carotenoid pool (Figure 4B) did not show significant differences between the studied seedling variants grown under optimal or post-stress recovery conditions, detailed analyses of selected carotenoid antioxidants such as lutein, zeaxanthin, and β-carotene (Figure 6A–C) indicated that high-temperature stress was a factor stimulating their synthesis in all seedling variants grown under post-stress recovery conditions. In plants, lutein and zeaxanthin are well-documented for their roles in protecting chlorophyll and facilitating photosynthesis. β-carotene is the precursor of zeaxanthin, a xanthophyll that indirectly impacts the synthesis of the stress hormone jasmonic acid [64]. Under abiotic stress conditions, such as excessive light intensity or oxidative stress, plants increase zeaxanthin accumulation as a protective mechanism against photosystem damage [64,65]. Lutein and other carotenoids also play a role in photoprotection, and changes in their levels have been associated with adaptation to environmental stress [66]. Ding et al. 2022 [65] demonstrated that exogenous zeaxanthin improves plant tolerance to light stress and low temperatures by acting as an antioxidant and a component of the photoprotective system. Studies by Hong et al. 2022 [67] on the microalga Mychonastes sp. showed that increased lutein and zeaxanthin production is induced by changes in light intensity and salinity, indicating that they act as stress metabolites. Rossi and Huang, 2022 [68] studied heat stress in Agrostis stolonifera and found that the exogenous application of β-carotene reduced leaf senescence and increased antioxidant activity, indicating its role in thermal tolerance. In Cicer arietinum, genotypes that were more resistant to high temperatures exhibited higher zeaxanthin concentrations and superior photochemical indices [69]. In Zea mays, an increase in β-carotene accumulation was observed in response to a temperature of 40 °C [70]. These studies demonstrate that high temperatures can modify the carotenoid content of plants.
ATR–FTIR spectra obtained from leaf samples provide valuable insights into their chemical composition, internal structure, and physiological condition [71,72]. In samples from seedlings grown under optimal conditions, the ATR–FTIR spectra of the H-Ker variant, in which coriander seeds were treated with keratin, showed qualitative differences compared with the control (C), particularly in the shape of the bands at 1372 cm−1 and 1016 cm−1. These observed differences may be associated with variations in the composition of cell wall polysaccharides, particularly cellulose and hemicellulose, as well as potential changes in the content or organisation of proteins present in the leaves. As demonstrated by the data presented above, the peak at 1372 cm−1 is attributed to the deformation vibrations of methyl (CH3) and methylene groups (CH2). These groups may be components of proteins, as well as groups characteristic of the structure of cellulose and hemicellulose [73]. The observed changes in the shape of the 1016 cm−1 peak may also reflect differences related to the structure of cellulose or hemicellulose, which are the building blocks of the cell wall [74]. Although data in this area remain fragmentary, these observations are consistent with previous findings regarding the use of keratin hydrolysates as biostimulants. In maize plants treated with keratin hydrolysate via foliar application, changes were observed in the intensities of amide I and II bands and in the so-called ‘fingerprint region’, mainly involving modifications in the composition of cellulose, proteins, and phenolic compounds, as determined based on shifts, intensity changes, and variations in band shapes [75]. Sahin et al. 2025 [76] demonstrated through rigorous experimentation that the application of keratin hydrolysate to soil significantly enhances the growth and mineral nutrition of lettuce, spinach, and radish plants. This finding substantiates the hypothesis that keratin hydrolysate possesses the qualities of an organic biostimulant. In the recorded spectra of all test variants exhibiting a recovery effect after stress (H, H-Col, H-Ker), qualitative differences were observed in the ATR–FTIR spectra in the form of bands at 1369 cm−1 and 1417 cm−1 compared to the control sample. Changes in the shape of these bands, associated with deformation vibrations of CH3 and CH2 groups, may indicate alterations in the content or structure of proteins or other organic compounds containing CH2/CH3 groups, including polyphenols. It should be noted that the presented ATR–FTIR spectra represent averaged measurements, and the analysis is qualitative in nature. Therefore, the observed spectral differences are interpreted as indicative trends rather than statistically confirmed changes.

4. Materials and Methods

4.1. Plant Material

Coriandrum sativum L. seeds obtained from TORAF (Maciejów, Poland) were primed using the hydroconditioning method with water (H), 0.5% fish collagen/water solutions (H-Col), or 1% keratin/water solutions (H-Ker). Non-primed seeds were used as control (C).

4.2. Primer Agents and Seed Priming Method

Hydrolysed proteins, such as collagen and keratin, were used as priming agents. The fish collagen was sourced from the skin of specific species of freshwater fish (INVENTIA Polish Technologies, Żuławka, Poland). The keratin hydrolysate came from sheep’s wool (PROTEINA Natural Protein Factory, Łódź, Poland). The chemical properties of the hydrolysed proteins used in the study are provided in the Supplementary Materials (Table S1). The amino acid content of these samples was determined using liquid chromatography (LC–MS/MS) according to the accredited analytical method PB 5.4, ed. 4 (30 June 2013). First, in order to set the hydro-priming conditions, the initial and final water content of the seeds was determined experimentally [14,77]. Based on the obtained results, the required amount of water for hydro-priming for a given portion of seeds [mL/g] could be calculated (to achieve the desired final seed moisture content). The seeds were primed in glass bottles and rotated on a STR 4 DRIVE rotor (BioCote, England, UK) in order for them to absorb fluid evenly. The hydro-priming temperature was ambient. At one-hour intervals, determined portions of water or aqueous solutions of protein hydrolysates were added. This procedure took approximately six hours, consistent with the absorption kinetics of coriander seeds at room temperature (these parameters were also determined experimentally). The seeds were then left to dry in open air at room temperature for a further three days. Secondary desiccation allowed them to regain their initial water content. All seeds (controlled and primed) were stored in plastic boxes at room temperature in darkness before they were used in the experiments.

4.3. Germination Tests

The seeds were placed on 7 cm-diameter Petri dishes, with a single layer of white filter paper wetted with distilled water. The germination test was conducted on samples of 100 seeds with 25 seeds per dish at four replicates. Pilot germination tests were conducted within the temperature range of 10–40 °C. A seed was considered germinated when its coat was broken and a radicle was visible. The number of seeds germinating was counted daily for up to 14 days. The results presented are the means of the values obtained in four replicates, with standard error of the mean (SEM).
The optimal conditions for coriander seed germination were experimentally determined to be darkness at 20 °C. Because none of the tested seeds germinated at 35 °C for nine days, these conditions were considered high-temperature stress. Subsequent transfer of the seeds to optimal conditions allowed for monitoring the post-stress germination effects.

4.4. Seedlings Growth Conditions

All variants of Coriandrum sativum L. seeds were placed in plastic boxes containing moistened cotton wool and germinated in darkness at 20 °C for three days. The young, etiolated seedlings were then transplanted into plastic pots containing a mixture of sterilised universal soil and perlite. They were then cultivated for 21 days in a breeding room at a constant temperature of 25 °C under a fixed photoperiod of 16 h of light and 8 h of darkness, with a light intensity of 150 μmol m−2 s−1.
To observe post-stress effects, seeds in plastic boxes containing moistened cotton wool were first incubated nine days in darkness at 35 °C then transferred to 20 °C for three days. The young, etiolated seedlings were then transplanted into plastic pots, and cultivation was continued as mentioned above.
Because the seeds imbibed but did not germinate during the 9-day incubation period at 35 °C, despite the different durations of the two experimental conditions (optimal conditions 3 + 21 = 24 days; post-stress recovery 9 + 3 + 21 = 33 days), measurements and biochemical assays were conducted on developmentally similar 24-day-old seedlings.

4.5. Growth Parameters

The following growth parameters of 24-day-old seedlings were determined: numbers of branches and leaves per plant, shoot and root lengths [cm], and fresh weight (FW) and dry weight (DW) of shoots and roots [g].
The total numbers of leaves and branches per plant were counted and recorded. To determine shoot and root length, the seedlings were thoroughly washed to remove soil. The roots and shoots of the plants were separated, and their lengths were then measured. Organ FW was weighted immediately after harvest. The drying of the plant material—at least 24 h at 100 °C—was performed to determine the DW.

4.6. Elemental Components Analysis

The methodology used to determine the elemental composition of plant samples has previously been described by Skwarek et al. [78]. Samples of coriander (shoots and roots) weighing 0.01–0.15 g were placed in a Teflon vessel containing 6 cm3 of 65% HNO3 (supplied by Chempur, Piekary Ślaskie, Poland). Aqua regia was used to test the soil for mineralisation and determine its metal content. The samples were then mineralised using a Magnum II microwave mineraliser (Ertec, Wrocław, Poland). This process involved running the samples at maximum microwave power for three 20 min cycles at 300 °C and a maximum pressure of 45 bar. The clear mineralised solution was transferred to 25 cm3 volumetric flasks and topped up with demineralised water. A reagent blank was also prepared. The samples were analysed using an ICP-OES 5110 spectrometer (Agilent, Santa Clara, CA, USA). Argon (HenDuKol, Łódź, Poland) was used to generate the plasma. The spectrometer parameters during the analysis were as follows: generator power of 1400 W, plasma gas flow of 12 dm3/min, auxiliary burner cooling gas flow of 1 dm3/min, nebuliser gas flow of 0.7 dm3/min, OneNeb nebuliser type and Double Pass Cyclonic Chamber nebuliser type, measurement time of 3 × 10 s, sample flow rate of 1.4 cm3/min, and limit of correlation coefficient of 0.9990. The measuring method was calibrated using chemical standards with different concentrations of the component. The instrument was calibrated using standard metal solution. The metals under test were analysed using standard curves prepared from calibration solutions of the individual metals (Ca, Cu, Fe, K, Mg, Mn, Mo, Na, and Zn in 2–5% HNO3; P in H2O; Chem-Lab, Zedelgem, Belgium). The standard solutions with a concentration 1000 mg/dm3 were diluted with HNO3 to match the acid concentration of the samples after the mineralisation process. The calibration solutions for ICP-OES analysis ranged from 0.005 to 200 mg/dm3 for the trace elements Ca, Cu, Fe, K, Mg, Mn, Mo, Na, P, and Zn. The procedure was repeated four times.

4.7. Determination of Nitrogen Content Using the Kjeldahl Method

The total nitrogen content of the plant material tested was determined using the modified Kjeldahl method based on the procedure described in the literature [79]. Separately dried aerial parts of coriander (0.4 g) and roots (0.2 g) were collected for analysis. The samples were mineralised in a Kjeldahl Digestor Hanon SH420F mineraliser in the presence of 10 cm3 of concentrated sulphuric acid (H2SO4) and a Kjeldahl catalytic tablet at 380 °C for one hour. After mineralisation, the nitrogen content was determined using a Hanon K9860 automatic Kjeldahl analyser, with a titrated 0.1 M hydrochloric acid (HCl) solution as the titrant. The analyses were performed in triplicate for each sample.

4.8. Nitrate and Nitrite Reductases Activity

The modified method described by Debouba et al. 2007 [80] was used to determine nitrate (NR) and nitrite (NiR) reductase activity. To prepare the plant extracts for determining enzyme activities, 0.5 g of frozen shoots were homogenised in 2.5 cm3 of 100 mM potassium phosphate buffer solution (pH 7.4) containing 7.5 mM cysteine, 1 mM EDTA, and 1.5% casein (w/v). The resulting homogenate was centrifuged (4 °C/15 min/15,000× g), and the obtained supernatant was used immediately as the extract for enzymatic activity estimation.
For the determination of nitrate reductase activity, the extract was incubated in a reaction mixture containing a 100 mM potassium phosphate buffer (pH 7.4), 10 mM EDTA, 0.15 mM NADH, and 0.1 M KNO3 at 30 °C for 30 min. The reaction was terminated by the addition of 100 µL of 1 M zinc acetate.
For the determination of nitrite reductase activity, the extract was incubated in a reaction mixture containing 100 mM potassium phosphate buffer (pH 7.4), 15 mM NaNO2, 5 mM methyl viologen, and 86.2 mM sodium dithionite in 190 mM NaHCO3 at 30 °C for 30 min. The reaction was terminated by vigorous shaking using a vortex mixer.
Subsequently, for both enzymatic assays, diazotisation of nitrite ions was induced by collecting 100 µL of the reaction mixture and mixing it in a 1:1 (v/v) ratio with the Griess reagent. The reagent was freshly prepared by combining a solution of 5.8 mM sulfanilamide and 0.8 mM N-(1-naphthyl)ethylenediamine dihydrochloride (NNEDD). Absorbance was measured at 540 nm. A standard curve (concentration range 0–80 µM) prepared for NaNO2 was used to perform the calculations. The activities of the NR and NiR were expressed as U/mg of protein.
The protein contents in extracts were determined using the 1976 Bradford [81] method, with a standard curve prepared using bovine serum albumin.

4.9. Determination of Photosynthetic Pigment and Its Precursor Contents

To determine the contents of chlorophyll, carotenoids, and total porphyrins, 0.1 g of leaves were placed in test tubes, and 15 cm3 of 96% ethanol was added [82]. The samples were then incubated in a water bath at 80 °C until the plant material was fully discoloured (3–4 h). The absorbance of chlorophyll a and b was then measured at 665 and 649 nm, respectively. Carotenoid absorption was measured at a wavelength of 440 nm. To measure total porphyrins, the absorbances at 575 and 628 nm were determined. These are the absorption peaks of protoporphyrin and protochlorophyllide, respectively. All the calculation formulas used in this analysis were presented in the previous work by Pipiak et al. 2024 [83].

4.10. Determination of β-Carotene, Lutein, and Zeaxanthin Levels

The methods of Marinova and Ribarova (2007) [84], Thirupathi et al. (2017) [85], and Rahman et al. (2023) [86] were used to determine the levels of β-carotene, lutein, and zeaxanthin. Each plant sample was homogenised using a laboratory grinder (IKA® A10 Basic, Ika Poland Sp. z o.o. Warszawa, Poland). Approximately 100–150 mg of each sample was suspended in 10 cm3 of water and saponified with 20 cm3 of 10% potassium hydroxide in methanol for two hours at room temperature and in the dark. The carotenoids were then extracted from the KOH/methanol phase by shaking with 20 cm3 of hexane. The lower KOH/methanol/aqueous phase was removed and upper-extracted twice more with 20 cm3 of hexane. The hexane was then evaporated at 35 °C using a rotary evaporator. The residue was redissolved in 10 cm3 of 96% ethanol, filtered, and diluted to 25 cm3. The obtained extract was used directly for HPLC analysis. Analysis was performed using a Prominence-i LC 2030 HPLC system (Shimadzu, Kyoto, Japan) consisting of a diode array detector. Chromatographic separation was achieved using a C18 column (150 mm × 4.6 mm, 3.0 µm, Arion® Plus, Chromservis s.r.o, Praha, Czech Republic) in isocratic elution mode. The mobile phase was an acetonitrile:methanol mixture (85:15 v/v). Separation was performed at room temperature with a flow rate of 1.4 mL/min and a run time of 40 min. The injection volume was 0.020 cm3 and the detection wavelengths were set to 447 and 452 nm. Beta-carotene, lutein, and zeaxanthin standards were purchased from Sigma-Aldrich (Sigma-Aldrich, Saint Louis, MO, USA). HPLC-grade methanol and acetonitrile, n-hexane, 96% ethanol, and potassium hydroxide were purchased from Chempur (Chempur, Piekary Śląskie, Poland).

4.11. ATR–FTIR Analysis

Fourier transform infrared spectroscopy with attenuated total reflection (ATR–FTIR) was used as part of the analysis to evaluate the structural changes that occurred in the dried plant material after 24 h at 70 °C [87,88]. During the measurements, leaves of the individual experimental variants were placed directly on a diamond measurement crystal. Five spectra were recorded for each sample within the 600–4000 cm−1 wavelength range using a PerkinElmer FTIR/NIR Spectrometer Spectrum 3 with an ATR accessory featuring a temperature-controlled diamond window (MIR ATR Diamond). Each measurement consisted of 32 scans at a spectral resolution of 4 cm−1. A uniform gauge force of 100–110 was applied to the plant samples under test. The averages of the recorded spectra were used for analysis.

4.12. Statistical Analysis

For each of the assayed parameters, the results/data are presented as the average value obtained from at least three biological replicates of each experimental variant. Statistical significance was assumed at α < 0.05, as determined by a two-way analysis of variance (ANOVA) followed by a Tukey’s post hoc test. All statistical analyses were performed using Statistica 13.3 software (TIBCO Software, Palo Alto, CA, USA).

5. Conclusions

The presented results confirmed the phytobiostimulatory potential of fish collagen and sheep keratin hydrolysates. These industrial by-products, applied at low concentrations, can be used to improve seed quality as agents enhancing the hydro-priming process. Germination tests showed that coriander seeds of the H-Col and H-Ker variants best withstood high-temperature stress during imbibition. They did not germinate at 35 °C, but all of these seeds capable of germinating in the tested population (i.e., 90%) germinated after the stress was removed. In contrast, in the H variant, germination after stress decreased by 12–15% (from 90% to 78–75%), whereas in the C variant, it decreased by about 30% (from 90% to 60%).
Observation of the development of seedlings derived from the different seed variants also provided valuable information on anti-stress strategies that are strengthened by seed-applied Col and Ker. These include an increase in the pool of chlorophylls and their precursors and in nitrogen content (in the H-Col variant), which resulted in the best growth of these seedlings.
The applied high-temperature stress also acted as a priming factor for the strongest individuals (those that were not eliminated): it changed the elemental profile of plants and stimulated the biosynthesis of carotenoid antioxidants (lutein, zeaxanthin, β-carotene).
The results obtained using modern ATR–FTIR technology are particularly intriguing and indicate directions for future research. Changes in the structure and composition of cell walls, probably induced by protein hydrolysates (especially H-Ker), should be investigated in detail, and the polyphenol profile should also be analysed.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27052188/s1.

Author Contributions

Conceptualisation, M.S.-F. and M.M.P.; methodology, M.S.-F., P.P., K.S., M.K. and M.M.P.; software, M.S.-F.; validation, M.S.-F., P.P., K.S. and M.K.; formal analysis, M.S.-F.; investigation, M.S.-F., P.P., K.S. and M.K.; resources, M.S.-F., P.P., K.S., M.K. and M.M.P.; data curation, M.S.-F.; writing—original draft preparation, M.S.-F., P.P. and M.M.P.; writing—review and editing, M.S.-F., P.P., K.S., M.K. and M.M.P.; visualisation, M.S.-F.; supervision, M.S.-F. and M.M.P.; project administration, M.S.-F.; funding acquisition, M.S.-F., P.P., K.S., M.K. and M.M.P. All authors have read and agreed to the published version of the manuscript.

Funding

M.S.-F. and M.M.P. (authors of the study idea) acknowledge the Łukasiewicz Research Network—Lodz Institute of Technology for analytical support using resources funded by the Ministry of Science and Higher Education (00/BCW/01/00/1/3/0401).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to also thank Magdalena Lasoń-Rydel for performing the compositional analysis of the hydrolysates (see Supplementary Materials), as well as INVENTIA Polish Technologies and PROTEINA Natural Protein Factory for providing the collagen and keratin hydrolysates, respectively.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Seed germination test under optimal conditions (darkness, 20 °C) (A) and its recovery effect after initial high-temperature stress (9-day incubation in darkness at 35 °C, and then germination test at 20 °C) (B). Tested seed variants: C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds. Values: mean ± SEM followed by different letters are significantly different (p < 0.05; two-way ANOVA for germination day 14 followed by Tukey’s post hoc test; samples of 100 seeds; n = 4).
Figure 1. Seed germination test under optimal conditions (darkness, 20 °C) (A) and its recovery effect after initial high-temperature stress (9-day incubation in darkness at 35 °C, and then germination test at 20 °C) (B). Tested seed variants: C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds. Values: mean ± SEM followed by different letters are significantly different (p < 0.05; two-way ANOVA for germination day 14 followed by Tukey’s post hoc test; samples of 100 seeds; n = 4).
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Figure 2. Total nitrogen (N) content in the shoots (A) and roots (B) of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C—opt; marked as plain coloured bars) or pretreated for 9 days at 35 °C (post-stress recovery—rec; marked as striped bars). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 3).
Figure 2. Total nitrogen (N) content in the shoots (A) and roots (B) of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C—opt; marked as plain coloured bars) or pretreated for 9 days at 35 °C (post-stress recovery—rec; marked as striped bars). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 3).
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Figure 3. Activity of nitrate reductase, NR (A), and nitrite reductase, NiR (B), in the shoots of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C—opt; marked as plain coloured bars) or pretreated for 9 days at 35 °C (post-stress recovery—rec; marked as striped bars). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 5–8).
Figure 3. Activity of nitrate reductase, NR (A), and nitrite reductase, NiR (B), in the shoots of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C—opt; marked as plain coloured bars) or pretreated for 9 days at 35 °C (post-stress recovery—rec; marked as striped bars). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 5–8).
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Figure 4. The content of chlorophyll a + b (Chl a + b) (A), carotenoids (Cars) (B), and ratio of chlorophyll a + b to carotenoids (Chl a + b/Cars) (C) determined in the leaves of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C—opt; marked as plain coloured bars) or pretreated for 9 days at 35 °C (post-stress recovery—rec; marked as striped bars). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 6).
Figure 4. The content of chlorophyll a + b (Chl a + b) (A), carotenoids (Cars) (B), and ratio of chlorophyll a + b to carotenoids (Chl a + b/Cars) (C) determined in the leaves of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C—opt; marked as plain coloured bars) or pretreated for 9 days at 35 °C (post-stress recovery—rec; marked as striped bars). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 6).
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Figure 5. The content of porphyrins: protoporphyrin (Proto) (A) and protochlorophyllide (Pchilide) (B) determined in the leaves of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C—opt; marked as plain coloured bars) or pretreated for 9 days at 35 °C (post-stress recovery—rec; marked as striped bars). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 6).
Figure 5. The content of porphyrins: protoporphyrin (Proto) (A) and protochlorophyllide (Pchilide) (B) determined in the leaves of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C—opt; marked as plain coloured bars) or pretreated for 9 days at 35 °C (post-stress recovery—rec; marked as striped bars). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 6).
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Figure 6. The content of lutein (A), zeaxanthin (B), and β-carotene (C) determined in the leaves of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C—opt; marked as plain coloured bars) or pretreated for 9 days at 35 °C (post-stress recovery—rec; marked as striped bars). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 4).
Figure 6. The content of lutein (A), zeaxanthin (B), and β-carotene (C) determined in the leaves of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C—opt; marked as plain coloured bars) or pretreated for 9 days at 35 °C (post-stress recovery—rec; marked as striped bars). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 4).
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Figure 7. ATR–FTIR spectra of leaves in 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds—green line; H—hydro-primed seeds—blue line; H-Col—collagen-primed seeds—black line; H-Ker—keratin-primed seeds—pink line) germinated and grown under optimal conditions 20 °C.
Figure 7. ATR–FTIR spectra of leaves in 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds—green line; H—hydro-primed seeds—blue line; H-Col—collagen-primed seeds—black line; H-Ker—keratin-primed seeds—pink line) germinated and grown under optimal conditions 20 °C.
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Figure 8. ATR–FTIR spectra of leaves in 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds—green line; H—hydro-primed seeds—blue line; H-Col—collagen-primed seeds—black line; H-Ker—keratin-primed seeds—pink line) after post-stress recovery.
Figure 8. ATR–FTIR spectra of leaves in 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds—green line; H—hydro-primed seeds—blue line; H-Col—collagen-primed seeds—black line; H-Ker—keratin-primed seeds—pink line) after post-stress recovery.
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Table 1. Representative 24-day-old seedlings of coriander grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C) or pretreated for 9 days at 35 °C (post-stress recovery).
Table 1. Representative 24-day-old seedlings of coriander grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C) or pretreated for 9 days at 35 °C (post-stress recovery).
ConditionsSeedling Variants
CHH-ColH-Ker
OptimalIjms 27 02188 i001Ijms 27 02188 i002Ijms 27 02188 i003Ijms 27 02188 i004
Post-stress
recovery		Ijms 27 02188 i005Ijms 27 02188 i006Ijms 27 02188 i007Ijms 27 02188 i008
Table 2. Growth parameters of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated at optimal conditions (20 °C) or pretreated for 9 days at 35 °C (post-stress recovery). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 10).
Table 2. Growth parameters of 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated at optimal conditions (20 °C) or pretreated for 9 days at 35 °C (post-stress recovery). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 10).
ConditionsGrowth ParameterSeedling Variants
CHH-ColH-Ker
OptimalShoot Length [cm]9.00 ± 1.50 ab8.80 ± 1.69a9.10 ± 1.72 abc8.70 ± 1.97 a
No. of Leaves/Plant4.00 ± 2.16a3.80 ± 1.48a4.20 ± 2.04 ab4.30 ± 2.45 ab
No. of Branches/Plant2.70 ± 0.82 a3.10 ± 0.74 ab3.10 ± 0.74 ab3.40 ± 0.70 ab
Fresh Weight of Shoots [g]0.27 ± 0.05 a0.28 ± 0.05 ab0.44 ± 0.09 bc0.36 ± 0.15 abc
Dry Weight of Shoots [g]0.04 ± 0.02 ab0.03 ± 0.01 a0.04 ± 0.02 ab0.05 ± 0.03 ab
Root Length [cm]11.0 ± 2.18a11.7 ± 2.08 ab11.9 ± 2.13 ab12.2 ± 0.87 ab
Fresh Weight of Roots [g]0.07 ± 0.03a0.12 ± 0.08 ab0.23 ± 0.08 bc0.31 ± 0.25 bc
Dry Weight of Roots [g]0.01 ± 0.00 a0.02 ± 0.01 a0.02 ± 0.01 a0.02 ± 0.01 a
Post-stress
recovery		Shoot Length [cm]11.2 ± 1.14 bc10.6 ± 1.17 abc11.3 ± 1.42 c10.5 ± 1.78 abc
No. of Leaves/Plant6.20 ± 1.75abc6.30 ± 1.49 abc6.70 ± 2.11 bc7.00 ± 1.49 c
No. of Branches/Plant3.70 ± 0.67ab3.90 ± 1.10 b3.70 ± 0.82 ab3.80 ± 0.79 ab
Fresh Weight of Shoots [g]0.29 ± 0.12ab0.42 ± 0.13 abc0.49 ± 0.13 c0.47 ± 0.15 c
Dry Weight of Shoots [g]0.06 ± 0.01 abc0.06 ± 0.02 bc0.06 ± 0.02 abc0.08 ± 0.03 c
Root Length [cm]11.3 ± 1.69a13.2 ± 1.62 ab13.6 ± 2.72 ab14.5 ± 2.84 b
Fresh Weight of Roots [g]0.22 ± 0.06abc0.30 ± 0.09 c0.29 ± 0.12 c0.31 ± 0.08 c
Dry Weight of Roots [g]0.01 ± 0.00 a0.02 ± 0.00 a0.02 ± 0.01a0.02 ± 0.01 a
Table 3. Elemental composition of shoots in 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C) or pretreated for 9 days at 35 °C (post-stress recovery). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 4 and one-way ANOVA only for Cu in recovery effect after stress).
Table 3. Elemental composition of shoots in 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C) or pretreated for 9 days at 35 °C (post-stress recovery). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 4 and one-way ANOVA only for Cu in recovery effect after stress).
ConditionsElementsSeedling Variants
CHH-ColH-Ker
OptimalMacroelements [g kg−1 DW]Ca11.6 ± 0.81 a11.9 ± 0.97 a11.1 ± 1.36 a11.9 ± 1.42 a
K40.7 ± 3.54 c36.3 ± 1.72 bc33.4 ± 2.16 b34.0 ± 2.87 b
Mg1.61 ± 0.09 a1.46 ± 0.26 a1.94 ± 0.30 a1.96 ± 0.27 a
Na0.94 ± 0.12 a1.07 ± 0.25 ab1.15 ± 0.27 ab0.83 ± 0.17 a
P6.09 ± 0.66 a5.68 ± 1.13 a5.56 ± 0.82 a5.59 ± 1.12 a
Post-stress
recovery		Ca16.5 ± 1.75 bc14.6 ± 0.84 ab16.1 ± 2.16 bc19.0 ± 2.96 c
K16.7 ± 0.04 a13.5 ± 1.75a13.7 ± 4.89 a13.6 ± 2.65 a
Mg4.90 ± 0.96 b3.96 ± 0.13 b3.99 ± 0.43 b4.63 ± 1.27 b
Na6.82 ± 0.57 d1.32 ± 0.03 ab3.91 ± 0.43 c1.70 ± 0.07 b
P5.75 ± 0.25 a4.65 ± 0.60a4.33 ± 1.39 a5.15 ± 1.67 a
OptimalMicroelements [mg kg−1 DW]CuND ND ND ND
Fe113 ± 9.81 a68.7 ± 11.79a85.2 ± 8.95 a67.4 ± 10.8 a
Mn83.2 ± 7.45 a83.7 ± 12.65 a83.9 ± 19.1 a83.8 ± 11.07 a
Mo1.22 ± 0.03 e1.35 ± 0.04 f1.45 ± 0.01 g2.43 ± 0.02 h
Zn36.2 ± 5.84 a32.8 ± 5.29 a32.8 ± 5.19 a42.0 ± 1.96 ab
Post-stress
recovery		Cu4.78 ± 2.35 a4.61 ± 1.88 a2.29 ± 0.37 a2.71 ± 0.08 a
Fe106 ± 26.42 a81.7 ± 20.0 a69.6 ± 32.5 a93.5 ± 58.8 a
Mn157 ± 1.48 d66.2 ± 0.48 a108 ± 4.18 b133 ± 2.23 c
Mo0.36 ± 0.03 a0.99 ± 0.05 c0.53 ± 0.03 b1.11 ± 0.03 d
Zn47.2 ± 7.03 b47.6 ± 3.66 b41.8 ± 2.25 ab43.2 ± 3.26 ab
ND—not detected (below the detection threshold of the method used).
Table 4. Elemental composition of roots in 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C) or pretreated for 9 days at 35 °C (post-stress recovery). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 4).
Table 4. Elemental composition of roots in 24-day-old coriander seedlings grown from all tested seed variants (C—control non-primed seeds; H—hydro-primed seeds; H-Col—collagen-primed seeds; H-Ker—keratin-primed seeds) germinated under optimal conditions (20 °C) or pretreated for 9 days at 35 °C (post-stress recovery). Values: mean ± SD followed by different letters are significantly different (p < 0.05; two-way ANOVA followed by Tukey’s post hoc test; n = 4).
ConditionsElementsSeedling Variants
CHH-ColH-Ker
OptimalMacroelements [g kg−1 DW]Ca11.6 ± 0.66a11.2 ± 1.49a12.9 ± 0.74ab10.2 ± 0.51a
K33.3 ± 7.36bc39.5 ± 043c28.1 ± 2.84b35.4 ± 5.01bc
Mg3.29 ± 0.44ab3.36 ± 0.50ab3.34 ± 0.25ab3.76 ± 0.53bc
Na8.45 ± 0.37bc11.5 ± 2.51d9.66 ± 2.38cd12.0 ± 0.96d
P7.16 ± 0.83b7.35 ± 1.34b7.40 ± 0.86b8.03 ± 1.26b
Post-stress
recovery		Ca17.7 ± 5.21bc19.4 ± 3.04c22.2 ± 1.20c18.2 ± 0.88c
K6.66 ± 2.84a5.43 ± 2.31a3.81 ± 1.25a5.86 ± 1.58a
Mg4.35 ± 0.33cd4.12 ± 0.25bc2.57 ± 0.14a5.11 ± 0.30d
Na2.46 ± 0.07a1.98 ± 0.02a0.97 ± 0.03a6.17 ± 0.04b
P5.12 ± 2.42ab3.24 ± 1.57a2.34 ± 1.09a3.49 ± 0.58a
OptimalMicroelements [mg kg−1 DW]Cu8.15 ± 0.84b5.47 ± 0.84a10.3 ± 0.57c5.49 ± 0.71a
Fe341 ± 35.2ab400 ± 62.5bc338 ± 55.1ab210 ± 15.1a
Mn178 ± 14.3b193 ± 34.0bc221 ± 6.78c301 ± 22.5d
Mo6.25 ± 0.04f3.89 ± 0.05d3.03 ± 0.62c4.57 ± 0.11e
Zn297 ± 22.9d176 ± 15.2b135 ± 14.7ab252 ± 15.6cd
Post-stress
recovery		Cu9.22 ± 1.20bc7.91 ± 0.34b8.03 ± 0.55b7.81 ± 0.46b
Fe329 ± 20.9ab524 ± 16.2cd552 ± 130d425 ± 71.9bcd
Mn213 ± 2.03bc191 ± 2.18bc80.3 ± 3.48a86.7 ± 0.26a
Mo2.35 ± 0.02b2.63 ± 0.07bc1.11 ± 0.03a2.22 ± 0.24b
Zn195 ± 30.1bc130 ± 25.4ab133 ± 62.0ab107 ± 10.6a
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Skwarek-Fadecka, M.; Pipiak, P.; Sieczyńska, K.; Krępska, M.; Posmyk, M.M. Evaluation of the Biostimulatory Potential of Waste Protein Hydrolysates in Coriander (Coriandrum sativum L.). Int. J. Mol. Sci. 2026, 27, 2188. https://doi.org/10.3390/ijms27052188

AMA Style

Skwarek-Fadecka M, Pipiak P, Sieczyńska K, Krępska M, Posmyk MM. Evaluation of the Biostimulatory Potential of Waste Protein Hydrolysates in Coriander (Coriandrum sativum L.). International Journal of Molecular Sciences. 2026; 27(5):2188. https://doi.org/10.3390/ijms27052188

Chicago/Turabian Style

Skwarek-Fadecka, Monika, Paulina Pipiak, Katarzyna Sieczyńska, Małgorzata Krępska, and Małgorzata M. Posmyk. 2026. "Evaluation of the Biostimulatory Potential of Waste Protein Hydrolysates in Coriander (Coriandrum sativum L.)" International Journal of Molecular Sciences 27, no. 5: 2188. https://doi.org/10.3390/ijms27052188

APA Style

Skwarek-Fadecka, M., Pipiak, P., Sieczyńska, K., Krępska, M., & Posmyk, M. M. (2026). Evaluation of the Biostimulatory Potential of Waste Protein Hydrolysates in Coriander (Coriandrum sativum L.). International Journal of Molecular Sciences, 27(5), 2188. https://doi.org/10.3390/ijms27052188

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