Hempseed Press Cake-Derived Protein Hydrolysate–Zn(II) Complex as a Seed Coating Improves Germination and Early Seedling Establishment in Hot Pepper (Capsicum annuum L.)

Abstract

Uniform germination and rapid seedling establishment are essential for efficient hot pepper production. This study evaluated a hempseed press cake-derived protein hydrolysate–zinc(II) complex (HPH–Zn) as a seed coating designed to enhance early crop performance via formulation-based Zn delivery. The HPH–Zn complex was synthesized via peptide–Zn chelation, physicochemically characterized, and applied at 0.25, 0.50, and 1.00 mg Zn g⁻¹ seed (HPH–Zn0.25, HPH–Zn0.50, and HPH–Zn1.00, respectively). Seed performance was assessed by laboratory germination, accelerated aging, and greenhouse tests. Compared with uncoated, polymer-only, and ZnSO₄ treatments, HPH–Zn significantly improved germination, post-aging performance, field emergence, and early seedling growth in a dose-dependent manner. Relative to uncoated seeds, HPH–Zn1.00 increased laboratory germination, post-aging germination, and field emergence by 10.9, 12.3, and 20.3%, respectively. These responses were associated with stronger soluble sugar accumulation and α-amylase activity during early imbibition. PCA ranked HPH–Zn1.00 as the highest-performing treatment, characterized by greater emergence, biomass, and Zn uptake. However, HPH–Zn0.50 still improved germination and early growth at lower Zn input, whereas HPH–Zn1.00 maximized emergence and Zn accumulation, indicating a performance–input trade-off. These results support waste-derived HPH–Zn as a formulation-based seed coating for improving early seedling establishment in hot pepper.

1. Introduction

Seed germination and early seedling growth are complex physiological and biochemical processes that play a fundamental role in determining crop establishment by regulating emergence uniformity, stand density, and seedling tolerance to transient post-sowing stress [1,2,3]. In hot pepper (C. annuum L.), inconsistent germination and early growth often lead to heterogeneous transplant quality, delayed production schedules, and reduced nursery efficiency. Therefore, strategies that improve seed performance during the earliest stages of development are agronomically important [4,5].

Efficient reserve mobilization during germination depends on the tight coordination of enzymatic activation, membrane integrity, and redox homeostasis, all of which are strongly influenced by micronutrient availability [6,7]. Among these micronutrients, zinc (Zn) plays a central role as a cofactor for hydrolytic and respiratory enzymes, a structural component of antioxidant systems, and a stabilizer of cellular membranes [8,9,10,11]. Accordingly, seed-applied Zn formulations are increasingly recognized for their ability to enhance germination and early growth. They may also support seedling resilience during critical stages of establishment [12]. Seed coating provides a strategically efficient platform for localized Zn delivery at the seed-substrate interface, where coating composition and release behavior may influence early micronutrient use efficiency, seedling establishment, and the risk of local toxicity from highly soluble Zn sources [2,11,13,14,15]. However, conventional inorganic Zn salts such as ZnSO₄ dissolve rapidly during imbibition. Rapid dissolution during imbibition may result in localized Zn surges, thereby narrowing the effective dose window and increasing the risk of phytotoxicity, particularly in sensitive horticultural seeds [16,17].

Recent advances in seed coating technology have shifted coatings from passive carriers to functional delivery platforms capable of modulating early seed metabolism and seedling establishment [14,15,18]. At the same time, sustainability concerns associated with conventional coating materials have intensified interest in bio-based alternatives derived from agro-industrial residues [19]. Nevertheless, most waste-derived materials have been explored empirically as bulk substitutes rather than engineered into chemically defined, performance-oriented formulations [20]. Protein hydrolysates (PHs), composed of low-molecular-weight peptides and amino acids, represent a particularly promising class of waste-derived biostimulants because of their involvement in germination-related metabolism and early growth regulation [21,22]. When used as seed-coating matrices, PHs can provide both biostimulant activity and coordination frameworks capable of stabilizing and modulating micronutrient delivery through peptide–metal interactions [5]. Hempseed press cake is a protein-rich by-product of the hemp oil industry with a complete essential amino acid profile and strong potential as a raw material for PH production [23,24]. Its conversion into a defined HPH–Zn complex therefore represents a value-added strategy for integrating circular bioeconomy principles with micronutrient delivery beyond a simple Zn dose effect. Peptide–Zn complexation has been proposed as a strategy to stabilize Zn, buffer its release, and improve physiological compatibility during early seedling development [25].

Systematic studies on waste-derived PH–Zn complexes designed for seed coating remain limited, particularly under iso-zinc conditions that allow formulation effects to be distinguished from Zn dose effects [14]. This distinction is critical because improvements in seed performance may result from enhanced Zn bioavailability and compatibility associated with chelation rather than from Zn concentration alone [9,16]. In horticulture, seed performance under stress-like conditions is especially important because seeds often encounter suboptimal storage and establishment environments. Accelerated aging assays provide a useful framework for assessing oxidative resilience and post-aging seed performance beyond standard germination tests, yet they have rarely been integrated with physicochemical characterization when evaluating nutrient-biostimulant seed-coating systems [7,26]. Therefore, the objectives of this study were to develop HPH–Zn as a seed coating for hot pepper, compare its performance with ZnSO₄ at an iso-Zn dose, and evaluate its effects on germination, post-aging seed performance, and early seedling establishment under laboratory, accelerated aging, and greenhouse conditions. We hypothesized that HPH–Zn would improve germination and early seedling growth relative to ZnSO₄ at an equivalent Zn dose.

2. Materials and Methods

2.1. Preparation and Characterization of HPH–Zn

HPH–Zn was prepared following previously reported methods with minor modifications [25]. Hempseed press cake-derived protein hydrolysate (HPH), which was produced from hempseed press cakes by using protease enzymatic hydrolysis [23], was dissolved in deionized water at a concentration of 20 mg mL⁻¹. A 100 mM ZnSO₄·7H₂O solution was then slowly added dropwise to the peptide solution to achieve a final HPH-to-zinc ratio of 1:1 (g: mmol). The mixtures were incubated in a shaking water bath at 60 °C for 60 min. Ethanol was subsequently added to the incubated solution until the ethanol concentration reached 80% (v/v) to precipitate the HPH–Zn. After standing for 60 min at room temperature, the mixture was centrifuged at 10,000 g for 10 min to obtain the precipitates. The white precipitates were washed three times with 80% ethanol to remove unbound Zn(II) ions. Finally, the HPH–Zn was collected and freeze-dried for structural characterization.

Attenuated total reflection–Fourier transform infrared (ATR–FTIR) spectra were recorded using a Nicolet™ iS50 FTIR spectrophotometer (Thermo Scientific, Waltham, MA, USA) equipped with a diamond crystal window over the range of 4000–400 cm⁻¹. The UV-visible spectra were recorded over the wavelength range of 200 to 800 nm by a UV-Vis spectrophotometer (Cole-Parmer^®, model SP-800 Series, Vernon Hills, IL, USA). Particle size distribution and zeta potential (ZP) were determined using a Zetasizer Nano ZS (Malvern Panalytical Ltd., Malvern, UK) based on dynamic light scattering (DLS) to confirm nanoscale dimensions and colloidal stability. The morphologies of HPH and HPH–Zn were analyzed using scanning electron microscopy coupled with energy-dispersive X–ray spectroscopy (SEM–EDS; Leo 1450 VP, Leo, North Billerica, MA, USA). Finally, Zn content was quantified using an Agilent 200 Series atomic absorption spectrophotometer (AAS; Agilent Technologies, Inc., Santa Clara, CA, USA) at the Soil Chemistry and Fertility Laboratory, Department of Soil Science, Kasetsart University, Thailand.

2.2. Seed Material and Experimental Design

A hot pepper seed lot (C. annuum L. cv. Spark) (Tiger Seeds Co., Ltd., Nonthaburi, Thailand) with 90% germination and 98% physical purity was used.

The experiment was conducted as a completely randomized design (CRD) with four replications. Six seed-coating treatments were evaluated: (i) uncoated seeds as a control; (ii) seeds coated with polymer only (HPH–Zn0); (iii) seeds coated with ZnSO₄ at 0.25 mg Zn g⁻¹ seed (ZnSO₄–0.25); and (iv–vi) seeds coated with the HPH–Zn complex delivering 0.25, 0.50, and 1.00 mg Zn g⁻¹ seed (HPH–Zn0.25, HPH–Zn0.50, and HPH–Zn1.00, respectively). ZnSO₄·7H₂O and HPH–Zn at 0.25 mg Zn g⁻¹ seed provided an iso-Zn comparison, and the ZnSO₄ level was selected from a preliminary screening as a non-phytotoxic dose that enhanced germination and seed performance.

2.3. Seed Coating Procedure

For each treatment, 10 g of seeds were coated with a polymer solution containing 0.1% (w/v) carboxymethyl cellulose (CMC) (Kingsun Chemical Ltd., Bangkok, Thailand) and 2.0% (w/v) red food coloring (Adinop Co., Ltd., Bangkok, Thailand) using a laboratory seed coater (Centricoater; model SKK10, Modern Seed Technology Center, Maejo University, Chiang Mai, Thailand). The Zn content of the HPH–Zn complex was 36.159 g kg⁻¹ (3.6159%, w/w), whereas ZnSO₄·7H₂O (Zn fraction = 0.2274, w/w) served as the inorganic Zn control. The mass of each Zn source was calculated from the target Zn dose using $m=Z{n}_{\mathrm{target}}/f_{\mathrm{Zn}}$ and then added to the polymer solution, while keeping the coating volume constant at 200 mL kg⁻¹ seed (2.0 mL 10 g⁻¹ seed batch). The coating drum was operated at 30 rpm, and the polymer solution was applied at a rate of 200 mL kg⁻¹ seed. After coating, seeds were dried using a forced dry-air seed dryer (Ceres International Co., Ltd., Bangkok, Thailand) at 33 °C for approximately 6 h, or until the seed moisture content approached the initial moisture level prior to coating. Seed moisture content was monitored using a seed moisture meter (Kett Electric Laboratory Co., Ltd., model PM-650 Type 6515, Tokyo, Japan), and drying was terminated when moisture content was <10%.

2.4. SEM–EDS Analysis of Uncoated and Coated Seed Morphology and Zn Distribution

Seed morphology and Zn distribution at the hilum region were also assessed using SEM–EDS. Two seeds per treatment were randomly selected for analysis, and four EDS acquisition points were recorded per seed to characterize Zn presence at the hilum and testa regions. The intended Zn dose per seed was calculated based on coating formulations; Zn deposition on coated seeds was additionally verified qualitatively by SEM–EDS.

2.5. Seed Germination and Early Seedling Establishment

2.5.1. Evaluation of Laboratory Germination

Fifty seeds from the uncoated and coated treatments were tested for germination using the top of paper (TP) method in plastic boxes (25 × 17 × 8 cm). Seeds were placed on moist, sterilized paper and incubated under controlled environmental conditions (20 °C, 12 h light/12 h dark photoperiod, and 70% relative humidity) with fluorescent lighting providing a photosynthetically active radiation (PAR) intensity of 13.92 µmol m⁻² s⁻¹. Each box was irrigated daily with 10 mL of distilled water. Seeds were considered germinated when radicle protrusion reached ≥2 mm, and germinated seeds were counted daily from the start of the experiment. Germination percentage, germination index (GI), and mean germination time (MGT) were calculated according to the International Seed Testing Association (ISTA) guidelines [27].

2.5.2. Evaluation of Seed Vigor After Accelerated Aging

Post-aging seed performance was assessed using an accelerated aging test (AA test) following the ISTA procedure [28]. The experiment was conducted with four replications, with 50 seeds per replicate. Seeds were placed on a wire mesh screen inside a plastic aging box, ensuring that seeds were exposed to a saturated humid atmosphere without direct contact with liquid water. The boxes were then incubated in a temperature-controlled chamber at 41 °C and 100% relative humidity for 72 h. Following the aging treatment, seeds were re-dried in a desiccation chamber containing silica gel for 6 h until the moisture content fell below 10%. After equilibration, germination and post-aging seed performance were evaluated using the same laboratory procedures described in Section 2.5.1.

2.5.3. Evaluation of Field Emergence and Seedling Establishment

Field emergence and early seedling growth under greenhouse conditions were evaluated by sowing uncoated or coated seeds in peat moss growing medium (Klasmann-Deilmann GmbH, Geeste, Germany) in plastic seedling trays (340 × 340 × 60 mm) containing 105 cells per tray, with one seed sown per cell. Each treatment comprised four replicates of 100 seeds. Seedlings were irrigated as required to maintain appropriate substrate moisture throughout the experiment.

Seeds were considered germinated when the hypocotyl protruded by at least 2 mm, and germination percentage was then calculated using the same formula described for the laboratory test (Section 2.5.1). On day 14 after sowing, ten seedlings per replicate were randomly sampled to determine fresh weight, seedling height, and root length. Shoots and roots were then oven-dried at 80 °C for 72 h and weighed after cooling to room temperature. Dried samples were ground into a fine powder using a variable-speed rotor mill (Pulverisette 14, Fritsch, Idar-Oberstein, Germany) prior to Zn content analysis. Then, Zn content was also quantified similarly to the protocol described in Section 2.1.

2.6. Biochemical Analyses of Soaked Seeds

Biochemical changes in seeds after soaking were assessed at 12 and 24 h using four replications per treatment. Briefly, soaked seed samples were frozen in liquid nitrogen, freeze-dried and then ground to a fine powder prior to analysis.

2.6.1. Determination of Total Soluble Protein

Total soluble protein was determined using a modified Bradford method [29]. Ground seed powder (0.5 g) was extracted in 2 mL of 100 mM sodium phosphate buffer (pH 7.8) containing PVP (0.4 g), kept on ice, and centrifuged (15,000× g, 4 °C, 15 min). The supernatant (25 µL) was mixed with 1250 µL Bradford reagent, and absorbance was read at 595 nm by a microplate reader (Revvity, model EnVision XCite 2105, Waltham, MA, USA). Protein concentration was calculated from a BSA standard curve and expressed as mg g⁻¹ DW.

2.6.2. Determination of Total Soluble Sugars

Total soluble sugars were determined using the phenol-sulfuric acid method [30]. Ground seed powder (0.5 g) was extracted with 10 mL of 80% ethanol for 24 h and centrifuged (2000 rpm). The supernatant (0.1 mL) was reacted with 1.0 mL of 5% phenol and 5.0 mL of 98% H₂SO₄, incubated at 30 °C for 20 min, and absorbance was measured at 490 nm by a microplate reader. Sugar concentration was determined using a D-glucose standard curve and expressed as mg g⁻¹ DW.

2.6.3. Analysis of α-Amylase Activity

α-amylase activity was analysed using an α-amylase assay kit (Ceralpha Method) (Reference code: K-CERA; SKU: 700004273; Megazyme, Bray, Ireland), including the kit extraction buffer, according to a previous study [31]. Ground seed powder (3.0 g) was extracted using the kit extraction buffer. The enzyme extract was reacted with the Ceralpha substrate, and absorbance was measured spectrophotometrically following the kit protocol. α-amylase activity was calculated from the kit calibration and expressed as Ceralpha Unit g⁻¹ DW.

2.7. Statistical Analysis

Statistical analyses were performed using Statistix software (version 10.0; Analytical Software, Tallahassee, FL, USA). Treatment effects were evaluated using analysis of variance (ANOVA), and mean comparisons were carried out using Tukey’s honestly significant difference (Tukey’s HSD) test at the 0.05 probability level. In addition, planned contrast analysis was conducted in Microsoft Excel to test specific a priori comparisons among selected treatments. PCA with 95% confidence ellipses was performed using R software (version 4.5.1; R Core Team, Vienna, Austria).

3. Results

3.1. Characterization of HPH–Zn

ATR–FTIR spectra of HPH–Zn showed clear shifting peaks relative to HPH (Figure 1), indicating coordination between Zn(II) ions and functional groups within the peptide matrix. The N–H stretching band shifted slightly from 3274 to 3279 cm⁻¹, while the –OH stretching region shifted from 2930 cm⁻¹ in HPH to peaks at 2962 and 2932 cm⁻¹ after complexation. In the amide region, the Amide I band (C=O stretching) shifted from 1646 to 1628 cm⁻¹ and the Amide II band (N–H bending) shifted from 1542 to 1515 cm⁻¹, indicating the involvement of peptide backbone groups in Zn(II) binding. Additional changes were also detected in the C–O stretching band (1046 to 1079 cm⁻¹) and in the low-wavenumber region assigned to Zn–O/Zn–N vibrations (452 to 456 cm⁻¹).

The UV–Vis spectra of HPH showed characteristic absorption within 220–300 nm, including a shoulder at ~265 nm (Figure S1). After Zn(II) complexation, no new peaks or major wavelength shifts were observed; instead, the ~265 nm band decreased in intensity and the spectrum became smoother between 250 and 300 nm. DLS analysis showed that the mean hydrodynamic diameter increased from 6.0 nm in HPH to 19.8 nm in HPH–Zn (Figure S2a), while the zeta potential shifted from +132.8 mV to −161.0 mV (Figure S2b), indicating marked surface charge reorganization of the nanoscale assemblies. SEM micrographs revealed that native HPH consisted of loosely aggregated irregular particles, whereas HPH–Zn formed more compact and organized clusters without visible crystalline deposits (Figure S3). The total Zn content of HPH–Zn averaged 36,159 mg kg⁻¹ (36.159 g kg⁻¹). Thus, these results indicate the formation of a structurally reorganized peptide–Zn(II) complex. These physicochemical characteristics provided the basis for evaluating the surface deposition behavior and biological performance of HPH–Zn as a seed-coating formulation.

3.2. Effects of HPH–Zn Seed Coating on Seed Performance and Early Seedling Establishment

3.2.1. Morphology and Zn Distribution of Uncoated and Coated Seeds

Surface morphology at the hilum region differed among treatments (Figure 2). Uncoated seeds exhibited an uneven and grooved seed-coat surface (Figure 2a,g), whereas polymer-only coating formed a thin film that partially smoothed the surface (Figure 2b,h). In ZnSO₄–0.25-coated seeds, ZnSO₄ appeared as plate- or flake-like crystalline deposits with uneven distribution across the hilum region (Figure 2c,i). In contrast, seeds coated with HPH–Zn at 0.25–1.00 mg Zn g⁻¹ seed showed no large crystalline structures but instead formed a more uniform coating layer with finely dispersed particles distributed across the hilum surface (Figure 2d–f,j–l). EDS analysis supported these morphological observations by confirming significant Zn enrichment on coated seed surfaces (Table S1). At the hilum, Zn concentration increased dose-dependently in HPH–Zn treatments, reaching 3.04% at 1.0 mg Zn g⁻¹ seed, whereas ZnSO₄–0.25 showed Zn levels comparable to HPH–Zn0.25 but with more visibly crystalline and heterogeneous surface deposition. Across all treatments, Zn concentration remained higher at the hilum than at the testa, suggesting preferential Zn deposition at the hilum region. These findings suggested that HPH–Zn coatings promoted a more homogeneous Zn deposition pattern at the hilum, an important region of the seed involved in the early stage of imbibition.

3.2.2. Laboratory Germination Test

Seed coating significantly affected the percentage of laboratory germination (

Table 1. Effect of seed coating with HPH–Zn on laboratory germination performance of hot pepper.

Treatments 1	Germination (%)	GI 2	MGT (Days)
Uncoated	86.00 ± 0.00 d 3	3.74 ± 0.01 d	10.84 ± 0.10 ab
HPH–Zn0	85.78 ± 0.16 d	3.93 ± 0.09 d	11.01 ± 0.20 a
ZnSO4–0.25	89.33 ± 0.47 c	4.13 ± 0.05 c	10.94 ± 0.08 ab
HPH–Zn0.25	90.67 ± 0.47 c	4.30 ± 0.10 bc	10.43 ± 0.04 bc
HPH–Zn0.50	94.67 ± 0.47 b	4.37 ± 0.03 b	10.19 ± 0.14 c
HPH–Zn1.00	96.89 ± 0.42 a	4.57 ± 0.00 a	10.14 ± 0.06 c

¹ Uncoated: uncoated seeds as control; HPH–Zn0: seeds coated with polymer only; ZnSO₄–0.25: seeds coated with ZnSO₄ at 0.25 mg Zn g⁻¹ seed; HPH–Zn0.25, HPH–Zn0.50, and HPH–Zn1.00: seeds coated with the HPH–Zn complex delivering 0.25, 0.50, and 1.00 mg Zn g⁻¹ seed, respectively. ² GI: germination index; MGT: mean germination time. ³ Means ± SE followed by the same letters in the same column are not significantly different at p = 0.05 as determined by Tukey’s HSD.

). Polymer-only coating (HPH–Zn0) did not improve germination or related traits relative to uncoated seeds. However, Zn-based coatings increased germination percentage and GI, while also reducing MGT, with the HPH–Zn complex showing a clear dose-dependent response. HPH–Zn1.00 showed the highest germination percentage and GI, at 96.89% and 4.57, respectively, followed by HPH–Zn0.50 with 94.67% and 4.37, respectively. MGT was lowest in HPH–Zn1.00 (10.14 days), although it did not differ significantly from HPH–Zn0.50 and HPH–Zn0.25. At the iso-Zn dose, HPH–Zn0.25 showed numerically higher germination and GI and a lower MGT than ZnSO₄–0.25.

3.2.3. Accelerated Aging Test

Accelerated aging markedly reduced seed performance in the control treatments; however, Zn-based seed coatings significantly improved post-aging germination and related performance traits (

Table 2. Effect of seed coating with HPH–Zn on post-aging seed performance of hot pepper after accelerated aging.

Treatments 1	Germination (%)	GI 2	MGT (Days)
Uncoated	76.67 ± 0.47 c 3	3.34 ± 0.05 d	11.00 ± 0.05 a
HPH–Zn0	74.44 ± 0.31 d	3.47 ± 0.09 cd	10.87 ± 0.03 ab
ZnSO4–0.25	80.17 ± 0.17 b	3.77 ± 0.04 c	10.66 ± 0.04 b
HPH–Zn0.25	81.89 ± 0.34 b	3.88 ± 0.02 b	10.32 ± 0.04 c
HPH–Zn0.50	88.67 ± 0.47 a	4.23 ± 0.00 ab	10.24 ± 0.01 cd
HPH–Zn1.00	88.92 ± 0.50 a	4.50 ± 0.07 a	10.00 ± 0.05 d

¹ Uncoated: uncoated seeds as control; HPH–Zn0: seeds coated with polymer only; ZnSO₄–0.25: seeds coated with ZnSO₄ at 0.25 mg Zn g⁻¹ seed; HPH–Zn0.25, HPH–Zn0.50, and HPH–Zn1.00: seeds coated with the HPH–Zn complex delivering 0.25, 0.50, and 1.00 mg Zn g⁻¹ seed, respectively. ² GI: germination index; MGT: mean germination time. ³ Means ± SE followed by the same letters in the same column are not significantly different at p = 0.05 as determined by Tukey’s HSD.

). HPH–Zn0 did not alleviate aging-induced deterioration relative to uncoated seeds. In contrast, both ZnSO₄ and HPH–Zn treatments increased germination percentage and GI after aging, while reducing MGT, with the HPH–Zn complex showing a clear dose-dependent response. After aging, HPH–Zn0.50 and HPH–Zn1.00 showed similarly high germination (88.67–88.92%), GI (4.23–4.50), and short MGT (10.00–10.24 days). At the iso-Zn dose, HPH–Zn0.25 and ZnSO₄–0.25 showed comparable germination percentage; however, HPH–Zn0.25 exhibited a higher GI and lower MGT, indicating stronger post-aging performance.

3.2.4. Greenhouse Test

Seed coating significantly affected field emergence, seedling growth, and seedling Zn content under greenhouse conditions (

Table 3. Effect of seed coating with HPH–Zn on field emergence, seedling growth, and seedling Zn content of hot pepper under greenhouse conditions.

Treatments 1	Field Emergence (%)	Shoot Height (cm)	Root Length (cm)	Dry Weight (mg Plant−1)	Zn Content (mg kg−1 Seedling DW)
Uncoated	73.67 ± 1.25 e 2	4.30 ± 0.25 e	3.51 ± 0.02 e	7.36 ± 0.33 e	20.57 ± 0.17 d
HPH–Zn0	78.67 ± 0.62 d	4.51 ± 0.20 de	4.16 ± 0.02 d	7.85 ± 0.10 d	20.44 ± 0.59 d
ZnSO4–0.25	80.50 ± 0.20 cd	5.10 ± 0.05 cd	4.37 ± 0.02 c	9.70 ± 0.06 c	33.31 ± 0.37 c
HPH–Zn0.25	82.67 ± 0.47 c	5.36 ± 0.04 bc	5.13 ± 0.02 b	10.42 ± 0.07 b	37.63 ± 0.12 b
HPH–Zn0.50	87.50 ± 0.20 b	5.73 ± 0.02 ab	5.66 ± 0.04 a	10.69 ± 0.04 ab	38.67 ± 0.14 b
HPH–Zn1.00	94.00 ± 0.82 a	6.06 ± 0.08 a	5.74 ± 0.06 a	10.96 ± 0.01 a	42.21 ± 0.63 a

¹ Uncoated: uncoated seeds as control; HPH–Zn0: seeds coated with polymer only; ZnSO₄–0.25: seeds coated with ZnSO₄ at 0.25 mg Zn g⁻¹ seed; HPH–Zn0.25, HPH–Zn0.50, and HPH–Zn1.00: seeds coated with the HPH–Zn complex delivering 0.25, 0.50, and 1.00 mg Zn g⁻¹ seed, respectively. ² Means ± SE followed by the same letters in the same column are not significantly different at p = 0.05 as determined by Tukey’s HSD.

). Field emergence was highest at HPH–Zn1.00 (94.00%), followed by HPH–Zn0.50 (87.50%), compared with 73.67% in uncoated seeds. Seedling growth was also enhanced by HPH–Zn coatings. HPH–Zn0.50 and HPH–Zn1.00 formed the high-performance group for shoot height, root length, and dry weight. Seedling Zn content increased markedly in all Zn-treated seeds relative to uncoated and polymer-only treatments. At the iso-Zn dose, HPH–Zn0.25 showed higher Zn content than ZnSO₄–0.25, and the highest Zn accumulation was observed at HPH–Zn1.00. Overall, these results indicate a clear dose-dependent response of HPH–Zn under greenhouse conditions, with progressive gains in emergence, growth, and Zn accumulation.

Although Tukey’s HSD did not separate ZnSO₄–0.25 and HPH–Zn0.25 for all endpoints, planned contrast analysis (Table S2) provided a targeted iso-Zn comparison and detected significant formulation effects at the equivalent Zn dose (0.25 mg Zn g⁻¹ seed). HPH–Zn0.25 differed from ZnSO₄–0.25 in laboratory germination percentage (Table S2a), GI (Table S2b), and MGT (Table S2c); post-aging germination percentage (Table S2d) and MGT (Table S2f); field emergence (Table S2g); root length (Table S2i); seedling dry weight (Table S2j); and seedling Zn content (Table S2k). However, seedling height (Table S2h) was not affected and post-aging GI (Table S2e) showed only a marginal effect. Across the HPH–Zn series (0.25–1.00 mg Zn g⁻¹ seed), all studied traits showed a significant linear dose response, whereas the quadratic component was generally non-significant, except for Zn content.

3.3. Biochemical Contents of Uncoated and Coated Hot Pepper Seeds After Soaking for 12 and 24 h

Across all treatments, imbibition from 12 to 24 h was characterized by a consistent decline in total soluble protein together with increases in total soluble sugar and α-amylase activity (Figure 3), indicating progressive reserve mobilization. The magnitude of these changes, however, differed among formulations. Among the HPH–Zn treatments, HPH–Zn1.00 showed a clearer increase in sugar accumulation and α-amylase activity between 12 and 24 h, whereas HPH–Zn0.50 showed no significant change in total soluble sugar during this period despite maintaining relatively high levels. In contrast, ZnSO₄–0.25 and uncoated seeds exhibited comparatively smaller temporal changes. Therefore, Figure 3 shows the within-treatment biochemical responses during the first 24 h of imbibition.

Treatment differences within each time point are presented in Table S3. At 12 h, ZnSO₄–0.25 showed the lowest total soluble protein, whereas HPH–Zn0.50 had the highest total soluble sugar and α-amylase activity. At 24 h, HPH–Zn0.50 maintained the highest total soluble protein and total soluble sugar, while HPH–Zn1.00 showed the highest α-amylase activity. HPH–Zn treatments were generally associated with higher sugar accumulation and enzyme activity than ZnSO₄–0.25 and uncoated seeds. Figure 3 therefore complements Table S3 by showing within-treatment changes over time, whereas Table S3 presents between-treatment differences at each sampling time.

3.4. Principal Component Analysis

PCA integrating laboratory germination, post-aging seed performance, field emergence, seedling growth, and seedling Zn content separated treatments primarily along PC1, which explained 89.90% of the total variance, whereas PC2 explained 3.50% (Figure 4). HPH–Zn treatments showed a clear dose-dependent shift along PC1, with HPH–Zn1.00 positioned at the far positive side of PC1 and HPH–Zn0.50 also grouped within the positive PC1 region (Figure 4a). In contrast, uncoated seeds and HPH–Zn0 were located on the negative side of PC1, whereas ZnSO₄–0.25 occupied an intermediate negative position and HPH–Zn0.25 was separated near the center-right of the ordination. This distribution indicates an overall gradient in treatment performance across the multivariate space.

The loading plot showed that GP, AA_GP, RL, AA_GI, FE, SH, Zn, DW, and GI were positively associated with PC1, whereas MGT and AA_MGT were negatively associated with this axis (Figure 4b). Accordingly, treatments located on the positive side of PC1 were associated with higher germination, field emergence, seedling growth, and Zn accumulation, whereas treatments on the negative side were associated with longer germination time. Thus, PCA was used to provide an integrative summary of how multiple traits collectively differentiated treatments across the experimental conditions evaluated in this study.

4. Discussion

The combined physicochemical evidence demonstrates that Zn was successfully incorporated into the HPH matrix through peptide-mediated chelation rather than simple physical association. FTIR analysis showed coordinated shifts in amide- and oxygen-containing bands together with the appearance of Zn–O/Zn–N vibrations (Figure 1), indicating interaction of Zn(II) ions with peptide carboxylate, amide, and hydroxyl groups, a coordination pattern widely reported for peptide–metal complexes [32,33,34]. The UV–Vis response, characterized by attenuation of the ~265 nm region without the appearance of new bands (Figure S1), is consistent with modification of the peptide electronic environment without major chromophore disruption, consistent with carbonyl- and amide-mediated Zn(II) coordination [25,35,36]. These molecular changes were accompanied by nanoscale structural reorganization, as Zn complexation increased particle size from 6.0 to 19.8 nm (Figure S2a,c) and shifted the zeta potential from +132.8 to −161.0 mV (Figure S2b,d), indicating the formation of highly charged peptide–Zn assemblies with strong colloidal stability [25,37]. SEM revealed surface restructuring relative to native HPH (Figure S3), corroborating metal-induced conformational assembly rather than passive aggregation [25,38]. Collectively, these spectroscopic, nanoscale, and morphological data support the formation of a stable HPH–Zn(II) chelate. These physicochemical properties may be associated with more compatible Zn delivery during early seed hydration and may help explain the enhanced biological performance observed in the coating assays [25,35,36].

These formulation-level differences were also reflected in distinct deposition patterns on the seed surface. ZnSO₄ formed discrete crystalline deposits, producing heterogeneous surface coverage and localized Zn-rich zones (Figure 2), a pattern similar to that reported for inorganic Zn seed coatings [11]. In contrast, HPH–Zn formed a smoother and more homogeneous nanoscale coating layer across the hilum region, consistent with peptide–Zn chelation giving rise to loosely aggregated structures rather than large crystals [25]. This deposition pattern is especially relevant in hot pepper because the seed coat exhibits structurally diverse micromorphological features [39]. SEM–EDS revealed that Zn accumulation was consistently greater at the hilum than at the seed testa (Table S1), likely reflecting anatomical heterogeneity of the seed coat; in C. annuum, elongated epidermal cells and undulating lateral walls generate a rough topology that can influence particle adhesion [40]. Given the recognized role of the hilum in water uptake, gas exchange, and solute transport during imbibition [41,42,43,44,45], the more continuous and homogeneous HPH–Zn deposition created a more uniform Zn interface than crystalline ZnSO₄. A similar surface-level advantage of nanoscale Zn delivery has been reported in soybean, where Zn nanocoated seeds improved germination speed and early seedling development relative to conventional Zn sources [11]. Accordingly, the superior performance of HPH–Zn under iso-Zn conditions may reflect not only the chemical effect of chelation, but also differences in surface distribution that could influence Zn availability at early seed entry sites.

The biochemical responses during imbibition provide a physiological context linking coating properties and seed performance. The decline in total soluble protein from 12 to 24 h (Figure 3a) reflects the hydrolysis of storage proteins into peptides and amino acids to support embryo development [46]. Zinc-coated seeds may have supported this process because Zn serves as a catalytic component of hydrolases, including amylases and proteases involved in reserve mobilization [47,48]. Concurrent increases in total soluble sugar and α-amylase activity indicate progressive starch degradation and metabolic activation during the first 24 h of imbibition. In the present study, HPH–Zn0.50 and HPH–Zn1.00 showed the strongest increases in sugar accumulation and α-amylase activity (Figure 3b,c), consistent with enhanced reserve mobilization. HPH–Zn treatments also maintained comparatively higher soluble protein levels at 24 h, suggesting regulated protein turnover and a possible metabolic buffering role of PH [49,50]. Because soluble proteins and sugars are positively associated with seed performance and germination capacity [51,52], these biochemical adjustments suggest that HPH–Zn was associated with earlier and more coordinated metabolic readiness during imbibition. This biochemical pattern is consistent with the higher germination percentage, faster GI, and lower MGT recorded in HPH–Zn–coated seeds. The formulation-dependent trends detected within individual time points (Table S3) also suggested that these responses were associated with differences in early metabolic coordination and were not attributable to Zn presence alone.

In this study, laboratory germination represented seed performance under controlled conditions, accelerated aging provided an assessment of post-aging physiological stability, and greenhouse testing assessed emergence and early seedling establishment under substrate-based nursery conditions. In laboratory tests, HPH–Zn increased germination from 86.00% in uncoated seeds to 96.89% at 1.00 mg Zn g⁻¹ seed, together with a higher germination index and reduced MGT (Table 1). This response is consistent with the established role of Zn as a cofactor for hydrolytic and respiratory enzymes, including α-amylase and dehydrogenases, required for reserve mobilization during germination [10,46]. During imbibition, Zn supplied through seed coating may enter metabolically active tissues through the micropyle and hilum, thereby supporting early metabolic activation [53,54]. The enhanced post-aging performance of HPH–Zn–coated seeds indicates improved protection against oxidative deterioration (Table 2). Accelerated aging promotes seed deterioration through increased respiration and excessive reactive oxygen species (ROS) production, which can damage membranes and delay germination [26,54]. Oxidative injury to membranes may also increase solute leakage and impair metabolic reactivation during imbibition [55,56]. Zn contributes to antioxidant defense through its structural role in superoxide dismutase, while peptides derived from PH may support redox regulation and membrane stabilization [21,57,58]. These responses may reflect enhanced antioxidant buffering and better preservation of membrane functionality during rehydration, consistent with HPH–Zn1.00 showing field emergence of 94.00% compared with 73.67% in uncoated seeds, together with markedly enhanced seedling Zn accumulation and growth (Table 3). Importantly, the formulation effect was supported statistically by planned contrast analysis under iso-Zn conditions, which detected significant differences between HPH–Zn0.25 and ZnSO₄–0.25 across multiple germination-related, post-aging, growth, and Zn accumulation traits (Table S2), despite conservative Tukey separation for some endpoints [59]. A previous study has shown that slow-release seed coatings can reduce local toxicity and improve seedling establishment relative to soluble coatings at comparable micronutrient doses [60]. A significant linear response was observed across the HPH–Zn series, with no significant quadratic component, indicating a proportional formulation effect rather than a simple Zn concentration effect. HPH–Zn1.00 maximized emergence and Zn uptake, whereas HPH–Zn0.50 still provided substantial gains in germination, post-aging performance, and early growth relative to ZnSO₄ and polymer-only treatments, indicating a practical performance–input trade-off.

PCA integrating laboratory germination, accelerated aging traits, field emergence, seedling growth, and Zn content indicated that the superior performance of HPH–Zn–coated seeds was driven by formulation-dependent effects rather than Zn dose alone (Figure 4a). The separation of treatments along PC1, together with the progressive shift from HPH–Zn0.25 to HPH–Zn1.00, indicated coordinated improvement in early establishment. Traits related to germination, seedling growth, field emergence, and seedling Zn content were positively associated with PC1, whereas MGT and post-aging MGT were negatively associated, linking faster germination with better overall performance (Figure 4b). The close clustering of uncoated, polymer-only, and ZnSO₄ treatments suggests a limited system-level effect of low-dose inorganic Zn, consistent with previous studies reporting narrower effective dose windows and weaker physiological integration for conventional Zn coatings [7,9,16]. These results suggest that waste-derived protein hydrolysate–Zn complexes may function as more than passive Zn carriers, potentially integrating micronutrient delivery with early physiological regulation at the seed-soil interface [2,14,61,62]. This finding is consistent with the broader concept that bio-based seed coatings can improve seedling establishment and stress tolerance beyond nutrient supply alone [17,22,26]. HPH–Zn appears to be a promising circular bioeconomy input for horticultural seed technology, and future work should examine its performance under abiotic stress conditions, field-scale production settings, and longer-term soil–rhizosphere interactions.

5. Conclusions

The results show that HPH–Zn can function as a formulation-based seed coating, improving germination, post-aging performance, and early seedling establishment. Under laboratory conditions, HPH–Zn increased germination from 86.00% in uncoated seeds to 96.89% at HPH–Zn1.00, together with a higher GI and lower MGT. Post-aging germination was also maintained at 81.89–88.92% in HPH–Zn treatments, compared with 76.67% in uncoated seeds, indicating greater tolerance to oxidative deterioration. Under greenhouse conditions, field emergence increased from 73.67% to 94.00% at HPH–Zn1.00, while HPH–Zn0.50 still achieved 87.50%, indicating substantial gains at lower Zn input. Although the highest Zn rate maximized emergence and Zn accumulation, intermediate rates still provided strong benefits, highlighting a practical performance-input trade-off. Finally, these findings support the rationale of the proposed coating approach, in which peptide-based Zn complexation is used to improve early seed performance while allowing formulation effects to be distinguished from Zn dose effects. This protocol therefore provides a useful framework for developing waste-derived seed-coating systems for horticultural crops and for evaluating their performance under both controlled and nursery-like conditions.