Next Article in Journal
Plant Biotechnology: Applications in In Vitro Plant Conservation and Micropropagation
Previous Article in Journal
Investigation of the Impact of Soil Physicochemical Properties and Microbial Communities on the Successful Cultivation of Morchella in Greenhouses
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 11
Issue 4
10.3390/horticulturae11040357
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Effects of Protein Hydrolysate Derived from Hempseed By-Products on Growth, Mineral Contents, and Quality of Greenhouse Grown Red Oak Lettuce

by
Bhornchai Harakotr
1,*,
Thamonwan Trisiri
1,
Lalita Charoensup
1,
Ornprapa Thepsilvisut
1,
Panumart Rithichai
1,
Patcharaporn Suwor
2 and
Yaowapha Jirakiattikul
1
1
Department of Agricultural Technology, Faculty of Science and Technology, Thammasat University, Pathum Thani 12120, Thailand
2
Office of Administrative Interdisciplinary Program on Agricultural Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(4), 357; https://doi.org/10.3390/horticulturae11040357
Submission received: 23 February 2025 / Revised: 16 March 2025 / Accepted: 24 March 2025 / Published: 26 March 2025
(This article belongs to the Section Vegetable Production Systems)
Browse Figures
Versions Notes

Abstract

:
The use of biostimulants derived from protein hydrolysates (PH) is now recognized as an effective strategy to promote sustainable vegetable production. This study aimed to evaluate the effects of the foliar application of PH derived from hempseed press cakes on Red Oak lettuce cultivated under greenhouse conditions. Foliar applications of PH at concentrations ranging from 0% to 7.5% (w/v) were compared with water (control) and a commercial product (Germinate®), applied weekly until 35 days after transplanting (DAT). Growth parameters, including plant height, plant width, leaf number, and leaf length and width, were recorded at 14 DAT and subsequently recorded weekly until harvest. Moreover, the fresh and dry weight, chlorophylls, mineral contents, antioxidants, and their activities in the harvested plants were determined. The results showed the positive effects of the hempseed press cake-derived PH on growth, yield, mineral contents, antioxidants, and antioxidant activities. In particular, the foliar application of PH at a concentration of 7.5% (w/v) is recommended to improve the yield and nutritional values in Red Oak lettuce. This study reports the first detailed research on the use of PH derived from hempseed press cakes for lettuce and may offer a sustainable alternative for vegetable production.

Graphical Abstract

1. Introduction

The application of chemical fertilizer plays a significant role in enhancing plant production. However, excessive use to achieve accelerated plant growth and increased yields may result in negative impacts on soil health and soil-based ecosystem functions [1]. As a result, the growing awareness of environmental sustainability and the importance of good agricultural practices have led to an increased interest in biostimulants as an alternative to chemical fertilizers [2]. A biostimulant refers to any substance or microorganism that, when applied to plants, enhances nutrient uptake or efficiency, improves tolerance to abiotic stress, and improves crop quality and yield, independent of its nutrient composition [3]. The main types of biostimulants used in agricultural purposes are humic and fulvic acids, protein hydrolysates, seaweed extracts, chitosan, silicon, growth promoting fungi and bacteria [3]. Among these, the application of protein hydrolysates (PH) has become increasingly widespread, enhancing plant growth and quality through an innovative and eco-friendly approach [4,5]. Protein hydrolysates are derived from the by-products of the agri-food industry, including meat, poultry, fish, seafood, and dairy processing, as well as plant-based sources like soy, legumes, and pulses [6]. They comprise a mixture of amino acids and peptides of variable length, along with soluble carbohydrates, phenols, mineral elements, and other compounds [2]. Protein hydrolysates have been associated with stimulating the production of the metabolites involved in growth processes and activating hormone-like activities, which subsequently influence plant growth and development [7]. Several studies have shown that PH improve the quality and nutraceutical properties of plants. Its effects on plant growth, quality, and bioactive compound contents have been documented across various plant species, including spinach (Spinacia oleracea L.) [8], tomato (Solanum lycopersicum L.) [9], basil (Ocimum basilicum L.) [10], and apricot (Prunus armeniaca L.) [11]. Considering the benefits of PH in plant cultivation, further research on its effects is necessary. This study, therefore, focused on PH derived from hempseed press cakes, obtained from by-products of the hempseed oil industry through an enzymatic hydrolysis process that is both environmentally and economically friendly. Hempseed press cake is regularly utilized as animal feed [12]; however, it can also serve an alternative agricultural purpose as a PH for crop cultivation. House et al. [13] noted that hempseed press cake contains all essential amino acids, has a high protein content ranging from 35% to 50%, and is easily digestible. With these properties, it is worth exploring the impact of PH derived from hempseed press cakes on vegetable growth and quality, particularly in lettuce.
Lettuce (Lactuca sativa L.) is widely consumed as the leafy vegetable rich in fiber, folate, iron, vitamin C, and bioactive compounds such as flavonoids, phenolic acids, carotenoids, tocopherols, and sesquiterpene lactones [14]. Since lettuce is a highly demanded leafy green used in salads throughout the year, ongoing research focuses on increasing yields through sustainable agricultural practices and improving product quality to support healthier living. The effects of PH derived from various sources on lettuce growth, yield, and nutritional quality have been studied and documented [15,16,17]. For example, Yaseen and Hajos [15] reported that willow (Salix babylonica L.) bark extract and its combination with Bistep (a type of humic acid) were the most effective biostimulants in influencing macronutrient content and ion ratios. Al-Karakia and Othman [16] observed that applying the amino acid-based biostimulant PerfectoseTM (liquid) enhanced the leaf number, fresh and dry weight, nutrient contents of N, P, K, and Mg in leaves, phenol content, flavonoid content, and antioxidant activity. Solano Porras et al. [17] also discovered that the solid-state fermentation of green waste (wood chips and grass residues) inoculated with Trichoderma harzianum, both with and without L-tryptophan under irrigation, positively influenced lettuce plant height, leaf number, chlorophyll a, carotenoids, total pigments, and antioxidant activity. However, the beneficial effects observed in these studies vary depending on the type of biostimulants, plant species, environmental conditions, and the dosage and timing of application.
We hypothesize that PH derived from hempseed press cakes enhance nutrient uptake and polyphenol production, thereby improving growth and antioxidant activity in lettuce. Therefore, the objective of this research study was to evaluate the effects of PH derived from hempseed press cakes on the growth, mineral content, and quality of Red Oak lettuce cultivated under greenhouse conditions. The findings of this study will provide the potential application of PH derived from hempseed press cakes to a variety of plant species, including vegetables, fruits, flowers, and ornamental plants. This approach not only promotes plant growth, yield, and quality but also contributes to efficient industrial waste management while supporting environmental sustainability.

2. Materials and Methods

2.1. Preparation of Tested Protein Hydrolysate

The optimized protocols for the PH derived from hempseed press cakes were followed by Feyzi et al. [18], with slight modifications. RPF3, a popular genotype developed by the Highland Research and Development Institute (Public Organization), Thailand, was purchased from a group of licensed hemp farmers in Tak province, Thailand. In the first stage, the hempseed press cakes were milled into powder using a variable speed rotor mill (Pulverisette 14, Fritsch, Idar-Oberstein, Germany) and sieved through a 60-mesh sieve to produce a uniform sample. The sample was defatted in a mixture of petroleum ether with a ratio of 1:4 (w/v) and stirred for 3 h. After solvent defatting, samples were evaporated using a rotary evaporator (Rotavapor R-300, Buchi, Flawil, Switzerland). Defatted flour was dispersed in water (1:7, w/v), and the pH was adjusted to 9.5 with 1 M NaOH under constant stirring at 200 rpm. Subsequently, hydrolysis was performed by applying 2.0% protease (Protease P1000, Siam Victory, Bangkok, Thailand) for 3 h at 200 rpm and 60 °C, with the pH adjusted to 9.5 during the process. Heating at 90 °C for 2 min results in the inactivation of the enzymes. After cooling, the hydrolysis solution was centrifuged at 3000× g for 20 min to achieve phase separation, following which the supernatants were lyophilized. The PH powder was kept in an aluminum foil bag at −20 °C until it was analyzed and used.
The yield of PH was expressed as the weight of the lyophilized products, given as a percentage (w/w) relative to the weight of the defatted powder used to produce the hydrolysates. Amino acid quantification was determined by the Prominence Amino Acid Analysis System, L463 (Table S1).

2.2. Growing Conditions and Plant Materials

The study was set up in the greenhouse, which was covered with multifunctional film (MultiTech, MTEC, NSTDA, Pathum Thani, Thailand), at the Agricultural Technology Farming Center greenhouse, Faculty of Science and Technology, Thammasat University, Thailand (+14.07450, +0.6094167, and 7.3 masl), between January and March 2024. SHT31 sensors (Tony Space, Bangkok, Thailand) were used to monitor air temperature and relative humidity in the system. Light intensity was measured using custom-designed phototransistor sensors created at the Thai Microelectronics Center (TMEC, Chachoengsao, Thailand) [19]. The microclimatic conditions during the study included an average maximum and minimum temperature of 38.34 °C and 32.91 °C, respectively, an average relative humidity of 53.88%, and a light intensity of 529.25 µmol/m2/s PAR.
The seedlings of Red Oak lettuce cv. “Opalix” were obtained from a standardized nursery and used in this study. Each seedling was transplanted into a white HDPE plastic growing bag (5″ × 11″) filled with commercial growing media (Lop Buri, Thailand), consisting of soil, composted rain tree leaves, filtrate cake, chicken manure, coconut coir dust, chopped coconut husk, and rice husk ash in a ratio of 4:1:2:1.5:0.4:0.3:0.3:4 by volume. The chemical properties of growing media were as follows: pH 6.39, EC 3.00 dS/m, 35.13% organic matter, 1.31% total N, 2.27% total P, 1.32% total K, 1.59% total Ca, 0.53% total Mg, and a C/N ratio of 15.58 [19]. There were no nutrients supplied throughout the growing period. Cultivation was carried out at a planting density of 16 plants/m2 (0.25 m between rows and plants). The total number of lettuce plants made up each treatment and replicate were 10 plants. All plants were irrigated twice daily at 8 a.m. and 4 p.m. using a drip irrigation system controlled by a timer.

2.3. Treatment Description and Experimental Design

The formulations of active substances for the experimental trials were prepared following Michalak et al. [20] and Chrysargyris et al. [21], with some modifications (Table S2). Consequently, a preliminary test was conducted to determine the possible phytotoxicity effects of the PH on lettuce plants after application via spraying. Lettuce seedlings at a stage of 4–5 true leaves were transplanted to non-circulating hydroponic systems with the Resh Tropical Dry Summer solution and grown for 14 days. Five concentrations of the PH, ranging from 2.5 to 10.0% (w/v), were evaluated. Each concentration was tested in three replicates, with a total of 10 seedlings per replication in each treatment. Plants were monitored for phytotoxicity (marked spots) at 2-day intervals for a period of 14 days. Based on the results of the preliminary experiment, the concertation of 2.5 to 7.5% (v/v), which showed no phytotoxicity effects on the plants, were selected for further evaluation.
For the main experiments, plants were arranged based on a complete randomized block design (CRD) with three replications (total of 10 plants) per the following treatments. Six different treatments were applied as follows: (1) only water used (control), (2) commercial product, (3) PH0: active substance without PH, (4) PH2.5: active substance with PH 2.5% (w/v), (5) PH5.0: active substance with PH 5.0% (w/v), and (6) PH7.5: active substance with PH 7.5% (w/v). A commercial product (Germinate®, Giffarine Co., Ltd., Bangkok, Thailand) containing 835 ppm of free amino acids was used as a positive control. The plants were allowed to grow for 14 DAT before the application of the experimental trials to ensure proper adaptation to the pots. Spraying at a dose of 25 to 50 mL per pot was performed at weekly intervals until 35 DAT. All treatments were sprayed onto the plant’s foliage using a hand sprayer in the evening.

2.4. Sampling and Growth Measurements

Five selected plants per experimental unit were measured. The growth parameters, including plant height, plant width, leaf number, and leaf length and width, were recorded at 14 DAT and then repeated weekly until 35 DAT. At the end of the experiment, five uniform plants were separated into aboveground and root parts, and then they were weighed for fresh weight measurement. Consequently, both parts’ dry weights were measured after oven-drying at 60 °C until weights were constant. The dried above-ground samples were further processed using a variable speed rotor mill for chemical analysis.

2.5. Plant Mineral Contents Analysis

The oven-dried lettuce shoots were sieved through an 8-mesh sieve and then homogenized for nutrient analysis. Approximately 0.1 g of the ground sample was digested with 15 mL of HNO3 and HClO4 (6:1, v/v), then diluted with distilled water to a final volume of 50 mL. The samples were kept in plastic tubes at room temperature. Kjeldahl’s method was used to analyze nitrogen (N) content [22]. The Vanadomolybdate method was used for the determination of phosphorus (P) content [23]. The potassium (K) and sodium (Na) contents were analyzed using a flame photometer, while the calcium (Ca) and magnesium (Mg) contents were analyzed using an atomic absorption spectrometer. The combustion method was used to determine sulfur (S) content [24]. All analysis was performed at the Soil Science Lab, Department of Plant and Soil Sciences, Chiang Mai University, Thailand.

2.6. Determination of Chlorophyll Contents

The pigment contents were analyzed following the protocol of Nagata and Yamashita [25] and Lichtenthaler [26] with modifications. Approximately 0.2 g of the ground sample was extracted with 1 mL of 100% acetone for 24 h at 20 °C. After extraction, the samples were diluted and the absorbance was measured in a spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan) at 646.8 and 663.2 nm. Subsequently, these compounds’ contents were calculated using the following equations.
Chlorophyll a (µg/mL) = 12.25 × A663.2 − 2.79 × A646.8
Chlorophyll b (µg/mL) = 21.50 × A646.8 − 5.10 × A663.2
Total chlorophyll = Chlorophyll a + b
Finally, the calculated value was multiplied by the total volume (10 mL) and then divided by the total dry weight (0.2 g), which was expressed as µg/g DW.

2.7. Sample Extraction and Bioactive Compound Analysis

2.7.1. Sample Extraction

The method followed from Jirakiattikul et al. [27], with some modifications. A total of 5 g of the ground sample was extracted three times using 95% ethanol in a 1:3 ratio (w/v) over 72 h and then filtered. The pooled extracts were dried in a hot air oven for 72 h at 50 °C. The resulting dried extracts were stored at −20 °C for future analysis.

2.7.2. Determination of Total Phenolic Content, Total Flavonoid Content, and Antioxidant Activities

The total phenolic content (TPC) was analyzed using the Folin–Ciocalteu colorimetric method described in Folin and Ciocalteu [28], whereas the total flavonoid content (TFC) was investigated using a method from Kubola et al. [29]. A microplate reader (Power Wave XS, Biotek, CA, USA) was used to analyze the flavonoid and phenolic contents at 510 and 765 nm absorbances, respectively. TPC and TFC were expressed on a dry weight basis as milligram gallic acid equivalent per gram of dry weight (mg GAE/g DW) and milligram quercetin equivalent per gram of dry weight (mg QE/g DW), respectively.
The two most common radical scavenging assays were conducted using 2,2′-azino bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. The antioxidant activity was analyzed using an ABTS radical scavenging activity assay followed from Re et al. [30], whereas DPPH radical scavenging assay followed from Brand-Williams et al. [31]. Absorbance was also measured in a microplate reader at 520 and 734 nm for DPPH and ABTS radical scavenging activity assays, respectively. Trolox was used as the reference compound. The results are expressed in µmol of Trolox equivalents per gram of dry weight (mg TE/g DW).

2.7.3. Determination of Phenolic Acids and Flavonoids

The phenolic acid and flavonoid compositions were analyzed following the protocol of Mizzi et al. [32] with modifications. Briefly, the reversed phase HPLC analyses of both compositions were performed using a Shimadzu system (Shimadzu Co., Ltd., Tokyo, Japan) equipped with a binary pump (mod. LC-20AC pump) and a diode array detector (mod. SPD-M20A). The HPLC separation was performed by an Inert-Sustain® C18 column (250 mm × 4.6 mm, 5 µm; GL Sciences Inc., Tokyo, Japan). The operating conditions were as follows: flow rate of 0.5 mL/min, column temperature of 5 °C, injection volume of 20 µL, and a detection wavelength of 350–600 nm. The mobile phase consisted of a combination of A (acetonitrile) and B (orthophosphoric, pH 2). The linear gradient was from 95% to 80% B (v/v) at 15 min, to 70% B at 20 min, to 65% B at 30 min, to 60% B at 35 min, to 50% B at 40 min, to 30% B at 52 min, and to 95% B at 60 min. It was then returned to the initial condition in 10 min. The detection wavelengths for hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids were 280, 320, and 370 nm, respectively. These compounds in the extracted samples were identified based on the retention time and spectrum of their respective standards. The results for phenolics and flavonoids were expressed as milligrams per 100 g of dry weight (mg/100 g of DW).

2.8. Statistical Analysis

Statistical analysis was performed using Statistix program (ver. 10.0, Analytical Software, Tallahassee, FL, USA). An analysis of variance (ANOVA) was performed to determine significant differences between the groups. Assumptions of ANOVA were checked before, using the Shapiro–Wilk test (normality) and Levene’s test (homogeneity of variances). If the normality assumption was violated, the Kruskal–Wallis test was applied. The results were considered as statistically significant at p ≤ 0.05. Pairwise comparisons were performed using Duncan’s Multiple Range Test (DMRT) and Dunn’s test. Principal component analysis (PCA) was carried out on mineral content, pigments, bioactive compounds, and their activities that were most effectual in distinguishing among studied variables by employing JMP® Statistical Software (ver. Free trial, JMP Statistical Discovery LLC, Cary, NC, USA). The graphs were then plotted by GraphPad® [33].

3. Results

3.1. Growth Performance

The visual response of Red Oak lettuce to the active substance at varying PH concentrations is illustrated in Figure 1. Between the 14th and 35th DAT, lettuce showed a significant difference in the number of leaves per plant on the 28th DAT, followed by a gradual increase. The highest leaf count was observed at PH5.0 and PH7.5 on the 35th DAT (Figure 2a). The canopy width showed a steady increase from the 14th to the 35th DAT, with no significant difference on the 28th DAT. At harvest, the plants treated with PH 7.5 exhibited the widest canopy, whereas the smallest canopy widths were observed in treatments with water, the commercial product, and PH0 (Figure 2b). The plant height gradually increased throughout the experimental period. The greatest plant height was observed at PH7.5 on both the 28th and 35th DAT (Figure 2c). Leaf width showed a highly significant difference on the 35th DAT, with the greatest width observed at PH7.5, which was significantly higher than that of the other treatments (Figure 2d). The leaf length exhibited significant variation throughout the experimental period, with particularly notable differences on both the 28th and 35th DAT. The greatest leaf length was recorded for PH7.5 at the harvest, while the control group had the shortest length (Figure 2e). Notably, the highest values for these parameters were observed when the PH was consistently applied at 7.5% (w/v) (PH7.5).
Regarding the harvest, the results indicated that the foliar application of PHs signifi-cantly influenced shoot and root weights, except for root fresh weight, which was not significantly affected (Table 1). The highest values for shoot fresh weight, shoot dry weight, and root dry weight were observed with PH7.5, while the application of water (control) resulted in the significantly lowest shoot fresh weight.

3.2. Mineral Nutrient Contents

The foliar application of PH highly significantly influenced the macronutrient and sodium contents in the lettuce leaves, except for Mg (Table 2). PH5.0 resulted in the highest levels of N, P, and S. The highest Mg content was observed at PH2.5, though it was not significantly different from the commercial product, PH5.0, and PH7.5. The highest K levels were found at PH7.5, but they were not significantly different from those in the other treatments, except for PH0. The commercial product resulted in the highest Ca content, while the control treatment exhibited the highest Na content.

3.3. Chlorophyll Contents

The foliar application of PH significantly affected the photosynthetic pigments (Table 3). PH5.0 had the highest content of chlorophyll a, while PH0 and water had the lowest. Water application led to the highest chlorophyll b content, which was significantly higher than that of the other treatments. Furthermore, PH5.0 exhibited the highest total chlorophyll content, showing a significant difference from the other treatments.

3.4. Bioactive Compounds and Their Activities

The bioactive compounds and their activities differed significantly under various foliar applications of PH (Table 4). The highest TPC and DPPH antioxidant activities were observed with the foliar application of PH7.5, while the highest TFC and ABTS antioxidant activities were observed with the commercial product and PH2.5, respectively. Interestingly, the lowest values for these parameters were observed with water application (control), except that the commercial product resulted in the lowest level of DPPH antioxidant activity.
The contents of phenolic acids and flavonoids varied significantly under different foliar applications of PH (Table 5). The application of PH7.5 resulted in the highest levels of chlorogenic acid, caffeic acid, ferulic acid, cinnamic acid, and quercetin. Similarly, the highest levels of p-hydroxybenzoic acid, caffeic acid, p-coumaric acid, cinnamic acid, and rutin were observed with PH5.0. In contrast, the application of water and PH0 led to low levels of phenolic acids. Additionally, low flavonoid content was noted in both the control and commercial products.

3.5. Principal Component Analysis

The principal component analysis (PCA) of growth, macronutrients, antioxidants, and antioxidant activities is presented in Figure 3. The first two principal components (PCs) are related, with eigen values greater than 1, and explained 72.8% of the total variance, with PC1 and PC2 accounting for 51.4% and 21.4%, respectively. The biplot revealed a positive correlation between PC1 and all the characteristics studied, except for the Ca, Na, and chlb contents that exhibited a negative correlation with this principal component. Moreover, PC2 was positively correlated with SDW, macronutrients (except Mg and S), Na, chlorophylls (except chlb), and TFC, while it was negatively correlated with SFW, TPC, phenolic acids (except, FA and p-CA), flavonoids, and both antioxidant activities. All the foliar applications with PH derived from hempseed by-products were positioned on the positive side of PC1. Notably, PH5.0 and PH7.5 were located on the higher and right quadrant of the PCA score plot as it delivered Red Oak lettuce of premium quality with high yield, antioxidants, and their activity. Moreover, high levels of phenolic acids (except FA and p-CA), flavonoids, and both antioxidant activities were closed in PH7.5. The upper left quadrant included water (control) and the commercial product, which all exhibited high Ca and Na contents, respectively. Finally, the lower left quadrant comprised the application of active substance without a PH (PH0), which was characterized by low levels of all the studied traits, except Chlb.

4. Discussion

Nowadays, plant biostimulants, particularly protein hydrolysates (PH), are emerging as eco-friendly tools to enhance vegetable production and quality under favorable or unfavorable growth conditions [34]. Our findings show that the new biostimulant, PH derived from hempseed by-products, stimulated lettuce growth and had a positive effect on production. However, the results reveal variations in growth performance and biomass depending on the applied concentrations of PH (Figure 1 and Figure 2, and Table 1). The application of the PH derived from hempseed press cake, especially at a concentration of 7.5% (w/v), significantly improved the growth and yield parameters of Red Oak lettuce. These improvements included an increase in the number of leaves, canopy width, plant height, leaf width and length, shoot and root fresh weight, and shoot dry weight. The positive effects of PH have been attributed to their composition of essential amino acids and soluble peptides, which can act as signaling molecules for polyphenol metabolites involved in plant growth processes and trigger activities similar to those induced by auxin and gibberellin hormones [7]. Ciriello et al. [2] explained that the essential amino acids serve as crucial precursors to indole-3-acetic acid (IAA), likely contributing to the enhanced growth and development of shoots and roots. These processes enhance plant metabolism and protein synthesis, both of which are essential for cell development, thereby improving crop yield and quality [35,36]. Similar findings have been reported in the previous studies using various types of PH on lettuce [16,17,37,38], basil [10], and primrose (Primula vulgaris) [39].
High chlorophyll content, functioning as a key photosynthetic pigment, could also be responsible for promoting the growth of Red Oak lettuce. It is likely that the higher the contents of chlorophyll a and total chlorophyll following the application of PH5.0 and PH7.5, the better the growth parameters (Table 3). Ertani et al. [40] revealed that the application of alfalfa-derived PH enhanced chlorophyll production and the upregulation of genes encoding components of the photosynthetic electron transfer chain (ferredoxin-2, the light-harvesting complex protein LHCA5), as well as Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), in tomatoes, leading to the synthesis of sugars crucial for plant growth and development. In addition, a direct relationship between photosynthetic pigment concentration and production was suggested, as higher pigment concentrations support the synthesis of carbohydrates essential for plant productivity [41]. The increase in chlorophyll content and the energy supplied for cellular metabolism through PH application may enhance N absorption by the plant thus promoting improved plant growth [42]. Colla et al. [43] reported that the PH derived from legumes positively influenced tomato growth parameters by promoting nitrogen uptake. They also found that the increased leaf nitrogen content may enhance photosynthesis and improve the translocation of photosynthates to the sinks, contributing to the higher biomass observed in PH-treated plants. Our results confirmed that both PH5.0- and PH7.5-treated plants, which had greater growth and photosynthetic pigments, had a high N content (Table 2 and Table 3). The positive effects of PH applications on leaf nitrogen content have also been observed on several vegetable crops such as lettuce, radish, and red pepper [44,45].
In this study, high mineral nutrition contents were observed in the PH-treated plants, except for Ca, which was higher in lettuce treated with the commercial product (Table 2). This indicated that the PH derived from a hempseed press cake enhanced the macronutrient absorption of Red Oak lettuce. Ertani et al. [40,46] noted that an alfalfa-based PH improved macronutrient uptake in tomatoes by stimulating the expression of genes encoding macronutrient transporters in cell membranes. This finding is consistent with that of Osman et al. [47], who reported that the foliar application of PH enhanced the uptake of N, P, and K by Pisum sativum. The foliar treatment with PH derived from legumes also showed higher K and Mg in tomatoes compared to the non-treated plants [35,48,49]. The positive correlation between PH and plant mineral nutrition can be partly attributed to the chelating ability of key amino acids they contain [50]. Amino acids play a vital role in agriculture by acting as chelating agents for metal ions and microelements. When chelated with amino acids, these form small, electrically neutral molecules that enhance their transport and absorption in plants [51]. These results indicate that the PH enhanced nutrient absorption in crops, thereby increasing crop productivity and suggesting its potential usefulness in sustainable crop production systems by reducing the need for inorganic fertilizers.
Lettuce is well known to be rich in nutrients and contain bioactive compounds that have been linked to health benefits [13]. In the present study, we found that PH-treated plants had greater antioxidant levels and activities compared to the non-PH-treated plants (Table 4). Additionally, the PH-treated plants exhibited higher phenolic acid and flavonoid compositions (Table 5), except for p-CA and CA contents, which showed no significant difference compared to the commercial products. Notably, foliar-applied PH7.5 resulted in significantly higher CGA, the predominant phenolic acid in lettuce, with levels approximately 3.77 and 7.85 times greater than those in water-treated and PH0-treated plants, respectively. These findings suggest that this protein hydrolysate could serve as a sustainable farming practice for producing high-quality lettuce. Several studies have reported the effect of PH on the content of bioactive compounds and the antioxidant activity in plants. Caruso et al. [52] found that the PH derived from legumes significantly enhanced antioxidant compounds and activity in perennial wall rocket compared to the non-treated control. Similarly, Zhou et al. [53] reported that the foliar application of a pig blood-derived PH notably increased phenolic contents and antioxidant properties in lettuce without reducing yield. Ertani et al. [54] noted enhanced ascorbate, CGA, p-CA, and capsaicin concentrations, as well as antioxidant activity, of Capsicum chinensis L. in greenhouse conditions. Vasantharaja et al. [55] reported that applying a 3% Sargassum swartzii extract enhanced the phenol and antioxidant activity of Vigna unguiculata. The accumulation of antioxidants, including phenols and flavonoids, has been linked to the biostimulant effects of PHs on the modification of primary and secondary plant metabolism [56]. Kulkarni et al. [57] reported the positive effect of biostimulant application in increasing spinach content in phenylalanine ammonia lyase. This enzyme plays a crucial role in the biosynthesis of phenolic acids [58]. Notably, plant-based PH activate secondary metabolism by upregulating the expression of some genes (PAL, CHS, F3H, DFR, F35H, and UFGT) involved with phenolic biosynthesis [53]. Our findings clearly suggest that PH application in lettuce is an effective strategy to enhance antioxidant levels, which are beneficial for various aspects of human health.
The effectiveness of PCA in elucidating the effects of biostimulants on productivity and quality of several horticultural crops has been reported [49,59,60]. In the present study, PCA is effective in plotting biomass, macronutrients, antioxidants, and antioxidant activities in relation to the active substance with different PH concentrations (Figure 3). The first two components accounted for about 72.8% of the total variation, providing a clear understanding of the structure underlying the analyzed variables. The PH derived from a hempseed press cake at a concentration of 2.5 to 7.5% (w/v) was positioned on the positive side of PC1, which accounted for 51.4% of the cumulative variance. This result confirmed that the PH derived from a hempseed press cake corresponded to all findings in lettuce, except Na, Ca, and Chlb. Moreover, both PH5.0 and PH7.5 were in the upper right quadrant of the PCA score plot, indicating high values for the studied traits. However, the PH7.5 treatment, situated in the right quadrants, was associated with increased yield, S, Mg, TPC, phenolic acids (except FA and p-CA), flavonoids, and both antioxidant activities. Therefore, the foliar application of hempseed press cake-derived PH at a concentration of 7.5% (w/v) is recommended for producing Red Oak lettuce with high yield and quality.
Future research should investigate the effects of this new biostimulant on other types of lettuce or crops than Red Oak lettuce. Similarly, the synergistic action of the PH derived from a hempseed press cake and/or microorganisms on crops should also be explored.

5. Conclusions

The results of this study showed that the use of PH derived from a hempseed press cake significantly improved the yield, mineral nutrition, antioxidants, and antioxidant activities of Red Oak lettuce grown under greenhouse conditions. These positive results were obtained when the PH was foliar applied successively at a concentration of 7.5% (w/v). Therefore, utilizing this biostimulant offers a promising and sustainable approach to achieving high crop yields while enhancing nutritional value.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11040357/s1, Table S1: Amino acid content of the protein hydrolysate derived from hempseed press cake was used in this study; Table S2: The formulations designed for foliar application containing the protein hydrolysate derived from hempseed press cake.

Author Contributions

Conceptualization, B.H.; methodology, B.H.; software, B.H.; validation, B.H.; formal analysis, B.H., T.T., L.C. and O.T.; investigation, B.H., T.T. and L.C.; resources, B.H.; data curation, B.H., T.T. and L.C; writing—original draft preparation, B.H. and Y.J.; writing—review and editing, B.H., Y.J., P.R., O.T. and P.S.; visualization, B.H., P.R. and P.S.; supervision, Y.J., P.R. and P.S.; project administration, B.H.; funding acquisition, B.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research project was supported by the Thailand Science Research and Innovation Fundamental Fund fiscal year 2024, Thammasat University (Grant no. TUFF 10/2567).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors also extend their gratitude to the Department of Agricultural Technology, Faculty of Science and Technology, Thammasat University, Thailand, for providing research facilities.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Krasilnikov, P.; Taboada, M.A.; Amanullah. Fertilizer use, soil health and agricultural sustainability. Agriculture 2022, 12, 462. [Google Scholar] [CrossRef]
  2. Ciriello, M.; Campana, E.; De Pascale, S.; Rouphael, Y. Implications of vegetal protein hydrolysates for improving nitrogen use efficiency in leafy vegetables. Horticulturae 2024, 10, 132. [Google Scholar] [CrossRef]
  3. Du Jardin, P. Plant biostimulants: Definition, concept, main categories and regulation. Sci. Hortic. 2015, 196, 3–14. [Google Scholar] [CrossRef]
  4. Colla, G.; Rouphael, Y.; Lucini, L.; Canaguier, R.; Stefanoni, W.; Fiorillo, A.; Cardarelli, M. Protein hydrolysate-based biostimulants: Origin, biological activity and application methods. In Proceedings of the II World Congress on the Use of Biostimulants in Agriculture, Florence, Italy, 16–19 November 2015; p. 1148. [Google Scholar] [CrossRef]
  5. Petropoulos, S.A. Practical applications of plant biostimulants in greenhouse vegetable crop production. Agronomy 2020, 10, 1569. [Google Scholar] [CrossRef]
  6. Czelej, M.; Garbacz, K.; Czernecki, T.; Wawrzykowski, J.; Waśko, A. Protein hydrolysates derived from animals and plants—A review of production methods and antioxidant activity. Foods 2022, 11, 1953. [Google Scholar] [CrossRef]
  7. Colla, G.; Hoagland, L.; Ruzzi, M.; Cardarelli, M.; Bonini, P.; Canaguier, R.; Rouphael, Y. Biostimulant action of protein hydrolysates: Unraveling their effects on plant physiology and microbiome. Front. Plant Sci. 2017, 8, 2202. [Google Scholar] [CrossRef]
  8. El-Nakhel, C.; Cozzolino, E.; Ottaiano, L.; Petropoulos, S.A.; Nocerino, S.; Pelosi, M.E.; Rouphael, Y.; Mori, M.; Di Mola, I. Effect of biostimulant application on plant growth, chlorophylls and hydrophilic antioxidant activity of spinach (Spinacia oleracea L.) grown under saline stress. Horticulturae 2022, 8, 971. [Google Scholar] [CrossRef]
  9. Ávila-Pozo, P.; Parrado, J.; Caballero, P.; Tejada, M. Use of a biostimulant obtained from slaughterhouse sludge in a greenhouse tomato crop. Horticulturae 2022, 8, 622. [Google Scholar] [CrossRef]
  10. Ciriello, M.; Formisano, L.; El-Nakhel, C.; Corrado, G.; Rouphael, Y. Biostimulatory action of a plant-derived protein hydrolysate on morphological traits, photosynthetic parameters, and mineral composition of two basil cultivars grown hydroponically under variable electrical conductivity. Horticulturae 2022, 8, 409. [Google Scholar] [CrossRef]
  11. Cirillo, A.; Izzo, L.; Ciervo, A.; Ledenko, I.; Cepparulo, M.; Piscitelli, A.; Di Vaio, C. Optimizing apricot yield and quality with biostimulant interventions: A comprehensive analysis. Horticulturae 2024, 10, 447. [Google Scholar] [CrossRef]
  12. Helstad, A.; Forsén, E.; Ahlström, C.; Labba, I.C.M.; Sandberg, A.S.; Rayner, M.; Purhagen, J.K. Protein extraction from cold-pressed hempseed press cake: From laboratory to pilot scale. J. Food Sci. 2022, 87, 312–325. [Google Scholar] [CrossRef] [PubMed]
  13. House, J.D.; Neufeld, J.; Leson, G. Evaluating the quality of protein from hemp seed (Cannabis sativa L.) products through the use of the protein digestibility-corrected amino acid score method. J. Agric. Food Chem. 2010, 58, 11801–11807. Available online: https://pubs.acs.org/doi/10.1021/jf102636b (accessed on 10 March 2024). [PubMed]
  14. Yang, X.; Gil, M.I.; Yang, Q.; Tomás-Barberán, F.A. Bioactive compound in lettuce: Highlighting the benefits to human health and impacts of preharvest and postharvest practices. Compr. Rev. Food. Sci. Food. Saf. 2022, 21, 4–45. [Google Scholar] [CrossRef]
  15. Yaseen, A.A.; Hajos, M.T. The effect of plant biostimulants on the macronutrient content and ion ratio of several lettuce (Lactuca sativa L.) cultivars grown in a plastic house. S. Afr. J. Bot. 2022, 147, 223–230. [Google Scholar] [CrossRef]
  16. Al-Karakia, G.N.; Othman, Y. Effect of foliar application of amino acid biostimulants on growth, macronutrient, total phenol contents and antioxidant activity of soilless grown lettuce cultivars. S. Afr. J. Bot. 2023, 154, 225–231. [Google Scholar] [CrossRef]
  17. Solano Porras, R.C.; Ghoreishi, G.; Sanchez, A.; Barrena, R.; Font, X.; Ballardo, C.; Artola, A. Solid-state fermentation of green waste for the production of biostimulants to enhance lettuce (Lactuca sativa L.) cultivation under water stress: Closing the organic waste cycle. Chemosphere 2025, 370, 143919. [Google Scholar] [CrossRef]
  18. Feyzi, S.; Varidi, M.; Zare, F.; Varidi, M.J. A comparison of chemical, structural and functional properties of fenugreek (Trigonella foenum graecum) protein isolates produced using different defatting solvents. Int. J. Biol. Macromol. 2017, 105, 27–35. [Google Scholar] [CrossRef] [PubMed]
  19. Kumsong, N.; Thepsilvisut, O.; Imorachorn, P.; Chutimanukul, P.; Pimpha, N.; Toojinda, T.; Trithaveesak, O.; Ratanaudomphisut, E.; Poyai, A.; Hruanun, C.; et al. Comparison of different temperature control systems in tropical-adapted greenhouses for green romaine lettuce production. Horticulturae 2023, 9, 1255. [Google Scholar] [CrossRef]
  20. Michalak, I.; Chojnacka, K.; Dmytryk, A.; Wilk, R.; Gramza, M.; Rój, E. Evaluation of supercritical extracts of algae as biostimulants of plant growth in field trials. Front. Plant Sci. 2016, 7, 1591. [Google Scholar] [CrossRef]
  21. Chrysargyris, A.; Charalambous, S.; Xylia, P.; Litskas, V.; Stavrinides, M.; Tzortzakis, N. Assessing the biostimulant effects of a novel plant-based formulation on tomato crop. Sustainability 2020, 12, 8432. [Google Scholar] [CrossRef]
  22. Bremner, J.M.; Mulvaney, C.S. Nitrogen-Total. In Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties; Page, A.L., Miller, R.H., Keeney, D.R., Eds.; American Society of Agronomy, Soil Science Society of America: Madison, WI, USA, 1982; pp. 595–624. [Google Scholar] [CrossRef]
  23. AOAC. Official Method of Analysis. In AOAC Official Methods of Analysis, 18th ed.; AOAC International: Washington, DC, USA, 2005. [Google Scholar]
  24. Kalra, Y. Handbook of Reference Methods for Plant. Analysis; CRC Press: Boca Raton, FL, USA, 1997. [Google Scholar] [CrossRef]
  25. Nagata, M.; Yamashita, I. Simple method for simultaneous determinations of chlorophyll and carotenoids in tomato fruit. Nippon Shokuhin Kogyo Gakkaish 1992, 39, 925–928. [Google Scholar] [CrossRef]
  26. Lichtenthaler, H.K.; Wellburn, A.R. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 1983, 1, 591–592. [Google Scholar] [CrossRef]
  27. Jirakiattikul, Y.; Ruangnoo, S.; Sangmukdee, K.; Chamchusri, K.; Rithichai, P. Enhancement of plumbagin production through elicitation in in vitro-regenerated shoots of Plumbago indica L. Plants 2024, 13, 1450. [Google Scholar] [CrossRef] [PubMed]
  28. Folin, O.; Ciocalteu, V. On tyrosine and tryptophane determinations in proteins. J. Biol. Chem. 1927, 73, 627–650. [Google Scholar] [CrossRef]
  29. Kubola, J.; Siriamornpun, S.; Meeso, N. Phytochemicals, vitamin C and sugar content of Thai wild fruits. Food Chem. 2011, 126, 972–981. [Google Scholar] [CrossRef]
  30. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [Google Scholar] [CrossRef]
  31. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. Leb. Wiss Technol. 1995, 28, 25–30. [Google Scholar] [CrossRef]
  32. Mizzi, L.; Chatzitzika, C.; Gatt, R.; Valdramidis, V. HPLC analysis of phenolic compounds and flavonoids with overlapping peaks. Food Technol. Biotechnol. 2020, 58, 12–19. [Google Scholar] [CrossRef]
  33. Motulsky, H.J. GraphPad Statistics Guide. 2016. Available online: http://www.graphpad.com/guides/prism/10/statistics/index.htm (accessed on 15 June 2024).
  34. Sabatino, L.; Consentino, B.B.; Rouphael, Y.; De Pasquale, C.; Iapichino, G.; D’Anna, F.; La Bella, S. Protein hydrolysates and mo-biofortification interactively modulate plant performance and quality of ‘Canasta’ lettuce grown in a protected environment. Agronomy 2021, 11, 1023. [Google Scholar] [CrossRef]
  35. Colla, G.; Cardarelli, M.; Bonini, P.; Rouphael, Y. Foliar application of protein hydrolysate, plant and seaweed extracts increase yield but differentially modulate fruit quality of greenhouse tomato. HortScience 2017, 52, 1214–1220. [Google Scholar] [CrossRef]
  36. Tadros, M.J.; Omari, H.J.; Turk, M.A. The morphological, physiological and biochemical responses of sweet corn to foliar application of amino acids biostimulants sprayed at three growth stages. Aust. J. Crop Sci. 2019, 13, 412–417. [Google Scholar] [CrossRef]
  37. El-Nakhel, C.; Cristofano, F.; Colla, G.; Pii, Y.; Lucini, L.; Rouphael, Y.A. Graminaceae-derived protein hydrolysate and its fractions provide differential growth and modulate qualitative traits of lettuce grown under non-saline and mild salinity conditions. Sci. Hortric. 2023, 319, 112130. [Google Scholar] [CrossRef]
  38. Dass, S.M.; Chai, T.-T.; Cao, H.; Ooi, A.L.; Wong, F.C. Application of enzyme-digested soy protein hydrolysate on hydroponic-planted lettuce: Effects on phytochemical contents, biochemical profiles and physical properties. Food Chem. 2021, 12, 100132. [Google Scholar] [CrossRef]
  39. Tütüncü, M. Effects of protein hydrolysate derived from anchovy by-product on plant growth of primrose and root system architecture analysis with machine learning. Horticulturae 2024, 10, 400. [Google Scholar] [CrossRef]
  40. Ertani, A.; Schiavon, M.; Nardi, S. Transcriptome-wide identification of differentially expressed genes in Solanum lycopersicon L. in response to an alfalfa-protein hydrolysate using microarrays. Front. Plant Sci. 2017, 8, 1159. [Google Scholar] [CrossRef] [PubMed]
  41. Parađiković, N.; Vinković, T.; Vrček, I.V.; Žuntr, I.; Bojić, M.; Medić-Šaric, M. Effect of natural biostimulant on yield and nutritional quality: An example of sweet yellow pepper (Capsicum annum L.) plants. J. Sci. Food Agric. 2011, 91, 2146–2152. [Google Scholar] [CrossRef]
  42. Francesca, S.; Cirillo, V.; Raimondi, G.; Maggio, A.; Barone, A.; Rigano, M.M. A novel protein hydrolysate-based biostimulant improves tomato performances under drought stress. Plants 2021, 10, 783. [Google Scholar] [CrossRef]
  43. Colla, G.; Rouphael, Y.; Canaguier, R.; Svecova, E.; Cardarelli, M. Biostimulant action of a plant-derived protein hydrolyzates produced through enzymatic hydrolysis. Front. Plant Sci. 2014, 5, 448. [Google Scholar] [CrossRef]
  44. Liu, X.Q.; Lee, K.S. Effect of mixed amino acids on crop growth. In Agricultural Science; Aflakpui, G., Ed.; InTech Europe Publisher: Rijeka, Croatia, 2021; pp. 119–158. [Google Scholar] [CrossRef]
  45. Tsouvaltzis, P.; Koukounaras, A.; Siomos, A.S. Application of amino acids improves lettuce crop uniformity and inhibits nitrate accumulation induced by the supplemental inorganic nitrogen fertilization. Int. J. Agric. Biol. 2014, 16, 951–955. [Google Scholar]
  46. Ertani, A.; Schiavon, M.; Trentin, A.; Malagoli, M.; Nardi, S. Effect of an alfalfa plant-derived biostimulant on sulfur nutrition in tomato plants. In Molecular Physiology and Ecophysiology of Sulfur, Proceedings of the 9th International Workshop on Sulfur Metabolism in Plants, Freiburg-Munzigen, Germany, 14–17 April 2014; Springer: Berlin/Heidelberg, Germany, 2015; pp. 215–220. [Google Scholar] [CrossRef]
  47. Osman, A.; Merwad, A.-R.M.; Mohamed, A.H.; Sitohy, M. Foliar spray with pepsin-and papain-whey protein hydrolysates promotes the productivity of pea plants cultivated in clay loam soil. Molecules 2021, 26, 2805. [Google Scholar] [CrossRef]
  48. Rouphael, Y.; Colla, G.; Giordano, M.; El-Nakhel, C.; Kyriacon, M.C.; De Pasacale, S. Foliar applications of a legume-derived protein-hydrolysate elicit dose-dependent increases of growth, leaf mineral composition, yield and fruit quality in two greenhouse tomato cultivars. Sci. Hortic. 2017, 226, 353–360. [Google Scholar] [CrossRef]
  49. Rouphael, Y.; Giordano, M.; Cardarelli, M.; Cozzolino, E.; Mori, M.; Kyriacou, M.C.; Bonini, P.; Colla, G. Plant- and seaweed-based extracts increase yield but differentially modulate nutritional quality of greenhouse spinach through biostimulant action. Agronomy 2018, 8, 126. [Google Scholar] [CrossRef]
  50. Popko, M.; Michalak, I.; Wilk, R.; Gramza, M.; Chojnacka, K.; Górecki, H. Effect of the new plant growth biostimulants based on amino acids on yield and grain quality of winter wheat. Molecules 2018, 23, 470. [Google Scholar] [CrossRef] [PubMed]
  51. Sun, W.; Shahrajabian, M.H.; Kuang, Y.; Wang, N. Amino acids biostimulants and protein hydrolysates in agricultural sciences. Plants 2024, 13, 210. [Google Scholar] [CrossRef]
  52. Caruso, G.; De Pascale, S.; Cozzolino, E.; Giordano, M.; El-Nakhel, C.; Cuciniello, A.; Cenvinzo, V.; Colla, G.; Rouphael, Y. Protein hydrolysate or plant extract-based biostimulants enhanced yield and quality performances of greenhouse perennial wall rocket grown in different seasons. Plants 2019, 8, 208. [Google Scholar] [CrossRef]
  53. Zhou, W.; Zheng, W.; Lv, H.; Wang, Q.; Liang, B.; Li, J. Foliar application of pig blood-derived protein hydrolysates improves antioxidant activities in lettuce by regulating phenolic biosynthesis without compromising yield production. Sci. Hortic. 2022, 291, 110602. [Google Scholar] [CrossRef]
  54. Ertani, A.; Pizzeghello, D.; Francioso, O.; Sambo, P.; Sanchez-Cortes, S.; Nardi, S. Capsicum chinensis L. growth and nutraceutical properties are enhanced by biostimulants in a long-term period: Chemical and metabolomic approaches. Front. Plant Sci. 2014, 5, 375. [Google Scholar] [CrossRef]
  55. Vasantharaja, R.; Abraham, L.S.; Inbakandan, D.; Thirugnanasambandam, R.; Senthilvelan, T.; Jabeen, S.K.A.; Prakash, P. Influence of seaweed extracts on growth, phytochemical contents and antioxidant capacity of cowpea (Vigna unguiculata L. Walp). Biocatal. Agric. Biotechnol. 2019, 17, 589–594. [Google Scholar] [CrossRef]
  56. Cristofano, F.; El-Nakhel, C.; Colla, G.; Cardarelli, M.; Pii, Y.; Lucini, L.; Rouphael, Y. Tracking the biostimulatory effect of fractions from a commercial plant protein hydrolysate in greenhouse-grown lettuce. Antioxidants 2023, 12, 107. [Google Scholar] [CrossRef]
  57. Kulkarni, M.G.; Rengasamy, K.R.R.; Pendota, S.C.; Gruz, J.; Plačková, L.; Novák, O.; Doležal, K.; Van Staden, J. Bioactive molecules derived from smoke and seaweed Ecklonia maxima showing phytohormone-like activity in Spinacia oleracea L. New Biotechnol. 2019, 48, 83–89. [Google Scholar] [CrossRef]
  58. Hyun, M.W.; Yun, Y.H.; Kim, J.Y.; Kim, S.H. Fungal and plant phenylalanine ammonia-lyase. Mycobiology 2011, 39, 257–265. [Google Scholar] [CrossRef] [PubMed]
  59. Di-Vaio, C.; Cirillo, A.; Cice, D.; El-Nakhel, C.; Rouphael, Y. Biostimulant application improves yield parameters and accentuates fruit color of Annurca apples. Agronomy 2021, 11, 715. [Google Scholar] [CrossRef]
  60. Distefano, M.; Steingass, C.B.; Leonardi, C.; Giuffrida, F.; Schweiggert, R.; Mauro, R.P. Effects of a plant-derived biostimulant application on quality and functional traits of greenhouse cherry tomato cultivars. Food Res. Int. 2022, 157, 111218. [Google Scholar] [CrossRef]
Horticulturae 11 00357 g001
Figure 1. The appearance of Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of a protein hydrolysate (PH). (a) water; (b) commercial product; (c) active substance without PH (PH0); (d) active substance with PH at 2.5% (w/v) (PH2.5); (e) active substance with PH at 5.0% (w/v) (PH5.0); (f) active substance with PH at 7.5% (w/v) (PH7.5).
Figure 1. The appearance of Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of a protein hydrolysate (PH). (a) water; (b) commercial product; (c) active substance without PH (PH0); (d) active substance with PH at 2.5% (w/v) (PH2.5); (e) active substance with PH at 5.0% (w/v) (PH5.0); (f) active substance with PH at 7.5% (w/v) (PH7.5).
Horticulturae 11 00357 g001
Horticulturae 11 00357 g002
Figure 2. Growth of Red Oak lettuce grown under greenhouse conditions between 14 and 35 DAT, as influenced by the foliar application of protein hydrolysates (PH) in the active substance. (a) number of leaves per plant; (b) canopy width; (c) plant height; (d) leaf width; (e) leaf length. (PH0: active substance without PH; PH2.5: active substance with PH at 2.5% (w/v); PH5.0: active substance with PH at 5.0% (w/v); PH7.5: active substance with PH at 7.5% (w/v). ns: not significant. Means with the same letters at the same time are not significantly different at p ≤ 0.05, as determined by the DMRT.
Figure 2. Growth of Red Oak lettuce grown under greenhouse conditions between 14 and 35 DAT, as influenced by the foliar application of protein hydrolysates (PH) in the active substance. (a) number of leaves per plant; (b) canopy width; (c) plant height; (d) leaf width; (e) leaf length. (PH0: active substance without PH; PH2.5: active substance with PH at 2.5% (w/v); PH5.0: active substance with PH at 5.0% (w/v); PH7.5: active substance with PH at 7.5% (w/v). ns: not significant. Means with the same letters at the same time are not significantly different at p ≤ 0.05, as determined by the DMRT.
Horticulturae 11 00357 g002
Horticulturae 11 00357 g003
Figure 3. Principal component loading plot and scores of PCA of growth, macronutrients, antioxidants, and antioxidant activities in Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of protein hydrolysate (PH) in the active substance. (PH0: active substance without PH; PH2.5: active substance with PH at 2.5% (w/v); PH5.0: active substance with PH at 5.0% (w/v); PH7.5: active substance with PH at 7.5% (w/v).
Figure 3. Principal component loading plot and scores of PCA of growth, macronutrients, antioxidants, and antioxidant activities in Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of protein hydrolysate (PH) in the active substance. (PH0: active substance without PH; PH2.5: active substance with PH at 2.5% (w/v); PH5.0: active substance with PH at 5.0% (w/v); PH7.5: active substance with PH at 7.5% (w/v).
Horticulturae 11 00357 g003
Table 1. Fresh weight and dry weight of Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of protein hydrolysate (PH) in the active substance.
Table 1. Fresh weight and dry weight of Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of protein hydrolysate (PH) in the active substance.
TreatmentsShoot (g/Plant)Root (g/Plant)
Fresh WeightDry WeightFresh WeightDry Weight
Water (control)119.53 ± 1.22 e 2/4.68 ± 0.12 c10.61 ± 0.340.21 ± 0.04 b
Commercial product125.07 ± 0.95 d5.01 ± 0.28 c10.20 ± 0.590.21 ± 0.01 b
PH0 1/124.27 ± 1.85 d5.08 ± 0.21 bc10.49 ± 0.310.21 ± 0.01 b
PH2.5132.80 ± 0.87 c4.90 ± 0.33 c10.15 ± 0.690.21 ± 0.01 b
PH5.0139.73 ± 0.31 b5.45 ± 0.19 ab10.23 ± 0.450.22 ± 0.02 b
PH7.5148.00 ± 3.89 a5.70 ± 0.22 a10.22 ± 0.240.26 ± 0.01 a
F-test****ns*
C.V. (%)1.454.524.501.37
ns: not significant; *, ** significantly difference at p ≤ 0.05 and 0.01, respectively. 1/ PH0: active substance without PH; PH2.5: active substance with PH at 2.5% (w/v); PH5.0: active substance with PH at 5.0% (w/v); PH7.5: active substance with PH at 7.5% (w/v). 2/ Means followed by the same letters in the same column are not significantly different at p ≤ 0.05 as determined by DMRT.
Table 2. Mineral nutrient contents (%) of Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of protein hydrolysate (PH) in the active substance.
Table 2. Mineral nutrient contents (%) of Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of protein hydrolysate (PH) in the active substance.
TreatmentsNPKCaMgSNa
Water (control)3.78 ± 0.02 d 2/0.77 ± 0.01 ab 3/5.63 ± 0.63 ab 3/1.35 ± 0.01 b 2/0.09 ± 0.01 c 2/0.24 ± 0.03 bc 2/0.34 ± 0.03 a 2/
Commercial product4.07 ± 0.03 b0.77 ± 0.01 ab6.27 ± 0.54 ab1.41 ± 0.02 a0.11 ± 0.01 abc0.21 ± 0.01 c0.26 ± 0.02 b
PH0 1/3.72 ± 0.03 e0.75 ± 0.01 b4.37 ± 0.71 b1.35 ± 0.02 b0.10 ± 0.02 bc0.20 ± 0.01 c0.15 ± 0.01 c
PH2.53.92 ± 0.02 c0.78 ± 0.01 ab5.99 ± 0.52 ab1.27 ± 0.02 c0.13 ± 0.00 a0.25 ± 0.01 b0.16 ± 0.03 c
PH5.04.13 ± 0.00 a0.84 ± 0.02 a6.03 ± 0.37 ab1.25 ± 0.04 c0.11 ± 0.01 abc0.32 ± 0.02 a0.23 ± 0.03 b
PH7.53.92 ± 0.03 c0.78 ± 0.01 ab6.63 ± 0.26 a1.34 ± 0.03 b0.12 ± 0.01 ab0.31 ± 0.01 b0.26 ± 0.00 b
F-test*************
C.V. (%)0.804.074.562.378.913.8813.35
*, ** significantly difference at p ≤ 0.05 and 0.01, respectively. 1/ PH0: active substance without PH; PH2.5: active substance with PH at 2.5% (w/v); PH5.0: active substance with PH at 5.0% (w/v); PH7.5: active substance with PH at 7.5% (w/v). 2/ Means followed by the same letters in the same column are not significantly different at p ≤ 0.05 as determined by DMRT. 3/ Medians followed by the same letters in the same column are not significantly different at p ≤ 0.05 as determined by Dunn’s test.
Table 3. Chlorophyll contents (µg/g DW) of Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of protein hydrolysate (PH) in the active substance.
Table 3. Chlorophyll contents (µg/g DW) of Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of protein hydrolysate (PH) in the active substance.
Treatments Chlorophyll aChlorophyll bTotal chlorophyll
Water (control)16.81 ± 0.48 c 2/37.92 ± 0.30 a54.74 ± 0.73 b
Commercial product18.02 ± 0.20 b37.19 ± 0.20 bc55.22 ± 0.22 b
PH0 1/16.87 ± 0.46 c37.32 ± 0.10 bc54.20 ± 0.40 b
PH2.517.19 ± 0.45 bc37.35 ± 0.24 bc54.55 ± 0.22 b
PH5.019.35 ± 0.70 a36.93 ± 0.23 c56.29 ± 0.89 a
PH7.517.25 ± 0.33 bc37.47 ± 0.21 b54.73 ± 0.35 b
F-test******
C.V. (%)2.650.590.97
** significantly difference at p ≤ 0.01. 1/ PH0: active substance without PH; PH2.5: active substance with PH at 2.5% (w/v); PH5.0: active substance with PH at 5.0% (w/v); PH7.5: active substance with PH at 7.5% (w/v). 2/ Means followed by the same letters in the same column are not significantly different at p ≤ 0.05 as determined by DMRT.
Table 4. Total phenolic content (TPC), total flavonoid content (TFC), and their antioxidant activities of Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of protein hydrolysate (PH) in the active substance.
Table 4. Total phenolic content (TPC), total flavonoid content (TFC), and their antioxidant activities of Red Oak lettuce grown under greenhouse conditions at 35 DAT, as influenced by the foliar application of protein hydrolysate (PH) in the active substance.
Treatments TPCTFC Antioxidant Activities
(mg TE/g DW) 3/
(mg GAE/g DW)(mg QE/g DW)DPPHABTS
Water (control)27.33 ± 1.05 e 2/77.85 ± 10.50 b 3/6.53 ± 0.08 ab 3/27.20 ± 2.40 b 3/
Commercial product30.36 ± 0.56 d166.85 ± 7.50 a6.10 ± 1.01 b29.30 ± 1.40 ab
PH0 1/29.19 ± 0.79 de87.35 ± 2.50 ab8.02 ± 1.49 ab41.40 ± 4.40 ab
PH2.537.79 ± 1.26 b98.35 ± 8.50 ab11.38 ± 0.55 ab49.90 ± 3.00 a
PH5.035.03 ± 1.40 c151.60 ± 9.50 ab17.04 ± 1.72 ab47.80 ± 2.40 ab
PH7.542.04 ± 1.87 a106.35 ± 6.50 ab18.37 ± 1.87 a45.30 ± 5.00 ab
F-test********
C.V. (%)1.442.332.381.68
** significantly difference at p ≤ 0.01. 1/ PH0: active substance without PH; PH2.5: active substance with PH at 2.5% (w/v); PH5.0: active substance with PH at 5.0% (w/v); PH7.5: active substance with PH at 7.5% (w/v). 2/ Means followed by the same letters in the same column are not significantly different at p ≤ 0.05 as determined by DMRT. 3/ Medians followed by the same letters in the same column are not significantly different at p ≤ 0.05 level as determined by Dunn’s test.
Table 5. Phenolic acids and flavonoids (mg/100 g DW) in Red Oak lettuce grown under greenhouse conditions at 35 DAT as dictated by the foliar application of protein hydrolysate (PH) in the active substance.
Table 5. Phenolic acids and flavonoids (mg/100 g DW) in Red Oak lettuce grown under greenhouse conditions at 35 DAT as dictated by the foliar application of protein hydrolysate (PH) in the active substance.
TreatmentsHydroxybenzoic AcidsHydroxycinnamic AcidsFlavonoids
CGA 2/PHBACFAFAp-CACARTQE
Water (control)41.26 ± 1.23 e 3/1.85 ± 0.13 e4.93 ± 0.13 c11.52 ± 0.46 d3.86 ± 0.35 b1.80 ± 0.18 c23.63 ± 0.73 d2.55 ± 0.24 d
Commercial product67.24 ± 9.64 d3.08 ± 1.00 d9.19 ± 0.84 b11.05 ± 0.92 d4.84 ± 0.38 a2.78 ± 0.54 ab27.18 ± 1.73 d2.77 ± 0.22 d
PH0 1/19.81 ± 0.60 f7.32 ± 0.30 c3.52 ± 0.19 c2.55 ± 0.18 e1.55 ± 0.06 c2.40 ± 0.09 b33.92 ± 0.15 c3.81 ± 0.12 b
PH2.5129.97 ± 1.96 c7.75 ± 0.75 c15.09 ± 0.59 a16.76 ± 0.12 c5.10 ± 0.07 a2.47 ± 0.04 b33.46 ± 0.93 c2.88 ± 0.01 d
PH5.0140.37 ± 3.90 b12.55 ± 0.42 a13.91 ± 3.47 a18.87 ± 0.49 b5.35 ± 0.06 a2.97 ± 0.05 a45.36 ± 0.34 a3.34 ± 0.13 c
PH7.5155.54 ± 6.31 a9.67 ± 0.49 b15.99 ± 0.29 a22.13 ± 0.55 a4.20 ± 0.56 b3.28 ±0.31 a42.37 ± 1.21 b4.35 ± 0.27 a
F-test****************
C.V. (%)5.488.4414.253.807.6810.302.915.76
** significantly difference at p ≤ 0.01. 1/ PH0: active substance without PH; PH2.5: active substance with PH at 2.5% (w/v); PH5.0: active substance with PH at 5.0% (w/v); PH7.5: active substance with PH at 7.5% (w/v). 2/ CGA, chlorogenic acid; PHBA, p-hydroxybenzoic acid; CFA, caffeic acid; FA, ferulic acid; p-CA, p-coumaric acid; CA, cinnamic acid; RT, rutin; QE, quercetin. 3/ Means followed by the same letters in the same column are not significantly different at p ≤ 0.05 as determined by DMRT.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Harakotr, B.; Trisiri, T.; Charoensup, L.; Thepsilvisut, O.; Rithichai, P.; Suwor, P.; Jirakiattikul, Y. Effects of Protein Hydrolysate Derived from Hempseed By-Products on Growth, Mineral Contents, and Quality of Greenhouse Grown Red Oak Lettuce. Horticulturae 2025, 11, 357. https://doi.org/10.3390/horticulturae11040357

AMA Style

Harakotr B, Trisiri T, Charoensup L, Thepsilvisut O, Rithichai P, Suwor P, Jirakiattikul Y. Effects of Protein Hydrolysate Derived from Hempseed By-Products on Growth, Mineral Contents, and Quality of Greenhouse Grown Red Oak Lettuce. Horticulturae. 2025; 11(4):357. https://doi.org/10.3390/horticulturae11040357

Chicago/Turabian Style

Harakotr, Bhornchai, Thamonwan Trisiri, Lalita Charoensup, Ornprapa Thepsilvisut, Panumart Rithichai, Patcharaporn Suwor, and Yaowapha Jirakiattikul. 2025. "Effects of Protein Hydrolysate Derived from Hempseed By-Products on Growth, Mineral Contents, and Quality of Greenhouse Grown Red Oak Lettuce" Horticulturae 11, no. 4: 357. https://doi.org/10.3390/horticulturae11040357

APA Style

Harakotr, B., Trisiri, T., Charoensup, L., Thepsilvisut, O., Rithichai, P., Suwor, P., & Jirakiattikul, Y. (2025). Effects of Protein Hydrolysate Derived from Hempseed By-Products on Growth, Mineral Contents, and Quality of Greenhouse Grown Red Oak Lettuce. Horticulturae, 11(4), 357. https://doi.org/10.3390/horticulturae11040357

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop