Valorization of Expired Milk into Protein Hydrolysate as a Plant Biostimulant: Characterization and Application on Hydroponically Grown Cos Lettuce

Abstract

To meet global food demand, agricultural systems must enhance crop performance, productivity, and sustainability. Biostimulants have emerged as a promising strategy, particularly in vegetable production, due to their ability to enhance plant growth and resilience. This study characterized milk-derived protein hydrolysates (MPH) produced from expired milk and evaluated their potential as biostimulants for hydroponic cos lettuce. Hydrolysis of expired milk with hydrochloric acid achieved 94.55% hydrolysis and yielded 80.77% free amino acids. MPH was applied at volumes of 0, 1, 3, and 5 mL L⁻¹ in combination with Hoagland and Arnon nutrient solution. The 1 mL L⁻¹ (MPH1) treatment significantly increased shoot and root biomass and canopy size while reducing nitrate accumulation and enhancing total flavonoid and ascorbic acid content, as well as antioxidant capacity. HPLC analysis showed that MPH1 treatment promoted the accumulation of key metabolites, including vanillic acid, para-coumaric acid, salicylic acid, ferulic acid, gallic acid, syringic acid, quercetin, myricetin, and naringenin. MPH1 improved uptake of phosphorus, potassium, calcium, magnesium, and iron, contributing to mineral biofortification and nutritional quality. These results demonstrate that MPH at 1 mL L⁻¹ is an effective biostimulant, improving yield and quality while reducing nitrate levels in hydroponically grown cos lettuce, offering a sustainable solution for food waste valorization.

1. Introduction

As the global population rises, the food and agricultural industry faces mounting challenges related to agricultural waste and resource inefficiencies [1]. Approximately two billion tons of agricultural waste are generated globally each year, with projections indicating that this volume could nearly double by 2025 and almost triple by 2100, creating a major economic and environmental burden [2]. These byproducts contain a considerable organic component (20–70% of the waste content), comprising nutrients such as organic acids, amino acids, cellulose, and phenolics [3]. Global raw milk production reached 965.2 million tonnes in 2023, with estimates suggesting that up to 19% of wasted food consists of dairy products [4]. These discarded materials, including sterilized milk, represent a substantial yet underutilized source of nutrients, as sterilized milk retains almost all of its protein content during storage, decreasing only slightly from 3.10% to 3.00%, and remains free of microbial contamination both before and after storage [5]. Transforming food waste into value-added products is crucial for the circular economy, as it contributes to reducing environmental impact and promoting sustainability. Therefore, the need to reduce agricultural waste necessitates the development of innovative solutions for upcycling these materials into high-value products, such as plant biostimulants.

Plant biostimulants differ from fertilizers and pesticides in their capacity to stimulate natural plant processes, hence improving nutrient use efficiency, stress resilience, and crop quality through mechanisms that extend beyond mere nutrient provision or pest management [6]. Protein hydrolysates (PHs) from vegetal and animal-based sources have shown potential as effective plant biostimulants. Vegetal-derived PH, especially legume-derived, improves root development and nutrient uptake by modulating hormone-like activities in lettuce [7], rocket leaves [8], and tomatoes [9]. Similarly, animal-derived PH, including blood- [10] and fish-derived PH [11], enhances plant growth and stress tolerance by improving nitrogen utilization and enhancing antioxidant activity in crops such as snapdragon, maize, tomato, and sorghum. Lastly, PHs based on amino acids are involved in many physiological processes, like enhancing plant nutrient uptake and acting as mineral biofortification, thereby improving macro- and micronutrient status. Consequently, the consumption of such biofortified crops can help address nutrient deficiencies and malnutrition problems, particularly in developing countries [12].

Recent research highlights the feasibility of recycling dairy byproducts as agricultural inputs; for example, hydrolysates of whey and casein have been shown to improve nitrogen utilization efficiency, growth, and metabolic activity in crops such as wheat, pea, tobacco, and tomato [13,14,15,16]. Moreover, acid hydrolysis is a novel and effective method for converting milk proteins into smaller peptides and free amino acids, substantially increasing the degree of hydrolysis and soluble amino acid yield [17]. Protein hydrolysate enriched in free amino acids is better absorbed by plants, supporting enhanced growth and metabolic activity [8,18]. However, the use of hydrolysates from expired milk as biostimulants in hydroponic lettuce remains largely unexplored. Thus, the systematic characterization and optimization of expired milk-derived hydrolysate use in hydroponic lettuce is urgently needed to establish efficacy, safety, and optimal application rates for such biostimulants in soilless cultivation systems.

Hydroponics has emerged as a sustainable soilless farming method that reduces water usage, accurately distributes nutrients, optimizes spatial efficiency, and enables the production of crops year-round [19,20]. Hydroponic lettuce (Lactuca sativa L.) is widely cultivated for its short growing cycles, superior nutritional quality, and notable production capacity [20]. However, climate change-related factors, including elevated temperatures and heightened plant stress, have led to reduced lettuce production, while growing demand has encouraged the widespread implementation of extensive agricultural practices and substantial fertilizer usage [19]. Unlike fertilizers and pesticides, plant biostimulants can be applied in small amounts to sustainably improve crop quality and yield, serving as a complementary strategy to conventional fertilization for modern nutrient management [19]. We hypothesize that expired milk-derived protein hydrolysate (MPH), when used as a biostimulant in hydroponically grown lettuce, can enhance crop growth, yield, and nutritional quality while simultaneously contributing to food waste reduction. This study systematically evaluates the chemical characteristics of MPH, investigates dose–response effects on plant performance, and identifies optimal application rates for hydroponic systems over two independent growing trials, potentially providing a novel approach to improve crop yield and nutritional quality in hydroponically grown cos lettuce.

2. Materials and Methods

2.1. Milk-Derived Protein Hydrolysate Preparation and Characteristics

MPH was prepared based on a modified acid hydrolysis method [17]. Commercial UHT milk that was six months past its expiration date was used, with the initial content of 3.29% total fat and 7.00% total protein, as determined using the AOAC method [21] prior to hydrolysis. For hydrolysis, the expired milk was suspended in 6 N hydrochloric acid (1:5), heated at 95 °C for 6 h, and cooled to 20 °C, and the pH was adjusted to 6.5 using calcium hydroxide. The MPH was centrifuged at 15,000× g at 4 °C for 20 min (using a Kubota 6000 centrifuge device; Kubota Corp., Tokyo, Japan), filtered through Whatman No.1 filter paper, and stored at 4 °C until further analysis.

The physicochemical properties of MPH, including color, total salt content, specific gravity, electrical conductivity, pH, degree of hydrolysis, and solubility, were assessed according to the method described by Sonklin et al. [22]. The soluble protein content was assessed using the Bradford assay [23], which involved mixing diluted MPH (1:10) with a protein reagent containing Coomassie Brilliant Blue G-250 dye solution at 0.01% (comprising 4.7% ethanol and 8.5% phosphoric acid) and measuring absorbance at 595 nm. Bovine serum albumin (BSA) was used as a standard and reported in grams per liter (g L⁻¹).

2.2. Amino Acid Profiles of MPH

Free and total amino acids were analyzed using the AOAC method [21]. Free amino acids were derivatized directly; total amino acids underwent acid hydrolysis (6 N HCl, 110 °C, 22 h) before AccQ-Fluor reagent derivatization. HPLC analysis was performed using a Waters Alliance 2695 system (Milford, MA, USA) with a fluorescence detector (JASCO FP2020; excitation 250 nm, emission 395 nm) and a Poroshell C18 column (2.7 µm, 100 × 4.6 mm). The mobile phase utilized (A) sodium acetate buffer (pH 4.90) and (B) 60% acetonitrile, with a 1.2 mL/min⁻¹ flow rate; 5 µL injection volume; 40 °C column temperature; and a gradient elution program (time range in min, B%) of 0–3, 6; 3–10, 14; 10–15, 20; 15–20, 30; 20–25, 60; 25–35, 100. Amino acids were identified using a 17-standard mixture, including aspartic acid, glutamic acid, alanine, proline, valine, leucine, isoleucine, phenylalanine, methionine, lysine, arginine, histidine, glycine, tyrosine, serine, threonine, and cysteine, and quantified as grams per 100 g protein in the sample solution (g 100 g⁻¹ protein).

2.3. Plant Materials and Growth Conditions

Cos lettuce (Lactuca sativa L.) was transplanted using the nutrient film technique (NFT) at a spacing of 16 cm, following a 14-day nursery period. The nutrient solution concentration (NSC) was prepared according to Hoagland and Arnon [24], containing several elements (in mg L⁻¹), including 210.0 N, 31.0 P, 195.0 K, 200.0 Ca, 48.0 Mg, and 64.0 S in terms of macronutrients, along with 5.00 Fe, 1.00 B, 1.10 Mn, 0.10 Zn, 0.04 Cu, and 0.02 Mo in terms of micronutrients. Thereafter, milk-derived protein hydrolysate (MPH) treatments were added at concentrations of 0 mL L⁻¹ (MPH0), 1 mL L⁻¹ (MPH1), 3 mL L⁻¹ (MPH3), and 5 mL L⁻¹ (MPH5), resulting in electrical conductivity (EC) values of 2.20, 2.40, 3.40, and 4.50 dS m⁻¹, respectively. The pH of NSC was adjusted to 6.5–7.0. MPH treatments were applied to the nutrient solution four times at five-day intervals, beginning three days after transplanting, across two growing trials. Lettuce plants were harvested 25 days after transplanting. For analysis, the second to fifth mature leaves were collected from three randomly selected plants per replicate, with eight biological replicates per treatment. Environmental conditions during the two growing trials (February to May 2024) were recorded as follows: a 12 h photoperiod, average temperature of 35 ± 10 °C, relative humidity of 60 ± 30%, and light intensity of 5000 ± 1000 lux. All samples were cryogenically ground, with HPLC samples further freeze-dried at −80 °C and stored at −20 °C.

2.4. Lettuce Biomass and Plant Canopy

Fresh shoots (g) and roots (g) were weighed on the day of harvest. The plant canopy area (cm²) was determined by capturing digital images of each lettuce sample and analyzing them using ImageJ 1.52a software (National Institutes of Health, Bethesda, MD, USA).

2.5. Determination of Bioactive Compounds

The extraction of bioactive compounds was performed based on the method described by Bhatt et al. [25]. Briefly, the sample tissue (3 g) was homogenized using 5 mL of 100% methanol, extracted overnight at 25 °C, and centrifuged at 15,000× g at 4 °C for 20 min. Total phenolic content (TPC) was measured using a modified Folin–Ciocalteu method [26]. Methanolic extract (0.1 mL) was mixed with 1.55 mL distilled water, 0.1 mL Folin–Ciocalteu reagent, and 300 µL of 20% sodium carbonate, incubated at 40 °C for 30 min, measured at 765 nm, and reported as mg GAE g⁻¹ DW. Total flavonoid content (TFC) was assessed using a colorimetric aluminum chloride method [27]. Methanolic extract (1 mL) was mixed with 0.15 mL of 5% sodium nitrite, 2 mL of 1 M aluminum chloride, 1 mL of 1 M sodium hydroxide, and 1.2 mL of distilled water, incubated for 30 min, measured at 510 nm, and reported as mg QE g⁻¹ DW.

2.6. Analysis of Phenolic and Flavonoid Profiles

Phenolic and flavonoid profiles were quantified according to the method described by Bhatt et al. [25]. Freeze-dried lettuce leaves (0.5 g) were extracted with 10 mL of 100% methanol (25 °C, 24 h), centrifuged (15,000× g, 4 °C, 20 min), and filtered through nylon syringe filters (0.45 µm). Analysis was performed using a Shimadzu LC-40 HPLC system (Kyoto, Japan) with a PDA detector (SPD-M40) and an Inertsil^® ODS-3 C18 column (Octadecyl Silane; particle size: 5 µm; length × inner diameter: 250 × 4.6 mm). The mobile phase utilized (A) water and acetic acid (100:1) and (B) methanol, acetonitrile, and acetic acid (95:5:1) as follows: 1.0 mL min⁻¹ flow rate; 20 µL injection volume; 30 °C column temperature; and a gradient elution program (time range in min, B%) of 0–2, 5; 2–10, 25; 10–20, 40; 20–30, 50; 30–45, 100. Phenolic and flavonoid compounds (mg 100 g⁻¹ DW) were quantified using a 14-compound standard mixture at 325 and 280 nm, including gallic acid, chlorogenic acid, caffeic acid, vanillic acid, para-coumaric acid, syringic acid, ferulic acid, sinapic acid, rosmarinic acid, quercetin, epicatechin, myricetin, and naringenin.

2.7. Determination of Antioxidant Capacity and Ascorbic Acid Content

Antioxidant capacity was assessed via 2, 2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging rate [28] by mixing 0.5 mL of the methanolic extract of the sample tissue with 2.5 mL of 0.1 mM DPPH•, incubating in the dark for 30 min, and measuring absorbance at 517 nm.

Ascorbic acid content was determined using the 2,4-dinitrophenylhydrazine (DNPH) method [29]. Frozen leaf tissue (1 g) was homogenized in 10 mL of 5% cold metaphosphoric acid and centrifuged (10,000× g, 4 °C, 10 min), and 0.5 mL of extract was mixed with 0.2 mL of 0.02% indophenol, 0.4 mL of 2% thiourea, and 0.2 mL of 2% DNPH (dissolved in 10 N sulfuric acid), incubated (50 °C, 1 h), mixed with 1 mL of 85% sulfuric acid, evaluated at 540 nm, and reported as mg AsA 100 g⁻¹ DW.

2.8. Determination of Soluble Nitrate Content

Nitrate content was measured using the salicyl-sulfuric acid method [30]. Frozen leaf tissue (1 g) was homogenized in 10 mL of phosphate buffer saline (pH 7.0) and centrifuged (4000× g, 4 °C, 10 min), and the supernatant (1 mL) was mixed with 0.4 mL of 5% salicylic acid and 9.5 mL of 2 M NaOH. The absorbance was measured at 410 nm (mg g⁻¹ DW) using potassium nitrate as a standard.

2.9. Mineral Profiles of MPH and Lettuce Leaves

Samples for mineral analysis were prepared using a modified method described by Wolf [31]. Oven-dried leaf tissue (5 g) underwent a furnace treatment at 500 °C for 3 h. MPH (5 mL) and ash leaf material (0.1 g) were digested in a mixture of distilled water, concentrated nitric acid, and concentrated sulfuric acid (1:1:1:1) at 100 °C for 6 h to prepare the samples for ionic element analysis. The mineral contents, specifically phosphorus, potassium, calcium, magnesium, and iron, were determined using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) (Avio 220 Max ICP-OES Scott/Cross-Flow Configuration, PerkinElmer, MA, USA) and assessed via comparison with standards using Syngistix for ICP Software version 5.1 (PerkinElmer, Shelton, CT, USA). Total nitrogen was quantified using the AOAC method [21]. MPH (5 mL) and ash leaf material (0.1 g) were digested in concentrated sulfuric acid (1:20) mixed with 1 g of catalyst using the macro-Kjeldahl digester system (KjelDigester K-446, Buchi, Thailand) and an automatic distillation and titration unit (MultiKjel K-365, Buchi, Thailand).

2.10. Statistical Analysis

All tests were conducted in a completely randomized design in four independent replicates to confirm the reproducibility of the results. The data report was presented as the mean ± standard error (SE). Normality and homogeneity of data variance were checked using Shapiro-Wilk test and Levene test, respectively. Analysis of variance (ANOVA) was performed to assess growth, bioactive compounds, and the phenolic and flavonoid contents. Mineral was analysed via t-test. Duncan’s multiple range test was used to determine significant differences at the 95% confidence level (p < 0.05) using SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA), and all graphical figures were analyzed and created using OriginPro 2024 (OriginLab Corporation, Northampton, MA, USA).

3. Results

3.1. Physicochemical Properties and Amino Acid Composition of Milk-Derived Protein Hydrolysate

The milk-derived protein hydrolysate shows potential as a plant biostimulant due to its nutrient profiles and physicochemical properties. Results showed that the milk-derived protein hydrolysate exhibited a high degree of hydrolysis (94.55%), a nearly neutral pH (6.51), and suitable specific gravity (1.01 g cm⁻³) and electrical conductivity (192.00 dS m⁻¹), as shown in

Table 1. Physicochemical characteristics of milk-derived protein hydrolysate (MPH).

Properties	MPH Compositions
Degree of hydrolysis (%)	94.55 ± 1.75
Soluble protein (g L−1)	2.43 ± 0.03
Total salt (%)	14.27 ± 0.22
Specific gravity (g cm−3)	1.10 ± 0.00
Electrical conductivity (dS m−1)	192.00 ± 2.53
pH	6.51 ± 0.04
Total nitrogen (g L−1)	6.10 ± 0.00
P (g L−1)	0.10 ± 0.01
K (g L−1)	0.31 ± 0.00
Ca (g L−1)	8.01 ± 0.12
Fe (mg L−1)	0.90 ± 0.04
Mg (g L−1)	0.64 ± 0.01

All data values represent means ± standard error.

. The acidic hydrolysis of milk proteins results in substantial protein degradation, yielding a free amino acid-to-total amino acid ratio of approximately 0.74 in MPH (

Table 2. Amino acid profiles of milk-derived protein hydrolysate.

Amino Acids	Total Amino Acids (g 100 g−1 Protein)	Free Amino Acids (g 100 g−1 Protein)
Essential amino acid
Lysine	4.05 ± 0.03	4.07 ± 0.01
Histidine	1.48 ± 0.01	0.88 ± 0.01
Threonine	3.06 ± 0.02	1.94 ± 0.03
Valine	4.43 ± 0.01	1.52 ± 0.01
Leucine	6.35 ± 0.01	4.11 ± 0.01
Isoleucine	3.54 ± 0.01	1.42 ± 0.01
Phenylalanine	3.91 ± 0.01	2.47 ± 0.03
Methionine	ND *	1.39 ± 0.01
Non-essential amino acid
Aspartic Acid	7.09 ± 0.01	6.37 ± 0.04
Glutamic Acid	13.81 ± 0.01	11.30 ± 0.01
Arginine	3.58 ± 0.01	2.66 ± 0.01
Glycine	3.00 ± 0.01	2.57 ± 0.03
Tyrosine	1.69 ± 0.04	1.67 ± 0.02
Serine	4.35 ± 0.01	3.31 ± 0.01
Cystine	ND *	3.42 ± 0.01
Alanine	2.98 ± 0.02	2.55 ± 0.01
Proline	5.30 ± 0.01	3.78 ± 0.01

* ND—not detected. All data values represent means ± standard error.

). The primary amino acids in milk-derived protein hydrolysate included non-essential amino acids, such as glutamic acid, aspartic acid, and proline, while the most prevalent essential amino acids were leucine, valine, and lysine.

3.2. Effect of MPH on Lettuce Biomass and Plant Canopy

Notable concentration-dependent effects on lettuce growth and biomass were observed following MPH applications, as shown in

Table 3. Effect of MPH concentrations on the growth of hydroponically grown cos lettuce.

Treatments	Shoot FW (g)	Root FW (g)	Plant Canopy (cm3)
MPH0	92.86 ± 5.83 b	14.47 ± 0.77 c	899.25 ± 97.89 bc
MPH1	118.76 ± 8.65 a	17.83 ± 0.88 a	1140.98 ± 164.49 a
MPH3	97.66 ± 11.17 b	16.09 ± 0.78 b	964.11 ± 125.95 b
MPH5	81.46 ± 9.55 c	14.32 ± 1.07 c	876.72 ± 113.78 c
p-value	*	*	*

All data values represent means ± standard error. Values with different letters indicate significant differences represented as * (p < 0.05).

. The lowest concentration treatment (MPH1) resulted in the most significant enhancements in shoot and root fresh weights (1.28-fold and 1.23-fold, respectively) compared to the untreated control. While MPH3 showed minor enhancements, the highest concentration (MPH5) had an adverse effect on growth. The plant canopy followed a similar pattern, with MPH1 showing the highest increase (1.27-fold), followed by MPH3 (1.07-fold) and MPH5 (0.97-fold), as visually demonstrated in lettuce morphology, where cos lettuce treated with MPH1 exhibits the largest and most vigorous canopy and root system compared to the other treatments (Figure 1).

3.3. Effect of MPH on Antioxidant Capacity and Ascorbic Acid Content

The most pronounced effect on DPPH• radical scavenging activities was observed in MPH1-treated lettuce, which showed a 1.15-fold increase compared to the untreated control. However, MPH3 treatments did not significantly alter the activity relative to the control (Figure 2a). The ascorbic acid content (AsA) showed a response to MPH treatments, with MPH1 and MPH3 resulting in significantly higher AsA levels (1.23-fold and 1.20-fold increases, respectively) compared to the control. In contrast, the AsA content in MPH5-treated lettuce did not significantly differ from that of the control. Notably, no statistically significant differences in AsA levels were observed among the three MPH treatments (MPH1, MPH3, and MPH5), suggesting that lower concentrations of MPH were sufficient to enhance AsA content without additional benefits from higher concentrations (Figure 2b).

3.4. Effect of MPH on Bioactive Compounds and Phenolic and Flavonoid Content Profiles

The application of MPH on cos lettuce resulted in varying responses in the total phenolic content (TPC) and total flavonoid content (TFC). Lower concentrations of MPH, particularly MPH1 and MPH3, led to non-significantly higher TPCs (1.21- and 1.04-fold increases, respectively) compared to the control (Figure 3a). However, the highest MPH concentration (MPH5) significantly reduced TPCs compared to MPH1. TFCs exhibited a more pronounced response, with MPH1 treatment resulting in significantly higher values (1.36-fold increase) compared to the control (Figure 3b). In contrast, higher MPH concentrations (MPH3 and MPH5) resulted in decreased TFCs, with MPH5 treatment producing significantly lower levels.

The phenolic compounds exhibited varying responses to MPH treatments, with vanillic acid showing the most significant enhancement (4.72-fold) in MPH1, followed by para-coumaric acid (3.15-fold), salicylic acid (2.88-fold), ferulic acid (2.44-fold), gallic acid (2.10-fold), and syringic acid (2.08-fold), compared to the control (

Table 4. Effect of MPH concentrations on phenolic and flavonoid profiles of hydroponically grown cos lettuce.

Parameters	Phenolic and Flavonoid Profiles (mg 100 g−1 DW)				p-Value
	MPH0	MPH1	MPH3	MPH5
Phenolic profiles
Gallic acid	0.61 ± 0.40 b	1.28 ± 0.67 a	0.90 ± 0.37 ab	0.58 ± 0.19 b	*
Chlorogenic acid	14.02 ± 13.71 ab	27.55 ± 30.23 a	18.84 ± 19.74 ab	6.84 ± 6.76 b	*
Caffeic acid	1.01 ± 0.60 ab	1.98 ± 1.40 a	1.56 ± 0.86 ab	0.80 ± 0.47 b	*
Vanillic acid	3.22 ± 1.67 b	15.2 ± 12.83 a	8.88 ± 7.69 ab	5.15 ± 5.04 b	*
Para-coumaric acid	2.05 ± 0.99 b	6.46 ± 3.97 a	3.71 ± 1.79 b	1.97 ± 0.71 b	*
Syringic acid	0.61 ± 0.24 b	1.27 ± 0.83 a	0.87 ± 0.43 ab	0.52 ± 0.22 b	*
Ferulic acid	1.02 ± 0.41 b	2.49 ± 2.06 a	1.92 ± 1.17 ab	0.92 ± 0.29 b	*
Sinapic acid	0.72 ± 0.45 ab	1.42 ± 1.15 a	0.92 ± 0.47 ab	0.43 ± 0.24 b	*
Rosmarinic acid	5.70 ± 4.37 ab	13.52 ± 14.73 a	7.96 ± 5.13 ab	4.39 ± 2.57 b	*
Salicylic acid	2.58 ± 1.23 b	7.43 ± 7.52 a	3.99 ± 2.02 ab	2.10 ± 1.12 b	*
Flavonoid profiles
Quercetin	2.80 ± 1.35 b	6.01 ± 4.43 a	4.36 ± 3.19 ab	2.65 ± 1.56 b	*
Epicatechin	14.87 ± 10.28 ab	29.52 ± 24.25 a	24.32 ± 15.53 ab	11.02 ± 7.14 b	*
Myricetin	5.08 ± 2.30 b	10.88 ± 7.79 a	8.17 ± 6.09 ab	4.42 ± 2.59 b	*
Naringenin	1.33 ± 0.79 b	2.62 ± 1.45 a	1.78 ± 1.02 ab	1.05 ± 0.45 b	*

All data values represent means ± standard errors. Values with different letters indicate significant differences represented as * (p < 0.05).

). Other phenolic compounds, including chlorogenic acid, caffeic acid, sinapic acid, and rosmarinic acid, were not significantly different between MPH3 and MPH5. Flavonoids, with the exception of epicatechin, were significantly enhanced in MPH1-treated samples, with quercetin showing the highest increase (2.15-fold), followed by myricetin (2.14-fold) and naringenin (1.97-fold), compared to the control. The enhanced flavonoid accumulation gradually diminished in MPH3 and MPH5, with values not significantly different from the control.

3.5. Effect of MPH on Soluble Nitrate Content

A clear pattern in soluble nitrate content was observed across different MPH treatments (Figure 4). MPH1 demonstrated the most pronounced reduction (1.60-fold decrease), followed by MPH3 (1.48-fold decrease), although no significant differences were observed between these treatments. This pattern suggests a non-linear response to the MPH treatment, where the lowest concentration (MPH1) causes a substantial decrease in soluble nitrate content, while the highest concentration (MPH5) results in partial recovery toward control levels, with the smallest reduction (a 1.25-fold decrease).

3.6. Effect of MPH on Mineral Content Profiles

The mineral profile analysis, focusing solely on MPH at 1 mL L⁻¹ (MPH1) as the optimal treatment, revealed that MPH1 significantly enhanced the mineral content of hydroponically grown cos lettuce compared to the control (

Table 5. Effect of MPH concentrations on mineral content profiles of hydroponically grown cos lettuce.

Mineral Composition	Nutrient Solutions		t-Test
	MPH0	MPH1
P (mg g−1 DW)	0.22 ± 0.10	0.39 ± 0.14	*
K (mg g−1 DW)	4.79 ± 0.29	5.65 ± 0.18	*
Ca (mg g−1 DW)	0.86 ± 0.04	1.25 ± 0.04	*
Fe (µg g−1 DW)	2.70 ± 0.60	5.10 ± 2.40	*
Mg (mg g−1 DW)	0.40 ± 0.01	0.59 ± 0.16	*

All data values represent means ± standard error. The level of significance was represented as * (p < 0.05).

). Among the minerals analyzed, iron (Fe) showed the greatest increase (1.89-fold), followed by phosphorus (P) (1.77-fold). Magnesium (Mg) and calcium (Ca) also exhibited substantial improvements (1.47- and 1.45-fold, respectively), while potassium (K) showed the smallest enhancement (1.18-fold).

4. Discussion

4.1. Implications of Physicochemical Characteristics and Amino Acid Profiles for Biostimulant Potential

Biostimulants can enhance plant growth and development, partly through beneficial mineral elements; however, their relatively low concentrations suggest that bioactive compounds, particularly peptides and amino acids, are the primary drivers of most biostimulant effects [11,13,14,15]. The milk-derived protein hydrolysate (MPH) used in this study exhibited a high degree of hydrolysis (94.55%) and a substantial soluble protein content, resulting in the production of abundant free amino acids. Normally, the molecular size of free amino acids varies, with an average molecular weight of approximately 110–138 Daltons [32]. Its neutral pH and suitable specific gravity facilitate nutrient uptake by plants, although its elevated salt content and electrical conductivity necessitate proper dilution before its use in agriculture. Key amino acids, such as glutamic acid, aspartic acid, and proline, which are abundant in MPH, play crucial roles in stress tolerance, osmoprotection, and metabolic regulation [15,18,33]. Meanwhile, essential amino acids like leucine, valine, and lysine further promote protein synthesis and plant vigor [15]. Similar amino acid profiles have been observed in enzymatic hydrolysates of cow and buffalo milk whey proteins, as well as soybean-derived hydrolysates, all of which demonstrate biostimulant activity and enhanced antioxidant function [13,14,16,34]. In contrast, fish-derived hydrolysates exhibit a distinct composition, with higher levels of phenylalanine, serine, and other specific amino acids [11]. These comparisons support the potential of milk-derived protein hydrolysate as a biostimulant for hydroponic lettuce, although optimal application conditions require further study.

4.2. MPH-Induced Changes in Cos Lettuce Biomass and Canopy

MPH application led to notable increases in cos lettuce biomass and canopy, likely due to its high content of amino acids and bioactive peptides that promote plant growth and development [13,14,16]. These findings are consistent with studies on animal-derived biostimulants, such as casein and whey protein hydrolysates, that stimulate overall biological yield in crops like tobacco, tomatoes, and peas [14,15,16]. Similarly, hydrolysates from chicken feathers and fish have demonstrated the capacity to enhance root and shoot growth and increase fresh and dry biomass in wheat seedlings, tomato, and sorghum [11,35]. The effectiveness of protein hydrolysates is strongly dependent on both their sources and applied rate, as similar improvements in lettuce biomass have been observed with plant-derived biostimulants at optimal concentrations [33,36]. The growth-promoting action of MPH involves complementary mechanisms, including providing readily available amino acids and peptides that supplement plant nutrition and improve nitrogen utilization [10,11,13,14,15,16], exerting hormone-like activity that modulates cell division and root development, such as indole-3-acetic acid (IAA) [8,9,37,38,39], and supplying osmoprotective amino acids such as proline and glycine to maintain osmotic balance under stress [15,18,33]. These combined actions help explain consistent improvement in growth, biomass, and productivity following optimal MPH application, as corroborated by similar results in lettuce, Chinese cabbage, and maize [7,37,40].

However, higher concentrations of MPH (MPH3 and MPH5) reduced cos lettuce growth and biomass, likely due to elevated electrical conductivity (EC) of the nutrient solution (4.5 dS m⁻¹), reaching levels detrimental for this moderately salt-sensitive species [18]. Similar growth inhibition at excessive concentrations of animal- and vegetal-derived protein hydrolysates has been reported in lettuce, Chinese cabbage, and both green and red baby lettuce [18,40]. This suppression may result from general amino acid inhibition, leading to a series of cellular disruptions, including amino acid imbalance, energy depletion, reduced nitrate uptake, and increased apoptosis sensitivity [6]. High concentrations of protein hydrolysate can also induce osmotic stress, impair root water and mineral absorption, and elevate proline synthesis, ultimately compromising plant health [18]. Therefore, optimizing both the concentration and electrical conductivity is essential for maximizing the plant growth-promoting effects of protein hydrolysate while avoiding adverse impacts associated with over-application.

4.3. MPH-Induced Changes in Antioxidant Capacity and Ascorbic Acid Content

The antioxidant capacity and ascorbic acid content of cos lettuce responded differently to MPH treatments, with the former increasing at the optimal MPH1 treatment and the latter declining at higher concentrations. This biphasic effect reflects a hormesis mechanism in which low MPH levels induce beneficial eustress that stimulates antioxidant synthesis, while excessive levels cause metabolic disruption and toxicity [6,41]. At optimal concentrations, bioactive peptides function as signaling molecules, upregulating stress-response pathways, enhancing carbon metabolism, and improving resource allocation [6,41,42]. These actions promote nitrogen use efficiency and carbon fixation, thereby expanding precursor pools for the phenylpropanoid and shikimate pathways [6,42,43]. Protein hydrolysate also induces the biosynthesis of key phytohormones such as IAA and salicylic acid (SA), thereby regulating antioxidant defenses [9,15,44]. These mechanisms enhance redox balance, as indicated by elevated AsA/DHA and GSH/GSSG ratios, leading to improved antioxidant capacity and redox homeostasis [35]. Similar improvements occur with other biostimulants, including pig blood-derived PH, seaweed extracts, and legume-derived PH in lettuce and baby rocket [8,10,33]. However, excessive MPH can trigger feedback inhibition of biosynthetic enzymes, osmotic stress from peptide aggregation, and the overproduction of ROS, leading to antioxidant depletion. This biphasic effect is confirmed by studies showing that supraoptimal concentrations reduce phenolic, flavonoid, and ascorbic acid contents, as well as antioxidant activity in leafy greens [8,10,33,36].

4.4. MPH-Induced Changes and Alterations in Bioactive Compounds and Phenolic and Flavonoid Content Profiles

The application of MPH on cos lettuce exhibited a clear hormetic response in the accumulation of phenolics and flavonoids, with lower concentrations (MPH1) significantly enhancing the total phenolic (TPC) and flavonoid (TFC) contents and higher concentrations (MPH5) suppressing these metabolites. This concentration-dependent pattern is consistent with previous studies showing similar biphasic responses in lettuce treated with pig blood-derived PH [10], seaweed extracts [33], and legume-derived PH [36]. Phenolic compounds, characterized by one or more hydroxylated aromatic rings, including hydroxybenzoic acids from the shikimate pathway and hydroxycinnamic acids from the phenylpropanoid pathway, are key targets influenced by MPH treatment [45]. With MPH1 treatment, enhanced carbon fixation provides essential intermediates via the shikimate pathway and increases in aromatic amino acids such as phenylalanine and tyrosine, thereby feeding the phenylpropanoid metabolism [10,42]. These conserved mechanisms have also been reported in tomatoes, lettuce, and mint following protein hydrolysate application [10,44,46].

As represented in the pathway diagram (Figure 5), MPH stimulates metabolic flux through the phenylpropanoid pathway, driving the coordinated biosynthesis of key phenolic acids and flavonoids via efficient resource allocation [10]. This optimal stimulation produces compounds such as vanillic acid, which stimulates antioxidant enzymes; para-coumaric acid, which promotes growth and modulates photosynthesis; and salicylic acid, a defense phytohormone that enhances resistance and supports root development under suboptimal conditions [44]. Other compounds, including ferulic acid, gallic acid, and syringic acid, further enhance plant defense by detoxifying reactive oxygen species (ROS) [47], while flavonoids strengthen protection by conferring UV defense, metal chelation, and scavenging free radicals [47,48]. However, at higher concentrations (MPH3, MPH5), reduced PAL activity and decreased enzyme sensitivity reflect concentration-dependent feedback inhibition [49], a hallmark of hormesis in biostimulant responses [6,41], which restricts the accumulation of late-acting flavonoids while allowing early-pathway metabolites related to trans-cinnamic acid synthases to persist [49]. These bioactive compounds not only enhance plant resilience but may also confer human health benefits, including antioxidant, antimicrobial, cardioprotective, antidiabetic, and anticancer effects [48]. Therefore, optimizing the MPH concentration is critical for balancing precursor availability and enzymatic thresholds, thereby ensuring a metabolite profile that benefits both crop performance and nutritional value.

4.5. MPH-Induced Changes in Soluble Nitrate Content

The reduction in soluble nitrate levels with MPH treatments is particularly noteworthy, as it aligns with studies indicating that protein hydrolysate can sequentially lower nitrate levels by providing amino acids that signal for nitrate regulation and enhance nitrogen uptake in the root zone [16,39,43]. In this study, MPH treatments significantly reduced nitrate content in cos lettuce to 210–220 mg kg⁻¹ of fresh weight. These levels fall within the acceptable daily intake (ADI) range of 220–440 mg for a 60 kg individual [50], thereby improving food safety by reducing nitrate accumulation to levels compliant with dietary guidelines. This beneficial effect is consistent with previous studies showing that applications of seaweed extracts [33] and protein hydrolysate [37,43] decreased nitrate concentration in many crops, such as lettuce, maize, and tomatoes, likely through the modulation of root zone signalling and the activation of nitrogen-related enzymes involved in nitrate metabolism.

4.6. MPH-İnduced Changes in Mineral Content Profiles

The enhancement in mineral content in MPH1-treated cos lettuce aligns with findings from previous biostimulant applications, including those using tropical plant extracts, seaweed extracts, and legume-derived protein hydrolysates [6,7,43]. This improvement is largely attributed to amino acids and peptides that stimulate phytohormone biosynthesis and root system development, thereby increasing the plant’s capacity for mineral uptake [38,39]. Protein hydrolysate has been shown to induce amino acid auxin permease (AAAP) transporters, thereby enhancing nutrient use efficiency and promoting the accumulation of nitrogen-based metabolites [37]. Additionally, it upregulates nitrate (NRT), ammonium (AMT), and amino acid transporters (AAT) to improve ammonium translocation [43]. Additionally, MPH-derived compounds activate key enzymes in carbon and nitrogen metabolism, including isocitrate dehydrogenase, malate dehydrogenase, citrate synthase, glutamine synthetase (GS), glutamate synthase (GOGAT), nitrate reductase (NR), nitrite reductase (NiR), aspartate aminotransferase (AST), ferredoxin–glutamate synthase (GLT), and glutamate dehydrogenase (GDH), collectively supporting enhanced mineral uptake and assimilation [16,39,43]. These physiological effects not only increase biomass but also contribute to agronomic biofortification strategies aimed at improving the nutritional quality of leafy vegetables [12]. As a result, MPH1-treated lettuce exhibits elevated levels of essential minerals, where potassium promotes cardiovascular health and electrolyte balance, calcium, phosphorus, and magnesium contribute to bone strength, and iron is essential for oxygen transport and cognitive function [12,51]. Therefore, regular consumption of such mineral-rich lettuce may enhance dietary mineral intake, supporting both plant productivity and human health.

5. Conclusions

This study demonstrates that expired milk-derived protein hydrolysate (MPH) at 1 mL L⁻¹ (MPH1) enhances plant biomass, nutritional quality, antioxidant capacity, phenolic and flavonoid profiles, and mineral composition of hydroponically grown cos lettuce while reducing nitrate accumulation. These findings highlight MPH1 as a sustainable biostimulant that supports high-quality lettuce production and promotes circular economy practices by repurposing expired milk. To advance its application, future studies should test MPH across diverse cultivars and growing conditions, clarify its mode of action in various environments, and compare its field performance with commercial biostimulants. Furthermore, the large-scale use of MPH will require standardized production and application guidelines to ensure consistent performance and environmental safety.