Protein Hydrolysates—Production, Effects on Plant Metabolism, and Use in Agriculture

Abstract

Agriculture is facing challenges to produce more food in a climate scenario that works in the opposite direction. To amend this, agriculture has to invent new ways of making more with less. Interest in using by-products and finding new ways to utilize them has been increasing in recent years. The use of protein-rich sources for protein hydrolyzation and the use of these protein hydrolysates as biostimulants in plant production have been increasing. These mixtures are mainly produced by chemical and enzymatic hydrolysis from agro-industrial protein-rich by-products of animal, plant, and algal origins. The application of PHs has the potential to alleviate environmental stress; improve plant growth; and increase productivity, fruit yield, and abiotic-stress tolerance in agricultural crops. The use of these biostimulants offers a way to reduce the use of agrochemicals and agrees with the “do more with less” task in the future of agricultural production. This review gives an insight into the production of PHs, referring to sources of raw materials and methods of hydrolysis, the uptake and translocation of PHs, their effect on plant growth, the development and physiology, their role in alleviating stressful conditions, and their use in agriculture. The beneficial effects of PHs on different aspects of plant physiology, metabolism, and plant functioning under stressful conditions are evident. Inconveniently, crops, and sometimes even cultivars, are affected differently based on the way that PH is applied, the timing, and the concentration applied. Further research is needed to elucidate the mechanisms by which the components of PHs modify plant physiology and metabolism.

1. Introduction

One of the first definitions of biostimulants was coined by Zhang and Schmidt in the web journal Grounds Maintenance in 1997 as “materials that, in minute quantities, promote plant growth” [1]. This definition was later officially (i.e., in a peer-reviewed journal) modified to “materials, other than fertilizers, that promote plant growth when applied in small quantities” by [2]. Per this definition, biostimulants are therefore explicitly distinguished from nutrients and soil amendments that are usually applied in larger quantities. Ideally, biostimulants are derived from natural sources, so they are compatible with organic farming practices and, hence, follow the organic certification standards. Regulation for biostimulant use in agriculture within the EU was finally legally defined in 2019 by the European Parliament (EU REGULATION 1009/2019).

The growing demand for sustainably produced basic agricultural products requires the optimization of the use of natural resources (e.g., water and soil) whilst reducing the negative environmental impact. The improvement, development, implementation, and application of sustainable agricultural practices resulting in increased product yield and quality despite limited nutrient availability and (a)biotic stresses are therefore of large interest to growers worldwide [3]. Under these circumstances, biostimulants emerge as promising tools in ensuring improved crop productivity, enhancing crop yield and quality, and inducing tolerance to different stresses [1]. The global market for biostimulants was projected to reach USD 2.2 billion in 2022 [4] and USD 7.6 billion in 2029, with an annual growth rate of 12%, with the largest market being in Europe (http://www.marketsandmarkets.com accessed on 1 August 2024). Two large consortia of biostimulant industries already exist in Europe and North America (the European Biostimulants Industry Council (EBIC) and Biostimulant Coalition, respectively) whose members are some of the largest companies that manufacture products for crop nutrition and protection (https://biostimulants.eu/members/ and http://www.biostimulantcoalition.org/ accessed on 1 August 2024).

Three types of biostimulants are recognized: humic substances (HSs), hormone-containing products (HCPs), and amino acid-containing products (AACPs) [2]. The latter are of particular interest because of their demonstrated beneficial effects on crop productivity [5], mitigation of abiotic stress [6], and enhancement of microbiome diversity [7]. In addition, they can be obtained from the by-products of agro-industry through protein hydrolysis, reducing the impact of agriculture on the environment whilst being economically attractive [8,9]. Indeed, in open-field agriculture and protected cultivation, protein hydrolysates (PHs) are gaining attention, which is followed by the formulation of several commercial products obtained from the PHs of plant or animal origin, such as Trainer^®, Vegamin©, and Hicure^®, in the last two decades.

Based on their contents, PHs can be divided into two categories: (i) mixture of peptides and amino acids of plant or animal origin; and (ii) mixture of individual amino acids such as glutamate, glutamine, proline, and others [10]. Besides peptides and amino acids, PHs can contain carbohydrates, minerals, phenols, and phytohormones, as well as some other organic substances, whose concentration is usually negligible [5]. Due to food safety concerns regarding PHs from animal sources, mainly linked to bovine spongiform encephalopathy, their use was prohibited by the EU in 2014 (European Regulation No. 354/2014). Moreover, there were reports of phytotoxic effects and growth depression after repeated application of PHs of animal origin [11,12]; however, they have not been consistent. A study by [13] concluded that animal-based PHs do not have any negative effects on eukaryotic cells or soil ecosystems and can be safely used in organic or conventional farming.

PHs can be applied to plants in several different ways—foliar, root application, mixed with nutrient solution, and even seeds can be primed with PHs (Figure 1). This mode of application can influence the effects which PHs have on the plant. For example, root uptake of amino acids is dependent on specialized transporters, while leaf absorption is a passive process which is dependent on different climate conditions that affect the absorption through biological responses of the plant [14].

The aim of this review article is to evaluate recent findings on PHs’ use in agriculture in terms of their effects on plant metabolism and their effects on important agronomic traits and to summarize their production (sources of raw materials) and classification.

2. Production of Protein Hydrolysates

Besides the chemical characteristics, source material and type of hydrolysis can influence the biological activity of the final product. Sources of proteins can vary greatly, and some of the most common ones are shown in Figure 2. Besides peptides and amino acids, PHs can contain carbohydrates, minerals, phenols, and phytohormones, as well as some other organic substances, whose concentration is usually negligible [5].

2.1. Sources of Raw Materials for Obtaining Protein Hydrolysates

Large quantities of by-products from agriculture and the food industry are produced every year. Valorization of these by-products is essential to achieve sustainability goals. Animal sources of PHs can vary greatly. It is estimated that 54 billion pounds of livestock by-products is generated annually worldwide [15], and about 47% out of 158 million tons of worldwide-produced whey is disposed of in the environment [16]. Animal by-products can vary from cow connective tissue [17], tanned leather waste [18], bones [19], casein [20], blood meal [21], and even feathers [22]. The fish industry is a large by-product generator, as more than 60% of fish biomass becomes by-products [23]. PHs from fish sources can be skin collagen [24], fins and backbones [25,26], head, trimmings, viscera, and frames [27]; practically every fish by-product has been made into PHs. Recently, insect-based PHs have attracted much attention, even though their whole biomass is used for hydrolyzation. The most-often used insects are crickets and mealworms [28].

For plant-derived PHs, legumes are a common source of raw materials. Defatted meal, seeds, and flour have already been used from soy [29], mungbean [30], black bean [31], and alfalfa [32]. In addition, seeds from Cucurbitaceae—watermelon, calabash, and pumpkins—have been proposed as promising sources for PH production [33]. Seed cake, a by-product of oil production from seeds (especially pumpkin, sesame, flax, and rapeseed), as well as cereal grain, is a rich source of proteins. Cucurbitaceae seeds are especially interesting: after the extraction of oil, which presents about half of the seeds’ weight [34], the seed cake is rich in carbohydrates and contains 60–65% w/w of proteins [35]. Not only seeds, but leaves of alfalfa [36], jackfruit [37], pumpkin [38], palms [39], and olive leaves [40] can be used in the production pf PHs. Olive leaves can be used in production of PHs, but their crude protein contents are typically small (70–129 g/kg dry weight), and the contents of interfering compounds (pigments and polyphenols) are high [41].

PHs from animal and plant sources generally differ significantly in the amount and type of amino acids. Animal-based PHs usually have higher amounts of total amino acids, with collagen-based PHs being richer in glycine and proline, while fish-based PHs have larger percentages of aspartic and glutamic acid. Similarly to the latter, legume-based PHs are rich in aspartic and glutamic acids [42,43], although large variability exists. Some reports state that PHs from soybean, lima bean, pigeon pea, and mung bean have large amounts of glutamic acid and asparagine, while Persian lime seed protein isolates are characterized by a high content of hydrophobic amino acids, such as Cys, Leu, Ala, Pro, Met, and Val [44,45]. In general, the contents of essential amino acids in plant-based PHs were lower than in animal-based PHs, ranging from approximately 20% for oat, lupin, and wheat to 30–40% in PHs derived from whey, milk, casein, egg, and muscle. Out of those, methionine, leucine, and lysine contents were typically lower in plant-based PHs compared to animal-based PHs [46]

Algae represent interesting sources of material for PH production, as their protein content can be relatively high, especially in microalgae and cyanobacteria, where it reaches up to 63%, with amino acids constituting up to 48% of total protein contents [47,48]. Some species, e.g., Spiruliina platensis, can contain high amounts of tryptophan and arginine, which act as precursors for auxins and polyamines [49]. In addition, algal-based PHs have been shown to contain signaling molecules (e.g., betaine), vitamins, polyamines, and polysaccharides (e.g., beta-glucan) [50].

2.2. Type of Hydrolysis

In addition to the protein source, the production process of PHs itself also affects the chemical, physical, technological, and biological characteristics of PHs. The most widely used methods for the production of PHs at the industrial scale are chemical and biochemical methods. Fermentation is another option for protein hydrolyzation, but it will not be covered in this review. Methods used in the production process of protein hydrolysates are shown in Figure 3. Chemical methods include acid and alkaline hydrolysis, while biochemical methods rely on autolysis and enzymatic hydrolysis [23]. While chemical methods are simple and easy to operate, their weakness comes from the harsh reaction conditions and unspecific cleavage of peptide bonds, resulting in a very heterogenous mixture of peptides and a reduction of the nutritional quality of the product [51]. Autolysis relies on the action of endogenous proteolytic enzymes (endo- and exo-proteases) on the animal proteins, but the process also generates heterogenous hydrolysate. Enzymatic hydrolysis uses exogenous enzymes, which allow for better control of the cleavage of peptide bonds, resulting in a more homogenous final product.

2.2.1. Chemical Hydrolysis

Two options for chemical hydrolysis are available—acid and alkaline. Acid hydrolysis typically makes use of hydrochloric and sulfuric acid. Factors affecting the final product in acid hydrolysis are the type and concentration of acids, temperature (ranges between 120–140 °C), pressure (220–130 kPa), time of hydrolysis (2–8 h), and concentration of the protein (50–60%) [52]. Acid hydrolysis destroys some of the essential amino acids, such as tryptophan, methionine, cystine, and cysteine, while glutamine and asparagine are converted to glutamic and aspartic aid [53]. A substantial amount of salt is left in the final product and is often partially or completely removed by precipitation, nanofiltration, and ion-exchange resins. Because acid hydrolysis is non-specific, all peptide bonds are a potential target, and the end result is a product with a high content of free amino acids. The downsides of acid hydrolysis are the high salt content, destruction of some amino acids, destruction of thermolabile compounds (for example vitamins), and conversion of free amino acids from L-from to D-form, which makes them unusable directly as a nitrogen source [54] and potentially toxic [11]. In addition, it is challenging to tailor the molecular weight of produced peptides, and the molecular weight is an important factor for the biological activity of the PHs [55].

Similarly, alkaline hydrolysis is a rather simple process which includes heating to solubilize the protein, followed by the addition of alkaline reagents. The most commonly used alkaline reagents are Ca, Na and K hydroxides. The advantage over acid hydrolysis is that the temperature is set to 25–55 °C. This temperature range is, however, kept for several hours until the set degree of hydrolysis is achieved, during which the partial degradation of the amino acid profile occurs, mainly via the destruction of serine and threonine amino acids [52].

2.2.2. Enzymatic Hydrolysis

Better control over the final product is one of the advantages of enzymatic hydrolysis. This process also has low energy requirements, and it offers the ability to control cleavage sites, thus enabling control over the resulting physicochemical characteristics (solubility, diffusion, and absorption). In addition, it is also a much “greener” technology compared to chemical hydrolysis (e.g., no need to create harsh reaction conditions, no aggressive chemicals are used, heating the mixture to lower temperature, reaction takes a few hours, etc.). Enzymatic hydrolysis is based on the addition of different proteases to the protein source, and these proteases are the key factors of almost all PH characteristics, including the biostimulant [56], biological, and functional properties of the final PH product [42,57,58]. Enzymatic hydrolysis includes several independent variables: protein substrate concentration, enzyme-to-substrate ratio, pH, temperature, and time [59]. Reaction conditions must be adjusted to optimize the activity of the selected proteolytic enzyme(s) (e.g., temperature, pH, and the ratio of enzyme and substrate) [60]. Other than these factors, the protein type, pretreatments, solid–liquid ratio can impact biological activity of produced peptides [61].

The process of enzymatic hydrolysis usually starts with a pretreatment of 5–20 min at 85–95 °C to inactivate endogenous proteases, which may interfere with added proteases. This makes the process fully controllable. Several types of proteases have been used to produce bioactive peptides, such as flavourzyme, alcalase, neutrase, umamizyme, and protamex, which are derived from microorganisms; enzymes from plant sources, such as papain, ficin, bromelain and cucumisin; and pepsin trypsin and chymotrypsin from animal sources [62]. The fundamental parameter in PH production is the degree of hydrolysis, which is defined as the percentage of broken protein in relation to the original protein. The degree of hydrolyzation, in most part, is determined by conditions of hydrolysis, as well as by the nature of the protease(s), as characterized by its type and activity. Proteases can differ in their preference for cleavage site and mechanism of cleavage. For example, pepsin cleaves in hydrophobic and aromatic regions, while trypsin cleaves C terminal to arginine or lysine. Proteases can be used singularly or in combination with each other; the combinations determine the degree of hydrolysis, the ratio of amino acids to peptides, and the molecular weight of peptides in the PH mixture. For example, [63] showed that alcalase-treated oat protein resulted in the majority of peptides being in the range 30–7.2 kDa, while treating the mixture further with flavourzyme reduced the molecular weight of peptides significantly, with major peaks in 5.7 and 1.5 kDa. When the desired degree of hydrolysis is achieved, the reaction is stopped by heating or acidification, after which the fractions are separated (sludge of solids and non-soluble proteins, aqueous layer, and lipid phase at the top). The lipid phase is removed, and the soluble phase is collected [64]. The final step is the drying of the PH by spray- or freeze-drying.

3. Mode of Action of PHs on Plants

3.1. Uptake and Translocation

Nitrogen is one of the most important elements in plant development, as it is a major constituent of vital metabolites for plant metabolism, such as proteins, nucleic acids, coenzymes, chlorophyll, amino acids, phytohormones, signaling molecules, etc. A limited supply of nitrogen adversely affects plant growth and metabolism [65,66,67]. Plants take up N usually in the form of NO₃⁻ and NH₄⁺, but the uptake of N in the form of amino acids and peptides is also possible through roots and leaves [68,69]. The uptake of amino acids and peptides by roots is often hindered by reduced phytoavailability induced by microbial activity [70]; the importance of amino acids for N nutrition depends on the plant species and the type of the soil [71]. For root uptake, high- and low-affinity systems have been identified, such as AtAAP1, which belongs to the amino acid permease (AAP) family, demonstrated to transport glutamate and neutral amino acids into plant cells [72]. AtAATP5 belongs to the same family, but it dictates the uptake of cationic amino acidy L-lysine and L-arginine, as shown by [73]. Some transporters have been implicated in amino acid uptake in both root epidermis and leaf mesophyll, such as the high-affinity transporter AtLHT1 [74].

The foliar application of PHs can increase amino acid and peptide availability by eliminating extensive competition with microorganisms for organic N [75]. Indeed, successful case studies have been reported. The foliar application of a biostimulant produced via the enzymatic hydrolysis of chicken feathers increased the contents of macro- and microelements in maize leaves and increased the protein content in the grain [76]. Texeira et al. [69] showed that the foliar application of amino acids and seed treatment of soybean with amino acids increased the N content in leaves in the V6 phase, applied either alone or combined. However, the amount of applied amino acids presented less than 1% of all amino acids already present in the leaf, suggesting that the applied amino acids were not a source of N for the plant but rather acted as signaling molecules that induced greater N uptake. By contrast, [77] measured the absorption of foliar-applied ¹⁵N-labelled inorganic and organic sources, namely glycine, L-glutamic acid, and L-proline. They showed that the uptake of the N compounds was 31–56% of the nitrogen applied 8 h after application. Although the authors concluded that amino acids alone should not be considered as a standalone N fertilization source, combinations of amino acids and different nitrogen sources could enhance the foliar uptake of N. After the uptake, either by the roots or through the leaf, amino acids and peptides are reduced for direct assimilation or transported through the plant vascular system to the shoot, where they can be stored, metabolized, or translocated to other N sinks, such as developing leaves, fruits, and seeds [78]. Santi et al. [17] showed that the application of PHs or amino acids to the nutrient solution of hydroponically grown maize induced transcriptional changes in different processes, such as cell-wall organization, transport processes, hormone metabolism, and stress responses, indicating that there is a metabolic response to these organic N sources.

3.2. Effects on Plant Development

3.2.1. Effects on Root Growth and Development

Root growth is one of the most important survival mechanisms under stress conditions [79]. Root development is crucial for maximizing water- and nutrient-use efficiency, and improvements in the development of the roots would significantly enhance plant productivity and help alleviate stressful conditions. Positive effects of PHs on root growth and development have been well documented in different plant species, such as maize [80], tomato [81], lily [82], tea plants [83], primrose [84], and perennial wall rocket [85]. As PHs are applied at low concentrations, their beneficial effects on plant growth and development are not due to a fertilization effect but due to auxin and gibberellin-like activities as signaling molecules [86]. Indeed, Colla et al. [87] confirmed the auxin-like activity of PH on maize coleoptile elongation, which induced a response similar to the application of inodole-3-acetic acid (IAA) alone. In the same research, similar effects were observed on tomato cuttings, in which the root dry weight, root length, root area, and shoot parameters were up to 26% higher than control plants. The auxin-like activity presumably arose from the high tryptophan content (1.4%) in the PH. Tryptophan is the main precursor of IAA in plants’ biosynthesis pathways. Certain peptides could also induce IAA-like activity, especially from plant-derived PHs, as reported by Matsumiya [88] for a root hair-promoting peptide from degraded soymeal product. Raguraj et al. [83] showed that chicken-feather protein hydrolysate at a dosage of 2 g/L increased root length by 94%, root surface area by 15%, and root dry biomass by 152% in tea plants. Similar results were obtained for chickpeas by Paul et al. [89], using also chicken-feather hydrolysate. A microarray analysis of maize seedlings treated with PH revealed that the positive effect on plant growth and metabolism is due to the combination of the activation of transcription factors, the differential expression of cell-wall components, stress, and hormonal metabolism [17]. As reported by Santi et al. [16], the glutamate receptor involved in root growth and C/N signaling was affected by PH, and also genes related to metal uptake and translocation, which could be due to the ability of peptides to chelate metal ions. This could facilitate the uptake of metals, which may decrease the energy expense for the biosynthesis of phytosiderophores. The higher contents of Cu, Mn, and Zn in their research could be attributed to this thesis. This research also revealed that there is a difference in activity of animal-based PHs and reconstituted mixture of amino acids, as PHs could regulate the glutamate receptor involved in C/N signaling and root growth, while reconstituted amino acids could not.

3.2.2. Shoot Growth and Development

The application of PHs has been shown to increase biomass and yield in plants (e.g., strawberry, tomato, maize, and lettuce) [90,91,92,93]. Gibberellin (GA)-like activity has been observed in several plant species treated with PHs. Colla et al. [87] showed that gibberellin-deficient pea plants had increased their shoot length by 33% after treatment with plant-based PH, indicating GA-related interference. Indeed, genes related to the activation/deactivation of GAs, primarily through the upregulation of GA 3-beta-dioxygenase, which converts inactive GAs to their active form; downregulation of GA 2-oxidase, which has a role in GA deactivation; and involvement in the GA signaling pathway, were differentially expressed in plants treated with PHs [17]. The upregulation of cytokinin (CK) riboside 5′-monophosphate phosphoribohydrolase, which is involved in producing the free-form cytokinins that play a role in modifying root architecture, has also been observed as a result of PH treatments [94]. Ceccarelli et al. [95] showed that Solanaceae- and Malvaceae-based PHs were able to reprogram the phytohormone profile of tomato plants by modulating GA and CK synthesis, as they stimulated the accumulation of IAA and GA precursors (4-(indol-3-yl) butanoate (IBA), tryptamine, and ent-7α-hydroxykaur-16-en-19-oate), as well as cytokinins, by inducing the production of zeatin riboside. The positive effect of PH treatment on shoot biomass in maize seedlings was attributed to the release of signaling molecules and hormone-like activity (auxin and GA-like activity) and the increase in activity of enzymes such as glutamine synthetase and nitrate reductase [92]. In the tomato, PH-treatment induced the accumulation of a direct ethylene precursor (1-aminocyclopropane-1-carboxylate), affecting its biomass [96]. Ethylene is usually associated with the senescence and ripening of fruits, but other regulatory effects of ethylene have been reported, such as flowering, plant growth, and cell division [97,98].

4. The Application Effects of PHs on Plants

4.1. Effect on N and C Status in Plants

The promotion of root architecture that has been observed upon treatment with PHs has been accompanied by an increased uptake and assimilation of minerals and N [10,87,99,100]. Sestili et al. [101] showed that the N status of tomato plants was affected by PH application, either foliar or by substrate drench, but the response depended on the N supply. Under suboptimal conditions, there was a downregulation of genes for nitrate assimilation (NR, nitrate reductase) which corresponded to the reduction in the transcript levels of ammonium and nitrate transporters. Due to the increase in N content, the authors hypothesize that PH acted as an N source, which was further supported by increase in amino acid transporter gene (AAT1) expression in leaves and roots, suggesting the re-allocation of amino acids in tomato plants. By contrast, under high N regime-upregulated transcript levels of ammonium and amino acid transporters, nitrate and nitrite reductase and ferredoxin-dependent glutamate synthase were upregulated upon PH treatments, which, in turn, improved the nutritional status of the nitrogen in tomato plants. Increased activity of enzymes involved in N metabolism (glutamine synthetase (GS) and glutamate synthase (GOGAT) was observed in salt-stressed maize plants treated with alfalfa hydrolysate-based biostimulant [86], where the activity of GOGAT increased by 28% in salt-stressed plants cultivated with PH compared to the ones cultivated without the biostimulant under salt stress. Studies have suggested that individual amino acids may play a part in the regulation of N uptake by roots, as exemplified by the increased glutamine supply, which reduced the influx of nitrate and ammonium, and also reduced the transcription levels of transporter genes in barley roots [102,103].

Alfalfa-based PH applied to hydroponically grown maize plants increased the activity of several enzymes in the tricarboxylic acid (TCA) cycle, namely malate dehydrogenase, isocitrate dehydrogenase, and citrate synthase, along with enzymes involved in N reduction and assimilation [32]. The interaction between carbon (C) and N metabolism pathways has an important role in plants, as the increased production of carbon skeletons can increase N assimilation in plants [104]; hence, metabolites such as sucrose or amino acids playing a role in the regulation of enzymes involved in N assimilation is no surprise. Similar results were obtained by Ertani et al. [86], as the activities of citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase increased in maize plants treated with meat-based PH, and their transcripts were also upregulated. These enzymes are an important part of the production of carbon skeletons, which help regulate the carbon–nitrogen interaction and nitrogen assimilation-involved enzymes in plants, and their upregulation reveals the connection between carbon and nitrogen metabolic pathways and the regulation of gene transcripts mediated by the application of PHs [86].

4.2. Effects on Photosynthesis

Positive effects of PHs on photosynthesis have been documented using various tools to assess these effects. The application of a commercial PH product has been shown to increase the CO₂ net assimilation rate (by 17.6%), stomatal conductance, and transpiration rate in a dose-dependent way in basil [105]. Similar results were obtained by Raguraj et al. [83], as they showed an increase in chlorophyll content and photosynthetic and transpiration rates, which increased by 9.65 and 26.5% when tea plants were treated with PH. Fish-based PH applied on lettuce resulted in higher chlorophyll content (which increased from 9.6 to 12.2 mg g⁻¹ DW) and improved photosynthetic rate (increased from 12.2 to 16.9 μmol CO₂ m⁻² s⁻¹), stomatal conductance, and transpiration rates (increased from 0.39 to 0.62 and from 2.9 to 4.0 9 μmol H₂O m⁻² s⁻¹, respectively) [106]. Several factors could be attributed to these observations, such as the hormone-like action of PH, higher water-use efficiency, and increase in potassium (K) content, which could increase stomatal reactivity. Higher amounts of photosynthetic pigments enable the plant to use light more efficiently [105,107]. The application of amino acids resulted in a 10 to 25% increase in chlorophyll a and b content in wheat plants, while tomato plants treated with amino acids showed an increase in the SPAD index [100,108].

A transcriptome analysis performed in maize revealed that components of photosynthetic electron transport (ferredoxin-2, the light-harvesting complex protein LHCA5, and chloroplast ATP synthase chain precursor), along with RuBisCo, are upregulated via the application of alfalfa-based PH, which resulted in the increase in accumulation of soluble sugars. Moreover, the upregulation of a gene related to photosystem II 22 kDa protein, involved in light harvesting and non-photochemical quenching and reducing reactive oxygen species generation, was observed [100]. These findings indicate a delicate involvement of PHs in photosynthesis, primary C metabolism and N assimilation and metabolism, altogether resulting in an increase in plant biomass and plant growth.

4.3. Effects Related to Plant Stress

The application of PHs has been shown to alleviate stress in plants by adjusting their metabolomic response, mainly through amino acids and peptides boosting their tolerance to various heavy metals. Proline is one example of an amino acid that accumulates in many plant species under heavy metal stress, and some heavy-metal-tolerant plants have a constitutively higher content of proline (i.e., also in unstressed conditions). Proline provides several functions that ameliorate conditions that arise during heavy-metal stress, such as water deficit and an increase in ROS, as it can chelate heavy metals and act as a regulator of plant metabolism [109]. Other amino acids have been proposed as chelators of Zn, Ni, Cu, As, and Cd, such as asparagine, glutamine, cysteine, and peptides such as glutathione [109,110].

Tomato plants treated with a commercial PH-based biostimulant showed better water status and pollen viability under limited water availability (50% irrigation), resulting in higher yields by 112%. This was paired with higher antioxidant activity, measured via FRAP assay [111]. It was speculated that exogenous molecules, such as glutamic acid, phenylalanine, glycine, and proline, could be responsible for this, as they can perform antioxidant activity, and they were present in the biostimulant formulation [111]. Research on pepper corroborates this effect of PHs on mitigating plant stress, as peppers under drought stress and early recovery showed lower levels of hydrogen peroxide, probably due to higher catalase activity or higher non-enzymatic antioxidant scavenger levels, which resulted in increased aboveground growth and yield [112]. Similarly, Leporino et al. [113] showed that Malvaceae-based PH-treated plants exhibit a high capacity for recovery after drought stress, and this capacity was speculated to be due to dipeptides containing glucogenic amino acids (Arg-Leu, Arg-Phe, Asp-Leu, Asp-Phe, Glu-Phe, Gly-Leu, PyroGlu-Val, Thr-Val, Val-Leu, and Val-Pro). As the plants exhibited photosynthesis-limiting conditions due to drought stress, glucogenic acids acted as a carbon source; moreover, signaling for ROS also decreased, which could be due to reactive oxygen-scavenging properties of some dipeptides [114]. Spinach plants under drought stress with the application of fish-based PH with kelp extract had higher activities of antioxidant enzymes (ascorbate peroxidase and guaiacol peroxidase), as well as increased contents of osmoprotectants (proline and total soluble sugars, increased by 20% and 37%, respectively), chlorophyll (14% increase), and carotenoids (up to 19% increase) [115]. All of this resulted in increased growth parameters in spinach plants under drought stress.

Pumpkin-seed PH reduced the effects of salt stress (induced by NaCl) in the common bean, where PH-treated plants showed increased activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX), helping the plants to tolerate the effects of salt stress [116]. This increased antioxidant capacity could be due to the high DPPH radical scavenging activity of the PH used. Higher levels of glutathione and reduced malondialdehyde (MDA) content in the follow-up treatment of plants with PH suggested that the applied PH provided an increase in antioxidant activity probably due to antioxidants present in the hydrolysate. In addition, the combined increase in proline and soluble sugar content helped adjust the osmotic potential of salt-stressed plants treated with PH. Relative water content (RWC) and membrane stability index were partially recovered by application of PH. Indeed, exogenous proline has been shown to increase RWC and cell membrane integrity in drought-stressed onion, along with the increase in osmoprotectants (free proline, soluble sugars, and total free amino acids) [117].

Combined drought and heat stress were alleviated by foliar application of plant-based PH on offseason crops—soybean, chili, and chickpea [118]. Here, the application of PH improved the membrane stability index, RWC, total chlorophyll, and proline content in leaves and, ultimately, yield (although the effects were dependent on the dose and on the plant culture). Ertani et al. [86] showed that the application of alfalfa-based PH on salt-treated maize plants stimulated flavonoid biosynthesis, visible as an increase in flavonoids that was consistent with the activity of phenylalanine ammonia lyase (PAL), which is an important enzyme in biosynthesis of phenolics. In this work, PAL has been shown to be sensitive to salt stress, while its gene expression and activity are affected by PHs under salt stress. This upregulation of PAL could be due to auxins in the PH, which could induce PAL expression (as shown by Schiavon et al. [119]), improvement of nitrogen assimilation, or, alternatively, a pathway that involves the proline-linked pentose-phosphate pathway through which the increase in proline would, in part, be responsible for the increase in phenolic compound content [120]. The possibility of stimulation of the PAL enzyme by PHs could be an important aspect of their application as PAL and phenylpropanoid biosynthetic pathway are activated under abiotic (such as drought, heavy-metal stress, heat stress, salinity, etc.) and biotic-stress conditions [121,122,123,124], resulting in the accumulation of various phenolic compounds. Different effects of PHs on plant root, shoot, and stress alleviation are summarized in Figure 4.

5. Protein Hydrolysates in Agriculture

The effects of different kinds of PHs have been extensively studied on many plant species, such as apple, basil, banana, celery, chickpea, tomato, grapevine, lettuce, maize, melon, rice, rapeseed, etc., with varying but generally beneficial observed effects [89,99,106,125,126,127,128,129,130,131,132]. An extensive list of effects produced by different types of PHs applied to various plant species is given in

Table 1. Effects of PH application on different plant species.

Plant Species	Type of PH	Application	Experimental Conditions	Effects	References
Lettuce (Lactuca sativa L.)	Legume-based (Trainer®)	Foliar	Greenhouse	Growth stimulation of epiphytic bacteria with plant growth-promoting activity and/or biological-control activity against pathogens	[7]
	Legume-based (Trainer®)	Foliar and root	Greenhouse	Higher yield and increase in SPAD index and photosynthetic parameters. Responses were cultivar-specific	[14]
	Animal-based (pig blood hydrolysate)	Foliar	Hydroponic system	Increased phenolic content, antioxidant activity, and upregulation of genes related to phenolic biosynthesis	[130]
	Legume-based (Trainer®)	Foliar	Pot trial in greenhouse	Increased nutrient-use efficiency, root dry weight, and leaf area. Dose-dependent positive effects on photosynthetic activity and root growth	[93]
	Legume-based (Trainer®)	Foliar and root	Pot trial in greenhouse	Improvement in nutrient use and uptake efficiency; increase in water-use efficiency, chlorophyll content, and antioxidant activity; and enhanced yield and quality	[133]
Pea (Pisum sativum L.)	Legume-based (Trainer®)	Foliar	Greenhouse	On average, 33% increase in shoot length in gibberellin-deficient plants; increase in plant biomass, SPAD index, and leaf nitrogen content	[87]
	Whey protein hydrolysate (hydrolyzed by papain and pepsin)	Foliar	Field trial	Improvement in nutrient status (N, P, and K); and increased content of photosynthetic pigments and, ultimately, yield	[134]
Tomato (Solanum lycopersicum L.)	Legume-based (Trainer®)	Fertigation	Greenhouse	Increase in SPAD index and leaf nitrogen content; and increase in shoot, root, and total dry biomass	[87]
	Sugar-cane molasses and yeast extract (CycoFlow®)	Fertigation	Field trial in tunnels	Significant stimulation of growth and number of fruits under heat stress, as well as increase in antioxidant content in leaf and fruit	[91]
	Legume-based (Trainer®)	Foliar	Field experiment	Increase in marketable yield (cultivar specific), increase net assimilation of CO2, improved nutritional status seen as increase in K and Mg, and increase in antioxidant activity and total soluble solids and bioactive molecules	[135]
	Legume-based (Trainer®)	Immersion	Laboratory bioassay	Auxin-like effect; and increase in root and shoot dry weight, root length, and area by 21, 35, 24, and 26%	[87]
	Vegetable-based, soy meal	Substrate drenching	Greenhouse, pot and plastic plug tray trials	Increased plant growth and fruit production; enhanced glycine content; and increase in expression levels of defense-related genes	[136]
	Legume-based (Trainer®)	Foliar and root	Pot trial in greenhouse	Enhanced shoot dry weight and marketable yield; and increased chlorophyll content and antioxidant activity	[133]
	Animal-based (PEPTON 85/16®) and seaweed-based (Acadian Suelo®)	Combination of foliar spray and irrigation	Field trial	Increases in plant height, stem diameter, leaf length, number of leaves per plant, root length and diameter, and yield. PEPTON 85/16 showed larger positive effect on yield than Acadian	[81]
Banana (Musa sp. Cv. G9)	Derived from chicken feathers	Foliar and root	Field trial	Increased content of proteins, amino acids, reducing sugars, total chlorophyll, and proline content. Promotes earlier flowering	[126]
Wall rocket (Diplotaxis tenuifolia L.)	Legume-based (Trainer®) and tropical-plant extract (Auxim®)	Foliar	Greenhouse	Enhanced growth and productivity; increased leaf dry matter content, oxalic and citric acids, concentrations of Ca and P, phenols and ascorbic acid, and antioxidant activity	[85]
Strawberry (Fragaria × ananassa cv. Diamante)	Animal-based (PEPTON 85/16®)	Soil injection	Field trial in tunnels	When exposed to cold stress, application of PH increased root biomass, earlier flowering, and early production of fruit	[90]
Grapevine (Vitis vinifera L.)	Legume-based (Trainer® and Stimtide®)	Foliar	Pot trial	Induction of changes in leaf proteome and metabolome, delay of physiological maturity, and maintained higher acidity in water-stressed plants	[129]
Persimmon (Diospyros kaki L.)	Animal origin, PH stabilized with calcium salts (Stressal®)	Irrigation	Orchard	Lower chloride uptake and resulting decreased level of necrosis and lower leaf-water potential under salt stress. Increased synthesis of proline, glycine betaine, and salt stress-response proteins	[137]
Spinach (Spinacia oleracea L.)	Legume-based (Trainer®) and animal based (Isabion®)	Foliar	Soilless cultivation system in climate chamber	Increased growth and yield, modulation of root architecture, and increase in N uptake and photosynthetic activity, but dependent on the available N levels	[138]
Chickpea (Cicer arietinum L.)	Animal-based, chicken feathers	PH mixed with soil	Pot trial	Increased germination rate, seedling growth, and nodule formation. Improved soil fertility (alteration of N, P, K, and C/N ratio)	[89]
Sweet basil (Ocimum basilicum L.)	Legume-based (Trainer®) and collagen-based Siapton®	Foliar	Greenhouse	Improvement of water-use efficiency, CO2 assimilation, and fresh weight with legume-based PH. Negative effects were observed using collagen-based PH at higher doses (decrease in photosynthetic parameters, growth, and biomass production)	[139]
Broccoli (Brassica oleracea L.)	Animal-based (Isabion®) and seaweed extract (Wokozim®)	Foliar	Field trial	Enhanced growth and yield, and increase in the content of antioxidant enzymes	[140]
	Vegetable-based	Foliar	Field trial	Increase in fresh and dry mass, head diameter, and broccoli yield	[141]
Olive (Olea europaea L.)	Vegetable-based (Sinergon Bio®)	Fertigation	Pot and field trial	Increased plant growth; higher leaf photosynthetic rates and stomatal conductance; and increased biomass of roots, stem, shoots, and leaves	[142]
Sweet potato (Ipomoea batatas [L.] Lam.)	Animal-based and whey-protein hydrolysate (hydrolyzed by trypsin)	Foliar	Field trial	Combination with potassium fertilization increased shoot dry weight; N, P, and K uptake; average tuber root weight; yield per plant; and marketable yield	[143]
Mung bean (Vigna radiata L.)	Animal-based, feathers	Soil application	Field trial	Increase in pod size and seed weight, and higher yields	[144]
Tea (Camellia sinensis L.)	Derived from chicken feathers	Soil drench	Trial in poly bags	Increase in plant height, leaf and dry biomass, root length and surface, dry root biomass, and chlorophyll a and b; increase in photosynthetic rate and stomatal conductance; and increase in leaf nitrogen, phosphorous, manganese, and copper contents	[83]
Wheat (Triticum aestivum L.)	Fish-based protein hydrolysate (Agromoree®)	Mixed with soil	Pot trial	Increase in wheat ears length, quantity of grains and seed weight, increase in the numbers of productive stems, increase in yield, increase in gluten content and mass fraction of protein	[145]
		Used as fertilizer	Field trial
	Whey-protein hydrolysate	Foliar	Field trial	Increase in grain yield, yield attributes, nitrogen accumulation, flag leaf area, and spike number	[146]
Maize (Zea mays L.)	Vegetable-based, alfalfa	Not specified	Hydroponic in climate chamber	Gibberellin- and auxin-like activity, enhanced plant growth and leaf sugar accumulation, and induction of enzymes involved in carbon metabolism	[32]
	Based on tanning residues	Root	Hydroponic in climate chamber	Enhancement of plant growth and microelement concentrations in maize seedlings; and increased activity of enzymes involved in nitrogen and carbon metabolism	[147]
	Plant-based, alfalfa	Root	Hydroponic in climate chamber	Increased activity of enzymes involved in nitrogen metabolism; and increase in plant biomass, antioxidant enzymes’ activity, and phenolics production	[86]
	Chickpea and Spirulina platensis	Root	Hydroponic in climate chamber	Auxin- and gibberellin-like activity, positive influence on plant growth and nitrogen assimilation, increased activity of peroxidase and esterase, and increased accumulation of micro- and macroelements	[80]
	Legume-based (Trainer®)	Immersion	Laboratory bioassay	Auxin-like effect and significant elongation of the coleoptile	[87]
Primrose (Primula acaulis cv. Danova F1)	Animal-based, anchovy head and viscera	Root	Pot trial	Increase in dry weight and leaf area, chlorophyll content, and root area	[84]
Petunia (Petunia × hybrida Hort.)	Animal-based, erythrocyte hydrolysate (Hydrostim®)	Foliar and root	Greenhouse	Depending on the mode of application, a significant effect on visual quality has been observed (increased number of flowers and leaves and leaf area), increase in total aboveground weight, P and K content, photosynthetic parameters, root length, and total root surface area number of root tips and crossings	[148]
Patchouli (Pogostemon cablin Benth)	Animal-based, feathers	Soil application	Poly bags, field trial	When used with 50% fertilizer, increased leaf area, dry weight, and chlorophyll content	[144]
Soybean (Glycine max [L.] Merril)	Animal-based (acid hydrolysis of collagen, alkalized with KOH)	Foliar	Pot trial	Increased nitrate, amino acid, and ureides content in soybean leaves; altered gene expression; and increased plant productivity	[149]
Hemp (Cannabis sativa L.)	Legume-derived	Foliar	Pot trial	Protective effect under saline irrigation, improvement of seed yield and residual biomass (fiber production)	[150]

.

Different modes of application of PHs, types of hydrolysates, and experimental conditions have been used to evaluate the effects of hydrolysates, and, likewise, many different species have been used. The most common types of application of PHs on plants were foliar and root application, while plant-based PHs were investigated more compared to animal-based ones. In many cases, the use of PHs has resulted in a biomass increase and yield increase. For example, wheat plants treated with rainbow trout-based PH were monitored for growth and yield. PH was used in a concentration of 3000–4000 L/ha in liquid form. The results obtained showed an increase in the length and quantity of wheat ears, the quantity of grains in the ear and seed weight, and a productivity increase of up to 6.3% compared to untreated control, while gluten content in seed increased a little over 5% [145]. These improvements in yield and quality parameters could indicate that a reduction in nitrogen fertilization could be achieved by using PH. Similar results were obtained for tomato and lettuce treated with plant-based PHs applied as a foliar spray or as root drench [133]. Root drenching showed better results, as yield, photosynthesis, water-use efficiency, and antioxidant activity were increased regardless of the N levels or plant species. The application of PHs in the form of a root drench increased lettuce dry weight by 31% and fruit dry weight by 22%, and the fresh marketable yields of lettuce and tomato by 21 and 32%, respectively. This indicates that root drenching could be an effective way of reducing N fertilizer inputs and leaching in greenhouse production and, hence, reducing the costs and, particularly, negative effects on the environment from leached N [151]. Whey PH, in combination with K fertilization, was tested for the effect on sweet potato productivity and quality [143]. The interaction of foliar-applied PH and potassium fertilization increased the dry shoot weight; N, P, and K uptake; and, ultimately, average tuber root weigh, yield, and marketable yield, with the most efficient bio-stimulatory effect of PH being when it was applied as a 0.20% solution.

Foliar application of tropical-plant extract (obtained by water extraction and fermentation of tropical plants) showed that it could represent an alternative to the exogenous application of synthetic hormones in sweet cherry cultivation, as it improved yield components (number of fruits per tree and fruit size; however, the effect depended on the cultivar) and qualitative traits such as soluble solids content, fruit firmness, anthocyanin concentration, post-harvest performances of the fruit, and resistance to cracking [152]. Similar results were obtained by Soppelsa et al. [125] on apple cv. Jonathan. The PH in this experiment significantly improved fruit quality, registered as improvements in intensity and extension of the red coloration at harvest (increase from 12.54 to 31.15 relative units, with a higher value meaning a more intense red coloration), as well as increased anthocyanin content in the skin (increase from 58.07 to 125.50 mg CGE 100 g⁻¹ DW). Fertilizer based on PH from chrome-tanned leather waste at a dose of 3 t/ha showed similar effects to commercial fertilizer-treated soybean plants, recorded as plant height, number of leaves, number of pods, and seed weight [153]. Kocira et al. [154] used the foliar application of commercial PH in concentrations of 0.3 and 0.5%, which modified the yield and quality of soybean during a three-year experiment. Yield was improved by 25%, with a 32% increase in the number of pods and seeds, 38% increase in plant height, and 34 and 74% increases in phenolic and flavonoid contents.

6. Conclusions and Future Prospects

Climate change and the growing population will challenge agricultural production to produce more food in unfavorable conditions. To overcome this, it will be necessary to move from traditional approaches and include new practices with a concept that is environmentally friendly. PHs offer an alternative to plant nutrition that is formulated from by-products. There are many factors that affect the final product in PH production, and there is a variety of protein sources that can be used with different techniques of production, all of which can influence the properties of PHs and the way they affect the plant. The beneficial effects of PHs are related to many processes of plant physiology, and the mechanisms that affect them are still not well understood. The use of -omics could greatly benefit the understanding of the underlying mechanisms, as well as the connections between them, by which PHs trigger plant responses. Are the effects of PHs based on their action alone, due to their hormone-like properties, or do they act as stimulants? Do PHs produced from different plants or animals have the same mechanisms by which they reduce ROS or affect plant nitrogen assimilation? These and other similar questions still need to be cleared up.

The increase in the acceptance of farmers towards the use of PHs has resulted in their increased production, the production of different formulations, the development of commercial products, and an increase in research based on different starting materials for hydrolysis and the investigations on the effect their use has on plant growth, development, and yield parameters. Studies show positive effects of PHs on different aspects of plant physiology and metabolism and their product yield and quality, and their use especially affects plant functioning under stressful conditions in a positive manner. Crops, and sometimes even cultivars, react differently based on way that PHs are applied, the timing of timing application, and the concentration applied. Further research is needed, especially to understand the underlying mechanisms by which the constituents of PHs affect the plant metabolism. The mechanisms of the root uptake of PHs are more elucidated than the mechanisms by which foliar-applied PHs enter and transport inside the leaves. Although there has been an increase in PH-related research in the last decade, the pool of species that have been subjected to intensive research on PHs’ effects is still pretty narrow.