Utilization of Protein Hydrolysates from Animal Waste for the Production of Biostimulants in Wheat Cultivation (Triticum aestivum L.)

The dynamic growth of the world’s population requires a steady increase in the production of crops for human and animal consumption [1]. It is estimated that the world’s population will reach 9.1 billion by 2050, 34% more than today. To provide sufficient food and nutrition for such a large population, global cereal consumption will need to increase from 2.6 billion tonnes to 2.9 billion tonnes by 2027 [2]. It is beyond dispute that wheat, maize and rice represent the most significant members of the cereal family, which encompasses a range of crops employed for human consumption, animal fodder and industrial purposes. [1]. Wheat (Triticum aestivum L.) is one of the world’s most important cereal crops. It is a primary food source for many societies, and as a source of straw it is a significant contributor to animal feed. [3]. Wheat ranks third (after maize and rice) in world cereal production. According to the Food and Agriculture Organization of the United Nations, world wheat production reached 760 million tonnes in 2020 [4]. Europe’s leading position in wheat production is due to the extensive area designated to wheat and the high yield potential of this cereal [5]. In Poland, winter wheat is the most important cereal grown, which contributed to the selection of this cereal for the study. According to data from of the Central Statistical Office for 2021, the total area designated to wheat in Poland was 2.39 million hectares, with 2.17 million hectares to winter wheat [6]. Sustainable food production is crucial for the global economy. Ensuring food access for all is one of humanity’s greatest challenges.

With increasing urbanisation, higher crop yields will be achieved mainly by improving productivity per unit area rather than by increasing the area under cultivation. This is one of the reasons why new compounds and techniques are being sought to improve plant growth and yield. A good example is the use of environmentally friendly substances such as biostimulants, which can affect the metabolism of plants, thereby improving the efficiency of nutrient uptake, as well as root and shoot growth thus increasing yields [7,8,9,10,11,12]. One type of biostimulant is based on protein hydrolysates. There are several methods to produce protein hydrolysates: enzymatic, chemical and thermal hydrolysis of a variety of animal and plant biomass [13,14,15]. In the context of implementing sustainable development principles in agriculture, the recovery of valuable biopolymers such as keratin, collagen and elastin from waste materials produced by the meat, tanning and fish industries has become particularly important [16]. The disposal of animal waste is a global environmental problem. The leather industry generates approximately 6 million tonnes of waste per year, and 23% of the meat sector’s production is wasted, including 20% at the production stage. Its efficient use can reduce negative environmental impacts and provide raw materials for the production of biopolymers [17,18]. Collagen and keratin and its hydrolisates serve as valuable sources of essential amino acids that can promote plant growth. Some of the studies described that tested the influence of protein hydrolysates showed that they increased plant biomass and stimulated the productivity of fruits and vegetables [19,20]. Other work has shown that protein hydrolysate-based biostimulants can improve the production efficiency of cereals such as winter wheat or maize [12,21]. The integration of crop protection products and biostimulants in agriculture offers economic and organisational benefits, as well as the potential for fewer treatments. Efforts are being made to enhance protein biostimulants with bioactive compounds.

Selected bioactive ingredients such as salicylic acid derivatives or organic titanium salts can be added to enhance the effect of biostimulants. Salicylic acid is an endogenous growth regulator involved in the regulation of physiological processes in plants [22]. Woznica et al (2014) confirmed the beneficial effect of salicylic acid and its derivatives in reducing the effects of abiotic stresses on plant growth, development and yield [23]. Titanium is also considered to be a beneficial element for plant growth [24,25]. The effect of titanium on plants is a multifactorial process, and it is therefore necessary to select the appropriate dose of fertilisation with this element in order to achieve optimum results. This element has been shown to improve plant performance by stimulating the activity of selected enzymes, increasing chlorophyll content and nutrient uptake, and improving plant productivity [24].

In accordance with the principles of sustainable agriculture, it is imperative to implement strategies that mitigate the environmental impact of agricultural practices while promoting the efficient and responsible utilisation of resources. It is noteworthy that any treatment to protect, maintain or fertilise crops increases production costs. Implementation of the combined use of plant protection products and biostimulants allows better organisation of work, saving of energy materials and significant reduction of production costs [26]. In addition, numerous studies have shown that foliar application is more effective than soil application [27,28]. Therefore, the combined application of foliar biostimulants with crop protection products is advantageous because it reduces production costs while improving the efficacy of crop protection products [29]. As a result, pesticides can be used at minimal doses [26,30,31]. These issues are of particular significance in the context of seeking pro-ecological methods to enhance the yield of economically important crops in a way that aligns with the principles of sustainable development.

In this work, the effects of three biostimulant formulations differing in the content of collagen or keratin hydrolysates and active ingredients such as sodium salicylate and titanium ascorbate were evaluated. The main objective was to analyse the effect of the simultaneous application of a fungicide and a biostimulant on the growth of winter wheat, as well as that on the macro- and micronutrient content of the soil and the amino acid content of the plants. According to current knowledge, the effect of simultaneous foliar application of biostimulants and fungicides on the growth and nutrient uptake of winter wheat has not yet been studied in detail.

The effect of the tested foliar preparations on selected growth parameters, such as the length of wheat shoots and roots, is shown in Figure 2.

Fig. 2.

Length of winter wheat shoots and roots in response to foliar application of the protein preparation (vertical bars indicate the mean ± SE; n = 3)

Grow box experiments showed that the application of the new products influenced the increase in seedling length (9.6%, 10% and 15.9% for the application of preparations 1, 4a & 8, respectively) compared to the control group without the biostimulant. This confirms the slight biostimulatory properties of the formulations tested. This is most evident for sample 8 (with collagen and keratin hydrolysates and the addition of sodium salicylate), where the value of the length of the wheat sprout was equal to 31.3 ± 3.8 cm, while that of this parameter for the control was approximately 27.2 ± 2.8 cm. The value of this parameter for sample 4a (with collagen hydrolysate and the addition of titanium ascorbate) and sample 1 (with collagen hydrolysate and the addition of sodium salicylate) was 29.6 ± 2.7 cm and 29.7 ± 2.2 cm, respectively. This means a slightly higher effect of sample 8’s composition on winter wheat growth compared to samples 1 and 4a. In addition, minimal effects of the biostimulants tested on root length measurements were observed. The length of the roots varied slightly from 20.5 ± 1.5 cm in the control experiment to 21.7–22.6 cm in the other cases. In addition, foliar application was found to be an effective method of delivering biostimulants to plants, which has been confirmed in several reports [27,28,29]. This study has shown that it is possible to use biostimulants and fungicides simultaneously. It seems necessary to select an appropriate dose of biostimulant No. 8 to achieve statistically greater differences in seedling and plant root growth. Similar observations can be found in literature [26,30]. In order to describe the effect of the biostimulants on plant growth more precisely, the fresh shoot and root weights of winter wheat were determined (Figure 3).

Fig. 3.

Fresh weight of the shoots and roots of the winter wheat in response to foliar applications of a protein preparation (vertical bars indicate the mean ± SE; n = 3)

It was observed that the application of the new products slightly increased the fresh shoot weight (7.4%, 7.4 cm and 9.0%, respectively, for the application of formulations 1, 4a & 8) compared to the control group without a biostimulant. The most pronounced effect was observed for formulation 8, with collagen and keratin hydrolysate and the addition of sodium salicylate. However, in order to achieve statistically greater differences in crop yield, it seems necessary to select an appropriate dose of biostimulant No. 8. In addition, a minimal effect of the biostimulants tested was observed on root fresh weight measurements. This parameter ranged from 61.6 g to 67.3 g for the preparations tested and was equal to 67.0 g for the control. The results obtained for formulation 8 are most likely due to the biostimulatory properties of the substances used. Salicylic acid has been shown to be an important molecule that can influence a number of different processes in plants, including seed germination [37], ion migration, growth rate and regulation of photosynthesis [38]. Woznica et al. (2020) investigated the influence of a salicylic acid formulation on the stimulation of winter wheat growth [23]. The formulation was found to reduce the dynamic surface tension and contact angle of spray droplets, resulting in improved wetting of hydrophobic surfaces. At the same time, many studies focusing on the biostimulatory properties of salicylic acid have confirmed the influence of this molecule on prolonged flag leaf viability in winter wheat and a significant increase in grain yield [39,40]. Titanium is also considered to be a beneficial element for plant growth [24,25]. It has been confirmed that titanium has a stimulating effect only in carefully selected doses [24]. Kováčik et al. (2018) studied the effect of the application of titanium ascorbate on the chlorophyll content in leaves and the yield of grain and straw of winter wheat [41]. The results show that both the first and second (repeated) application of Mg titanate stimulated the growth of winter wheat and tended to increase the content of total chlorophyll in leaves. In addition, the higher single dose had a more positive effect than a lower single dose [40]. The role of titanium in the plant’s metabolism is not yet fully understood. It is possible that the biostimulatory effect of titanium ascorbate in preparation 4a could be improved by using higher doses in a future study. Biostimulants containing these molecules are known to have positive effects on various plants by increasing their biomass and boosting the natural immunity of plants to biotic stresses [34].

Collagen and keratin hydrolysates derived from animal waste containing peptides, oligopeptides and a mixture of free amino acids (e.g. glutamic acid, alanine, leucine, proline and hydroxyproline) also play an important role in stimulating the growth of cereals, including winter wheat [18]. Popko et al. (2018) investigated the influence of new plant growth stimulants based on selected amino acids upon, for example, yield and the macro- and micronutrient content in winter wheat. The active ingredient was a mixture of amino acids with short peptides obtained by acid hydrolysis of keratin material. Field trials showed that foliar biostimulation with an amino acid-based preparation increased grain yield in winter wheat by 5.4% and 11%, depending on the type of preparation [12]. It has been confirmed that exogenously applied amino acids can influence biological processes that act as signalling molecules in the regulation of plant growth and development [15]. Colla et al. (2015) found that amino acids as well as small peptides are absorbed by leaves and roots and then transported within the whole plant [42]. Mironenko et al. (2022) showed that liquid protein hydrolysate can contribute to an increase in the length and number of wheat ears and seed weight [43]. At the same time, many studies describing research on amino acid biostimulants have shown that they increase the efficiency of conventional mineral fertilisers [44]. In connection with these reports, the amino acid content of wheat seedlings harvested on the 31st day of cultivation was examined. This allowed us to assess whether the application of biostimulants containing peptides, oligopeptides and a mixture of free amino acids affected the amino acid content in the plants tested. The results of the amino acid analysis are summarised in Table 2.

The results show that the amino acid content of plants treated with biostimulants increased slightly. The application of biostimulant 8 resulted in a notable increase in amino acid content in wheat seedlings, with the majority of amino acids exhibiting higher levels compared to the control trial. It is worth noting that with the application of biostimulants 1 and 8, increased amounts of amino acids leucine and isoleucine were observed in wheat seedlings, 3.01 g/kg and 1.62–1.63 g/kg, respectively. Due to the low amount of these amino acids in wheat protein [45], the results obtained may indicate a potentially good direction in wheat cultivation. The levels of aspartic acid and threonine were also found to be higher in plants treated with biostimulants. The aspartic acid content was highest in the trial after biostimulant 1 treatment and was 5.63 g/kg. In contrast, the highest threonine content was found in plants after the application of biostimulant 8 (1.83 g/kg). Both aspartic acid and threonine promote the germination and uniform emergence of cereals, further influencing the development of the root system [12]. Analyses showed that alanine, valine and arginine levels also increased in the plants after the application of each of the biostimulants tested. The alanine content in the samples tested was 2.39–2.51 g/kg. Alanine stimulates chlorophyll synthesis, and is involved in the hormone metabolism and resistance to low temperatures [46,47]. On the other hand, valine, which increases plant resistance to stress conditions [48], was determined to be 2.18–2.27 g/kg. An increase in arginine content was also observed in plants treated with biostimulants to a level of 2.21–2.45 g/kg. The increase in arginine content in wheat seedlings is important because this amino acid is involved in resistance to low temperatures and is a precursor of polyamines [46].

In addition, the concentrations of selected micro- and macro-nutrients in wheat shoots and soil were analysed (Table 3, Table 4). Analysing the results presented in Table 3, it was found that the application of the new biostimulants had no or little effect on the mineral content in the leaves of winter wheat, with the exception of preparation No. 8 (containing collagen and keratin hydrolysates with the addition of sodium salicylate). However, none of the biostimulants applied had a significant effect on the concentration of N, P, K, Mg and Ca in winter wheat seedlings. The contents of sodium, phosphorus, potassium, magnesium and calcium in all samples tested ranged from 6.76% to 6.85%, from 1.18% to 1.27%, from 7.19% to 7.46%, from 0.28% to 0.29% and from 0.65% to 0.68%, respectively. Compared to the control (winter wheat without foliar biostimulation), winter wheat treated with formulation No. 8 showed higher contents with respect to its dry mass of boron (5.86±1.95 mg/kg), iron (112.0±35.3 mg/kg), manganese (128.0±23.9 mg/kg), zinc (52.4±18.7 mg/kg) and sodium (110.0±33.9 mg/kg). This means that there was an increase of 34.8% for iron, 13.3% for manganese, 7.2% for zinc and 4.8% for sodium. With regard to the controls, the mineral content in the leaves of variants 1 and 4a was characterised by lower contents of the elements listed, except for manganese. Of particular note is the high iron content of the wheat leaves used in formulation 8. Iron is one of the micronutrients essential for proper plant growth and development. Maintaining adequate levels of iron in the plant is extremely important for the functioning of key processes such as photosynthesis, cellular respiration, nucleotide metabolism and chlorophyll synthesis, which, in turn, translates in agriculture into obtaining adequate biomass and nutritional quality of crops [49]. It can therefore be concluded that the results of the pot trials showed that only biostimulant 8 increased the levels of selected minerals, including iron, in wheat seedlings. A similar relationship was also found by Popko et al (2015). These authors demonstrated that the use of this fertiliser increased the content of Mn by 386%, Zn by 177% and Fe by 21% [50]. The increased efficacy of preparation No. 8 is therefore related to the presence of amino acids obtained as a result of the hydrolysis of keratin and collagen. Amino acids are known to be good chelating agents and act as carriers of microelements [12].

Table 4 shows the average content of microelements in the soil as a function of the preparation applied. Analysing these results, it was found that the application of the new biostimulants had little or no effect on the mineral content of the soil after winter wheat cultivation. Salinity values in all samples ranged from 1.91 to 2.46 g KCl/kg. Concentrations of elements such as P, K and Mg were slightly too high due to the type of soil used. The contents of copper and iron were lower in the soils of the stimulated plants (preparation No. 8) than in the soils of the unstimulated plants, by 13.8% and 4.5%, respectively, but these differences were not statistically significant. It can only prove the slightly increased uptake of these components from the soil, which is necessary to produce increased plant biomass. On the other hand, manganese ranged from 4.41 to 5.92 mg/kg, zinc from 3.47 to 5.04 mg/kg, and boron from 2.52 to 5.93 mg/kg for the samples tested. Organic carbon and nitrogen contents also did not differ significantly among the soil samples, ranging from 44.2 to 50.4% and 1.05 to 1.15%, respectively. The results substantiate the value of such studies in evaluating the compatibility and practicality of combining biostimulants and crop protection products.

The implementation of sustainability principles in agriculture is becoming a requisite practice due to the increasing degradation of ecosystems and the depletion of natural resources. Consequently, any attempt to reuse waste products in line with the principles of a circular economy is gaining importance. Research into the possibility of recovering valuable biopolymers from animal waste and attempts to use them as growth promoters for crops is aligned with this trend. As foliar fertilisation has recently become increasingly important in plant growth and development, the use of protein hydrolysates derived from animal waste in the formulation of these preparations appears to be a significant avenue of research. These formulations are environmentally friendly and can partially supplement the effects of the nutrients applied. The study demonstrated that the new products had a beneficial impact on the growth of wheat (Triticum aestivum L.) seedlings, as evidenced by increases in both the plant length and fresh weight. The strongest effect was observed with formulation 8, which contained collagen and keratin hydrolysates and sodium salicylate. This formulation increased the nutrient and amino acid content of the seedlings compared to the control. The results demonstrated a positive impact of the treatments, suggesting their potential as an added-value and additional method for stimulating plant growth. Certainly, further research is needed to determine the effect of higher concentrations of the individual components of the preparations tested on winter wheat growth. With increasing environmental awareness and advances in biotechnology, the use of protein hydrolysates from animal waste in plant cultivation is likely to grow.