Changes in Biochemistry and Yield in Response to Biostimulants Applied in Bean ( Phaseolus vulgaris L.)

: Biostimulants are preparations that favorably impact the growth, development, and yield of plants. The research objective was to examine the e ﬀ ect of the frequency of use of Kelpak, Terra Sorb Complex and Fylloton biostimulants on improving the yield and nutritional properties of beans. Been seeds (variety Oczko) were sown in the ﬁrst week of May in 2015, 2016, and 2017. During the growing season, Fylloton (1%), Terra Sorb Complex (0.5%), and Kelpak (1%) biostimulants were applied by single (BBCH 12-13) and double spraying of plants (BBCH 12-13, BBCH 61). All variants of treatment with biostimulants were compared with the control. Single application of Kelpak had a positive e ﬀ ect on increasing the number of pods. The double application of Kelpak increased the number and yield of seeds and protein contents. Double application of Fylloton increased the number of seeds, and application of Terra Sorb Complex increased the protein content in the beans. Application of all biostimulants increased the ﬂavonoid content. Biostimulants containing seaweed (Kelpak– Ecklonia maxima extract) or amino-acid extracts (Fylloton– Ascophyllum nodosum extract and amino acids or Terra Sorb Complex–amino acids) increased the seed yield, while improving its quality by increasing the content of protein, polyphenols, and ﬂavonoids. It was found that the double application of Kelpak biostimulant stimulated the yield and quality of beans to a greater extent. yield of bean variety Oczko. Fylloton; of Fylloton; TS_1, single spraying of Terra Sorb Complex; TS_2, double spraying of Terra Sorb Complex; single spraying of double spraying of study


Introduction
Agricultural production seeks technological solutions to improve the quality of yields. Therefore, biostimulants are increasingly popular as preparations that favorably impact the growth, development, and yield of plants [1,2], and they are safe for humans and environmentally friendly at the same time. Du Jardin [3] defines biostimulants as "any substance or microorganism applied to plants with the aim of enhancing nutrition efficiency, abiotic stress tolerance, and/or crop quality traits, regardless of its nutrient content". In addition, in order to develop legal provisions regarding the registration of biostimulants based on their specificity of operation, the European Biostimulants Industry Council (EBIC) was created. Currently, however, their registration is based on legal provisions on fertilizers and pesticides, and, for some of them, there is a marketing gap in many European Union (EU) member states [3][4][5][6]. responsible for stimulating the growth and development of the root system, which in turn contributes to better uptake and translocation of macro-and microelements, and it is associated with a significant increase in crops [48].
Biostimulants based on protein products and protein hydrolysates consist of a mixture of peptides, animal or vegetal amino acids, and single amino acids. Amino acids are a building material for proteins, but they are also precursors of phytohormones. They are involved in the synthesis of, e.g., vitamins, enzymes, terpenes, amines, purines, pyrimidines, and alkaloids [49,50]. They also play an important role in the process of pollination and fruit formation [51]. The application of exogenous amino acids, which are active in metabolic signaling (glutamate, histidine, proline, glycine, betaine), induces plant defense mechanisms by increasing their resistance to abiotic stress factors [8,52]. Due to the presence of specific peptides and precursors of phytohormone biosynthesis (tryptophan, which is the main precursor of IAA (Indole-3-acetic acid) biosynthesis and bioactive peptides), protein hydrolysates affect the hormonal balance of plants, which is related to stimulating plant growth [53]. The use of these biopreparations positively impacts the quality of agricultural produce, increasing the content of carotenoids, flavonoids, polyphenols, and ascorbic acid [54][55][56][57], and reducing the amount of undesirable compounds, e.g., nitrates [56].
The beneficial effect of biostimulants with seaweed extracts or amino acids on plant growth and development, and on the quantity and quality of the yield, regardless of its developmental stage, was confirmed in numerous studies [13,34,35,[39][40][41][57][58][59][60][61][62]. An interesting solution is to combine these two components in a single preparation, as in the case of the Fylloton biostimulant (Biolchim Poland), which contains the extract of Ascophyllum nodosum (L.) Le Jolis, as well as vegetal amino acids. The composition of this preparation includes Ascophyllum nodosum extract, amino acid complexes of vegetal origin 37.5%, organic nitrogen 6%, organic carbon of biological origin 11%, and organic substance 35%.
Bean is an economically important legume that is sensitive to low temperatures in the early stages of its development and flowering. The use of biostimulants that positively affect the metabolic processes occurring in the plant, especially in the time of climate change, which causes stress factors for this sensitive plant, can be one of the elements contributing to the improvement in the quantity and quality of bean yield. Plant response to the biostimulant often depends on the variety, as demonstrated in earlier studies [15,39,59,60]. There are also no reports regarding the reaction of two-colored coat seed of bean to treatment with biostimulants. In view of the above and based on the importance being given to the improving crop yields, the research objective was to investigate the effect of the use of Kelpak, Terra Sorb Complex, and Fylloton biostimulants on improving the yield and nutritional properties of common bean (Phaseolus vulgaris L.) variety Oczko.

Plant Materials and Growth Conditions
The research material came from field studies carried out in the year 2015-2017 in Perespa (50 • 66' north (N); 23 • 63' east (E)), Poland, on common beans (Phaseolus vulgaris L.), variety Oczko. The experiment was established in a random block system, in four replications, on an area of 10 m 2 . The experiment was established on an alkaline (pH in 1 M KCl-7.4) soil of the brown rendzina subtype. Soil fertility level was as follows: phosphorus medium (12.6-14.2 mg P 2 O 5 in 100 g of soil), potassium medium (15.3-17.1 mg K 2 O in 100 g of soil), and magnesium medium (6.2-6.8 mg Mg in 100 g of soil).
In each year of research, the forecrop for common beans was winter wheat. Tillage for bean was carried out in accordance with good agricultural practice [63]. Pre-winter plowing was performed in the first week of November. In the spring, soil treatment combined with mineral fertilization was performed. Mineral fertilizers were in the following doses: 30 kg N·ha −1 , 60 kg P 2 O 5 ·ha −1 , and 120 kg K 2 O·ha −1 . Mineral fertilization was used at a constant level throughout all experimental combinations. Beans of common bean, variety Oczko (with a red and white bean coat), were sown with a mechanical precision bean drill in the first week of May (2 May in 2015, 2016, and 2017) at a depth of 3-4 cm, in rows 45 cm apart, using 30 plants per 1 m 2 . Biostimulants Terra Sorb Complex, Kelpak, and Fylloton were used during the growing season, according to the experiment design (Table 1), and the obtained results were compared with the control, in which pure water was used for double spraying the plants. In individual years of research, biostimulants were used in time frames dependent on the development phase of plants, as shown in Table 1. Plants single sprayed with the biostimulant in BBCH 12-13, in the second period (plant stage of BBCH 61), were sprayed with pure water. The plants were sprayed using a GARLAND FUM 12B backpack sprayer. The Lechler LU 120-03 atomizer was used, at a working pressure of 0.30 MPa, using 300 L of the working liquid per 1 ha. All variants of treatment with biostimulants were compared with the control, where plants were treated with the same volume of water (no biostimulant was applied). No pesticides were used in the cultivation, as pathogens, pests and weeds did not exceed the damage threshold. Plants were weeded manually. The average temperature and rainfall during the bean growing season are shown in Table 2. The weather station (W200P, Vector Instruments Ltd., Rhyl, UK) was located in the experimental field, in which the experiment was carried out, at 210 m above sea level.
After harvesting plants in the third week of August (22 August 2015; 27 August 2016; 24 August 2017), 20 plants were randomly selected from each plot, and the number of pods, number of seeds, seed yield, and weight of one thousand seeds was determined. The beans obtained from each plot were dried, ground in a laboratory mill, and sieved with a 0.310-mm sieve. The flours were stored at −20 • C and used for further chemical analysis.

Determination of Polyphenols
A ground sample of bean seeds of 0.25 g was weighed, to which 4 cm 3 of extraction solution (acetone:water:hydrochloric acid 70:29:1) was added. The solutions were shaken for 1 h. Then, 100 µL of distilled water and 0.4 mL of Folina-Ciocalteu reagent were added to 100 µL of extract, and, after 10 min, 2 mL of 10% Na 2 O 3 solution was added. After 30 min, the absorbance of methanol was measured at λ = 725 nm. The polyphenol content was calculated in mg·100 g −1 , from the gallic acid calibration curve (1 mg·mL −1 ).

Determination of Flavonoids
A ground sample of bean seeds of 0.25 g was weighed, to which 4 cm 3 of extraction solution (acetic acid:methanol 1:19) was added. The solutions were shaken for 1 h. Then, 0.1 mL of a 2% AlCl 3 ·6H 2 O methanolic solution was added to 1 mL of the extract, together with 1.4 mL of CH 3 COOH methanolic solution (1:19). The sample was then incubated at 20 • C for 30 min. Absorbance was measured at λ = 425 nm against methanol. The flavonoid content was calculated in mg·100 g −1 , from the calibration curve for quercetin (0.2 mg·mL −1 ).

Determination of Proteins
The protein content of bean extracts was determined using the Bradford reagent, according to the method of Redmile-Gordon et al. [57] with modifications. The Bradford reagent (150 µL) was applied on a microplate and 50 µL of assay or standard protein (BSA, bovine serum albumin) was added. The samples were shaken at room temperature for 15 min. Absorbance at 595 nm was measured using an Epoch Microplate Spectrophotometer (BioTek-USA). The resulting protein was expressed in mg·g −1 of dry weight (DW).

Statistical Analysis
The statistical analysis was performed using the Statistica 10PL program by StatSoft ® . The normal distribution of variables was tested using the Shapiro-Wilk test. The one-way (for 2015, 2016, and 2017) and the two-way (for average 2015-2017) analysis of variance was used. The significance of the mean was determined using the Tukey test, at a significance level of p < 0.05.

Results
Based on the two-way ANOVA analysis, the effect of the number of applications and biostimulant treatment on seed yield, number of seeds, and phenol content in bean seeds was found ( Table 3). The effect of biostimulant treatment on the number of pods, the weight of one thousand seeds, and the content of proteins and flavonoids in bean seeds were demonstrated. The interaction of biostimulant treatment with its number of applications regarding the impact on the number of seeds, as well as the content of protein and phenols in bean seeds, was found. Table 3. Two-way ANOVA of number of applications and treatment of bean variety Oczko (average 2015-2017). Treating plants with the biostimulants had a positive effect on increasing the number of pods (Table 4). In 2015, no significant effect of the biostimulants on the studied trait was identified; however, a tendency to increase the number of pods was observed after foliar application of biostimulants, especially Kelpak. On the other hand, in 2016, after treating the plants with biostimulants based on seaweed extract, i.e., both after a single application of Kelpak (Ecklonia maxima extract) and after a single or double application of Fylloton (Ascophyllum nodosum extract, amino acids), a significant increase in this trait was identified (increases by 36%, 28%, and 33%, respectively, as compared to the control). Treatment of plants with Kelpak in 2017 significantly increased the number of pods, regardless of the number of applications (increase by 18% for a single application, and by 19% for a double application, as compared to the control). A synthesis of the three years of research (2015-2017) confirmed that a single spraying of plants with the Kelpak biostimulant in the BBCH 12-13 phase significantly increased the number of pods (by 26%), as compared to the control. Analysis of variance showed that, regardless of the number of applications, the treatment of plants with Fylloton significantly increased the number of seeds in 2015, as did the double spraying with Kelpak (increases by 29%, 34%, and 32% respectively, as compared to the control) ( Table 4). In the second year of research, the best results were obtained after double spraying with biostimulant based on the extract of Ecklonia maxima, with a significant increase of 37%, as compared to the control. In 2017, it was found that spraying plants with biostimulants containing seaweed extracts, i.e., Fylloton and Kelpak, significantly increased the number of seeds, by 30% and 27%, respectively, as compared to the control. The average of three years of research demonstrated that an increase of this trait, by 31% and 32%, respectively, as compared to the control, was obtained after a double foliar application of Fylloton and Kelpak.

Degrees of Freedom
Double application of Kelpak, in 2015 and 2016, had the most beneficial effect on seed yield, increasing this trait by 22% and 38%, respectively, as compared to the control (Figure 1). In 2017, the best effects in increasing the bean crop were obtained after double treatment of plants with Fylloton and Kelpak biostimulants, when a 25% increase of this trait was obtained, as compared to the control. A synthesis of the three years of research demonstrated that double spraying plants with Kelpak Analysis of variance showed that the use of biostimulants in bean cultivation resulted in a reduction of the weight of one thousand seeds (Figure 2). In the first year of research, a significant increase in the weight of one thousand seeds was obtained in the control, by 11%-15%, as compared to the combination with Fylloton or Terra Sorb Complex. However, in 2016 there were no significant differences in the weight of one thousand seeds between the combinations that included the biostimulants and the control. Plants in the control plot in 2017 were characterized by a higher weight of one thousand seeds by 8%, as compared to the double application of the Terra Sorb Complex. In turn, the average of three years of research confirmed that the highest weight of one thousand seeds (an increase of 9%, as compared to the double use of Fylloton) was obtained in the control.  Analysis of variance showed that the use of biostimulants in bean cultivation resulted in a reduction of the weight of one thousand seeds (Figure 2). In the first year of research, a significant increase in the weight of one thousand seeds was obtained in the control, by 11%-15%, as compared to the combination with Fylloton or Terra Sorb Complex. However, in 2016 there were no significant differences in the weight of one thousand seeds between the combinations that included the biostimulants and the control. Plants in the control plot in 2017 were characterized by a higher weight of one thousand seeds by 8%, as compared to the double application of the Terra Sorb Complex. In turn, the average of three years of research confirmed that the highest weight of one thousand seeds (an increase of 9%, as compared to the double use of Fylloton) was obtained in the control.
Foliar application of biostimulants increased the protein content in the seeds (Table 5). In 2015 and 2017, the best effects were obtained after applying Terra Sorb Complex as a single or double spraying of plants, which increased this trait by 12% and 13% (in 2015), and by 9% and 10% (in 2017), respectively, as compared to the control. Double treatment of plants with the Terra Sorb Complex biostimulant in 2016 had the most beneficial effect on increasing the protein content by 13%, as compared to the control. A synthesis of the three years of research showed that, regardless of the number of applications, the use of an amino acid-based biostimulant significantly increased the protein content, as did the double application of the biostimulant containing the of the Ecklonia maxima extract (11%, 12%, and 10% increases, respectively, as compared to the control). to the combination with Fylloton or Terra Sorb Complex. However, in 2016 there were no significant differences in the weight of one thousand seeds between the combinations that included the biostimulants and the control. Plants in the control plot in 2017 were characterized by a higher weight of one thousand seeds by 8%, as compared to the double application of the Terra Sorb Complex. In turn, the average of three years of research confirmed that the highest weight of one thousand seeds (an increase of 9%, as compared to the double use of Fylloton) was obtained in the control.   Double application of biostimulants containing seaweed extracts most favorably impacted the content of phenols in the seeds (Table 5). In 2015, an increase in the phenolic compound content in beans was noted after a double application of Fylloton (an increase of this trait by 82%, as compared to the control). However, after a single application of Kelpak, this trait was found to be reduced, as compared to the control. In turn, in 2016 and 2017, the best effects in increasing the phenol content were observed after a double application of Kelpak (increases by 42% and 41%, respectively, as compared to the control). No significant differences were found for this trait in the average for the years 2015-2017, but only a tendency to increase after a double application of biostimulants containing seaweed.
The application of preparations containing seaweed had the most beneficial effect on increasing the flavonoid content in the seeds (Table 5). In the first year of research, this characteristic was increased by 87%, as compared to the control, after a double application of Kelpak. In 2016, a single application of Fylloton had the most beneficial effect on the increase of the flavonoid content (by 98%, as compared to the control). On the other hand, in 2017, a significant increase in the flavonoid content was noted both with a single application of Fylloton and double of Kelpak (increases by 90% and 82%, respectively, as compared to the control). The average of three years of research showed that the application of all biostimulants significantly increased the flavonoid content, by 64%-88%, as compared to the control.

Discussion
The results of our research show a positive effect of natural biostimulants on bean yield, as well as on its quality. A more beneficial effect in modifying the yield components (number of pods and seeds, seed yield) was obtained after using biostimulants based on seaweed extract, especially upon double application of the Ecklonia maxima (Kelpak) extract. Only in the case of the weight of one thousand seeds was a reduction of this trait observed as a result of using biostimulants. The use of other biostimulants, i.e., Fylloton, which contains an Ascophyllum nodosum extract and amino acids, as well as Terra Sorb Complex containing amino acids, also had a positive effect on the yield components. In turn, the conditions favorable for setting pods and seeds, and increasing the weight of seeds were the most beneficial in 2016, when the double use of Kelpak resulted in the largest increase in these characteristics compared to control. This year, there were also favorable temperature and humidity conditions conducive to flowering, and setting pods and seeds. The significant increase in protein content was most positively influenced by weather conditions in 2015 and 2016, when the highest value of this feature was obtained after double application of Terra Sorb Complex. The application of biostimulants based on marine algae had a positive effect on the flavonoid content in all years of research. In 2015, however, a significant increase in polyphenols was found after double application of Fylloton.
Numerous studies conducted on arable crops confirmed the beneficial effect of seaweed extracts on increasing yield components [11,12,47,[64][65][66]. Previous research on other bean and soybean varieties confirmed the stimulating effect of the Ecklonia maxima or Ascophyllum nodosum extracts on the number of pods and seeds, and the weight of beans [39][40][41]67]. The lack of effect of Kelpak on the one-thousand-seed weight of bean was also confirmed by previous studies on common bean [66]. Use of extracts from Kappaphycus alvarezii and A. nodosum increased the number of pods, seeds, and yield in soybean [68,69], even under stress conditions (reduced NPK fertilization) [70]. Bean plants reacted favorably to the foliar application of Caulerpa racemosa and A. nodosum extract by increasing the number of pods and seeds, one thousand weight of bean, and seed yield in common beans [71,72], mung beans [73], and broad beans [74].
The foliar application of amino acids positively affects the yield of many plants, even growing under stress [11,75,76]. In previous studies, the studied bean responded positively to foliar application of the Terra Sorb Complex; however, the yielding effect depended on the variety, concentration, and number of applications of the biostimulant, as well as climatic conditions prevailing in a given study year [59]. The Aura variety (with white seeds) responded more favorably to a single application of a 0.5% concentration, and the Toska variety (with red seeds) responded more favorably to a single application of a 0.3% concentration of this biostimulant. The plants increased the number of pods and seeds, as well as the seed yield; however, no effect of this biostimulant was found for the weight of one thousand seeds. Foliar application of biostimulants containing amino acids increased the number of pods and seeds, weight of one thousand seeds, and yield in seeds of beans [71,77], peas [78], and broad beans [79,80].
The positive effect of seaweed extracts on plant growth and development and, as a result, on the increase in yield is undoubtedly associated with the presence of phytohormones, especially cytokinins [81]. Together with auxins, cytokinins regulate many physiological processes, including those affecting plant growth and development [82][83][84]. Aremu et al. [85] and Masondo et al. [86] also observed a positive effect of Kelpak on increasing the content of cytokinins. In addition, Kulkarni et al. [87] found an increase in the content of cis-zeatin, dihydrozeatin, and isopentenyladenine after using the Ecklonia maxima extract (Kelpak). The many active substances and compounds included in Kelpak suggest that it is not only cytokinin that is responsible for the growth and development of plants, but also probably the cross-reactions of these compounds with other bioactive molecules included in biostimulants that are based on seaweed extracts [70].
Thanks to the content of endogenous auxins, seaweed extracts have a positive effect on the growth and development of the root system [88]. This improves the uptake of water and nutrients and, in effect, stimulates the growth and development of plants, contributing to the improvement of yield quantity and quality [81]. The application of biostimulants based on seaweed extracts also has a positive effect on plant growth and development due to the content of gibberellins (GA1, GA3, GA4, GA5, GA6, GA7, GA13) [14], which affect seed germination, stem elongation, leaf expansion, and flower and seed development [89][90][91], as well as of gibberellin-like substances, e.g., terpenoids and tocopherol [92,93]. In the seaweed extract (Kelpak), the presence of brassinosteroids, brassinolide and castasterone [10], was identified. As phytohormones, brassinosteroids promote cell division and elongation, stimulate stem and root growth, and initiate flowering and flower development, as well as fruit development and increases in seed yield. Under stress conditions, they protect plants against abiotic and biotic stress [94,95].
The positive effect of amino acid-based preparations on plant growth and development probably results from the fact that, at the molecular level, they stimulate the plant's defense response to biotic and abiotic stress factors [96]. The amino acids contained in them are easily absorbed by plants. They participate in the synthesis of a number of organic compounds and affect the uptake of macro-and microelements [97]. Garcia et al. [98] showed that foliar application of amino acids and peptides together with nutrients increases the content of potassium, calcium, magnesium, iron, copper, and zinc in leaves, affecting their nutritional condition and promoting improved growth and development of plants. Applied on a leaf, preparations of this type exhibit phytohormone-like effects, comparable to that of auxin and gibberellin [54]. They also contribute to increasing the content of phytohormones (gibberellins, cytokinins, auxins) [76]. In addition, the use of protein hydrolysates in plant cultivation has a beneficial effect on the uptake of water and nutrients, resulting in increasing crop yielding thanks to the increased microbial and enzymatic activity of the soil, improved mobility and solubility of microelements (iron, zinc, manganese, copper), modified structure of the root system (its length, compaction, and number of lateral roots), or the increased synthesis of nitrate and glutamine reductase, as well as the activity of iron reductase [11,54,[99][100][101][102]. Numerous reports confirmed that protein hydrolysates, such as auxins and gibberellins, have hormone-like effects, stimulating root and shoot growth. This, in turn, has a positive effect on crop productivity [11,53,54,100,[102][103][104][105][106].
In our research, the use of biostimulants based on amino acids and seaweed extracts had a positive effect on the nutritional value of beans by increasing their protein content. Application of seaweed extract and amino acids had a positive effect on increasing the protein content in bean, pea, and faba bean seeds [59,71,74,78,80]. This was confirmed by Rouphael [62]; in their research, the increase in protein content in spinach plants was obtained after using biostimulants containing an extract of Ecklonia maxima and Ascophyllum nodosum and legume-derived protein hydrolysate. The protein content of legumes leaves was also determined by seaweed extracts. Numerous authors found an increase in this trait after the application of Ulva rigida, Fucus spiralis, Hypnea musciformis, and Colpomenia sinuosa extracts in bean leaves [16,107,108]. However, the use of biostimulants is not always beneficial for the protein content in beans [15,109]. Schubert and Mengel [110] demonstrated that amino-acid uptake is an important mechanism for recovering carbon and nitrogen that was lost in the rhizosphere. Ertani et al. [100] found that the stimulation of nitrogen assimilation is responsible for accelerating the growth and metabolism of nitrogen in plants treated with protein hydrolysates. This is due to an increase in the activity of two key enzymes, nitrate reductase and glutamine synthetase, thereby contributing to increasing protein firmness.
After the application of biostimulants containing seaweed extracts, researchers observed an increase in the phenolic content. Ertani [56] showed that the treatment of plants with biostimulants stimulates numerous metabolic pathways in plants. The pathways are also associated with the synthesis of secondary metabolites, including phenolic compounds, which play an important role in protecting plants against stress factors. In turn, a frequent indicator of plant resistance to biotic factors is the content of phenolic compounds, which are precursors to more complex phenolic structures, such as flavonoids and lignins [60,111]. The presence of bioactive compounds in biostimulants, including phytohormones, amino acids, protein, and phenols, is responsible for the physiological response of plants treated with these preparations [56,112,113]. Eckol (phenolic compound) found in seaweed extracts affects the phenylpropanoid pathway in the biochemical synthesis of phenolic acids [114]. Aremu et al. [85,115] showed that the timing of eckol plants increases the content of phenolic compounds, such as p-hydroxybenzoic and ferulic acids. In turn, the Kelpak application increases the content of caffeic acid, ferulic acid [85,116], protocatechuic acid, p-hydroxybenzoic acid, gentisic acid, p-coumaric acid, and trans-cinnamic acid in Eucomis autumnalis [85]; however, the content of phenolic compounds depended on the biostimulant concentration. In turn, Rouphael et al. [62] found an increased content of phenolic compounds after the treatment of plants with biostimulants containing alginians, fucoidans, and laminarins that affect endogenic hormonal homeostasis [117]. Treatment of plants with eckol also had a positive effect on the flavonoid content, increasing the amount of kempferol in plants several times. However, foliar application of Kelpak increased the content of kaempferol in tubers and entire plants, as well as taxifolin in leaves of Eucomis autumnalis [85,115]. Increasing the content of bioactive compounds in plants treated with biostimulants is associated with a mechanism that includes the stimulation of the chalcone isomerase enzyme, involved in the biosynthesis of flavanone precursors [117].
Paul et al. [118] found that tomato plants treated with protein hydrolysates were characterized by a higher content of, e.g., low-molecular-weight phenolic compounds, phytohormones (polyamines), hydroxy-carotenoids, poly-hydroxy fatty acids, and membrane lipids (glycoand phospholipids). They suggest that the metabolic changes caused by treating plants with protein hydrolysates can be correlated with a relatively small number of processes that converge toward the ROS-related (reactive oxygen species-related) plant signaling network. Increasing the content of secondary metabolites, such as phenols and carotenoids, which play a key role in protecting plants against oxidative stress [62,102,119], suggests fine-tuning of ROS signaling in plants after the application of protein hydrolysates [118]. In addition, the use of animal protein hydrolysates had a positive effect on the content of protein, phenols, and flavonoids in bananas [120], and vegetal protein hydrolysates stimulated an increase in the content of phenolic compounds and anthocyanins in grape [55].
So far, there are few reports confirming the beneficial effect of a preparation consisting of a combination of seaweed extract and amino acids. In our research, combining the Fylloton biostimulant, which induces the extract's effect, with Ascophyllum nodosum and amino acids increased the yield components, particularly the number of seeds after its double application. Moreover, previous studies, conducted on three soybean varieties, confirmed that this preparation has a positive effect on the number of pods and seeds, as well as seed yield [40]. The application of Fylloton in the cultivation of winter oilseed rape positively influenced the increase in the number of pods, the yield of seeds, and the weight of one thousand seeds, especially after applying the biostimulant together with the Perfektmikro micronutrient fertilizer containing EDTA-chelated (ethylenediaminetetraacetic acid-chelated) manganese, copper, iron, and zinc, as well as molybdenum, boron, and nitrogen [121].

Conclusions
All studied biostimulants had a positive effect on quantity and quality of bean yield. Double application of Kelpak biostimulant (Ecklonia maxima extract) stimulated morphological features and seed yield, as well as the content of polyphenols and flavonoids in seeds to a greater extent. In turn, the biostimulant containing amino acids (Terra Sorb Complex) significantly increased the protein content in beans. In contrast, Fylloton containing Ascophyllum nodosum extract and amino acids had also a more favorable effect on the number of seeds. In 2015 and 2017, biostimulants containing seaweed extracts had the most beneficial effect on bean yield and its quality. On the other hand, in 2016, treatment of plants with Kelpak had a more beneficial effect on the studied features. Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflicts of interest.