Foliar Spray with Pepsin-and Papain-Whey Protein Hydrolysates Promotes the Productivity of Pea Plants Cultivated in Clay Loam Soil

Papain and pepsin-hydrolyzed whey protein (PAH and PEH, respectively) were prepared and characterized for its degree of hydrolysis, chemical constituents (amino acid and peptides) and antioxidant activity. A field experiment was conducted at El Salheya El Gedida City, Sharqia, Egypt, during the seasons 2019 and 2020, to investigate the biological action of the foliar spray of PAH and PEH on the growth and yield of pea plants cultivated in a clay loam soil. Foliar application of the papain and pepsin-hydrolyzed whey protein (PAH and PEH, respectively) at 1000 and 2000 mg/L was applied three times after 25, 35 and 45 days from planting. All protein foliar spray treatments had significant positive effects on the uptake of N, P and K, simultaneously increasing the contents of all the photosynthetic pigments (Chlorophyll a, Chlorophyll b and Carotenoids) in a concentration-dependent manner. The most conspicuous increase was seen in Chlorophyll b (105% increase), followed by Carotenoids (91% increase). Generally, the favorable increases caused by the second level of application (2000 mg/L) were nearly 2–3 times that of the low level (1000 mg/L). Pod growth and formation indicators, e.g., no. of pod/plant, pod length and no. of seeds/pod, responded more evidently to the hydrolyzed than the intact form of whey protein treatments. Hydrolyzed whey protein foliar spray treatments achieved significantly higher increases in the global field yield components of Pisum sativum plants than the intact form, where peptic hydrolysates were significantly superior to papain hydrolysate. The treatment PEH (2000 mg/L) can be recommended as the most effective bio-stimulating foliar spray treatment for higher plant productivity when applied 25, 35 and 45 days after planting.


Introduction
Modern agriculture is criticized for increasing environmental pollution, particularly in vegetable production systems where the soil fertilizer application has often been associated with this phenomenon [1]. To tackle these rising challenges, plant biostimulants have been the most promising effective technologies. They consist of natural substances, other than fertilizers and pesticides, that are capable of promoting plant growth, yield and quality when applied to the crop in low quantities [2,3]. Significant quantities of by-products generated from the industrial processing of agricultural and food products must be disposed of continuously. While the effective implementation of green chemistry principles is essential for reducing waste, it is impossible to eliminate waste output. Thus, efforts are currently   However, ESI-MS revealed big numbers of the peptidic components in the peptic WPH ( Figure 1 and Table 1), and 53 and 183 were detected in positive and negative ions mode, respectively.
The extent of protein modification by pepsin and papain was estimated by assessing the degree of hydrolysis (DH) and the data are presented in Figure 2. Papain-whey protein hydrolysates recorded the relatively lower degree of hydrolysis at all the time points of comparison, i.e., 5%, 8%, 11%, 15%, 21%, 25%, 30 and 32%. Pepsin-whey protein hydrolysates (PEH) obtained after 8 h degradation had the highest DH (48%). Lower degrees of hydrolysis were recorded at less incubation time periods, i.e., of 8%, 12%, 20%, 25%, 33%, 38%, and 42%, after 1, 2, 3, 4, 5, 6 and 7 h, respectively, after the same time periods, respectively. Free amino acids analysis identified the presence of 15 amino acid in the composition of PEH and PAH after 8 hydrolysis ( Table 2). The content of the hydrophobic amino acid residues (Gly, Ala, Val, Ile, Leu, and Phe) was around 31.9% of the total amino acids in PEH against 25.5% in PAH.
The hydrolysates were tested for the antioxidant activity using the DPPH radical scavenging activity method and the results presented in Figure 2 indicate a directly proportional relationship between the hydrolysis time, the degree of hydrolysis and the antioxidant activity. The maximum antioxidant activities of 500 µg hydrolyzed protein/mL of pepsin and papain after 8 h hydrolysis, reached 68% and 52%, respectively.

Field Experiment
The data in Table 3 present the uptakes of N, P and K by Pisum sativum plants under normal clay loam soil conditions, when treated with foliar spray of native whey protein (NAP), papain (PAH) or pepsin (PEH) hydrolyzed whey protein at two different concentrations; 1000 and 2000 mg L −1 , as compared to normal control (CNT). It is evident that all protein foliar spray treatments have significant positive effects on the uptake of the three elements in a concentration-dependent manner. It can also be noticed that the hydrolyzed forms of whey protein are more influential than the intact form, especially the peptic hydrolysate. This enhancing effect on the mineral uptake was most pronounced in the case of phosphorus, followed by nitrogen. The maximum enhancing effect on mineral uptake was recorded with PEH 2000 achieving relative increases, amounting to 269, 228 and 214 in P, N and K uptake, as related to the respective values of the control, respectively. This trend of mineral uptake was also reflected in the accumulation of the three minerals in the seeds of plants receiving foliar sprays of whey protein or its pepsin and papain hydrolysates. Generally, all treatments induced significant increases in the value of the three elements: N, P and K. Moreover, the increases induced by the hydrolyzed when protein were significantly higher than those that achieved the intact whey protein. In accordance with the trend of mineral uptake by the shoots, the plants treated with PEH 2000 foliar spray achieved the highest significant seed contents of P, N and K with increases over the control amounting to 335%, 298% and 259%, respectively.
The results in Table 4 reveal the photosynthetic pigment contents in Pisum sativum leaves after foliar spray with native, NAP, PEH and PAH, as compared to CNT. Protein spray treatments have significantly increased the contents of all the photosynthetic pigments (Chlorophyll a, Chlorophyll b and Carotenoids), as compared to the control in a concentration-dependent manner. The most conspicuous increases are seen in case of Chlorophyll b (105% increase), followed by Carotenoids (91% increase). It is also observable that the increase in the second level of application (2000 mg L −1 ) is nearly 2-3 times the increase induced by the low level of application (1000 mg L −1 ).  Mean values in the same column for each trait followed by the same lower-case italic letter is not significantly different according to Duncan's multiple range test at p ≤ 0.05. Table 5 presents the growth and yield parameters of Pisum sativum plants grown in clay loam soil and treated with a foliar spray of native and hydrolyzed whey protein at two different concentrations, as compared to the normal control. The plant growth parameters, plant height and leaf area responded positively to all treatments in a concentrationdependent manner, showing more responsiveness in the case of the protein hydrolysates than the intact one. The traits of pod growth and formation, no. of pod/plant, pod length and no. of seeds/ pod responded more evidently to the different whey protein treatments with more responsiveness to the hydrolyzed forms. PEH 2000 achieved maximum increases in the three mentioned traits of pod formation, amounting to 110%, 102% and 134% relative to the control, respectively. Table 5. Growth and yield parameters of Pisum sativum plants grown in poor clay loam soil and treated with foliar spray of native whey protein (NAP), papain-hydrolyzed whey protein (PAH) or pepsin-hydrolyzed whey protein (PEH) at two different concentrations; 1000 and 2000 mg L −1 , as compared to normal control (CNT). It is evident in Figure 3 that all protein spray treatments caused different increases in seed protein content compared to the control, receiving only foliar water spray instead of the protein solutions. The first level of foliar spray (1000 mg L −1 ) of whey protein, papain hydrolyzed whey protein (PAH) and pepsin hydrolyzed whey protein (PEH) caused increases in the seed protein content, amounting to 21%, 32% and 44% of the control, respectively, against 61%, 78% and 93% in the case of the second level (2000 mg L −1 ). The SDS-PAGE electropherograms of the different plant samples, receiving different kinds (NAP, PAH, and PEH) and concentrations (1000-2000 mg L −1 ) of foliar sprays of protein substances are not different from each other. It is evident in Figure 3 that all protein spray treatments caused different increases in seed protein content compared to the control, receiving only foliar water spray instead of the protein solutions. The first level of foliar spray (1000 mg L −1 ) of whey protein, papain hydrolyzed whey protein (PAH) and pepsin hydrolyzed whey protein (PEH) caused increases in the seed protein content, amounting to 21%, 32% and 44% of the control, respectively, against 61%, 78% and 93% in the case of the second level  The data in Table 6 depict the global field yields of Pisum sativum plants cultivated under normal clay loam soil conditions and treated with a foliar spray of native whey protein NAP, PAH and PEH at two different concentrations; 1000 and 2000 mg L −1 , as compared to normal control that received no foliar spray. It is evident that all intact or hydrolyzed whey protein foliar spray treatments achieved significant increases in field global yield parameters of Pisum sativum plants, i.e., the weight of 100 seed, Pods fresh weight, shoots, seed, and biological yield (dry weight). The pattern of effect is similar to that observed on the growth parameters, i.e., the whey hydrolysates were more effective The data in Table 6 depict the global field yields of Pisum sativum plants cultivated under normal clay loam soil conditions and treated with a foliar spray of native whey protein NAP, PAH and PEH at two different concentrations; 1000 and 2000 mg L −1 , as compared to normal control that received no foliar spray. It is evident that all intact or hydrolyzed whey protein foliar spray treatments achieved significant increases in field global yield parameters of Pisum sativum plants, i.e., the weight of 100 seed, Pods fresh weight, shoots, seed, and biological yield (dry weight). The pattern of effect is similar to that observed on the growth parameters, i.e., the whey hydrolysates were more effective than the intact whey protein and pepsin hydrolysate was the most effective treatment. Foliar spray of pepsin hydrolysate at 2000 mg L −1 (PEH 2000) recorded the maximum yield of dry seed weight incurring a relative increase about 105% over the control alongside with 75% increase in shoot dry weight and 90% increase in the biological yield over the control. These high increases may represent a considerable economic gain. Table 6. Global field yields of Pisum sativum plans cultivated under normal clay loam soil conditions and treated with foliar spray of native whey protein (NAP), papain-hydrolyzed whey protein (PAH) or pepsin-hydrolyzed whey protein (PEH) at two different concentrations; 1000 and 2000 mg L −1 , as compared to normal control (CNT) that received no foliar spray. Mean values in the same column for each trait followed by the same lower-case italic letter is not significantly different according to Duncan's multiple range test at p ≤ 0.05.

Discussion
Cow whey protein was hydrolyzed with one of two enzymes (pepsin and papain) for 8 h at their optimal conditions using the same enzyme/substrate ratio, as well as the hydrolysis times. Thus, the change in the degree of hydrolysis in the current study was dependent on the ability of every enzyme to hydrolyze whey proteins. A higher maximum degree of peptic hydrolysis (48%) occurred after 8 h. This variation between the two studied enzymes could be due to the slightly broader specificity of pepsin at pH 2 and its preferential cleavage beside the more frequent hydrophobic amino acids, unlike papain, which preferentially cleaves beside the basic amino acid. This influence of hydrophobic amino acids on the peptic hydrolysis was also proven by the result that methylated whey proteins were more susceptible to peptic hydrolysis [36] and is confirmed by more released free hydrophobic amino acids in the peptic hydrolysate. The less susceptibility of whey protein to papain may be due to its narrower specificity and to the hydrophobic nature of whey proteins in accordance with Reference [36], reporting that increasing hydrophobicity by esterification further limited the susceptibility of whey protein to trypsin, which is similar to papain. As both pepsin and papain are specific towards hydrophobic amino acids, the hydrolyzed fragments become exposed, facilitating electron transfer to radicals upon cleavage [37]. Based on this fact, the results indicated that whey protein hydrolysates (after 8 h) had a considerable potential antioxidant activity where pepsin hydrolyzed protein showed the highest one. Similar results were obtained when using microgranules enriched with protein [38,39].
The increased mineral uptake of N, P and K by foliar spray with whey protein and its enzymatic hydrolysate agree with previous results [40,41], testing the influence of some hydrolysates on the growth and yield of strawberry plants grown under limited nutrient supplies. The supply of these protein hydrolysates as free or bound amino acids may promote the uptake other minerals by osmotic mechanism or though electrostatic binding to these elements to the charges on the amino acids or the short peptides [20]. The reflection of the accelerated uptake of the three macro-nutrients on their accumulation in the seeds agree with [16] and may promote the seed nutritional value.
The observed concentration-dependent promoting action of foliar spray with either intact or hydrolyzed whey protein on the photosynthetic pigments of Pisum sativum and this effect was more pronounced, particularly in the hydrolyzed forms may refer to higher metabolic activity of the smaller protein moieties. These small moieties can be used as bio-stimulants or as building blocks in the synthesis of the photosynthetic pigments, in accordance with References [18,42,43]. The greater increases in the pigments by the high level of the applied protein (2000 mg L −1 ) may suggest their use as building blocks.
The noticed increases in the plant growth and yield parameter may be a direct reflection of the promoting action on the level of the photosynthetic pigments, which generally drives the whole machinery plant metabolism. Additionally, the direct provision of nitrogenous blocks from the intact or hydrolysate whey protein may have its influence on driving the anabolic processes inside the plants [18][19][20]. It can also be concluded that that more hydrolyzed proteins may respond more to the plant requirements of nitrogenous compounds as they may either directly or a after few transformation processes enter in the synthetic pathways supporting plant growth and maturity.
The recorded high protein content in the seeds of Pisum sativum plants in response to foliar spray with whey protein (1000-2000 mg L −1 ) in a concentration-dependent manner may indicate the action of this protein either in its native form or its enzyme-hydrolyzed ones to promote protein synthesis and accumulation in the plant seeds. The concentrationeffect may indicate that the sprayed protein substances are absorbed and assimilated by plants to build up its own proteins. This may confirm the potentiality of using protein or protein hydrolysates as a biofertilizer foliar spray. The higher increasing action on protein content by the hydroyzed protein may indicate that the plants use the simple peptides of free amino acids more efficiently than the intact protein molecules, which may need further processing to enter the protein synthesis machinery of the plants, in accordance with Reference [44]. The higher effectiveness of pepsin than papain hydrolysate may be due to the difference in the degree of hydrolysis and thus the quicker incorporation of the smaller peptide fractions in the protein synthesis than the longer ones, as well as the more available free amino acids [45]. In summary, spraying plants with pepsin-hydrolyzed whey protein can nearly double the protein content in the seed, which is quite a good result. The similarity of the SDS-PAGE electropherograms among the different plant samples, receiving different kinds may mean that foliar spray with these substances did not induce major changes in the pathways of the protein synthesis and accumulation in the seeds and their role is mainly to stimulate the process or providing bricks required for protein synthesis.

Materials
Cow milk whey was obtained from the Food Science Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt, and freeze-dried. Papain (from Carica papaya) and pepsin (from porcine gastric mucosa) were obtained from Sigma (St. Louis, MO, USA). All other used chemicals were of analytical grade.

WPH Production
Enzymatic hydrolysis was performed using papain or pepsin at their optimal conditions; pH 6 at 37 • C and pH 2 at 37 • C for papain and pepsin, respectively, as described by Reference [46]. The whey protein concentrate was dissolved in 0.1 M phosphate buffer and 0.1 M glycine-HCl buffer for papain and pepsin, respectively at 100 g/L and the enzyme was added at a ratio (w/v) of 1:200 (enzyme: substrate).

Degree of Hydrolysis Estimation
The degree of hydrolysis was measured every 1 h, during 8 h hydrolysis. At the end of hydrolysis, samples were heated in a boiling water bath for 10 min to inactivate the enzyme. The hydrolysate was centrifuged at 4000× g for 15 min and the supernatant was lyophilized and stored at −20 • C for further analysis or use.

Antioxidant Activity Evaluation
Whey protein hydrolysates (300 µg/mL) were evaluated for antioxidative activity after 1, 2, 3, 4, 5, 6, 7 and 8h. The DPPH radical scavenging activity was estimated as described by [48] with slight modifications. One milliliter of hydrolysate was combined with 3 mL 0.15 mM DPPH (in 95% ethanol), shaken vigorously using a mixer and incubated for 30 min in the darkness at room temperature before measuring color absorbance at 517 nm. Ethanol was used as a control. The radical scavenging capacity of the samples was estimated as the decrease in the color absorbance according to the following equation.

Free Amino Acids Estimation
Free amino acids were analyzed by reverse-phase HPLC after derivatization using diethyl ethoxymethylene manolate.

Field Experiment
A field experiment was conducted at El Salheya El Gedida City, Sharqia Governorate, Egypt (30.642045 • N, 31.862875 • E) during the seasons October 1st, 2019 and 2020 in clay loam soil, to investigate the impact of foliar spraying pea plants (Pisum sativum L., Master B) with native, papain-and pepsin-hydrolysed whey protein. The presented results were the means of these two seasons. The cultivated land was located in the east Delta with an average altitude of 1000 m above sea level. The weather during the 2019 and 2020 seasons in El Salhiya, Egypt, was nearly sunny with a maximum temperature of 18 • C and minimum temperature of 12 • C. Precipitation falling was in the range 8.8-9.1 mm, Wind was 8 km/h ENE, Humidity was 70%, Cloud 21% and pressure was about 1019 mb. The physical and chemical properties of used soil were evaluated according to References [49][50][51] and presented in Table 7.
The experiment followed a randomized complete block design in a factorial arrangement using three replicates. Pea seeds were planted in 10.5 m 2 plots (five rows); 3 m long × 0.7 m width. Plots of all treatments were fertilized with 30 kg P ha −1 as ordinary superphosphate (65 g P kg −1 ) and 100 kg K ha −1 as potassium sulphate (410 g K kg −1 ) before sowing. Mineral nitrogen was added at a rate of 50 kg N ha −1 as ammonium sulphate (205 g N kg −1 ) in two equal doses, after thinning and before the 2nd irrigation. During the experiment period water was applied to the crop through drip irrigation on three-day interval to meet the crop water requirement Seven treatments were prepared including the control receiving solely distilled water used in the other treatments. The other six treatments included native whey protein at 1000 and 2000 mg L −1 (NAP 1000 and NAP 2000), papain hydrolyzed whey protein at the same concentrations (PAH 1000 and PAH 2000) and also pepsin hydrolyzed whey protein (PEH 1000 and PEH 2000). These different treatments (0.6 and 1.2 kg PAH or PEH/600 L water/ha) were sprayed on growing pea plants at 25, 35 and 45 days after planting. One row (20 cm wide) was left between treatments as buffering area. At 70 days, ten plants were randomly selected (per year) from each treatment for the measurement of growth (plant height, leaf area, and pod length) and photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) according to Reference [52]. At harvest, plant samples were separated into shoot and pods, dried at 70 • C for 72 h, weighed, digested with concentration H 2 SO 4 /HClO 4 and analyzed for total nitrogen, phosphorus and potassium content [53]. Plant total nitrogen was determined using the micro-Kjeldahl method [53]. Plant potassium was determined by flame photometer [53] and plant total phosphorus was colourimetrically assessed using the ascorbic acid method [54]. Protein per cent "yield quality" in seeds was calculated by multiplying N% × 6.25 [55]. The relative change in the N, P, K-uptake of Pisum sativum plants, photosynthetic pigments, growth and yield parameters was calculated according to the following equation: The value o f the treatment − The value o f the control The value o f the control × 100 Statistical Analysis All of the obtained data were statistically analyzed(LSD at 0.05) according to the method described by Russell [56]. Significant statistical differences among means were compared at p ≤ 0.05 by Duncan's multiple range test. The analysis was implemented statistically by MSTAT C computer software, version 6.303 (Berkeley, CA, USA).

Conclusions
The considerable increases in the global field parameters are a direct reflection of the amelioration in the growth parameters previously mentioned, confirming the final positive impact of using whey protein hydrolysate as a foliar spray treating Pisum sativum plants grown in poor clay loam lands. The considerable highest increases in the global dry seed weight by the foliar spray with pepsin hydrolysate at 2000 mg L −1 achieved the maximum yield of dry seed weight agrees with the previous analysis on the plant growth traits. These high increases may represent a considerable economic gain and can be recommended as an efficient treatment to enable the cultivation of Pisum sativum in poor clay loam lands, while achieving considerable biological yield and dry seeds.