Biostimulant application of whey protein hydrolysates and potassium fertilization enhances the productivity and tuber quality of sweet potato

Utilizing biostimulants like protein hydrolysates is one of the most creative and promising approaches to improving nutritional efficiency, abiotic stress tolerance, or crop quality traits. In the present study, whey protein was hydrolysed with trypsin for 4 h at an enzyme/substrate ratio (1/300, w/w). The obtained whey protein hydrolysates (WPH) were chemically characterized, and their antioxidant activity was estimated. WPH, which was produced using trypsin for 4 hours and presented the highest antioxidant activity. Therefore, it was selected as a bio stimulant with potassium fertilization for enhancing the productivity and tuber quality of sweet potatoes. A field experiment was carried out during the two successive summer seasons, at a private vegetable farm in Faques City, Sharkia Governorate, Egypt, to study the effect of different potassium rates (50, 75, and 100 kg K2O/fad) and WPH at 0.10 and 0.20% as a foliar application compared to unsprayed plants (control). The interaction between K2O at 100 kg /fad and spraying with WPH at 0.15% increased shoot dry weight/ plant, N, P, and K uptake by shoots, yield/plant, marketable yield, and total yield/fad, as well as average tuber root weight. It was concluded that the most efficient bio-stimulating foliar spray treatment for increasing sweet potato productivity was WPH (0.20%).


Introduction Introduction Introduction Introduction
Sweet potatoes (Ipomoea batatas [L.] Lam.) are a common food that people eat daily. Due to its nutritional properties, adaptability to various edaphoclimatic conditions, and quick yield, this crop is important to society and the economy (Amaro et al., 2017). Poor fertility, particularly low levels of potassium (K), phosphorus (P), nitrogen (N), sulfur (S), and micronutrients, limit sweet potato productivity (Uwah et al., 2013). Potassium influences the quality and growth of vegetables by increasing biomass production and leaf area and reducing sugar concentration (Mukhongo et al., 2017). The dry weight of biomass is significantly increased by fertilizing sweet potato with potassium (Taffouo et al., 2017), boosting production and its components (Hasanah et al., 2021), and gave the best tuber root quality (Nedunchezhiyan et al., 2010). The development of systems that are both environmentally friendly and sustainable in order to meet the need to feed the world's growing population is one of agriculture's greatest obstacles (Anitha, 2020). Utilizing bio stimulants like protein hydrolysates from various sources is one of the most creative and promising approaches to addressing these significant issues El-Sanatawy et al., 2021;Osman et al., 2021a;Osman et al., 2021b). Regardless of its nutrient content, any substance or microorganism that is applied to plants with the intention of improving nutritional efficiency, abiotic stress tolerance, or crop quality traits is a bio stimulant, it is regulate or improve the physiological processes of plants (Du Jardin, 2015). Bio stimulants include peptides, vitamins, enzymes, substances with hormone-like effects (derived from algal extracts), antioxidants, humic and fulvic acids, silicon, and other minerals, as well as some strains of microorganisms (Almadi et al., 2020). Various studies have been carried out on the effect of protein hydrolysates as a bio stimulant on plant growth and production (Colla et al., 2015;Colla et al., 2017;Almadi et al., 2020;Shahrajabian et al., 2022). Some crops' yields have improved when bio stimulants are used in agriculture. However, there aren't many studies on how to use these products on crops like sweet potatoes, whose commercialized parts are the roots (Rós et al., 2015). In the present study, whey protein was hydrolysed with trypsin for 4 h at an enzyme/substrate ratio (1/300, w/w). The obtained whey protein hydrolysates (WPH) were chemically characterized, and their antioxidant activity was estimated. WPH, which was produced using trypsin for 4 hours and presented the highest antioxidant activity. Therefore, it was selected as a bio stimulant with potassium fertilization for enhancing the productivity and tuber quality of sweet potatoes.

Whey protein hydrolysates preparation and characterization
Trypsin was utilized for the enzymatic hydrolysis of WPH (buffer, 0.1 M phosphate buffer; pH, 8.8; temperature of 37 °C), and their optimal conditions are a ratio of enzyme to substrate of 1:300 w/w. Enzyme and substrate were thoroughly mixed. At the optimal temperature and with constant stirring, the mixture was incubated for four hours. To inactivate the enzyme, the mixture was then heated for ten minutes in a boiling water bath at 100 °C. Hydrolysate was clarified by centrifugation at 5000 xg for 15 min at 5 °C to remove insoluble substrate fragments, and the supernatant was lyophilized and frozen at -20 °C until further used (Abdel-Hamid et al., 2017). The percent of trichloroacetic acid (TCA) ratio was used to determine the degree of hydrolysis as described by (Hoyle and Merrltt, 1994). Degree of hydrolysis (DH) was calculated using the formula below: DH (%) = (Soluble nitrogen in TCA 10%/ Total nitrogen in the sample) × 100 Using the DPPH radical scavenging activity assay, the antioxidant activity of WPH (500 µg/mL) produced with trypsin at various times (0, 1, 2, 3 and 4 h) was estimated to determine the optimal time for producing antioxidant peptides (Göçer et al., 2011). The radical scavenging capacity of the samples was measured as a decrease in the absorbance of DPPH radicals, and it was calculated using the following equation: Radical scavenging activity (%) = [(A control-A sample)/A control] × 100 A= absorbance at 517 nm. Electrospray ionization mass spectrometry (ESI-MS) with positive and negative ions was used to analyse the protein hydrolysate with the highest antioxidant activity that had been obtained after 4 hours (Al-Mohammadi et al., 2020).
Plant growing conditions, (plant materials) treatments, and experimental design A field experiment was carried out at a private vegetable farm in Faques City, Sharkia Governorate, Egypt to investigate the effects of various potassium rates (120, 180, and 240 kg K2O/ha and whey protein hydrolysates (WPH) at 0.10 and 0.20% as foliar applications alongside unsprayed plants (as a control) on growth, yield, and tuber root quality of sweet potato cv. 'Buregard'. These treatments were arranged in a splitplot design in a randomized complete block design with three replications. Potassium rates were randomly distributed in the main plots, while the concentrations of WPH were randomly arranged in the sub-plots.
The physical and chemical properties of experimental soil in the two seasons showed that it was clay in texture and had 1.99 and 1.92% organic matter, 8.05 and 8.04 pH, 2.08 and 2.05 mmhos/cm EC, 8.41 and 8.92 ppm are available N, 0.048 and 0.044% available P and 0.93 and 0.89% available K, respectively.
Stem cuttings of sweet potato (about 20 cm length) were transplanted at 25 cm apart on 15 and 17 th April in the 1 st and 2 nd seasons, respectively. The experimental unit area was 12.6 m 2 . It contains three lines with 6m length each and 70 cm distance between each two lines. One line was used for taking the samples to measure the growth and chemical traits and the other two lines were used for yield determinations. Potassium rates were applied as soil application into three equal portions beginning 45 days after transplanting every two weeks in the form of potassium sulphate (48.5% K2O). Plants were sprayed with the different concentrations of WPH or water four times at 15 days intervals beginning 45 days after transplanting in both seasons. Each plot was received 2 L. solutions of each concentrations using spreading agent in all treatments to improve adherence of the spray to the plant foliage for increasing WPH absorption by the plants. The untreated plants were sprayed with water and spreading agent. One line was left between each two experimental plots without spraying as a guard row to avoid the overlapping of spraying salutation. All treatments received equal amounts of ammonium sulphate (20.5 % N) and calcium superphosphate (15.5% P2O5) at a rate of 480 and 360 kg/ha, respectively.

Sampling and measurements
One line was used for taking the samples to measure the growth and chemical traits in the two growing seasons to measure leaf area/plant and the dry weight of shoots (leaves and branches) and Nitrogen, phosphorus, and potassium in shoots were determined in both seasons according to the methods described by (Reynolds and toxicology, 1989) and N, P and K uptake by shoots were calculated (mg/shoot). At harvest time, (at 150 days after transplanting), all tuber roots of each treatment were classified into two grades (marketable and non-marketable roots), then weighed to determine the total yield/ha (ton). Marketable tuber roots have a weight of about 100 to 250 g, while non-marketable roots have a weight of less than 100 g or more than 250 g. In addition, the average tuber root weight was calculated. Potassium use efficiency (KUE) was estimated by dividing the yield/ha, by the potassium quantity/fad., and expressed as kg tuber roots/kg K2O according to Clark (CLARK and RB 1982). Quality characters of sweet potato (tuber yield, dry matter, nitrogen content, protein content, starch content, sugar content, potassium content, and carotene content) were recorded at the time of harvest.

Statistical analysis
The recorded data were subjected to the statistical analysis of variance according to Hoshmand (2017), and means separation was done according to Duncan (1955) test. Figure 1 depicts the percentage of whey protein hydrolysed after being treated for four hours with trypsin (E/S ratio of 1:300). Over time, the degree of enzymatic degradation gradually rises. Variations in DH are typically brought about by the enzymatic reaction time, which influences the breaking of peptide bonds. For hydrolysates obtained from whey protein by enzymatic hydrolysis using trypsin, antioxidant activity using the DPPH assay was assessed. The results are shown in Figure 1. The results recorded an increase in antioxidant activity along with an increase in the degree of enzymatic hydrolysis. Whey protein hydrolysate prepared with trypsin increased its DPPH radical scavenging activity by up to 50% with increasing DH (4 h).  The protein hydrolysate obtained after 4h with the highest antioxidant activity (50%) was subjected to electrospray ionization mass spectrometry (ESI-MS) with positive and negative ions for molecular weight estimation and results are presented in Figure 2 and Table 1. The hydrolysate was composed on 35 peptides for positive ion with the molecular weight ranging from 141 Da to 859 Da, and 35 peptides for negative ion with the molecular weight ranging from 159 Da to 802 Da.  Vegetative growth characteristics The effect of potassium fertilizer at different levels (120, 180, and 240 Kg K2O/ha) and foliar whey protein hydrolysate at different concentrations (0, 0.1, and 0.2%) on vegetative growth characteristics during the 2020 and 2021 seasons are presented in Figure 3A, and B. Figure 3A depicts the positive effects that potassium fertilizer had on sweet potato plants. The treatment that received 240 kg K2O/ha) had the highest values of vegetative growth characteristics (leaf area and dry weight of shoots). Figure 3B depicts the positive effects that WPH had on sweet potato plants. The treatment that received 0.2% had the highest values of vegetative growth characteristics (leaf area and dry weight of shoots). It is clear from Figure 3 that potassium played an important role in vegetative growth characteristics (leaf area and dry weight of shoots). The increasing rate of potassium fertilizers from 120 to 240 kg K2O/ha resulted in a significant effect on the vegetative parameters. Also, the same results were observed with WPH foliar application. The effect of interaction between potassium fertilizer at different levels (120, 180 and 240 Kg K2O/ha) and foliar application of WPH at different concentrations (0, 0.1, and 0.2%) on vegetative growth characteristics during 2020 and 2021 seasons are presented in Table 2. The highest value of interaction effect was recorded with treatment that received 180 Kg K2O/ha with 0.2% WPH in both seasons.

Minerals uptake (N, P and K)
The data in Figure 4 present the uptake of N, P, and K by sweet potato plants when treated with potassium fertilizer at different levels (120, 180, and 240 Kg K2O/ha) and foliar whey protein hydrolysate at different concentrations (0, 0.1, and 0.2%). Increased rate of potassium caused significant increment in N, P and K uptake in sweet potato shoots in both seasons. Plants that received 240 kg K2O/ha had the highest N, P and K contents in comparison with other treatments. Results illustrated in Figure 4 presented that WPH had a positive effect on N, P and K uptake in sweet potato shoots in both seasons. Increasing rat of WPH foliar application up to 0.2% resulted in a significant effect on minerals uptake.
The effect of interaction between potassium fertilizer at different levels (120, 180, and 240 Kg K2O/ha) and foliar application of WPH at different concentrations (0, 0.1, and 0.2%) on N, P, and K uptake (mg/plant) during 2020 and 2021 seasons are presented in Table 3. The highest minerals (N, P, and K) uptake values of interaction effect were recorded with treatment that received 240 Kg K2O/ha with 0.2% WPH in both seasons. Table 3. Table 3. Table 3. Table 3. Effect of interaction between potassium fertilizer at different levels (120, 180, and 240 Kg K2O/ha) and foliar application of WPH at different concentrations (0, 0.1, and 0.2%) on N, P, and K uptake (mg/plant) during 2020 and 2021 seasons Treatments Treatments Treatments Treatments N uptake N uptake N uptake N uptake P uptake P uptake P uptake P uptake K uptake K uptake K uptake K uptake K K K K2 2      Yields and its components of sweet potato Effect of potassium fertilizer at different levels (120, 180, and 240 Kg K2O/ha) on yield and its components (marketable, unmarketable, and total yield, weight of root and yield/plant) of sweet potato during 2020 and 2021 seasons are presented in Figure 5. Marketable yield, and total yield (ton/ha) were significantly increased with increasing potassium rates from 120 to 240 K2O (kg/ha). Increasing the K rates from 120 to 240 kg/fed increased marketable and total yield from 24 to 32 tons/ha and from 26 to 33 tons/ha, respectively 9 in the 1st season. the same trend was observed in the 2nd season. Unmarketable yield (ton/ha) was significantly decreased with increasing potassium rates from 120 to 240 kg K2O/ha in both seasons. In the 1 st season, the weight of root/plant increased significantly from 115 to 127 g/plant when the K rates were increased from 120 to 240 kg /ha, while in the 2 nd season increased significantly from 111.9 to 121.8 g/plant when the K rates were increased from 120 to 180 kg/ha and nonsignificant differences was observed between 180 and 240 kg K2O/ha. Also, the same results were observed for yield/plant in both seasons.    The results illustrated in Figure 6 presented that WPH at different concentrations (0.1 and 0.2%) had a positive effect on marketable yield, total yield, the weight of root/plant, and yield/plant when compared to control. On average, foliar application of WPH up to 0.2% significantly increased marketable yield from 24 ton/ha to 32 tons/ha compared to control in both seasons. The same trend was observed in the total yield, the weight of root/plant, and yield/plant. Unmarketable yield (ton/ha) was significantly decreased with increasing WPH levels from 0 to 0.2% in both seasons.
The effect of interaction between potassium fertilizer at different levels (120, 180, and 240 Kg K2O/ha) and foliar application of WPH at different concentrations (0, 0.1, and 0.2%) on yield and its components of sweet potato during 2020 and 2021 seasons are presented in Table 4. Results reported that in Tables 4 showed that the highest total yield, the weight of root/plant, and yield/plant was recorded with when K was applied at the rate of 240 Kg/ha. With WPH at rate of 0.2% in both seasons. Mean values in the same column for each trait followed by the same lower-case letter is not significantly different according to Duncan's multiple range test at p ≤ 0.05.

Tuber root quality
Data in Table 4 showed that there are significant increases in dry matter by application of K fertilization at rats of 120, 180, and 240 Kg K2O/ha. The dry matter of tuber root was increased from 21.17 g at 120 kg/ha to 23.19 g, and 24.99 g for 180, and 240 kg/ha, respectively for the 1st growing season and from 23.55 g to 26.99 g and 28.4 g for the same treatments at the second growing season. Data also showed that application of K fertilization significantly increased starch, total sugar, carotene, and K content for the two growing seasons. In the 2nd season, total protein was significantly increased with increasing K fertilization rats while, in the 1st season nonsignificant differences was observed between 180 and 240 kg/ha. Quality of sweet potato tubers expressed as dry matter, total protein, starch, total sugar, carotene, and K content was increased with increasing WPH foliar application rats (Table 5). Mean values in the same column for each trait followed by the same lower-case letter is not significantly different according to Duncan's multiple range test at p ≤ 0.05.
The effect of interaction between potassium fertilizer at different levels (120, 180, and 240 Kg K2O/ha) and foliar application of WPH at different concentrations (0, 0.1, and 0.2%) on tuber root quality during 2020 and 2021 seasons are presented in Table 6. The highest total protein was recorded with when K was applied at the rate of 180 Kg/ha. With WPH at rate of 0.2% in both seasons. The highest values of dry matter, starch, and total sugar were recorded when K were applied at 240 kg/ha with WPH at 0.2% in both seasons. The highest value of carotene was obtained at 240 kg/ha with WPH at 0.1, and 0.2% in both seasons. The highest amount 12 of K content was recorded at 180 kg/ha with WPH at 0.2% in the 1st season, and at 240 kg/ha with WPH at 0.2% in the second season. Mean values in the same column for each trait followed by the same lower-case letter is not significantly different according to Duncan's multiple range test at p ≤ 0.05.

Discussion Discussion Discussion
Sweet potatoes (Ipomoea batatas [L.] Lam.) are a common food for human use. Due to its nutrition qualities, adaptability to various edaphoclimatic conditions, and high yield within a short period of time, this crop is of significant socioeconomic value (Da Silva et al., 2022). Fertilization with potassium (K) is an essential step in the formation of sweet potatoes. K is necessary for photosynthesis, transport of sugar, movement of water and nutrients, synthesis of protein, and formation of starch in sweet potatoes (Harvey et al., 2022). This study found that increasing the potassium fertilizer rate promoted vegetative growth as measured by leaf area and shoot dry weight. This can be accounted for by the idea that raising K improves N uptake. Previous reports by El-Baky et al. (2010) and Cecílio Filho et al. (2016) noted the increase in vegetative development and strong yield response to K fertilization. In the present study, marketable, total yield, weight of root and yield/plant were increased with increasing the rate of potassium fertilizer. This result could be as a result of potassium's crucial involvement in increasing photosynthate synthesis and delivery to roots (Mengel and Kirkby, 1987). According to El-Baky et al. (2010), the critical function of potassium in the energy status of the plant, the translocation and storage of assimilates, and the maintenance of tissue water relations can all be ascribed to rising roots production of plants as a result of increasing potassium application rates. In the current study, increased potassium levels significantly increased the uptake of N, P, and K by sweet potato shoots during both seasons. According to the research of Clarkson and Hanson (1980) this might be caused by the plant's high K nutrient mobility. Based on the beneficial impact of translocation of assimilates, it is possible to explain the observed improvement in the fruit quality metrics (total protein, starch, total sugar, carotene, and K content) as altered by potassium nutrition (Mengel, 1997). Today's agriculture must continually overcome obstacles to produce more and better food for a population that is expanding in a climate that is changing. Agricultural practices must develop novel approaches to managing crop nutrition, crop productivity and plant health. It is also required to develop novel chemicals or biological agents that can enhance plant yield (by controlling plant physiology, metabolism, and crop performance) as well as agro-product quality. Agents having these qualities have been proposed for sustainable agriculture throughout the past ten years. It has been demonstrated that 13 these substances, which are referred to as biostimulants, enhance plant nutrition, quality, yield, and abiotic tolerance in a variety of agricultural crops (Moreno-Hernández et al., 2020). In the present study, whey protein was hydrolysed with trypsin for 4 h at an enzyme/substrate ratio (1/300, w/w). The obtained whey protein hydrolysates (WPH) were chemically characterized, and their antioxidant activity was estimated. WPH, which was produced using trypsin for 4 hours and presented the highest antioxidant activity, Therefore, it was selected as a biostimulant with potassium fertilization for enhancing the productivity and tuber quality of sweet potatoes (Kosaric and Asher, 2005). Trypsin hydrolysis of whey proteins produced peptides having biological properties (Ballatore et al., 2020). With 6%-7% total solids, whey is a considerable by-product of the cheesemaking process. Whey proteins, a soluble by-product of cheese production, make up almost 20% of all milk proteins (Kosaric and Asher, 2005). In the present study, the responses of vegetative growth, yield and roots quality of sweet potato were statistically significant with increasing WPH foliar application. Protein hydrolysates have been shown experimentally to increase plant root and shoot biomass, increasing crop productivity in both greenhouse and open field settings for a variety of crops, including tomato, lettuce, kiwifruit, papaya, pepper, lily, passionfruit, pea, wheat, and corn (Colla et al., 2017;El-Sanatawy et al., 2021;Osman et al., 2021b). By improving nutrient use efficiency, protein hydrolysates can assist plants in performing their functional functions more effectively when nutrients are scarce (Planques et al., 2012). As a result, it has been demonstrated that the foliar application of protein hydrolysates from various sources (animal and plant) increases the vegetative development and productivity of various fruit trees. Recently, it was suggested that protein hydrolysates could be a novel and effective way to bio-stimulate plant metabolism and production by improving nutrient absorption and inducing physiological and molecular processes that lessen the effects of various abiotic stresses . The following are effects of biostimulation activity may be due to: (i) the release of key enzymes involved in N assimilation and C metabolism (citrate synthase, malate, and isocitrate dehydrogenase); (ii) increased promoted auxin and gibberellin-like activities; and (iii) increased antioxidant enzymatic activity, pigment biosynthesis, and production of secondary metabolites Sestili et al., 2018).

Conclusions Conclusions Conclusions
K plays a vital role in sweet potato production. In the current study, increased potassium levels significantly increased the uptake of N, P and K by sweet potato shoots during both seasons as well as vegetative growth and tuber quality. Whey protein hydrolysates as a bio stimulant with potassium fertilization was subjected for enhancing the productivity and tuber quality of sweet potatoes. The recommended dose for enhancing sweet potato vegetative growth, yield, and tuber quality was recorded at 240 Kg K2O/ha with 0.2% WPH.
Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable.