Biostimulants combination enhance the metabolites content of Brassica oleracea var. botrytis curds cultivated in the desert region of Basrah governorate

WuroodHantooshNeamah1✉Emailwurood.neamah@uobasrah.edu.iq

ZainabA.Al-sudani2

FatimahAliHasan1

FanarHashumAl-Hashemi3

Medicinal and Aromatic Plants Unit, College of AgricultureUniversity of Basrah61001BasrahIraq

2Horticultue and Landscape Design, College of AgricultureUniversity of Basrah61001BasrahIraq

3Horticultue and Landscape Design, College of Agriculture and ForestryUniversity of Mosul41002MosulIraq

Wurood Hantoosh Neamah¹, Zainab A. Al-sudani², Fatimah Ali Hasan¹, Fanar Hashum Al-Hashemi³

¹Medicinal and Aromatic Plants Unit, College of Agriculture, University of Basrah, 61001Basrah, Iraq

²Horticultue and Landscape Design, College of Agriculture, University of Basrah, 61001Basrah, Iraq

³Horticultue and Landscape Design, College of Agriculture and Forestry, University of Mosul, 41002 Mosul, Iraq

Corresponding author: wurood.neamah@uobasrah.edu.iq

Abstract

Among Brassica vegetables, cauliflower (Brassica oleracea var. botrytis) is widely recognized as a consumer favorite due to its rich and healthy content of nutrients and bioactive compounds. The enhancement of productivity and quality of preferred crops has gained highly importance in recent decades, driven by increasing customers demand for affordable and healthy food. Away from chemical materials and their risky side effects, biostimulants and biofertilizers could be a suitable alternative for traditional fertilizers under desert conditions. In a current study the extract of Liquorice root LRE and bread yeast BYE applied both with 0, 10, and 20 g/L on Brassica plant to explore their influence in curds metabolites. Obtained results showed that while carbohydrates reduced in curds of treated plants, protein increased with both stimulants application. The non-significant increasing in phenols and flavonoids content was observed in curds of LRE and BYE-treated plants with an elevation in antioxidant activity in their methanolic extracts. Both LRE and BYE applications promoted the curds content of macronutrients and caused a notable alteration in active compounds profile. Noteworthy, the values of acquired outcomes increased or decreased according to applied concentrations of individual or combination of both biostimulants. Based on our finding, the foliar application with LRE and BYE enhanced the primary and secondary metabolites of Brassica curds. However, the combination of 10 g/L of LRE and 20 g/L of BYE is recommended due to its act in promoting of flavonoids content, antioxidant capacity, potassium content and active compounds profile including organosulfur, troponoid, methyl 9-cis,11-trans-octadecadienoate, 1H-Indole-3-acetonitrile and 9,12,15-Octadecatrienoic acid.

Key words:

Biostimulants

Liquorice root extract

Bread yeast extract

Cauliflower curds

Metabolites. Desert region

1- Introduction

To achieve sustainability in the agricultural sector, it is necessary to eliminate synthetic fertilizers and apply instead biostimulants (BIOs) to enhance both the quantity and quality of crops. Biostimulants are substances or/and microorganisms that optimize plant growth through enhance nutrition efficiency, soil fertility, abiotic stress tolerance, and crop quality without direct effect on the nutrients content (Du Jardin, 2015). BIOs are commercially available in numerous forms and could be designated as product contain mixture of both substances and microorganism. Patrick du Jardin divided biostimulants into seven categories: humic and fulvic acids, protein hydrolysates (PHs), oligopeptides and polypeptides, seaweed and botanical extracts, biopolymers, and beneficial fungi and bacteria (Du Jardin, 2015). BIOs derived from plants have received a notable attention in the recent years and researchers highly investigated their potential effect on plants growth under normal and stress conditions (Han et al., 2024; keya Tudu et al., 2022). Among plants that used their extracts as BIOs, Liquorice (Glycyrrhiza glabra) root extract (LRE) is used particularly due to the rich content of osmoprotectants, phytohormones and antioxidants (E. Desoky et al., 2019). LRE has been reported as a promising natural biostimulants due to its role in enhancement the plant ability to uptake nutrients (Salih et al., 2021), promote a good growth and yield (Elrys & Merwad, 2017), and improve abiotic stress tolerance (E.-S. M. Desoky, A. S. Elrys, et al., 2019; E.-S. M. Desoky, A. I. ElSayed, et al., 2019). Numerous active compounds exist in LRE extract such as antioxidants, phenols, flavonoids, triterpenoid and saponins and help to stimulate its favourable role when applied on crops (Mamedov & Egamberdieva, 2019). Likewise, recent utilization of bread yeast Saccharomyces cerevisiae extract (BYE) as biostimulants have garnered attention for their potential to enhance plant growth and contribute to sustainable agriculture (Vargas et al., 2024). BYE derived from yeast cells is rich in effective components such as low-molecular-weight organic substances, amino acids, nucleotides, peptides, nitrogen, phosphorus, and trace elements with absence of chemical hormones and toxic ingredients (Vieira et al., 2016). The foliar application of BYE on plants can stimulate plant metabolism and enhance the vitality, leading to boost nutrient uptake, increase biomass, improv biochemical traits and consequently optimize the high value productivity in cabbage and tomato crops (Dima et al., 2023; Dima et al., 2020).

White cauliflower Brassica oleracea var. botrytis is annual plant belongs to the Brassica family, is an edible and highly desirable crop among Iraqi consumers. The edible parts of the plants are immature inflorescences, heads, or curds. Curds are rich with a privileged content of nutrients and active constituents such as fibres, soluble sugars, minerals, carotenoids, vitamins, glucosinolates, polyphenols, and flavonoids (Fouad A Ahmed & Rehab FM Ali, 2013). White cauliflower is one of crops that are voracious for fertilizers special the nitrogen. Achieving high and good productivity of crop could be required massive amounts of fertilizers that are costly, polluted, and not ensuring the sustainable requirements. Thus, exploring suitable alternatives such as biofertilizers and biostimulants is necessary to reduce the agriculture cost, optimize the quality and quantity of yield, minimize global carbon footprint and protect our environment (Díaz-Pérez et al., 2024).

Current study is aimed to investigate the effect of LRE and BYE combination as biostimulants on nutrient value and active component profile and their antioxidant activity in Brassica oleracea var. botrytis curds that grown in desert area in Basrah.

2- Materials and methods

2.1. Cultivation and treatments

The verity cauliflower hybrid F1 Alnahar seeds from Monarch seeds, Noord-Scharwoude, Holland, cultivated in trays with total peat moss substrate. After 40 days of cultivation, seedlings transferred to permanent field, treatment with biostimulants started after 30 days of transferring. Biostimulants treatments were performed weakly with LRE and BYE at three concentrations (0, 10, 20) g/L, the period between each treatment was 2 weeks.

2.2. Biochemical traits of curds

The cruds of treated plants were dried at room temperature and used to estimate the content of carbohydrates, protein, phenols, flavonoids, and elements as well as their active constituent’s profile

2.2.1. Carbohydrates content

Carbohydrates content was determined by Phenol-Sulphuric Acid method according to (DuBois et al., 1956). From dried samples 0.5 g homogenized with 70 ml of distilled water at 70°C for 1 hr. Then, 5 ml of filtrated extract placed in a volumetric flask and made up to 30 ml by distilled water. From diluted extract, 1 ml placed in a new tube and homogenized with 1m of 5% phenol and 5 ml of pure sulfuric acid by vortex. After brining the mixture to the room temperature, the absorption recorded by spectrophotometer at 490 nm and carbohydrates concentration was valuated from glucose standard curve and expressed as mg of glucose equivalents per gram of sample dry weight (mg GU/g dw).

2.2.2. Protein content

Protein content was determined in dried plant samples following Kjeldahl method. From dried samples 0.20 g was digested with 5 ml of pure sulfuric acid at 350°C for 30 minutes. After the samples cooling, 3 ml of the mixture of 96 ml sulfuric acid and 6 ml perchloric acid was added to the sample solution and placed on digestion plate at 350°C until the solution being clear. The volume of sample solution was made up to 50 ml by distilled water. From diluted sample 10 ml was mixed with 10 ml of 6 N of sodium hydroxide in the tube and placed in Kjeldahl analyzer instrument. The distillate was collected in a conical flask containing 2% of Boric acid and 2% of bromocresol green and methyl red indicator solution (0.099 g + 0.066 g in 100 ml Ethanol). The collection was titrated with 2% HCl until the solution has a slightly violet color, the percentage of nitrogen was calculated through the equation:

$\:\text{N}=\left(\text{V}\:\text{X}\:\text{N}\:\text{X}\:\text{M}\text{W}\:\text{X}\:\text{T}\text{V}\:\right)100/1000\:\text{X}\:\text{S}\text{W}\:\text{X}\:\text{V}\text{S})$

%N = Percentage of nitrogen

V = Volume of titration acid (HCl)

N = Normality of HCl

MW = Molecular weight of nitrogen

TV = Total volume of digestion solution

SW = Weight of plant sample

VS = Volume of digestion solution

The percentage of protein was calculated following the question:

$\:\text{\%}\text{P}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}=\text{\%}\text{N}\:\text{X}\:6.25$

(conversion factor)

2.2.3. Total phenols content

Cruds content of phenols estimated by Folin–Ciocalteu method according to (Porter, 2012). With 5 ml of 80% methanol, 0.25 g of dry sample was homogenized and kept 48 hrs in dark, 24 hrs at room temperature and 24 hrs at 4°C. To 0.5 ml of the methanolic extract, 0.5 ml of Folin–Ciocalteu reagent was added and followed with 8 ml of distilled water. After 5min of incubation, 1ml of 20% sodium carbonate (Na2CO3) was added. After 30 mins incubation at ambient temperature in dark, an intense blue color developed, and its absorbance was measured at 760 nm. Gallic acid was used as a standard by preparing stock solution at concentration 1 mg/ml and used for the calibration. The results were expressed as mg of gallic acid equivalents per gram of the dry weight sample (mg GAE/g dw)

2.2.4. Total flavonoids content

Total flavonoids content in cruds of control and treated plants was evaluated following (Samidha Kamtekar, 2014). To 0.5 ml of extract that placed in 10 ml tubes, 0.5 ml of 80% methanol added and followed by the adding of 10 ml of distilled water and 0.3 ml of 5% sodium nitrite (NaNO3). After incubation for 5 mins, 0.3 ml of 10% aluminum chloride (AlCl3) and 2 ml of 1M sodium hydroxide (NaOH) were added. The volume was made up to 10 ml with distilled water and vortexed, the absorbance was recorded spectrophotometry at 510 nm after orange-yellowish color development. Quercetin was used as a standard by preparing stock solution at concentration 1 mg/ml and used for the calibration. The results were expressed as mg of quercetin equivalent per gram of the dry weight sample (mg QE/g dw) .

2.2.4. Elements composition

Potassium, phosphors, and sulfur were determined by the dry ash method (Akinyele & Shokunbi, 2015). In porcelain crucibles, 1 g of cruds samples were placed and incinerated at an initial temperature of 200^◦C for 1h on a hot plate until the smoke disappears. Then, samples kept in Nabertherm muffle furnace at 556°C for 5 hrs for total incineration. Next, ashes were dissolved by a mixture of 4 ml of nitric acid (65%) and 10 ml of distilled water on a hot plate. The solution was filtered and made up to 25ml with distilled water and used for elements (P, K, S) analysis by atomic absorption/emission spectroscopy. The calibration curves were generated using the standards of each element, and the results were expressed as mg of element per gram of the dry weight sample.

2.2.5. GC-MS analysis

2.2.5.1. Samples preparation

From dried cruds, 0.250 g homogenized with 5 ml of 80% methanol, vortexed to achieve a good mixing and incubated in dark at room temperature overnight. Next day, supernatant collected carefully by micropipette and sent for injection in GCMC instrument

2.2.5.2 GCMC conditions

GC-MS analysis was carried out by using an Agilent Technologies, 7890B GC system coupled to an Agilent Technologies 5977A MSD with EI Signal detector, using HP-5ms 5% phenyl, 95% methyl siloxane (30m*250um*0.25). The oven temperature was set at 40°C hold for 5 mins then raised to 8°C/min to 300°C for 20 mins, Helium carrier gas flow rate was 1 ml/min and purge flow of 3 ml/min. The injection mode was pulsed Splitless with injection temperature 290°C and the injection sample volume was 0.5 µl. The mass spectrometer used Ion Source Temperature 230°C, with scan speed 1562(N2), and the mass range 44–750 m/z. Compound identification was implemented using mass spectral data and retention indices, in comparison with authentic standards and spectral libraries, including the NIST 2014, 2020 database.

3- Results

Fig. 1

illustrates the effect of biostimulants spray on curds content of carbohydrates and proteins, the spray with LBE and BYE at different concentration caused reduce the carbohydrates content of curds compared to the control plants, the range of values between 19.32 mg GU/g dw and 22.86 mg GU/g dw. On the other hand, protein proportion increased remarkably in curds of 10 g/L of BYE-treated plants (15.89%) and plants that sprayed with 20 g/L of LRE and 10 g/L of BYE combination (14.87%) (Fig. 1A&B).

Figure (1): The effect of foliar application with Liquorice root extract and Bread yeast extract on carbohydrates and protein content in Brassica curds. (A) Representative the effect of Liquorice root extract and Bread yeast extract on the carbohydrates content of Brassica curds. L0B0 control or untreated plants. L0B1treated plants with Bread yeast extract at 10 g/L. L0B2 treated plants with Bread yeast extract at 20 g/L. L1B0 treated plants with 10 g/L of Liquorice root extract. L1B1treated plants with 10 g/L of Liquorice root extract and 10 g/L of Bread yeast extract. L1B2 treated plants with 10 g/L of Liquorice root extract and 20 g/L of Bread yeast extract. L2B0 treated plant with Liquorice root extract. L2B1 treated plants with 20 g/L of Liquorice root extract and 10 g/L of Bread yeast extract. L2B2 treated plants with 20 g/L of Liquorice root extract and Bread yeast extract. (B) Representative the effect of Liquorice root extract and Bread yeast extract on the protein content of Brassica curds. A multiple ANOVA was performed using ordinary one-way ANOVA multiple comparisons to compare the averages of treatments with L0B0. Significance was designated as follows: *p < 0.05, ***p < 0.0001.

Total phenolic content and total flavonoids content in curds exhibited non-significant increase due to foliar application of both extracts on Brassica plants. The increase of phenols content was in curds of 20 g/L of BYE-treated plants (17.03 mg GAE/g dw), while flavonoids content increased in curds of combination of 10 g/L of LRE and 20 g/L of BYE-treated plant (16.54 mg QE/g dw) (Fig. 2A&B). Antioxidants activity of curds extracts against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals reinforced in curds methanolic extract of LRE-sprayed plants and BYE-sprayed plants and their combination-sprayed plants recorded 75.00%, 77.59%, and 74.09% respectively (Fig. 2C).

Figure (2): The effect of foliar application with Liquorice root extract and Bread yeast extract on phenols and flavonoids content and DPPH scavenging in Brassica curds. (A) Representative the effect of Liquorice root extract and Bread yeast extract on the phenols content of Brassica curds. L0B0 control or untreated plants. L0B1treated plants with Bread yeast extract at 10 g/L. L0B2 treated plants with Bread yeast extract at 20 g/L. L1B0 treated plants with 10 g/L Liquorice root extract. L1B1treated plants with 10 g/L of Liquorice root extract and 10 g/L of Bread yeast extract. L1B2 treated plants with 10 g/L of Liquorice root extract and 20 g/L of Bread yeast extract. L2B0 treated plant with Liquorice root extract. L2B1 treated plants with 20 g/L of Liquorice root extract and 10 g/L of Bread yeast extract. L2B2 treated plants with 20 g/L of Liquorice root extract and Bread yeast extract. (B) Representative the effect of Liquorice root extract and Bread yeast extract on the flavonoids content of Brassica curds. (C) Representative the effect of Liquorice root extract and Bread yeast extract on the DPPH scavenging of Brassica curds extract. A multiple ANOVA was performed using ordinary one-way ANOVA multiple comparisons to compare the averages of treatments with L0B0. Significance was designated as follows: *p < 0.05, **P < 0.01, ****p < 0.0001.

Elements composition including nitrogen, phosphors, calcium and sulfur displayed the effect of foliar application of biostimulants on macro elements content in Brassica curds. Nitrogen portion increased in curds of 10 g/L BYE-treated plants and 20 g/L LRE and 10 g/L BYE-treated plants (2.54, 2.38) % (Fig. 3A). Phosphor content was impacted with individual and combination applications of LRE and BYE, P content promoted in curds of 10 g/L of LRE and BYE-treated plants (5.67 mg/g), 20 g/L LRE-treated plants (6.00 mg/g) and 20 g/L of LRE and BYE-treated plants (6.10 m/g) of dry weight (Fig. 3B). Furthermore, potassium content increased subsequently the foliar spray with LRE and BYE. Curds of 20 g/L BRE-treated plants (28.83 mg/g), 10 g/L of LRE-treated plants (23.31 mg/g) and the combination 10 g/L of LRE and 20 g/L of BRE-treated plants (28.52 mg/g) of dry weight (Fig. 3C). Sulfur content in Brassica curds was boosted in curds of plants that treated with 10 g/L of LRE and the combination of 20 g/L of LRE and 10 g/L of

BRE that both recorded 3. 60 mg/g of dry weight (Fig. 3D).

Figure (3): The effect of foliar application with Liquorice root extract and Bread yeast extract on macro elements content in Brassica curds. (A) Representative the effect of Liquorice root extract and Bread yeast extract on the nitrogen content of Brassica curds. L0B0 control or untreated plants. L0B1treated plants with Bread yeast extract at 10 g/L. L0B2 treated plants with Bread yeast extract at 20 g/L. L1B0 treated plants with 10 g/L Liquorice root extract. L1B1treated plants with 10 g/L of Liquorice root extract and 10 g/L of Bread yeast extract. L1B2 treated plants with 10 g/L of Liquorice root extract and 20 g/L of Bread yeast extract. L2B0 treated plant with Liquorice root extract. L2B1 treated plants with 20 g/L of Liquorice root extract and 10 g/L of Bread yeast extract. L2B2 treated plants with 20 g/L of Liquorice root extract and Bread yeast extract. (B) Representative the effect of Liquorice root extract and Bread yeast extract on the phospho content of Brassica curds. (C) Representative the effect of Liquorice root extract and Bread yeast extract on the potassium content of Brassica curds. (D) Representative the effect of Liquorice root extract and Bread yeast extract on the sulfur content of Brassica curds A multiple ANOVA was performed using ordinary one-way ANOVA multiple comparisons to compare the averages of treatments with L0B0. Significance was designated as follows: *p < 0.05, ****p < 0.0001.

Table (1): GC-MS analysis profile of Brassica methanolic extract

R.T.	Compound name	Chemical class	L0B0	L0B1	L0B2	L1B0	L1B1	L1B2	L2B0	L2B1	L2B2
9.26	Dimethyl trisulfide	Organosulfur	3.68	3.58	3.13	4.06	2.87	4.98	2.81	2.79	2.89
18.43	Phenol, 4-ethenyl-2,6-dimethoxy-	Phenolic compounds	1.47	0.72	0.75	1.00	0.83	0.60	0.52	0.58	0.64
19.5	4-Ethoxy-2-(methylamino) tropone Furan-2-carboxaldehyde, 5-(1-piperidyl)-	Troponoid Volatile compound	8.48 -	5.79 -	6.16 -	- 7.23	- 6.37	4.10 -	5.29 -	5.84 -	- 6.54
20.72	d-Proline	Amino acid	0.97	1.46	1.55	2.19	1.86	-	3.31	2.69	-
21.26	1H-Indole-3-acetonitrile	Glycosides	1.65	1.53	1.55	1.53	1.68	1.83	1.15	1.10	1.03
22.19	Hexadecanoic acid	Sat. Fatty acid	1.23	3.26	2.67	2.27	4.21	5.07	4.59	7.44	4.43
23.82	Methyl 9-cis,11-trans-octadecadienoate	Methyl ester	-	2.59	2.04	1.56	2.032	4.65	3.18	4.73	2.89
23.89	9,12,15-Octadecatrienoic acid	Unsat. Fatty acid	1.24	10.12	9.35	7.42	10.83	21.10	13.11	15.30	9.99
27.33	Glycerol monopalmitate	Fatty acid	1.33	1.18	1.51	1.57	2.18	1.38	2.18	1.73	0.86
33.29	.gamma.-Sitosterol	Sterol	0.45	0.51	0.76	2.11	-	-	1.93	2.00	1.53

4- Discussion

The utilization of botanical extract as biofertilizer or biostimulants to enhance plant growth or plant tolerance to stress conditions has gained considerable attention in recent years. Obtained results showed that carbohydrates content in curds decreased when foliar spray applied on Brassica plants which could be attributed to exploiting carbohydrates to increase the growth parameters of sprayed plants including plant high, leaves number, leaf area and biomass (data not shown). Furthermore, total carbohydrates were utilized also by treated plants to increase the productivity traits such as yield, diameter, biomass, and weight of Brassica cruds (data not shown). Under similar conditions, instead of carbohydrates accumulation in induvial organs, plant direct total carbohydrates to promote its growth and yield (Van den Ende, 2014). On the other hand, protein percentage exhibited an increase in curds of L0B1-treated plants and L2B1-treated plants. The role of protein in the plant cover expanded ambit through their structural, enzymatic, metabolic, regulatic and storage functions (Rasheed et al., 2020). Foliar application with biostimulants that are rich with protein such as BYE (Demirgul et al., 2022) or amino acid such as LRY (Wang et al., 2015) or both could increase the curds content of protein and promote yield quality from the healthy components.

Brassica curds are good source for phenolic compounds particularly flavonoids (F. A. Ahmed & R. F. Ali, 2013). Phenols including flavonoids are natural compounds that are synthesized by the phenylpropanoid pathway and widely distributed in plants to promote and maintain its growth and development. Besides, they have several medical benefits such as anticancer, antiinflammation and antioxidant properties (Kuljarusnont et al., 2024). In our study, the total of phenols and flavonoids in curds have not shown significantly affected due to the foliar application with LRY or BYE. However, an insignificant increase was observed in phenols and flavonoids content in curds of L0B2-treated plants and L1B2-treated plants respectively. This increase may be related to the role of LRY and BYE in boosting and stimulating secondary metabolites production including total phenols and flavonoids, conforming with obtained outcomes by Younes in Allium cepa (Younes et al., 2021) and Dawood in Glycine max (Dawood et al., 2013).

In the antioxidant context, previous literature indicated the active constituents in Brassicaceae vegetables and their related antioxidant properties (Mulțescu et al., 2021). The antioxidants activity in Brassica curds is correlated to various compounds such as phenols, flavonoids, sulfurs compounds, glucosinolates and others. This activity may be influenced extremely by several factors including cultivar, maturity, growing and harvesting conditions and storge circumstances (Podsędek, 2007). Following the effect of LRE and BYE on antioxidant activity of curds methanol extract, L0B2-treated plant and L1B0-treated plants recorded a significant activity. Likewise, curds extract of plants sprayed with combination of both LRY and BYE concentrations recorded also antioxidant activity against DPPH. The augmentation of DPPH scavenging efficiency due to LRE implementing was observed previously in the extract of Stevia rebaudiana bertoni under normal conditions (Khashan & AL-Taweel, 2024) and Phaseolus vulgaris L. under salinity conditions (Rady et al., 2019), both found LRE promoted antioxidant activity in plants due to increase the non-enzymatic antioxidant (e.g. proline, phenols, tocopherols, carotenoids, etc). In the current study, the capacity of curds extract to scavenging DPPH radicals could be increased regarding the increase of Dimethyl trisulfide, d-Proline, 9,12,15-Octadecatrienoic acid and gamma.-Sitosterol (Table 1). Likewise, BYE application boosted antioxidants properties in Allium sativum L. as a tolerance strategy against drought stress through increasing of antioxidant enzymes and antioxidants compounds such as proline and ascorbic acid (Abdelaal et al., 2021). Our result indicated that BYE enhanced the scavenging of free radicals in curds extract of treated plants which could be imputed to induced role of BY in promoting compounds with antioxidants features including Dimethyl trisulfide, d-Proline, 9,12,15-Octadecatrienoic acid, Methyl 9-cis,11-trans-octadecadienoate, Glycerol monopalmitate and gamma.-Sitosterol.

These compounds increased also in curds extract of sprayed plants with combination of LRE and BYE according to GC-MS analysis (Table 1) which consequently enhanced its antioxidants efficiency.

Microelements (N, P, K, S) increased compatibly with biostimulants foliar application. Brassica curds have been reported to contain a high macro elements content (Ekholm et al., 2007). However, cultivars, growing and environmental conditions, and harvesting and post harvesting circumstances influence curds content of macro and micro elements (Kalisz et al., 2019; Kisko et al., 2021). In a present study potassium recorded the highest content in Brassica curds and the content increased remarkably with LRE and BRE at L0B2 and L1B2-treated plants. Likewise, other macronutrients including N, P, and S increased in curds of treated plants: for N at L0B1 and L2B1, for P at L1B1, L2B0, and L2B1, and for S at L1B0 and L2B1. The increase of macro elements in curds may be referred to a rich content of elements in LRE (Gormez & Sengul, 2014) and BRE (Tao et al., 2023), these elements either move through or interact with the apoplast space which subsequently effect on their translocation to different plant parts including the curds (Fernández & Brown, 2013). Additionally, this increase may be linked to enhanced root capacity for nutrients uptake from the soil post biostimulants application (Halpern et al., 2015). It has been reported that LRE and BYE increased minerals content in mango fruit and potato tuber when biostimulants applied on the plants (Orján et al., 2023; Yousif et al., 2024). Macro minerals almost participate in all human body functions and act significantly in metabolic processes. For example, phosphorus serve important role in the bone, teeth, phosphoproteins and nucleic acids formation, fat and starch metabolism and heartbeat and kidney function normalization (Elser, 2012).

GC-MS is mostly favoured technique to identify the quality and quantity of compounds in complex mixture due to its sensitivity and selectivity. Numerous agents influence GC-MS analysis results, some are related to plants (plants species, growing conditions, extraction methods) and others are related to GC-MS instrumentation conditions (temperature program, injection technique, detector sensitivity). Under our experimental conditions, GC-MS result of curds methanolic extract indicated high alteration in the peak area of most important detected compounds. Several compounds increased due to LRE or BYE or both applications such as Dimethyl trisulfide (L1B0, L1B2), d-Proline (L0B1, L0B2, L1B0, L1B1, L2B0, L2B1), 1H-Indole-3-acetonitrile (L1B1, L1B2), Hexadecanoic acid (all treatments), Methyl 9-cis,11-trans-octadecadienoate (all treatments), Glycerol monopalmitate (all treatment except L2B2) and gamma.-Sitosterol (L0B1, L0B2, L1B0, L2B0, L2B1, L2B2). On the contrary, several compounds decreased due to individual or combination of foliar applications such as Dimethyl trisulfide (L0B1, L0B2, L1B0, L1B1, L2B0, L2B1, L2B2), Phenol, 4-ethenyl-2,6-dimethoxy- (all treatments), 4-Ethoxy-2-(methylamino) tropone (L0B1, L0B2, L1B2, L2B0, L2B1), 1H-Indole-3-acetonitrile (L0B1, L0B2, L1B0, L2B0, L2B1, L2B2), and Glycerol monopalmitate (L2B2). Interestingly, some compounds disappeared due to LRE or BYE or both applications such as 4-Ethoxy-2-(methylamino) tropone (L1B0, L1B1, L2B2), Furan-2-carboxaldehyde, 5-(1-piperidyl)- (L0B0, L0B1, L0B2, L1B2, L2B0, L2B1), d-Proline (L1B2, L2B2), and gamma.-Sitosterol (L1B1, L1B2). The alteration of active compounds in Brassica curds extract with LRE foliar spray may be correlated to rich content of LRE from sugars, vitamins, mineral nutrients, amino acids, selenium, and phytohormones which effect on secondary metabolites biosynthesis pathways such as sulfurs-containing compounds pathway (Nakai & Maruyama-Nakashita, 2020), shikimate/phenylpropanoid pathway (Tak et al., 2023), hormone biosynthesis pathways (Bajguz & Piotrowska-Niczyporuk, 2023), and sterol biosynthesis pathway (Du et al., 2022). Likewise, BYE application caused a variation in peak areas of secondary compounds in Brassica curds extract which may be referred to the BYE content of effective components including amino acids, vitamins, proteins and hormones (Medani & Taha, 2015). Some of these components serve as precursors for secondary metabolites biosynthesis pathways such as amino acids (Trovato et al., 2021), while some act as enzymes cofactors, signaling molecules, and genes expression regulators such as vitamins and hormones (Parra et al., 2018; Rui et al., 2020). These active compounds in Brassica curds have crucial role in maintaining human health when incorporated into the daily dietary due to their therapy functions as antioxidants, antiinflammation, anticancer, antiviral, anti-obesities, antihypertensive, and antigenotoxicity (Zhang et al., 2025). For example, 1H-Indole-3-acetonitrile displayed a broad-spectrum antiviral potential against influenza A, HSV-1, VSV and SARS-CoV-2 (Hui et al., 2023; Zhao et al., 2021). Thus, the increase of their amount after LRE and BYE application may enhance the nutritional, health-promoting and therapeutic value of brassica curds.

5- Conclusion

In summary, we found that the combination of biostimulants including LRE and BYE could be affected prominently on metabolites content of Brassica curds. When two biostimulants are applied as combination, their act may complement each other or act synergistically to enhance the overall effect, resulting in improving plants yield quantity and quality in the region that has desert soil properties. Our results indicated an increase in primary and secondary metabolites in Brassica curds after foliar application of LRE and BYE concentrations. Although the induvial treatments had an obvious impact on experimental parameters, the combination of study agents also acted synergistically to boost these parameters. In crops cultivation under desert conditions, obtained findings could be considered a promising result within the sustainable required direction. The remarkable function of LRE and BYE in improving the quality components of Brassica curds including phenols, flavonoids, macronutrients, and active compounds provides compelling evidence of the alternative role that biostimulants may play in eco-friendly agriculture. This improvement not only increase the nutritional value of Brassica curds but also enrich them with active compounds that contribute to the promoting of healthy foods sources.

Author Contribution

B designed research studies and performed field work, A and C performed lab experiments and data analysis. A and D wrote/edited the manuscript. All authors have read and approved the final version of the manuscript

Declarations

Ethical approval and consent to participate

Not applicable

Consent for publication

Not applicable

Funding

The research was not supported by any funding

clinical trial

Not applicable

References

Abdelaal K, Attia KA, Niedbała G, Wojciechowski T, Hafez Y, Alamery S, Alateeq TK, Arafa SA. Mitigation of Drought Damages by Exogenous Chitosan and Yeast Extract with Modulating the Photosynthetic Pigments, Antioxidant Defense System and Improving the Productivity of Garlic Plants. Horticulturae. 2021;7(11):510.

Ahmed FA, Ali RF. (2013). Bioactive compounds and antioxidant activity of fresh and processed white cauliflower. Biomed Res Int, 2013, 367819. https://doi.org/10.1155/2013/367819

Ahmed FA, Ali RF. Bioactive compounds and antioxidant activity of fresh and processed white cauliflower. Biomed Res Int. 2013;2013(1):367819.

Akinyele IO, Shokunbi OS. Comparative analysis of dry ashing and wet digestion methods for the determination of trace and heavy metals in food samples. Food Chem. 2015;173:682–4. https://doi.org/https://doi.org/10.1016/j.foodchem.2014.10.097.

Bajguz A, Piotrowska-Niczyporuk A. Biosynthetic Pathways of Hormones in Plants. Metabolites. 2023;13(8). https://doi.org/10.3390/metabo13080884.

Dawood MG, El-Lethy SR, Sadak MS. Role of methanol and yeast in improving growth, yield, nutritive value and antioxidants of soybean. World Appl Sci J. 2013;26(1):6–14.

Demirgul F, Simsek O, Sagdic O. Amino acid, mineral, vitamin B contents and bioactivities of extracts of yeasts isolated from sourdough. Food Bioscience. 2022;50:102040.

Desoky E-SM, Elrys AS, Rady MM. Integrative moringa and licorice extracts application improves Capsicum annuum fruit yield and declines its contaminant contents on a heavy metals-contaminated saline soil. Ecotoxicol Environ Saf. 2019;169:50–60.

Desoky E-SM, ElSayed AI, Merwad A-RM, Rady MM. Stimulating antioxidant defenses, antioxidant gene expression, and salt tolerance in Pisum sativum seedling by pretreatment using licorice root extract (LRE) as an organic biostimulant. Plant Physiol Biochem. 2019;142:292–302.

Desoky E, Elrys AS, Rady MM. (2019). Licorice root extract boosts Capsicum annuum L. production and reduces fruit contamination on a heavy metals-contaminated saline soil. Int Lett Nat Sci, 73.

Díaz-Pérez M, Moreno JMM, García JJH, Callejón-Ferre Á-J. Application of microalgae in cauliflower fertilisation. Sci Hort. 2024;337:113468.

Dima Ș-O, Constantinescu-Aruxandei D, Tritean N, Ghiurea M, Capră L, Nicolae C-A, Faraon V, Neamțu C, Oancea F. Spectroscopic analyses highlight plant biostimulant effects of Baker’s yeast vinasse and selenium on cabbage through foliar fertilization. Plants. 2023;12(16):3016.

Dima S-O, Neamțu C, Desliu-Avram M, Ghiurea M, Capra L, Radu E, Stoica R, Faraon V-A, Zamfiropol-Cristea V, Constantinescu-Aruxandei D. Plant biostimulant effects of baker’s yeast vinasse and selenium on tomatoes through foliar fertilization. Agronomy. 2020;10(1):133.

Du Jardin P. Plant biostimulants: Definition, concept, main categories and regulation. Sci Hort. 2015;196:3–14.

Du Y, Fu X, Chu Y, Wu P, Liu Y, Ma L, Tian H, Zhu B. Biosynthesis and the Roles of Plant Sterols in Development and Stress Responses. Int J Mol Sci. 2022;23(4). https://doi.org/10.3390/ijms23042332.

DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric Method for Determination of Sugars and Related Substances. Anal Chem. 1956;28(3):350–6. https://doi.org/10.1021/ac60111a017.

Ekholm P, Reinivuo H, Mattila P, Pakkala H, Koponen J, Happonen A, Hellström J, Ovaskainen M-L. Changes in the mineral and trace element contents of cereals, fruits and vegetables in Finland. J Food Compos Anal. 2007;20(6):487–95.

Elrys A, Merwad A-R. Effect of alternative spraying with silicate and licorice root extract on yield and nutrients uptake by pea plants. Egypt J Agron. 2017;39(3):279–92.

Elser JJ. Phosphorus: a limiting nutrient for humanity? Curr Opin Biotechnol. 2012;23(6):833–8.

Fernández V, Brown PH. From plant surface to plant metabolism: the uncertain fate of foliar-applied nutrients. Front Plant Sci. 2013;4:289.

Gormez A, Sengul M. Total phenolics, mineral contents, antioxidant and antibacterial activities of Glycyrrhiza glabra L. roots grown wild in Turkey Volume. In: December; 2014.

Halpern M, Bar-Tal A, Ofek M, Minz D, Muller T, Yermiyahu U. The use of biostimulants for enhancing nutrient uptake. Adv Agron. 2015;130:141–74.

Han M, Kasim S, Yang Z, Deng X, Saidi N, Uddin M, Shuib E. Plant extracts as biostimulant agents: A promising strategy for managing environmental stress in sustainable agriculture. Phyton. 2024;93(9):2149.

Hui X, Yu X, Huang K, Xu T, Cao L, Zhang Y, Zhao L, Zhao Y, Lv C, Feng s, Jiang Y, Liu L, Jin M. In vitro and in vivo effects of 3-indoleacetonitrile—A potential new broad-spectrum therapeutic agent for SARS-CoV-2 infection. Antiviral Res. 2023;209:105465. https://doi.org/https://doi.org/10.1016/j.antiviral.2022.105465.

Kalisz A, Sękara A, Smoleń S, Grabowska A, Gil J, Komorowska M, Kunicki E. Survey of 17 elements, including rare earth elements, in chilled and non-chilled cauliflower cultivars. Sci Rep. 2019;9(1):5416. https://doi.org/10.1038/s41598-019-41946-z.

keya Tudu C, Dey A, Pandey DK, Panwar JS, Nandy S. (2022). Role of plant derived extracts as biostimulants in sustainable agriculture: a detailed study on research advances, bottlenecks and future prospects. New future developments Microb Biotechnol Bioeng, 159–79.

Khashan NS, AL-Taweel SK, IN STEVIA PLANT AS EFFECTED BY SHADING AND FOLIAR SPRAYING OF MORINGA AND LIQUORICE EXTRACT: ANTIOXIDANTS ACTIVITY IN STEVIA PLANT AS EFFECTED BY SHADING AND FOLIAR SPRAYING OF MORINGA AND LIQUORICE EXTRACT. ANTIOXIDANTS ACTIVITY. Iraqi J Market Res Consumer Prot. 2024;16(2):188–99.

Kisko MFK, Kadhum NJ, Ali ZA, Abid NS. Effects of Nitrogen and Sulfur Sprays on the Growth and Production of Broccoli Brassica Oleracea var. Italica L. Baghdad Sci J. 2021;18(3):25.

Kuljarusnont S, Iwakami S, Iwashina T, Tungmunnithum D. Flavonoids and Other Phenolic Compounds for Physiological Roles, Plant Species Delimitation, and Medical Benefits: A Promising View. Molecules. 2024;29(22):5351.

Mamedov NA, Egamberdieva D. (2019). Phytochemical constituents and pharmacological effects of licorice: a review. Plant and human health, Volume 3: Pharmacology and therapeutic uses, 1–21.

Medani R, Taha R. (2015). Improving growth and yield of caraway (Carum carvi L.,) plants by decapitation and/or active dry yeast application.

Mulțescu M, Susman I-E, Burnichi F, Israel-Roming F. (2021). Antioxidant activity in selected Brassicaceae vegetables. Sci Bull Ser F Biotechnologies, 25(1).

Nakai Y, Maruyama-Nakashita A. Biosynthesis of Sulfur-Containing Small Biomolecules in Plants. Int J Mol Sci. 2020;21(10):3470.

Orján EM, Kormányos ES, Fűr GM, Dombi Á, Bálint ER, Balla Z, Balog BA, Dágó Á, Totonji A, Bátai ZI. The anti-inflammatory effect of dimethyl trisulfide in experimental acute pancreatitis. Sci Rep. 2023;13(1):16813.

Parra M, Stahl S, Hellmann H. Vitamin B₆ and Its Role in Cell Metabolism and Physiology. Cells. 2018;7(7). https://doi.org/10.3390/cells7070084.

Podsędek A. Natural antioxidants and antioxidant capacity of Brassica vegetables: A review. LWT-Food Sci Technol. 2007;40(1):1–11.

Porter Y. Antioxidant properties of green broccoli and purple-sprouting broccoli under different cooking conditions. Bioscience Horizons: Int J Student Res. 2012;5. https://doi.org/10.1093/biohorizons/hzs004.

Rady M, Desoky E-S, Elrys A, Boghdady M. Can licorice root extract be used as an effective natural biostimulant for salt-stressed common bean plants? South Afr J Bot. 2019;121:294–305.

Rasheed F, Markgren J, Hedenqvist M, Johansson E. Modeling to Understand Plant Protein Structure-Function Relationships-Implications for Seed Storage Proteins. Molecules. 2020;25(4). https://doi.org/10.3390/molecules25040873.

Rui Z, Xiu-Juan W, Wei G. Regulation mechanism of plant hormones on secondary metabolites. Zhongguo Zhong yao za zhi = Zhongguo Zhongyao Zazhi = China J Chin Materia Med. 2020;45(17):4205–10.

Salih II, Mohammed KH, Hassan FA. Effect of spraying of algazon and licorice root extract on chemical traits of myrtus (Myrtus communis L). Indian J Ecol. 2021;48(15):127–30.

Samidha Kamtekar VK, Patil V. (2014). Estimation of Phenolic content, Flavonoid content, Antioxidant and Alpha amylase Inhibitory Activity of Marketed Polyherbal Formulation (Vol. Volume: 4;). Issue: 9.

Tak Y, Kaur M, Gautam C, Kumar R, Tilgam J, Natta S. Phenolic biosynthesis and metabolic pathways to alleviate stresses in plants. Plant phenolics in abiotic stress management. Springer; 2023. pp. 63–87.

Tao Z, Yuan H, Liu M, Liu Q, Zhang S, Liu H, Jiang Y, Huang D, Wang T. Yeast Extract: Characteristics, Production, Applications and Future Perspectives. J Microbiol Biotechnol. 2023;33(2):151–66. https://doi.org/10.4014/jmb.2207.07057.

Trovato M, Funck D, Forlani G, Okumoto S, Amir R. Amino acids in plants: Regulation and functions in development and stress defense. Volume 12. Frontiers Media SA; 2021. p. 772810.

Van den Ende W. Sugars take a central position in plant growth, development and, stress responses. A focus on apical dominance. Front Plant Sci. 2014;5:313. https://doi.org/10.3389/fpls.2014.00313.

Vargas MF, Mestre MV, Vergara C, Maturano P, Petrignani D, Pesce V, Vazquez F. Residual brewer’s Saccharomyces cerevisiae yeasts as biofertilizers in horticultural seedlings: towards a sustainable industry and agriculture. Front Industrial Microbiol. 2024;2:1360263.

Vieira EF, Carvalho J, Pinto E, Cunha S, Almeida AA, Ferreira IM. Nutritive value, antioxidant activity and phenolic compounds profile of brewer’s spent yeast extract. J Food Compos Anal. 2016;52:44–51.

Wang L, Yang R, Yuan B, Liu Y, Liu C. The antiviral and antimicrobial activities of licorice, a widely-used Chinese herb. Acta Pharm sinica B. 2015;5(4):310–5.

Younes NA, Rahman MM, Wardany AA, Dawood MFA, Mostofa MG, Keya SS, Latef A, A. A. H., Tran LP. Antioxidants and Bioactive Compounds in Licorice Root Extract Potentially Contribute to Improving Growth, Bulb Quality and Yield of Onion (Allium cepa). Molecules. 2021;26(9). https://doi.org/10.3390/molecules26092633.

Yousif KH, Mohammed GH, Gulli WGH, Saeid AJI. (2024). Impact of Bread Yeast Extract and Humic Acid on Vegetative Growth, Quantity and Nutrient Content of Two Cultivars of Potato (Solanum tuberosum L). J Kirkuk Univ Agricultural Sci, 15(4).

Zhang X, Jia Q, Jia X, Li J, Sun X, Min L, Liu Z, Ma W, Zhao J. Brassica vegetables-an undervalued nutritional goldmine. Hortic Res. 2025;12(2):uhae302. https://doi.org/10.1093/hr/uhae302.

Zhao X, Zhao L, Zhao Y, Huang K, Gong W, Yang Y, Zhao L, Xia X, Li Z, Sheng F. 3-Indoleacetonitrile is highly effective in treating influenza a virus infection in vitro and in vivo. Viruses. 2021;13(8):1433.

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