Physico-Chemical Characterization,Biological Attributes and Sensory Evaluation of Commercial Ketchup

Abstract

Objectives/methodology

The present study focuses on the evaluation of antioxidant potential (AP), total phenolic (TP) and total flavonoid (TF), antimicrobial activity (AA), antidiabetic capacity (AC), heavy metals contents (HMC) and sensory evaluation of commercial ketchup samples.

Results

The AP, TP and TF were found to be in the range of 35.51 to 77.92%, 2.66 to 14.98 mg GAE/100 g and 1.69 to 4.23 mg QE/g, respectively. The results of AA showed that commercial ketchup (CK) samples showed inhibition of microbial growth ranged from 18.96 to 30.67 mm against Staphylococcus aureus (S. aureus) and 19.00 to 31.14 mm against Escherichia coli (E. coli). The AC (involving alpha-amylase assay) was determined and represented as IC₅₀ value of 23.12 to 62.76 µg/mL. According to the sensory evolution, all the samples were acceptable for use with overall acceptability <50.

Conclusions

The current study concluded that ketchup exhibited good antioxidant, antimicrobial and antidiabetic potential, however, the level of heavy metals in tested ketchup were required to be controlled.

Keywords

Commercial ketchup antioxidant activity antimicrobial activity antidiabetic activity heavy metals

Introduction

Ketchup is the term used to describe products made from a variety of vegetables and fruits pulp, usually seasoned with different spices.¹ Ripe tomatoes are the most famous and popular ketchup components, typically flavoured with salts, sugar, vinegar, and various spices. Ketchup is a relatively stable product that is preserved by adding preservatives to extend its shelf life.^2,3 It is a popular sauce among young people and families and is mostly served with fast foods, including burgers, sandwiches, French fries and chicken wings.⁴ Ketchup consumption is high because of its low-calorie content and convenience. Its popularity originates from its distinctive taste, which it imparts to a variety of foods.⁵ Many brands of ketchup are available in the market; therefore, commercial ketchup can vary significantly in composition, and almost all manufacturers have their own formula based on composition or the amount of ingredients, spices, and flavours they use. Thus, it is challenging to know which parameters are key to its quality.⁶

Ketchup is also called a vegetable sauce because its primary constituent, tomato, is considered a vegetable. After Great Britain and Germany, Poland ranks as Europe's third-largest ketchup market. Ketchup is the main source of carotenoids, where lycopene is found to be most abundant.⁷ Almost 80% of tomatoes are consumed in processed forms such as ketchup, tomato puree and tomato juice.⁶ From a physical perspective, ketchup is a two-phase system; one phase is colloidal, while the other is dispersed. The solid particles of spices and tomato pulp are distributed in a continuous colloidal phase which consists of organic acids, salt, sugar, soluble pectin fractions and extracts of other compounds dissolved in water. Polysaccharide hydrocolloids are mostly used as a thickening agent for the formation of ketchup, so the viscosity of ketchup depends on the thickening of the continuous phase due to this thickener agents.^8–10

Tomatoes and their by-products contain vitamin E, folate, carotene, and lycopene.¹¹ Ketchup has been beneficial for different health conditions including cardiovascular disease, human papillomavirus persistence, hepatic fibro genesis and certain types of cancer. According to a recent report, it may also be helpful for fatal growth and improve lung functions. Ketchup possesses antioxidant activity due to its carotenoids and lycopene which are derived from tomatoes.^12–14

Materials and Methods

Chemicals and Reagents

Folin-Ciocalteu phenol reagent, nitric acid, perchloric acid, hydrogen peroxide, DPPH (2,2-diphenyl-1-picrylhydrazyl), gallic acid, quercetin, ascorbic acid distilled water, ethanol, methanol and sulphuric acid chemicals were procured from Merck (Darmstadt, Germany). All the substances that were used in the research were of analytical grade.

Sample Collection

The five local brands and one international brand of ketchup were used for research purposes. The selected ketchup brands were popular among people. All the ketchups were purchased from the Metro Cash and Carry store, Faisalabad, Punjab, Pakistan. All ketchup samples were preserved at −4 °C for different analyses. The local ketchup samples were indicated by K1- K5 and the international brand ketchup sample was indicated as K6.

Preparation of Ketchup Extract

The ketchup extracts were prepared following the method described¹⁵ with slight changes. Briefly, each sample of ketchup (40 g) was dissolved in 400 mL methanol (80%) and shaken on rotary orbital shaker (Scilogex SCI-0180–S digital orbital shaker, Scilogex, China) at 200 rotations per minute (rpm) for 48 h. Then the mixture was filtered using filter paper (Whitman's number 1) and the filtrates were concentrated using water bath (WB-1R2H7, Infitek, China) at 45 °C. Then stock solutions were prepared for further analysis by mixing samples (6 mg) with methanol (25 mL) in a mixer (LMS7-H5507, Zhengzhou LABOAO, China) which were stored at −4 °C for further analysis.

Scavenging Capacity of DPPH Radical

The appraisal of AP of commercial ketchup samples was done by DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay with slight alternation.¹⁶ Briefly, DPPH assay was carried out by mixing DPPH (1.9 mg) with methanol (50 mL). One millilitre of each extract (20% w/v) was subjected to mix with 4 mL of DPPH solution and placed at room temperature for 30 min in dark place to record its absorbance at 517 nm using UV-VIS spectrophotometer (Lambda EZ 201, Perkin Elmer, USA). Ascorbic acid (30 μg/mL) was employed as reference in each treatment to record the results. The inhibition of DPPH was determined using formula (1).

Inhibition (%) = (A_{b} - A_{s} / A_{b}) \times 100

(1)

A_b and A_s were the absorbance of the control and sample, respectively.

Estimation of TP

TP was assessed using Folin-Ciocalteu reagent method.¹⁶ Briefly, 1 mL of each sample crude extract was mixed with 7.5 mL of double de-ionized water. Then, 0.5 mL of Folin-Ciocalteu phenol reagent and 1 mL of 5% Na₂CO₃ were added. The resultant solutions were placed for in dark place at room temperature for 90 min to measure the absorbance at 765 nm using spectrophotometer (Lambda EZ 201, Perkin Elmer, USA). The calibration curve of gallic acid was plotted to express the obtained results in mg GAE/100 g using following formula:

C = C_{1} \times V / m

Where C = total phenolic content in mg/g in GAE (Gallic acid equivalent), C₁= concentration of gallic acid established from calibration curve in mg/mL, V = volume of sample in mL.

Calculation of TF

The total flavonoid contents were determined using an aluminum trichloride method with slight alternation.¹⁷ Briefly, 2 mL of each sample extract was subjected to mix with the 2 mL of AlCl₃ (2%) and placed in a dark place at room temperature for 15 min to record its absorbance at 430 nm. The quercetin was employed to construct a calibration curve to determine TF as mg QE/100 g.

C = C_{1} \times V / m

Where C = total flavonoid content in mg/g in GAE (gallic acid equivalent), C₁= concentration of gallic acid established from calibration curve in mg/mL, V = volume of sample in mL.

Evaluation of AA Using Disc Diffusion Assay (DDA)

The AA of tested ketchup extract (0.9 units/mL) was estimated by DDA protocol.¹⁸ The cultures of microbial strains were prepared by autoclaving (2840 ELC-G, Thermo fisher scientific, US) the growth media at 121 °C for 15 min at 1 atm. The sterilized growth medium was inoculated with pure microbial strain followed by incubation at 37 °C in the dark place in incubator (Heratherm Compact microbiological incubator, 18L, Thermo Fisher Scientific, US) for 24 h. The inoculums with optimal growth (10⁸ CFU/mL) were stored at −4 °C for further analysis.

Nutrient agar (Oxoid) was dissolved in distilled water at a ratio of 28 g/L to make a homogenous mixture followed by sterilization in an autoclave for 15 min at 121 °C. The prepared inoculation with optimal growth was then added to the sterilized medium and poured into a sterilized petri dish. The sterilized discs of filter paper were immersed with 100 µL of crude methanol extracts and placed on a growth medium, followed by incubation for 24 h at 37 °C. The discs of extracts showed clear zones representing their inhibiting potential. The zone reader was used for the determination of inhibition zones (mm)

AC by Alpha-Amylase Assay

The AC of ketchups was estimated following a method.¹⁶ Briefly, 0.5 mL of each extract was mixed with 0.5 mL of alpha-amylase and placed in a dark place at room temperature for 10 min. The resultant solution was mixed with 0.5 µL of starch solution and placed again in a dark place at room temperature for 10 min, followed by the addition of 1 mL DNSA solution. The resultant solution was heated in the water bath. After 5 min, the mixture was cooled and then diluted by adding 10 mL of distilled water to record its absorbance at 540 nm. Inhibition (%) was determined by the given equation

Inhibition (%) = 100 * [(A_{b} - A_{s}) / A_{b}]

Where, A_b and A_s were absorbance of control and sample, respectively

Determination of Heavy Metals in Tomato Ketchup

Estimation of heavy metals in ketchup samples was performed using the wet digestion method.¹⁹ Accurately, 1 g of each ketchup extract was placed in a digestion flask and then added 10 mL of concentrated HNO₃ and heated for 10 min at 60–70 °C in a fume hood on the hot plate. The resultant mixture was allowed to cool and after cooling, 5 mL of HClO₄ was added to heat at 60–70 °C on the hot plate to reduce the volume to 2–3 mL which was cooled again at room temperature and finally diluted by adding double de-ionized water to desired volume and stored in sample bottles. The concentration of heavy metals was measured by atomic absorption spectrophotometer (121-0032 spectrophotometer, Hitachi instruments Inco, Japan).

Sensory Evaluation

Six panellists, from the University of Agriculture Faisalabad, Pakistan ranging in age from 20 to 50 years, participated in this study. The sensory quality of ketchup was evaluated using a set of 25-point hedonic measures.²⁰

Statistical Analysis

All the analyses and tests were performed in triplicates, and the results were expressed as mean ± standard deviation. Analysis of variance was employed to assess significant differences among the samples with a predefined level of statistical significance (p < 0.05).²¹ The Minitab software was used to evaluate the data.

Results

Antioxidant Potential

The antioxidant potential of the samples was estimated by the free radical scavenging method. The values of AP of commercial ketchups were ranged from 35.51–77.92%. After six months of preservation, the highest inhibition 77.92% was shown by K6 sample, while the lowest inhibition was indicated by the K4 sample which was 35.51% after 06 months of preservation. Ascorbic acid used as a reference was found to be 85.58%. The significant level was p < 0.05. The detailed results were expressed in Table 1. All the results were compared with the literature. Previously, the antioxidant activity of seven commercially available Korean kinds of ketchup was found to be 45.10 to 90.87%, which conforms to our results.¹⁵

Table 1.

Antioxidant Activity, Total Phenolic and Flavonoid Contents of Different Commercial Brands of Ketchups.

Storage time	Commercial brands of ketchup	Antioxidant activity (% inhibition)	Total phenolic contents (mg GAE/100 g)	Total flavonoid contents (mg QE/g)	Antibacterial activity (mm)
Storage time	Commercial brands of ketchup	Antioxidant activity (% inhibition)	Total phenolic contents (mg GAE/100 g)	Total flavonoid contents (mg QE/g)	Escherichia coli	Staphylococcus aureus
06 months storage	K1	58.38 ± 0.05^c	2.93 ± 0.02^c	1.76 ± 0.03^b,c	20.50 ± 0.05^e	27.86 ± 0.11^b
	K2	73.79 ± 0.81^b	2.92 ± 2.54^c	1.77 ± 0.32^b,c	30.00 ± 0.18^b	18.96 ± 0.25^d
	K3	55.11 ± 1.58^d	2.73 ± 1.74^c,d	1.69 ± 1.72^c	19.00 ± 0.43^e	24.93 ± 0.09^c
	K4	35.51 ± 2.42^f	2.66 ± 0.35^d	1.79 ± 0.15^b,c	24.16 ± 0.01^d	29.00 ± 0.11^b
	K5	44.03 ± 0.91^e	3.48 ± 0.59^b	2.01 ± 0.05^b	27.03 ± 0.07^c	29.90 ± 0.16^b
	K6	75.80 ± 0.07^b	10.70 ± 0.08^a	3.81 ± 0.94^a	28.00 ± 0.02^c	26.03 ± 0.01^c
24 h storage	K1	60.21 ± 0.04^c	3.01 ± 0.32^c	2.43 ± 0.04^b	21.01 ± 0.43^e	28.43 ± 0.04^c
	K2	74.67 ± 0.17^b	2.99 ± 0.19^d	1.92 ± 0.01^d	31.14 ± 0.17^b	19.22 ± 0.11^e
	K3	55.34 ± 0.05^d	2.89 ± 0.08^d	1.78 ± 0.01^d	19.28 ± 0.21^f	25.04 ± 0.32^d
	K4	36.98 ± 0.01^f	2.70 ± 0.01^d	2.02 ± 0.27^c	25.00 ± 0.14^d,e	30.10 ± 0.09^b
	K5	44.21 ± 0.05^e	4.67 ± 0.07^b	2.21 ± 0.19^b,c	27.12 ± 0.42^c	30.67 ± 0.35^b
	K6	77.92 ± 0.34^b	14.98 ± 0.23^a	4.23 ± 0.11^a	28.05 ± 0.24^c	27.93 ± 0.16^c
	Standard	85.58 ± 0.01^a	–	–	43.66 ± 0.01^a	45.29 ± 0.08^a

K1-K5: National brands of ketchup. K6: International brand of ketchup.

Standard for antioxidant activity: Ascorbic acid.

Standard for antibacterial activity: Acarbose.

Assessment of TP and TF

The total phenolic contents of commercial ketchups were determined using Folin-Ciocalteu calorimetric assay and its values were found to be in the range of 2.66–14.98 mg GAE/100 g. The K6 sample showed the highest phenolic contents 14.98 mg GAE/100 g after 24 h’ preservation while K4 sample showed minimum phenolic contents 2.66 mg GAE/100 g after 06 months of preservation (Table 1). The statistical significance was set at p < 0.05. The range of total phenolic contents of seven Korean ketchups was 1.37–2.77 mg GAE/g.¹⁵

The TF contents were estimated by AlCl₃ methodology, and the results of our study were in the range of 1.69–4.23 QE mg/g. The K6 sample presented a maximum TF content 4.23 QE mg/g after 24 h’ preservation while K3 sample presented a minimum TF 1.69 QE mg/g after 06 month's preservation (Table 1). The statistical significance was set at p < 0.05. The results of TF of our research were compared with literature. TF in different ketchups which were supplemented by natural plant sources ranged from 38.3 to 302.3 QE mg/100g.²²

Estimation of AA of Ketchup

The antimicrobial activity of ketchup samples was estimated by DDA and in our study two types of bacterial strains were used, one was gram-positive was S. aureus and the second was gram-negative which was E. Coli. The AA against S. aureus where inhibition of zone were ranged from 18.96–30.67 mm. The K5 sample showed the highest inhibition which was 30.67 mm after 24 h’ preservation while K2 sample showed the lowest AA against S. aureus which was 18.96 mm after 06 months of preservation. Ampicillin used as a standard whose zone of inhibition was found to be 45.29 mm. In the case of gram-negative bacteria E. Coli the values of inhibition were 19.00–31.14 mm. The highest result was shown by K3 (31.14 mm) while the lowest result was displayed by K3 (19.00 mm) against E. coli. The AA was compared with ampicillin which was referred as standard (Table 1).

Determination of AC and Sensory Evaluation

AC was estimated using alpha-amylase and the results was estimated as IC₅₀ value whose values were ranged from 3.12–23.12 µg/mL. The K6 sample was shown the highest inhibition (IC₅₀= 62.76 µg/mL) while K1 sample was shown lowest inhibition (IC₅₀= 23.12 µg/mL) while Acrobose as standard was found to lowest (IC₅₀= 21.14 µg/mL).

According to consumer opinion, the score lies in the range of 62.48–78.23. All the ketchup samples were acceptable for use as the overall acceptability <50. The K6 sample was more appreciated by consumers and got the highest score 78.23 while the lowest score 62.48 was given to K1, further, detailed results are discussed in (Table 2). The sensory evaluation of tomato seed oil indicated highly appreciated results by consumers as compared to control.²⁰

Table 2.

Sensory Evaluation of Commercial Ketchup.

Sample	Sensory attribute
	Odor	Appearance	Color	Taste	Overall acceptability
	(25)	(25)	(25)	(25)	(100)
K1	12.78 ± 0.01^e	15.92 ± 0.28^d	15.10 ± 0.04^c	14.45 ± 0.12^c	62.48 ± 0.15
K2	20.32 ± 0.17^b	19.69 ± 0.06^b	17.45 ± 0.29^b	21.27 ± 0.01^b	21.27 ± 0.01^b
K3	14.37 ± 0.43^d	17.12 ± 0.21^c	12.22 ± 0.06^d	11.71 ± 0.25^d	63.23 ± 0.24^d
K4	17.21 ± 0.07^c	15.03 ± 0.09^d	14.89 ± 0.17^c,d	15.67 ± 0.06^c	69.12 ± 0.19^c
K5	19.18 ± 0.34^c	19.87 ± 0.11^b	21.09 ± 0.41^a	22.42 ± 0.17^b	71.19 ± 0.02^b
K6	23.08 ± 0.05^a	22.11 ± 0.01^a	21.67 ± 0.23^a	23.21 ± 0.09^a	78.23 ± 0.11^a

K1-K5: National brands of ketchup. K6: International brand of ketchup.

Heavy Metals in Commercialized Brands of Ketchup

In our study, heavy metals were estimated both qualitatively and quantitatively. Total eight heavy metals were determined in all ketchups and the amount of Pb, Cd, Zn, Cu, Fe, Co, Mn and Ni were 0.04–0.96, 0.16–0.56, 22.00–40.16, 2.00–4.03, 8.00–18.50, 0.10–3.00, 0.90–1.36 and 0.26–2.76 ppm, respectively (Table 3) and most of them were under the acceptable range of EU or USA standard.^23,24 The commercial ketchup at the local market in Bangladesh contained different heavy metals (lead, zinc, and copper) within the permissible limit according to BSTI standards. The ketchup contained cadmium, iron and nickel (0.043 and 41.66 ppm).²⁵ The manganese was present 40.1 ppm in tomato pomace²⁶

Table 3.

Determination of Heavy Metals in Commercialized Ketchup Samples.

Sample	Concentration of heavy metals (ppm)
Sample	Pb	Cd	Zn	Cu	Fe	Co	Mn	Ni
K1	0.86 ± 0.06^b	0.27 ± 0.01^d	40.16 ± 0.08^a	4.03 ± 0.11^a	18.50 ± 0.08^a	2.50 ± 0.07^b	1.03 ± 0.08^a,b	0.63 ± 0.01^b,c
K2	0.05 ± 0.02^e	0.46 ± 0.11^b	25.80 ± 0.41^c	2.00 ± 0.04^d	8.00 ± 0.19^f	2.83 ± 0.03^a,b	1.00 ± 0.13^b	0.26 ± 0.06^c
K3	0.17 ± 0.14^d	0.43 ± 0.03^b	36.33 ± 0.06^b	2.53 ± 0.16^b	14.03 ± 0.06^d	0.10 ± 0.02^d	1.30 ± 0.27^a,b	1.16 ± 0.39^b
K4	0.04 ± 0.08^e	0.56 ± 0.06^a	37.03 ± 0.44^b	2.46 ± 0.24^b	16.90 ± 0.07^b	1.96 ± 0.41^c	1.36 ± 0.06^a	2.76 ± 0.13^a
K5	0.96 ± 0.02^a	0.36 ± 0.13^c	23.00 ± 0.23^d	2.06 ± 0.08^c	15.03 ± 0.01^c	1.50 ± 0.15^c	1.26 ± 0.17^a,b	0.30 ± 0.08^c
K6	0.34 ± 0.07^c	0.16 ± 0.09^e	22.00 ± 0.24^e	2.00 ± 0.04^d	9.33 ± 0.09^e	3.00 ± 0.03^a	0.90 ± 0.44^c	0.26 ± 0.06^c

K1-K5: National brands of ketchup. K6 International brand of ketchup.

Discussion

Ketchup serves as a promising source of antioxidants and these antioxidants come from tomatoes.²⁷ Plants (vegetables and fruits) are the best source of antioxidant compounds. These antioxidants are very useful for humans as they possess potential against different diseases such as cancer, cardiovascular disease, anti-viral and anti-bacterial.^17,21

The phenolic compounds have been documented to impart a very important role in the sensory and nutritional quality of different vegetables and fruits.²⁸ Phyto-chemicals contain mostly phenolic compounds which act as antioxidants, pollinator attractive, anti-feedants etc²⁹ These phytochemicals play a very important role in plants for production and protection from pathogens.³⁰ These phyto-chemicals are used for the preservation of foods and are beneficial against different diseases such as brain disinfection, cancer etc²²

Almost 4000 flavonoids have so far been identified and this number is continuously increasing.³¹ Flavanols, anthocyanin and isoflavonols are the different forms of flavonoids containing greater redox potential which is responsible for quenching oxygen free radicals, as a reducing agent and is used as a hydrogen donor.³² Vegetables and fruits are excellent sources of flavonols which is the main class of flavonoids. The flavonoid contents depend upon different conditions such as climate, growing conditions, cooking, storage etc³³ Fruits, tomatoes, some aromatic plants and herbs are the main sources of flavanones, whether some additional fruits like grapes contain naringenin, oranges fruits contain hesperetin and lemons contain eriodictyol.³⁴ The role of flavonoids in plants is unclear although several functions have been suggested such as being potent against microbial attack and also protecting from the UV-B radiation. Higher use of fruits and vegetables can decrease the risk of cancer due to non-nutritive compounds like flavonoids.³³

The AA of ketchup using anacardic acid (preservative) against S. aureus bacterial strain was 4.32 log cfu/g after six months of preservation while 4.30 log cfu/g was recorded at initial time. In case of E. coli 4.52 log cfu/g inhibition was observed after six month's preservation, while 4.32 log cfu/g inhibition was indicated at initial time.²⁰ The microbial infection creates health disorders.³⁵ The presence of microorganisms in food might be attributed to different factors including harvesting, manufacturing, transport, and storage or by their natural microflora. Microorganisms affect the food quality which cause spoilage of food and different food-borne diseases. Foodborne diseases and food spoilage might be linked to economic losses. Developing countries observed 10–25% food loss due to microbial spoilage. Due to the presence of microorganisms, the colour, texture and taste of food may be changed.³⁶

The detailed results are discussed in Figure 1. The different solvent extracts against Heliotropium strigosum showed different antidiabetic activities and the range of antidiabetic activity was 10.45 to 100.¹⁶

Figure 1.

Antidiabetic activity of commercial ketchup.

In the foods, the sensory evaluation was used as an opinion from the consumer point of view and investigated the consumer behaviour toward food which was used for analysis.

Metals and metalloids available in the earth crust, biosphere, atmosphere and in food are stable and accumulate in the tissues of plants and humans. Eliminating heavy metals from the body is very slow; hence, they transfer in the form of more toxic substances. The effects of heavy metals and trace elements on nutrition and human health have been studied extensively in past recent years. A few elements like Fe, Zn and copper Cu are used as nutrients and beneficial for human health at appreciable limits but Cd, Ni, As, Hg and Pb are regarded as toxic and harmful elements for human health as well as for plants even in very small concentration.^37,38 The contamination of food by metals has raised the interest of scientists and the public as they pose severe health problems. The presence of heavy metals in food badly affects its quality. Heavy metals find two different ways to get in food. The first way is an endogenous process, in this process, metals are produced by plant metabolism and inter food chain; the second way is through the insertion of metals in food during processing, storage and packaging.^25,26 This study acknowledges certain limitations that may impact the generalizability of the findings, including a) They are contaminated with heavy metals, b) Their major limitation is their questionable drug bioavailability, c) their drug-drug interactions, which decreases their efficacy also add to their limitations and d) Another disadvantage to be listed is their false advertisement. Future research should address these limitations by exploring broader variables or alternative methodologies to enhance the robustness of the results.

Conclusions

The present study concludes that the ketchup is beneficial and potential food against different diseases as it contains a significant number of phytochemicals including but not limited to phenolic and flavonoid contents which lead to its good antioxidant, antidiabetic and antimicrobial attributes but it is necessary for the concentration of heavy metals should be controlled because they are highly carcinogenic. The K6 sample showed the highest AP, TP and TF as well as AC. The K6 sample was also highly appreciated by consumers as compared to other ketchups. Whether in the case of AA the K5 ketchup sample exhibited maximum inhibition against S. aureus while in case of E. coli the maximum inhibition was shown by K2 sample but the concentration of heavy metals was different in all ketchups.

Footnotes

Acknowledgements

The authors are thankful to the Deanship of Graduate Studies and Scientific Research at University of Bisha for supporting this work through the Fast-Track Research Support Program. The authors express their gratitude to Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R158), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. Authors are thankful to the staff of Central Hi-Tech Laboratory, University of Agriculture, Faisalabad, Pakistan for their help and unprecedented support towards this research work.

ORCID iDs

Munawar Iqbal

Naveed Ahmad

Arif Nazir

Statement of Human and Animal Rights

This article does not contain any human or animal studies.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Statement

This article does not contain any studies with human or animal participants.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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