Determination of aflatoxin M1 (AFM1) in dairy sheep, goats and camel milk in Hail Region, Kingdom of Saudi Arabia and evaluation of different methods reducing its concentrations in milk

ABSTRACT

This study aimed to determine aflatoxin M1 (AFM1) in dairy sheep, goats, and camel milk in Hail, Saudi Arabia, and evaluate methods for reducing AFM1. A total of 67 milk samples were collected from Ghazala and Al-Shweimis villages, Hail. AFM1 in milk samples was analyzed using enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) with fluorescence detection. Results revealed concentration of AFM1 in goat and camel milk samples from Ghazala was higher than that in goat and camel milk samples from Al-Shweimis with a significant difference (p<0.05) using both ELISA and HPLC methods. The mean AFM1 concentration using ELISA for goat milk was (12.96 ± 2.23 ng/l), while the AFM1 concentration for camel milk was (1.54 ± 0.29 ng/l). Microwave heating and boiling are effective methods to reduce the AFM1 in milk. ‏‎‏‎‏The highest AFM1 removal percent was 42.96% by microwave and 12.32 % by boiling milk samples.      

KEYWORDS:

• Mycotoxins
• HPLC
• ELISA
• milk
• AFM1, Saudi Arabia

Introduction

Aflatoxins (AFs), poisonous secondary metabolites from Aspergillus flavus, A. parasiticus, and A. nomius, may result into threats to humans and animals as well as they affect food safety [1,2]. Of the several mycotoxins, four major toxins have been characterized which are B1, B2, G1 and G2. The most toxic aflatoxin is AFB1which is produced by A. flavus. The major metabolite of AFB1 is AFM1which mainly found in milk and can be transferred to man through consumption of milk and other products contaminated by AFB1 [3,4]. So far, more than 20 types of Aflatoxins have been identified among which aflatoxins M1, G1, G2, B1 and B2 receive the most importance in the field of food hygiene. AFB1 is recognized as the most dangerous aflatoxins, and infected livers metabolize it, producing aflatoxin M1 and M2, which may spread through milk [5]. AFB1excretion in milk varies from 1% to 2%, with levels reaching high levels 12–24 h after consumption, dropping to undetectable after 72 h [6].

For human ingestion, the acceptable limit for AFs is between 4 and 30 μg/kg. In almost any product intended for direct use, the European Union has the strictest standard level, with AFB1 and total AFs must not exceed 2 μg/kg and 15 μg/kg in any product [7–9]. Likewise, in the United States, the highest amount of AFs allowed is 20 μg/kg [10]. In Saudi Arabia, the maximum tolerable levels of AFs set by Saudi Standardization Organization is 10 µg/kg depending on the type of diet [11].

Milk is essential for a balanced diet, but it contains disease-causing dietary pollutants like Aflatoxin M1, posing a global problem [3]. AFM1 contamination poses a serious risk to people, especially young children, and can be passed from mother to baby through milk [12]. Controlling AFM1 in milk is crucial for human health and trade. Contamination can be reduced by lowering AFM1 content in contaminated milk or indirectly by lowering AFB1 contamination in dairy animals’ feed. Mitigation strategies involve good agricultural practices, storage, and decontamination of feed crops [13].

A linear correlation exists between AFB1 in animals’ diet and AFM1 in milk [14], leading to a focus on reducing or eliminating AFB1 from feedstuffs. Special feed rules are implemented in many countries to limit AFM1 in liquid milk and dairy products. Although eliminating AFs from human and animal diets is challenging, implementing regulatory limits and official monitoring programs can reduce exposure risks through standardized analytical procedures Biocontrol can indirectly reduce AFB1 contamination in dairy animals’ feed during growth and storage [15]. Control of fungal contamination in cow feed using AFM1-containing milk treatment, is challenging in some countries [13]. Prevention and reduction of AFM1 concentrations in milk is brought about through chemical, physical, and biological techniques, including irradiation, extraction, sequestering, and heat treatments. Research shows pasteurization at 62°C for 30 min reduces AFM-1 concentration by 32%, while heating potentially reducing concentration from 12% to 35% [16]. Recently, the use of clay minerals like; bentonite and hydrated sodium calcium aluminosilicate (HSCAS) have been found to be very useful and effective in reducing the bioavailability of AFB1 in the animal feed [17]. The use of the anti-aflatoxin effect of Bifidobacterium bifidum and Saccharomyces cerevisiae was tried using different concentrations of the organisms at different temperatures for 24 h [18]. Although time-consuming and expensive it resulted into reduction of 90% of aflatoxins in milk spiked with 0.5 μg/mL when treated with 10¹⁰ colony-forming unit per ml (cfu/ml) of both organisms at 37°C for 24 h.

In some countries, like Spain; the level of AFM1 was measured in raw cow’s milk and was tested using ELISA and HPLC and has been shown that 3.3% of total samples were positive for AFM1 [19]. In Iran, however, the concentration of AFM1 in dairy cow (high milk) was higher than other dietary elements (13.16 ppb). For instance, in India and Korea a concentration of (50–3000 ng/l) and 55.7% of the samples were contaminated in both countries, respectively, [20,21]. In Yemen, a serious threat was detected in processed cheese as a result of demonstrating considerably high level of AFM1 reaching 5.95 ug/kg in 79.12% of the samples collected from supermarkets using HPLC and ELISA methods [22]. In all these studies, there was no significant difference between the results obtained using HPLC or ELISA, however, HPLC showed higher sensitivity and specificity.

There are several dairy farms around Hail city and required samples can be obtained and local people will be educated by the importance of AFM1 and how to avoid this. Therefore; this study aims to determine AFM1 contamination in milk samples from different dairy animals (including camel, sheep, and goats) in two localities in Hail, Saudi Arabia. Furthermore, different physical methods involving boiling and microwaving were attempted to reduce the contamination with AFM1 in milk samples.

Materials and methods

Samples collection

A total of 67 milk samples were collected from camel (18), goat (25), and sheep (24) from two localities near Hail city: Ghazala (40 km north) and Al-Shweimis (300 km south). A total of 30 milk samples were collected from different animal species from Ghazala, whereas 37 samples were collected from different animal species from Al-Shweimis. Raw milk was obtained from small farms in designated areas specified for Elisa and HPLC techniques. Samples were collected in a sterile containers.

Sample preparation ELISA analysis

The ELISA kits used a 50 ng/ml concentrated aqueous solution and daily standard working solutions for method validation. The standard solutions remained at −20°C throughout the process, while the standard working solutions were prepared daily. All solutions were protected from light and milk samples spiking to prevent AFM1 inactivation. The reference curves were prepared using standard solutions of known AFM1 concentrations.

The concentrations of AFM1 were determined using the Ridascreen® aflatoxin M1 test kit, a competitive enzyme immunoassay based on antigen–antibody reaction (R-Biopharm AG, Darmstadt, Germany). Milk samples were centrifuged for 10 min at 3500 g to remove the fatty layer. A volume of 100 µl skimmed milk was used for quantitative testing. Standards and samples were placed into their respective wells in the microtiter plate. The ELISA method was conducted following the manufacturer’s instructions, with 100 µl of standard solutions and samples added in separate wells and incubated for 60 min at room temperature in the dark. The liquid was poured off the wells and the micro well holder was tapped upside down vigorously (three times). All the wells were filled with 250 μl of washing buffer and emptied. The washing procedure was repeated twice. Then, 100 µl of the enzyme conjugate (secondary antibody) was added and incubated for 1 hr at room temperature in the dark. The washing was repeated three times. Substrate and chromogen solutions was added to each well and mixed thoroughly (50 μl each), followed by incubation for 30 min at room temperature in the dark. After the incubation time, 100 µl of stop reagent was added to each well and mixed thoroughly. The reaction is measured using ELISA plate reader at wavelength 450 nm. The concertation of the AFM1 will be calculated in reference to the controls included in the test.

Sample preparation HPLC-FL analysis

Milk samples were prepared in 50 ml aliquots and stored at −20°C. They were placed in a water bath at 35–37°C. Samples were shaken manually for 5 min, then centrifuged at 3500 g for 15 min to separate and discard fat. The fatless samples were filtered using Whatman filter paper, and 50 ml of the sample was transferred to the cleanup step.

Standard solution preparation

Chromatographic analysis involved preparing working standard solutions of each analyte at a 50 ng/ml concentration using a MeOH:water solution. AFs solutions were stable at −18°C and prepared daily through further dilution at different concentrations.

HPLC-FL analysis

The skimmed milk was passed through an AFM1 immuno-affinity column at a rate of 1–2 drops/second until air passes through. The column headspace was filled with water, and the column was washed twice with 10 ml of purified water. The elution involved passing 1.25 ml of ACN (Acetonitrile):MeOH (3:2) solution and 1.25 ml of purified water. The eluent was collected, filtrated, and evaporated to 2 ml under a nitrogen stream. Aliquots of 100 μl of the resulting samples were injected into the HPLC system. For fat-containing milk samples, they were centrifuged at 3500 g for 15 minutes for fat removal.

HPLC system which was used in the current study is a Shimadzu Class VP, equipped with a multi-λ fluorescence detector (FD) with an excitation wavelength of 365 nm and an emission wavelength of 435 nm. The chromatographic column was C18 5 mm (4.6 × 250 mm) (HS, Bellefonte, U.S.A.). The mobile phase (water: acetonitrile: methanol; 68: 24: 8, v/v/v) was run for 15 min at 30°C with a flow rate of 1 ml/min. Calibration curve was prepared from either peak heights or peak areas by injecting 20 μl of a series of standard solutions of AFM1 with concentrations of 0.05, 0.1, 0.5, 1.0, 2.5, and 10 μg/l to ensure linear relationship. The retention time for AFM1 was 6.2–6.7 min and the standard AFM1 concentrations was obtained from Sigma Aldrich (Germany).

Reducing contamination experiments

Milk samples known to show detectable levels of AFM1 were undergone detoxification using boiling and microwave irradiation. Milk samples were boiled at 100°C for 10 min in order to study the effect of boiling in reducing the concentrations of AFM1. Milk samples with known concentrations of AFM1 were also exposed to microwave radiation in microwave oven at high energy level for 2 min following the method of Zhao et al. [23].

Statistical analysis

The prevalence data for AFM1 determined by HPLC was expressed as no. and percent and compared using the Chi-square test (ꭓ2). The other comparisons (variables) of AFM1 concentration (ng/l) determined by HPLC or ELISA were checked by Levene test for homogeneity, which indicated non-homogenized variables, therefore the suite comparisons are non-parametric tests. The Games-Howell multi-comparison test and Mann-Whitney U test were used with different animals’ milk or locations, respectively. All comparison were conducted using SPSS V. 21 with a p value less than 0.05.

Results

AFM1 were detected in all samples collected using the ELISA method whereas, 30 (44.78) samples showed detectable levels of AFM1 using HPLC method. There was obviously significant difference between the methods having the ELISA method more reliable (p < 0.001). The minimum and maximum levels of AFM1 as measured by ELISA were 0.29 and 20.25 ng/l while those detected by HPLC method were 1.16 and 21.1 ng/l, respectively.

The AFM1 concentration determined by ELISA in animal milk samples collected from Ghazala located in the Hail Region. The highest concentration of AFM1 (12.96 ± 2.23 ng/l) was detected from goat milk, while the lowest concentration of AFM1 (1.54 ± 0.29 ng/l) was detected in camel milk was (Table 1). There was a significant difference between the groups studied (p > 0.001).

Table 1. Concentration of AFM1 (mean ± SE, ng/l) in milk of different animals in the village of Ghazala determined by ELISA. The post-hoc comparison was determined by Games-Howell test at p ≤0.05.

AFM1 (ng/l) determined by ELIZA				Games-Howell test p value
Samples	Mean ± SE	Minimum	Maximum
Sheep milk (n = 10)	5.74 ± 0.34	3.23	7.14	<0.001
Goat milk (n = 10)	12.96 ± 2.23	4.29	20.25
Camel milk (n = 10)	1.54 ± 0.29	0.29	3.18

In Al-Shweimis village, the mean AFM1 concentrations for sheep milk was (7.49 ± 1.00 ng/l), while the mean AFM1 concentrations for camel’s milk was (2.34 ± 0.34 ng/l). The differences between the studied groups were significant (p > 0.001) (Table 2). There was a significant difference in concentration of AFM1 (ng/l) in sheep milk collected from Al-Shweimis village determined by ELISA compared to that of Ghazala village p ≤0.05 (Table 3).

Table 2. Differences in the concentration of AFM1 (ng/l) in milk of different animals in the village of al-Shweimis determined by ELISA.

Aflatoxin M1 (ng/l) as determined by ELISA				Games-Howell test p value
Samples	Mean ± SE	Minimum	Maximum
Sheep milk (n = 14)	7.49 ± 1.00	5.34	20.15	<0.001
Goat milk (n = 15)	4.59 ± 0.31	1.88	6.10
Camel milk (n = 8)	2.34 ± 0.34	1.46	3.89

Table 3. Differences in the concentration of AFM1 (ng/l) in milk from different animals in the villages of Ghazala and al-Shweimis as determined by HPLC.

	Ghazala Village			Al-Shweimis Village
Samples	Mean±SE	Minimum	Maximum	Mean±SE	Minimum	Maximum	Mann-Whitney U test (p value)
Sheep Milk	2.33 ± 0.78	1.16	5.14	4.27 ± 1.09	1.14	12.55	0.004 (Ghazala) 0.093 (Al-Shweimis)
Goat Milk	12.4 ± 2.59^a	3.24	21.1	1.33 ± 0.14	1.17	1.61
Camel Milk	-	-	-	-	-	-

The concentration of AFM1 in sheep collected from Ghazala village was found to be 5.74 ± 0.34 (3.23–7.14) whereas, that of sheep in Al-Shweimis village was 7.49 ± 1.00 (5.34–20.15). While the concentration of AFM1 in goats in Ghazala village was 12.96 ± 2.23 (4.29–20.25), the AFM1 concentration in goats in AL-Shweimis was 4.59 ± 0.31(1.88–6.10). The levels of AFM1 in camels, however, were the lowest in both villages being 1.54 ± 0.29 in Ghazala and 2.34 ± 0.34 in Al-Shweimis (Tables 1 and 2).

Only one sample of sheep milk obtained from Al-Shweimis village exceeded EC regulation (50 ng/kg) by recording 74.37 ng/l AFM1 determined by ELISA method. None of the samples with AFM1 exceeded US Regulation (500 ng/kg).

The effect of boiling and microwaving was evaluated in reducing the level of milk contamination with AFM1 was determined by both methods ELISA and HPLC. Using ELISA, boiling has reduced the concentration of AFM1 by 12.55% whereas microwave irradiation has reduced the AFM1 concentration by 42.96% (Table 4). When using HPLC, boiling has reduced the concentration of AFM1 by 82.63% whereas microwave irradiation has reduced the AFM1 concentration by 63.24% (Table 5).

Table 4. Effect of boiling and microwave treatments on AFM1 concentration (ug/ml) in milk as determined by ELISA.

Treatment	Number of samples	AFM1 concentration (ug/ml)		Percent of reduction (%)	Sign test^1
		Before	After		No of negative differences^2	No of positive differences^3	P value
Boiling	8	15.23 ± 4.35	13.32 ± 6.31	12.55	4	4	0.492
Microwaving	8	23.03 ± 9.62	13.13 ± 7.41	42.96	6	2	0.037

Means in the same row bearing with different letters means a significant differences at P value ≤ 0.05 of paired sample t-test.

1. Sign test: AFM1 concentration before treatment – AFM1 concentration after treatment.

2. Number of negative differences = No of samples indicated lower AFM1 concentration after treatment.

3. Number of positive differences = No of samples indicated higher AFM1 concentration after treatment.

Table 5. Effect of boiling and microwave treatments on AFM1 concentration (ug/ml) in milk as determined by HPLC.

Treatment	Number of samples	AFM1 concentration (ug/ml)		Percent of reduction (%)	Sign test^1
		Before	After		No of negative differences^2	No of positive differences^3	P value
Boiling	13	17.38 ± 6.34	3.02 ± 0.81	82.63%	10	3	0.032
Microwaving	12	3.18 ± 0.75	1.17 ± 0.04	63.24%	8	4	0.023

Means in the same row bearing with different letters means a significant differences at P value ≤ 0.05 of paired sample t-test.

1. Sign test: AFM1 concentration before treatment – AFM1 concentration after treatment.

2. Number of negative differences = No of samples indicated lower AFM1 concentration after treatment.

Discussion

A healthy diet is essential for good health, and milk is a nutrient-dense food for growth, development, and body maintenance [24]. Middle East and Arab countries consume camel, sheep, and goat milk for local consumption, but some markets handle it for local consumption. AFM1 is a dietary toxins concern, causing concern for over 30 years [25]. AFM1 poses health risks, particularly in liver cancer development from milk and dairy products [26]. Moreover, AFM1 can result in hepatotoxic, carcinogenic, and immunosuppressive effects [27]. AFM1 levels in milk samples have been reported in a number of different reports [28–30]. AFM1 prevalence in animal milk in the Hail Region, Kingdom of Saudi Arabia, was reported in Ghazala and Al-Shweimis villages, with limited data on its occurrence.

On previous studies, high AFM1 contamination was reported in goat and sheep milk samples, with higher levels found in Jeddah, Saudi Arabia, and Thailand [28,31,32].

The amount of AFM1 in milk depends on the amount of AFB1 in the food consumed. The type of feed used and environmental conditions affect the amount of AFM1 in milk. Camels are primarily feed by grazing, while sheep and goats follow an intermediate feeding schedule. They are sent out to graze daily, then returned for milking and given a prepared ration in the evening. This variance in AFM1 levels can be attributed to the animal species’ feeding habits [33].

AFM1 concentration in milk samples from Ghazala and Al-Shweimis villages in Saudi Arabia is nearly similar both geographical locations, possibly due to similar environmental conditions, livestock management, and dairy processing systems. Studies worldwide have shown that high AFM1 contamination in milk poses a problem in countries with dry and cool climates, including Brazil [34], Egypt [35], Iran [36], and Pakistan [37]. High humidity conditions are favorable for the development of fungi and the formation of mycotoxin in feed and feed components [38].

The goat milk in Ghazala village reported the highest concentration of AFM1 (12.96 ng/l) while, sheep milk (7.49 ng/l ±1.00) was the highest among the three types of milk in Al-Shweimis village. Moreover, goat milk collected from Ghazala village is the highest contamination level among different animal milk samples and locations. Oezdemir [39] detected AFM1 in 93 (84.54%) of 110 goat milk samples in Turkey and AFM1 levels in 7 (6.36%) of 110 milk samples were found to be higher than the maximum tolerable limit (50 ng/l). Zheng et al. [40] demonstrated that, 76.0% of goat milk samples in China contained AFM1 in concentrations ranging from 0.005 to 0.135 µg/l (mean: 0.022 ± 0.025 µg/l, median is 0.012 µg/l). Furthermore, they proved that, contamination with AFM1 in goat milk is higher than camel milk. Both feeding practices and the kinds of foods have an impact on the levels of AFM1 in goat milk [32]. Geographical area, nation, farming practices, and seasons significantly affect AFM1 levels in milk [39]. Camel milk has lower levels compared to other animal milk samples in the two villages studied. Similar results were also reported from Jordan [41], Qatar [42], and Iran [43]. The lower incidence of AFM1 in camel milk compared to other milk may be due to (a) lower dietary intake of parent AFB1, as camels are offered less feed concentrate than bovine feeding regimes; (b) activity of camel’s ruminal microflora that leads to more degradation of AFB1; (c) intestinal morphological differences preventing AFB1 absorption in camel; or (d) hepatic microsomal enzyme activity that causes AFB1 in camels to be biodegraded into other biotransformed metabolites other than AFM1 [42]. In the same context Kamkar [44] and Dashti et al. [45] revealed that, camels spend most of the year grazing and eating wild vegetables. Other animals are fed artificial foodstuffs created from a variety of stored grain products and agricultural industry byproducts. The latter feed is vulnerable to a fungus infestation and subsequent aflatoxin contamination while being held. According to our research, camel milk is guaranteed to be safe in terms of AFM1 contamination in Hail.

Accurate detection of AFM1 in milk is crucial due to regulatory requirements. Challenges include proper sample preparation and low concentration identification. Various techniques are used for detecting AFM1 in milk and dairy products, but ELISA and HPLC are preferred for more confirmation. HPLC-FL with post-column derivatization is the reference method for determining mycotoxins qualitatively and quantitatively [46]. This is now the approach that is most frequently used to determine the presence of AFM1 in milk [47]. Additionally, the HPLC approach demands a difficult and complicated sample preparation process that uses a lot of chemical solvents [48]. ELISA is a technique that is mostly utilised in screening control because it provides precise, rapid responses with the ability for mass repetition [49]. Routine analysis prefers this quick, low-cost method due to its fewer preparatory steps and smaller sample volumes compared to HPLC [50,51]. ELISA’s accuracy depends on mycotoxin type, sample preparation, and material type. However, prior separation enables the accuracy and reproducibility of the procedure to be improved [52]. The results from the ELISA kit were in agreement with those reported by the HPLC method taking into account the quality control of the milk samples. Moreover, the results revealed a high detection efficiency of the ELISA technique over HPLC, whereas the AFM1 was detected in all milk samples examined by ELISA. Similarly, Maggira et al. [49] The ELISA kit is a faster and reliable alternative to HPLC for routine milk AFM1 determination, according to evaluation. Moreover, Rodriguez Velasco et al. [19] verified that AFM1 concentrations found using the ELISA approach were higher than those found using the HPLC-FLD procedure. Determination of AFM1 in milk was conducted using an enzyme-linked immunosorbent assay (ELISA) in more recent studies [24,27].

There are specific regulations for lowering the level of aflatoxin M1 in products involving milk in many states throughout the world due to its unfavorable effects on human health [50]. Due to the impact that financial matters have on each country, these regulation limits may vary [53]. The European Commission sets a maximum AFM1 limit of 50 ng/kg in liquid and dry milk products, based on ALARA (as low as reasonably attainable). However, the US rules and Codex Alimentarius Commission set a 500 ng/kg limit [54,55]. Within those two points, Syria’s regulation level is restricted to 200 ng/kg [56]. Our data reported that, only one sample of sheep milk obtained from Al-Shweimis village exceeded the EC regulation (50 ng/kg) by recording 74.37 ng/l AFM1 determined by ELISA methods. Meanwhile, no sample exceeded U.S. Regulation (500 ng/kg). Gürbay et al. [57] used HPLC to discover AFM1 in 16 (59.3%) of the milk samples, and they discovered that only one sample exceeded Turkey’s maximum allowed limit for AFM1 in Ankara. AFM1 levels were found to be greater than the maximum tolerated limit (50 ng/l) authorised by the Turkish Food Codex in 7 (6.36%) of 110 milk samples [39]. Alborzi et al. [58] determined that the maximum tolerance level for AFM1 (50 ng/l) approved by the European Union was exceeded in 17.8% of the samples. The prevalence of AFM1 contamination in the raw milk tested in Portugal was 80.6%; low levels (5–10 ng/l) were found in 17 samples (54.8%), levels between 11 and 20 ng/l were found in two samples (6.5%), and levels between 21 and 50 ng/l were found in six samples (19.3%) [59]. AFM1 levels in all samples of milk in peri-urban Nairobi, Kenya, were higher above the detecting limit (5ng/kg). Also, they were above 50 ng/kg in two-thirds of the samples, and 500 ng/kg was exceeded in 7.5% of the samples [60]. The overall AFM1 incidence of the milk samples analysed in this study was lower than many other studies.

Although AFM1 contamination is not significantly above the legal limit in our study, it could negatively impact food quality and harm human health. As a result, dairy animals should be handled with care during milking, fed high-quality food, and public health should create efficient monitoring programs to reduce aflatoxin contamination. Recent research focuses on lowering levels of AFs in feeds and food, using biological, physical, and chemical techniques to mitigate AFs [61]. Overall, the results demonstrated that AFM1 levels in naturally contaminated milk samples were reduced by boiling and microwaving methods, as determined by ELISA. Yosef et al. [62], found a significant decrease in AFM1 concentrations in milk from Al-Riyadh farms across treatment strategies compared to real positive controls. The ranking of the decrease rate was as follows: Boiling treatment (23.93%) is followed by pasteurisation (12.90%) and then exposure to microwave radiation (52.08%). AFM1 was degraded by 12.21% after 15 min of milk sterilisation at 121°C, compared to 14.50% when milk was boiled [63]. Moreover, Deveci [64] found pasteurization partially reduces AFM1 in milk, influenced by heat treatment time and temperature, affecting AFM1 degradation. Conversely, Kuboka et al. [60] concluded that, boiling had no effect on the levels of AFM1 contamination in collected milk samples. Finally, more studies are needed to study other powerful detoxification agents, such as probiotics, plant extracts to reduce the toxin, and their benefits for health and financial conditions.

Availability of data and materials

All the datasets generated or analyzed during this study are included in this published article.

Acknowledgements

This study was supported by the Researchers Supporting Project (RSP2023R94), King Saud University, Riyadh, Saudi Arabia.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia. [(RSP2023R94)]; Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia [(RSP2023R94)].