Microcapsules loaded with date seed extract and its inhibitory potential to modulate the toxic effects of mycotoxins in mice received mold-contaminated diet

Mycotoxins are the secondary fungal metabolites generally produced by wide range of fungi including aflatoxins (AF), ochratoxin A (OTA), fumonisins (FB), zearalenone (ZEN), and deoxynivalenol (DON). Nowadays, they are main concern to food and agricultural commodities due to undesirable health and socio-economic effect. This investigation was designed to synthesized microcapsules loaded the bioactive compounds of date seed and evaluated its inhibitory activities in mice received mold-contaminated diet. The finding revealed that the developed microcapsule is homogenous and mostly spherical with size of 2.58 μm with acceptable PDI of 0.21. The main phytochemical has been confirmed by HPLC analysis were xylose, fructose, mannose, glucose, and galactose with the respective values of 41.95%, 2.24%, 5.27%, and 0.169%. The in vivo analyses manifested that the mice received date seed microcapsules significantly (p < 0.05) improved the average daily weight gain, feed intake, liver enzymes (ALT, ALP, and AST), and lipid peroxidation values compare to mice group received mycotoxin-contaminated diet. Furthermore, encapsulation date seed bioactive compounds notably up-regulated the expression of GPx, SOD, IFN-γ, and IL-2 genes while down-regulated the iNOS gene. Consequently, the novel microcapsules loaded date seed is suggested to be considered as a promising mycotoxin inhibitor.


Introduction
Mycotoxins are carcinogenic toxins, widely occur in human foods, animal feed, and famous as the secondary fungal metabolites (De Ruyck et al. 2015). Due to their chemical makeup, they are readily absorbed by the body from the lungs and intestines into the bloodstream, where they can then travel to other organs like the kidneys and liver (Adam et al. 2017). Furthermore, if they reach to cellular genome, they are able to cause the mutagenic alteration, immunosuppressive condition, and cancer development (Adam et al. 2017). Mycotoxins come in a variety of forms, with aflatoxin B1, zearalenone, and ochratoxin A being the most frequently found in contaminated agricultural products, feed, and food due to their highly toxic and carcinogenic effects on human and animal cells (Zhou et al. 2017). All the mentioned mycotoxins are highly resistant to heat due to the presence of at least one benzene ring in their chemical structure so their removal and decomposition in the normal range of cooking are not feasible (Adam et al. 2017;Benkerroum, 2016).
Physical, chemical, and microbial methods were all used to lessen the amount of mycotoxins in food and feed (Adeyeye and Samuel 2020; Luo et al. 2018;Vila-Donat et al. 2018). One of the promising approach to control and eliminate mycotoxin is the use of natural bioactive constitutes of plants as the outstanding alternative to limit exposure, mitigating and related infection of mycotoxins (Makhuvele et al. 2020). Medicinal plants are one of the oldest and most widespread forms of medication in different parts of the world because they contain pharmacological and natural bioactive component including functional carbohydrates including monosaccharides (glucose, fructose, and galactose), oligosaccharides (fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, mannan oligosaccharides), and polysaccharides (cellulose, starch, and glycogen) (Saini et al. 2022). One of the advantages of using natural carbohydrates as compared to other methods is related to their environmental friendly and safety to control the mycotoxins (Makhuvele et al. 2020).
Date seeds are high in natural bioactive compounds like functional carbohydrates and phenolic compounds, but are rarely viewed as having commercial value or potential as functional food ingredients. Moreover, they have a wide range of biological properties under in vivo and in vitro conditions, and their potential for use in the cosmetic and nutraceutical industries is widely acknowledged (Habib et al. 2014;Sirisena et al. 2015). One of the promising technologies for capturing and effectively delivering of natural bioactive constitutes is microencapsulation using spray drying (Abrahão et al. 2019). This technique is an efficient method due to enhancement in the oxidative stability and protection of these compounds against the environmental condition (Rigon et al. 2016). Since one of the paramount interests in the food safety is search for alternative approach to control and detoxification of mycotoxin, this research is conducted to encapsulate the bioactive components extracted from date seeds and to assess its potential immunobiotic and health promoting activity in the mice received mold-contaminated feed.

Materials
The dates used in this study came from the Mashhad, Iran, market and were of the Mazafati cultivar. For the encapsulation walls, we used modified starch (HI-CAP® 100, Ingredion, Humberg, Germany), maltodextrin Foodchem,China), and whey protein concentrate (60% protein content). Sigma supplied the Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the high-performance liquid chromatography (HPLC) standards (glucose, fructose, mannose, and xylose) (Germany). The majority of the study's solvents and chemicals came from a German company called Merck.

Extraction procedure
The seeds were extracted from the date fruit and dried in a 50 °C oven for 72 h. Using a laboratory grinder, the seeds were ground to a fine powder. The following steps were taken to make the crude extract of date seed: After shaking the mixture for 10 min and autoclaving it at 121 °C for 20 min, 100 g of dried date seed were successfully rehydrated. After centrifuging at 10,000 g for 10 min at room temperature (RT), insoluble materials were filtered out using Whatman filter paper No. 1. (Maidstone, UK). Hydrophobic groups were washed away by combining 500 mL of extract with 400 mL of a chloroform/methanol mixture (2:1), shaking the mixture for 5 min, and letting it sit for 20 min at room temperature. After that, the water/ethanol mixture was transferred to another bottle, and the process was repeated twice more. After mixing 350 mL of the extract with 650 mL of acetonitrile, shaking for 5 min, and centrifuging at 10,000 g for 10 min at RT, the proteins could be removed by separating the supernatant from the precipitated protein (Jahromi et al., 2017) . To obtain the extract enriched by monomeric and oligomeric saccharides, the water content was removed at 40 °C using a Rotary-evaporator (Heidolph Instruments GmbH & Co. KG, Germany), and the residue was freezedried (FreeZone 6 Liter Benchtop, Labconco Corporation, Kansas City, MO, USA).

Microencapsulation of date seed extract
In a spray dryer on the scale of a pilot plant with countercurrent air flow, the microencapsulation was carried out. For the wall material composition, the carrier agents maltodextrin (70%), modified starch (20%), and whey protein concentrate (10%) were used. In a nutshell, 100 mL of water was combined with the crude extract (8 g), tween 80 (3 g), and 32 g of wall material in a 1:4 ratio. The solution was then left overnight to completely rehydrate. Prior to spray drying, the solution was homogenized at 10,000 rpm for 5 min. Spray dryer pressure was set at 0.4 kg/cm 2 , temperature was set between 130 and 80 °C, and flow rate was set to 8 mL/min. One hundred milliliters of the obtained solution was then saved for later research (Rigon et al. 2016).

Characterization of microcapsules
Zetasizer examined the microcapsules' size and polydispersity index (PDI) (Malvern Instruments Ltd., UK). Scanning electron microscopy (SEM) was used to analyze the morphology of microcapsules.

Microcapsules' overall phenolic content and antioxidant activity
Using a visible spectrophotometer (Novaspec II Visiblespectro, Japan), the total phenolic content was assessed calorimetrically at 765 using the Folin-Ciocalteu assay. The results were reported as mg gallic acid equivalent (GA eq.)/g dry crude extract (Karimi et al. 2018). Additionally, DPPH scavenging activity was used to measure the free radical scavenging capacity of microcapsules (Karimi et al. 2019).

HPLC for oligosaccharide and monosaccharide detection
According to the procedure outlined by Jahromi et al. (2017), the concentrations of saccharides in the microcapsules were determined using HPLC (2690, Waters, USA) with a COSMOSIL Sugar-Dcolumn (250 4.6 mm i.d., 5 m). Acetonitrile and water were the main components of the mobile phase, which had a flow rate of 0.7 mL/min and a column temperature of 35 °C. Saccharides were detected using a Refractive Index Detector (2414, Waters, USA) at a temperature of 30 °C and a sensitivity of 1024. The running time was 20 min, and the sample injection volume was 20 L. Date seeds contained 580 g/kg of mannan in their cell walls, according to Jaafar and Jarvis (1992), and Zhang et al. (2009) reported extracting up to 48.8% of D-mannose from seeds, indicating that mannan constitutes the majority of the seeds' saccharides. For the assay of oligosaccharides in this study, five pure MOS-mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexaose-from Megazyme, Ireland, were used as standards. The same procedure used to detect oligosaccharides was used to detect monosaccharides, with the exception that the mobile phase was changed to 80% acetonitrile in water (as opposed to 65% for oligosaccharides) and the flow rate was changed to 1 mL/min. Standards included glucose, feructose, mannose, and xylose from Sigma-Aldrich in St. Louis, Missouri, USA.

Animal trails
The current research was conducted after approval from the Islamic Azad University of Mashhad. The 30 white balb/c mice with respective weight of 20-25 g were purchased from Razi Vaccine and Serum Research Institute of Mashhad. They were kept in the cage for a week at 58% humidity, 10% relative humidity, 23 °C, and 1 °C with 12-h light/dark cycles in order to adapt to the animal house environment. The mice were then divided into three groups at random. As the control group, group 1 (T1) received a typical regimen. The diets of the other two groups (T2 and T3) were supplemented with microcapsules containing either mycotoxin-contaminated diets alone or mycotoxin-contaminated diets combined with date seed extract (3 mg/g diet), respectively. Water and food were available at all times. Table 1 displays the experimental diets. The mycotoxin-contaminated diet ( Table 2) was made using the methods described below. The diet was moistened with sterile water up to a 20% level. For about 2 weeks, the feed was cultured in natural conditions (temperature of 25-30 °C, humidity of 70%). The food was pelleted and air-dried. The mice were sacrificed and put to death on day 28 of the experiment. Samples of the ileum, liver, and blood were taken for additional examination.

Liver enzymes potential and lipid peroxidation assay
Alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase potential were examined using an automated chemistry analyzer (Roche, Hitachi 902 analyzer, Japan). Based on a prior similar study, the lipid peroxidation of the liver under various treatments was carried out using the malondialdehyde (MDA) content in the liver tissue lysate by thiobarbituric acid reactive substances (Garcia et al. 2005).

Histopathology and morphometric analyses
When the mice were scarified (days 28), the liver, kidney, and jejunum tissues were collected and cleaned using physiological serum. They were initially fixed in buffered formalin (10% formalin in 0.1 M sodium phosphate buffer, pH7). The next step is to paraffinize, slice, and stain the tissue using the hematoxylin and eosin method (Shafaei et al. 2020). The prepared slides were then examined with a light microscope at a 20× magnification. The villus height, villus width, crypt depth, and goblet cell count were measured as morpho-structural features of the ileum (Navarrete et al. 2015).

Gene expression analysis
The liver tissue was preserved in liquid nitrogen after the in vivo study was completed for gene expression profiling. To achieve this, liver tissue was minced and prepared for RNA extraction using an RNeasy Mini kit from Qiagen in Hilden, Germany, in accordance with the kit's instructions. A Quantitect Reverse Transcription kit was then used to create the cDNA (Qiagen, Hilden, Germany). The temperature settings for the real-time PCR program were 95 °C for 5 min (1×), 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 30 s (35×). Finally, the expressions of genes related to antioxidants (GPx, SOD), inflammation (iNOS), and immune modulation (IFN-, IL-2) were assessed. Gene expressions were normalized using the reference gene -actin. (Kathirvel et al. 2010). The list of primers performed is listed in Table 3.

Characterization of microcapsules
In the encapsulation process, the bioactive compounds present in the date seed extract are coated by wall materials. The encapsulation protects the bioactive material from oxidation, degradation, and any chemical and physical reactions to maintain the biological and functional properties of the material in the core. The obtained results exhibited that spray-dried technique could produce microcapsules loaded by date seed extract with size of 2.58 μm with acceptable 0.21 PDI (Table 4). The zeta potential indicated moderate stability behavior in the colloidal dispersions (moderate stability behavior: ± 30 to ± 40 mv) (Smith et al. 2017). In this study, the moderate stability behavior of the colloidal dispersions indicated that there was a balance between attractive and repulsive forces between particles, which prevented them from aggregating or settling down.
The morphology of the microcapsules is one of the critical parameters in encapsulation process as any cracked or damaged structure could compromise the stability and effectiveness of the encapsulated materials (Choudhury et al. 2021).
In the current study, the SEM photomicrographs demonstrated the mostly spherical, homogenous, and smooth surface of microcapsules (Fig. 1). The SEM photomicrographs presented in this study showed that the microcapsules had a mostly spherical shape with a smooth surface, indicating good structural integrity. Any cracked or damaged structure could compromise the stability and effectiveness of encapsulated materials.

HPLC analysis and biological potential of microcapsules
The HPLC analysis of microcapsules demonstrated that xylose, fructose, mannose, glucose, and galactose as the main component of the date seed microcapsules with the respective values of 41.95 ± 2.35, 2.24 ± 0.07, 5.27 ± 0.08, and 0.169 ± 0.04% (Table 5). The early study conducted by Ghnimi et al. (2017) reported that the date seeds store carbohydrates mainly as mannan in the form of gluco-and galactomannans, and others are glucose, arabinose, rhamnose, and xylose. Furthermore, the total phenolic compounds of date  Ghnimi et al. (2017) reported the very high levels of phenolic antioxidants (3100-4400 mg gallic acid equivalent/100 g) rendering it as potential antioxidant giving 580-930 μM Trolox equivalents antioxidant activity (TEAC). The carbohydrates such as mannose, fructose, glucan, fructooligosaccharides, and mannan oligosaccharides are potent in binding the mycotoxins in the gastrointestinal tract (Nahle et al. 2022). In the study conducted by Aravind et al. (2003) showed that the esterified glucomannans alleviated the adverse effects of several combinations of mycotoxins (168 ppb AFB1 + 8.4 ppb OTA + 53 ppb ZEA + 32 ppb T-2 toxin) naturally found in feed on productivity and serum biochemical and hematological parameters of broilers.
The other study conducted by Avantaggiato et al. (2014) revealed that the phenolic compounds in the dehydrated grape pomace are potent adsorbent of the aflatoxin B1, zearalenone, ochratoxin A, and fumonisin. Hence, the bioactive compounds detected in the microcapsules loaded by date seed extract could render it as potential adsorbent of mycotoxins.

Animal trial
The results of average daily weight gain and average daily feed intake are presented in Table 6. When compared to the group that was not given the mycotoxin-contaminated food (T2), the mice who were given the mycotoxin-contaminated diet (T2) showed a significant decrease (p < 0.05) in both their final weight and their total feed consumption (T1). This finding is alarming as it suggests that mycotoxins can have a detrimental effect on the growth and development of animals. On the other hand, the food containing date seed microcapsules significantly (p < 0.05) improved the average daily weight gain and average daily feed intake (T3).
The results observed in this study were in agreement with the results of Aravind et al. (2003) who reported the adverse effects of feeds contaminated by several combinations of mycotoxins (168 ppb AFB1 + 8.4 ppb OTA + 53 ppb ZEA + 32 ppb T-2 toxin) on weight gain and feed intake in broiler chickens. The broiler chickens' growth parameters and feed intake were improved by the addition of glucomannans at a concentration of 0.05%.

Liver enzyme and lipid peroxidation assay
The liver enzymes (ALT, ALP, and AST) together with lipid peroxidation values are summarized in Table 7. Major biomarkers of liver health and function include the liver enzymes ALT, AST, ALP, and lipid peroxidation (MDA). The rise in these parameters' values suggested oxidative stress, inflammation, and liver dysfunction (Goodarzi et al. 2020). Overall, the liver enzymes (ALT, ALP, and AST) and lipid peroxidation values were significantly (p < 0.05) increased in the mice fed mycotoxin-contaminated feed (T2) when compared to the control group (T1). Microcapsules containing date seed extract as a dietary supplement significantly (p < 0.05) reduced liver enzyme levels (ALT, ALP, and AST) and lipid peroxidation values (T3). This finding leads to the hypothesis that the mycotoxins decreased appetite  Table 6 The averages of mice body weight changes and feed intake during experiment receiving different treatments T1: control, T2: mycotoxin-contaminated diet, T3: mycotoxin-contaminated diet + date seed extract microcapsules (3 mg/g diet) Different letters in the same raw indicated significant difference (p < 0.05) The analyses were performed in triplicates  Table 7 The liver enzymes biochemical assay T1: control, T2: mycotoxin-contaminated diet, T3: mycotoxin-contaminated diet + date seed extract microcapsules (3 mg/g diet) Different letters in the same column indicated significant difference (p < 0.05) The analyses were performed in triplicates and caused weight loss as a result of gastroenteritis. Inducing systemic inflammation and oxidative stress, the mycotoxins decreased appetite while raising liver enzymes and lipid peroxidation. The date seed extract contained in microcapsules and used in this study helped the mice given mycotoxin-contaminated food gain weight, eat more, and produce fewer liver enzymes and less lipid peroxidation. Similar experiment indicated that date seed extract had antioxidant and anti-inflammatory properties, which could help protect against oxidative stress and inflammation-related diseases (Zhang et al. 2013). Another study found that date seed extract had hypoglycemic effects in diabetic rats, suggesting that it may have potential as a natural treatment for diabetes (El Fouhil et al. 2013). This observation is most likely explained by the fact that the date seed extract contains phenolic compounds and carbohydrates with adsorbent potential, antioxidant properties, and anti-inflammatory properties. These outcomes were in line with earlier studies showing that phenolic compounds and carbohydrates may be able to lessen the negative effects of mycotoxins in various animals (Ahmed et al. 2022;Boudergue et al. 2009;Vila-Donat et al. 2018). While more research is needed to fully understand its mechanisms of action and potential applications in human health, these findings are certainly promising. Additionally, further research could investigate the optimal dosage and duration of supplementation with date seed extract for maximum benefit.

Histopathology and morphometric analyses
In contrast to the control group, mice given mycotoxin-contaminated feed (T2 & T3) did not exhibit any histopathological changes in their liver, kidney, or jejunum tissues, as shown in Fig. 2. Table 8   consuming date seed extract in microcapsules significantly (p < 0.05) increased villus height, villus width, number of goblet cells, and decreased crypt depth. The number of goblet cells increased in the mice that were fed a diet contaminated with mycotoxins as a defense mechanism against the mycotoxins. The results obtained were consistent with the results of Poloni et al. (2020) who observed the impair in the villus height, villus width, crypt depth and the atrophy, hyperplasia of goblet cells, prominent inflammatory infiltrate, and edema when broiler chickens received aflatoxin B1-contaminated diet. The intestinal morphological characteristics have been improved when the mannooligosaccharide and beta glucan have been supplemented in the dietary regimen. The potential absorption of carbohydrates and phenolic compounds found in the microcapsules loaded by the date seed extract is thought to be the cause of the improvement in the intestinal morphological characteristics.

Gene expression analysis
The GPx and SOD genes are involved in the antioxidant enzyme system (Moslemi et al. 2022). Additionally, the IFN-and IL-2 genes are the main activators of macrophages and stimulate natural killer cells and neutrophils. A study have been done by Habib and Ibrahim (2011) revealed that date seed extract supplementation significantly increased the expression of antioxidant enzyme genes (including GPx and SOD) while decreasing the expression of inflammatory genes (including iNOS). The iNOS gene is a gene that causes inflammation (Becerril et al. 2018). Our funding exhibited that mycotoxin suppressed (p < 0.05) the expression of GPx, SOD, IFN-, and IL-2 genes while up-regulating (p < 0.05) the expression of the iNOS gene, according to the main marker genes involved in the antioxidant, inflammatory, and immunomodulation systems. Date seed extract that was loaded into microcapsules significantly (p < 0.05) improved the expression of the genes and up-regulated the expression of the GPx, SOD, IFN-, and IL-2 genes while down-regulating the expression of the iNOS gene (Table 9). The results of lipid peroxidation as biomarkers of oxidative stress and inflammation were consistent with gene expression findings.

Conclusion
The findings suggested that date seed extract microcapsules might function as an immunobiotic and protective agent to lessen the toxic effects of mycotoxins in mice given a diet contaminated with mycotoxins. Consequently, the developed microcapsules loaded by the date seed extract could be considered as a promising mycotoxin inhibitor to be used in animal feed industry. Further research is needed to determine its efficacy in different animal species and under different conditions. Table 9 The expression analysis of antioxidant-, inflammation-, and immunomodulation-related genes in mice received different treatments T1: control, T2: mycotoxin-contaminated diet, T3: mycotoxin-contaminated diet + date seed extract microcapsules (3 mg/g diet) Different letters in the same column indicated significant difference (p < 0.05) The analyses were performed in triplicates