# Anti-acetylcholinesterase and antidiabetic activities of raw and heated camel milk

Maha Hamouda, Amel Sboui, Jalloul Bouajila, Abir Omrani, Mohamed Dbara, Mohamed Hammadi, Touhami Khorchani

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Maha Hamouda, Amel Sboui, Jalloul Bouajila, Abir Omrani, Mohamed Dbara, et al. Anti-acetylcholinesterase and antidiabetic activities of raw and heated camel milk. Cogent Food & Agriculture, 2024, 10 (1), 10.1080/23311932.2024.2434641. http://05185082

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# Anti-acetylcholinesterase and antidiabetic activities of raw and heated camel milk

Maha Hamouda, Amel Sboui, Jalloul Bouajila, Abir Omrani, Mohamed Dbara, Mohamed Hammadi & Touhami Khorchani

**To cite this article:** Maha Hamouda, Amel Sboui, Jalloul Bouajila, Abir Omrani, Mohamed Dbara, Mohamed Hammadi & Touhami Khorchani (2024) Anti-acetylcholinesterase and antidiabetic activities of raw and heated camel milk, Cogent Food & Agriculture, 10:1, 2434641, DOI: 10.1080/23311932.2024.2434641

**To link to this article:** https://doi.org/10.1080/23311932.2024.2434641

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# Anti-acetylcholinesterase and antidiabetic activities of raw and heated camel milk

Maha Hamoudaª, Amel Sbouiª, Jalloul Bouajilaᵇ, Abir Omraniª, Mohamed Dbaraª, Mohamed Hammadiª and Touhami Khorchaniª

ªLivestock and Wildlife Laboratory, Arid Lands Institute of Mednine, University of Gabes, Mednine, Tunisia; ᵇLaboratoire de Génie Chimique, Université de Toulouse, CNRS, INPT, UPS, Toulouse, France

## ABSTRACT

This study aimed to investigate the effect of different heat treatments on camel milk's *in vitro* anti-Alzheimer and anti-diabetic potential. Camel milk was subjected to three heat treatments: low and high-heat pasteurization and boiling. Unheated camel milk was used as a control. Camel milk caseins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after separation by fast ion exchange liquid chromatography (FPLC). The concentration of insulin-like substances was measured using an ELISA kit. The *in vitro* anti-Alzheimer activity was evaluated using acetylcholinesterase (AChE) inhibitory assay and the *in vitro* antidiabetic activity was evaluated using alpha-amylase and alpha-glycosidase inhibitory assay. The unheated camel milk and caseins exhibited significantly higher anti-AChE activity than the heated ones (p < 0.05) and showed higher antidiabetic potential than the heated samples at all concentrations tested. There was also a significant (p < 0.05) difference in insulin-like substance content in the heat-treated groups compared with the control group. Heat treatment could affect the anti-diabetic and anti-AChE properties of camel milk. Therefore, among these different heat treatments, pasteurization at 63°C showed the strongest inhibition of these activities and the highest insulin content in camel milk. Moreover, at all concentrations and activities tested, the percentage of inhibition of beta-casein (β-CN) was more pronounced than that of alpha-casein (α-CN).

## ARTICLE HISTORY

Received 3 March 2024
Revised 18 September 2024
Accepted 25 October 2024

## KEYWORDS

Camel milk; caseins; heat treatment; anti-acetylcholinesterase inhibitory; antidiabetic activities

## SUBJECTS

Food Additives & Ingredients; Agriculture and Food; Biology; Biochemistry

## Introduction

Diabetes mellitus (DM) is a group of metabolic disorders of carbohydrate metabolism in which blood glucose levels are abnormally high, resulting in hyperglycemia (Bunsroem et al., 2022). This hyperglycemia is the result of one or more defects in insulin secretion and/or action. DM can cause generalized anxiety and lead to several life-threatening complications such as heart disease, high blood pressure, stroke and other cardiovascular diseases (Bunsroem et al., 2022). Current treatments for diabetes include oral antidiabetic drugs and insulin. Some commonly used drugs to treat DM patients include dipeptidyl peptidase-IV (DPP-IV) inhibitors, glucagon-like peptide-1 (GLP-1) analogs and α-glucosidase inhibitors (Zhang et al., 2009). On the other hand, continuous use of these drugs can lead to insulin resistance

and side effects (Wang et al., 2016). Therefore, the demand for effective, affordable and non-toxic drugs has gained more attention, and natural alternative foods, such as milk and milk proteins, have been used for some purposes. Alzheimer's disease (AD) is a chronic and irreversible neurodegenerative disease with defects in the central nervous system and cognitive function (Atri, 2019). Patients with AD suffer from memory loss, thinking problems, cognitive decline and neuroinflammation (Ringman, 2017). Estimates of the number of people affected by AD are 50 million, equally divided between developed and developing countries (Souiei et al., 2023). The exact pathogenesis of AD is still not fully understood, and the disease could be described as a neurological disorder caused by several factors, such as decreased levels of acetylcholine (ACh) in synaptic clefts and oxidative stress (Lee et al., 2016). Acetylcholinesterase

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /></figure>

(AChE) belongs to the family of acetyl hydrolases and is responsible for the hydrolysis of ACh to choline and acetate in the synaptic cleft. This enzyme catalysis the breakdown of ACh so that it can function as a neurotransmitter. AChE is found in neuromuscular junctions and in cholinergic-like chemical synapses, where its main activity at these sites is to inhibit synaptic transmission (Chen et al., 2019). In patients with dementia and AD, the activity of AChE damages the cognitive function of the brain due to an irregularity in cholinergic activity. AChE has attracted much attention as a drug target. It is a critical enzyme in AD, responsible for defects in the cholinergic pathway and the formation of β-amyloid plaques. Therefore, inhibition of AChE helps to restore cognitive function in these patients (Liu et al., 2018). Camel milk is known for its distinctive composition and potential health benefits, making it a valuable resource in different cultures (Salami et al., 2011). Heat treatment, such as pasteurization and boiling, is one of the most important processes in food preparation. It is often used to eliminate harmful bacteria to make camel milk safe for human consumption and to increase the shelf life of dairy products. The heat stability of camel milk is different from that of cow's milk. In fact, camel milk is known to have different properties from cow's milk, including its poor stability at high temperatures (Metwalli et al., 2020). Various authors attribute this instability to micelle caseins and the lack of κ-casein and β-lactoglobulin in camel milk.

Therefore, the effect of heating on the biological activities of camel milk is an interesting topic that has gained attention in recent years. While heat treatment is necessary to ensure the safety of dairy products, it is important to strike a balance between safety and preserving the potential health benefits of camel milk. Therefore, the aim of the current study was to evaluate the effect of heat treatment on the *in vitro* anti-Alzheimer and anti-diabetic potential of camel milk.

# Materials and methods

## Milk samples

Raw camel milk was obtained from a herd of camels (*Camelus dromedarius*) belonging to the Livestock and Wildlife Laboratory of the Arid Regions Institute (IRA) (Medenine, Tunisia) and kept at refrigerated temperatures (4°C) until analysis.

The milk samples were subjected to three heat treatments:

* Pasteurized camel milk at 63°C for 30 min.

* Pasteurized camel milk at 90°C for 3 min.

* Boiled camel milk at 100°C for 3 min.

## Purification of caseins

Caseins were prepared by adjusting the pH of the defatted milk samples to 4.2 with 1M HCL. The mixture was then centrifuged at 3000g for 20min. The precipitated caseins were separated from the whey supernatant. It was then, washed with distilled water to remove the whey residue and solubilized at pH 7 with 1 M NaOH. All prepared samples were lyophilized and stored at -20°C. The separation of caseins into alpha and beta caseins was performed according to Chaoui-Kherouatou and Attia (2008), using fast protein liquid chromatography (FPLC AKTA purifier system) with a GL 5/50 Mono-Q exchange column.

## Electrophoresis SDS-PAGE

SDS-PAGE was performed according to Laemmli and Favre (1973) using 15% (w/v) separating gel and 5% (w/v) stacking gel.

## Anti-acetylcholinesterase activity

AChE activity was assessed by the Ellman method as reported by Dawra et al. (2022) with some modifications. A volume of 200 µL of buffer A (0.1 M Na₂HPO₄; pH = 8) containing 300 µL of dithiobisnitrobenzoate (DTNB) is added to 200 µL of sample and 100 µL of AChE. After incubation for 15 min with stirring, 100 µL of acetylthiocholine iodide (ACTHi) is added. The enzyme AChE cleaves ACTHi into acetate and thiocholine. The thiocholine then reacts with DTNB to form a compound called 5-thio-2-nitrobenzoate (TNB). TNB is a yellow colored compound with an absorbance peak at 412 nm which can be measured using a spectrophotometer. The absorbance is proportional to the enzymatic activity of AChE. AChE inhibitory activity was calculated using the following formula $I\% = [(Ac-As)/Ac] \times 100$

where Ac is the absorbance of the control and As is the absorbance of the sample.

## Insulin content

The determination of insulin concentration in milk samples was performed using a DEMEDITEC Insulin ELISA kit based on a solid phase sandwich immunoenzymatic reaction (Domeditec, Germany).
# Antidiabetic activities

## Inhibition of alpha amylase

Alpha-amylase inhibition was determined according to Kim et al. (2011) with some minor modifications. The mixture containing 100 µL of each sample and 100 µL alpha-amylase (1U/mL) was incubated at 25°C for 15 min. Then 200 µL of starch (1%) was added as substrate and left for 3 min at 25°C. The reaction was completed by adding 200 µL of DNS solution (3,5-dinitrosalicylic acid 96 mM, 15% sodium potassium tartrate in NaOH (2M)) and placed in a boiling water bath for 10 min. The reaction mixture was diluted with 1.8 mL of PBS buffer solution in an ice bath. The absorbance was then measured at 540 nm. Acarbose, a commercial inhibitor, was used as a positive control at a concentration of 150 mg.L⁻¹. The percentage of enzymatic inhibition of the samples was calculated as follows:

$$ \text{Alpha-amylase inhibition (\%)} = \left[ \frac{(Ac - As)/A \text{ control}}{*100} \right] $$

where Ac is the absorbance of the control and As is the absorbance of the sample.

## Inhibition of alpha glycosidase

The α-glycosidase activity was determined according to the method of Shalaby et al. (2014) with slight modifications. The effect of different samples on the α-glycosidase activity was studied using 4-nitrophenyl-α-D-glycopyranoside (pNPG) as substrate. An aliquot of 100 µL phosphate buffer (0.1 M, pH 6.9), 200 µL alpha-glycosidase (1 U/mL) and 200 µL sample were incubated at 25°C for 10 min. An aliquot of 100 µL of 5 mM PNP-G was added and the mixture was incubated for 5 min at 25°C and Do was determined at 405 nm. The percentage of enzyme activity was calculated as follows: % inhibition = $100\%((A c - A s)/Ac)$.

## Statistical analysis

All experiments were performed at least in triplicate. Results were obtained using statistical software, XLSTAT version 2014.5.03 (Addinsoft, Pearson edition, Waltham, MA). One-way analysis of variance (ANOVA) was performed, followed by Tukey's test for multiple comparisons of means. A p value of 0.05 was used to indicate a significant difference.

# Result and discussion

## Separation of caseins by FPLC

The chromatographic profiles of the camel casein fraction are shown in Figure 1. After 40 min,

complete separation of camel milk casein was achieved in the form of two major peaks. These results were also confirmed by Farah and Farah Reisen who isolated two major peaks and were in discordance with the results of Ochirkhuyag et al. (1997) who revealed three caseins with molecular masses estimated at 27.5; 35.3 and 26.3 kDa identified as homologs of β, αS1 and αS2 caseins of cow's milk, respectively. A decrease in peak intensity was observed mainly on heating to 100°C.

## Electrophoretic profile of camel milk casein

The SDS-PAGE profile of α and β caseins is shown in Figure 2. Migration of each fraction revealed a single well band with a molecular weight of 35 kDa for peak a and 38 kDa for peak b. The absence of a protein band corresponding to kappa-casein is due to its low content in camel milk as previously shown (Ho et al., 2022).

It was observed that heating affected the intensity of the casein protein bands. From these results, a reduction in protein content was observed with heating at 90°C. Which is in accord with Clement (2018) who also reported that alpha and beta caseins were affected by heating temperature and times. It was reported also that camel milk caseins are less stable than bovine ones at higher temperature (100–130°C) (Farah & Atkins, 1992).

## Acetylcholinesterase activity

The AChE inhibition percentages of camel milk and casein are shown in Figures 3 and 4, respectively. Camel milk showed anti-Alzheimer's activity with an AChE inhibition of 37.34%. This result was also confirmed by Abdulkadir et al. (2021) who showed that camel milk can modulate biochemical and neurobehavioural changes in AD induced by aluminum chloride in rats. Indeed, this activity was significantly affected by heat ($p < 0.05$); boiling at 100°C reduced the AChE inhibitory potency of camel milk from 37.34% to 26.79%. Indeed, Rasheed (2017) attributed this activity to the heavy chain of camel milk immunoglobulin, which is used in immunotherapy for patients suffering from various diseases such as cancer and AD.

Deficiency of the Ach neurotransmitter due to hyperactivity of the enzyme AChE is the major cause of AD (Chen et al., 2019).

The ability to inhibit AChE depends on the concentration of the sample. In this study, caseins showed a sample dose-dependent inhibition of the AChE enzyme. In fact, AChE inhibition increases with increasing sample concentration tested.
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>**Figure 1.** FPLC profiles of different casein fractions under different heat treatment: (A) unheated casein, (B) pasteurized casein at 63°C, (C) pasteurized casein at 90°C and (D) boiled casein.</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>**Figure 2.** SDS-PAGE profile of camel milk casein fraction under different heat treatment. Lane M=molecular mass marker, lane 1= Unheated α-CN, lane 2= pasteurized α-CN (63°C), lane 3= pasteurized α-CN (90°C), lane 4 = boiled α-CN, lane 5 = unheated β-CN, lane 6 = pasteurized β-CN (63°C), lane 7 = pasteurized β-CN (90°C), lane 8=boiled β-CN.</figcaption></figure>
<figure><img src="image_18.png" /></figure>

Camel alpha- and beta-caseins (β-CN) exhibit greater AChE inhibitory activity than total caseins at all concentrations used. Fractionation of caseins can release active peptides hidden in the native protein with the ability to inhibit AChE. At a concentration of 40 mg/mL, β-CN exhibited the highest AChE

inhibitory capacity (70.82%), followed by alpha-casein (α-CN) (61.40%) and total caseins with 45.34%. AChE inhibition was significantly decreased in the different camel caseins due to high temperature as shown in Figure 4. This decrease can be attributed to the degradation of caseins after heat treatment, possibly due to the sensitivity of different peptides/amino acids to high thermal processing (Y. Zhang et al., 2021). Ho et al. (2022) have also reported that the absence of β-lactoglobulin and the lack of kappa-casein are responsible for the low stability of camel milk at high temperatures.

To our knowledge, this study is the first to report the effect of heat treatment on the AChE inhibitory activity of camel milk caseins.

<figure><img src="image_19.png" /><figcaption>**Figure 3.** Effect of heat treatment on the acetylcholinesterase activity of camel milk.</figcaption></figure>

## Insulin concentration

Camel milk has anti-hyperglycemic properties due to its higher concentration of insulin-like protein (Hammam,

<figure><img src="image_20.png" /><figcaption>**Figure 4.** Effect of heat treatment on the acetylcholinesterase activity of camel milk caseins (a) Total caseins, (b) α-CN, (c) β-CN.</figcaption></figure>
<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>**Figure 5.** Effect of heat treatment on the insulin concentration of camel milk.</figcaption></figure>

2019). Previous research suggests that camel milk and insulin may serve as effective complementary therapies for people with type I diabetes and for streptozotocin-induced diabetic albino rats (Raj et al., 2023). Thus, the concentration of insulin-like substances of the control and the three heat-treated milk samples were 55.49 ± 6.91, 42.21 ± 3.73, 27.86 ± 2.50 and 25.47 ± 2.06 µIU/mL, respectively (Figure 5). In previous work, the mean concentration of insulin-like proteins in camel milk was 24.35 ± 0.59 µIU/mL (Ansari et al., 2020) and 41.9 ± 7.38 µIU/mL (Wernery et al., 2003), whereas it was higher than in cow milk (16.32 ± 5.98 U/L) (Agrawal et al., 2020). Different concentrations of insulin-like substances have also been reported in breast milk (Young et al., 2017). In this study, the mean concentration of insulin-like substances in fresh camel milk was 55.49 µIU/mL. This variable insulin content could be due to the difference in technique and/or protocol used, as well as breed/region and stage of lactation (Anwar et al., 2022; Ansari et al., 2020). In fact, increasing the heating of camel milk resulted a significant decrease in the content of insulin-like proteins (Figure 5). Wernery et al. (n.d.) reported that pasteurization of camel milk at 72°C for 5 min and boiling at 98°C for 5 min resulted a significant decrease in insulin concentration in the milk. However Ansari et al. (2020) and Ollikainen (2013) did not find a significant decrease in insulin concentration in bovine milk after pasteurization at (63°C/30 min) and at 72°C, although reduced immunochemical activity was reported at higher temperatures. Agrawal et al. also reported a decrease in insulin-like proteins after cooking camel milk. Therefore, the decrease in insulin concentration observed in Figure 5 may be related to denaturation of insulin homologous proteins after heating.

Camel milk is known to contain an insulin-like protein that has several beneficial properties, including resistance to gastric acid and the potential to help

maintain glucose homeostasis (Raj et al., 2023). The presence of such an insulin-like protein in milk was supported by the fact that the amino acid sequence of some camel milk proteins was rich in half cystine, like some peptides of the insulin family (Beg et al., 1985). However, other authors reported that the amino acid sequence of camel milk insulin is the same as that of bovine milk insulin (Malik et al., 2012). Both have been predicted to be sensitive to gastrointestinal enzymatic digestion in a computer model, which has the same number of cleavage sites for trypsin, pepsin and chymotrypsin. It has been reported (Malik et al., 2012) that digestive enzymes can degrade camel milk insulin before entering the bloodstream. Therefore, the anti-diabetic effect of camel milk may be due to other factors that protect insulin from digestion. Wernery et al. (n.d.) reported that the lack of coagulation of camel milk in an acidic environment was considered to be the main factor for its therapeutic effect on insulin-dependent diabetic patients (Agrawal et al., 2020). Indeed, examination of the fatty acid composition shows that camel milk contains larger fat globules than bovine milk. Malik et al. (2012) suggested that camel milk insulin could be encapsulated in lipid nanoparticles, protecting it from degradation and facilitating its absorption into the bloodstream. In addition, the high levels of zinc in camel milk compared to other milks such as cow's milk may significantly improve the interaction of insulin with its receptor (Shori, 2015).

## Alpha amylase and alpha glycosidase activities

Alpha-amylase and alpha-glycosidase inhibitory activities are indirect indicators to evaluate the anti-diabetic activity of camel milk. It has been reported that inhibition of alpha-amylase and alpha-glycosidase reduces the hydrolysis of carbohydrates, thereby reducing the possibility of sugar absorption by the human intestine (Layer et al., 1986).

The results of α-glycosidase and α-amylase inhibition as indicators of the antidiabetic activity of camel milk are shown in Figure 6. Heat treatment did not have a significant effect on the level of inhibition of α-glycosidase (Figure 6). In this context, several authors reported the inhibition of alpha glycosidase and α-amylase by fresh camel milk (Akan, 2021; Baba et al., 2021), fermented milk (Ayyash et al., 2018) and yoghurt camel milk (Shori & Baba, 2014). Different studies have also shown that feeding animals and patients with diabetes restores the production and release of insulin compared to control animals and patients (Ayoub et al., 2018). Nevertheless, a significant decrease in the percentage of alpha-amylase
<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>**Figure 6.** Effect of heat treatment on the antidiabetic activity of camel milk through the inhibition capacity of alpha glycosidase (A) and alpha amylase (B).</figcaption></figure>

inhibition was demonstrated using boiled camel milk. The inhibitory effect of the different caseins against α-glycosidase is shown in Figure 7. The results showed that camel caseins have an antidiabetic activity. Akan (2021), reported similar results, who showed that peptides derived from donkey milk casein and camel milk caseins had better antidiabetic activity compared to peptides derived from milk whey proteins.

According to the results presented in Figures 7 and 8, pasteurization at 63°C did not show any significant effect on the inhibition of alpha-amylase and alpha-glycosidase and therefore on the antidiabetic property of camel milk. This finding is in agreement with the results presented by Sboui, et al. (2012) who showed that pasteurized milk had the same therapeutic effect as fresh milk on diabetic dogs induced by alloxan. While boiling at 100°C led to a significant decrease in the antidiabetic activity of caseins. As shown in Figure 8, samples of caseins treated at 100°C showed the lowest activity at different concentrations. This can be attributed to the fact that heat treatment of the milk decreased the abundance of camel milk caseins. This is in agreement with Mohamed et al. (2022) who previously reported that camel caseins are less stable than cow's milk caseins at high temperatures (100–130°C). As shown in Figure 8(B,C), at the same concentration and heat treatment, β-CN showed a higher percentage of alpha-amylase inhibition than α-CN. A recent study has shown that camel milk proteins are an interesting source of bioactive peptides with the potential to inhibit the activity of metabolic enzymes, DPP-IV, which are associated with diseases such as diabetes and obesity (Mudgil et al., 2019; Nongonierma et al., 2018). It has also been reported that camel milk proteins can restore and increase insulin secretion from

pancreatic beta cells by either stimulating glucose-mediated insulin secretion, inhibiting glucagon secretion from pancreatic alpha cells and its function in the liver, and/or inhibiting the key enzymes that indirectly control insulin secretion such as DPP-IV (Ayoub et al., 2018). In addition, it has been mentioned that the anti-inflammatory effect (Limon et al., 2014) and the presence of antioxidants (vitamins E and C (1.5 g.L⁻¹), proteins) (Alhaj et al., 2022) in camel milk can reduce/prevent damage to metabolic pathways and the risk of diabetic diseases by destroying free radicals in the body and triggering the body's natural antioxidant defenses (Izadi et al., 2019; Raj et al., 2023). In fact, camel milk is rich in zinc (2.2 mg.L⁻¹) (Alhaj et al., 2022), which is an essential mineral for various biological functions, including the proper functioning of the pancreas and insulin secretion (Raj et al., 2023). The presence of insulin or an insulin-like substance in camel milk is one of the theories that have been proposed to explain the hypoglycemic effect of camel milk observed in various *in vivo* studies on type 1 and type 2 diabetes using animal models as well as diabetic patients (Malik et al., 2012; Shori, 2015). Previous studies have shown that the existence of such insulin-like proteins was supported by the fact that the amino acid sequence of some camel milk proteins was found to be rich in cysteine residues similar to those of the insulin peptide family (Beg et al., 1985; Rasheed, 2017). Active molecules that directly or indirectly target and modulate insulin secretion and/or insulin action pathways may mediate the antidiabetic effect of camel milk. Regarding insulin secretion, many *in vivo* and *in vitro* studies have reported that camel milk increases insulin secretion. This may involve an effect on pancreatic β-cells and their functions such as stimulating insulin synthesis and release and/or
<figure><img src="image_25.png" /><figcaption>A)</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>B)</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>C)</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>**Figure 7.** Effect of heat treatment on the alpha glycosidase activity of camel milk caseins (a) Total caseins (b) α-CN, (c) β-CN.</figcaption></figure>

protecting them from damage and apoptotic pathways (Anwar et al., 2022). However, this important aspect requires further experimental and technical analysis and refinement to achieve optimal fractionation and identify the real potential agents.

## Conclusion

Camel milk is increasingly recognized for its nutritional properties and health benefits. The current study supports camel milk proteins as a promising functional food for the treatment of hyperglycemia and AD. The results of this study show that camel milk has potent anti-Alzheimer and anti-diabetic effects. According to our results, the anti-diabetic and anti-Alzheimer effects of camel milk and caseins depend on the intensity and time of heating.

Raw camel milk showed high inhibition of AChE, alpha-amylase and alpha-glucosidase. Heating at 63°C for 30 min gave the best results for AChE, alpha-amylase and alpha-glucosidase inhibition, while heating at 100°C for 3 min gave the lower

activities. Therefore, camel milk heated at 63°C for 30 min may be used in the future as a potential anti-diabetic and anti-Alzheimer's substance in the form of a dietary supplement for diabetics and/or people with AD. Further research is needed to understand the effects of heat treatment and to optimize processing methods to maximize the retention of the biological activities of the milk. In addition, future studies will aim to identify the different bioactive peptides responsible for the anti-Alzheimer and anti-hyperglycemic effects of camel milk caseins.

## Authors' contributions

Maha Hamouda: Conceptualization, Formal analysis, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. Amel Sboui: Conceptualization, Supervision, Validation, Visualization, Writing – review & editing. Jalloul Bouajila: Methodology, Supervision, Writing – review & editing. Abir Omrani: Software. Mohamed Dbara: Methodology. Mohamed Hammadi: Project administration. Touhami Khorchani: Conceptualization, Supervision, Visualization.

All authors have approved the final manuscript.
<figure><img src="image_29.png" /><figcaption>A)</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>b)</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>c)</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>**Figure 8.** Effect of heat treatment on the alpha amylase activity of camel milk caseins (a) Total caseins, (b) α-CN, (c) β-CN.</figcaption></figure>

## Disclosure statement

The author declares no conflict of interest.

## Funding

The author acknowledges the University of Gabes for financial support with a PhD scholarship. The authors would like to express their gratitude to the P2ES Scientific Research Excellence Encouragement project. This study is part of the P2ES 2023-D4P1 project entitled "Innovation on the Valorization of Camel Milk and Its Dairy Products: Sensory Analysis and Marketing."

## About the authors

**Mrs Maha Hamouda** is currently doing PhD in the Department of Livestock and Wildlife Laboratory, Arid Lands Institute (IRA), University of Gabes, Tunisia.

**Dr Amel Sboui** is a biochemist at the Livestock and Wildlife Laboratory, Arid Lands Institute (IRA), Tunisia. She is researcher and academic author with a prolific career in

the field of dairy and food science, particularly focusing on the properties and health benefits of camel milk. Dr. Sboui's research has significantly contributed to the understanding of camel milk's biochemical properties, its potential therapeutic applications, and its role in nutrition and health.

**Pr Jalloul Bouajila** is a Doctor of Analytical Chemistry at the Université Paul Sabatier, Faculty of Pharmacy and CNRS. He has published more than 170 scientific articles. His research interests include bioactive compounds from plants and herbs, as well as their biological activities.

**Dr Abir Omrani** has done her doctorate in in Biological and Biotechnology Sciences from the Higher Institute of Biotechnology of Monastir, Tunisia.

**Mr Mohamed Dbara** Chemistry technician in the livestock and wildlife laboratory of Arid Land Institute of Mednine Tunisia.

**Pr Mohamed Hammadi** is the head of the Livestock and Wildlife Laboratory of Arid Land Institute of Mednine Tunisia.

**Pr Touhami Khorchani** has a doctorate in Applied Biological Sciences (Animal Production) from the University of Ghent in Belgium. He is currently a senior researcher at the Arid Zone
<figure><img src="image_33.png" /></figure>

Research Institute (Institut des Régions Arides) (IRA) (Médenine, Tunisia) and he served as the head of the Livestock and Wildlife Laboratory from 2002 to 2017 and Director of Information from 2017 to 2023. He is currently the Director of International Cooperation at the same institute.

## ORCID

Maha Hamouda http://orcid.org/0000-0001-5617-3021

## Data availability statement

All data related to this manuscript will be provided from the corresponding author upon reasonable request.

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