Chitosan-Coated Probiotic Nanoparticles Mitigate Acrylamide-Induced Toxicity in the Drosophila Model

Chitosan-Coated Probiotic Nanoparticles Mitigate Acrylamide-Induced Toxicity in the Drosophila Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chitosan-Coated Probiotic Nanoparticles Mitigate Acrylamide-Induced Toxicity in the Drosophila Model Swetha Senthil Kumar, Sahabudeen Sheik Mohideen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4780644/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract The novelty of this study lies in the development of an effective therapeutic agent using natural components—specifically, low molecular weight chitosan and L. fermentum —utilizing the Drosophila model. The design and formulation of chitosan-coated probiotic nanoparticles (CSP NPs) aim to enhance the bioavailability of probiotics in the gut, thereby improving their efficacy against ACR-induced toxicity. Nanoencapsulation, a vital domain of the medical nanotechnology field plays a key role in targeted drug delivery, bioavailability, multi-drug load delivery systems and synergistic treatment options. Chitosan, known for its non-toxic nature, offers additional benefits such as anti-inflammatory properties and immune system stimulation. Lactobacillus fermentum , incorporated for its cholesterol-lowering and potent immunomodulatory effects, also plays a significant role in influencing behavioural and developmental mechanisms in Drosophila . The synergistic effect of chitosan and L. fermentum ensures the stability and sustained release of microbial load and its secondary metabolites, facilitating prolonged exposure in the gut. This slow-release mechanism allows for an extended duration of action, effectively combating the detrimental effects of process-induced toxins like acrylamide. By optimizing bioavailability through nanoencapsulation, this study demonstrated the efficiency of the formulation in rescuing ACR-induced behavioural and biochemical deficits. Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Developmental biology Biological sciences/Microbiology Earth and environmental sciences/Environmental sciences Health sciences/Medical research Health sciences/Risk factors Gut microbiota Nanoencapsulation Acrylamide Bioactives Chitosan Lactobacillus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Gut health has gained considerable attention in recent years and has become an essential part of most fitness regimes. The gut microbiota, consisting of trillions of microorganisms residing in the gastrointestinal tract, plays a crucial role in maintaining human health and well-being. This microbial community is not just a passive bystander but actively contributes to numerous physiological processes essential for human health including mental fitness, immunological and metabolic mechanisms. However, this niche of vital microbes gets easily disrupted due to several factors including the consumption of foods lacking fibre content, lack of exercise, stress, excessive intake of antibiotics, proton pump inhibitors (PPIs), and non-steroidal anti-inflammatory drugs (NSAIDs), etc. Among these factors affecting the gut microbiome, the food we consume plays a major role in regulating our health [ 1 ]. The healthy food items or supplements we consume undergo several physical and chemical changes during digestion in our gastrointestinal tract before the nutrients from them get absorbed into our body. These nutrients or bioactives present in our present in vegetables, herbs, spices, or fruits serve as cofactors, vitamins, or essential factors for better functioning of our body. However, their absorption in the small intestine is often affected by several factors including chemical form, matrix, and metabolic pathways [ 2 ]. Therefore, it is essential to develop a suitable delivery matrix to ensure the bioavailability of essential bioactives for a healthy population. Advances in nanotechnology offer us vast opportunities to manipulate the chemical, physical and biological properties of various compounds to suit our goals. One such development is the synthesis of nano-delivery systems made with biocompatible materials that ensure slow-release mechanisms for enhanced bioavailability [ 3 ]. Probiotics are microbes that improve health by modulating the gut microbiota. Some probiotic species, such as Bifidobacterium , Lactobacillus , and Akkermansia , interact with the gut microbiota, increasing beneficial bacteria and reducing pathogenic species. This interaction can help restore the gut microbiota from dysbiosis. Studies have shown that Bifidobacterium pseudolongum can help recover gut microbiota dysbiosis in obese mice, increasing the abundance of Butyricimonas and Bifidobacterium [ 4 ]. Probiosis is a process where bacteria, such as Lactobacillus strains, can alter the composition and function of the intestinal microbiome. These bacteria produce antimicrobial agents or metabolic compounds that suppress the growth of other microorganisms or compete for receptors and binding sites with other intestinal microbes. They can also enhance the integrity of the intestinal barrier, resulting in immune tolerance and decreased translocation of bacteria across the mucosa [ 5 ]. Probiotics can also modulate intestinal immunity and alter the responsiveness of intestinal epithelia and immune cells to microbes in the intestinal lumen. Studies have shown that probiotic treatment can reduce pain and flatulence in patients with IBS, but not many have demonstrated associations with altered microbiota [ 6 , 7 ]. Probiotic supplements have been shown in studies to boost gut microbiota quantity, but they cannot alter microbial metabolites on their own accord. Synbiotics, a mix of probiotics and prebiotics, have been created to enhance the gut microbiota by modifying certain species, whereas prebiotics feed the microbiota and impact metabolite formation, notably short-chain fatty acids (SCFA) [ 8 , 9 ]. Our study fabricated chitosan-coated probiotic nanoparticles (CSP NPs) to ensure the bioavailability of the probiotic load ( L. fermentum ) in the gut with a sustained release profile. The chitosan coating doubles as a prebiotic (non-digestible fibre that enhances the functionality of probiotics in the gut) and as a delivery system with innate immunomodulatory and antioxidant effects. Consequently, we analysed the efficiency of this synthesised synbiotic formulation against common heat-processed toxins – acrylamide (ACR) using the Drosophila melanogaster model. ACR is a white crystalline compound used in textiles, paper, cosmetics, cement, and mining processes. It has been classified as hazardous and probable carcinogenic to humans due to its presence in industrial effluents. In humans, ACR has been observed to cause muscle weakness, impaired cognition, and peripheral neuropathy in employees exposed via inhalation or skin absorption [ 10 , 11 ]. Several food and drug associations have set standards and mitigation strategies to reduce ACR formation or presence in food products. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) set 4–5 µg/kg bw/day acrylamide from food as the highest tolerable intake level in 2005 [ 12 , 13 ]. In India, the major source of acrylamide is the consumption of deep-fried food products ranging from 82.0 to 4245.6 µg/kg [ 14 ]. Exposure studies by the US CDC organization in 2019 showed that children aged 3–11 had 108 − 93.8 pmol/g haemoglobin, while older adults had 223–257 pmol/g haemoglobin [ 15 ]. The post-pandemic state has aggravated the situation, with young children being twice as impacted as adults due to their eating habits, rapid metabolism, and low body weight. Fast food intake has also grown, which may raise the risk of infertility [ 16 , 17 ]. ACR's negative effects have been explored in model organisms such as rats, mice, zebrafish, and Drosophila , with findings indicating circadian clock disturbance, developmental delays, and neurotoxic effects [ 18 – 24 ]. Drosophila melanogaster is used as a model organism to study and understand various diseases, their molecular mechanisms, and their effects on humans. It is a widely preferred model due to its short life cycle, genetic similarity to humans, and high fecundity rate. Additionally, lactic acid bacteria (LAB) have been reported to have nutrient-dependent regulatory control on Drosophila development and can regulate growth hormones like ecdysone and insulin receptors [ 25 ]. Storelli et al. also reported the influence of LAB on the developmental mechanisms in Drosophila under nutrient-scarce conditions [ 26 ]. Furthermore, studies with Lactobacillus strains have reported that these bacteria can alleviate the ACR-induced oxidative stress in vivo models and reduce the bioavailability of dietary acrylamide under different simulated gastrointestinal conditions ( in vitro ) [ 22 , 27 ]. Therefore, in this study, we have discussed the formulation process of chitosan-coated probiotic nanoparticles, Further, our study analyses the efficiency of these synbiotic formulations against ACR-induced toxicity using the fruit fly model. Our results demonstrate the extended bioactivity of the probiotic load as well as its protective mechanisms against ACR-induced toxicity. 2. Materials and Methods 2.1. Chemicals and Cultures Acrylamide (3x Crystalline), low molecular weight chitosan, glacial acetic acid, yeast extract, propionic acid, orthophosphoric acid, nitro blue tetrazolium salt (NBT), phenazonium Methosulphate (PMS), nicotinamide adenine dinucleotide (reduced) disodium salt (NADH), potassium chloride, glutathione reduced (GSH), tetrasodium pyrophosphate (TSPP), ethanol, 5, 5-dithiobis 2-nitrobenzoic acid (DTNB), Abcam TMRE-mitochondrial membrane potential assay kit and, 1-chloro-2,4-dinitrobenzene (CDNB) were procured from Sisco Research Laboratories, India. Agar-Agar Type − 1, dextrose, sodium phosphate monobasic, sodium phosphate dibasic, bovine serum albumin (BSA), phosphate buffer saline (PBS) and methylparaben were purchased from Himedia Pvt. Ltd., India. All chemicals used in this study were of analytical grade. Lactobacillus fermentum (MCC 2760) stock was obtained from NCCS, Pune and periodically subcultured in MRS broth (pH 6.5) at 37°C. 2.2. Fabrication and Characterisation of Chitosan Coated Probiotic Nanoparticles (CSP NPs) Chitosan-coated probiotic nanoparticles (CSP NPs) were synthesised following a previously reported study with slight modifications [ 28 ]. Briefly, a 0.5% solution of low molecular weight chitosan was prepared with 1% acetic acid and 3 ml of L. fermentum broth (8 log CFU/ml) was mixed with constant stirring. To this, a stoichiometric amount of 1mg/ml sodium tripolyphosphate (TPP) was added dropwise with constant stirring for 2 hours at room temperature. The mixture was then sonicated for 30 minutes and centrifuged at 10,000 rpm for 15 minutes. The pellet was then lyophilised and stored at 4℃. The preparation of chitosan nanoparticles (CS NPs) followed a similar method excluding the addition of the probiotic culture. The size and morphology of synthesised CS and CSP NPs were examined using JEM-2100 Plus Hi-Resolution Transmission Electron Microscope (HRTEM), JEOL Japan. The surface charge and particle size of CSP NPs in an aqueous system were then estimated using Malvern/Nano ZS-90 Zeta Sizer. 2.3 Drosophila Rearing and Maintenance The Drosophila Oregon K wild-type strain was maintained at 25 ± 2°C and 60% humidity under a 12-hour dark-light cycle. Flies were cultured on a standard cornmeal agar medium consisting of cornflour, agar-agar type 1, D-glucose, sugar, and yeast extract. To prevent microbial contamination, the medium was autoclaved and supplemented with antifungal agents including propionic acid, Tego (methyl para hydroxy benzoate dissolved in ethanol), and orthophosphoric acid at 55°C. Additionally, 2 mM acrylamide (ACR), 10 µg/ml CSP NPs, and 10 µg/ml CSP NPs + ACR (CSPA) treatment groups were prepared by the stoichiometric addition of respective compounds to the media at 50–55°C. 2.4. Survival and Behavioural Assay The lifespan of flies was estimated by transferring twenty-five healthy newborn adult male flies to freshly prepared treatment media. The flies were then constantly monitored and the mortality rate was calculated by tallying the number of dead flies every 24 hours. To assess the locomotor functions at the larval stage, male and female flies were added to the respective media for 5 days. The parent flies were then discarded and the vials were carefully maintained till the development of third instar larvae. These larvae were then transferred to an agar plate placed on top of a graph paper and allowed to crawl for one minute. The number of 1mm grids traversed per minute was represented as the crawling activity for statistical analysis. Similarly, the adult climbing activity was determined by exposing thirty male flies to the respective treatment media for 5 days. The treated flies were then transferred to a climbing assay setup with 3 cm marked vials. The vials were tapped moderately and the percentage of active flies was reported as the number of flies above the 3 cm mark after 10 seconds [ 29 ]. 2.5. Estimation of Oxidative Stress Parameters Biochemical analysis was carried out with twenty newborn male flies post-5-day exposure to respective treatment groups. Protein estimation of the treated flies was performed following Lowry’s method with bovine serum albumin as the standard. Additionally, reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione-s-transferase (GST) activities were estimated following the previously reported study [ 30 , 31 ]. Briefly, the treated flies were homogenised in Tris and sodium phosphate buffer for ROS and antioxidant enzyme activity analysis respectively. Stoichiometric quantities of respective reagents were mixed with 100 µl homogenate for further analysis. The fluorescence and absorbance readings were further analysed for statistical analysis. 2.6. Estimation of Ovarian Mitochondrial Membrane Potential Thirty female flies were treated with control and treatment media, and their ovaries were dissected to determine their mitochondrial membrane potential. The experiment was slightly modified from the Abcam TMRE-Mitochondrial Membrane Potential experiment Kit. The ovaries were dissected in Schneider's insect medium, stained with 200 nM TMRE for 15 minutes in the dark, washed with PBS, and observed using a Leica DM6 fluorescent microscope. 2.7. Statistical Analysis All experiments performed in this study were carried out in triplicates to establish statistical significance. Experimental data are expressed as mean ± SEM unless stated otherwise, Statistical analysis of these data was performed using Graph Pad Prism 6.0 software following Dunnett's multiple comparison tests to compare the significance of treatment groups with control and ACR, respectively. The significance level for the datasets was set to P < 0.05. 3. Results 3.1. Characterisation of CSP NPs Chitosan and chitosan encapsulated probiotic nanoparticles were analysed using a transmission electron microscope (TEM), the size of the coated particles (CSP NPs) depicted an average size of 565 nm whereas the CS NPs without the probiotic load showed an average particle size of 110 nm. The TEM images (Fig. 1 ) illustrate the spherical chitosan molecules encapsulating rod-shaped Lactobacillus fermentum cells. Figure 2 illustrates the particle size and charge of the synthesised CSP NPs. The average zeta potential of the coated probiotic NPs was 4.38 ± 15.8 mV with a Z-average of 663.4 d. nm and a polydispersity index of 0.427 ± 0.87. 3.2. Viability of Synthesised CSP NPs CSP NPs bacterial load viability was determined using the pour plate method as illustrated in Fig. 3 . The synthesised nanoparticles indicated an average viability of 7.93 log CFU/ml when stored at room temperature for 49 days. Furthermore, the viability and release profile of the synthesised CSP NPs under simulated gastrointestinal fluid systems demonstrated subsequent stability of the probiotic load (SGF 120 = 9.07 log CFU/ml; SIF 180 = 7 log CFU/ml) compared to the free probiotic culture with SGF 120 = 8 log CFU/ml and SIF 180 = 5.87 log CFU/ml. 3.3. Effect of CSP NPs on Survival and Behavioural Parameters in ACR-treated Flies The synthesised CSP NPs were orally administered to the fruit fly model to assess their efficiency against common environmental and food-borne toxins like acrylamide (ACR). ACR-treated flies demonstrated low survival capacity (12 days), reduced larval crawling (37 mm/min) and declined climbing activity in adult male flies (75.55%) as illustrated in Fig. 4 A, B and C respectively. Alternatively, the control (55 days; 68.25 mm/min; 96.29%) and co-treated flies (CSPA = 48 days; 67.03 mm/min; 85.55%) demonstrated a significant level of activity and survival capacity. 3.4 CSP NP modulates ACR-induced Redox Stress Factors in D.mel Male flies post-exposure to respective treatment groups were homogenised in suitable buffers to estimate redox stress parameters. ACR-treated flies exhibited increased ROS, SOD and GST activities as depicted in Fig. 5 A, B and C respectively. Furthermore, ACR exposure indicated a negative influence on protein levels (Fig. 5 D). On the other hand, the CSPA-treated group demonstrated a significant recovery in ROS and antioxidant enzyme activity levels. Comparing the data with the control group revealed the prominent effect of CSPA treatment in restoring the alterations in vital biochemical factors and thereby curbing the toxic influence of ACR within the fruit fly model. 3.5 CSP NPs Reverse ACR-induced Mitochondrial Membrane Depolarisation in D.mel Ovaries Ovaries dissected from treated female flies were stained with TMRE solution to estimate the influence of ACR and CSPA treatment on mitochondrial membrane potential (MMP) as illustrated in Fig. 6 . TMRE dye accumulation in control and CSPA was observed indicating the presence of an active mitochondrial population. Whereas, the ACR-treated samples were noted to have a substantial reduction in TMRE fluorescence indicating the role of ACR to induce mitochondrial depolarisation. This result indicates the functionality of CSPA treatment in protecting against ACR-induced mitochondrial depolarisation and subsequent apoptosis mechanisms at the cellular level. 4. Discussion Acrylamide has been one of the most researched compounds since 2002 due to its dual role as a prominent environmental and food-based toxin. Contamination of water bodies due to industrial discharge is one aspect of toxin exposure, however, a more serious concern arises because of its formation in commonly consumed high-temperature processed food items. According to a 2005 study, the amount of ACR in food products varies with storage conditions and length of time, but the important thing to remember is that throughout long-term storage, their levels do not fully diminish. Consequently, multiple other studies have discerned the toxic effects of other food-processing toxins like bisphenol-A, heterocyclic amines, methylimidazole, etc., on humans. Therefore, it is imperative to formulate a suitable dietary supplement that can reduce, restore, or protect our physiological systems from these food-processed toxins. In the vast scope of food-based research, our chitosan-coated probiotic nanoparticles emphasise the benefits of incorporating simple naturally available dietary supplements to counteract the negative impacts of dietary toxins. The concept of ‘gut-brain interaction’ has been highflying in recent times. Many have realised the importance of the enteric system commonly known as the second brain of the human body and its influence on overall well-being. The gut microbiome is one of the key regulators of the enteric system and aids in the gut-brain crosstalk, influencing mental, metabolic, and immunological health. Previous studies reported ACR-induced cognitive impairments observed in mice caused due to dysregulation in the gut-brain axis. The study reported that the ACR treatment reduced occludin protein expressions, increased gut permeability, and increased LPS and pro-inflammatory factor levels in the gut and serum during the night phase leading to worse performance in spatial and working memory [ 32 ]. Another study investigated the effects of AA on female Sprague Dawley rats and found that it increased blood glucose levels, decreased insulin levels, and produced intestinal barrier damage. It also demonstrated gut microbial dysbiosis and a bile acid metabolism challenge, with lower Lactobacillus and Bacteroides abundance and higher cholic acid levels. These findings point to the possibility of reducing ACR toxicity by manipulating the gut microbiota [ 33 ]. In this study, we have exploited the physical and biological properties of a natural biopolymer, chitosan to develop sustained-release probiotic supplements. Probiotics such as Bifidobacterium and Lactobacillus species are commonly known and are easily sourced from milk, curd, and fermented products. Studies on Bifidobacterium animalis , Lactobacillus acidophilus , Akkermansia muciniphila , Bifidobacterium longum , and Lactobacillus rhamnosus have demonstrated recovery of gut dysbiosis, colonic inflammation, improved microbiota diversity, etc. However, probiotic supplements in combination with prebiotics like inulin, chitosan, arabinoxylan, melanoids, flavonoids, etc have significant modulatory effects on the gut microbiota. The prebiotics generally influence the gut microbes and aid in the production of short-chain fatty acids (SCFA). The synbiotic formulations further enhance the accumulation of beneficial gut microbiota, reduce the presence of harmful colonic bacteria, and impart positive modulatory effects via SCFA-mediated systemic reactions [ 4 ]. We fabricated CSP NPs by coating the Lactobacillus fermentum culture with low molecular weight chitosan in the presence of TPP. The characterisation of these NPs confirmed the nano-size range via the TEM technique and additionally, the positive zeta charge and low polydispersity index pointed toward the narrow particle size distribution. Therefore, we could confirm the easy permeability of this formulation across the physiological barrier [ 34 ]. Furthermore, data from the viability assays indicated long-term storage of synthesised NPs at room temperature and sustained probiotic release profile under gastric and intestinal pH conditions. Our experimental data confirmed with other research on synbiotic NP formulations [ 35 – 37 ]. Our experimental data in the fruit fly model demonstrated the efficiency of CSP NPs against ACR by enhancing behavioural functions at larval and adult fly stages. The treated flies exhibited increased survival capacity, normalised ROS levels, and no fluctuations in antioxidant enzyme activities. Furthermore, the MMP in CSPA-treated ovaries demonstrated substantial TMRE intensity indicating the presence of an active mitochondrial population. The in vivo study confirms the interplay between synbiotic supplements and ACR toxicity mitigation strategy. ACR-induced behavioural changes analysed through locomotor functions are a characteristic feature of the fruit fly model. The interaction between environmental cues, neuromuscular coordination and subsequent impact on development and behaviour enables us to comprehend the impact of xenobiotics at larval and adult stages. The decrease in locomotor functions at the larval stage affects feeding rate, memory, habitat selection and survival. This in turn when assayed at the adult stage similarly exhibits reduced climbing activity, mating abnormalities, fecundity issues, etc. The biochemical data further confirms the impact of ACR within the fruit fly model. These results align with the previously reported studies on ACR toxicity using D.mel models [ 38 – 41 ]. SOD and GST belonging to the enzymatic antioxidant defence mechanism play a vital role in xenobiotic clearance and redox stress normalization. Oxidative stress species like free oxygen/nitrogen radicals, H 2 O 2 , etc. are produced during normal cellular functioning and in response to xenobiotic triggers. It is, therefore, in the control of these enzymatic antioxidant defences to regulate the cellular redox status. Elevation in ROS, SOD and GST activity implies active interaction between ACR and subsequent detoxification mechanisms via GST mediated biotransformation method [ 42 , 43 ]. The ovarian mitochondrial depolarisation can also be linked to our earlier findings reporting low survival rate and high redox stress. Unfavourable conditions and ACR’s direct influence on MMP trigger the apoptotic pathways leading to non-functional ovaries and teratogenic attributes of ACR. However, the CSPA (CSP NPs + ACR) treatment demonstrated no significant toxicity at adult and larval stages. Therefore, we postulate that the cotreatment of synbiotics with ACR enhances the gut barrier by modulating the composition and activity of the gut microbiota. Chitosan NPs coating serving as a protective matrix ensures the delivery and controlled release of the probiotic load in the gut and aids in intestinal mucosal adhesion. Additionally, the L. fermentum load in interaction with CS NPs can produce SCFAs and antimicrobial peptides. This interaction can stimulate gut-associated lymphoid tissue (GALT), enhance mucosal immunity, regulate inflammatory responses, and promote tolerance to harmless antigens, thereby reducing inflammation and improving intestinal barrier function [ 44 – 47 ]. By maintaining gut integrity and reducing intestinal permeability, they prevent systemic inflammation and the translocation of toxins (like ACR metabolites – glycidamide) into circulation. In conclusion, our study highlights the effectiveness of synthesised chitosan-coated L . fermentum nanoparticles against ACR-induced toxicity. Our experimental findings indicate that our synbiotic formulation successfully preserved gut integrity and microbiota, preventing the manifestation of toxic symptoms associated with ACR exposure in fruit flies. This highlights the significant interaction between ACR and gut microbiota within our experimental model. Prospects of our study include a detailed investigation into the protective mechanisms exhibited by the synbiotic formulation via gene and protein expression estimation. Declarations Author Contributions – Swetha Senthilkumar – Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing, Software, Validation; Sahabudeen Sheik Mohideen – Conceptualization, Methodology, Validation, Writing - review & editing and Supervision. Acknowledgment The authors acknowledge the SRM SCIF and DBT platform for Advanced Life Science’s support in TEM, fluorescence imaging studies and Zeta analysis. The authors also recognize the support provided by the Department of Science and Technology, Ministry of Science and Technology, Government of India through the INSPIRE Fellowship program. Conflict of Interests – The authors disclose no conflicts of interest. Ethical Approval – Not Applicable Consent to Participate – Not Applicable Consent to Publish – Not Applicable Data Availability Statement – All data sets used and/or generated in this work are obtainable from the corresponding author upon reasonable request. Funding – The authors also recognize the support provided by the Department of Science and Technology, Ministry of Science and Technology, Government of India through the INSPIRE Fellowship program (No. DST/INSPIRE Fellowship/2021/IF210062). The funders played no part in the study's methodology, data collection and analysis, publication decision, or drafting of the manuscript. References S. Pedroza Matute, S. Iyavoo, Exploring the gut microbiota: lifestyle choices, disease associations, and personal genomics, Front Nutr 10 (2023). https://doi.org/10.3389/FNUT.2023.1225120. M.J. Rein, M. Renouf, C. Cruz-Hernandez, L. Actis-Goretta, S.K. Thakkar, M. da Silva Pinto, Bioavailability of bioactive food compounds: a challenging journey to bioefficacy, Br J Clin Pharmacol 75 (2013) 588. https://doi.org/10.1111/J.1365-2125.2012.04425.X. T. Zhang, C. Shang, T. Du, J. Zhuo, C. Wang, B. Li, J. Xu, M. Fan, J. Wang, W. Zhang, Cytoprotection of probiotics by nanoencapsulation for advanced functions, Trends Food Sci Technol 142 (2023) 104227. https://doi.org/10.1016/J.TIFS.2023.104227. K. Bauer-Estrada, C. Sandoval-Cuellar, Y. Rojas-Muñoz, M.X. Quintanilla-Carvajal, The modulatory effect of encapsulated bioactives and probiotics on gut microbiota: improving health status through functional food, Food Funct 14 (2023) 32–55. https://doi.org/10.1039/D2FO02723B. D.M. Saulnier, J.K. Spinler, G.R. Gibson, J. Versalovic, Mechanisms of Probiosis and Prebiosis: Considerations for Enhanced Functional Foods, Curr Opin Biotechnol 20 (2009) 135. https://doi.org/10.1016/J.COPBIO.2009.01.002. P. Hemarajata, J. Versalovic, Effects of probiotics on gut microbiota: mechanisms of intestinal immunomodulation and neuromodulation, Therap Adv Gastroenterol 6 (2013) 39. https://doi.org/10.1177/1756283X12459294. V.C. Goodoory, M. Khasawneh, C.J. Black, E.M.M. Quigley, P. Moayyedi, A.C. Ford, Efficacy of Probiotics in Irritable Bowel Syndrome: Systematic Review and Meta-analysis, Gastroenterology 165 (2023) 1206–1218. https://doi.org/10.1053/J.GASTRO.2023.07.018. N. Joseph, K. Vasodavan, N.A. Saipudin, B.N.M. Yusof, S. Kumar, S.A. Nordin, Gut microbiota and short-chain fatty acids (SCFAs) profiles of normal and overweight school children in Selangor after probiotics administration, J Funct Foods 57 (2019) 103–111. https://doi.org/10.1016/J.JFF.2019.03.042. P. Markowiak-Kopeć, K. Śliżewska, The Effect of Probiotics on the Production of Short-Chain Fatty Acids by Human Intestinal Microbiome, Nutrients 12 (2020). https://doi.org/10.3390/NU12041107. L. Hagmar, M. Törnqvist, C. Nordander, I. Rosén, M. Bruze, A. Kautiainen, A.L. Magnusson, B. Malmberg, P. Aprea, F. Granath, A. Axmon, Health effects of occupational exposure to acrylamide using hemoglobin adducts as biomarkers of internal dose, Scand J Work Environ Health 27 (2001) 219–226. https://doi.org/10.5271/sjweh.608. A. Koszucka, A. Nowak, I. Nowak, I. Motyl, Acrylamide in human diet, its metabolism, toxicity, inactivation and the associated European Union legal regulations in food industry, Crit Rev Food Sci Nutr 60 (2020) 1677–1692. https://doi.org/10.1080/10408398.2019.1588222. L.C. Dolan, R.A. Matulka, G.A. Burdock, Naturally Occurring Food Toxins, Toxins (Basel) 2 (2010) 2289. https://doi.org/10.3390/TOXINS2092289. V. Matoso, P. Bargi-Souza, F. Ivanski, M.A. Romano, R.M. Romano, Acrylamide: A review about its toxic effects in the light of Developmental Origin of Health and Disease (DOHaD) concept, Food Chem 283 (2019) 422–430. https://doi.org/10.1016/j.foodchem.2019.01.054. L. Shamla, P. Nisha, Acrylamide in deep-fried snacks of India, Food Addit Contam Part B Surveill 7 (2014) 220–225. https://doi.org/10.1080/19393210.2014.894141. CDC, Nceh, DLS, Od, Fourth National Report on Human Exposure to Environmental Chemicals Update, n.d. S.M. Lai, Z.T. Gu, M.M. Zhao, X.X. Li, Y.X. Ma, L. Luo, J. Liu, Toxic effect of acrylamide on the development of hippocampal neurons of weaning rats, Neural Regen Res 12 (2017) 1648–1654. https://doi.org/10.4103/1673-5374.217345. S. Stagi, S. De Masi, E. Bencini, S. Losi, S. Paci, M. Parpagnoli, F. Ricci, D. Ciofi, C. Azzari, Increased incidence of precocious and accelerated puberty in females during and after the Italian lockdown for the coronavirus 2019 (COVID-19) pandemic, Ital J Pediatr 46 (2020) 165. https://doi.org/10.1186/s13052-020-00931-3. H. Wang, P. Huang, T. Lie, J. Li, R.J. Hutz, K. Li, F. Shi, Reproductive toxicity of acrylamide-treated male rats, Reproductive Toxicology 29 (2010) 225–230. https://doi.org/10.1016/j.reprotox.2009.11.002. Z. Huang, S. Wang, Y. Yang, J. Lou, Z. Liu, Z. Liu, H. Yong, S. Shan, F. Song, Mitochondrial dysfunction promotes the necroptosis of Purkinje cells in the cerebellum of acrylamide-exposed rats, Food and Chemical Toxicology 171 (2023) 113522. https://doi.org/10.1016/J.FCT.2022.113522. B.O. Yilmaz, N. Yildizbayrak, Y. Aydin, M. Erkan, Evidence of acrylamide- and glycidamide-induced oxidative stress and apoptosis in Leydig and Sertoli cells, Hum Exp Toxicol 36 (2017) 1225–1235. https://doi.org/10.1177/0960327116686818. M. Huang, J. Jiao, J. Wang, Z. Xia, Y. Zhang, Characterization of acrylamide-induced oxidative stress and cardiovascular toxicity in zebrafish embryos, J Hazard Mater 347 (2018) 451–460. https://doi.org/10.1016/j.jhazmat.2018.01.016. S. Zhao, X. Zhao, Q. Liu, Y. Jiang, Y. Li, W. Feng, H. Xu, M. Shao, Protective effect of Lactobacillus plantarum ATCC8014 on acrylamide-induced oxidative damage in rats, Appl Biol Chem 63 (2020). https://doi.org/10.1186/s13765-020-00527-9. M.I. Alkhalaf, Diosmin protects against acrylamide-induced toxicity in rats: Roles of oxidative stress and inflammation, J King Saud Univ Sci 32 (2020) 1510–1515. https://doi.org/10.1016/J.JKSUS.2019.12.005. A.A. Alturfan, A. Tozan-Beceren, A.Ö. Şehirli, E. Demiralp, G. Şener, G.Z. Omurtag, Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats, Mol Biol Rep 39 (2012) 4589–4596. https://doi.org/10.1007/s11033-011-1249-5. S. Westfall, U. Iqbal, M. Sebastian, G.M. Pasinetti, Gut microbiota mediated allostasis prevents stress-induced neuroinflammatory risk factors of Alzheimer’s disease, 1st ed., Elsevier Inc., 2019. https://doi.org/10.1016/bs.pmbts.2019.06.013. G. Storelli, A. Defaye, B. Erkosar, P. Hols, J. Royet, F. Leulier, Lactobacillus plantarum promotes drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing, Cell Metab 14 (2011) 403–414. https://doi.org/10.1016/j.cmet.2011.07.012. L. Rivas-Jimenez, K. Ramírez-Ortiz, A.F. González-Córdova, B. Vallejo-Cordoba, H.S. Garcia, A. Hernandez-Mendoza, Evaluation of acrylamide-removing properties of two Lactobacillus strains under simulated gastrointestinal conditions using a dynamic system, Microbiol Res 190 (2016) 19–26. https://doi.org/10.1016/j.micres.2016.04.016. G. Gunasangkaran, A.K. Ravi, V.A. Arumugam, S. Muthukrishnan, Preparation, Characterization, and Anticancer Efficacy of Chitosan, Chitosan Encapsulated Piperine and Probiotics (Lactobacillus plantarum (MTCC-1407), and Lactobacillus rhamnosus (MTCC-1423) Nanoparticles, Bionanoscience 12 (2022) 527–539. https://doi.org/10.1007/S12668-022-00961-7/FIGURES/5. V. Sundararajan, P. Dan, A. Kumar, G.D. Venkatasubbu, Applied Surface Science Drosophila melanogaster as an in vivo model to study the potential toxicity of cerium oxide nanoparticles, Appl Surf Sci 490 (2019) 70–80. https://doi.org/10.1016/j.apsusc.2019.06.017. S. Senthilkumar, R. Raveendran, S. Madhusoodanan, M. Sundar, S.S. Shankar, S. Sharma, V. Sundararajan, P. Dan, S.S. Mohideen, Developmental and behavioural toxicity induced by acrylamide exposure and amelioration using phytochemicals in Drosophila melanogaster, J Hazard Mater 394 (2020) 122533. https://doi.org/10.1016/j.jhazmat.2020.122533. Y.H. Siddique, M. Haidari, W. Khan, A. Fatima, S. Jyoti, S. Khanam, F. Naz, Rahul, F. Ali, B.R. Singh, T. Beg, Mohibullah, A.H. Naqvi, Toxic potential of copper-doped ZnO nanoparticles in Drosophila melanogaster (Oregon R), Toxicol Mech Methods 25 (2015) 425–432. https://doi.org/10.3109/15376516.2015.1045653. X. Tan, J. Ye, W. Liu, B. Zhao, X. Shi, C. Zhang, Z. Liu, X. Liu, Acrylamide aggravates cognitive deficits at night period via the gut–brain axis by reprogramming the brain circadian clock, Arch Toxicol 93 (2019) 467–486. https://doi.org/10.1007/S00204-018-2340-7/FIGURES/9. Z. Yue, Y. Chen, Q. Dong, D. Li, M. Guo, L. Zhang, Y. Shi, H. Wu, L. Li, Z. Sun, Acrylamide induced glucose metabolism disorder in rats involves gut microbiota dysbiosis and changed bile acids metabolism, Food Research International 157 (2022) 111405. https://doi.org/10.1016/J.FOODRES.2022.111405. S. Biswas, P.K. Mukherjee, A. Kar, S. Bannerjee, R. Charoensub, T. Duangyod, Optimized piperine–phospholipid complex with enhanced bioavailability and hepatoprotective activity, Pharm Dev Technol 26 (2021) 69–80. https://doi.org/10.1080/10837450.2020.1835956. G. Gunasangkaran, A.K. Ravi, V.A. Arumugam, S. Muthukrishnan, Preparation, Characterization, and Anticancer Efficacy of Chitosan, Chitosan Encapsulated Piperine and Probiotics (Lactobacillus plantarum (MTCC-1407), and Lactobacillus rhamnosus (MTCC-1423) Nanoparticles, Bionanoscience 12 (2022) 527–539. https://doi.org/10.1007/S12668-022-00961-7/FIGURES/5. P. Ebrahimnejad, M. Khavarpour, S. Khalili, Survival of Lactobacillus acidophilus as probiotic bacteria using chitosan nanoparticles, International Journal of Engineering, Transactions A: Basics 30 (2017) 456–463. https://doi.org/10.5829/IDOSI.IJE.2017.30.04A.01. A.G. Alkushi, A. Abdelfattah-Hassan, H. Eldoumani, S.T. Elazab, S.A.M. Mohamed, A.S. Metwally, E. S.El-Shetry, A.A. Saleh, N.A. ElSawy, D. Ibrahim, Probiotics-loaded nanoparticles attenuated colon inflammation, oxidative stress, and apoptosis in colitis, Scientific Reports 2022 12:1 12 (2022) 1–19. https://doi.org/10.1038/s41598-022-08915-5. G.K. Pratap, D. Ananda, C.G. Joshi, M. Shantaram, Ameliorative activity of medicinal plant fraction for neuroprotection against acrylamide-induced neurotoxicity in Drosophila melanogaster—a comparative study, The Journal of Basic and Applied Zoology 2021 82:1 82 (2021) 1–10. https://doi.org/10.1186/S41936-021-00240-Z. S.N. Prasad, Mitigation of acrylamide-induced behavioral deficits , oxidative impairments and neurotoxicity by oral supplements of geraniol ..., Chem Biol Interact 223 (2014) 27–37. https://doi.org/10.1016/j.cbi.2014.08.016. N.K. Tripathy, K.K. Patnaik, M.J. Nabi, Acrylamide is genotoxic to the somatic and germ cells of Drosophila melanogaster, Mutation Research/Genetic Toxicology 259 (1991) 21–27. https://doi.org/10.1016/0165-1218(91)90105-U. P.P. Kumar, H.P. Harish, Low Molecular Weight Chitosan (∼20 kDa) protects acrylamide induced oxidative stress in D. melanogaster by restoring dopamine and KIF5B levels, Carbohydr Polym 222 (2019) 115005. https://doi.org/10.1016/j.carbpol.2019.115005. N. Kahkeshani, S. Saeidnia, M. Abdollahi, Role of antioxidants and phytochemicals on acrylamide mitigation from food and reducing its toxicity, J Food Sci Technol 52 (2015) 3169–3186. https://doi.org/10.1007/S13197-014-1558-5. S. Dasari, M.S. Ganjayi, B. Meriga, Glutathione S-transferase is a good biomarker in acrylamide induced neurotoxicity and genotoxicity, Interdiscip Toxicol 11 (2018) 115. https://doi.org/10.2478/INTOX-2018-0007. P. Poinsot, A. Penhoat, M. Mitchell, V. Sauvinet, L. Meiller, C. Louche-Pélissier, E. Meugnier, M. Ruiz, M. Schwarzer, M.C. Michalski, F. Leulier, N. Peretti, Probiotic from human breast milk, Lactobacillus fermentum, promotes growth in animal model of chronic malnutrition, Pediatr Res (2020) 1–8. https://doi.org/10.1038/s41390-020-0774-0. S. Westfall, N. Lomis, S. Prakash, Ferulic Acid Produced by Lactobacillus fermentum Influences Developmental Growth Through a dTOR-Mediated Mechanism, Mol Biotechnol 61 (2019) 0. https://doi.org/10.1007/s12033-018-0119-y. Y.J. Jang, W.K. Kim, D.H. Han, K. Lee, G. Ko, Lactobacillus fermentum species ameliorate dextran sulfate sodium-induced colitis by regulating the immune response and altering gut microbiota, Gut Microbes 10 (2019) 696–711. https://doi.org/10.1080/19490976.2019.1589281. S.C.R. Thumu, P.M. Halami, In vivo safety assessment of Lactobacillus fermentum strains, evaluation of their cholesterol-lowering ability and intestinal microbial modulation, J Sci Food Agric 100 (2020) 705–713. https://doi.org/10.1002/jsfa.10071. Additional Declarations No competing interests reported. Supplementary Files CSPNPsJCS.png Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 11 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 18 Aug, 2024 Reviews received at journal 15 Aug, 2024 Reviews received at journal 12 Aug, 2024 Reviews received at journal 04 Aug, 2024 Reviewers agreed at journal 02 Aug, 2024 Reviewers agreed at journal 01 Aug, 2024 Reviewers agreed at journal 31 Jul, 2024 Reviewers agreed at journal 30 Jul, 2024 Reviewers invited by journal 30 Jul, 2024 Editor assigned by journal 30 Jul, 2024 Editor invited by journal 26 Jul, 2024 Submission checks completed at journal 26 Jul, 2024 First submitted to journal 22 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4780644","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":341724925,"identity":"f450177a-b7bb-49fa-9b0b-21b99467343e","order_by":0,"name":"Swetha Senthil Kumar","email":"","orcid":"","institution":"SRM Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Swetha","middleName":"Senthil","lastName":"Kumar","suffix":""},{"id":341724926,"identity":"3de44176-ea2f-4b63-a9f1-ddb31b8905cb","order_by":1,"name":"Sahabudeen Sheik Mohideen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIie2OoQrCUBSGjwizTFaVgb7CgQsrC3uVXQRXhmgxm65FsOpbaBHjRDBtWo9YNGhasygueCcm0U2b4X5cDufC+fh/AIXiL9GhCPIZ6e7Cc7rfKNXe4xh/UDB4/BDy7qHejxbnjrAdtomOk32SgFHyEQ7zzwqGrYY5Fh6fkWcRFwjVQYzAwwwFfDTLYulapGnEewhIMoWLjGLDmN2k4rDhSiM3QXDyFCDfSlMKE2hKRZMplRwFKbbs0drjI5IKF0yvhKd2kF3MZ7t213YMWWx7SWo1o9+YHq5ZxV7R0xH8ICgUCoXiDXd15lA5sAqOxAAAAABJRU5ErkJggg==","orcid":"","institution":"SRM Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Sahabudeen","middleName":"Sheik","lastName":"Mohideen","suffix":""}],"badges":[],"createdAt":"2024-07-22 09:21:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4780644/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4780644/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-72200-w","type":"published","date":"2024-09-11T15:57:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63030606,"identity":"d05ac6ec-7dc4-461d-9c30-34b1c9eb09f9","added_by":"auto","created_at":"2024-08-22 09:18:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":345668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy. \u003c/strong\u003eChitosan and chitosan-coated probiotic nanoparticles were imaged using the TEM technique\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4780644/v1/e8bf7105ac2ed8727b9b5280.png"},{"id":63031939,"identity":"e615888e-0a65-4f54-aab2-5c846434cd3b","added_by":"auto","created_at":"2024-08-22 09:34:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZeta Size Distribution Analysis. \u003c/strong\u003eThe size and particle charge of chitosan-coated probiotic nanoparticles were analysed using the zeta potential technique\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4780644/v1/9caf0ac48a49fd44112040d1.png"},{"id":63031280,"identity":"5be4455d-df04-43c5-905e-e808c0b17694","added_by":"auto","created_at":"2024-08-22 09:26:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViability of CSP NPs. \u003c/strong\u003eThe viability of chitosan-coated \u003cem\u003eL. fermentum\u003c/em\u003e cells was analysed periodically to determine the efficacy of the synthesised CSP NPs. Simulated gastrointestinal systems were utilised to determine the survival and release profile of the fabricated CSP NPs\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4780644/v1/e2e9a065b6df296e303e36f5.png"},{"id":63030608,"identity":"874c3f0c-174a-405d-b9ab-9edc3374d0b0","added_by":"auto","created_at":"2024-08-22 09:18:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":88819,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival and Behavioural Parameters of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eD.mel\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe effects of CSP NP were tested against ACR-induced survival (A) and behavioural deficits (B and C) using the fruit fly model\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4780644/v1/508aa5789562182305a5e622.png"},{"id":63031281,"identity":"55287ae1-d36c-4115-acf8-6a9d933781e8","added_by":"auto","created_at":"2024-08-22 09:26:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":104953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiochemical Factors of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eD.mel\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe effects of CSP NP were tested against ACR-induced ROS levels (A), antioxidant enzyme activity (SOD – B and GST – C) and protein levels (D) using the fruit fly model\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4780644/v1/0f8c0ba8610fe534b192e8cb.png"},{"id":63030609,"identity":"fe9e17a3-9c3e-4d2e-9cde-b7617599e283","added_by":"auto","created_at":"2024-08-22 09:18:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":394613,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial Membrane Potential in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eD.mel\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ovaries. \u003c/strong\u003eTMRE staining of fruit fly ovaries showing changes in mitochondrial membrane potential post-treatment. Increased fluorescence intensity was observed in control and CSPA groups than in ACR-treated flies\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4780644/v1/d66e992b9a88d7c9c585a7ca.png"},{"id":64619506,"identity":"10ff6370-4e28-4af8-8bf5-2a230d0e1ce4","added_by":"auto","created_at":"2024-09-16 16:15:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1630206,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4780644/v1/7ce08d9f-9b1a-4829-b258-bfd254d49d6e.pdf"},{"id":63030607,"identity":"ab9caf88-7f8a-4761-bc7b-724bb3a1a098","added_by":"auto","created_at":"2024-08-22 09:18:51","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":146960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"CSPNPsJCS.png","url":"https://assets-eu.researchsquare.com/files/rs-4780644/v1/93915df6a5f576919e898c1c.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chitosan-Coated Probiotic Nanoparticles Mitigate Acrylamide-Induced Toxicity in the Drosophila Model","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGut health has gained considerable attention in recent years and has become an essential part of most fitness regimes. The gut microbiota, consisting of trillions of microorganisms residing in the gastrointestinal tract, plays a crucial role in maintaining human health and well-being. This microbial community is not just a passive bystander but actively contributes to numerous physiological processes essential for human health including mental fitness, immunological and metabolic mechanisms. However, this niche of vital microbes gets easily disrupted due to several factors including the consumption of foods lacking fibre content, lack of exercise, stress, excessive intake of antibiotics, proton pump inhibitors (PPIs), and non-steroidal anti-inflammatory drugs (NSAIDs), etc. Among these factors affecting the gut microbiome, the food we consume plays a major role in regulating our health [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe healthy food items or supplements we consume undergo several physical and chemical changes during digestion in our gastrointestinal tract before the nutrients from them get absorbed into our body. These nutrients or bioactives present in our present in vegetables, herbs, spices, or fruits serve as cofactors, vitamins, or essential factors for better functioning of our body. However, their absorption in the small intestine is often affected by several factors including chemical form, matrix, and metabolic pathways [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, it is essential to develop a suitable delivery matrix to ensure the bioavailability of essential bioactives for a healthy population. Advances in nanotechnology offer us vast opportunities to manipulate the chemical, physical and biological properties of various compounds to suit our goals. One such development is the synthesis of nano-delivery systems made with biocompatible materials that ensure slow-release mechanisms for enhanced bioavailability [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProbiotics are microbes that improve health by modulating the gut microbiota. Some probiotic species, such as \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, and \u003cem\u003eAkkermansia\u003c/em\u003e, interact with the gut microbiota, increasing beneficial bacteria and reducing pathogenic species. This interaction can help restore the gut microbiota from dysbiosis. Studies have shown that \u003cem\u003eBifidobacterium pseudolongum\u003c/em\u003e can help recover gut microbiota dysbiosis in obese mice, increasing the abundance of \u003cem\u003eButyricimonas\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Probiosis is a process where bacteria, such as \u003cem\u003eLactobacillus\u003c/em\u003e strains, can alter the composition and function of the intestinal microbiome. These bacteria produce antimicrobial agents or metabolic compounds that suppress the growth of other microorganisms or compete for receptors and binding sites with other intestinal microbes. They can also enhance the integrity of the intestinal barrier, resulting in immune tolerance and decreased translocation of bacteria across the mucosa [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProbiotics can also modulate intestinal immunity and alter the responsiveness of intestinal epithelia and immune cells to microbes in the intestinal lumen. Studies have shown that probiotic treatment can reduce pain and flatulence in patients with IBS, but not many have demonstrated associations with altered microbiota [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Probiotic supplements have been shown in studies to boost gut microbiota quantity, but they cannot alter microbial metabolites on their own accord. Synbiotics, a mix of probiotics and prebiotics, have been created to enhance the gut microbiota by modifying certain species, whereas prebiotics feed the microbiota and impact metabolite formation, notably short-chain fatty acids (SCFA) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study fabricated chitosan-coated probiotic nanoparticles (CSP NPs) to ensure the bioavailability of the probiotic load (\u003cem\u003eL. fermentum\u003c/em\u003e) in the gut with a sustained release profile. The chitosan coating doubles as a prebiotic (non-digestible fibre that enhances the functionality of probiotics in the gut) and as a delivery system with innate immunomodulatory and antioxidant effects. Consequently, we analysed the efficiency of this synthesised synbiotic formulation against common heat-processed toxins \u0026ndash; acrylamide (ACR) using the \u003cem\u003eDrosophila melanogaster\u003c/em\u003e model.\u003c/p\u003e \u003cp\u003eACR is a white crystalline compound used in textiles, paper, cosmetics, cement, and mining processes. It has been classified as hazardous and probable carcinogenic to humans due to its presence in industrial effluents. In humans, ACR has been observed to cause muscle weakness, impaired cognition, and peripheral neuropathy in employees exposed via inhalation or skin absorption [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Several food and drug associations have set standards and mitigation strategies to reduce ACR formation or presence in food products. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) set 4\u0026ndash;5 \u0026micro;g/kg bw/day acrylamide from food as the highest tolerable intake level in 2005 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In India, the major source of acrylamide is the consumption of deep-fried food products ranging from 82.0 to 4245.6 \u0026micro;g/kg [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Exposure studies by the US CDC organization in 2019 showed that children aged 3\u0026ndash;11 had 108\u0026thinsp;\u0026minus;\u0026thinsp;93.8 pmol/g haemoglobin, while older adults had 223\u0026ndash;257 pmol/g haemoglobin [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe post-pandemic state has aggravated the situation, with young children being twice as impacted as adults due to their eating habits, rapid metabolism, and low body weight. Fast food intake has also grown, which may raise the risk of infertility [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. ACR's negative effects have been explored in model organisms such as rats, mice, zebrafish, and \u003cem\u003eDrosophila\u003c/em\u003e, with findings indicating circadian clock disturbance, developmental delays, and neurotoxic effects [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eDrosophila melanogaster\u003c/em\u003e is used as a model organism to study and understand various diseases, their molecular mechanisms, and their effects on humans. It is a widely preferred model due to its short life cycle, genetic similarity to humans, and high fecundity rate. Additionally, lactic acid bacteria (LAB) have been reported to have nutrient-dependent regulatory control on \u003cem\u003eDrosophila\u003c/em\u003e development and can regulate growth hormones like ecdysone and insulin receptors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Storelli et al. also reported the influence of LAB on the developmental mechanisms in \u003cem\u003eDrosophila\u003c/em\u003e under nutrient-scarce conditions [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Furthermore, studies with \u003cem\u003eLactobacillus\u003c/em\u003e strains have reported that these bacteria can alleviate the ACR-induced oxidative stress in \u003cem\u003evivo\u003c/em\u003e models and reduce the bioavailability of dietary acrylamide under different simulated gastrointestinal conditions (\u003cem\u003ein vitro\u003c/em\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, in this study, we have discussed the formulation process of chitosan-coated probiotic nanoparticles, Further, our study analyses the efficiency of these synbiotic formulations against ACR-induced toxicity using the fruit fly model. Our results demonstrate the extended bioactivity of the probiotic load as well as its protective mechanisms against ACR-induced toxicity.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Chemicals and Cultures\u003c/h2\u003e\n \u003cp\u003eAcrylamide (3x Crystalline), low molecular weight chitosan, glacial acetic acid, yeast extract, propionic acid, orthophosphoric acid, nitro blue tetrazolium salt (NBT), phenazonium Methosulphate (PMS), nicotinamide adenine dinucleotide (reduced) disodium salt (NADH), potassium chloride, glutathione reduced (GSH), tetrasodium pyrophosphate (TSPP), ethanol, 5, 5-dithiobis 2-nitrobenzoic acid (DTNB), Abcam TMRE-mitochondrial membrane potential assay kit and, 1-chloro-2,4-dinitrobenzene (CDNB) were procured from Sisco Research Laboratories, India. Agar-Agar Type \u0026minus;\u0026thinsp;1, dextrose, sodium phosphate monobasic, sodium phosphate dibasic, bovine serum albumin (BSA), phosphate buffer saline (PBS) and methylparaben were purchased from Himedia Pvt. Ltd., India. All chemicals used in this study were of analytical grade. \u003cem\u003eLactobacillus fermentum\u003c/em\u003e (MCC 2760) stock was obtained from NCCS, Pune and periodically subcultured in MRS broth (pH 6.5) at 37\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Fabrication and Characterisation of Chitosan Coated Probiotic Nanoparticles (CSP NPs)\u003c/h2\u003e\n \u003cp\u003eChitosan-coated probiotic nanoparticles (CSP NPs) were synthesised following a previously reported study with slight modifications [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Briefly, a 0.5% solution of low molecular weight chitosan was prepared with 1% acetic acid and 3 ml of \u003cem\u003eL. fermentum\u003c/em\u003e broth (8 log CFU/ml) was mixed with constant stirring. To this, a stoichiometric amount of 1mg/ml sodium tripolyphosphate (TPP) was added dropwise with constant stirring for 2 hours at room temperature. The mixture was then sonicated for 30 minutes and centrifuged at 10,000 rpm for 15 minutes. The pellet was then lyophilised and stored at 4℃. The preparation of chitosan nanoparticles (CS NPs) followed a similar method excluding the addition of the probiotic culture. The size and morphology of synthesised CS and CSP NPs were examined using JEM-2100 Plus Hi-Resolution Transmission Electron Microscope (HRTEM), JEOL Japan. The surface charge and particle size of CSP NPs in an aqueous system were then estimated using Malvern/Nano ZS-90 Zeta Sizer.\u003c/p\u003e\u003cspan\u003e\n \u003ch2\u003e2.3 Drosophila Rearing and Maintenance\u003c/h2\u003e\n \u003c/span\u003e\n \u003cp\u003eThe \u003cem\u003eDrosophila\u003c/em\u003e Oregon K wild-type strain was maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60% humidity under a 12-hour dark-light cycle. Flies were cultured on a standard cornmeal agar medium consisting of cornflour, agar-agar type 1, D-glucose, sugar, and yeast extract. To prevent microbial contamination, the medium was autoclaved and supplemented with antifungal agents including propionic acid, Tego (methyl para hydroxy benzoate dissolved in ethanol), and orthophosphoric acid at 55\u0026deg;C. Additionally, 2 mM acrylamide (ACR), 10 \u0026micro;g/ml CSP NPs, and 10 \u0026micro;g/ml CSP NPs\u0026thinsp;+\u0026thinsp;ACR (CSPA) treatment groups were prepared by the stoichiometric addition of respective compounds to the media at 50\u0026ndash;55\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Survival and Behavioural Assay\u003c/h2\u003e\n \u003cp\u003eThe lifespan of flies was estimated by transferring twenty-five healthy newborn adult male flies to freshly prepared treatment media. The flies were then constantly monitored and the mortality rate was calculated by tallying the number of dead flies every 24 hours. To assess the locomotor functions at the larval stage, male and female flies were added to the respective media for 5 days. The parent flies were then discarded and the vials were carefully maintained till the development of third instar larvae. These larvae were then transferred to an agar plate placed on top of a graph paper and allowed to crawl for one minute. The number of 1mm grids traversed per minute was represented as the crawling activity for statistical analysis. Similarly, the adult climbing activity was determined by exposing thirty male flies to the respective treatment media for 5 days. The treated flies were then transferred to a climbing assay setup with 3 cm marked vials. The vials were tapped moderately and the percentage of active flies was reported as the number of flies above the 3 cm mark after 10 seconds [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Estimation of Oxidative Stress Parameters\u003c/h2\u003e\n \u003cp\u003eBiochemical analysis was carried out with twenty newborn male flies post-5-day exposure to respective treatment groups. Protein estimation of the treated flies was performed following Lowry\u0026rsquo;s method with bovine serum albumin as the standard. Additionally, reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione-s-transferase (GST) activities were estimated following the previously reported study [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Briefly, the treated flies were homogenised in Tris and sodium phosphate buffer for ROS and antioxidant enzyme activity analysis respectively. Stoichiometric quantities of respective reagents were mixed with 100 \u0026micro;l homogenate for further analysis. The fluorescence and absorbance readings were further analysed for statistical analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Estimation of Ovarian Mitochondrial Membrane Potential\u003c/h2\u003e\n \u003cp\u003eThirty female flies were treated with control and treatment media, and their ovaries were dissected to determine their mitochondrial membrane potential. The experiment was slightly modified from the Abcam TMRE-Mitochondrial Membrane Potential experiment Kit. The ovaries were dissected in Schneider\u0026apos;s insect medium, stained with 200 nM TMRE for 15 minutes in the dark, washed with PBS, and observed using a Leica DM6 fluorescent microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e\n \u003cp\u003eAll experiments performed in this study were carried out in triplicates to establish statistical significance. Experimental data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM unless stated otherwise, Statistical analysis of these data was performed using Graph Pad Prism 6.0 software following Dunnett\u0026apos;s multiple comparison tests to compare the significance of treatment groups with control and ACR, respectively. The significance level for the datasets was set to P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Characterisation of CSP NPs\u003c/h2\u003e\n \u003cp\u003eChitosan and chitosan encapsulated probiotic nanoparticles were analysed using a transmission electron microscope (TEM), the size of the coated particles (CSP NPs) depicted an average size of 565 nm whereas the CS NPs without the probiotic load showed an average particle size of 110 nm. The TEM images (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) illustrate the spherical chitosan molecules encapsulating rod-shaped \u003cem\u003eLactobacillus fermentum\u003c/em\u003e cells.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the particle size and charge of the synthesised CSP NPs. The average zeta potential of the coated probiotic NPs was 4.38\u0026thinsp;\u0026plusmn;\u0026thinsp;15.8 mV with a Z-average of 663.4 d. nm and a polydispersity index of 0.427\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Viability of Synthesised CSP NPs\u003c/h2\u003e\n \u003cp\u003eCSP NPs bacterial load viability was determined using the pour plate method as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The synthesised nanoparticles indicated an average viability of 7.93 log CFU/ml when stored at room temperature for 49 days. Furthermore, the viability and release profile of the synthesised CSP NPs under simulated gastrointestinal fluid systems demonstrated subsequent stability of the probiotic load (SGF\u003csub\u003e120\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9.07 log CFU/ml; SIF\u003csub\u003e180\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7 log CFU/ml) compared to the free probiotic culture with SGF\u003csub\u003e120\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8 log CFU/ml and SIF\u003csub\u003e180\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.87 log CFU/ml.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Effect of CSP NPs on Survival and Behavioural Parameters in ACR-treated Flies\u003c/h2\u003e\n \u003cp\u003eThe synthesised CSP NPs were orally administered to the fruit fly model to assess their efficiency against common environmental and food-borne toxins like acrylamide (ACR). ACR-treated flies demonstrated low survival capacity (12 days), reduced larval crawling (37 mm/min) and declined climbing activity in adult male flies (75.55%) as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B and C respectively. Alternatively, the control (55 days; 68.25 mm/min; 96.29%) and co-treated flies (CSPA\u0026thinsp;=\u0026thinsp;48 days; 67.03 mm/min; 85.55%) demonstrated a significant level of activity and survival capacity.\u003c/p\u003e\u003cspan\u003e\n \u003ch2\u003e\u003cstrong\u003e3.4 CSP NP modulates ACR-induced Redox Stress Factors in\u003c/strong\u003e \u003cstrong\u003eD.mel\u003c/strong\u003e\u003c/h2\u003e\n \u003c/span\u003e\n \u003cp\u003eMale flies post-exposure to respective treatment groups were homogenised in suitable buffers to estimate redox stress parameters. ACR-treated flies exhibited increased ROS, SOD and GST activities as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, B and C respectively. Furthermore, ACR exposure indicated a negative influence on protein levels (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). On the other hand, the CSPA-treated group demonstrated a significant recovery in ROS and antioxidant enzyme activity levels. Comparing the data with the control group revealed the prominent effect of CSPA treatment in restoring the alterations in vital biochemical factors and thereby curbing the toxic influence of ACR within the fruit fly model.\u003c/p\u003e\u003cspan\u003e\n \u003ch2\u003e\u003cstrong\u003e3.5 CSP NPs Reverse ACR-induced Mitochondrial Membrane Depolarisation in\u003c/strong\u003e \u003cstrong\u003eD.mel\u003c/strong\u003e \u003cstrong\u003eOvaries\u003c/strong\u003e\u003c/h2\u003e\n \u003c/span\u003e\n \u003cp\u003eOvaries dissected from treated female flies were stained with TMRE solution to estimate the influence of ACR and CSPA treatment on mitochondrial membrane potential (MMP) as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. TMRE dye accumulation in control and CSPA was observed indicating the presence of an active mitochondrial population. Whereas, the ACR-treated samples were noted to have a substantial reduction in TMRE fluorescence indicating the role of ACR to induce mitochondrial depolarisation. This result indicates the functionality of CSPA treatment in protecting against ACR-induced mitochondrial depolarisation and subsequent apoptosis mechanisms at the cellular level.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAcrylamide has been one of the most researched compounds since 2002 due to its dual role as a prominent environmental and food-based toxin. Contamination of water bodies due to industrial discharge is one aspect of toxin exposure, however, a more serious concern arises because of its formation in commonly consumed high-temperature processed food items. According to a 2005 study, the amount of ACR in food products varies with storage conditions and length of time, but the important thing to remember is that throughout long-term storage, their levels do not fully diminish. Consequently, multiple other studies have discerned the toxic effects of other food-processing toxins like bisphenol-A, heterocyclic amines, methylimidazole, etc., on humans. Therefore, it is imperative to formulate a suitable dietary supplement that can reduce, restore, or protect our physiological systems from these food-processed toxins.\u003c/p\u003e \u003cp\u003eIn the vast scope of food-based research, our chitosan-coated probiotic nanoparticles emphasise the benefits of incorporating simple naturally available dietary supplements to counteract the negative impacts of dietary toxins. The concept of \u0026lsquo;gut-brain interaction\u0026rsquo; has been highflying in recent times. Many have realised the importance of the enteric system commonly known as the second brain of the human body and its influence on overall well-being. The gut microbiome is one of the key regulators of the enteric system and aids in the gut-brain crosstalk, influencing mental, metabolic, and immunological health.\u003c/p\u003e \u003cp\u003ePrevious studies reported ACR-induced cognitive impairments observed in mice caused due to dysregulation in the gut-brain axis. The study reported that the ACR treatment reduced occludin protein expressions, increased gut permeability, and increased LPS and pro-inflammatory factor levels in the gut and serum during the night phase leading to worse performance in spatial and working memory [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Another study investigated the effects of AA on female Sprague Dawley rats and found that it increased blood glucose levels, decreased insulin levels, and produced intestinal barrier damage. It also demonstrated gut microbial dysbiosis and a bile acid metabolism challenge, with lower \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBacteroides\u003c/em\u003e abundance and higher cholic acid levels. These findings point to the possibility of reducing ACR toxicity by manipulating the gut microbiota [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we have exploited the physical and biological properties of a natural biopolymer, chitosan to develop sustained-release probiotic supplements. Probiotics such as \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e species are commonly known and are easily sourced from milk, curd, and fermented products. Studies on \u003cem\u003eBifidobacterium animalis\u003c/em\u003e, \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e, \u003cem\u003eBifidobacterium longum\u003c/em\u003e, and \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e have demonstrated recovery of gut dysbiosis, colonic inflammation, improved microbiota diversity, etc. However, probiotic supplements in combination with prebiotics like inulin, chitosan, arabinoxylan, melanoids, flavonoids, etc have significant modulatory effects on the gut microbiota. The prebiotics generally influence the gut microbes and aid in the production of short-chain fatty acids (SCFA). The synbiotic formulations further enhance the accumulation of beneficial gut microbiota, reduce the presence of harmful colonic bacteria, and impart positive modulatory effects via SCFA-mediated systemic reactions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe fabricated CSP NPs by coating the \u003cem\u003eLactobacillus fermentum\u003c/em\u003e culture with low molecular weight chitosan in the presence of TPP. The characterisation of these NPs confirmed the nano-size range via the TEM technique and additionally, the positive zeta charge and low polydispersity index pointed toward the narrow particle size distribution. Therefore, we could confirm the easy permeability of this formulation across the physiological barrier [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, data from the viability assays indicated long-term storage of synthesised NPs at room temperature and sustained probiotic release profile under gastric and intestinal pH conditions. Our experimental data confirmed with other research on synbiotic NP formulations [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our experimental data in the fruit fly model demonstrated the efficiency of CSP NPs against ACR by enhancing behavioural functions at larval and adult fly stages. The treated flies exhibited increased survival capacity, normalised ROS levels, and no fluctuations in antioxidant enzyme activities. Furthermore, the MMP in CSPA-treated ovaries demonstrated substantial TMRE intensity indicating the presence of an active mitochondrial population. The \u003cem\u003ein vivo\u003c/em\u003e study confirms the interplay between synbiotic supplements and ACR toxicity mitigation strategy.\u003c/p\u003e \u003cp\u003eACR-induced behavioural changes analysed through locomotor functions are a characteristic feature of the fruit fly model. The interaction between environmental cues, neuromuscular coordination and subsequent impact on development and behaviour enables us to comprehend the impact of xenobiotics at larval and adult stages. The decrease in locomotor functions at the larval stage affects feeding rate, memory, habitat selection and survival. This in turn when assayed at the adult stage similarly exhibits reduced climbing activity, mating abnormalities, fecundity issues, etc. The biochemical data further confirms the impact of ACR within the fruit fly model. These results align with the previously reported studies on ACR toxicity using \u003cem\u003eD.mel\u003c/em\u003e models [\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. SOD and GST belonging to the enzymatic antioxidant defence mechanism play a vital role in xenobiotic clearance and redox stress normalization.\u003c/p\u003e \u003cp\u003eOxidative stress species like free oxygen/nitrogen radicals, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, etc. are produced during normal cellular functioning and in response to xenobiotic triggers. It is, therefore, in the control of these enzymatic antioxidant defences to regulate the cellular redox status. Elevation in ROS, SOD and GST activity implies active interaction between ACR and subsequent detoxification mechanisms via GST mediated biotransformation method [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The ovarian mitochondrial depolarisation can also be linked to our earlier findings reporting low survival rate and high redox stress. Unfavourable conditions and ACR\u0026rsquo;s direct influence on MMP trigger the apoptotic pathways leading to non-functional ovaries and teratogenic attributes of ACR.\u003c/p\u003e \u003cp\u003eHowever, the CSPA (CSP NPs\u0026thinsp;+\u0026thinsp;ACR) treatment demonstrated no significant toxicity at adult and larval stages. Therefore, we postulate that the cotreatment of synbiotics with ACR enhances the gut barrier by modulating the composition and activity of the gut microbiota. Chitosan NPs coating serving as a protective matrix ensures the delivery and controlled release of the probiotic load in the gut and aids in intestinal mucosal adhesion. Additionally, the \u003cem\u003eL. fermentum\u003c/em\u003e load in interaction with CS NPs can produce SCFAs and antimicrobial peptides. This interaction can stimulate gut-associated lymphoid tissue (GALT), enhance mucosal immunity, regulate inflammatory responses, and promote tolerance to harmless antigens, thereby reducing inflammation and improving intestinal barrier function [\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. By maintaining gut integrity and reducing intestinal permeability, they prevent systemic inflammation and the translocation of toxins (like ACR metabolites \u0026ndash; glycidamide) into circulation.\u003c/p\u003e \u003cp\u003eIn conclusion, our study highlights the effectiveness of synthesised chitosan-coated \u003cem\u003eL\u003c/em\u003e. \u003cem\u003efermentum\u003c/em\u003e nanoparticles against ACR-induced toxicity. Our experimental findings indicate that our synbiotic formulation successfully preserved gut integrity and microbiota, preventing the manifestation of toxic symptoms associated with ACR exposure in fruit flies. This highlights the significant interaction between ACR and gut microbiota within our experimental model. Prospects of our study include a detailed investigation into the protective mechanisms exhibited by the synbiotic formulation via gene and protein expression estimation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions \u0026ndash; Swetha Senthilkumar \u0026ndash;\u003c/strong\u003e Conceptualization, Methodology, Data\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecuration, Writing - original draft, Writing - review \u0026amp; editing, Software,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eValidation; \u003cstrong\u003eSahabudeen Sheik Mohideen \u0026ndash;\u003c/strong\u003eConceptualization, Methodology, Validation, Writing - review \u0026amp; editing and Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the SRM SCIF and DBT platform for Advanced Life Science\u0026rsquo;s support in TEM, fluorescence imaging studies and Zeta analysis. The authors also recognize the support provided by the Department of Science and Technology, Ministry of Science and Technology, Government of India through the INSPIRE Fellowship program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests \u0026ndash;\u0026nbsp;\u003c/strong\u003eThe authors disclose no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e \u0026ndash; Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e \u0026ndash; Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e \u0026ndash; Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement \u0026ndash;\u0026nbsp;\u003c/strong\u003eAll data sets used and/or generated in this work are obtainable from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e \u0026ndash; The authors also recognize the support provided by the Department of Science and Technology, Ministry of Science and Technology, Government of India through the INSPIRE Fellowship program\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(No. DST/INSPIRE Fellowship/2021/IF210062). The funders played no part in the study\u0026apos;s methodology, data collection and analysis, publication decision, or drafting of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eS. Pedroza Matute, S. Iyavoo, Exploring the gut microbiota: lifestyle choices, disease associations, and personal genomics, Front Nutr 10 (2023). https://doi.org/10.3389/FNUT.2023.1225120.\u003c/li\u003e\n\u003cli\u003eM.J. Rein, M. Renouf, C. Cruz-Hernandez, L. Actis-Goretta, S.K. Thakkar, M. da Silva Pinto, Bioavailability of bioactive food compounds: a challenging journey to bioefficacy, Br J Clin Pharmacol 75 (2013) 588. https://doi.org/10.1111/J.1365-2125.2012.04425.X.\u003c/li\u003e\n\u003cli\u003eT. Zhang, C. Shang, T. Du, J. Zhuo, C. Wang, B. Li, J. Xu, M. Fan, J. Wang, W. Zhang, Cytoprotection of probiotics by nanoencapsulation for advanced functions, Trends Food Sci Technol 142 (2023) 104227. https://doi.org/10.1016/J.TIFS.2023.104227.\u003c/li\u003e\n\u003cli\u003eK. Bauer-Estrada, C. Sandoval-Cuellar, Y. Rojas-Mu\u0026ntilde;oz, M.X. Quintanilla-Carvajal, The modulatory effect of encapsulated bioactives and probiotics on gut microbiota: improving health status through functional food, Food Funct 14 (2023) 32\u0026ndash;55. https://doi.org/10.1039/D2FO02723B.\u003c/li\u003e\n\u003cli\u003eD.M. Saulnier, J.K. Spinler, G.R. Gibson, J. Versalovic, Mechanisms of Probiosis and Prebiosis: Considerations for Enhanced Functional Foods, Curr Opin Biotechnol 20 (2009) 135. https://doi.org/10.1016/J.COPBIO.2009.01.002.\u003c/li\u003e\n\u003cli\u003eP. Hemarajata, J. Versalovic, Effects of probiotics on gut microbiota: mechanisms of intestinal immunomodulation and neuromodulation, Therap Adv Gastroenterol 6 (2013) 39. https://doi.org/10.1177/1756283X12459294.\u003c/li\u003e\n\u003cli\u003eV.C. Goodoory, M. Khasawneh, C.J. Black, E.M.M. Quigley, P. Moayyedi, A.C. Ford, Efficacy of Probiotics in Irritable Bowel Syndrome: Systematic Review and Meta-analysis, Gastroenterology 165 (2023) 1206\u0026ndash;1218. https://doi.org/10.1053/J.GASTRO.2023.07.018.\u003c/li\u003e\n\u003cli\u003eN. Joseph, K. Vasodavan, N.A. Saipudin, B.N.M. Yusof, S. Kumar, S.A. Nordin, Gut microbiota and short-chain fatty acids (SCFAs) profiles of normal and overweight school children in Selangor after probiotics administration, J Funct Foods 57 (2019) 103\u0026ndash;111. https://doi.org/10.1016/J.JFF.2019.03.042.\u003c/li\u003e\n\u003cli\u003eP. Markowiak-Kopeć, K. Śliżewska, The Effect of Probiotics on the Production of Short-Chain Fatty Acids by Human Intestinal Microbiome, Nutrients 12 (2020). https://doi.org/10.3390/NU12041107.\u003c/li\u003e\n\u003cli\u003eL. Hagmar, M. T\u0026ouml;rnqvist, C. Nordander, I. Ros\u0026eacute;n, M. Bruze, A. Kautiainen, A.L. Magnusson, B. Malmberg, P. Aprea, F. Granath, A. Axmon, Health effects of occupational exposure to acrylamide using hemoglobin adducts as biomarkers of internal dose, Scand J Work Environ Health 27 (2001) 219\u0026ndash;226. https://doi.org/10.5271/sjweh.608.\u003c/li\u003e\n\u003cli\u003eA. Koszucka, A. Nowak, I. Nowak, I. Motyl, Acrylamide in human diet, its metabolism, toxicity, inactivation and the associated European Union legal regulations in food industry, Crit Rev Food Sci Nutr 60 (2020) 1677\u0026ndash;1692. https://doi.org/10.1080/10408398.2019.1588222.\u003c/li\u003e\n\u003cli\u003eL.C. Dolan, R.A. Matulka, G.A. Burdock, Naturally Occurring Food Toxins, Toxins (Basel) 2 (2010) 2289. https://doi.org/10.3390/TOXINS2092289.\u003c/li\u003e\n\u003cli\u003eV. Matoso, P. Bargi-Souza, F. Ivanski, M.A. Romano, R.M. Romano, Acrylamide: A review about its toxic effects in the light of Developmental Origin of Health and Disease (DOHaD) concept, Food Chem 283 (2019) 422\u0026ndash;430. https://doi.org/10.1016/j.foodchem.2019.01.054.\u003c/li\u003e\n\u003cli\u003eL. Shamla, P. Nisha, Acrylamide in deep-fried snacks of India, Food Addit Contam Part B Surveill 7 (2014) 220\u0026ndash;225. https://doi.org/10.1080/19393210.2014.894141.\u003c/li\u003e\n\u003cli\u003eCDC, Nceh, DLS, Od, Fourth National Report on Human Exposure to Environmental Chemicals Update, n.d.\u003c/li\u003e\n\u003cli\u003eS.M. Lai, Z.T. Gu, M.M. Zhao, X.X. Li, Y.X. Ma, L. Luo, J. Liu, Toxic effect of acrylamide on the development of hippocampal neurons of weaning rats, Neural Regen Res 12 (2017) 1648\u0026ndash;1654. https://doi.org/10.4103/1673-5374.217345.\u003c/li\u003e\n\u003cli\u003eS. Stagi, S. De Masi, E. Bencini, S. Losi, S. Paci, M. Parpagnoli, F. Ricci, D. Ciofi, C. Azzari, Increased incidence of precocious and accelerated puberty in females during and after the Italian lockdown for the coronavirus 2019 (COVID-19) pandemic, Ital J Pediatr 46 (2020) 165. https://doi.org/10.1186/s13052-020-00931-3.\u003c/li\u003e\n\u003cli\u003eH. Wang, P. Huang, T. Lie, J. Li, R.J. Hutz, K. Li, F. Shi, Reproductive toxicity of acrylamide-treated male rats, Reproductive Toxicology 29 (2010) 225\u0026ndash;230. https://doi.org/10.1016/j.reprotox.2009.11.002.\u003c/li\u003e\n\u003cli\u003eZ. Huang, S. Wang, Y. Yang, J. Lou, Z. Liu, Z. Liu, H. Yong, S. Shan, F. Song, Mitochondrial dysfunction promotes the necroptosis of Purkinje cells in the cerebellum of acrylamide-exposed rats, Food and Chemical Toxicology 171 (2023) 113522. https://doi.org/10.1016/J.FCT.2022.113522.\u003c/li\u003e\n\u003cli\u003eB.O. Yilmaz, N. Yildizbayrak, Y. Aydin, M. Erkan, Evidence of acrylamide- and glycidamide-induced oxidative stress and apoptosis in Leydig and Sertoli cells, Hum Exp Toxicol 36 (2017) 1225\u0026ndash;1235. https://doi.org/10.1177/0960327116686818.\u003c/li\u003e\n\u003cli\u003eM. Huang, J. Jiao, J. Wang, Z. Xia, Y. Zhang, Characterization of acrylamide-induced oxidative stress and cardiovascular toxicity in zebrafish embryos, J Hazard Mater 347 (2018) 451\u0026ndash;460. https://doi.org/10.1016/j.jhazmat.2018.01.016.\u003c/li\u003e\n\u003cli\u003eS. Zhao, X. Zhao, Q. Liu, Y. Jiang, Y. Li, W. Feng, H. Xu, M. Shao, Protective effect of Lactobacillus plantarum ATCC8014 on acrylamide-induced oxidative damage in rats, Appl Biol Chem 63 (2020). https://doi.org/10.1186/s13765-020-00527-9.\u003c/li\u003e\n\u003cli\u003eM.I. Alkhalaf, Diosmin protects against acrylamide-induced toxicity in rats: Roles of oxidative stress and inflammation, J King Saud Univ Sci 32 (2020) 1510\u0026ndash;1515. https://doi.org/10.1016/J.JKSUS.2019.12.005.\u003c/li\u003e\n\u003cli\u003eA.A. Alturfan, A. Tozan-Beceren, A.\u0026Ouml;. Şehirli, E. Demiralp, G. Şener, G.Z. Omurtag, Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats, Mol Biol Rep 39 (2012) 4589\u0026ndash;4596. https://doi.org/10.1007/s11033-011-1249-5.\u003c/li\u003e\n\u003cli\u003eS. Westfall, U. Iqbal, M. Sebastian, G.M. Pasinetti, Gut microbiota mediated allostasis prevents stress-induced neuroinflammatory risk factors of Alzheimer\u0026rsquo;s disease, 1st ed., Elsevier Inc., 2019. https://doi.org/10.1016/bs.pmbts.2019.06.013.\u003c/li\u003e\n\u003cli\u003eG. Storelli, A. Defaye, B. Erkosar, P. Hols, J. Royet, F. Leulier, Lactobacillus plantarum promotes drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing, Cell Metab 14 (2011) 403\u0026ndash;414. https://doi.org/10.1016/j.cmet.2011.07.012.\u003c/li\u003e\n\u003cli\u003eL. Rivas-Jimenez, K. Ram\u0026iacute;rez-Ortiz, A.F. Gonz\u0026aacute;lez-C\u0026oacute;rdova, B. Vallejo-Cordoba, H.S. Garcia, A. Hernandez-Mendoza, Evaluation of acrylamide-removing properties of two Lactobacillus strains under simulated gastrointestinal conditions using a dynamic system, Microbiol Res 190 (2016) 19\u0026ndash;26. https://doi.org/10.1016/j.micres.2016.04.016.\u003c/li\u003e\n\u003cli\u003eG. Gunasangkaran, A.K. Ravi, V.A. Arumugam, S. Muthukrishnan, Preparation, Characterization, and Anticancer Efficacy of Chitosan, Chitosan Encapsulated Piperine and Probiotics (Lactobacillus plantarum (MTCC-1407), and Lactobacillus rhamnosus (MTCC-1423) Nanoparticles, Bionanoscience 12 (2022) 527\u0026ndash;539. https://doi.org/10.1007/S12668-022-00961-7/FIGURES/5.\u003c/li\u003e\n\u003cli\u003eV. Sundararajan, P. Dan, A. Kumar, G.D. Venkatasubbu, Applied Surface Science Drosophila melanogaster as an in vivo model to study the potential toxicity of cerium oxide nanoparticles, Appl Surf Sci 490 (2019) 70\u0026ndash;80. https://doi.org/10.1016/j.apsusc.2019.06.017.\u003c/li\u003e\n\u003cli\u003eS. Senthilkumar, R. Raveendran, S. Madhusoodanan, M. Sundar, S.S. Shankar, S. Sharma, V. Sundararajan, P. Dan, S.S. Mohideen, Developmental and behavioural toxicity induced by acrylamide exposure and amelioration using phytochemicals in Drosophila melanogaster, J Hazard Mater 394 (2020) 122533. https://doi.org/10.1016/j.jhazmat.2020.122533.\u003c/li\u003e\n\u003cli\u003eY.H. Siddique, M. Haidari, W. Khan, A. Fatima, S. Jyoti, S. Khanam, F. Naz, Rahul, F. Ali, B.R. Singh, T. Beg, Mohibullah, A.H. Naqvi, Toxic potential of copper-doped ZnO nanoparticles in Drosophila melanogaster (Oregon R), Toxicol Mech Methods 25 (2015) 425\u0026ndash;432. https://doi.org/10.3109/15376516.2015.1045653.\u003c/li\u003e\n\u003cli\u003eX. Tan, J. Ye, W. Liu, B. Zhao, X. Shi, C. Zhang, Z. Liu, X. Liu, Acrylamide aggravates cognitive deficits at night period via the gut\u0026ndash;brain axis by reprogramming the brain circadian clock, Arch Toxicol 93 (2019) 467\u0026ndash;486. https://doi.org/10.1007/S00204-018-2340-7/FIGURES/9.\u003c/li\u003e\n\u003cli\u003eZ. Yue, Y. Chen, Q. Dong, D. Li, M. Guo, L. Zhang, Y. Shi, H. Wu, L. Li, Z. Sun, Acrylamide induced glucose metabolism disorder in rats involves gut microbiota dysbiosis and changed bile acids metabolism, Food Research International 157 (2022) 111405. https://doi.org/10.1016/J.FOODRES.2022.111405.\u003c/li\u003e\n\u003cli\u003eS. Biswas, P.K. Mukherjee, A. Kar, S. Bannerjee, R. Charoensub, T. Duangyod, Optimized piperine\u0026ndash;phospholipid complex with enhanced bioavailability and hepatoprotective activity, Pharm Dev Technol 26 (2021) 69\u0026ndash;80. https://doi.org/10.1080/10837450.2020.1835956.\u003c/li\u003e\n\u003cli\u003eG. Gunasangkaran, A.K. Ravi, V.A. Arumugam, S. Muthukrishnan, Preparation, Characterization, and Anticancer Efficacy of Chitosan, Chitosan Encapsulated Piperine and Probiotics (Lactobacillus plantarum (MTCC-1407), and Lactobacillus rhamnosus (MTCC-1423) Nanoparticles, Bionanoscience 12 (2022) 527\u0026ndash;539. https://doi.org/10.1007/S12668-022-00961-7/FIGURES/5.\u003c/li\u003e\n\u003cli\u003eP. Ebrahimnejad, M. Khavarpour, S. Khalili, Survival of Lactobacillus acidophilus as probiotic bacteria using chitosan nanoparticles, International Journal of Engineering, Transactions A: Basics 30 (2017) 456\u0026ndash;463. https://doi.org/10.5829/IDOSI.IJE.2017.30.04A.01.\u003c/li\u003e\n\u003cli\u003eA.G. Alkushi, A. Abdelfattah-Hassan, H. Eldoumani, S.T. Elazab, S.A.M. Mohamed, A.S. Metwally, E. S.El-Shetry, A.A. Saleh, N.A. ElSawy, D. Ibrahim, Probiotics-loaded nanoparticles attenuated colon inflammation, oxidative stress, and apoptosis in colitis, Scientific Reports 2022 12:1 12 (2022) 1\u0026ndash;19. https://doi.org/10.1038/s41598-022-08915-5.\u003c/li\u003e\n\u003cli\u003eG.K. Pratap, D. Ananda, C.G. Joshi, M. Shantaram, Ameliorative activity of medicinal plant fraction for neuroprotection against acrylamide-induced neurotoxicity in Drosophila melanogaster\u0026mdash;a comparative study, The Journal of Basic and Applied Zoology 2021 82:1 82 (2021) 1\u0026ndash;10. https://doi.org/10.1186/S41936-021-00240-Z.\u003c/li\u003e\n\u003cli\u003eS.N. Prasad, Mitigation of acrylamide-induced behavioral deficits , oxidative impairments and neurotoxicity by oral supplements of geraniol ..., Chem Biol Interact 223 (2014) 27\u0026ndash;37. https://doi.org/10.1016/j.cbi.2014.08.016.\u003c/li\u003e\n\u003cli\u003eN.K. Tripathy, K.K. Patnaik, M.J. Nabi, Acrylamide is genotoxic to the somatic and germ cells of Drosophila melanogaster, Mutation Research/Genetic Toxicology 259 (1991) 21\u0026ndash;27. https://doi.org/10.1016/0165-1218(91)90105-U.\u003c/li\u003e\n\u003cli\u003eP.P. Kumar, H.P. Harish, Low Molecular Weight Chitosan (\u0026sim;20 kDa) protects acrylamide induced oxidative stress in D. melanogaster by restoring dopamine and KIF5B levels, Carbohydr Polym 222 (2019) 115005. https://doi.org/10.1016/j.carbpol.2019.115005.\u003c/li\u003e\n\u003cli\u003eN. Kahkeshani, S. Saeidnia, M. Abdollahi, Role of antioxidants and phytochemicals on acrylamide mitigation from food and reducing its toxicity, J Food Sci Technol 52 (2015) 3169\u0026ndash;3186. https://doi.org/10.1007/S13197-014-1558-5.\u003c/li\u003e\n\u003cli\u003eS. Dasari, M.S. Ganjayi, B. Meriga, Glutathione S-transferase is a good biomarker in acrylamide induced neurotoxicity and genotoxicity, Interdiscip Toxicol 11 (2018) 115. https://doi.org/10.2478/INTOX-2018-0007.\u003c/li\u003e\n\u003cli\u003eP. Poinsot, A. Penhoat, M. Mitchell, V. Sauvinet, L. Meiller, C. Louche-P\u0026eacute;lissier, E. Meugnier, M. Ruiz, M. Schwarzer, M.C. Michalski, F. Leulier, N. Peretti, Probiotic from human breast milk, Lactobacillus fermentum, promotes growth in animal model of chronic malnutrition, Pediatr Res (2020) 1\u0026ndash;8. https://doi.org/10.1038/s41390-020-0774-0.\u003c/li\u003e\n\u003cli\u003eS. Westfall, N. Lomis, S. Prakash, Ferulic Acid Produced by Lactobacillus fermentum Influences Developmental Growth Through a dTOR-Mediated Mechanism, Mol Biotechnol 61 (2019) 0. https://doi.org/10.1007/s12033-018-0119-y.\u003c/li\u003e\n\u003cli\u003eY.J. Jang, W.K. Kim, D.H. Han, K. Lee, G. Ko, Lactobacillus fermentum species ameliorate dextran sulfate sodium-induced colitis by regulating the immune response and altering gut microbiota, Gut Microbes 10 (2019) 696\u0026ndash;711. https://doi.org/10.1080/19490976.2019.1589281.\u003c/li\u003e\n\u003cli\u003eS.C.R. Thumu, P.M. Halami, In vivo safety assessment of Lactobacillus fermentum strains, evaluation of their cholesterol-lowering ability and intestinal microbial modulation, J Sci Food Agric 100 (2020) 705\u0026ndash;713. https://doi.org/10.1002/jsfa.10071.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Gut microbiota, Nanoencapsulation, Acrylamide, Bioactives, Chitosan, Lactobacillus","lastPublishedDoi":"10.21203/rs.3.rs-4780644/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4780644/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe novelty of this study lies in the development of an effective therapeutic agent using natural components—specifically, low molecular weight chitosan and \u003cem\u003eL. fermentum\u003c/em\u003e—utilizing the \u003cem\u003eDrosophila\u003c/em\u003e model. The design and formulation of chitosan-coated probiotic nanoparticles (CSP NPs) aim to enhance the bioavailability of probiotics in the gut, thereby improving their efficacy against ACR-induced toxicity. Nanoencapsulation, a vital domain of the medical nanotechnology field plays a key role in targeted drug delivery, bioavailability, multi-drug load delivery systems and synergistic treatment options. Chitosan, known for its non-toxic nature, offers additional benefits such as anti-inflammatory properties and immune system stimulation. \u003cem\u003eLactobacillus fermentum\u003c/em\u003e, incorporated for its cholesterol-lowering and potent immunomodulatory effects, also plays a significant role in influencing behavioural and developmental mechanisms in \u003cem\u003eDrosophila\u003c/em\u003e. The synergistic effect of chitosan and \u003cem\u003eL. fermentum\u003c/em\u003e ensures the stability and sustained release of microbial load and its secondary metabolites, facilitating prolonged exposure in the gut. This slow-release mechanism allows for an extended duration of action, effectively combating the detrimental effects of process-induced toxins like acrylamide. By optimizing bioavailability through nanoencapsulation, this study demonstrated the efficiency of the formulation in rescuing ACR-induced behavioural and biochemical deficits.\u003c/p\u003e","manuscriptTitle":"Chitosan-Coated Probiotic Nanoparticles Mitigate Acrylamide-Induced Toxicity in the Drosophila Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 09:18:46","doi":"10.21203/rs.3.rs-4780644/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-18T14:29:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-15T18:22:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-12T06:33:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-04T13:42:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138980072703565361934422812605256264322","date":"2024-08-02T09:12:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"108887439128421869096002210549522204954","date":"2024-08-01T17:54:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230340340110528621150521367439607798423","date":"2024-07-31T11:29:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255428616708882516790635963837336959930","date":"2024-07-30T22:11:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-30T16:17:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-30T16:13:59+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-26T14:03:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-26T13:18:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-22T09:18:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"345cc801-66a8-4403-8fb0-44b9f7dc9252","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36205117,"name":"Biological sciences/Biological techniques"},{"id":36205118,"name":"Biological sciences/Biotechnology"},{"id":36205119,"name":"Biological sciences/Developmental biology"},{"id":36205120,"name":"Biological sciences/Microbiology"},{"id":36205121,"name":"Earth and environmental sciences/Environmental sciences"},{"id":36205122,"name":"Health sciences/Medical research"},{"id":36205123,"name":"Health sciences/Risk factors"}],"tags":[],"updatedAt":"2024-09-16T16:08:59+00:00","versionOfRecord":{"articleIdentity":"rs-4780644","link":"https://doi.org/10.1038/s41598-024-72200-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-09-11 15:57:51","publishedOnDateReadable":"September 11th, 2024"},"versionCreatedAt":"2024-08-22 09:18:46","video":"","vorDoi":"10.1038/s41598-024-72200-w","vorDoiUrl":"https://doi.org/10.1038/s41598-024-72200-w","workflowStages":[]},"version":"v1","identity":"rs-4780644","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4780644","identity":"rs-4780644","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}