
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39261567
72200
10.1038/s41598-024-72200-w
Article
Chitosan-coated probiotic nanoparticles mitigate acrylamide-induced toxicity in the Drosophila model
Senthil Kumar Swetha
Sheik Mohideen Sahabudeen sahabuds@srmist.edu.in

grid.412742.6 0000 0004 0635 5080 Developmental Biology Lab, Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, 603203 Tamil Nadu India
11 9 2024
11 9 2024
2024
14 2118222 7 2024
4 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Acrylamide (ACR) with its extensive industrial applications is a classified occupational hazard toxin and carcinogenic compound. Its formation in fried potatoes, red meat and coffee during high-temperature cooking is a cause for consideration. The fabrication of chitosan-coated probiotic nanoparticles (CSP NPs) aims to enhance the bioavailability of probiotics in the gut, thereby improving their efficacy against ACR-induced toxicity in Drosophila melanogaster. 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. Our study exploited the nanoencapsulation technology to coat Lactobacillus fermentum (probiotic) with chitosan (prebiotic), both with substantial immunomodulatory effects, to ensure the stability and sustained release of microbial load and its secondary metabolites in the gut. The combination of pre-and probiotic components, called synbiotic formulations establishes the correlation between the gut microbiota and the overall well-being of an organism. Our study aimed to develop a potent synbiotic to alleviate the impacts of heat-processed dietary toxins that significantly influence behaviour, development, and survival. Our synbiotic co-treatment with ACR in fruit flies normalised neuro-behavioural, survival, redox status, and restored ovarian mitochondrial activity, contrasting with several physiological deficits observed in the ACR-treated model.

Keywords

Gut microbiota
Nanoencapsulation
Acrylamide
Bioactives
Chitosan
Lactobacillus
Subject terms

Biological techniques
Biotechnology
Developmental biology
Microbiology
Environmental sciences
Medical research
Risk factors
DST SERB INSPIREIF210062 Senthil Kumar Swetha issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Gut health has gained considerable attention recently 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 health1.

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 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 pathways2. 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 bioavailability3.

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 Bifidobacterium4. 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 mucosa5.

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 microbiota6,7. 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 absorption10,11. 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 200512,13. In India, the major source of acrylamide is the consumption of deep-fried food products ranging from 82.0 to 4245.6 µg/kg14. 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 haemoglobin15.

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 infertility16,17. 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 effects18–24. 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 receptors25. Storelli et al. also reported the influence of LAB on the developmental mechanisms in Drosophila under nutrient-scarce conditions26. 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.

Results

Characterisation of CSP NPs

Chitosan and chitosan encapsulated probiotic nanoparticles were analysed using a transmission electron microscope (TEM). The synthesised CS NPs without the probiotic load were small and spherical with an average size of 110 nm. The nanoencapsulation process of L. fermentum within chitosan NPs showed a significant increase in size (565 nm) and elongated morphology. The increase in size and morphological changes indicate the successful encapsulation of probiotics within the chitosan matrix. Figure 1 (CSP NPs) shows a rod-shaped bacterial cell tightly surrounded by small spherical chitosan particles that may act as a barrier to protect the probiotic load in acidic conditions. Furthermore, the TEM image of L. fermentum before encapsulation was observed to be 1000–1060 nm, to ensure effective nanoencapsulation the bacterial cells are sonicated to reduce the size and prevent aggregates. The fact that the encapsulated nanoparticles are 565 nm, significantly larger than the 110 nm CS NPs but smaller than the original 1060 nm probiotic cells, suggests that the encapsulation and lyophilization process may compress or compact the probiotic cells to some extent.Fig. 1 Transmission electron microscopy. Probiotic, chitosan, and chitosan-coated probiotic nanoparticles were imaged using the TEM technique.

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. Overall, high absolute zeta potential, nanoscale hydrodynamic diameter, and narrow size distribution range ensure the stability of the particles in suspensions. FTIR analysis detects the presence of functional bonds and peak shifts occur when interacting with other compounds. Chitosan comprises primarily O–H and N–H stretching, C–H stretching, amide bands, and C–O–C stretching among other peaks. The presence of these functional groups is characterised by 3320, 1451, 1576, and 989 cm−1 respectively28. Figure 3 illustrates the slight shift in the amide bonds characterised by peaks at 1535 cm−1 in CS and CSP NPs and can be related to the interaction between chitosan and TPP29. Similarly, the peak corresponding to the C–O stretch indicated a shift from 989 to 1004.47 cm−1 in CS NPs but was retained in CSP NPs. This can be explained by the glycosidic bonds present in polysaccharides, present in chitosan as well as the bacterial cell wall of Lactobacillus fermentum. With chitosan (CS) as a reference peak, we can confirm the presence of chitosan in the synthesised NPs and mild shifts in existing peaks could be linked to interactions between the bacterial components.Fig. 2 Zeta size distribution analysis. The size and particle charge of chitosan-coated probiotic nanoparticles were analysed using the zeta potential technique.

Fig. 3 Fourier transform infrared spectroscopy (FTIR). The presence of functional groups in standard chitosan and synthesised NPs (CS and CSP) were analysed to ensure purity.

Viability of synthesised CSP NPs

CSP NPs bacterial load viability was determined using the pour plate method as illustrated in Fig. 4. The synthesised nanoparticles indicated an average viability of 7.93 log CFU/ml 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 (SGF120 = 9.07 log CFU/ml; SIF180 = 7 log CFU/ml) compared to the free probiotic culture with SGF120 = 8 log CFU/ml and SIF180 = 5.87 log CFU/ml.Fig. 4 Viability of CSP NPs. The viability of chitosan-coated L. fermentum 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.

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. 5A–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. Baseline control groups like CS (59 days; 67.1 mm/min; 85.92%), CS NPs (39 days; 62.03 mm/min; 86.7%) and probiotics (55 days; 70.07 mm/min; 95.55%) treatments were also assessed for their effect on fly survival and behaviour parameters. These controls did not indicate any negative impact on Drosophila lifespan and locomotor functions as evidenced by the data.Fig. 5 Survival and Behavioural Parameters of D.mel. The effects of CSP NP were tested against ACR-induced survival (A) and behavioural deficits (B, C) using the fruit fly model.

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. Following the behaviour analysis, baseline control groups (CS, CS NPs and probiotics) along with control, ACR and CSPA groups were tested for fluctuations in the ROS levels. As illustrated in Fig. 6A, there were no significant variations in ROS levels between the control and baseline control groups. The observed ROS levels are commonly attributed to normal cellular activity, however, a marked increase in ROS intensity was noticed in flies treated with ACR thereby indicating the presence of xenobiotic-induced oxidative stress and activation of antioxidant mechanisms. Owing to the non-toxic nature of the baseline control groups (CS, CS NPs and probiotics) as evidenced by long survival period, high locomotor function at larval and fly stages and no significant induction of ROS levels, further experiments were performed with control, ACR, CSP NPs and CSPA treatment groups.Fig. 6 Biochemical Factors of D.mel. The 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.

ACR-treated flies exhibited increased ROS, SOD and GST activities as depicted in Fig. 6A–C respectively. Furthermore, ACR exposure indicated a negative influence on protein levels (Fig. 6D). 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.

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. 7. 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.Fig. 7 Mitochondrial Membrane Potential in D.mel ovaries. TMRE 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.

Discussion

Heat-processed contaminants (HPC) are compounds formed during the thermal processing of food items, such as frying, baking, and grilling, to enhance texture and flavour. These compounds, such as acrylamide, furans, bisphenol-A, and chloropropanols, are found in various baked and fried food products. While health and safety organizations have established strict regulations on HPC concentrations in industry-manufactured food products, HPCs are hidden dangers that we are exposed to through our daily dietary intake30. ACR, formed through the Maillard reaction, is more prominent in fried potatoes, roasted coffee, fried red meat, and baked confectionaries. ACR has been widely studied and several therapeutic formulations aiming to curb the redox stress to control ACR-induced toxic effects have been reported using varied animal models19,20,31–34. However, it is important to understand the involvement of the gut when analysing food-based toxicological studies. Animal studies have reported ACR-induced cognitive impairments via the gut-brain axis, intestinal barrier damage, gut microbial dysbiosis, bile acid metabolism challenges, and higher cholic acid levels35–37. Additionally, ACR was observed to cause changes in intestinal morphology, disrupt the enteric nervous system, increase apoptotic markers in the GI tract, aggravate mucosal inflammation and damage tight junctions36,38–40. Therefore, it is crucial to formulate dietary supplements that can reduce, restore, or protect our physiological systems from these food-processed toxins.

In the extensive field of food-based research, our chitosan-coated probiotic nanoparticles emphasise the benefits of using simple, naturally accessible dietary supplements to mitigate the detrimental effects of dietary contaminants. The notion of 'gut-brain interaction' has recently gained popularity. Many people have recognised the importance of the gastrointestinal system, sometimes known as the second brain of the human body, and its impact on general well-being. The gut microbiota is an important regulator of the enteric system and contributes to gut-brain crosstalk, which influences mental, metabolic, and immunological health4. This study uses chitosan, a natural biopolymer, to create sustained-release probiotic supplements. Probiotics like Bifidobacterium and Lactobacillus are easily sourced from milk, curd, and fermented products. Studies show recovery of gut dysbiosis, colonic inflammation, and improved microbiota diversity. Combining probiotic supplements with prebiotics like inulin, chitosan, and arabinoxylan can significantly modulate gut microbiota, enhancing the accumulation of beneficial gut microbiota and reducing harmful colonic bacteria4.

Lactobacillus fermentum is a probiotic strain that can enhance gut health by strengthening the intestinal barrier, amplifying the gut response, reducing inflammation, and modulating the immune system. It increased mucus production, stimulated the release of anti-microbial peptides, and produced secretory immunoglobulin A. It was also reported to maintain the gut epithelium barrier and colon tissue integrity41,42. L. fermentum can also reduce inflammation-associated tumorigenesis and the release of pro-inflammatory cytokines. It also supports the immune system by increasing epithelial barrier integrity. Additionally, L. fermentum can improve gut microbiota composition, reduce blood cholesterol, and increase antioxidant enzyme activity43–46. Chitosan is a non-plant-derived prebiotic that can enhance gut flora, boost probiotic activity, and inhibit pathogens. Chitosan has been permitted as food additives and active ingredients in several countries, including the European Union, Australia, New Zealand, and China. They are also classified as Generally Recognised as Safe (GRAS) by the United States Food and Drug Administration. Researchers claim that chitosan and its derivatives can improve animal gastrointestinal health, growth performance, productivity, nutrient absorption, and immunity47.

We fabricated CSP NPs by coating the Lactobacillus fermentum culture with low molecular weight chitosan in the presence of TPP. Gunasangakaran et al. 2022 reported the synthesis and characterisation of chitosan-probiotic nanoparticles with chitosan and TPP concentrations as 0.05 mg/ml and 0.1 mg/ml respectively. The developed synbiotic product showed a high surface charge and polydispersity index with a size range of 360–500 nm via SEM imaging48. Ebrahimnejad et al.49 prepared synbiotic NPs with chitosan concentrations ranging from 0.05 to 0.5 mg/ml with 0.1 mg/ml TPP to assess the difference in the nano-formulation size and bacterial load capacity. The study reported that with the increase in chitosan concentration, the bacterial encapsulation greatly decreased. The highest encapsulation load was reported in nano-formulation synthesized with 0.05 mg/ml chitosan49. Another study by Alkushi et al.50 reported the preparation of NPs with 0.4% chitosan and 20 g/L alginate as an effective therapeutic formulation against colon inflammation with a size range of 21–25 nm50. Multiple other synbiotic nano-formulations have been synthesised and characterised to demonstrate effective delivery of the probiotic load51,52. However, to the best of the authors’ knowledge, no other studies have reported the effectiveness of synbiotic nano-formulations against HPCs.

Based on data reported by Ebrahimnejad et al.49, the present study utilized 0.5% chitosan and 1 mg/ml TPP to synthesise the synbiotic nano-formulation, CSP NPs. Our TEM images of free probiotic culture, CS NPs and CSP NPs indicated the effect of nanoencapsulation wherein the 1µm size of free probiotics and 110 nm CS NPs transformed to 400–500 nm CSP NPs. Although the NPs size reported by existing studies is relatively smaller than the present formulation, it is important to note that our CSP NPs hold thrice the amount of probiotic load (3 ml; 8 log CFU/ml) comparatively49,50,53. Zeta potential reflects the surface charge of the nanoparticles, which is a critical factor in determining their stability in suspension. A zeta potential of 4.38 mV suggests that the nanoparticles have a moderate positive surface charge. This level of zeta potential indicates that the nanoparticles may have significant stability in suspension, with a low risk of aggregation over time54.

Additionally, PDI measures the distribution of particle sizes within a sample, with values ranging from 0 to 1. A PDI of 0.427 suggests that the particle size distribution is low polydispersity. This means that while the particles are not all the same size, there is not an extreme variation in size which conforms to moderate stability and low aggregation rates in suspensions55. Additionally, the nanoscale ensures potential permeability across the physiological barriers as the intestinal endothelial cells have a 1.2–2 µm intercellular distance56. Furthermore, FTIR data of CSP NPs illustrated the presence of chitosan characteristic peaks namely, O–H and N–H stretching, C–H stretching, amide bands, and C–O–C stretching. Slight shifts or changes in intensity compared to the spectrum of pure chitosan observed might occur due to the nanoparticle formulation process, interactions, or modifications. These characteristic peaks confirm the presence and structure of chitosan within the nanoparticles29. Our in vitro experiments also demonstrated the stability and viability of CSP NPs through repeated storage viability analysis at room temperature and under simulated gastrointestinal conditions. The storage viability demonstrated substantial probiotic viability after 49 days indicating a higher shelf life of CSP NPs when stored as lyophilised powder. Additionally, the main aspect of the encapsulation process is to offer considerable protection for the probiotic load for 6 h (digestion in stomach = 2 h; in intestine = 4 h) against acidic pH in the stomach and bile salts in the intestine. Subsequently, our simulated GI studies demonstrated significant viable probiotic load post-6-h thereby demonstrating the efficiency of chitosan nano-coating on the probiotic load and slow-release profile57.

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. Baseline groups such as probiotics, CS and CS NPs treatments were also analysed to rule out their influence on the fruit fly model. Behavioural and ROS levels indicated no noteworthy influence of the baseline groups on the fly physiology as evidenced by no significant variance compared to the control group.

The lifespan and behavioural parameters in the fruit fly model are vital life-history traits and are generally very closely associated with the interaction between environmental cues and neuromuscular coordination. While ACR treatment shortened the lifespan and reduced larval crawling and adult climbing behaviour, the CSPA treatment did not exhibit any alterations compared to the control group. The influence of synbiotics in suppressing the impact of ACR can be observed. However, to the best of the author’s knowledge, the present study serves as the first to analyse the effects of synbiotic formulation against ACR in the D.mel model. We can corroborate the present results with Gómez et al.58 who reported the impact of commensal microbiota on the behaviour of the fruit fly model. The study explored the effects of axenic (no gut microbiota) and probiotic-treated fly models and described the positive influence of probiotic treatment on fly development and behaviour58.

Furthermore, ACR treatment groups exhibited increased ROS and antioxidant enzyme activity (SOD and GST) while these levels were on par with control groups in the flies exposed to CSPA treatment groups. While ROS production is essential for cellular functioning, homeostasis sets in motion a positive feedback loop that effectively maintains a balance between redox stress levels and antioxidant mechanisms for healthy physiology. However, xenobiotic exposure disrupts this balance causing an increase in stress markers and subsequent activation of the antioxidant mechanisms. The normalised levels of ROS, SOD and GST in CSPA treatment imply a balance between the redox factors, once again demonstrating the protective influence of synbiotics at the cellular level. A similar study by Westfall et al.59, demonstrated the influence of synbiotics in normalising the redox stress factors in aged flies and correlated the data with cognitive impairments observed in Alzheimer’s disease.

Finally, 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, CSPA-treated ovaries exhibited highly active mitochondrial pools. We believe in addition to the synbiotic formulation, the probiotic strain used in this study—L. fermentum and its metabolite ferulic acid play a vital role in protecting the fly model against ACR-induced mitochondrial dysfunction and subsequent developmental toxicity. As reported by Westfall et al.60, the probiotic strain and its metabolite greatly influence dTOR and IlS signalling pathways. These pathways mainly target nutrient-mediated development processes, furthermore correlating this mechanism in humans implies its effect on hormonal regulations, puberty-related changes, obesity, diabetes and in some instances neurodegenerative disorders60.

Overall, the CSPA (CSP NPs + ACR) treatment demonstrated no significant toxicity at adult and larval stages within the fruit fly model. 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 potentially 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 function60–63. Therefore, we postulate that by maintaining gut integrity and reducing intestinal permeability, they prevent systemic inflammation and the translocation of toxins (like ACR metabolites—glycidamide) into circulation.

With positive outcomes of CSP NPs in the fruit fly model, it is imperative to comprehend the differences in the commercially available probiotic and synbiotic formulations. The strain origin and CFU count are information generally withheld as a part of trade secrets, therefore we compiled available literature relevant to commercial probiotic/synbiotic products. Stasiak-Różańska et al.57 compared five commercially available probiotic and synbiotic formulations in Poland, including Lactobacillus rhamnosus GG, Bifidobacterium BB-12 + FOS, Lactobacillus casei + inulin, Lactobacillus acidophilus + inulin, and Lactobacillus plantarum. The study tested these strains under simulated gastrointestinal conditions using a food matrix. Lactobacillus plantarum proved to be the most resilient, showing significant growth in both gastric and intestinal environments, while the other strains, even those with prebiotics like FOS and inulin, were less resistant57.

Another study by Naissinger da Silva et al.64, analysed 11 commercial formulations (lyophilized/capsules) segregated as industrial (IP), commercial (CP), manipulated (MP) and fermented probiotics (FM). It highlighted the IP formulations demonstrated high initial probiotic concentrations (8 log CFU g−1) and maintained good viability under simulated GI studies. Additionally, synbiotic formulations maintained high initial concentrations (around 10 log CFU g−1) and acceptable viability after digestion (around 7 log CFU g−1), suggesting they could effectively colonize the intestines64.

10 branded formulations (lyophilized/capsules) marketed in Italy were tested for their efficiency in vitro. Lactobacillus species generally resist acidic conditions, but this tolerance varies by species and strain. Single-strain Lactobacillus formulations showed expected resistance, while polymicrobial formulations like Yovis and VSL3 were also resilient, likely due to the complexity of their microbial consortia. In the intestine, probiotics face alkaline conditions and bile salts, which are more harmful to Gram-positive bacteria. Using artificial intestinal fluids to simulate gut conditions, seven probiotic formulations showed significant reductions in viable organisms, but Lactoflorene Plus, Yovis, and VSL3 were the most resistant to intestinal juice65.

Moreover, multiple other researches across the world have been conducted to test the viability and strain concentrations of the commercially produced probiotic/synbiotic formulations in India and Pakistan (5 formulations)66, the Republic of the Philippines and the Republic of Korea (10 formulations)67, United States and Canada (182 formulations)68, India (20 formulations)69. These reports have emphasised the need for manufacturers to ensure that their products maintain sufficient probiotic viability throughout shelf life and digestion, as required by regulatory standards.

In conclusion, the present study stands as preliminary research to formulate and test the efficacy of the synthesised synbiotic formulation prepared with 0.5% chitosan and 8 log CFU/ml L. fermentum culture against heat-processed contaminants like acrylamide (oral administration). Our data highlights the effectiveness of synthesised chitosan-coated L. fermentum nanoparticles against ACR-induced toxicity. Additionally, these 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. Additionally, although a plethora of commercial probiotic/synbiotic formulations are available in the market, our formulation differs in key aspects such as—nanoscale product, mono-strain formulation, reliable prebiotic coating with the addition of cryoprotectants, high initial probiotic load, stable shelf life, and resilient viability under simulated GI conditions. Despite the beneficial aspects of the synthesised nano-formulation, we understand the need for sequencing of strains and colonisation studies in higher model systems. The future prospects of our study include a detailed investigation into the protective mechanisms exhibited by the synbiotic formulation via gene and protein expression estimation in higher-order model systems before proceeding to clinical studies.

Materials and methods

Chemicals and cultures

Acrylamide (3 × Crystalline), low molecular weight chitosan (34.5 m Pas, 90.1% degree of deacetylation, ⁓ 50 k Da), 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.

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 modifications48. 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 1 mg/ml sodium tripolyphosphate (TPP) was added dropwise with constant stirring for 2 h at room temperature. The mixture was then sonicated for 30 min and centrifuged at 10,000 rpm for 15 min. The pellet was washed thrice with distilled water, then suspended in 10% sucrose and 1% glycerol solution (used as protectants) and stored at − 20 °C. The frozen samples were then lyophilized at − 40 °C and the freeze-dried product was stored at 4 °C for long-term storage. The preparation of chitosan nanoparticles (CS NPs) followed a similar method excluding the addition of the probiotic culture. The overall yield for the above-mentioned process was 220 mg and 370 mg for CSP and CS NPs respectively.

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 synthesised nanoparticles were suspended in distilled water and coated on the copper grid for further imaging. To analyse the purity of the synthesised nanoparticles, the samples were subjected to Fourier Transform Infrared Spectroscopy (FTIR). The nanoparticle samples in powder form were loaded into the FTIR instrument (Shimadzu, IR tracer 100, Japan) and analysed using ATR mode between 4000 and 400 cm⁻1. A standard chitosan sample was used as a reference to ensure the purity of the synthesised NPs.

The surface charge and particle size of CSP NPs in an aqueous system were then estimated using Malvern/Nano ZS-90 Zeta Sizer. The samples were dispersed in distilled water at 25 °C and suspensions were loaded into the Zeta Sizer for size analysis using dynamic light scattering method. The fluctuations in light scattering intensity caused by the movement of the nanoparticles are used to calculate the hydrodynamic diameter of the particles, which reflects their size distribution in the suspension. Additionally, the electrophoretic light scattering method was employed to measure the zeta potential, which is the electrical potential at the boundary layer surrounding the particles in a suspension. This potential is indicative of the surface charge of the particles and influences their suspension stability.

Viability and stability analysis of CSP NPs

The storage viability of CSP NPs at room temperature was estimated by suspending 1 g of probiotics nanoparticles in 0.01% sodium citrate solution (pH 6) for 10 min. The suspension was then serially diluted and plated on MRS agar plates for overnight incubation. The observed viable colonies were enumerated as log CFU/ml for statistical analysis. The above-mentioned method was repeated every 7th day to establish the storage stability of the synthesised formulation49.

The viability and stability of CSP NPs under simulated gastrointestinal conditions were analysed by suspending 1 g of synthesised nanoparticles and 1 ml of free probiotic culture in a 10 ml simulated gastric fluid (3 mg/ml pepsin; pH 2 adjusted with 1M HCl) with constant stirring on a magnetic stirrer. The release and viability profile of the nano-encapsulated probiotics were estimated by plating 1 ml of the mixture at 0, 60 and 120 h. Then, the nanoparticles and free suspension were pelleted (6000 g; 5 min) and incubated in 9 ml of simulated intestinal fluid (3 mg/ml pancreatin and 1% bile salt; pH 8). The subsequent stability profile was estimated by plating isolates on MRS agar plates post 0, 60, 120 and 180 h of incubation at 37 °C. The viable colonies were enumerated as log CFU/ml for statistical significance50,70.

Drosophila rearing and maintenance

The Drosophila Oregon K wild-type strain was maintained at 25 ± 2 °C and 60% humidity under a 12 h 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. As baseline control groups—0.5% chitosan (CS), 10 µg/ml CS NPs and 3 ml of L. fermentum culture (8 log CFU/ml) treatment groups were analysed to comprehend the impact on behaviour and ROS levels. 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.

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 h. 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 1 mm 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 s71.

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 study72,73. 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.

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 min in the dark, washed with PBS, and observed using a Leica DM6 fluorescent microscope.

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.

Acknowledgements

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.

Author contributions

S.S.—Conceptualization, Methodology, Data curation, Writing—original draft, Writing—review and editing, Software, Validation; S.S.M.—Conceptualization, Methodology, Validation, Writing—review and editing and Supervision.

Data availability

All data sets used and/or generated in this work are obtainable from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Pedroza Matute S Iyavoo S Exploring the gut microbiota: Lifestyle choices, disease associations, and personal genomics Front. Nutr. 2023 10 1225120 10.3389/fnut.2023.1225120 37867494
Pedroza Matute, S. & Iyavoo, S. Exploring the gut microbiota: Lifestyle choices, disease associations, and personal genomics. Front. Nutr. 10, 1225120 (2023).37867494 10.3389/fnut.2023.1225120
2. Rein MJ Bioavailability of bioactive food compounds: A challenging journey to bioefficacy Br. J. Clin. Pharmacol. 2013 75 588 10.1111/j.1365-2125.2012.04425.x 22897361
Rein, M. J. et al. Bioavailability of bioactive food compounds: A challenging journey to bioefficacy. Br. J. Clin. Pharmacol. 75, 588 (2013).22897361 10.1111/j.1365-2125.2012.04425.x
3. Zhang T Cytoprotection of probiotics by nanoencapsulation for advanced functions Trends Food Sci. Technol. 2023 142 104227 10.1016/j.tifs.2023.104227
Zhang, T. et al. Cytoprotection of probiotics by nanoencapsulation for advanced functions. Trends Food Sci. Technol. 142, 104227 (2023).10.1016/j.tifs.2023.104227
4. Bauer-Estrada K Sandoval-Cuellar C Rojas-Muñoz Y Quintanilla-Carvajal MX The modulatory effect of encapsulated bioactives and probiotics on gut microbiota: Improving health status through functional food Food Funct. 2023 14 32 55 10.1039/D2FO02723B 36515144
Bauer-Estrada, K., Sandoval-Cuellar, C., Rojas-Muñoz, Y. & Quintanilla-Carvajal, M. X. The modulatory effect of encapsulated bioactives and probiotics on gut microbiota: Improving health status through functional food. Food Funct. 14, 32–55 (2023).36515144 10.1039/D2FO02723B
5. Saulnier DM Spinler JK Gibson GR Versalovic J Mechanisms of probiosis and prebiosis: Considerations for enhanced functional foods Curr. Opin. Biotechnol. 2009 20 135 10.1016/j.copbio.2009.01.002 19243931
Saulnier, D. M., Spinler, J. K., Gibson, G. R. & Versalovic, J. Mechanisms of probiosis and prebiosis: Considerations for enhanced functional foods. Curr. Opin. Biotechnol. 20, 135 (2009).19243931 10.1016/j.copbio.2009.01.002
6. Hemarajata P Versalovic J Effects of probiotics on gut microbiota: Mechanisms of intestinal immunomodulation and neuromodulation Therap. Adv. Gastroenterol. 2013 6 39 10.1177/1756283X12459294 23320049
Hemarajata, P. & Versalovic, J. Effects of probiotics on gut microbiota: Mechanisms of intestinal immunomodulation and neuromodulation. Therap. Adv. Gastroenterol. 6, 39 (2013).23320049 10.1177/1756283X12459294
7. Goodoory VC Efficacy of probiotics in irritable bowel syndrome: Systematic review and meta-analysis Gastroenterology 2023 165 1206 1218 10.1053/j.gastro.2023.07.018 37541528
Goodoory, V. C. et al. Efficacy of probiotics in irritable bowel syndrome: Systematic review and meta-analysis. Gastroenterology 165, 1206–1218 (2023).37541528 10.1053/j.gastro.2023.07.018
8. Joseph N Gut microbiota and short-chain fatty acids (SCFAs) profiles of normal and overweight school children in Selangor after probiotics administration J. Funct. Foods 2019 57 103 111 10.1016/j.jff.2019.03.042
Joseph, N. et al. Gut microbiota and short-chain fatty acids (SCFAs) profiles of normal and overweight school children in Selangor after probiotics administration. J. Funct. Foods 57, 103–111 (2019).10.1016/j.jff.2019.03.042
9. Markowiak-Kopeć P Śliżewska K The effect of probiotics on the production of short-chain fatty acids by human intestinal microbiome Nutrients 2020 12 1107 10.3390/nu12041107 32316181
Markowiak-Kopeć, P. & Śliżewska, K. The effect of probiotics on the production of short-chain fatty acids by human intestinal microbiome. Nutrients 12, 1107 (2020).32316181 10.3390/nu12041107
10. Hagmar L Health effects of occupational exposure to acrylamide using hemoglobin adducts as biomarkers of internal dose Scand J Work Environ Health 2001 27 219 226 10.5271/sjweh.608 11560335
Hagmar, L. et al. Health effects of occupational exposure to acrylamide using hemoglobin adducts as biomarkers of internal dose. Scand J Work Environ Health 27, 219–226 (2001).11560335 10.5271/sjweh.608
11. Koszucka A Nowak A Nowak I Motyl I Acrylamide in human diet, its metabolism, toxicity, inactivation and the associated European Union legal regulations in food industry Crit. Rev. Food Sci. Nutr. 2020 60 1677 1692 10.1080/10408398.2019.1588222 30907623
Koszucka, A., Nowak, A., Nowak, I. & Motyl, I. Acrylamide in human diet, its metabolism, toxicity, inactivation and the associated European Union legal regulations in food industry. Crit. Rev. Food Sci. Nutr. 60, 1677–1692. 10.1080/10408398.2019.1588222 (2020).30907623 10.1080/10408398.2019.1588222
12. Dolan LC Matulka RA Burdock GA Naturally occurring food toxins Toxins (Basel) 2010 2 2289 10.3390/toxins2092289 22069686
Dolan, L. C., Matulka, R. A. & Burdock, G. A. Naturally occurring food toxins. Toxins (Basel) 2, 2289 (2010).22069686 10.3390/toxins2092289
13. Matoso V Bargi-Souza P Ivanski F Romano MA Romano RM Acrylamide: A review about its toxic effects in the light of developmental origin of health and disease (DOHaD) concept Food Chem. 2019 283 422 430 10.1016/j.foodchem.2019.01.054 30722893
Matoso, V., Bargi-Souza, P., Ivanski, F., Romano, M. A. & Romano, R. M. Acrylamide: A review about its toxic effects in the light of developmental origin of health and disease (DOHaD) concept. Food Chem. 283, 422–430. 10.1016/j.foodchem.2019.01.054 (2019).30722893 10.1016/j.foodchem.2019.01.054
14. Shamla L Nisha P Acrylamide in deep-fried snacks of India Food Addit. Contam. Part B Surveill. 2014 7 220 225 10.1080/19393210.2014.894141 25029406
Shamla, L. & Nisha, P. Acrylamide in deep-fried snacks of India. Food Addit. Contam. Part B Surveill. 7, 220–225 (2014).25029406 10.1080/19393210.2014.894141
15. CDC, Nceh, DLS & Od. Fourth National Report on Human Exposure to Environmental Chemicals Update.
16. Lai SM Toxic effect of acrylamide on the development of hippocampal neurons of weaning rats Neural Regen Res 2017 12 1648 1654 10.4103/1673-5374.217345 29171430
Lai, S. M. et al. Toxic effect of acrylamide on the development of hippocampal neurons of weaning rats. Neural Regen Res 12, 1648–1654 (2017).29171430 10.4103/1673-5374.217345
17. Stagi S 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. 2020 46 165 10.1186/s13052-020-00931-3 33148304
Stagi, S. et al. 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, 165 (2020).33148304 10.1186/s13052-020-00931-3
18. Wang H Reproductive toxicity of acrylamide-treated male rats Reprod. Toxicol. 2010 29 225 230 10.1016/j.reprotox.2009.11.002 19903525
Wang, H. et al. Reproductive toxicity of acrylamide-treated male rats. Reprod. Toxicol. 29, 225–230 (2010).19903525 10.1016/j.reprotox.2009.11.002
19. Huang Z Mitochondrial dysfunction promotes the necroptosis of Purkinje cells in the cerebellum of acrylamide-exposed rats Food Chem. Toxicol. 2023 171 113522 10.1016/j.fct.2022.113522 36417989
Huang, Z. et al. Mitochondrial dysfunction promotes the necroptosis of Purkinje cells in the cerebellum of acrylamide-exposed rats. Food Chem. Toxicol. 171, 113522 (2023).36417989 10.1016/j.fct.2022.113522
20. Yilmaz BO Yildizbayrak N Aydin Y Erkan M Evidence of acrylamide- and glycidamide-induced oxidative stress and apoptosis in Leydig and Sertoli cells Hum. Exp. Toxicol. 2017 36 1225 1235 10.1177/0960327116686818 28067054
Yilmaz, B. O., Yildizbayrak, N., Aydin, Y. & Erkan, M. Evidence of acrylamide- and glycidamide-induced oxidative stress and apoptosis in Leydig and Sertoli cells. Hum. Exp. Toxicol. 36, 1225–1235 (2017).28067054 10.1177/0960327116686818
21. Huang M Jiao J Wang J Xia Z Zhang Y Characterization of acrylamide-induced oxidative stress and cardiovascular toxicity in zebrafish embryos J. Hazard Mater. 2018 347 451 460 10.1016/j.jhazmat.2018.01.016 29353190
Huang, M., Jiao, J., Wang, J., Xia, Z. & Zhang, Y. Characterization of acrylamide-induced oxidative stress and cardiovascular toxicity in zebrafish embryos. J. Hazard Mater. 347, 451–460 (2018).29353190 10.1016/j.jhazmat.2018.01.016
22. Zhao S Protective effect of Lactobacillus plantarum ATCC8014 on acrylamide-induced oxidative damage in rats Appl. Biol. Chem. 2020 63 1 4 10.1186/s13765-020-00527-9
Zhao, S. et al. Protective effect of Lactobacillus plantarum ATCC8014 on acrylamide-induced oxidative damage in rats. Appl. Biol. Chem. 63, 1–4 (2020).10.1186/s13765-020-00527-9
23. Alkhalaf MI Diosmin protects against acrylamide-induced toxicity in rats: Roles of oxidative stress and inflammation J. King Saud. Univ. Sci. 2020 32 1510 1515 10.1016/j.jksus.2019.12.005
Alkhalaf, M. I. Diosmin protects against acrylamide-induced toxicity in rats: Roles of oxidative stress and inflammation. J. King Saud. Univ. Sci. 32, 1510–1515 (2020).10.1016/j.jksus.2019.12.005
24. Alturfan AA Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats Mol. Biol. Rep. 2012 39 4589 4596 10.1007/s11033-011-1249-5 21947844
Alturfan, A. A. et al. Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats. Mol. Biol. Rep. 39, 4589–4596 (2012).21947844 10.1007/s11033-011-1249-5
25. Westfall S Iqbal U Sebastian M Pasinetti GM Gut Microbiota Mediated Allostasis Prevents Stress-Induced Neuroinflammatory Risk Factors of Alzheimer’s Disease. Progress in Molecular Biology and Translational Science 2019 Elsevier
Westfall, S., Iqbal, U., Sebastian, M. & Pasinetti, G. M. Gut Microbiota Mediated Allostasis Prevents Stress-Induced Neuroinflammatory Risk Factors of Alzheimer’s Disease. Progress in Molecular Biology and Translational Science Vol. 168 (Elsevier, 2019).
26. Storelli G Lactobacillus plantarum promotes drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing Cell Metab. 2011 14 403 414 10.1016/j.cmet.2011.07.012 21907145
Storelli, G. et al. Lactobacillus plantarum promotes drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing. Cell Metab. 14, 403–414 (2011).21907145 10.1016/j.cmet.2011.07.012
27. Rivas-Jimenez L Evaluation of acrylamide-removing properties of two Lactobacillus strains under simulated gastrointestinal conditions using a dynamic system Microbiol. Res. 2016 190 19 26 10.1016/j.micres.2016.04.016 27393995
Rivas-Jimenez, L. et al. Evaluation of acrylamide-removing properties of two Lactobacillus strains under simulated gastrointestinal conditions using a dynamic system. Microbiol. Res. 190, 19–26 (2016).27393995 10.1016/j.micres.2016.04.016
28. Joseph JJ Sangeetha D Gomathi T Sunitinib loaded chitosan nanoparticles formulation and its evaluation Int. J. Biol. Macromol. 2016 82 952 958 10.1016/j.ijbiomac.2015.10.079 26522243
Joseph, J. J., Sangeetha, D. & Gomathi, T. Sunitinib loaded chitosan nanoparticles formulation and its evaluation. Int. J. Biol. Macromol. 82, 952–958 (2016).26522243 10.1016/j.ijbiomac.2015.10.079
29. Ahmad N Khan MR Palanisamy S Mohandoss S Anticancer drug-loaded chitosan nanoparticles for in vitro release, promoting antibacterial and anticancer activities Polymers (Basel) 2023 15 3925 10.3390/polym15193925 37835972
Ahmad, N., Khan, M. R., Palanisamy, S. & Mohandoss, S. Anticancer drug-loaded chitosan nanoparticles for in vitro release, promoting antibacterial and anticancer activities. Polymers (Basel) 15, 3925 (2023).37835972 10.3390/polym15193925
30. Mogol BA Gökmen V Thermal process contaminants: Acrylamide, chloropropanols and furan Curr. Opin. Food Sci. 2016 7 86 92 10.1016/j.cofs.2016.01.005
Mogol, B. A. & Gökmen, V. Thermal process contaminants: Acrylamide, chloropropanols and furan. Curr. Opin. Food Sci. 7, 86–92 (2016).10.1016/j.cofs.2016.01.005
31. Yousef MI El-Demerdash FM Acrylamide-induced oxidative stress and biochemical perturbations in rats Toxicology 2006 219 133 141 10.1016/j.tox.2005.11.008 16343728
Yousef, M. I. & El-Demerdash, F. M. Acrylamide-induced oxidative stress and biochemical perturbations in rats. Toxicology 219, 133–141 (2006).16343728 10.1016/j.tox.2005.11.008
32. Khiralla G Elhariry H Selim SM Chitosan-stabilized selenium nanoparticles attenuate acrylamide-induced brain injury in rats J. Food Biochem. 2020 44 e13413 10.1111/jfbc.13413 32748421
Khiralla, G., Elhariry, H. & Selim, S. M. Chitosan-stabilized selenium nanoparticles attenuate acrylamide-induced brain injury in rats. J. Food Biochem. 44, e13413 (2020).32748421 10.1111/jfbc.13413
33. Singh MP Jakhar R Kang SC BiotransformaMorin hydrate attenuates the acrylamide-induced imbalance in antioxidant enzymes in a murine model Int. J. Mol. Med. 2015 36 992 1000 10.3892/ijmm.2015.2306 26252199
Singh, M. P., Jakhar, R. & Kang, S. C. BiotransformaMorin hydrate attenuates the acrylamide-induced imbalance in antioxidant enzymes in a murine model. Int. J. Mol. Med. 36, 992–1000 (2015).26252199 10.3892/ijmm.2015.2306
34. Albalawi A Carnosic acid attenuates acrylamide-induced retinal toxicity in zebrafish embryos Exp. Eye Res. 2018 175 103 114 10.1016/j.exer.2018.06.018 29928899
Albalawi, A. et al. Carnosic acid attenuates acrylamide-induced retinal toxicity in zebrafish embryos. Exp. Eye Res. 175, 103–114 (2018).29928899 10.1016/j.exer.2018.06.018
35. Tan X Acrylamide aggravates cognitive deficits at night period via the gut–brain axis by reprogramming the brain circadian clock Arch Toxicol. 2019 93 467 486 10.1007/s00204-018-2340-7 30374679
Tan, X. et al. Acrylamide aggravates cognitive deficits at night period via the gut–brain axis by reprogramming the brain circadian clock. Arch Toxicol. 93, 467–486 (2019).30374679 10.1007/s00204-018-2340-7
36. Yue Z Acrylamide induced glucose metabolism disorder in rats involves gut microbiota dysbiosis and changed bile acids metabolism Food Res. Int. 2022 157 111405 10.1016/j.foodres.2022.111405 35761659
Yue, Z. et al. Acrylamide induced glucose metabolism disorder in rats involves gut microbiota dysbiosis and changed bile acids metabolism. Food Res. Int. 157, 111405 (2022).35761659 10.1016/j.foodres.2022.111405
37. Muszyński S Maternal acrylamide exposure changes intestinal epithelium, immunolocalization of leptin and ghrelin and their receptors, and gut barrier in weaned offspring Sci. Rep. 2023 2023 13 1 16
Muszyński, S. et al. Maternal acrylamide exposure changes intestinal epithelium, immunolocalization of leptin and ghrelin and their receptors, and gut barrier in weaned offspring. Sci. Rep. 2023(13), 1–16 (2023).
38. Schabacker J Schwend T Wink M Reduction of acrylamide uptake by dietary proteins in a Caco-2 gut model J. Agric. Food Chem. 2004 52 4021 4025 10.1021/jf035238w 15186132
Schabacker, J., Schwend, T. & Wink, M. Reduction of acrylamide uptake by dietary proteins in a Caco-2 gut model. J. Agric. Food Chem. 52, 4021–4025 (2004).15186132 10.1021/jf035238w
39. Amirshahrokhi K Acrylamide exposure aggravates the development of ulcerative colitis in mice through activation of NF-κB, inflammatory cytokines, iNOS, and oxidative stress Iran J. Basic Med. Sci. 2021 24 312 33995942
Amirshahrokhi, K. Acrylamide exposure aggravates the development of ulcerative colitis in mice through activation of NF-κB, inflammatory cytokines, iNOS, and oxidative stress. Iran J. Basic Med. Sci. 24, 312 (2021).33995942
40. Brownlee IA Uranga JA Palus K Dietary exposure to acrylamide has negative effects on the gastrointestinal tract: A review Nutrients 2024 16 2032 10.3390/nu16132032 38999779
Brownlee, I. A., Uranga, J. A. & Palus, K. Dietary exposure to acrylamide has negative effects on the gastrointestinal tract: A review. Nutrients 16, 2032 (2024).38999779 10.3390/nu16132032
41. Zhao Y Lactobacillus fermentum and its potential immunomodulatory properties J. Funct. Foods 2019 56 21 32 10.1016/j.jff.2019.02.044
Zhao, Y. et al. Lactobacillus fermentum and its potential immunomodulatory properties. J. Funct. Foods 56, 21–32 (2019).10.1016/j.jff.2019.02.044
42. Dempsey E Corr SC Lactobacillus spp. for gastrointestinal health: Current and future perspectives Front. Immunol. 2022 13 840245 10.3389/fimmu.2022.840245 35464397
Dempsey, E. & Corr, S. C. Lactobacillus spp. for gastrointestinal health: Current and future perspectives. Front. Immunol. 13, 840245 (2022).35464397 10.3389/fimmu.2022.840245
43. Lacerda DC Potential role of Limosilactobacillus fermentum as a probiotic with anti-diabetic properties: A review World J. Diabetes 2022 13 717 10.4239/wjd.v13.i9.717 36188141
Lacerda, D. C. et al. Potential role of Limosilactobacillus fermentum as a probiotic with anti-diabetic properties: A review. World J. Diabetes 13, 717 (2022).36188141 10.4239/wjd.v13.i9.717
44. De Gregorio A Protective Effect of Limosilactobacillus fermentum ME-3 against the Increase in Paracellular Permeability Induced by Chemotherapy or Inflammatory Conditions in Caco-2 Cell Models Int J Mol Sci 2023 24 6225 10.3390/ijms24076225 37047193
De Gregorio, A. et al. Protective Effect of Limosilactobacillus fermentum ME-3 against the Increase in Paracellular Permeability Induced by Chemotherapy or Inflammatory Conditions in Caco-2 Cell Models. Int J Mol Sci 24, 6225 (2023).37047193 10.3390/ijms24076225
45. Chou Y-C Ho P-Y Chen W-J Wu S-H Pan M-H Lactobacillus fermentum V3 ameliorates colitis-associated tumorigenesis by modulating the gut microbiome Am. J. Cancer Res. 2020 10 1170 32368393
Chou, Y.-C., Ho, P.-Y., Chen, W.-J., Wu, S.-H. & Pan, M.-H. Lactobacillus fermentum V3 ameliorates colitis-associated tumorigenesis by modulating the gut microbiome. Am. J. Cancer Res. 10, 1170 (2020).32368393
46. Ozen M Piloquet H Schaubeck M Limosilactobacillus fermentum CECT5716: Clinical potential of a probiotic strain isolated from human milk Nutrients 2023 15 2207 10.3390/nu15092207 37432320
Ozen, M., Piloquet, H. & Schaubeck, M. Limosilactobacillus fermentum CECT5716: Clinical potential of a probiotic strain isolated from human milk. Nutrients 15, 2207 (2023).37432320 10.3390/nu15092207
47. Guan Z Feng Q Chitosan and chitooligosaccharide: The promising non-plant-derived prebiotics with multiple biological activities Int. J. Mol. Sci. 2022 23 6761 10.3390/ijms23126761 35743209
Guan, Z. & Feng, Q. Chitosan and chitooligosaccharide: The promising non-plant-derived prebiotics with multiple biological activities. Int. J. Mol. Sci. 23, 6761 (2022).35743209 10.3390/ijms23126761
48. Gunasangkaran G Ravi AK Arumugam VA Muthukrishnan S Preparation, characterization, and anticancer efficacy of chitosan, chitosan encapsulated piperine and probiotics (Lactobacillus plantarum (MTCC-1407), and Lactobacillus rhamnosus (MTCC-1423) nanoparticles Bionanoscience 2022 12 527 539 10.1007/s12668-022-00961-7
Gunasangkaran, G., Ravi, A. K., Arumugam, V. A. & Muthukrishnan, S. Preparation, characterization, and anticancer efficacy of chitosan, chitosan encapsulated piperine and probiotics (Lactobacillus plantarum (MTCC-1407), and Lactobacillus rhamnosus (MTCC-1423) nanoparticles. Bionanoscience 12, 527–539 (2022).10.1007/s12668-022-00961-7
49. Ebrahimnejad P Khavarpour M Khalili S Survival of Lactobacillus acidophilus as probiotic bacteria using chitosan nanoparticles Int. J. Eng. Trans. A Basics 2017 30 456 463
Ebrahimnejad, P., Khavarpour, M. & Khalili, S. Survival of Lactobacillus acidophilus as probiotic bacteria using chitosan nanoparticles. Int. J. Eng. Trans. A Basics 30, 456–463 (2017).
50. Alkushi AG Probiotics-loaded nanoparticles attenuated colon inflammation, oxidative stress, and apoptosis in colitis Sci. Rep. 2022 12 1 19 10.1038/s41598-022-08915-5 34992227
Alkushi, A. G. et al. Probiotics-loaded nanoparticles attenuated colon inflammation, oxidative stress, and apoptosis in colitis. Sci. Rep. 12, 1–19 (2022).34992227 10.1038/s41598-022-08915-5
51. Pandey RP Gunjan H Mukherjee R Chang CM Nanocarrier-mediated probiotic delivery: A systematic meta-analysis assessing the biological effects Sci. Rep. 2024 14 1 11 10.1038/s41598-023-50972-x 38167627
Pandey, R. P., Gunjan, H., Mukherjee, R. & Chang, C. M. Nanocarrier-mediated probiotic delivery: A systematic meta-analysis assessing the biological effects. Sci. Rep. 14, 1–11 (2024).38167627 10.1038/s41598-023-50972-x
52. Alkushi AG Multi-strain-probiotic-loaded nanoparticles reduced colon inflammation and orchestrated the expressions of tight junction, NLRP3 inflammasome and caspase-1 genes in dss-induced colitis model Pharmaceutics 2022 14 1183 10.3390/pharmaceutics14061183 35745756
Alkushi, A. G. et al. Multi-strain-probiotic-loaded nanoparticles reduced colon inflammation and orchestrated the expressions of tight junction, NLRP3 inflammasome and caspase-1 genes in dss-induced colitis model. Pharmaceutics 14, 1183 (2022).35745756 10.3390/pharmaceutics14061183
53. Pupa P The efficacy of three double-microencapsulation methods for preservation of probiotic bacteria Sci. Rep. 2021 11 13753 10.1038/s41598-021-93263-z 34215824
Pupa, P. et al. The efficacy of three double-microencapsulation methods for preservation of probiotic bacteria. Sci. Rep. 11, 13753 (2021).34215824 10.1038/s41598-021-93263-z
54. Kumar N Preparation of probiotic-loaded solid lipid nanoparticles and in vitro survival in gastrointestinal conditions BIO Web Conf. 2024 86 01051 10.1051/bioconf/20248601051
Kumar, N. et al. Preparation of probiotic-loaded solid lipid nanoparticles and in vitro survival in gastrointestinal conditions. BIO Web Conf. 86, 01051 (2024).10.1051/bioconf/20248601051
55. Kim S Novel modified probiotic gold nanoparticles loaded with ginsenoside CK exerts an anti-inflammation effect via NF-κB/MAPK signaling pathways Arab. J. Chem. 2024 17 105650 10.1016/j.arabjc.2024.105650
Kim, S. et al. Novel modified probiotic gold nanoparticles loaded with ginsenoside CK exerts an anti-inflammation effect via NF-κB/MAPK signaling pathways. Arab. J. Chem. 17, 105650 (2024).10.1016/j.arabjc.2024.105650
56. Chang YN The high permeability of nanocarriers crossing the enterocyte layer by regulation of the surface zonal pattern Molecules 2020 25 919 10.3390/molecules25040919 32092877
Chang, Y. N. et al. The high permeability of nanocarriers crossing the enterocyte layer by regulation of the surface zonal pattern. Molecules 25, 919 (2020).32092877 10.3390/molecules25040919
57. Stasiak-Różańska L Berthold-Pluta A Pluta AS Dasiewicz K Garbowska M Effect of simulated gastrointestinal tract conditions on survivability of probiotic bacteria present in commercial preparations Int. J. Environ. Res. Public Health 2021 18 1 17 10.3390/ijerph18031108
Stasiak-Różańska, L., Berthold-Pluta, A., Pluta, A. S., Dasiewicz, K. & Garbowska, M. Effect of simulated gastrointestinal tract conditions on survivability of probiotic bacteria present in commercial preparations. Int. J. Environ. Res. Public Health 18, 1–17 (2021).10.3390/ijerph18031108
58. Gómez E Impact of probiotics on development and behaviour in Drosophila melanogaster -a potential in vivo model to assess probiotics Benef Microbes 2019 10 179 188 10.3920/BM2018.0012 30574803
Gómez, E. et al. Impact of probiotics on development and behaviour in Drosophila melanogaster -a potential in vivo model to assess probiotics. Benef Microbes 10, 179–188 (2019).30574803 10.3920/BM2018.0012
59. Westfall S Lomis N Prakash S Longevity extension in Drosophila through gut-brain communication Sci. Rep. 2018 10.1038/s41598-018-25382-z 29849035
Westfall, S., Lomis, N. & Prakash, S. Longevity extension in Drosophila through gut-brain communication. Sci. Rep.10.1038/s41598-018-25382-z (2018).29849035 10.1038/s41598-018-25382-z
60. Westfall S Lomis N Prakash S Ferulic acid produced by Lactobacillus fermentum influences developmental growth through a dTOR-mediated mechanism Mol. Biotechnol. 2019 61 1 10.1007/s12033-018-0119-y 30368647
Westfall, S., Lomis, N. & Prakash, S. Ferulic acid produced by Lactobacillus fermentum influences developmental growth through a dTOR-mediated mechanism. Mol. Biotechnol. 61, 1 (2019).30368647 10.1007/s12033-018-0119-y
61. Poinsot P Probiotic from human breast milk, Lactobacillus fermentum, promotes growth in animal model of chronic malnutrition Pediatr. Res. 2020 10.1038/s41390-020-0774-0 32023624
Poinsot, P. et al. Probiotic from human breast milk, Lactobacillus fermentum, promotes growth in animal model of chronic malnutrition. Pediatr. Res.10.1038/s41390-020-0774-0 (2020).32023624 10.1038/s41390-020-0774-0
62. Jang YJ Kim WK Han DH Lee K Ko G Lactobacillus fermentum species ameliorate dextran sulfate sodium-induced colitis by regulating the immune response and altering gut microbiota Gut Microbes 2019 10 696 711 10.1080/19490976.2019.1589281 30939976
Jang, Y. J., Kim, W. K., Han, D. H., Lee, K. & Ko, G. Lactobacillus fermentum species ameliorate dextran sulfate sodium-induced colitis by regulating the immune response and altering gut microbiota. Gut Microbes 10, 696–711. 10.1080/19490976.2019.1589281 (2019).30939976 10.1080/19490976.2019.1589281
63. Thumu SCR Halami PM In vivo safety assessment of Lactobacillus fermentum strains, evaluation of their cholesterol-lowering ability and intestinal microbial modulation J. Sci. Food Agric. 2020 100 705 713 10.1002/jsfa.10071 31599967
Thumu, S. C. R. & Halami, P. M. In vivo safety assessment of Lactobacillus fermentum strains, evaluation of their cholesterol-lowering ability and intestinal microbial modulation. J. Sci. Food Agric. 100, 705–713 (2020).31599967 10.1002/jsfa.10071
64. Naissinger da Silva M In vitro test to evaluate survival in the gastrointestinal tract of commercial probiotics Curr. Res. Food Sci. 2021 4 320 325 10.1016/j.crfs.2021.04.006 34095855
Naissinger da Silva, M. et al. In vitro test to evaluate survival in the gastrointestinal tract of commercial probiotics. Curr. Res. Food Sci. 4, 320–325 (2021).34095855 10.1016/j.crfs.2021.04.006
65. Vecchione A Compositional quality and potential gastrointestinal behavior of probiotic products commercialized in Italy Front. Med. (Lausanne) 2018 5 59 10.3389/fmed.2018.00059 29564327
Vecchione, A. et al. Compositional quality and potential gastrointestinal behavior of probiotic products commercialized in Italy. Front. Med. (Lausanne) 5, 59 (2018).29564327 10.3389/fmed.2018.00059
66. Patrone V Molinari P Morelli L Microbiological and molecular characterization of commercially available probiotics containing Bacillus clausii from India and Pakistan Int. J. Food Microbiol. 2016 237 92 97 10.1016/j.ijfoodmicro.2016.08.012 27543819
Patrone, V., Molinari, P. & Morelli, L. Microbiological and molecular characterization of commercially available probiotics containing Bacillus clausii from India and Pakistan. Int. J. Food Microbiol. 237, 92–97 (2016).27543819 10.1016/j.ijfoodmicro.2016.08.012
67. Dioso CM Do your kids get what you paid for? Evaluation of commercially available probiotic products intended for children in the Republic of the Philippines and the Republic of Korea Foods 2020 9 1229 10.3390/foods9091229 32899215
Dioso, C. M. et al. Do your kids get what you paid for? Evaluation of commercially available probiotic products intended for children in the Republic of the Philippines and the Republic of Korea. Foods 9, 1229 (2020).32899215 10.3390/foods9091229
68. Shehata HR Newmaster SG Combined targeted and non-targeted PCR based methods reveal high levels of compliance in probiotic products sold as dietary supplements in United States and Canada Front. Microbiol. 2020 11 531086 10.3389/fmicb.2020.01095
Shehata, H. R. & Newmaster, S. G. Combined targeted and non-targeted PCR based methods reveal high levels of compliance in probiotic products sold as dietary supplements in United States and Canada. Front. Microbiol. 11, 531086 (2020).10.3389/fmicb.2020.01095
69. Kesavelu D Rohit A Karunasagar I Karunasagar I Composition and laboratory correlation of commercial probiotics in India Cureus 2020 12 e11334 33173652
Kesavelu, D., Rohit, A., Karunasagar, I. & Karunasagar, I. Composition and laboratory correlation of commercial probiotics in India. Cureus 12, e11334 (2020).33173652
70. Golnari M Behbahani M Mohabatkar H Comparative survival study of Bacillus coagulans and Enterococcus faecium microencapsulated in chitosan-alginate nanoparticles in simulated gastrointestinal condition LWT 2024 197 115930 10.1016/j.lwt.2024.115930
Golnari, M., Behbahani, M. & Mohabatkar, H. Comparative survival study of Bacillus coagulans and Enterococcus faecium microencapsulated in chitosan-alginate nanoparticles in simulated gastrointestinal condition. LWT 197, 115930 (2024).10.1016/j.lwt.2024.115930
71. Sundararajan V Dan P Kumar A Venkatasubbu GD Applied surface science drosophila melanogaster as an in vivo model to study the potential toxicity of cerium oxide nanoparticles Appl. Surf. Sci. 2019 490 70 80 10.1016/j.apsusc.2019.06.017
Sundararajan, V., Dan, P., Kumar, A. & Venkatasubbu, G. D. Applied surface science drosophila melanogaster as an in vivo model to study the potential toxicity of cerium oxide nanoparticles. Appl. Surf. Sci. 490, 70–80 (2019).10.1016/j.apsusc.2019.06.017
72. Senthilkumar S Developmental and behavioural toxicity induced by acrylamide exposure and amelioration using phytochemicals in Drosophila melanogaster J. Hazard Mater. 2020 394 122533 10.1016/j.jhazmat.2020.122533 32279006
Senthilkumar, S. et al. Developmental and behavioural toxicity induced by acrylamide exposure and amelioration using phytochemicals in Drosophila melanogaster. J. Hazard Mater. 394, 122533 (2020).32279006 10.1016/j.jhazmat.2020.122533
73. Siddique YH Toxic potential of copper-doped ZnO nanoparticles in Drosophila melanogaster (Oregon R) Toxicol. Mech. Methods 2015 25 425 432 10.3109/15376516.2015.1045653 26000624
Siddique, Y. H. et al. Toxic potential of copper-doped ZnO nanoparticles in Drosophila melanogaster (Oregon R). Toxicol. Mech. Methods 25, 425–432 (2015).26000624 10.3109/15376516.2015.1045653
