A graphical abstract of the study is illustrated hereafter (Fig. S1).

Introduction

Helicobacter pylori, which in 1994 was categorized by the WHO “World Health Organization” as a group I carcinogen, is a Gram (–), microaerophilic, flagellated, helical bacteria that occupy the human stomach mucosa and duodenum, it is known as one of the most widespread infections globally, particularly within underdeveloped regions, and the primary reason of chronic gastritis, peptic ulcer disorders (PUD), as well as gastric cancer and gastric MALT lymphoma over time1,2. Helicobacter pylori is thought to infect 50–75% of the world’s population. Only one out of every ten people infected with this microbe was marked by the emergence of infection signs, the others are shown to remain asymptomatic carriers3.

Some external factors, such as increases in O2 concentration, changes in pH/temperature, and exposure to antimicrobial agents or any other unfavorable environmental conditions, may create pressure on H. pylori4,5. In order, H. pylori has evolved several morphological and metabolic modifications as a means of adapting to these stressors6.

The transition of H. pylori from its typical spiral-shaped bacillus to a coccoid form is one of the morphological alterations. The coccoid forms of H. pylori are typically in a less virulent, low activity of metabolism state described as VBNC “viable but non-culturable”7. It has been proven that bacteria in the VBNC state are capable of continuing to be pathogenic and can even shift back to active regrowth states. In addition, it has been demonstrated that these forms may defy immune reactions, accelerate the development of cancer, and tolerate high temperatures, lengthy incubation, aerobiosis, and treatments with antibiotics or proton pump inhibitors, and then contributing to the failure of therapy. Therefore, it is crucial to eradicate these forms of H. pylori7,8,9.

Even though H. pylori was discovered about four decades ago, there is no 100% effective medication10,11. The currently used H. pylori treatment consists of triple or quadruple therapeutic groups such as CTT “clarithromycin triple therapy”, ATT “amoxicillin triple therapy”, BQT “bismuth quadruple therapy”, and NBQT “nonbismuth quadruple therapy”. However, with the emergence of drug-resistant strains, a resistance rate of 20% to CTT and ATT and around 30% to BQT appeared12,13,14. According to the WHO, H. pylori ranks among the top 12 antibiotic-resistant bacteria that need to be targeted for drug development15.

Finding more efficient, safe, and affordable treatments has become increasingly crucial to overcome the pathogenicity of antibiotic-resistant H. pylori16,17. Plant compounds with anti- H. pylori action have been the subject of numerous studies18. It has been discovered that antibacterials derived from plant seeds can successfully suppress or limit microorganisms and address antibiotic resistance19. Naturally occurring plant-based biopolymers (glucans, gums, mucilage, and cellulose) are useful components for creating affordable, environmentally beneficial products. Plantaginaceae, Linaceae, Acanthaceae, and Brassicaceae are the families of plants that produce mucilage from their seed coats20. Compounds having antiviral, antibacterial, and anticancer activities are especially abundant in plants in the Brassicaceae family21. Among its relatives, broccoli has the largest quantity of secondary metabolites including phenolic compounds, flavonoids, and sulforaphane. Moreover, it has been reported that seeds contain the highest concentration of sulforaphane, an antibacterial agent that inhibits H. pylori22.

One of the more recent transdisciplinary areas that have significantly improved a range of applications is nanotechnology. A branch of nanotechnology called nanobiotechnology works with lifeforms to generate and manipulate nanoparticles (NPs)23. Metallic NPs have been extensively utilized in several biological fields, including as antibacterial, antioxidant, and anticoagulant agents. Due to its remarkable physicochemical qualities, which include little toxicity, chemical stability, and biocompatibility, selenium nanoparticles (SeNPs), have garnered a lot of attention24,25. It also has a broad variety of uses in the biomedical sector26. Because of their controllability, eco-friendliness, high yield, and affordability, green synthesized SeNPs have benefits over conventional physical and chemical procedures in the synthesis of nanomaterials of diverse sizes, shapes, and biological types27,28. Recently, SeNPs have proven to exhibit potent antibacterial activity against pathogenic microorganisms29.

The biopolymers have demonstrated enhanced superiority when it comes to their potential for use in drug encapsulation, or carrying applications30. Chitosan is a positively charged biopolymer molecule that is obtained from the bioactive deacetylated form of chitin. It may be effectively acquired from a variety of sources that include the skeletons of insects, fungi, and crustacean wastes27,31,32. Chitosan offers several benefits, including its antibacterial activity, mucoadhesivity, remarkable biocompatibility and biodegradability, nontoxicity, and low allergenicity33,34. Comparing chitosan in nanoform to bulk materials, it has been demonstrated to increase biopolymer bioactivities (such as drug transport, anticancer, antibacterial, toxicant adsorption, bioremediation, nanometals conjugation, and antioxidant and anti-inflammatory properties). One of the most adaptable polymers for the creation of antimicrobial chemotherapies in therapeutic research is chitosan nanoparticles (NCT), which have demonstrated strong antibacterial activity against a variety of microbes29,35. The most common theory regarding chitosan’s antibacterial activity is that the electrostatic force between positively charged chitosan and negatively charged bacteria cell walls encourages a closer interaction, disrupting the cell and changing the permeability of the membrane, which allows the drug to pass through the bacteria cell wall. then attaches itself to DNA, inhibiting DNA replication and ultimately leading to cell death33,36.

Consequently, the key goal of this research is to characterize and ascertain the effect of the nanocomposites built from greenly synthesized SeNPs from broccoli seed mucilage with NCT against H. pylori.

Materials and methods

The “Agricultural Research Center, Giza, Egypt” is where the broccoli “Brassica oleracea var. italica” seeds were attained. Unless specified otherwise, all used chemicals, media, and reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, United States). All methods were carried out in accordance with relevant guidelines.

Mucilage of broccoli seeds (MBS)

The seeds were thoroughly ground into a fine powder using a porcelain mortar before being soaked in distilled water (DW) at a 1:20 (w/v) ratio with continually stirred using a magnetic stirrer (650 × g) for 6 h at room temperature, then the mixture was filtered using a pair of filter paper (6 mm Ø). Ethanol (96%) was added to the filtrate in a 1:1 ethanol to filtrate ratio to obtain the mucilage at 3–4 °C for 3 h. When the mucilage precipitates, it is separated through centrifugation (4800 × g) and dried by vacuum drying oven at a temperature of 42 °C until the mucilage is completely dried.

Green-synthesis of SeNPs

An aqueous solution of sodium selenite (Na2SeO3) was prepared (0.17 g Na2SeO3 for every 100 ml of distilled water). for green synthesis of SeNPs, an equal volume of both aqueous solutions of MBS (0.1%, w/v) and sodium selenite were held on a magnetic stirrer (650 × g for 90 min) with the addition of Ascorbic acid as a reducing agent until a reddish-orange color change was noticed indicating the synthesis of MBS-SeNPs. Then it collected by centrifuging (10,000 g, 25 min), washed with DW to remove extra materials, centrifuged again, and then was dried.

Preparation and loading of NCT

A solution of chitosan with concentrations of 1% (w/v) was prepared. STPP (sodium tripolyphosphate) solution is dripped very gradually into a 1:1 ratio of chitosan solution and MBS/ SeNPs solution within the continuous magnetic stirring. Then collected by centrifuging, washing with DW, centrifuged again, and drying. Different concentrations of NCT/MBS-SeNPs were made as a study to determine the most effective combination for them, trial (T1) with 1:2 NCT: MBS-SeNPs ratio, trial (T2) with 1:1 NCT: MBS-SeNPs, and 2:1 NCT: MBS-SeNPs ratio for trial (T3).

A schematic diagram of the nanocomposite (NCT/MBS-SeNPs) synthesis process is provided hereafter (Fig. S2).

Characterization of NCT/MBS/SeNPs

FTIR analysis

The “Fourier transform infrared spectroscopy” examinations of the produced compounds powder (NCT, MBS, MBS/SeNPs, NCT/MBS/SeNPs) that were combined with 1% KBr then obtained from wavenumber range of 500–4000 cm−1 by using a FTIR spectroscopy, JASCO FT-IR-360, Tokyo, Japan. To determine the distinctive biochemical bonding/interactions in produced compounds. The resulting peaks were plotted with wavenumber (cm−1) on the X-axis and transmittance (%) on the Y-axis.

The particle sizes (Ps) and superficial charges (zeta potentiality)

Using zetasizer (Zeta plus, Brookhaven, USA), employing the DLS “Dynamic Light Scattering” methodology that analyzes the temporal fluctuations using the intensity/photon autocorrelation function, the Ps and zeta (ζ) potential of the produced nanomaterials or nanocomposites were appraised after dissolving it in DW and sonicated, their Ps/ ζ potentials were recorded between + 150 and − 150 mV at RT.

TEM “transmission electron microscope” and SEM “scanning electron microscope” imaging

The particle size, apparent shape, morphology, and distribution of the produced nanomaterials or NCs were screened using the “scanning electron microscope” (SEM; IT100, JEOL, Tokyo, Japan) and “transmission electron microscope” (TEM; Leica-Leo 0430; Cambridge, UK). For the SEM, DIW suspensions of prepared NCs (T1, T2, T3) were sonicated before inspection. These NC solutions were mounted onto self-adhesive SEM carbon discs, coated by palladium-gold (with Polaron coater: E5100 II, PA), and inspected at 15–20 kV acceleration and 30 kx magnification. The MBS-mediated SeNPs were inspected via TEM after dropping its sonicated solution (0.01%, w/v) onto TEM copper grids, dehydrating with vacuum for 33 min, and exposing to imaging at acceleration of 30 kV.

In vitro determination of antibacterial activity

Experiments, both qualitative and quantitative, were used to demonstrate the antibacterial activity of NCT/MBS/SeNPs against a standard strain of H. pylori (ATCC-700824), and H. pylori isolated from antral biopsy specimens of gastric ulcer patients at Kafr El-Sheikh University Hospital. After sampling, the specimens were minced and homogenized in a sterile saline solution (0.5 mL) before seeding on BHI “brain heart infusion” agar supplemented with 7% defibrinated horse blood. The plates were incubated for at most 10 days under microaerophilic conditions (5% O2, 10% CO2, and 85% N2) at thirty-seven degrees Celsius. Using Gram stain, urease/catalase/oxidase assays, API Campy (automated system mini-API, BioMérieux), and PCR (Eppendorf Thermocycler, Hamburg, Germany), suspected isolates were confirmed as H. pylori37. Assuming the same conditions, Amoxicillin was used as the standardized positive antibacterial.

Qualitative assay

The ZOI assay “zones of inhibition” using the “disc diffusion method” was carried out for preliminary examination of the activity of generated nanocomposites MBS, NCT, MBS/ SeNPs, and NCT/MBS/SeNPs (T1, T2, and T3) against H. pylori. A sterile paper disc (Whatman No. 1, 6 mm Ø) was immersed in a solution of each substance (10% in DIW) and placed onto a BHI plate that was inoculated with H. pylori. After 24–48 h of humid microaerophilic incubation at 37 °C, the ZOI diameters that initially appeared were measured, and their triplicate means were computed18,38.

Quantitative assay

The microdilution procedure was used to assess the MIC “minimum inhibitory concentration” of MBS, NCT, MBS/ SeNPs, and NCT/MBS/SeNPs (T1, T2, and T3) against H. pylori. Each substance was serially concentrated within the range of 10–100 g/mL, then, 1 mL of each solution was added to tubes containing 8 mL of Mueller Hinton broth with 7% of defibrinated horse blood. After that, 1 mL of a 1:1000 dilution of bacteria was added to make an overall volume of 10 ml. A positive control tube was prepared without any of the tested substances under the same conditions. The MIC was evaluated after 2–3 days of microaerophilic incubation at 37 °C37,39.

Microscopic observation of treated H. pylori

Images from SEM (JEOL JSMIT100, Tokyo, Japan) were used to see how the bacterial cells’ morphology and structure changed after being exposed to NCT/MBS/SeNPs for 6–12 h. A standardized procedure was used in the SEM bacterial imaging, 24-h-old grown bacteria in BHI were exposed to 100 μg/mL of composite while being incubated under microaerophilic conditions at 37 °C. Bacterial samples were centrifuged at 4700 g for 30 min, rinsed with NaCl solution, and then centrifuged again before being imaged using SEM. The SEM imaging protocol involved the dehydration of cells via successive ethanol concentrations followed by drying through critical-point drier “Auto-Samdri-815; Tousimis, Rockville, MD” and palladium/gold coating. The images were captured to detect distortions/deformations in cells structures at 20 kV acceleration and 30.000 × magnification.

Statistical analysis

For statistical computation, the SPSS package “V 17.0, SPSS Inc., Chicago, IL” was used. Experiments were carried out in triplicate, and data were reported as means SD. Results’ significances at p ≤ 0.05 were computed using a t-test and one-way ANOVA.

Results

Characterization of NCT/MBS/SeNPs

FTIR analysis

The FTIR spectrum of the NCT, MBS, MBS/ SeNPs, and NCT/MBS/SeNPs determines biological groups, bonds, and the interaction among each other, through assessing the absorbed amount of infrared radiation over a broad spectrum of wavelengths. The extent of chemical composition changes that result from the interaction and interference of materials with each other, confirming the development of a new compound with a new chemical composition, was determined.

In the FTIR spectrum of (NCT) (Fig. 1), the N–H stretching vibrations were identified as the quite wide band at 3422 cm−1, which overlapped the O–H stretching vibrations peak at that same location. The smaller band at 2921 cm−1 is ascribed to the stretching vibrations of chitosan’s –CH and –CH2, which is a typical polysaccharide band. The other absorption bands spotted at 1644 cm−1,1415 cm−1,1386 cm−1,1036 cm−1 are ascribed to stretched C=O in amide I, CH2 bending, CH3 symmetrical deformations and − OH vibrated stretching of C6, in that order30,40,41,42,43.

Figure 1
figure 1

FTIR spectra for NCT, MBS, MBS/ SeNPs, and NCT/MBS/SeNPs.

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In (Fig. 2) (MBS), Owing to the multiple bioactive compounds observed in broccoli, a peak with a wide width in the range of 3200–3500 cm−1 might be attributed to the stretching vibration of the phenol hydroxyl group, which also overlapped with the amines’ N–H. This is followed by a peak at 2850 cm−1 which has been pinpointed as the methylene symmetric and asymmetric C–H members of the aliphatic group. The bands present at 1749 and 1644 cm−1 reflect to C=O present in carboxylic acids and stretching vibration of carbonyl (C=O bonds derived from ester groups), in that order44,45,46,47.

Figure 2
figure 2

TEM image of the green synthesized SeNPs from MBS.

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According to the modifications noticed in Fig. 1 (MBS/ SeNPs) compared to the spectrum of (MBS), It appears that the SeNPs affect the bands’ strength, which shifts towards higher wavenumbers. Such as the band 3292 cm−1 which shifts to 3415 cm−1 due to the formation of nanoparticles. The 2915, 2850, and 1250 cm−1 bands appeared with lower intensity/ wideness than they were in the spectrum of (MBS). Bands at 3000, 1749, 1554,1458, 1310, 1144, 702, 626, and 536 m−1 vanished, indicating that these bonds were broken and occupied upon interaction with SeNPs, and other bands newly appeared, for example, at 3850, 1415 and 1332 cm−1, where the band 1415 cm−1 indicate fluoro compounds with C–F stretching46,48.

The (NCT/MBS/SeNPs) spectrum in Fig. 1 revealed numerous biochemical bonds/groups from both composing agents (NCT and MBS-SeNPs) that were present in the composites of (NCT/MBS/SeNPs), these groups are denoted in the figure by blue lines for groups derived from NCT and red lines for groups derived from MBS-SeNPs.

Ultrastructure assessment

For examining a nanoparticle’s charge, size, and morphology, zeta potential analysis and electron microscopy imaging were carried out (Table 1, Figs. 2 and 3). According to Table 1, using the DLS assessments of the zetasizer, the computed NCT ζ potential was highly positive (+ 39.62 mV) with a Ps range between 73.23 and 823.64 nm, while the MBS/SeNPs particles were negatively charged and much lower Ps (4.81–57.86 nm). Accordingly, when NCT was added to MBS/SeNPs particles in (T1), the charge increased to (− 12.45 mV), as NCT concentration grew, it increased to become moderately positive in (T2), then in (T3), where NCT concentration was highest, the ζ value became highly positive (+ 27.64 mV). The observed mean diameter of (T1), (T2), and (T3) was 204.13, 131.64, and 159.37, respectively. T2 represents the smallest particle size range (41.39–521.77 nm) and mean particle diameter (131.64 nm), with an equal quantity of NCT and MBS/SeNPs (1:1). In accordance with TEM (Fig. 2), the MBS-SeNPs spread uniformly and took on spherical forms, with Ps range between 3.6 and 42 nm. The uniform distribution of NPs and their stability with MBS during green synthesis were confirmed by the TEM images of the photosynthesized MBS-SeNPs. Also, the SEM (Fig. 3) images showed morphological characteristics revealed themselves as semispherical-shaped clusters with some aggregates present in addition to the individual particles with Ps range between 90.5 and 720 nm for T1 (A), and lower Ps range from 50.23 to 515.7 nm for T2 (B), and 40.7–670.8 nm for T3 (C), confirming the findings reported in Table 1.

Table 1 Zeta potential and Ps distributions of produced nanomaterials and nanocomposites.
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Figure 3
figure 3

SEM image of biopolymers nanocomposites, with A standing for T1, B for T2, and C for T3.

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In vitro determination of antibacterial activity

Qualitative and quantitative assays

The biopolymer nanocomposite's ability to eradicate both H. pylori strains (isolated and standard) in comparison to Amoxicillin (the standard anti-Helicobacter pylori) was assessed via MIC and ZOI tests. Furthermore, the ideal concentration for elimination was identified (Table 2). The isolated strain showed more sensitivity to the tested compounds than the standard strain based on MIC and ZOI resalted values. The compounds T1, T2, and T3 show the minimal MIC and broadest ZOI compared to NCT, MBS, and MBS/ SeNPs separately. The 1:1 NCT: MBS-SeNPs concentration in T2 outperformed the other two concentrations (T1 and T3) in terms of elimination of both isolated and standard H. pylori. Table 2 makes it evident that the third trial’s results, with a higher chitosan concentration, outperform the first one. This is because chitosan is positively charged, which makes it easier for it to attach to negatively charged cells. Nonetheless, MBS/SeNPs’s toxicity is still greater than NCT’s, based on this, the second trial gave better results, where the amount of chitosan is suitable for adhesion to cells, and the amount of selenium is appropriate for eliminating them. T2 also exhibits remarkable efficacy in comparison to the standard antibiotic (Amoxicillin), with 25.9 mm ZOI and 0.08 mg/L MIC against H. pylori I and 27.3 mm ZOI and 0.10 mg/L MIC against H. pylori ATCC-700824.

Table 2 Antibacterial activity of biopolymers nanocomposites against H. pylori.
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Microscopic observation of treated H. pylori

The SEM micrograph (Fig. 4) displays how the nanoparticles stick to the bacteria’s external surface (red arrows indicate this), the core reason for this adhesion is due to chitosan molecules’ potent positive charge, which allows them to bind to the negatively charged cell surface, thereby supporting the biopolymers nanocomposites permeability inside the cell before being destroyed. Figure 4A showed H. pylori’s ordinary morphology, smooth and intact cell walls with multiple stuck nanoparticles on it when the exposure time first started, after exposure for 5 h (B), the coccoid form of H. pylori was observed clearly, which reflects the pressure to which the bacteria were exposed, the bacterial walls of both phenotype (bacilli and cocci) became rougher, puffy and less intact, other morphological abnormalities were also observed. When NCT/MBS/SeNPs exposure was extended to 10 h (Fig. 4C), the cells were completely lysed, ruptured, and released their internal components with the NPs sticking on them. These results demonstrate the strong effectiveness of NCT/MBS/SeNPs in eliminating the two H. pylori phenotypes.

Figure 4
figure 4

Micrograph by SEM of H. pylori after exposure to NCT/MBS/SeNPs for (A) 0 h, (B) 5 h, (C) 10 h. *Some connected NPs to the bacterial cells are indicated by red arrows. Blue arrows indicate other morphological forms of H. pylori.

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Discussion

FTIR spectroscopy has proven to be a powerful tool to identify the extent to which components combine to generate new compounds with distinct features49. The primary distinguishing bands of the usual bands of natural chitosan are displayed in the FTIR spectrum of (NCT) (Fig. 1). STPP interactions with chitosan were primarily located in the range around 3422 cm−1, these interactions demonstrated a wider spacing and lower intensity compared to bulk Cht, which provides evidence for reduced − H bonding caused by the interactions with TPP cross-linkage30. Additionally, the functional group P=O which is located at 1036 cm−1 demonstrates the ionic bond-mediated cross-linkage between the phosphate groups of STPP and chitosan. These are all indications that a nanoparticle solution has been formed27,50. The ionic-gelation interaction used in the TPP cross-linking protocol to synthesize NCT was shown to be an effective operational protocol. The synthesized NCT with this protocol exhibited remarkable properties for practical application as nanocarriers for other bioactive constituents51. The MBS shows great potential for conjugating, reducing, and stabilizing SeNPs48. The band 3292 cm−1 in (MBS) spectrum shifts to 3415 cm−1 in (MBS/ SeNPs) spectrum indicating Se interaction with N–H and O–H groups, and the newly appeared 2915, 2850, and 1250 cm−1 bands, and the bands at 3000, 1749, 1554,1458, 1310, 1144, 702, 626, and 536 cm−1 that disappeared indicate the MBS role in SeNPs conjugation/reduction52. The noticeable changes in the (NCT/MBS/SeNPs) spectrum and the peaks that were different and shifted from those of their parent compounds (MBS/SeNPs and NCT), in addition to peaks that were detected from both spectrums reveal that there were biochemical and physical interactions occurring during MBS/SeNPs being trapped within NCT29.

The selection of electron microscopy approaches (e.g. TEM and SEM) based on the potency of TEM to appraise the actual precise size and shape of nanoparticles (especially the nanometals), whereas the SEM approach can effectually screen the surface morphology and alterations (particularly in nanopolymers and nanocomposites)25,31,36,41. Based on earlier research, the spherical shape observed in Fig. 2 is evidence of the presence of SeNPs. The Ps have a major impact on SeNPs’ biological activity53. The primary variables influencing the size of the particles are the host used in the synthesis process and the conditions of the synthesis. However, because SeNPs with a particle size of less than 200 nm easily infiltrate cells, participate in metabolic processes, and demonstrate increased biological activity, smaller nanoparticles are of the greatest interest for future biomedical usage53,54. This suggests that the Ps of SeNPs that MBS synthesizes (4.81–57.86 nm) is more than adequate for use as an antibiotic, as evidenced by comparison with previous studies that produced SeNPs with greater Ps29,53,55. According to Table 1, the sizes of MBS/SeNPs increased significantly after combining NCT in T1, T2, and T3, indicating their combination and integrations27. The data suggests that MBS/NCT/SeNPs tend to form aggregates rather than separate particles, resulting in a larger particle size range. The potential improvements or modifications to the experimental design that might address this in future studies could be suggested to employ ultra-sonication after the NCs preparation to disperse particles, adjust blending ratio of NCT to MBS-SeNPs for optimizing particles’ charges, and optimize the biosynthesis conditions (e.g. temperature, pH, stirring speed,…etc.)19,25,28,49,54.

Several fields, including biotechnology, food processing, pharmaceutics, and water treatment, have employed chitosan56. This biopolymer has also been applied as a strong antibiotic agent against bacteria and fungi57. The effectiveness of chitosan against Gram-negative bacteria is greater than Gram-positive58. One of the most contributing properties to chitosan’s antibacterial effect is the positive charges that possess on its surface. This makes it easier to attach to negatively charged cells and more effectively deliver active ingredients to them33,36. Chitosan gets significant advantages by being transformed from its basic form into nanoparticles since this enhances the surface area that interacts with bacteria and, thus, strengthens adhesion59. When looking for bioactive compounds that are more effective, less harmful, and less expensive, plants are the best option60. Broccoli’s antibacterial properties have been investigated by numerous scientists against Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, Bacillus cereus, and Listeria monocytogenes, either with application of plain plant extract or the synthesized nanomaterial with extract61,62,63,64. Numerous studies have demonstrated that nano-selenium particles have greater toxicity against pathogens than nano-chitosan27,31,41, but it is less adhering to the surface of bacteria because of its negative charge according to24,31,52, which repels the negative charge of bacteria as well. Therefore, using chitosan loaded with nano-selenium particles gives the best outcomes, as shown in previous studies, where the positive chitosan sticks to the negative cell, and the selenium then kills it29,31,41.

The in vitro determination of anti H. pylori activity in current investigation involved the usage of different techniques to appraise the potency and actions of screened agents/NCs26,32,33. The qualitative “ZOI” approach gives direct and general reflections about the potency of antimicrobials toward microbial species; the quantitative “MIC” assay provides exact results about the required concentrations from each agent to suppress or kill the microorganism, whereas the SEM microscopic observations screens the action of antimicrobials to disfigure /distort bacterial cells after exposure35,36,41,58,59.

As we previously explained, the deadly effect of NCT, SeNPs, and MBS separately has been proven in many studies against different types of bacteria, whether Gram-negative or Gram-positive. Therefore, in this study, the effect of NCT/MBS/SeNPs was tested as an anti-H. pylori and its effectiveness was successfully proven.

Conclusion

This study constructed nanocomposites of NCT/MBS/SeNPs, in which the green synthesis of MBS resulted in the successful production of selenium nanoparticles with a particle size range of 4.81–57.86 nm. In three trials, chitosan was also employed at varying concentrations: (T1) with a 1:2 NCT ratio for MBS-SeNPs, (T2) with a 1:1 NCT ratio for MBS-SeNPs, and (T3) with a 2:1 NCT ratio for MBS-SeNPs. FTIR, zetasizer, SEM, and TEM were used to characterize the nanocomposites, and their effective synthesis was demonstrated. MIC, ZOI, and microscopic examination of treated bacteria were also used to assess its anti-H. pylori efficacy, with greater success favoring the (T2). From what was achieved, the effectiveness of NCT/MBS/SeNPs in eliminating H. pylori was well-proven. We advise employing NCT/MBS/SeNPs as a potent anti-H. pylori agent in light of the study’s findings and conclusion.