Antioxidant and Antibacterial Activities of Nano-probiotics Versus Free Probiotics Against Gastrointestinal Pathogenic Bacteria

Abstract

Antibiotic-resistant pathogenic bacteria and the oxidative stress related to their infections are dangerous health problems. Finding new safe, effective antibacterial and antioxidant agents is an urgent global need. Probiotics are a strong candidate for possible antibacterial and antioxidant agents. The delivery of these probiotics without any effect on gastrointestinal digestion is the most important point for their application. The encapsulation of the probiotics on nanoparticles or other supports is a well-known method for the safe delivery of the probiotics. Little information is known about the effect of the probiotic encapsulation on its antibacterial and antioxidant activity. The present study tried to investigate the effect of probiotic encapsulation on nano-chitosan on its antioxidant activity and antibacterial activity against some pathogenic bacteria. We encapsulated some known probiotic species on nano-chitosan and investigated the antibacterial activity of the nano-probiotics and free probiotics against gastrointestinal pathogenic bacteria. The antioxidant characters of the free and encapsulated probiotics were investigated in terms of DPPH radicle scavenging activity, ferric ion chelating activity, hydroxyl radicle scavenging activity, superoxide anion radicle scavenging activity, and anti-lipid peroxidation activity. Results showed the superiority of the encapsulated probiotics as antibacterial and antioxidant agents over the free ones. The encapsulation improved the antibacterial activity of Sporolactobacillus laevolacticus against Bacteroides fragilis by 134% compared to the free one. Also, significantly, the encapsulation increased the hydroxyl radicle scavenging activity of Enterococcus faecium by about 180% compared to the free one. Nano-chitosan encapsulation synergistically increased the antioxidant and antibacterial activity of the studied probiotics. This can be promising for controlling pathogenic bacteria.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-023-01140-2.

Introduction

Infection with gastrointestinal pathogenic bacteria is one of the most dangerous bacterial infections that causes high morbidity and mortality worldwide [1]. This bacterial infection danger is represented by its antibiotic resistance, and thus, its infection causes chronic inflammation that can be carcinogenic to the gastrointestinal tract [2]. Besides bacterial antibiotic resistance, overconsumption of antibiotics has numerous side effects, such as allergies, nephritis, gastrointestinal problems, hematological problems, and disturbances in the nervous system. Thus, it is crucial to find new, safe, and cost-effective natural antibacterial agents [3]. With this huge demand for antibacterial agents worldwide, probiotics have gained the upper hand [4]. Probiotics are live microorganisms that can improve human health through the improvement of the immune system and/or the intestinal microflora in both quantitative and qualitative ways [5]. The antibacterial activity of the probiotics is attributed to their ability to produce antibacterial compounds such as bacteriocin or bacteriocin-like substances, lactic acid, and hydrogen peroxide [6]. In addition, Pruthviraj et al. [7] reported that probiotics have promising antibacterial activity due to their ability to produce many important active compounds that help stop the colonization of pathogenic bacteria. Besides, the competition between the probiotics and the pathogenic bacteria affects nutrition and adhesion sites [8]. Another important role of probiotics as anticancer agents is that they can detoxify toxins secreted by pathogenic bacteria [9]. One of the primary reasons people use probiotics is to help restore the balance of the gut microbiota when it is disrupted by factors such as antibiotics, diet, stress, or illness [10]. Gut dysbiosis is an imbalance in the composition and function of the gut microbiota, the community of microorganisms living in the digestive tract, which can lead to negative health effects Probiotics can help address gut dysbiosis by introducing beneficial microorganisms into the gut. [11]. Studies have shown that certain probiotic strains can help mitigate the effects of gut dysbiosis. They can reduce symptoms of gastrointestinal disorders, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and diarrhea caused by infections [12].

Pathogen infections combine with different disorders in the host cells. Increasing the reactive oxygen species in the cells causes oxidative stress, which is one of the most dangerous disorders, combined with pathogen infection [13]. This oxidative stress is the root of many chronic human diseases [14]. Thus, it is better to find antibacterial agents with high antioxidant capacity to be more effective in defending against pathogenic bacterial infection and the result of oxidative stress [15]. Antioxidant systems produced by microorganisms are a potential technique for preserving low levels of free radicals. These microbial characteristics help to promote health and, even more, to control various disorders. Probiotics bacteria antioxidant action is reported [16]. Probiotic bacteria treatment increases the hepatic antioxidant capacity [17]. Probiotic Lactobacillus spp. treatment increased the antioxidant activity of soy and almond milk [18]. Li et al. [19] reported that probiotics increased superoxide dismutase and antioxidant capacity. Probiotic bacteria are effective as antioxidants in meat processing instead of chemical antioxidants [20]. Probiotic lactic acid bacteria can antagonize oxidative stress by scavenging DPPH and ABTS radicals, besides suppressing nitric oxide production [21]. Zhou et al. [14] reported that Lactobacillus plantarum probiotics have eight antioxidant-related genes that give them high antioxidant capacity.

Prebiotics and postbiotics are components of the diet and gut microbiota that play important roles in influencing the composition and function of the gut microbiota. They interact with gut bacteria and have a significant impact on gut health [22]. Prebiotics are non-digestible dietary fibers, usually carbohydrates, that serve as a food source for beneficial gut bacteria. They promote the growth and activity of specific microorganisms in the gut. Prebiotics selectively nourish beneficial bacteria, such as Bifidobacterium and Lactobacillus. These bacteria are known for their positive effects on gut health. Prebiotics can enhance the overall diversity of the gut microbiota. A more diverse gut microbiota is associated with better gut health and a stronger immune system [23, 24]. Postbiotics are bioactive compounds produced by gut bacteria during the fermentation of prebiotics. These compounds can include short-chain fatty acids (SCFAs), vitamins, enzymes, bile acids, and various metabolic products [25]. These metabolites have been shown to play a role in reducing reactive oxygen species (ROS) and inducible nitric oxide synthase (iNOS) activity in the body through various mechanisms [26]. SCFAs, such as butyrate, acetate, and propionate, have anti-inflammatory properties. They can modulate the immune response by promoting the differentiation and activity of regulatory T cells (Tregs), which help control excessive inflammation. Reduced inflammation can lead to a decrease in ROS production, as chronic inflammation is a common source of ROS in the body. SCFAs can enhance the body's antioxidant defenses. For example, butyrate can upregulate the expression of antioxidant enzymes [27]. SCFAs contribute to the maintenance of the gut barrier. A healthy gut barrier prevents the translocation of harmful bacteria and their byproducts into the bloodstream, which prevents systemic inflammation and the increase in ROS production [28, 29]. Bile acids also play a role in reducing ROS production and iNOS activity. They are involved in the emulsification and absorption of dietary fats and fat-soluble vitamins, which can affect oxidative stress in the gut and the rest of the body. Bile acids can modulate the expression of genes involved in antioxidant defense. By regulating the immune response and inflammation, bile acids can indirectly impact the production of ROS and the activity of iNOS, which are often upregulated during inflammation [30, 31]. Butyrate, a type of SCFA, has been shown to inhibit the expression of iNOS in certain cell types. iNOS produces nitric oxide (NO), which, in excess, can contribute to oxidative stress and inflammation. By inhibiting iNOS, butyrate can help reduce the production of NO and, by extension, ROS [32].

Despite the numerous health benefits of probiotics, they are exposed to the danger of gastrointestinal digestion, which limits their effectiveness. Thus, it is important to find a safe way to deliver the probiotics without affecting gastrointestinal digestion [19]. El Sayed and Mabrouk [33] reported that probiotic encapsulation of sodium alginate and rice flour increased its viability under gastric conditions. Lactobacillus acidophilus encapsulation in alginate-galbanum enabled it to survive under stimulated gastric conditions [34]. Similarly, Barajas-Álvarez et al. [35] reported the success of the encapsulation of Lacticaseibacillus rhamnosus to save its viability in storage and gastric conditions. Encapsulation saves the probiotics from adverse conditions through the protection of their plasma membrane from damage, besides reducing the contact between the probiotics and the gastric solutions [36]. Encapsulation efficiency depends mainly on the type of encapsulation material and method. Nano-materials showed high efficiency for probiotic encapsulation [37]. Various studies showed the efficiency of encapsulation in saving the survival of the probiotics, but more studies are needed to explore the effect of encapsulation on the activity of the probiotics [38–41]. From this point of view, this study tries to investigate the effect of probiotic encapsulation on the antioxidant and antibacterial activity against some gastrointestinal pathogenic bacteria.

Materials and Methods

Preparation on Nano-probiotic

Probiotic Bacterial Species

Lactococcus lactis subsp. lactis (ATCC 11454) (formerly Streptococcus cremoris), Enterococcus faecium (ATCC BAA-2319), Lactiplantibacillus plantarum (ATCC 49445) (formerly, Lactobacillus plantarum), Sporolactobacillus laevolacticus (ATCC 23493) (formerly known as Bacillus laevolacticus), Latilactobacillus sakei (ATCC 15521) (Formerly Lactobacillus sakei), and Limosilactobacillus fermentum (ATCC 9338) (Formerly Lactobacillus fermentum) were purchased from the American Type Culture Collection. The bacterial cultures were prepared with 10¹⁰ CFU/mL cell concentrations.

Nano-chitosan Preparation

We used defined Nano-chitosan particles (NS6130-09-918) from Intelligent Materials Pvt. Ltd., USA. The nao-particles are with sizes 80–100 nm.

Encapsulation of Probiotics on Nano-chitosan

Nano-probiotics were prepared according to Shaim et al. [42]. We added autoclaved sterilized nano-chitosan particles to the bacterial culture (10¹⁰ CFU/mL) with 10% concentration (W/V) at room temperature to prepare stable nano-chitosan suspension as mentioned in the product instruction. The mixture of nano-chitosan and bacterial culture was incubated at shaking (100 rpm) for different times to determine the suitable incubation time for maximum encapsulation capacity (supplementary data Figure S1), after incubation, the suspension was centrifuged at 13,000 rpm, 4 °C for 30 min. The pellets were used as nano-probiotics bacteria (Figure S2).

Antioxidant Activity

The antioxidant activity of the 10% free probiotic bacteria in freshly prepared suspension (10 g of freshly centrifuged bacterial cells in 100 mL phosphate-buffer saline (PBS), pH 7.4) and freshly prepared 10% nano-probiotics bacteria (10 g nano-probiotic in 100 mL PBS).

DPPH radical scavenging activity of the two groups of probiotics was determined according to Duz et al. [43]. Freshly prepared 0.05 mmol/L DPPH solution (1 mL) and 1 mL of the samples stored were incubated in the dark for 1h at 37 °C. After incubation, the mixtures were centrifuged at 10,000×g for 10 min at 20 °C, and then the optical density of the supernatant was read at 517 nm using a Shimadzu UV-2550PC UV–Vis Spectrophotometer. Ascorbic acid from the Sigma Aldrich sample was used as a positive control. DPPH radical scavenging activity (%) was calculated according to the following equation [43]:

$$DPPHradiclescavengingactivity(\%)=\frac{ODb-ODs}{\mathit{ODb}}\times 100$$

where ODs is the optical density of the sample, ODb is the optical density of PBS and DPPH solutions.

Fe⁺² ion chelating activity for the free and nano-probiotic cells was determined according to Decker and Welch [44] were applied. The reaction mixture consists of 1 mL of the prepared free cells suspension or nano-probiotic suspension, 0.05 mL of a 2 mmol/L FeCl₂ solution, and 0.2 mL of 5 mmol/L ferrozine and the reaction mixture was incubated in dark at 37 °C for 10 min. another reaction mixture was conducted with 1mL EDTA (1mg/mL) (control) was used for comparison. After incubation, the mixtures were centrifuged at 10,000 × g for 10 min at 20 °C, and then the optical density of the supernatant was read at 562 nm using (Shimadzu UV-2550PC UV–Vis Spectrophotometer). The chelating activity was calculated according to the following equation [44]:

$$\mathrm{Ferric}\mathrm{ion}\mathrm{chelating}\mathrm{activity}\left(\%\right)=\frac{ODc-ODs}{\mathit{ODc}}\times 100$$

where ODc is the optical density of the control (EDTA), ODs is the optical density of the sample.

Hydroxyl radical scavenging activity was determined according to Wang et al. [45]. The reaction mixture consisted of 1 mL Brilliant blue (0.435 mmol/L), 2 mL FeSO₄ (0.5 mmol/L), 1.5 mL H₂O₂ (3%, w/v), and 1 mL of the prepared samples was added to the mixture. The tubes were incubated in the dark at 37 °C for 1 h. Ascorbic acid (1 mg/mL) was used for comparison as positive control. After incubation, the mixtures were centrifuged at the same previous conditions, and the optical density of the supernatant was read at 624 nm using (Shimadzu UV-2550PC UV–Vis Spectrophotometer). The hydroxyl radical scavenging activity was calculated according to the following equation [45]:

$$\mathrm{Hydroxyl}\mathrm{Radical}\mathrm{Scavenging}\mathrm{Activity}\left(\%\right)=\frac{ODs-ODb}{ODo-ODb}\times 100$$

where ODs: is the optical density of the sample, ODb: is the optical density of the blank (reaction mixture with 1mL of PBS instead of the sample), ODo: is the optical density of the Brilliant blue without sample and the Fenton reaction system.

The superoxide anion radical scavenging activity assay mixture consists of 0.1 mL of the prepared samples, and 4.5 mL Tris–HCl solution (0.05 M, pH 8.2). The mixture was incubated for 20 min at 25 °C in a water bath, after incubation 0.4 mL pyrogallol (0.25 M) was added and then incubated for 4 min at 25 °C. After incubation was stopped with the addition of 8M HCl (0.1 mL), and then the samples were centrifuged at the same conditions and the optical density was read at 320nm using (Shimadzu UV-2550PC UV–Vis Spectrophotometer). A blank tube was prepared with Tris–HCl buffer instead of the samples. Ascorbic acid and BHA were used as positive control. The superoxide radical scavenging activity was calculated as follows [46]:

$$\mathrm{Superoxide}\mathrm{anion}\mathrm{radical}\mathrm{scavenging}\mathrm{activity}\left(\%\right)=\frac{ODb-ODs}{\mathit{ODb}}\times 100$$

where ODs is the optical density of the samples, and ODb is the optical density of the blank.

We followed the method of Hsu et al. [47] to determine the anti-lipid peroxidation activity of the free and the nano-probiotic cells. The reaction mixture consisted of 1 mL egg yolk suspension, 0.5 mL prepared sample suspension, 1 mL PBS, and 1 mL of iron sulfate (25 mM). The mixture was incubated on shaking (100rpm) for 15 min. at 37 °C. After incubation, 1 mL of 20% trichloroacetic acid was added and the tubes remained stable for 10 min, and then centrifuged at the same previous centrifugation condition. After centrifugation, we added 2 mL of 0.8% Thiobarbituric acid (TBA) to 3 mL of the supernatant, and then the tubes were incubated in a shaking boiling water bath for 10 min. After incubation the tubes were cooled and centrifuged as previously mentioned and the supernatant optical density was read at 532nm using (Shimadzu UV-2550PC UV–Vis Spectrophotometer). A blank tube was prepared with 0.5 mL of PBS instead of the sample, and ascorbic acid (1 mg/ml) was used as positive control. The Lipid peroxide inhibition rate (%) was calculated according to the following equation [47]:

$$\mathrm{Lipid}\mathrm{peroxide}\mathrm{inhibition}\mathrm{rate}\left(\%\right)=\frac{ODb-ODs}{\mathit{ODb}}\times 100$$

where ODs: is the optical density of the samples, and ODb: is the optical density of the blank.

Antibacterial Activity

Pathogenic Bacterial Species

We used Campylobacter jejuni (ATCC 49943) (Formerly, Vibrio jejuni), Bacteroides fragilis (ATCC 29762), Escherichia coli (ATCC BAA-2471), Fusobacterium nucleatum (ATCC 25586), Helicobacter pylori (ATCC 51407) (Formerly, Campylobacter pylori), Neisseria gonorrhoeae (ATCC 43070), Porphyromonas gingivalis (ATCC 33277), and Salmonella enterica (ATCC 14028) (formerly Salmonella choleraesuis) as pathogenic bacteria to study the antibacterial activity of the free and nao-probiotics. These pathogenic bacteria were purchased from the American Type Culture Collection.

Antibacterial Activity Determination

To assess the antibacterial activity of free probiotic and nano-probiotic bacteria, we employed the disk diffusion agar method [48]. The obtained free probiotic and nano-probiotic bacteria suspensions were applied to sterile agar disks (8 mm in diameter) for 5 min, and then the disks were incubated at 37 °C for 15 min to dry. These disks were positioned on the Mueller–Hinton Agar medium inoculated with the pathogenic bacteria. For 24 h, the cultures were incubated at 37 °C. After incubation, a ruler was used to measure the growth inhibition zone diameter of the free and nano-probiotics.

Statistical Analysis

The statistical analysis was performed by SPSS software (version 14). Data were expressed as mean ± standard error. We used the T-test, P < 0.01for the comparison between the free probiotic and nano-probiotic bacterium in the antibacterial activity and the comparison between the free probiotic or nano-probiotic bacterium and the control in the antioxidant activity [49].

Results and Discussion

Antioxidant Activity

One of the most dangerous disorders is the imbalance between the production of reactive oxygen species (ROS) and their detoxification through the antioxidant machinery in the living cells, which causes oxidative stress. Numerous diseases, such as inflammation, cancer, neurological disorders, cardiovascular diseases, and diabetes can be brought on by oxidative stress [50]. Thus, it is important and continuous need to search for natural safe antioxidants to be used as a protective tool against oxidative stress. Nowadays probiotics are receiving more scientific attention for their natural antioxidants [18, 20]. Probiotics have antioxidant activity through different mechanisms like the formation of antioxidant compounds, metal ions chelating activity, hydroxyl chelating activity, superoxide chelating activity, and peroxide chelating activity [51].

The DPPH radical scavenging method is one of the most widely used antioxidant methods in comparison to other tests because of its simplicity, speed, sensitivity, and repeatability. DPPH radicle measures the ability of the compounds in the cells to scavenge free radicle through the reduction of the DPPH radical's purple color after its addition which is directly related to the strain's increased antioxidant activity [52]. We determined the DPPH radicle scavenging activity of the free probiotic and nano-probiotic in comparison with ascorbic acid as control are found in Fig. 1. The free probiotic bacteria have DPPH radicle scavenging activity from the lowest value was about 47% in S laevolacticus and the maximum activity was about 58% in L lactis which is significantly less than that of the control. On the other hand, the encapsulation of the probiotic on the nano-chitosan (nano-probiotic bacteria) raised the DPPH radicle scavenging activity of the nano-probiotic bacteria to be non-significantly different from the control. Rwubuzizi et al. [16] reported that The DPPH radicle scavenging of probiotic bacteria ranges from 50 to 60%. The DPPH antioxidant activity of the probiotics can be related to the peptides released through proteolysis and phenolic and flavonoid compounds [14, 21, 53].

Fig. 1.

DPPH radicle scavenging activity of free and nano-chitosan encapsulated probiotics. Values are mean of three replicates. The bars represent standard deviation. Columns followed by asterisk are significantly different from the control according to paired-T test (P < 0.01)

The release of reactive oxygen species like the hydroxyl radical and superoxide anion can be catalyzed by transition metals such as ferrous. Additionally, ferrous ions are capable of catalyzing the dissolution of lipid peroxides. Fenton reactions, one of the main sources of the free radicles, are catalyzed with Fe⁺² ions. Thus, the decrease in Fe⁺² concentration acts as a preventative measure against oxidative damage [43]. The probiotic bacteria have a ferrous ion chelating capacity [54]. All of the free-investigated probiotic strains in the current study were shown to have Fe⁺² ion chelating capacity ranging from 43 to 50% (Fig. 2). The Fe⁺² ions' chelating capacity of the probiotic can be related to the chelating actions of the cell wall of some probiotics in addition some probiotics can synthesize metal-chelating proteins such as lactoferrin [55].

Fig. 2.

Ferric ions chelating activity of free and nano-chitosan encapsulated probiotics. Values are mean of three replicates. The bars represent standard deviation. Columns followed by asterisk are significantly different from the control according to paired-T test (P < 0.01)

Hydroxyl and superoxide anion radicles are considered the main ROS [56]. Probiotics are known for their ability to capture the released hydroxyl and superoxide anion radicles [57]. The results of the present study ensured the ability of the probiotics to scavenge the hydroxyl and superoxide anion radicles. The scavenging of the hydroxyl and superoxide anions radicle was about 40% (Figs. 3 and 4 respectively). The ability of the probiotics to release antioxidant enzymes such as superoxide dismutase, oxidases, and peroxidases is the main source of the probiotic capacities for hydroxyl and superoxide anion radicle detoxification [17].

Fig. 3.

Hydroxyl radicle scavenging activity of free and nano-chitosan encapsulated probiotics. Values are mean of three replicates. The bars represent standard deviation. Columns followed by asterisk are significantly different from the control according to paired-T test (P < 0.01)

Fig. 4.

Superoixde anion scavenging activity of free and nano-chitosan encapsulated probiotics. Values are mean of three replicates. The bars represent standard deviation. Columns followed by asterisk are significantly different from the control according to paired-T test (P < 0.01)

Lipid peroxidation is the most common process caused because of the increase in ROS. Lipid peroxidation is highly toxic in living cells as it causes the oxidation of the cell membrane phosphor lipids to peroxide which causes the membrane deterioration [58]. Lipid peroxide inhibition is one of the most important items that definite the antioxidant power of the probiotics [53]. The free probiotics showed anti-lipid peroxidation ranged from 51 to 61% (Fig. 5). The role of probiotics in the inhibition of lipid peroxidation is related to antioxidant enzymes such as superoxide dismutase, peroxidases, and oxidases, besides the presence of antioxidant compounds such as phenols and flavonoids [17].

Fig. 5.

Anti-lipid peroxidation activity of free and nano-chitosan encapsulated probiotics. Values are mean of three replicates. The bars represent standard deviation. Columns followed by asterisk are significantly different from the control according to paired-T test (P < 0.01)

Antibacterial Activity

The antibacterial activity of probiotics is well-known [59]. The studied free probiotics showed antibacterial activity with an inhibition zone ranging its diameter from 14mm in the case of E. faecium against B. fragilis and E. coli, to 28 mm in the case of L. lactis against C. jejuni (Fig. 6). The antibacterial activity of the probiotics is related to the probiotic ability to produce antibacterial compounds such as bacteriocins, organic acids, and other secondary metabolites with antimicrobial activity such as antibiotics [60]. Acharjee et al. [61] reported the antibacterial activity of some commercial probiotics on pathogenic bacteria due to the secretion of antibacterial activity and synergized with the synthetic antibiotics. Keeratikunakorn et al. [62] reported the antibacterial activity of the cell-free suspension from different probiotics against pathogenic bacteria as these suspensions contain antibacterial compounds. Lactobacillus probiotics cell-free suspension showed antibacterial activity against different pathogenic bacteria due to the antimicrobial compounds that inhibit the pathogen's adhesion to the host and destroy its genetic materials [63]. Lactic acid bacteria isolated from infants' feces have antimicrobial activity due to their ability to secrete antimicrobial compounds [64]. The antibacterial mechanisms of probiotics against pathogenic bacteria can be one of the different pathways, as follows: probiotics compete with the pathogenic bacteria on energy and nutrients. Probiotics secrete metabolites that inhibit the pathogen's growth. Probiotics produce disruptors that interrupt the quorum sensing of the bacteria which suppresses the growth ability, and the environmental tolerance. Probiotics also, reduce the virulence of the pathogen [65–67].

Fig. 6.

Antibacterial activity of free and nano-chitosan encapsulated probiotics against different pathogenic bacteria. Values are mean of three replicates. The bars represent standard deviation. Asterisks represent the significant difference between the free and the nano-encapsulated probiotics according to paired-T test (P < 0.01)

Effect of the Encapsulation on the Antioxidant and Antibacterial Activity of the Probiotics

The encapsulation of probiotics enhanced their antioxidant and antibacterial activity (supplementary Table Sl, S2) (Figures l, 2, 3, 4, 5, 6). This is in the same line with González-Ferrero et al. [68] who reported that the probiotic encapsulation increased the protective effect of the probiotics. This enhancement is related to the synergetic effect between the probiotics and the nano-chitosan which itself has antioxidant and antibacterial activity [69]. Similarly, the encapsulation of probiotics synergistically controls intestinal inflammation [70]. Encapsulation not only works synergistically with the probiotics but also, has a protective effect on the probiotics and their exsogenous active compounds [37]. In addition, the encapsulation of the probiotics prevents its degradation [71]. Alehosseini et al. [72] reported that the encapsulation of the probiotics provides a protective safe environment for probiotic viability. Zaeim et al. [73] reported that encapsulation of probiotics protects it from the digestive and proteolysis enzymes.

Conclusion and Future Prospective

Nano-chitosan encapsulation synergistically increased the antioxidant and antibacterial activity of the studied probiotics. This can be promising for controlling pathogenic bacteria. In vivo, studies are necessary to test the efficiency of nano-probiotics antioxidant and antibacterial activity in the living cells. Besides, a detailed study for the characterization of the nano-probiotic and its release profile data is needed.

Supplementary Information

Below is the link to the electronic supplementary material.

Data Availability

All data generated or analyzed during this study are included in this published article.

Declarations

Conflict of interest

We declare that we have no competing interests.