Probiotic Bidobacterium Isolated from Chicken Intestine with PVP Showed Synergistic Effects on Reduction of AFB1

Food contamination with aatoxin is one of the most important concerns of health professionals. One of the best ways to reduce aatoxin content in food is to use probiotics. Therefore, this study was performed to isolate Bidobacterium from the chick's intestine and its probiotic activities and also its application with Polyvinylpyrrolidone (PVP) to reduce aatoxin B1 (AFB1) toxin in the medium were investigated. Samples were isolated from the chicken intestine. After preparing the samples, Bidobacterium was isolated and identied using biochemical and molecular methods. To measure probiotic activities, pH, bile, and salt tolerance tests were used. Then, the antimicrobial activity of isolate against gastrointestinal pathogens and the antibiotic susceptibility test were done. Then, the effect of selected isolate and PVP on reducing AFB1 in the medium was studied using ELISA and HPLC. Biochemical and molecular evaluations indicated isolation of B. bidum strain from chicken intestine. The selected strain showed antimicrobial activities on S. enterica, E. coli, and P. vulgaricus and was found to be resistant against Amikacin, Ampicillin, Erythromycin, and Ceftazidine antibiotics. The selected strain showed the ability to reduce the concentration of AFB1 in the medium (50% reduction) and when used in combination with PVP showed the synergistic effects in reducing the concentration of AFB1 from the medium (up to 90%). In conclusion, it was found that selected B. bidum strain together with PVP could have synergistic effects in reducing AFB1 toxin in medium up to 90%.


Introduction
Contamination of food products with a atoxins is one of the most serious concerns of the health system of today's societies (Mahato et al., 2019). A atoxin B1 (AFB1) is the most potent carcinogen whose hepatocarcinogenic effects have been studied (Chu et al., 2018). The most important biochemical effects of AFB1 are inhibition of DNA replication and RNA synthesis (Ferreira et al., 2019). In addition to the liver, a atoxins have been reported to cause signi cant pathological changes in other organs, such as the kidneys and spleen (Zeng et al., 2019).
Chemical, physical and biological methods have been used to remove AFB1 in food products (Sipos et al., 2021). Chemical methods are mainly based on the use of acids, alkalis, oxidants, and substances that can degrade mycotoxins, and of the physical methods, we can mention heat and UV treatments (Sipos et al., 2021). The use of many physical and chemical methods to remove mycotoxins from contaminated food is limited due to safety issues and the possibility of losing product quality, low e ciency, and high cost (Sipos et al., 2021). These problems have led researchers to focus on the use of microorganisms and plant extracts, nanoparticles, polymers, and biopolymers (Chaudhari et al., 2020;Makhuvele et al., 2020; Solis-Cruz, Hernandez-Patlan, Hargis, & Tellez, 2018). The biological approach, using microorganisms and their metabolites, is recommended as a promising alternative to the detoxi cation of mycotoxins (Guan et al., 2021). The use of bacteria to eliminate AFB1 is more practical due to some advantages such as more removal in a short period as well as no pigment production (Marrez, Shahy, El-Sayed, & Sultan, 2018). In recent years, various bacteria including lactic acid bacteria (LAB) such as Lactobacillus, Bi dobacterium, Propionibacterium, and Lactococcus in the AFB1 removal have been reported (Cuevas-González et al., 2020). The researchers showed that some species of LAB and bi dobacteria isolated from dairy products were able to remove a atoxins (Mahmood Fashandi, Abbasi, & Mousavi Khaneghah, 2018). It has been suggested that the binding of mycotoxins to LAB is a physical phenomenon related to the bacterial cell wall structure and that peptidoglycans and polysaccharides are two important factors for the binding of toxins to LAB (12). However, several components may be involved in the binding of AFB1 to LAB (Asurmendi, Gerbaldo, Pascual, & Barberis, 2020).
An important approach to prevent a atoxin toxicity is the use of absorbers (Wu et al., 2020). The adsorbents can bind to AFB1 and reduce the toxicity of the toxin. As a result, these adsorbents reduce the absorption of mycotoxins and reduce their distribution in the blood and target organisms. The adsorbents are high molecular weight compounds that can bind to mycotoxins and can form a stable mycotoxin adsorbent (Liu, Galani Yamdeu, Gong, & Or la, 2020). Polymers including cholestyramine, styrene, divinylbenzene, polyvinylpyrrolidone, and its modi ed form, polyvinylpolpyrrolidone are such adsorbents.
Polyvinylpyrrolidone (PVP) is one of the acetylene derivatives of vinylpyrrolidone monomer, which is supplied in powder and aqueous solution. Its powder form is white or milky in color, which can absorb moisture up to 40% of its weight (de Jesús Nava-Ramírez et al., 2021). Due to the presence of a highly polar amide ring and the possibility of hydrogen bond formation, this polymer dissolves in water and many other polar solvents (Čolović et al., 2019). The use of PVP has expanded rapidly in sensitive applications including pharmaceuticals and food industries due to its unique properties such as range of solubility and wide compatibility with other materials, physiological inertia, ability to form complexes with other materials, and inherent adhesion (Teodorescu, Bercea, & Morariu, 2019). Polyvinylpyrrolidone is recognized by the World Health Organization (WHO) as a polymer without side effects that is fully compatible with the human body (Kumar, Nehra, Dilbaghi, Tankeshwar, & Kim, 2018). This polymer can be used as an additive in all edible and non-edible products in solution, suspension, gel, and solid forms (tablet form) such as syrups and pharmaceutical tablets, gels, and creams. Therefore, due to the harmful effects of AFB1 on health, the present study was performed to study the effects of Bi dobacterium and PVP on the reduction of AFB1.

Sampling and culture of intestinal contents
Thirty local chicks over 15 days old and weighed over 100 g, were collected from the villages of Ardabil province, Iran. Sampling was performed by the Convenience Sampling method (Non-random Sampling). hrs at 37°C in anaerobic conditions and nally the colony-forming unit (CFU) was done.

pH Tolerance Test
The selected Bi dobacterium isolate was cultured in MRS broth culture medium with different pHs ranged from 1, 2, 3, 4, 5, 6, 7, 8, and 9 and incubated at 37°C for 48 hrs in anaerobic conditions. The growth of Bi dobacterium was measured with a spectrophotometer at 620 nm.

Bile tolerance test
The Bi dobacterium selected isolate was cultured on MRS broth supplemented with 0.1, 0.3, 0.6, 0.9, and 1.2% ox gall (pH6) and incubated at 37°C for 48hrs in anaerobic conditions. The growth of Bi dobacterium was measured with a spectrophotometer at 620 nm.

Salt tolerance test
The Bi dobacterium was cultured on MRS broth supplemented with 2.5%, 4.5%, 6.5%, and 8.5% NaCl and incubated at 37°C for 48hrs in anaerobic conditions. The growth of Bi dobacterium was measured with a spectrophotometer at 620 nm (KUSHARYATI et al., 2020).

Antibacterial activity and selected Bi dobacterium isolate
Antibacterial activities of Bi dobacterium produced metabolite were studied against gastrointestinal pathogens including Salmonella enterica, Escherichia coli, and Proteus vulgaricus using disc diffusion method, and the diameters of growth inhibition zone were measured.

Antibiotics resistance
The susceptibility and resistance of isolated bacteria to Amikacin, Fusidic acid, Ampicillin, Erythromycin, Ceftazidime, Amphotericin, and Chloramphenicol antibiotics were also assessed by disk diffusion method.

Molecular identi cation
Bacterial DNA extraction was performed using DBA extraction kit (SINACLON, Iran) and their quality was evaluated using the Nanodrop device (Nanodrop 2000c, Thermo Scienti c, Waltham, USA). The PCR reaction mixture consisted of Mastermix solution (Yekta Tajhiz Azma, Iran, Cat No: YT1553), enzyme buffer, MgCl 2 , and four dNTPs nucleotides. The 16S rRNA gene was used for molecular identi cation.
Agarose gel electrophoresis was used to evaluate the accuracy of PCR products and to determine the length of ampli ed fragments. Gene sequencing was performed at Fanavaran Gene Company (Tehran, Iran) (Nomoto et al., 2017).
2.6. In vitro study of the effect of Bi dobacterium and PVP on AFB1

Preparation of the standard AFB1
A atoxin B1 purchased from Sigma (Germany) was dissolved in the benzene-acetonitrile organic solvent according to manufacture instructions. Phosphate buffer was used to dilute the sample. To remove the organic solvent, a water bath was used at 80°C for 15 minutes.

Preparation of a atoxin from Aspergillus avus
First, A. avus (PTCC 5018) purchased from Iran Scienti c and Industrial Research Center was cultured in PDB medium in several asks and incubated at 26°C for two weeks. To extract a atoxin from the PDB medium, the contents of each ask were rst mixed uniformly. The contents of the asks were then passed through a Whatman 42 paper lter (with a porosity of 2 to 3 µm). For every 100 ml of ltered solution, 40 ml of chloroform solvent was added and the resulting mixture was stirred in a decanter funnel for 20 min. After 24 hrs, the lower phase containing chloroform solvent and a atoxin was isolated. 2.6.3. Measurement of free and Bi dobacterium bi dum attached a atoxin B1 Bi dobacterium selected isolate was cultured in broth MRS medium and after a maximum growth of 48 hours (growth was measured by spectrophotometry at 620 nm), the tubes containing the bacteria were centrifuged for 15 minutes at 3000 rpm. Bi dobacterium precipitate was washed 3 times each time with 5 ml of phosphate buffer solution (PBS) and added to 5 ml of standard a atoxin B1 solution extracted in separate vials. A atoxin B1 samples in PBS were used as a control. Falcons were incubated for 72 hrs at 37°C. The samples were collected at different time intervals (0, 24, and 48 hrs), then each centrifuged for 15 minutes at 4000 rpm. Free a atoxin was isolated for measurement. Samples were screened with an a atoxin B1 ELISA kit and the optimal sample was analyzed by HPLC and nally, the percentage of AFB1 bound to yeast was calculated (ELISA kit, ZellBio, Germany) (

Measurement of free and PVP-attached a atoxin B1
The PVP polymer solution was prepared according to international standards and serially diluted. After preparing different dilutions in each vial, 5 ml of standard and extracted AFB1 solution were added in separate vials for 48 hrs at 37°C. Samples were collected at different time intervals (0, 24, 48 hrs) and then each was centrifuged for 15 minutes at 4000 rpm to measure free a atoxin and supernatant were screened with a atoxin B1 ELISA kit and the optimal sample was analyzed by HPLC and nally the percentage of a atoxin bound to PVP was calculated.
To evaluate the effect of pH, the experiments were conducted at pH 5.5 and 8, and the percentage of polymer adsorption at different pHs was calculated by HPLC.
2.6.5. Measurement of the synergistic effect of Bi dobacterium bi dum and PVP in reducing the a atoxin B1 Bi dobacterium selected isolate was cultured in MRS broth medium and incubated for 48 hrs. The growth of Bi dobacterium was evaluated by spectrophotometry at 620 wavelength and then the medium centrifuged for 15 min at 3000 rpm. Bi dobacterium precipitate was added to 5 ml of standard and extracted AFB1 solution after washing three times with 5 ml of phosphate buffer solution (PBS). Also, PVP (with the optimal concentration obtained in the previous step) was added to the solution and incubated for 24 hrs at 37°C. Then centrifuged for 15 min at 4000 rpm and nally, the percentage of a atoxin bound to bacteria and PVP was calculated by HPLC.

Statistical analysis
Data were expressed as means± SD. A two-way analysis of variance was used for statistical analysis.
SPSS software version 26 was used for data analysis. Tukey's Multiple Range Test was used to compare the means. P <0.05 was considered as a signi cant level.

Isolation and identi cation of Bi dobacterium bi dum
In the present study, 25 isolates were isolated from 50 samples of chicken intestine samples that were studied using microbiological and biochemical tests. Using different probiotic tests, 4 strain was selected as the preferred Bi dobacterium strains. (Figure 1). The results showed that the isolated strains Bi dobacterium bi dum are gram-positive, pleomorphic rod shape, anaerobic, catalase-negative, capsule negative, agella negative, gelatin hydrolysis negative, indole negative, lacking motility, nitrate reduction negative, oxidase negative, spores negative, and Simmon Citrate negative.
The results of the sugar fermentation test showed that the isolated strains Bi dobacterium bi dum can ferment sugars such as Fructose-6-phosphate, Galactose, Glucose, Lactose, and Maltose but in the presence of Cellobiose, Inulin, Mannitol, Ra nose, Ribose, Salicin, Sorbitol, Starch, Sucrose, Trehalose, Xylan and Xylose sugars fermentation did not occur. Variable results were observed for the sugars including Amylose, Mannose, and Melibiose.

pH tolerance
At low pH (below 2) the isolated isolates did not show growth in acidic medium, but at higher pH (3≤pH≤5) bacterial growth was seen, and the highest growth of isolates was observed at pH 4.5 and 5 in acidic medium ( Figure 2). Also, the isolated Bi dobacterium strains grew in pH medium 8 and 9 and no growth arrest was observed at high pH. In general, these results indicate that the I3 isolated bacterial strain has a high tolerance to a wide range of pH, indicating its activity at the pH of the gastrointestinal tract is one of the important properties of probiotics.

NaCl tolerance
The growth of the selected bacterial strains at different NaCl concentrations was examined and the results showed that the selected I3 strain has a high tolerance to high concentrations of NaCl (Figure 3a), which indicates the appropriate probiotic activity of this selected strain.

Bile salt tolerance
Bi dobacterium strain (I3) showed good tolerance to different concentrations of Ox-gall from 0.1-1.2% ( gure 3b), which indicates a high tolerance of this strain to bile salt. Tolerance to different concentrations of bile salt is one of the important properties of probiotics and thus, the selected strain can be considered as a probiotic due to its high tolerance to bile salts.

Antimicrobial activities of selected Bi dobacterium isolate
Antibacterial activities of selected Bi dobacterium isolate were studied against S. enterica, E.coli, and P. vulgaricus using the disc diffusion method the results showed the highest growth of inhibition on S. enterica, but the lowest growth inhibition showed for E. coli (Figure 4a).

Antibiotics resistance
The results of antibiotic resistance test indicated that the selected strain of Bi dobacterium was resistant to Amikacin, Ampicillin, Erythromycin, and Ceftazidine antibiotics but was sensitive to Amphotericin and Chloramphenicol antibiotics (Figure 4b).

Molecular identi cation
DNA extraction of the selected Bi dobacterium strain was done and the sequencing results of 16S rRNA gene indicated that the isolated strain was from Bi dobacterium bi dum and showed 100% similarity to ZT-B1 strain ( Figure 5).

Detoxifying of AFB1 by selected Bi dobacterium strain and PVP
The selected Bi dobacterium strain caused a nearly two-fold decrease in standard and extracted AFB1 content over 24 hrs, indicating a high uptake of AFB1 into the surface of the strain wall (Figure 6a,  b). This optimal sample was analyzed by HPLC and the results showed that the isolate can adsorb and reduce standard and extracted AFB1 up to 48.32% and 47.47%, respectively.
The results of the present study showed a decrease in the concentration of standard and extracted AFB1 by PVP in the medium and also the relatively quickly binding of AFB1 to PVP occurred. After 24 hrs, a 50% decrease in standard and extracted AFB1 content was observed (Figure 6c, d).
The optimal sample, ie 2.5 mg/ml PVP during 24 hrs was analyzed by HPLC. HPLC results showed that PVP polymer with the above concentration can absorb and reduce a atoxin up to 52.78% and 55%, respectively.
The effect of using the isolated Bi dobacterium strain from the intestines of chickens and PVP (25 mg/ml) on the standard and extracted AFB1 content was studied and the results showed that it caused a severe reduction of the AFB1 (About 90%) content in the medium (Figure 6e, f). This indicates that the use of Bi dobacterium strain with PVP has a synergistic effect in reducing AFB1 content.
The results of AFB1 adsorption to PVP at pHs of 5.5 and 8 showed that there was no signi cant difference in terms of AFB1 adsorption to PVP polymer at different pHs.

Discussion
Identi cation of new species with probiotic activity is of great importance (de Melo Pereira, de Oliveira Coelho, Júnior, Thomaz-Soccol, & Soccol, 2018) and the probiotic activity of Bi dobacterium bi dum strains has been reported in various studies (Ku, Park, Ji, & You, 2016;Zanotti et al., 2015). In the present study, the Bi dobacterium bi dum strain was isolated from the intestine of chickens and was shown to have good probiotic activities. Also, the desired strain was able to reduce the concentration of AFB1 in the medium and when used in combination with PVP in the environment, synergistic effects were observed in reducing the concentration of AFB1 in the medium.
Although the use of biochemical methods is still widely used in the identi cation of bacteria (Marzan, Hossain, Mina, Akter, & Chowdhury, 2017), the use of molecular identi cation methods such as polymerase chain reaction (PCR) in the detection of bacterial strains allows identifying of them more accurately (Skowronek et al., 2020). In the present study, in addition to biochemical methods, the 16S rRNA gene ampli cation method was used to make a more accurate identi cation, and the results showed that the selected Bi dobacterium bi dum has 100% identity with ZT-B1.
As mentioned in recent studies, the antimicrobial properties of Bi dobacterium are important probiotic features (Monteiro et al., 2019; Yasmin et al., 2020) that showed in the present study in a selected strain isolated from chicken intestine, and antimicrobial effects were seen on all three pathogens S. enterica, E. coli, and P. vulgaricus using the disc diffusion method. Such a feature has been reported in other studies (Invernici et al., 2018;Prabhurajeshwar & Chandrakanth, 2019). This may be due to the production of bacteriocins and mechanisms including the production of bacterial inhibitory compounds, the regulation of intestinal pH, the strengthening of the immune system, the blockage of bacterial binding sites, competition for nutrient uptake, the production of organic acids such as acetic acid, propionic acid, phenyl lactic acid, formic acid or free fatty acids and so on ( The good resistance of selected bacterium strain to different concentrations of Ox-gall bile salt was observed in the present study, which can be attributed to the ability of the strain to reduce the detergent effects of bile salts (Rastogi, Mittal, & Singh, 2019). This indicates the probiotic activity of the selected strain that can resist and continue to grow at different concentrations of bile salts. Also, high resistance of the selected strain to a wide range of pHs was observed in the present study, which is considered an important characteristic of probiotics, indicating their resistance to gastrointestinal conditions (Balasingham, Valli, Radhakrishnan, & Balasuramanyam, 2017). Survival of other probiotic strains even at pH 1 has been reported in other studies (Maragkoudakis, Chingwaru, Gradisnik, Tsakalidou, & Cencic, 2010). Also, another feature of probiotics is their tolerance to different concentrations of NaCl, which was observed in the current study. Overall, these results indicate the appropriate probiotic activities of the selected strain that can be considered in future studies.
Aspergillus avus grows on a wide range of agricultural products and foodstuffs and infects them by producing AFB1 toxin. A atoxins, as one of the most toxic fungal metabolites, weaken the body's immune system and cause cancers and tumors in humans (Fouad et al., 2019). A atoxin contamination of crops is a devastating global problem in agriculture, food and livestock industries. The use of biological methods, such as the use of antagonistic bacteria, can inactivate and degrade a atoxins by producing certain biological compounds. The results of the current study showed a decrease in AFB1 concentration with the application of selected strain Bi dobacterium bi dum in the medium. Also, the greatest decrease in AFB1 occurred in the next 24 hrs, indicating that binding of the toxin to the selected strain is a rapid process. Speci c binding of a atoxin M1 in dairy products to LAB has been reported in studies (Ismail et al., 2016). In this study, the ability of bacteria to adsorb toxins in the medium was investigated and the results showed that the adsorption process is rapid. This adsorption appears to be due to the reaction of AFB1 with the bacterial surface wall without chemical modi cation of the toxin (Zolfaghari, Khezerlou, Ehsani, & Khosroushahi, 2020). The ndings of the present study show that Bi dobacterium bi dum has a high ability to adsorb high amounts of AFB1 and this can be attributed to the composition of the bacterial cell wall.
One of the most important strategies used to reduce the risk of a atoxin contamination is the use of toxin binders to reduce the adsorption of a atoxin in the gastrointestinal tract. In the present study, it was shown that PVP in a concentration-dependent manner could reduce the concentration of AFB1 in the medium and the highest percentage of AFB1 adsorb from the medium was observed at a concentration of 25 mg/ml. Polyvinylpyrrolidone, which dissolves in water and bio uids, is a non-toxic and indigestible polymer (Fadeeva, Barinov, & Fomin, 2020). With such special properties, this polymer has found wide applications in the preparation of pharmaceutical compounds and medical research (Fadeeva et al., 2020). The current study is the rst to investigate the effects of PVP on AFB1 contamination, and the results showed that it can reduce the AFB1 content in the medium that can be attributed to AFB1 binding to the structure of this compound.
The synergistic effect of simultaneous application of PVP and selected strains was observed in reducing standard and extracted AFB1 in the medium. It seems that the cell walls of selected bacterial strains together with the structural properties of PVP are effective in drastically reducing the concentration of AFB1 in the medium. To the best of our knowledge, this is the rst study to examine the effect of a combination of Bi dobacterium bi dum strain and PVP in reducing AFB1. However, more studies are needed to con rm this.
In conclusion, it can be concluded that the selected Bi dobacterium bi dum strain has probiotic properties and the ability to reduce AFB1 in the medium, and when used in combination with PVP, shows a synergic effect on reducing AFB1 in the medium. However, in vivo studies are needed to con rm its effectiveness.

Consent for publication
Not applicable Availability of data and materials All data generated or analyzed during this study are included in this published article [and its supplementary information les]. Raw sequence data on 16s RNA gene had been submitted to the NCBI Sequence Read Archive (SRA) with the accession number PRJNA730567 (https://www.ncbi.nlm.nih.gov/sra/PRJNA730567).
MRF and AAS managed the project and conceptualized it. FSM provided the samples for the study. SAA designed and performed the experiments, completed the data analysis, submitted sequence data to GenBank, and writing the manuscript. All authors reviewed and agreed to the published version of the manuscript. Figure 1 a The four selected Bi dobacterium spp. isolates colonies morphology on MRS Agar medium. b Microscopic image (1000 ×) of Gram-stained Bi dobacterium spp. smears illustrating the Gram-positive bacilli.

Figures
Page 16/19 Figure 2 The growth of four selected Bi dobacterium isolates after cultured 48 hours in the MRS broth with different pHs ranging from 1 to 9.   a The effects of selected Bi dobacterium spp. isolate on growth on three gastrointestinal pathogens S. enterica, E.coli, and P. vulgaricus. b Antibiotic resistance test Figure 5 The results of blasting of sequences of the 16S rRNA of the isolated Bi dobacterium bi dum strain with 9 strains.