Identification of the isolates and assessment of the pectinase activity
Isolates AGS3, gram-negative, non-acid-fast, aerobic, rod-shaped, catalase-positive, oxidase-positive, citrate positive, and motile and S4 gram-positive, non-acid-fast, aerobic, rod-shaped, catalase-negative, oxidase negative, citrate negative, and not motile (Fig. 1b) were selected according to their clear halo area radius (Fig. 1a). Optimal growth conditions for both isolates were at 30 °C and pH 6.8–7.0. Results of phylogenetic analysis which was performed using the maximum-likelihood method showed that isolate AGS3 belongs to the Proteobacteria phylum, Alcaligenaceae family, and Alcaligenes genus, and S4 was a member of Firmicutes phylum, Paenibacillaceae family, and the genus of Paenibacillus (Fig. 1c). Further, BLAST analysis revealed that AGS3 and S4 isolates belonged to the species Alcaligenes faecalis and Paenibacillus polymyxa respectively. Henceforth, the sequence of isolates A. faecalis AGS3 (MZ093052) and P. polymyxa S4 (MZ596260) was submitted to GenBank, The National Center for Biotechnology Information (NCBI).
Alcaligenes faecalis is best known for anaerobic respiration with nitrite, and degradation of microbial reserve polymers such as poly-(3-hydroxybutyrate) (PHB). A. faecalis has also been used in the production of nonstandard amino acids. Although A. faecalis is recognized to have a variety of hydrolytic enzymes that can be used in the biodegradation of plant wastes, the study on its potential needs more progress26.
Paenibacillus polymyxa is recognized for producing a wide variety of secondary metabolites, which enables it to resist various environmental stresses and makes it an auspicious biotechnological agent in agriculture and industrial processes. P. polymyxa has the nitrogen-fixing ability, as well as the production of plant growth-regulating factors, hydrolytic enzymes, and antibiotic compounds. P. polymyxa strains are known for producing several hydrolytic enzymes including proteases, β-1,3-glucanases, cellulases, xylanase, lipase, amylase, chitinases, and pectinase. Therefore, various studies investigated P. polymyxa strains’ capability in waste management and wastewater treatment. However, compounds produced by the hydrolytic activity of P. polymyxa enzymes have not been yet identified and need more investigation27.
Pectin degradation assay was employed using DNS reagent to determine the number of pectic oligo-saccharides (POS) produced by pectinase activity of A. faecalis AGS3 and P. polymyxa S4 isolates. The results of determination of the highest yield of POS release showed that A. faecalis AGS3 (Fig. 2a) and P. polymyxa S4 (Fig. 2b) isolates have their maximum concentration of released sugars after 20 h and 4 h incubation at 30 °C, 180 rpm respectively.
In the end, obtained POSs were lyophilized and stored for further analysis.
Thin-layer chromatography and LC–ESI–MS of obtained products
Thin-layer chromatography (TLC) was performed to verify the degradation of pectin by the isolates. TLC patterns confirmed the degradation of pectin to POS in both isolates (Fig. 3a). Further analysis of produced oligo-galacturonic acids was implemented by LC–ESI–MS of lyophilized samples. The LC–ESI–MS mass spectrums of A. faecalis AGS3 and P. polymyxa S4 isolates products fractions showed that different types of oligo-galacturonic acids were present in enzymatic products of pectin degradation in both isolates (Fig. 3b, c, Table 1). In the end, forms of mono galacturonic acid and unsaturated mono, di, tri, and penta galacturonic acid were identified. There are different types of pectinolytic enzymes. Among them reaction conducted by pectin lyase, produces unsaturated oligo-galacturonates, therefore considering products obtained in this study, it is assumed that pectinolytic activity of A. faecalis AGS3 and P. polymyxa S4 isolates are due to pectin lyase enzyme.
In general, most reported pectinases belong to the fungi such as Aspergillus, Alternaria, and Penicillium28,29,30,31. Nevertheless, there have been reports showing various types of pectinolytic enzymes in the bacteria; for instance, Acinetobacter guillouiae, Kosakonia sacchari, and Bacillus vallismortis have been reported to have polygalacturonases. Pectin and pectate lyases have been proven to be present in Streptomyces, Actinomycetes, Pseudomonas, and Bacillus species31,32. There are some endophytic pathogens such as Xanthomonas compestris, Ervinia chrysanthemi, Colletotrichum lindemuthianum, Pseudomonas siringea, and Phytophthora capsici which are reported to have pectinolytic activity31. Studies suggest that some enteric pathogens including Salmonella and Escherichia coli, despite their lack of pectinolytic enzymes, are capable of using oligomers that result from pectin degradation10,31. We believe that this study is the first to identify compounds obtained from the pectinolytic activity of A. faecalis and P. polymyxa.
Determination of antioxidative effects of POS
Radical scavenging activity (RSA) of obtained POSs, was tested by the DPPH reagent. The results for a range of concentrations from 1.25 to 80 mg/mL revealed that the antioxidative effects of POSs get higher by raising their concentrations. RSA of POS obtained from A. faecalis AGS3 isolate ranged from 18 to 81% for 1.25 to 80 mg/mL concentrations, and the results for P. polymyxa showed a range of RSA from 13 to 74% for the same concentrations (Fig. 4).
Oxidative stress is a concept that is associated with the loss of balance between pro-oxidants and antioxidants and is related to the physiology of common diseases. Antioxidants have their role in neutralizing reactive forms of oxygen with a negative impact on living cells33. Yeung et al.34 used the Fenton reaction to hydrolyze okra pectin and found that the antioxidant activity of obtained POS is concentration-dependent. The results of the DPPH assay of POS obtained by enzymatic hydrolyzation Streptomyces hydrogenases YAM1 showed that RSA increases in higher concentrations. Hosseini Abari et al.10 also demonstrated that enzymatically modified pectin has more antioxidative activity compared to untreated pectic polysaccharides. Similar studies corroborate the dose-dependency of POSs antioxidative activity. In this study, as shown in Fig. 6, regarding an increment of antioxidant properties of samples by concentration, the highest RSA was reported in 80 mg/mL of samples obtained from A. faecalis AGS3 and P. polymyxa S4. In 20 mg/mL of POS, results were 59% for P. polymyxa S4 and 69% for A. faecalis AGS3, which showed a 20–30% raise in RSA, compared to polygalacturonic acids as mentioned in Hosseini Abari et al. study10. These results show a significant increment in RSA of POS in comparison to the results of the antioxidative activity of pectic polysaccharides from previous studies. This is the first report of RSA in products of A. faecalis and P. polymyxa species.
Determination of the anticancer effects on MCF‑7 human breast cancer cells.
An assessment carried out by MTT assay and flowcytometry showed significant anticancer activity on MCF-7 cells for POS obtained from apple waste using pectinolytic enzymes of P. polymyxa S4 and A. faecalis AGS3 isolates. As mentioned in Fig. 5, results of the MTT assay showed a maximum cell viability reduction at 40 mg/mL of POSs obtained from A. faecalis AGS3 and P. polymyxa S4 with 93% and 91% respectively after 48 h incubation. Minimum cell viability reduction was obtained at 1.25 mg/mL of POS after 24 h of treatment with 17% and 37% for A. faecalis AGS3 and P. polymyxa S4, respectively.
Likewise, results of flow cytometric analysis at 5 and 40 mg/mL of obtained POSs after 48 h incubation demonstrated induction of apoptosis, with 84% and 100% for A. faecalis AGS3, and 90% and 98% for P. polymyxa S4 (Fig. 6b). In Fig. 6b M1 zone represents the distribution of living cells, not stained by the PI reagent, and the M2 zone demonstrates dead cells, stained by the PI reagent. As is visible in Fig. 6a, treated cells also were subject to morphological changes.
Cancer as one of the significant health problems in the world, has numerous physiological and biochemical inducers called carcinogens. The majority of synthetic drugs used in chemotropic treatments due to their side effects on non-cancerous cells and causing drug resistance of cancerous cells can originate more problems for patients, therefore using natural compounds with high anticancer properties has become a superior solution10,13,35. Studies have shown that enzymatically hydrolyzed citrus pectin fragments can affect the progression of proliferative prostate cancer by reducing serum PSA by 50% after 14 months of treatment. It has been proven that enzymatically treated citrus and apple pectins can inhibit growth and induce apoptosis in human intestinal cancer cells19. Low molecular weight modified apple pectin inhibits the cell cycle in colorectal tumors in human colorectal cancer cells (HT-29) in vitro and colitis-associated colorectal cancer in mice36. The same low molecular weight apple pectin reduced the risk of colon cancer tumors in mice and was reported to bind to galactin-318. Examination of the monosaccharide composition showed that galacturonic acid was the major component of the modified pectin structure and only small amounts of galactose were found in it, however, the monosaccharide composition is similar to that of pectasol monosaccharides. While MCP may not be rich in galactooligo-saccharides, it can be selectively absorbed in the small intestine compared to oligo-galacturonic acids37. In the study of Delphi et al. on MDA-MB-231 cancer cells, treatment with POS induced apoptosis and reduced survival of cancer cells38. The results of measuring the effect of pectic oligosaccharides and pectic polysaccharides on the HT29 cancer cell line by Li et al.39 showed that POSs are more effective in inhibiting cancerous cells in much lower concentrations than pectin. Hosseini Abari et al.10 demonstrated 92% apoptosis of MCF-7 cell line after 24 h treatment with 20 mg/mL POS which was significantly higher than pectin’s effect in the same concentration. Supporting previous results, our study obtained oligosaccharides that showed significant anticancer properties for both P. polymyxa S4 and A. faecalis AGS3 samples. To the best of our knowledge, there are no previous reports of anti-tumor properties for P. polymyxa and A. faecalis species products.
Determination of the cytotoxicity effects of POS on L-929 mouse fibroblast cells
Cytotoxicity effects of obtained POSs on L-929 cells were determined after 48 h incubation by MTT assay. L-929 cells were treated with 5 and 40 mg/mL of obtained products and untreated cells were used as control. As mentioned in Fig. 7a, the analysis showed no significant toxicity for A. faecalis AGS3 with less than 3% death, and P. polymyxa S4 with about 2% death. Morphology of treated cells as shown in Fig. 7b confirmed the results of the MTT assay.
Cytotoxicity of drugs used in treating cancer on non-cancerous cells is a major concern in chemotropic treatments. Opposing to previous results from Delphi et al. which showed toxicity on high concentrations of POS on HUVEC cells, in the current study after 48 h treatment of L-929 cells with 40 mg/mL of POSs, no significant toxicity was observed38. According to our information, no other study investigated the cytotoxicity of POS obtained from A. faecalis and P. polymyxa.