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Abstract The present study was conducted at Genetics and Cytology Department (National Research Centre), between 2018 and 2022, to address the persistent biofilm problem in dairy industries by researching for biofilm-eradicating agent(s) from microbial enzymes and biosynthesized nanomaterials. The enzymes were Actinomycetes protease serine-type and/or Bacillus deoxyribonuclease-B (NucB), selected based on the structural components of the biofilm matrix. The nanoparticles (NPs) was metal/metal oxide, e.g. Ag, CuO, and ZnO, that becomes a new focus of interest for treatment of the bacterial biofilms. At the beginning, isolation of the biofilm-forming bacteria on stainless steel surfaces in dairy industries was carried out to establish a biofilm model for the subsequent experiments. Microbial genetic techniques, including induction of mutation, protoplast fusion, and gene expression in heterologous system, were applied to improve the enzymes productivity. Statistical optimization for maximum possible yield was also applied. In addition, the most effective biosynthesized nanoparticles had subjected to the characterization techniques and toxicity assay. Combination study have been performed between enzyme and synthesized NPs to evaluate the synergistic benefits for biofilm eradication. The results could be summarized in the following points: 1. Isolation, identification and evaluation of nonstarter dairy biofilmforming bacteria post-pasteurization. A- Ten bacterial strains were isolated from five different sources and identified by means of the 16S rRNA gene sequences and the biochemical tests of API 50CH and 20E. Resultsrevealed that the ten isolates were members of Bacillus and related species and their sequences were deposited in NCBI GeneBank database under accession numbers from OM857595 to OM857604. B- The biofilm forming capability of the isolated strains was evaluated on foodgrade stainless steel (SS-316). Results reveled that only one strain exhibited Summary & Conclusion Radwan A. (2023). PhD., Fac. Sci., Ain Shams Univ. 106 high biofilm formation, three moderate strains, and six weak strains, while their mixed-species biofilm was increased 2.4-fold in comparison to the highest strain. 2. Activity-based screening for anti-biofilm protease and characterization of the effective. Fourteen extracellular crude proteases secreted by thermoalkali actinobacteria were screened on the basis of the anti-biofilm activity toward the 10 mixedspecies model of industrial dairy biofilms. The activity-based screening showed that two strains, i.e., Streptomyces sp. ACD/G413 and Streptomyces exfoliates 15/G710, produce anti-biofilm protease. characterization of these two proteases was carried out on different parameters including proteolytic inhibitors, optimum temperature and thermal stability, and pH activity and stability. The characterization results revealed that ACD/G413 and 15/G710 proteases were thermophilic alkaline proteases (TA-proteases) belong to serine-type. 3. Genetic improvement of thermophilic alkaline proteases strains and molecular analysis of the best products. A- St. exfoliates 15/G710 strain, which exhibited the most thermostable protease as well as highest removal activity toward the formed biofilm of dairy sporeformers, was subjected to UV irradiation and EMS mutagen at different exposure times. Results reveled that EMS induced changes in genetic material leads to over protease production. EMS mutagenic treatment increased the proteolytic activity of St. exfoliates 15/G710 strain by 1.96 fold. B- Interspecific protoplast fusion was conducted between Streptomyces exfoliates 15/G710_EMS.7 and Streptomyces sp. ACD/G413 strains. The best generated fusant (St-F.13) exhibited 1.97-fold than the highest parental strain (15/G710_EMS.7). The protease activity assayed under submerged culture of St-F.13 fusant, 15/G710_EMS.7 mutant, ACD/G413 parental strain, and 15/G710 parental strain were 395.89, 202.2, 135.74, and 102.09 U/mL, respectively. Thus, the estimated improvement for protease production after Summary & Conclusion Radwan A. (2023). PhD., Fac. Sci., Ain Shams Univ. 107 genetic techniques application was 2.9-fold than the highest parental strain (ACD/G413). C- Dendrogram based on RAPD-PCR analysis showed a genetic similarity between St-F.13 fusant and CD/G413 parent more than 15/G710_EMS.7 parent. SDS-PAGE and Zymogram assay indicated that 15/G710 parent might produce another protease along with that appeared at molecular mass ≈ 48 kDa which was similar to that detected in ACD/G413 parent and St-F.13 fusant. 4. Statistical optimization of reaction and production parameters for maximum possible protease yield of St-F13 fusant The statistical optimization were applied through Plackett–Burman design followed by a response surface methodology using MINITAB software. The optimized factors for increasing the reaction activity were pH, substrate concentration (Casein), temperature, and amount of enzyme per reaction, were selected from eight independent variables. However, the glucose, peptone (digest of casein), CaCO3, and inoculum size were the optimized factors for the production among of nine factors tested. Under the optimized conditions, protease productivity of St-F13 fusant increased from 395.89 to 505.13 U/mL, which represented about 1.27-fold. 5. Cloning of nucB gene in heterologous system for over-expression using vector pET 29a(+) and expression host E. coli BL21. The nucB (deoxyribonuclease-B) gene isolated from dairy biofilm-forming strain, B. paralicheniformis PMp/10, was amplified by PCR, cloned in vector pET 29a(+), and transformed into the expression host E. coli BL21 after propagation in DH5α. Expression analysis by SDS-PAGE showed unsatisfactory results related to overexpression, since high level of toxicity was observed after induction. At the same time, the analysis indicated activity of the constructed nucB gene of strain PMp/10, so another expression system required for such toxic protein. Gene sequence was deposited in GenBank database under accession number “OP712506” and title “Bacillus paralicheniformis str. PMp/10 NucB (nucB) gene, complete cds”. Summary & Conclusion Radwan A. (2023). PhD., Fac. Sci., Ain Shams Univ. 108 6. Biosynthesis of anti-biofilm nanomaterials, characterization, and evaluation of the best treatments. A- Ten different metal/metal oxide (Ag, CuO, and ZnO) NPS were synthesized microbially and screened for the anti-biofilm activity, individually and in combination (one-by-one) with proteinase K (Prot-K) and crude protease of St-F.13 fusant. The effective treatments were combination of the synthesized ZnO_G240 which improved the removal efficiency to 99.19% and 99.12%, for Prot-K and crude protease of St-F.13, respectively compared to un-treated biofilm. This ZnO_G240 NPs was characterized by TEM imaging, FTIR measurements, and XRD analysis and was evaluated in terms of toxicity using brine shrimp lethality assay B- Confirming biofilm eradication of the best treatments using CV stain and SEM for individual enzyme and combination treatments with ZnO_G240 NPs showed that the combination of the ZnO_G240 NPs with either Prot-K or StF.13 crude protease caused complete eradication of attached biofilms on stainless steel surface and confirmed the synergistic interaction between the ZnO_G240 NPs and both proteases. Conclusion: This study indicates the importance of using anti-biofilm degrading microbial enzymes in combination with biosynthesized nanoparticles alternative or complementary to the current clean in place (CIP) method to prevent recolonization by the released cells for biofilm eradication in dairy industries. |