الفهرس 
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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.