الفهرس 
Literature ReviewOnly 14 pages are availabe for public view
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Abstract
10. Summary
The first documentation of biofilm-forming bacteria was done roughly 80 years back in 1943. Biofilms which formed within just a few minutes, and followed by mature biofilms developing within a few days, accelerate growth on different material surfaces that resistant to disinfectants and sanitizers, remain a significant public health- safety related issue represent the major challenge and hazard on human health.
The existence of biofilm is the main carrier of microbial communities has been explored for several years in most industries such as food, beverages, dairy & IFM products and throughout drinking water distribution systems (DWDSs), significant impact on the safety of industrial products and drinking water.
Due to the qualitative and quantitative deficiencies of the neonatal innate immune system, IFM industry requires very high levels of microbiological quality and must conform to national
and international microbiological criteria. However, on the basis of the available manufacturing technologies, the generation of a completely sterile product remains impossible. Consequently, intrinsic contamination of IFM may be a cause of possibly serious illness in infants. Indeed, there are risks of bacterial infection associated with using IFM and many contamination incidents have occurred worldwide. The World Health Organization (WHO) recommends breast milk as the ideal food source for infant growth and development in 2020.
The present study provides insights into biofilm evolution over time interval to enhance the presently available treatment designing. Importantly, the treated surface with natural products retained its anti-biofilm ability, that provide an antimicrobial surface.
The purposes of this study are as follows:
Bacterial growth curve and confirm the ability of the tested bacterial strains to form biofilm on polystyrene tubes and PVC chips by tube adherence method for the detection of biofilm which can be an economical and effective method, using locally commercial mineral water, milk and juice as media.
Efficacy of mimicking mother’s behavior using locally available cleansing methods on the bacterial biofilm formation on feeding bottle’s different surfaces and feeding beverages such as IFM and baby drink as media.
Quantitively evaluation of the bacterial biofilm formation on various surfaces of feeding bottle PP, PC and SI teats using baby beverages.
Phytochemical analysis of natural products (Propolis and Chitosan).
Detection of the anti-bacterial efficacy of propolis and chitosan.
Detection the efficiency of propolis and chitosan as bacterial anti-biofilm agents.
Application of the natural products combination as anti-biofilm agents on various feeding bottle surfaces using IFM and baby drink.
Antifungal activity of propolis and chitosan.
The biochemical results of EEP and CS (invitro/in vivo) liver markers ALT, AST and kidney function; CRE were investigated.
The conclusion and application of this study as follows:
Log phase was from (2-16h) for P. aeruginosa ATCC27 27853, E. coli ATCC 8739 and L. monocytogenes ATCC19116.
All tested bacterial strains had the ability for the biofilm formation on polystyrene tubes that at zero-time, E. coli, for P. aeruginosa and L. monocytogenes were indicated as non-biofilm formation with OD600:0.008, 0.009 and 0.013, respectively except mixed species had moderate biofilm formation (OD600: 0.350) while, at 6h all tested bacterial strains and mixed species indicated as strongest biofilm formation.
Development and biofilm formation of the tested bacterial strains reveled that mixed species showed the highest biofilm formation using locally milk (OD600: 1.082) followed by P. aeruginosa in mineral water (OD600: 0.551) and E.coli in juice, (OD600: 0.462) at 6h.
While P. aeruginosa formed the highest biofilm on PVC chips using mineral water and juice with OD600:1.336, 0.876, respectively, followed by E. coli with milk as media (OD600: 1.207) at 6h, respectively.
The tested bacterial strains exhibited biofilm formation on various feeding bottle’s surfaces PP, PC & SI teats and the tendency of P. aeruginosa, E. coli and Dual spp. to form highly biofilm on SI teats surface with (OD600 :0.988, 0.933 and 0.742), respectively.
In case of IFM as medium , P. aeruginosa showed highly biofilm formation on PP and PC surfaces with OD600 :1.418 and 1.058, respectively while highly biofilm formation of E. coli and Dual spp. were detected on PP & PC surfaces with (OD600 : 0.647 and 0.837), respectively.
Notably, in case of baby drink, Dual spp. and P. aeruginosa revealed the highest biofilm formation on SI teats with OD600 :1.334 and 1.333, respectively.
Substantiation i.e., the different cleansing methods when mimic mother’s behavior were ineffective to eradicate biofilm formation of the tested bacterial strains on different surfaces of feeding bottles with tap water at room temperature, soapy water and boiled water either in IFM or baby drink.
Phytochemical analysis of natural product “propolis” that is one of natural preservatives considered as a potent antiseptic substance, successfully used in several clinical settings, without concomitant toxic/deleterious effects and all over the world, the ethanolic extracted propolis is considered pharmaceutical product and its chemical analysis has pointed to the presence of total phenolic content 0.807 - 0.801mg GAE/g , total flavonoid content 0.741- 0.745 mg CE/g and antioxidant power activity range 1.15 to 1.18 mg TE/g., showing antibacterial effect on P. aeruginosa and E.coli with inhibition diameter 21 and 15 mm, respectively.
In addition, “chitosan” (CS) which a natural polymer with MW (448877) and degree of deacetylation (75-85%), with antioxidant activity with IC50 = 10.21 ± 0.77 µg/ml has been widely used in medical, food packaging and other fields due to its antifungal and antibiofilm properties but showed non-antibacterial effect on the tested Gram- negative bacterial strains.
The antibiofilm activity of EEP (0.05gm/ml) and chitosan (0.02gm/ml) by pre-drenched the feeding bottle’s surfaces in for 24h, separately against the tested bacterial strains revealed that the inhibition of the bacterial biofilm formation on all treated surfaces.
It was noticed, the antibiofilm activity of EEP on PC treated surface was the highly efficacy on E.coli and P. aeruginosa biofilm formation with inhibition percentage (-7.6%) and (-4.5%), respectively. Also, PC treated surface with CS showed the highly antibiofilm activity on P. aeruginosa biofilm formation with (-6.3%), in case of IFM as medium.
Notably, PP treated surface with EEP exhibited the most potent efficacy of P. aeruginosa biofilm formation (-0.15%), in baby drink due to its natural antimicrobial ingredients
Synergism efficiency of different ratios combination of natural products on the bacterial biofilm formation revealed that all different ratios had antibiofilm property.
The most potent ratio showed highly efficacy on P. aeruginosa biofilm formation using IFM as medium was chitosan to propolis (v/v) [1:3] on PP, SI teats and PC treated surfaces inhibited by
(-0.2%), (-2.2%), and (-6.4%), respectively.
The efficacy ratio of chitosan to propolis was (v/v) [1:4] on PC, PP and SI teats treated surfaces inhibited by (-4.0%), (-2.9%), and
(-4.9%), using baby drink as medium, respectively.
The ratio of chitosan to propolis [1:4] and propolis to chitosan [1:1] had the highest inhibition of E.coli biofilm formation on different treated surfaces in case of IFM as medium.
Antifungal activity of different concentrations of EEP and CS proved inhibition of all tested fugal strains biomass and 0.0125gm/ml of EEP inhibit Penicillium spp. NRC4290 with IB (80.1%) while 0.025gm/ml of EEP inhibit Aspergillus terrreus KU21241 and Aspergillus niger ATCC 16404 with IB: 69.6 and 54%, respectively.
while, 0.005 ,0.02 and 0.01 gm/ml of CS shown the highest efficacy against Aspergillus niger ATCC 16404, Aspergillus terrreus and KU21241 Penicillium spp. NRC4290 with IB: 60.6, 51.4 and 47.9%, respectively.
Synergism effect of different ratios combination of the natural products exhibited that the most potent ratios was chitosan to propolis (v/v) [1:4] and its inversed ratio on the inhibition of the tested fungal strains with IB: 88.6, 75.9 and 61.6% for Penicillium spp. NRC4290, Aspergillus niger ATCC 16404 and Aspergillus terrreus KU21241, respectively.
Notably, the inhibition of Penicillium spp., NRC4290, Aspergillus terrreus KU21241 and Aspergillus niger ATCC 16404 with IB; 55.3, 51.1 and 47.7%, respectively for inversed one.
The toxicological effects of EEP and CS on Swiss albino mice were investigated that there was not any signs of acute toxicity and mortality during the first 24h, and hepatotoxicity[Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) [and renal biomarkers (Creatine)] were specifically targeted and assessed during the subacute treatment to investigate the harmful effects of the natural products.
After 7 days of daily treatment with both natural products. It was shown that there were no significant changes appeared on ALT activity, and it was estimated for EEP and CS treatment by 52.00 units/liter (-5.45%), 45.00units/liter (-18.18%), respectively.
While, CS treatment only had a substantial impact on the AST, which decreased by (-36.60%) compared to the control group (without treatment) and estimated by 170.33 unit/L and AST was estimated by 233.50 unit/L (-13.09%) for EEP treatment.
The ALT and AST levels were not significantly differed from control animals after a 14-day of recovery period and AST values after treatment with EEP and CS were 152.83 (24.25%) and 176.00 (43.09%) unit/L, respectively, whereas ALT values were 48.00 (-14.79%) and 49.50 (-12.12%) unit/L, respectively.
In vivo, the present results provide opportunities to advance the use of bee products like EEP and natural polymer like chitosan as promising medicinal sources as they have not cytotoxic effects.
Conclusions: Incorporating EEP and CS may be a promising medicament in reducing Gram negative P.aeruginosa and E.coli biofilm formation on various feeding bottle’s surfaces and also, antifungal activity against Aspergillus niger ATCC 16404, Aspergillus terrreus KU21241 and Penicillium spp. NRC4290.