Gıdalarda Kullanılmak Üzere Bakteriyofaj Temelli Enkapsülasyon Yöntemlerinin İncelenmesi, Optimizasyon ve Depolama Etkinliği Tayini
Abstract
It is known that bacteriophages are effective antimicrobial agents in biocontrol applications related to food preservation and safety. While exploiting this potential is an exciting future prospect, there is an urgent need to produce safe and effective bacteriophage products to be successful. The widespread use of bacteriophages requires advanced methodologies for medium and long-term storage and easy transportation. The characteristics of the chosen method, formulations, and high specificity of the process conditions to the bacteriophage to be treated bring the need to produce alternative methods. Within the scope of this thesis, research has been carried out under two main headings regarding the use of bacteriophages as biocontrol agents in food samples: i) drying of bacteriophages via freeze drying (lyophilization), spray drying and vacuum foam drying method, monitoring the drying process effect and investigation of storage stability, ii) investigation of antimicrobial activity of dried powders in raw chicken meat and bacteriophage-based edible coating for the biocontrol of Salmonella in strawberry samples. Within the scope of this thesis, for the first time in the literature, the effectiveness of powder bacteriophage formulations, which were stored for a certain time and temperature after drying, was tested in real food samples.
In this study, it is aimed to evaluate the use of freeze drying, spray drying and vacuum foam drying methods in the production of bacteriophage powders, formulation design, storage stability and activity at two different temperatures. In the lyophilization, 12 different formulations containing bacteriophage cocktail were prepared and the cryoprotectants at different concentrations used can be listed as follows: sucrose, mannitol, polyethlyneglycol, and sucrose gelatin mixture. During the lyophilization process, a decrease in the bacteriophage titer of 0.59-2.35 log PFU was observed in the cryoprotectants used. The phage-loaded 0.3 M sucrose gelatin mixture at 4±1 °Ϲ and 25±1 °Ϲ displayed significantly less phage titer loss (p<0.05) than the other excipients and liquid phage cocktail at the end of the 12 months storage. In the spray drying, phage cocktails containing different amounts of mannitol, casein, skimmed milk and maltodextrin was studied. During the spray drying process, 1.19-1.95 log PFU reduction in bacteriophage titer was observed in the excipients used. Among the excipients, the phage-loaded 15% maltodextrin and 5% casein displayed significantly less phage titer loss (p<0.05) than the other excipients and liquid phage cocktail at the end of the 12 months storage period at 4±1 °Ϲ and 25±1 °Ϲ, respectively. The effect of vacuum foam drying process, which allows drying under mild conditions, on drying process losses and bacteriophage titer was investigated. In this study, drying of S. Enteritidis F5-4 bacteriophage in two different formulations was studied using bovine serum albumin+glycerol and bovine serum albumin+sucrose formulations. For bovine serum albumin+sucrose formulation, decrease in bacteriophage titer was observed in three different process conditions and 0.68 log PFU titer reduction in formulation containing 15% (a/h) bovine serum albumin + 10% (h/h) sucrose under Process II (35±1 °Ϲ, 0.06 MPa, 5 h) conditions observed as the most successful result.
The effect of the most stable bacteriophage powder formulation on the biocontrol of Salmonella was investigated after 6 days of storage at 4±1 °C in raw chicken meat samples. In the lyophilization study, the results showed that there were significant reductions of Salmonella at the end of the storage in chicken meat for newly prepared phage powder (1.86 log CFU/cm2 and 2.18 log CFU/cm2), lyophilized phage powders stored at 4±1 °Ϲ (1.08 log CFU/cm2 and 1.26 log CFU/cm2) and stored at 25±1 °Ϲ (0.66 log CFU/cm2 and 1.00 log CFU/cm2) for 10 months at MOI 100 and 1000, respectively. In the spray drying study, the results showed that there were significant reductions of Salmonella at the end of the storage in chicken meat for newly prepared dried phage powder containing 15% maltodextrin (1.85 log CFU/cm2 and 2.13 log CFU/cm2) and 5% casein (1.78 log CFU/cm2 and 2.09 log CFU/cm2) and spray dried phage powders stored at 4±1 °Ϲ (1.09 log CFU/cm2 and 1.30 log CFU/cm2) and 25±1 °Ϲ (0.72 log CFU/cm2 and 0.95 log CFU/cm2) for 10 months at MOI 100 and 1000, respectively. Within the scope of the investigation of bacteriophage preparations prepared in film form, the physicochemical quality and the effectiveness of antimicrobial activity against Salmonella and phage stability of dip-coated strawberries were investigated using bacteriophage cocktail with four different biopolymers for a period of five days at 4±1°Ϲ. The biopolymers used in this study were whey protein concentrate (WPC), carboxymethyl cellulose, chitosan, and sodium alginate. The color, pH, and titratable acidity (TA) of strawberry samples and the stability and antimicrobial activity of bacteriophages during storage were also investigated. Amongst all the biopolymers, WPC coating showed the least escalation in pH and the least decrease in TA. During storage, 0.7 log PFU/g reduction in WPC coating with the bacteriophage cocktail was observed. The highest antimicrobial effect was observed in WPC with a reduction of 3.1 log CFU/g after 5 days at MOI 1000. In the light of the results, it was concluded that the phage loaded WPC coating had much better physicochemical and microbiological quality during refrigerated storage; therefore, it could be used effectively to extend the postharvest life of strawberries.
