Evaluating the efficacy of a phage cocktail against Pseudomonas fluorescens group strains in raw milk: microbiological, physical, and chemical analyses.

The objective of this study was to investigate the effectiveness of a phage cocktail against Pseudomonas fluorescens group and its effect on the microbial, physical and chemical properties of raw milk during different storage conditions. A phage cocktail consisting of Pseudomonas fluorescens, Pseudomonas tolaasii, and Pseudomonas libanensis phages was prepared. As a result, reductions in fluorescent Pseudomonas counts of up to 3.44 log units for the storage at 4 °C and 2.38 log units for the storage at 25 °C were achieved. Following the phage application, it is found that there was no significant difference in the total mesophilic aerobic bacteria and Enterobacteriaceae counts. However, it was observed that the number of lactic acid bacteria was higher in phage-treated groups. The results also showed that pH values in the phage added groups were lower than the others and the highest titratable acidity was obtained only in the bacteria-inoculated group. As a future perspective, this study suggests that, while keeping the number of target microorganisms under control in the milk with the use of phages during storage, the microbiota and accordingly the quality parameters of the milk can be affected. This work contributes to the development of effective strategies for maintaining the quality and extending the shelf life of milk and dairy products.

Isolation and characterization of yogurt starter cultures from traditional yogurts and growth kinetics of selected cultures under lab-scale fermentation.

The development of new starter cultures is a crucial task for the food industry to meet technological requirements and traditional products are important reservoirs for new starter cultures. In this respect, this study aimed to isolate, identify, and determine the technological characteristics of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains originated from traditional yogurt samples. Genotypic discrimination of 200 isolates revealed the presence of distinct 19 S. thermophilus and 11 Lb. delbrueckii subsp. bulgaricus strains as potential starter cultures. Strain-specific properties determined the acidification capacity of the yogurt starter cultures and a higher acidification capacity was observed for S. thermophilus strains compared to Lb. delbrueckii subsp. bulgaricus strains. Proteolytic activity was found between 0.012-0.172 and 0.078-0.406 for S. thermophilus and Lb. delbrueckii subsp. bulgaricus strains, respectively. 4 of S. thermophilus and 3 of Lb. delbrueckii subsp. bulgaricus strains were found resistant to all tested bacteriophages. The antibiotic susceptibility tests of the isolates revealed that a very low antibiotic resistance was observed for the yogurt starter cultures. Finally, the growth kinetics of selected strains were determined and the maximum specific growth rate of selected S. thermophilus and Lb. delbrueckii subsp. bulgaricus was calculated as 0.527 h-1 and 0.589 h-1, respectively.

Applications of Bacteriophage Cocktails to Reduce Salmonella Contamination in Poultry Farms.

Salmonella contamination is a critical problem in poultry farms, with serious consequences for both animals and food products. The aim of this study is to investigate the use of phage cocktails to reduce Salmonella contamination in poultry farms. Within the scope of the study, Salmonella phages were isolated from chicken stool. After the host range of phages was determined, morphological characterization was performed through transmission electron microscopy analysis. Then, replication parameters and adsorption rates were determined by one-step growth curves. After that, phage cocktail was prepared, and its effectiveness was tested in three environments, which were drinking water, shavings, and plastic surfaces. The results obtained have demonstrated that the phage cocktail can reduce Salmonella count up to 2.80 log10 units in drinking water, up to 2.30 log10 units on shavings, and 2.31 log10 units on plastic surfaces. It has been determined that phage cocktails could be a successful alternative in reducing Salmonella contamination in poultry environment. This work is the first study to investigate the use of phage cocktails for reducing Salmonella contamination in poultry water and on shavings, and it is presumed that the results obtained will contribute to the fight against pathogens by making them applicable to poultry farms.

Investigation of phage and molasses interactions for the biocontrol of E. coli O157:H7.

Resistance to antibiotics is one of the most critical health problems in the world. Therefore, finding new treatment methods to be used as alternatives to antibiotics has become a priority for researchers. Similar to phages, certain products containing antimicrobial components, such as molasses, are widely used to eliminate resistant bacteria. Molasses has a strong antimicrobial effect on bacterial cells, and this effect is thought to be due to the breakdown of the cytoplasmic cell membrane and cell proteins of the polyphenols in molasses. In the present study, phage-molasses interactions were investigated to examine the effects of concomitant use. It was found that molasses samples increased the size of phage plaques by up to 3-fold, and MIC and 1/2 × MIC concentrations of molasses increased the burst size of phages. Although no synergistic effect was found between the phage and molasses, the antimicrobial activities of the components and the effect of molasses on phage activity were demonstrated.

Determination of yolk:white ratio of egg using SDS-PAGE.

The aim of present study was to determine liquid egg adulteration based on yolk:white ratio of egg using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by measuring Brix. To construct a calibration model, liquid egg samples were prepared by mixing egg yolk and white (yolk:white) at different concentrations. A high coefficient of determination value (R 2 = 0.992) was obtained. Limit of detection and limit of quantification were estimated as 62 g/kg and 187 g/kg. The accuracy of the model was tested using eleven LWE samples, and the yolk ratios of 90.9% of these samples were predicted successfully. Liquid whole egg (LWE) containing additional egg white up to 30% was also predicted with a low relative error of less than 10%. Yolk:white ratio of LWE samples and authentication of the components of LWE (containing extra white or water) can be determined using proposed method.

SERS-based rapid assay for sensitive detection of Group A Streptococcus by evaluation of the swab sampling technique.

Beta-hemolytic, Group A Streptococcus pyogenes (GAS) is a life-threating pathogen and the reason for prominent disease, pharyngitis. The conventional analysis of GAS, gold standard, takes 48 hours and the related rapid tests lack in accuracy and sensitivity. In this study, firstly, the efficiency of swab sampling, which is a must in the GAS detection, was discussed with the proposed surface-enhanced Raman spectroscopy (SERS)-based batch assay and each step was controlled by the plate-counting method. Secondly, SERS-based lateral flow immunoassay (LFIA) test strips were constructed and the variation in the SERS intensity of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) was observed. Thus, a linear correlation was found with a R2 value of 0.9926 and the LOD was calculated to be 0.2 CFU mL-1 of GAS which could be counted as one cell. The combination of the gold standard with the LFIA-SERS technique enabled the fast and accurate pathogen detection. In addition, GAS was quantified with paper-based test strips up to 100 CFU ml-1 level of bacteria for the first time without any interference. Besides, this study was featured with the discussion of the whole cell and pretreated cell detection of pathogens with LFIAs. Therefore, this work enlightens the points that have never been discussed on pathogen detection with paper-based platforms.

Microencapsulation of phages to analyze their demeanor in physiological conditions.

Nowadays, phage therapy emerges as one of the alternative solutions to the problems arising from antibiotic resistance in pathogenic bacteria. Although phage therapy has been successfully applied both in vitro and in vivo, one of the biggest concerns in this regard is the stability of phages in body environment. Within the scope of this study, microencapsulation technology was used to increase the resistance of phages to physiological conditions, and the resulting microcapsules were tested in environments simulating body conditions. For this purpose, Bacillus subtilis, Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis), and Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium) phages were isolated from different sources and then microencapsulated with 1.33% (w/v) sodium alginate using a spray dryer to minimize the damage of physiological environment. Stability of microcapsules in simulated gastric fluid and bile salt presence was tested. As a consequence, the maximum titer decrease of microencapsulated phages after 2-h incubation was found to be 2.29 log unit for B. subtilis phages, 1.71 log unit for S. Enteritidis phages, and 0.60 log unit for S. Typhimurium phages, while free phages lost their viability even after a 15-min incubation. Similarly, microencapsulation was found to increase the stability of phages in the bile salt medium and it was seen that after 3 h of incubation, the difference between the titers of microencapsulated phages and free phages could reach up to 3 log unit.

A Raman-spectroscopy-based approach for detection and discrimination of Streptococcus thermophilus and Lactobacillus bulgaricus phages at low titer in raw milk.

In this study, a method combining Raman spectroscopy with chemometric analysis was developed for detection of phage presence in raw milk and discrimination of Streptococcus thermophilus and Lactobacillus bulgaricus phages which are among the main phages causing problems in dairy industry. For this purpose, S. thermophilus and L. bulgaricus phages were added into raw milk separately, and then some pretreatments such as fat separation, removal of casein, and filtration were applied to the raw milk samples. Raman spectra of the samples were collected and then analyzed using principal component analysis in order to discriminate these phages in raw milk. In the next step, dilutions of S. thermophilus phages in pretreated raw milk were prepared, and Raman spectra were collected. These spectra were analyzed by using partial least squares method to quantify phages in low titer. Consequently, it has been demonstrated that S. thermophilus and L. bulgaricus phages, which have titers sufficient to fail the fermentation (~ 107 pfu/mL) and have lower titers (102-103 pfu/mL), could be discriminated from antibiotic and each other. Additionally, low concentrations of S. thermophilus phages (102 pfu/mL) could be detected through Raman spectroscopy with a short analysis time (60 min) and high coefficient of determination (R2) values for both calibration (0.985) and validation (0.906) with a root mean square error of calibration of 70.54 and root mean square error of prediction of 165.47. However, a lower success was achieved with L. bulgaricus phages and the obtained coefficient of determination values were not sufficiently high (0.649).

A simple and fast method for discrimination of phage and antibiotic contaminants in raw milk by using Raman spectroscopy.

Phage and antibiotic in raw milk poses significant risks for starter culture activity in fermented products. Therefore, rapid detection of phage and antibiotic contaminations in raw milk is a crucial process in dairy science. For this purpose, a preliminary novel method for detection of phage and antibiotic was developed by using Raman spectroscopy. Streptococcus thermophilus phages and ampicillin which are quite important elements in dairy industry were used as model. The phage and antibiotic samples were added to raw milk separately, and Raman measurements were carried out. The obtained spectra were processed with a chemometric method. In this study, it has been demonstrated that the presence of phage has a titer sufficient to stop the fermentation (107 pfu/ml), and antibiotic in a concentration which inhibits the growth of starter cultures (0.5 µg/ml) in raw milk could be discriminated through Raman spectroscopy with a short analysis time (30 min).

Development of rolling circle amplification based surface-enhanced Raman spectroscopy method for 35S promoter gene detection.

In this study, we developed the genetically modified organism detection method by using the combination of rolling circle amplification (RCA) and surface-enhanced Raman spectroscopy (SERS). An oligonucleotide probe which is specific for 35S DNA promoter target was immobilised onto the gold slide and a RCA reaction was performed. A self-assembled monolayer was formed on gold nanorods using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and the second probe of the 35S DNA promoter target was immobilised on the activated gold coated slide surfaces. Probes on the nanoparticles were hybridised with the target oligonucleotide. Quantification of the target concentration was performed via SERS spectra of DTNB on the nanorods. SERS spectra of target molecules were enhanced through the RCA reaction and the detection limit was found to be 6.3fM. The sensitivity of the developed RCA-SERS method was compared with another method which had been performed without using RCA reaction, and the detection limit was found to be 0.1pM. The correlation between the target concentration and the SERS signal was found to be linear, within the range of 1pM to 10nM for the traditional assay and 100fM to 100nM for the RCA assay. For the developed RCA-SERS assay, the specificity tests were performed using the 35S promoter of Bt-176 maize gene. It was found out that the developed RCA-SERS sandwich assay method is quite sensitive, selective and specific for target sequences in model and real systems.