In this experimental study, we developed lateral flow immunoassay strips for the detection of Escherichia coli . For labelling, gold nanoparticles were prepared and modified with a Raman label. As a ...result of this, not only colorimetric responses but also Raman responses were obtained from the same strips. The color and Raman intensities were employed for the quantitative determination of Escherichia coli . The employment of two different detection methods strengthens the quantitative determination of Escherichia coli by lateral flow immunoassay, which is mostly used for qualitative detection or sometimes the semi-quantitative determination of samples. After careful optimization and validation steps, the developed strips allowed the highly sensitive determination of Escherichia coli in water and urine samples.
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.
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.
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 (~ 10
7
pfu/mL) and have lower titers (10
2
–10
3
pfu/mL), could be discriminated from antibiotic and each other. Additionally, low concentrations of
S. thermophilus
phages (10
2
pfu/mL) could be detected through Raman spectroscopy with a short analysis time (60 min) and high coefficient of determination (
R
2
) 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).
Thioredoxin reductase (TrxR, EC 1.6.4.5) is a ubiquitous flavoenzyme that is present from Archaea to humans, and it is the
only enzyme capable of catalyzing the reduction of thioredoxin (Trx) by ...nicotinamide adenine dinucleotide phosphate (NADPH).
Although TrxR has been purified and characterized from different bacteria, plants, and mammalian organisms, a survey of the
literature revealed no studies on the purification and characterization of TrxR from the mussel Mytilus galloprovincialis Lam.
In this study, TrxR was purified to homogeneity from the hepatopancreatic tissue of M. galloprovincialis Lam. by extraction,
ammonium sulfate precipitation, and DEAE-Sepharose CL-6B anion and 2’,5‘-ADP-agarose chromatographies, and some of
its kinetic properties were examined. Molar mass determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis
revealed only a single protein band corresponding to a molecular weight of 35 kDa. Optimum pH and temperature were
found to be 7.0 and 60°C, respectively. Km and Vmax values for NADPH were found to be 85 μmol and 4.82 μmol/min/mg,
respectively. For 5,5ʹ-dithiobis (2-nitrobenzoic) acid (DTNB), the Km and Vmax values were 193 μmol and 1.32 μmol/min/mg,
respectively. Increasing the knowledge on the kinetic properties of TrxR will significantly increase the prospects of enzyme
application as an oxidative stress biomarker in mussels and fishes for monitoring contamination in coastal environments.
Purpose
The aim of our study was to compare klotho in the serum and aqueous humor of patients with primary open-angle glaucoma (POAG), pseudoexfoliation glaucoma (PEXG) and pseudoexfoliation syndrome ...(PEX).
Materials and methods
18 POAG, 20 PEXG, 19 PEX and 20 control patients were included in our study. Aqueous humor and serum samples were collected at the time of cataract surgery. Samples were collected using enzyme-linked immunosorbent assay to evaluate the levels of Klotho protein.
Results
Klotho levels in the serum and aqueous humor of PEXG patients (34.45 ± 3.59, 0.20 ± 0.15 ng/ml), PEX (35.85 ± 4.26, 0.23 ± 0.20 ng/ml) patients and POAG patients (35.99 ± 3.73, 0.25 ± 0.20 ng/ml) were significantly lower than control group (40.14 ± 3.85, 0.53 ± 0.39 ng/ml) (PEXG,
P
< 0.001,
P
< 0.001; PEX,
P
= 0.002,
P
= 0.003; POAG,
P
= 0.006,
P
= 0.003, respectively). Both serum and aqueous levels of klotho in the PEXG and PEX patients were lower than POAG patients, but the difference did not reach statistical significance (PEXG & POAG
P
= 0.149,
P
= 0.696), (PEX & POAG
P
= 0.845,
P
= 0.775).
Conclusion
Klotho levels in the serum and aqueous humor decreased in PEX, PEXG and POAG groups compared to control group, but the reduction was most significant in PEXG group.
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.
Today, antibiotic resistance is one of the most crucial problems so that researchers have begun to search for alternatives to antibiotics. Among these alternatives, phage therapy and phytotherapy ...became prominent. The present study aims to put forward a different approach by combining these two methods. For this purpose, Staphylococcus aureus phages were isolated from commercial mixtures. After that, extraction of phenolic compounds from pomegranate peels (Ps), grape seeds (GSs), and black cumin (NS) was carried out and antimicrobial activities were determined. Phage‐phenolic interactions were investigated by examining the effect of phenolic extracts on phage plaque sizes and then on phage titer and bacteria count. As a result, it was found that only the NS extract had a positive effect on phage activity and increased the size of phage plaques. However, no synergistic effect was observed in experiments performed in liquid media. It was also found that P and GS extracts inhibited phage activity.
Practical Applications
The present study was focused on effects of extracts obtained from pomegranate peel, grape seed, and black cumin on Staphylococcus aureus phage activity. It is known that antibiotic resistance constitutes a major obstacle to the pathogen elimination. In this study, phage therapy and phytotherapy, which are prominent methods in pathogen elimination, have been combined and the effect of phenolic compounds isolated from natural sources on phage activity and bacteria count have been examined. Given the results of the current study, it has been demonstrated that the combined use of phages and antimicrobial compounds could be an important alternative to combating pathogens. It is thought that this combined method should be taken into consideration both in the practice of phage therapy on humans and animals, and in applications in the food industry.
Determination of yolk:white ratio of egg using SDS-PAGE Uysal, Reyhan Selin; Acar-Soykut, Esra; Boyaci, Ismail Hakki
Food Science and Biotechnology/Food science and biotechnology,
02/2020, Letnik:
29, Številka:
2
Journal Article
Recenzirano
Odprti dostop
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.
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.
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