Late blowing is a type of microbiological spoilage that causes unwanted changes in the taste, odour and texture of cheese due to the metabolic activities of Clostridium contaminating raw milk. This ...study aimed to develop a multiplex quantitative real-time polymerase chain reaction (PCR) analysis method for the rapid and simultaneous detection of Clostridium butyricum, Clostridium sporogenes and Clostridium tyrobutyricum using specific primers and probes. The optimised method was determined to work with high sensitivity and specificity to quantify late-blowing Clostridium species in cheese samples. The lowest detectable gene copy number was 4.9 × 101, 7.8 × 101 and 8.5 × 101 for C. tyrobutyricum, C. butyricum and C. sporogenes, respectively. These results demonstrated that this method is a powerful tool for detecting and quantifying late-blowing agents in cheese. This is also the first multiplex qPCR study involving C. butyricum for detecting Clostridia, a late-blowing agent in cheese.
•Late-blowing is one of the most important problems of cheese producers.•The culture method does not provide quantitative results at species level.•This qPCR method was able to detect Clostridium species with high sensitivity.•This assay has low detection limit ̴ log10 1.0 CFU/mL.•This assay has the potential as a molecular tool for analyses cheese by rapidly.
Late blowing, a microbiological spoilage in hard and semi-hard cheese caused by
Clostridium
spores in raw milk, results in high economic losses for cheese producers. This study compared the ...sensitivity of the newly developed multiplex qPCR method which employing novel oligonucleotide primers and fluorescent TaqMan probes, and the culture-based most probable number (MPN) method in detecting the late blowing agent Clostridium species in traditional Turkish cheese. A total of 50 naturally contaminated cheese samples obtained from producers were analysed by both methods.
Clostridium tyrobutyricum
was the most common species occurring in 74% of the cheese samples, followed by
C. butyricum
and
C. sporogenes
occurring in 50% and 16% of the samples, respectively. The results of the two methods were consistent in 42 out of the 50 (84%) cheese samples. Our results indicate that the multiplex qPCR method is more sensitive than the MPN method. The multiplex qPCR method provided a favourable alternative to traditional cultural methods. This alternative molecular method has great potential in the laboratory and in the field for the rapid detection of late blowing of cheese samples.
Betulinic acid (BA), a betulin derivative, is an important plant-based natural product. BA has many biological activities and attracts attention especially as a promising antitumour drug. Extraction ...from plants or semi-synthesis from betulin are the common methods to obtain BA, but these methods have some drawbacks. In recent years, microbial biotransformation of betulin to BA has been anticipated as an alternative method. Betulin is abundant in the outer bark of birch (Betula spp.) and obtained from these plants, however endophytes of Betula spp. have not been investigated for biotransformation of betulin to BA before. Therefore, this study aimed to investigate the culturable endophytes of Betula pubescens var. litwinowii (Doluch.) Ashburner & McAll and to determine their biotransformation capacity of betulin to BA. To this end, bacterial and fungal endophytes were isolated from the surface-sterilized root, stem, and branch samples. A total of 37 endophytes (11 bacteria and 26 fungi) were identified by polyphasic approach. All endophytes were screened for the biotransformation of betulin to BA. Betulin and BA content of the extracts were determined by RP-HPLC analysis. Two different bacterial and fungal biotransformation processes were designed. It was found that the fungus BRF59 produced BA via betulin biotransformation under the Fungal Biotransformation Process-2 when the fungus was acclimatized to betulin before biotransformation, and glycerol was used as a carbon source, instead of glucose, during bioprocess. The highest BA yield and BA concentration was obtained as 12.22±0.94 % and 80.85±2.64 µg/ml, respectively, under non-optimized conditions. The fungal isolate BRF59, belonging to the genus Fusarium according to ITS analysis, was further identified based on RPB2 sequence analysis and found that Fusarium lacertarum. In conclusion, this study demonstrated that betulin was biotransformed to BA by an endophytic fungus isolated from the genus Betula for the first time. F. lacertarum BRF59 might be a good candidate for production of BA through betulin biotransformation and, using glycerol as a carbon source during biotransformation might enable this by-product to convert value-added chemical.
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•Culturable endophytes of Betula pubescens var. litwinowii (Doluch) was investigated.•Biotransformation of betulin to betulinic acid by endophytes was investigated.•Fusarium lacertarum BRF59 produced betulinic acid via betulin biotransformation.•The highest betulinic acid production was 80.85±2.64 µg/ml.•Acclimatization to betulin and using glycerol induced betulinic acid production.
This study aimed to investigate the endophytes of Juniperus macrocarpa
collected from ?esme in ?zmir, Turkey, using a culture-dependent approach
and to evaluate their antimicrobial activity for the ...first time. Since
endophytes interact with phytochemicals of the host plant, in addition to
the standard culture media, a J. macrocarpa extract supplemented culture
media was also used for isolation to enhance the cultivability of the
endophytes. Six bacteria out of twelve and three fungi out of seven were
isolated from the plant extract supplemented culture media. The genotypic
identification of the bacterial and fungal isolates was determined based on
16S rDNA and Internal Transcribed Spacer (ITS) sequence analysis,
respectively. The genus Juniperus, which has ethnopharmacological uses, is
rich in phytochemicals with multiple bioactivities. Since Juniperus spp. is
listed as a priority natural habitat, it is necessary to find alternative
resources that could replace the bioactive compounds of these plants.
Endophytes of Juniperus spp. might be good candidates as antimicrobial
producers. From this point of view, the antimicrobial activity of the crude
fermentation liquid of the J. macrocarpa endophytes, and also aqueous and
methanolic extracts of J. macrocarpa, were evaluated using a disc diffusion
assay against a panel of test microorganisms, including antibiotic resistant
ones. One fungus and seven bacteria showed remarkable antimicrobial activity
against at least one test microorganism. These results indicated that some
endophytes of J. macrocarpa had antimicrobial properties like their host
plant and could substitute these plants as a source of antimicrobials.
Microbial secondary metabolites, which play a pivotal role in struggling with infectious diseases, are the new source for controlling bacterial contaminations and possess a strong antimicrobial ...potential. The present study is designed to evaluate the in vitro and in vivo bactericidal activities of prodigiosin against Staphylococcus aureus. For this purpose, Serratia marcescens was used to produce prodigiosin. Characterization of the prodigiosin was carried out using NMR. In addition, bioautographic detection of prodigiosin was detected by TLC. Antibacterial assays, in vivo epicutaneous infection tests, swap analyses, and histopathological examinations were determined. The results revealed that prodigiosin was detected by NMR and TLC. According to antimicrobial susceptibility tests, prodigiosin is an efficient bactericidal compound that demonstrated strong antibacterial activity toward S. aureus. In vivo, animal studies determined that the strong inhibition of S. aureus-caused epidermal infection occurs by prodigiosin at 48 h. Histopathological results showed that S. aureus + prodigiosin skin sections consist of improved and healthy tissues without any infection area compared with the S. aureus and control groups. The in vivo study verified the antibacterial results with swap analyses, and histopathological findings showed that prodigiosin is a promising microbial metabolite effective against S. aureus infection. This study proved that prodigiosin with excellent bioactivity exhibited antibacterial properties, which might possess massive potential for new therapeutic approaches using micro-organisms.
Phytases catalyze the hydrolysis of phytic acid into myo-inositol phosphates and inorganic phosphates and are used as animal feed additives. The main objective of this study is to discover a new ...phytase, which has high thermal stability, protease resistance, and pH stability for its usability in the feed industry. For this purpose, this study focused on purifying and characterizing the thermostable phytase derived from thermotolerant Aspergillus tubingensis TEM 37 strain at first time, which was isolated from a hot spring soil in the Gediz geothermal field (Turkey). The optimum pH and temperature of the phytase were determined as pH 2.0-5.5 and 45 °C, respectively. The molecular weight of the enzyme was determined as 48 kDa. The phytase preserved almost 100% of its activity at 80 °C for 3 h. The enzyme showed high resistance against K
+
, Ba
2+
, Cu
2+
, Mg
2+
, Zn
2+
and Tween 20, CTAB, and isooctane among tested compounds. The enzyme also showed high stability against proteases and retained its activity as 88% for pepsin and 98% for trypsin for 120 minutes. In conclusion, it was demonstrated that the thermotolerant A. tubingensis TEM 37 strain naturally produces the phytase that was thermostable and resistant to proteases as required by feed industry.
The qualitative and quantitative α-amylase production capacities of six thermophilic bacilli were screened. Geobacillus sp. D413 was selected for enzyme optimization, as it displayed higher α-amylase ...activity. The maximum enzyme activities of D413 and G. stearothermophilus ATCC 12980T were observed at the time of 72 h. While the optimal pH of medium for bacterial growth and enzyme production of D413 (pH 7.0) differed from ATCC 12980T (pH 8.0), the optimal temperature for enzyme production was 55°C for both. The effects of various carbon and nitrogen sources were determined by changing their concentrations. The highest bacterial growth and enzyme production were sustained by the starch and maltose containing medium. Both bacterial growth and enzyme production were inhibited by NH4Cl. D413 and ATCC 12980T amylases showed optimal activity at 65ºC, pH 9.0 and at 65ºC, pH 7.5, respectively. They remained active over temperature and pH ranges of 45-75ºC and 4.0-10.5. Their activities retained 65% and 54% when incubated at 75ºC for 10 min and 98-86.5% and 95-84.5% at pH 4.0-10.5 for 15 h at 37ºC. In conclusion, the α-amylase production conditions of D413 have been optimized which can be useful in biotechnological processes such as hydrolysis of starch to glucose.
Üçlü-faz ayırma (TPP) tekniği α-galaktozidazın Aspergillus lentulus’dan tek adımda kısmi saflaştırılması için ilk kez başarıyla kullanıldı. Yüksek aktivite ve saflaştırma katı elde etmek için enzimin ...ekstraksiyon etkinliğine amonyum sülfat konsantrasyonu, ekstrakt t-butanol oranı ve pH etkisi araştırıldı. Sistemin optimum saflaştırma parametreleri %55(w/v) amonyum sülfat konsantrasyonu, 1:1 (v/v) ham ekstrakt t-butanol oranı ve pH 5.5 olarak belirlendi. Bu optimize TPP sistemi α-galaktosidaz için 5.3 saflaştırma katı ile %178 aktivite verimi oluşturdu. pH 6.5 ve 50oC’de maksimum aktivite gözlendi. α-Galaktozidaz 25-60°C sıcaklık aralığında ve pH 2.6-5.5 aralığında oldukça iyi bir kararlılık gösterdi. KM ve Vmax değerleri sırasıyla 0.365 mM ve 0.093 U olarak belirlendi. Metal iyonları ve şekerler arasında Na2CO3 ve galaktoz enzim aktivitesi üzerinde kuvvetli inhibitor etkisi gösterdi. TPP ile A. lentulus’dan farklı biyokimyasal özelliklere sahip yeni bir α-galaktozidazın elde edilmesi onun çeşitli biyoteknolojik uygulamaları açısından ilgi çekici olacaktır.
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