Učinkovitost primjene reuterina te bakteriocina nizina i pediocina pri konzerviranju sirovog mlijeka s mliječnih farmi

Research background. In the current scenario of milk production in developing and developed countries, several factors influence the shelf-life of raw milk and add significant numbers of microbial contaminants that drastically lower the initial microbial quality leading to milk spoilage by the time it reaches the processing units. Experimental approach. The present study was undertaken to investigate the biopreservative efficacy of reuterin system along with different combinations of bacteriocins in controlling the initial microflora of raw milk at farm level. Lactobacillus reuteri strain LR47, having effective antimicrobial activity, was shortlisted from our previous study and further characterized for reuterin production and tested in raw milk system. Results and conclusions. Preliminary testing of the cell-free supernatant from L. reuteri LR47 demonstrated significant growth inhibition of the majority of the tested bacterial indicators of milk spoilage. Further genetic analysis of the L. reuteri LR47 revealed the presence of two genes (pduC and dhaB) involved in the utilization of glycerol to produce reuterin via two different pathways. The strain LR47 was also found to possess comparatively higher capacity to convert glycerol into reuterin when checked through colourimetric assay. In the raw milk biopreservation experiment with reuterin alone or in combination with bacteriocins, the highest level of growth suppression in the total bacterial load and coliform counts was observed in the sample that was treated with a combination of reuterin, nisin and pediocin. The treatment combining these three natural biopreservatives at specific concentrations was able to maintain the initial microbial quality and extend the shelf-life of raw milk by 6 h at 37 °C based on the microbial counts and physicochemical properties, viz. pH and titratable acidity. In conclusion, the results confirm that the use of reuterin in combination with bacteriocins is a promising approach for temporary control of the raw milk microflora and extension of its shelf-life until further processing. Novelty and scientific contribution. This study demonstrates for the first time the use of reuterin for the extension of shelf-life of raw milk as an alternative treatment method.Pozadina istraživanja. Pri današnjoj proizvodnji mlijeka u razvijenim zemljama te onima u razvoju, nekoliko čimbenika utječe na trajnost sirovog mlijeka i pojačava kontaminaciju patogenim mikroorganizmima, što bitno utječe na inicijalnu populaciju mikroorganizama i uzrokuje kvarenje mlijeka prije početka obrade. Eksperimentalni pristup. U ovom je radu ispitana učinkovitost reuterina kao biokonzervansa u različitim kombinacijama s bakteriocinima za kontrolu mikroflore sirovog mlijeka na farmi. U našem je prethodnom radu zbog izražene antimikrobne aktivnosti od nekoliko sojeva bakterija odabran soj Lactobacillus reuteri LR47, te je ispitana njegova sposobnost proizvodnje reuterina u sirovom mlijeku. Rezultati i zaključci. Preliminarni su testovi pokazali da je supernatant bez stanica bakterije L. reuteri LR47 bitno inhibirao rast većine ispitanih uzročnika kvarenja mlijeka. Daljnje genetičke analize soja otkrile su prisutnost dvaju gena (pduC i dhaB) odgovornih za iskorištenje glicerola tijekom dva različita metabolička puta proizvodnje reuterina. Osim toga, kolorimetrijskom je analizom otkriveno da soj LR47 ima veću sposobnost konverzije glicerola u reuterin od ostalih sojeva bakterija. Pri ispitivanju postupaka biološkog konzerviranja sirovog mlijeka pomoću reuterina ili reuterina u kombinaciji s bakteriocinima, najizraženije je svojstvo inhibicije ukupnog broja mikroorganizama i broja koliformnih bakterija zapaženo u uzorku mlijeka tretiranog kombinacijom reuterina, nizina i pediocina. Broj mikroorganizama i fizikalno-kemijska svojstva, kao što su pH-vrijednost i titracijska kiselost, pokazali su da se kombinacijom određenih koncentracija tih triju prirodnih biokonzervansa očuvala početna mikrobiološka kakvoća mlijeka, te produljila njegova trajnost za 6 sati pri 37 °C. Dobiveni rezultati potvrđuju da se primjenom reuterina u kombinaciji s bakteriocinima može privremeno kontrolirati mikroflora sirovog mlijeka, te produljiti njegova trajnost do daljnje prerade. Novina i znanstveni doprinos. U ovom je radu po prvi put opisana primjena reuterina kao alternativnog postupka produljenja trajnosti sirovog mlijeka

Investigation of phytochemical composition, evaluation of antioxidant, antibacterial activities and toxicity study of Emblica officinalis and Terminalia bellirica fruits

The present study deals with exploration of various beneficial potential of Emblica officinalis and Terminalia bellirica fruit extracts which includes phytochemical analysis and evaluation of total phenolics content (TPC), total flavonoids content (TFC) and antimicrobial activity. The antioxidant activity of the extracts was examined using diphenylpicrylhydrazyl (DPPH), TPC and TFC assay. The antimicrobial activity of the extracts was evaluated by agar well diffusion assay. Although, the extracts prevented the growth of both Gram positive and Gram negative bacteria. The EOA and EOM extracts both exhibit tannins, saponins, phenols and carbohydrates whereas, alkaloids and flavonoids are found only in EOM, and glycosides showed their presence in aqueous extract only. In contrast, TBA and TBM extracts, both contains alkaloids, tannins, coumarins, flavonoids and phenols. While carbohydrates are present barely in TBM, and TBA extract showed the presence of saponins and glycosides. The TPC results show that EOM (528±.013) has highest phenolic content in it followed by EOA (509±.003), TBM (284±.06) and TBA (280±.036). In TFC analysis too EOM (154±0) showed a higher concentration range of flavonoids followed by EOA (142±0),TBM (126±.017)  and TBA (119±.007). In DPPH scavenging assay EOM (86.88±.310) showed maximum % inhibition followed by EOA (85.32±.414), TBM (80.57±.569) and TBA (75.55±.362) respectively. Antibacterial assay showed varying results for different bacterial strains such as, for EOA (10±1)  demonstrated  highest inhibition against E. coli, EOM (11.5±0.5) exhibited maximum inhibition against S. aureus, for K. pneumoniae EOA (13±2) displayed maximum inhibition and in case P. aeruginosa too EOA (12.5±0.5) showed maximum inhibition while TBM and TBA too showed significant amount of inhibition against test bacterial strains. The antibacterial and antioxidant activities of the extracts were found to be positively associated with the total phenolic and flavonoid content of the extracts. This study was conducted to identify the phytochemical composition of the fruit extracts of the two plants, their antioxidant and antibacterial potential, alongwith their neuroprotective and nootropic role. In order to establish their neuroprotective role, acute toxicity study was conducted on wistar rats to establish the safety of the extracts. The current study demonstrates and compares the antioxidant, antibacterial activities of the fruit extracts of Terminalia bellirica and Emblica officinalis and acute toxicity study further demonstrates that the extracts used are safe to conduct neuroprotective studies. Keywords: TPC(Total phenolic content), TFC(Total flavonoids content), EOM (Emblica officinalis Methanolic fruit extract), EOA(Emblica officinalis aqueous fruit extract) TBM (Terminalia bellirica Methanolic fruit extract) and TBA (Terminalia bellirica aqueous fruit extract)

IN SILICO PREDICTION AND VALIDATION OF MICRORNAS FROM JAPANESE ENCEPHALITIS VIRUS (JEV)

Objective: MicroRNAs are endogenous, small, single stranded, non coding RNAs having 19-25 nucleotides. These miRNAs are complementary to their target messenger RNAs that bind principally to its 3' un translated regions (3' UTRs). Small RNAs play crucial roles in the regulation of gene expression in many eukaryotes; therefore it is important to predict potential viral miRNAs which might be involved in an establishment of Japanese Encephalitis virus (JEV) disease. Different computational approaches and methods were used for predicting viral microRNAs from the JEV genome in this work.Methods: In the present study, the use of genome-wide computational approach has been demonstrated to predict miRNAs and their target(s) in JEV genome. Two freely accessible softwares, MiPred and Genscan were used to predict the secondary structures of the potential miRNAs.Results: In all, 36 miRNAs were predicted and characterized by conducting genome-wide homology search against all the reported miRNAs. These miRNAs were further validated by performing phylogenetic analyses and using statistical tools.Further, attempt was made to predict the 3′ untranslated regions of mRNAs from whole genome of JEV which may prove helpful in finding putative targets of these miRNAs.Conclusion: This is the first study to identify and validate miRNAs in JEV which is an important step in identifying putative JEV miRNAs that utilize host cell machinery, and may play a crucial role in neuroinflammation and silencing of host genes, thus demonstrating the role of viral miRNAs in establishing viral pathogenesis.Â

Oral vaccines: new needs, new possibilities

Vaccination is an important tool for handling healthcare programs both in developed and developing countries. The current global scenario calls for a more-efficacious, acceptable, cost-effective and reliable method of immunization for many fatal diseases. It is hoped that the adoption of oral vaccines will help to provide an effective vaccination strategy, especially in developing countries. Mucosal immunity generated by oral vaccines can serve as a strong first line of defense against most of the pathogens infecting through the mucosal lining. Advances in elucidating the mechanism of action of oral vaccines will facilitate the design of more effective, new generation vaccines. There are promising developments in the use of different agents to effectively deliver the vaccine candidate. It is hoped that ongoing research may be able to set another cardinal point, after polio vaccine, in eradicating infectious diseases

Expression of anthrax lethal factor gene by osmolyte induction

The anthrax toxin consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA mediates the entry of LF and EF to the cytosol where they exert their effects. Although PA is the major component of the vaccines against anthrax, LF has also been found to play an important role in enhancing protective immunity. We have developed an osmolyte-inducible LF expression system. The protein expression system contributed no additional amino acids to the recombinant LF making it suitable for the human vaccine trials

Gln277 and Phe554 residues are involved in thermal inactivation of protective antigen of Bacillus anthracis

Protective antigen (PA) is the main component of all the vaccines against anthrax. The currently available vaccines have traces of other proteins that contribute to its reactogenicity. Thus, purified PA is recommended for human vaccination. PA loses its biological activity within 48 h at 37°C and its thermolability has been a cause of concern as accidental exposure to higher temperatures during transportation or storage could decrease its efficacy. In the present study, we have used protein engineering approach to increase the thermostability of PA by mutating amino acid residues on the surface as well as the interior of the protein. After screening several mutants, the mutants Gln277Ala and Phe554Ala have been found to be more thermostable than the wild-type PA. Gln277Ala retains ~45% and Phe554Ala retains 90% activity, even after incubation at 37°C for 48 h while in the same period wild-type PA loses its biological activity completely. It is the first report of increasing thermostability of PA using site-directed mutagenesis. Generation of such mutants could pave the way for better anthrax vaccines with longer shelf life

Empowering Cloud of Things with Edge Computing: A Comparative Analysis

Number of networks and their sizes in terms of connected devices are increasing very rapidly. These devices are producing massive amount of raw data for different progressive purposes that need to be processed and stored somewhere. Local data storage and complete processing of massive data will not be possible by resource constrained Internet of Things (IoT) enabling devices anymore in the future. Therefore, there is a need of data storage and processing capabilities to meet the desired goals of applications. Cloud computing and edge computing are the possible solution to circumvent limited data storage and computation issues of IoT devices. This paper presents a comparative study of popular architectures in which IoT devices are integrated with cloud. For this purpose, various IoT and Cloud of Things (CoT) architectures have been studied. Strengths and weaknesses of the studied IoT and CoT architectures are comprehensively analyzed on the basis of five comparison parameters namely; resource based segregation of IoT enabling devices, range based segregation of the communication network, segregation of security solutions, cloud connectivity, and edge computing services

Thermal inactivation of protective antigen of Bacillus anthracis and its prevention by polyol osmolytes

Protective antigen (PA) of Bacillus anthracis is the main immunogen of all anthrax vaccines. It is a highly thermolabile molecule and loses its activity rapidly when exposed to higher temperatures. Earlier some cosolvents had been used to stabilize PA with variable success but no study has been done to find out the primary cause of PA thermal inactivation. This study aims at elucidating the predominant cause of thermal inactivation of PA in order to develop more effective strategies for its thermostabilization. The prime cause for the loss of biological activity of PA at high temperature was its aggregation and an inverse correlation between PA activity and its aggregation on heating was observed. Inactivation of the protein by autolysis did not occur. This paper reports the use of a series of polyol osmolytes to stabilize PA. Different polyols stabilized PA to a different extent against thermal inactivation in a concentration dependent manner, with glycerol stabilizing to the maximum extent. Addition of NaCl to glycerol solution further enhanced the thermal stability of PA. An increase in the T<SUB>½</SUB> value, the temperature at which 50% of the activity is retained during short-term incubation, of more than 20°C was observed. The half-life (t<SUB>½</SUB>) of PA thermal inactivation at 40°C increased by more than 6 times in the presence of the mixture of glycerol and NaCl as compared to control. This study demonstrates for the first time that aggregation of the PA molecule is the predominant cause of its thermal inactivation, and can be very effectively prevented by the use of glycerol and other polyols to increase the shelf life of the recombinant vaccine against anthrax

A Comprehensive Review of Computing Paradigms, enabling Computation Offloading and Task Execution in Vehicular Networks

Road safety, optimized traffic management, and passenger comfort have always been the primary goals of the vehicle networking research community. Advances in computer and communication technology have made the dream of modern intelligent vehicles a reality through the use of smart sensors, cameras, networking devices, and storage capabilities. Autonomous operation of modern intelligent vehicles requires massive computations where tasks are outsourced. The research community has proposed various computing paradigms: mobile cloud computing, vehicle cloud computing, multi-access edge computing, vehicle edge computing, vehicle fog computing, and voluntary computing-based VANET (VCBV) to move computational power close to the user and handle the delay-sensitive applications of modern intelligent vehicles. In this study, we have provided a comprehensive overview of all computing paradigms related to vehicular networks. We have presented the architectural details, similarities, differences, and key features of each computing paradigm. Finally, we concluded the study with open research challenges in vehicular networks