Depiction of the In Vitro and Genomic Basis of Resistance to Hop and High Hydrostatic Pressure of Lactiplantibacillus plantarum Isolated from Spoiled Beer.

Among the beer-spoiling microorganisms, the dominant ones belong to the genera Lactobacillus, Leuconostoc, Oenococcus, and Pediococcus. It is assumed that resistance to hop bitters correlates with resistance to other factors and can significantly impact the brewing industry. Beer preservation with high hydrostatic pressure eliminates the spoiling microorganisms while preserving all desired properties of the beer. Here, we present comprehensive in vitro and genomic analysis of the beer-spoiling Lactiplantibacillus plantarum KKP 3573 capacity to resist hop and high hydrostatic pressure. Lp. plantarum KKP 3573 is a strain isolated from spoiled beer. Our finding suggests that the growth rate of the strain depends on the medium variant, where a small concentration of beer (5 IBU) stimulates the growth, suggesting that the limited concentration has a positive effect on cell growth. At the same time, increased concentrations of 20 IBU, 30 IBU, and pure beer 43.6 IBU decreased the growth rate of the KKP 3573 strain. We observed that higher extract content in the pressurized beer increased microbial survivability. The wort and Vienna Lager beer can stimulate the baroprotective effect. The taxonomy of the novel strain was confirmed after whole genome sequencing (WGS) and comparative genomic analysis. More specifically, it contains a chromosome of 3.3 Mb with a GC content of 44.4%, indicative of the Lp. plantarum species. Accordingly, it possesses high genomic similarity (>98%) with other species members. Annotation algorithms revealed that the strain carries several genes involved in resistance to stress, including extreme temperature, hop bitters and high pressure, and adaptation to the brewing environment. Lastly, the strain does not code for toxins and virulence proteins and cannot produce biogenic amines.

Characterization and Genome Study of a Newly Isolated Temperate Phage Belonging to a New Genus Targeting Alicyclobacillus acidoterrestris.

The spoilage of juices by Alicyclobacillus spp. remains a serious problem in industry and leads to economic losses. Compounds such as guaiacol and halophenols, which are produced by Alicyclobacillus, create undesirable flavors and odors and, thus, decrease the quality of juices. The inactivation of Alicyclobacillus spp. constitutes a challenge because it is resistant to environmental factors, such as high temperatures, and active acidity. However, the use of bacteriophages seems to be a promising approach. In this study, we aimed to isolate and comprehensively characterize a novel bacteriophage targeting Alicyclobacillus spp. The Alicyclobacillus phage strain KKP 3916 was isolated from orchard soil against the Alicyclobacillus acidoterrestris strain KKP 3133. The bacterial host's range and the effect of phage addition at different rates of multiplicity of infections (MOIs) on the host's growth kinetics were determined using a Bioscreen C Pro growth analyzer. The Alicyclobacillus phage strain KKP 3916, retained its activity in a wide range of temperatures (from 4 °C to 30 °C) and active acidity values (pH from 3 to 11). At 70 °C, the activity of the phage decreased by 99.9%. In turn, at 80 °C, no activity against the bacterial host was observed. Thirty minutes of exposure to UV reduced the activity of the phages by almost 99.99%. Based on transmission-electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the Alicyclobacillus phage strain KKP 3916 was classified as a tailed bacteriophage. The genomic sequencing revealed that the newly isolated phage had linear double-stranded DNA (dsDNA) with sizes of 120 bp and 131 bp and 40.3% G+C content. Of the 204 predicted proteins, 134 were of unknown function, while the remainder were annotated as structural, replication, and lysis proteins. No genes associated with antibiotic resistance were found in the genome of the newly isolated phage. However, several regions, including four associated with integration into the bacterial host genome and excisionase, were identified, which indicates the temperate (lysogenic) life cycle of the bacteriophage. Due to the risk of its potential involvement in horizontal gene transfer, this phage is not an appropriate candidate for further research on its use in food biocontrol. To the best of our knowledge, this is the first article on the isolation and whole-genome analysis of the Alicyclobacillus-specific phage.

Newly Isolated Virulent Salmophages for Biocontrol of Multidrug-Resistant Salmonella in Ready-to-Eat Plant-Based Food.

Due to irrational antibiotic stewardship, an increase in the incidence of multidrug resistance of bacteria has been observed recently. Therefore, the search for new therapeutic methods for pathogen infection treatment seems to be necessary. One of the possibilities is the utilization of bacteriophages (phages)-the natural enemies of bacteria. Thus, this study is aimed at the genomic and functional characterization of two newly isolated phages targeting MDR Salmonella enterica strains and their efficacy in salmonellosis biocontrol in raw carrot-apple juice. The Salmonella phage vB_Sen-IAFB3829 (Salmonella phage strain KKP 3829) and Salmonella phage vB_Sen-IAFB3830 (Salmonella phage strain KKP 3830) were isolated against S. I (6,8:l,-:1,7) strain KKP 1762 and S. Typhimurium strain KKP 3080 host strains, respectively. Based on the transmission electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the viruses were identified as members of tailed bacteriophages from the Caudoviricetes class. Genome sequencing revealed that these phages have linear double-stranded DNA and sizes of 58,992 bp (vB_Sen-IAFB3829) and 50,514 bp (vB_Sen-IAFB3830). Phages retained their activity in a wide range of temperatures (from -20 °C to 60 °C) and active acidity values (pH from 3 to 11). The exposure of phages to UV radiation significantly decreased their activity in proportion to the exposure time. The application of phages to the food matrices significantly reduced the level of Salmonella contamination compared to the control. Genome analysis showed that both phages do not encode virulence or toxin genes and can be classified as virulent bacteriophages. Virulent characteristics and no possible pathogen factors make examined phages feasible to be potential candidates for food biocontrol.

Genomic Analysis and In Vitro Investigation of the Hop Resistance Phenotype of Two Novel Loigolactobacillus backii Strains, Isolated from Spoiled Beer.

Loigolactobacillus backii is an important beer-spoiling species, exhibiting high hop tolerance. Here, we present the annotated whole genome sequence of two recently isolated strains, Lg. backii KKP 3565 and KKP 3566. Firstly, to study the genetic basis of the persistence of the two isolates in beer, a comprehensive bioinformatic analysis ensued. Their chromosome map was constructed, using whole-genome sequencing and assembly, revealing that the two strains carry genomes with a length of 2.79 Mb with a GC content of 40.68%. An average nucleotide identity (ANI) analysis demonstrated that the novel strains possess unique genomic sequences, also confirming their classification into the Lg. backii species. Their genome harbors numerous insertion sequences and plasmids, originating from other beer-spoiling species. Regarding their adaptation in brewery environment, homologous genes that confer resistance to hop were spotted, while the impact of hop bitters and pure beer on bacterial growth was investigated, in vitro. In brief, low hop concentrations were found to induce the proliferation of strains, while a higher concentration negatively affected their growth. Nonetheless, their ability to survive in pure beer indicated their tolerance to high hop concentrations. These results offer insight into the capacity of Lg. backii KKP 3566 and Lg. backii KKP 3566 to tolerate the extreme conditions prevalent in the brewery environment.

Research on the Electron Structure and Antimicrobial Properties of Mandelic Acid and Its Alkali Metal Salts.

This article investigated the structure, and the spectroscopic and antimicrobial properties of mandelic acid and its alkali metal salts. The electron charge distribution and aromaticity in the analyzed molecules were investigated using molecular spectroscopy methods (FT-IR, FT-Raman, 1H NMR, and 13C NMR) and theoretical calculations (structure, NBO, HOMO, LUMO, energy descriptors, and theoretical IR and NMR spectra). The B3LYP/6-311++G(d,p) method was used in the calculations. The antimicrobial activities of mandelic acid and its salt were tested against six bacteria: Gram-positive Listeria monocytogenes ATCC 13932, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, and Loigolactobacillus backii KKP 3566; Gram-negative Escherichia coli ATCC 25922 and Salmonella Typhimurium ATCC 14028, as well as two yeast species, Rhodotorulla mucilaginosa KKP 3560 and Candida albicans ATCC 10231.

Effectiveness of a Phage Cocktail as a Potential Biocontrol Agent against Saprophytic Bacteria in Ready-To-Eat Plant-Based Food.

This study aimed to evaluate the effectiveness of the phage cocktail to improve the microbiological quality of five different mixed-leaf salads: rucola, mixed-leaf salad with carrot, mixed-leaf salad with beetroot, washed and unwashed spinach, during storage in refrigerated conditions. Enterobacterales rods constituted a significant group of bacteria in the tested products. Selected bacteria were tested for antibiotic resistance profiles and then used to search for specific bacteriophages. Forty-three phages targeting bacteria dominant in mixed-leaf salads were isolated from sewage. Their titer was determined, and lytic activity was assessed using the Bioscreen C Pro automated growth analyzer. Two methods of phage cocktail application including spraying, and an absorption pad were effective for rucola, mixed leaf salad with carrot, and mixed leaf salad with beetroot. The maximum reduction level after 48 h of incubation reached 99.9% compared to the control sample. In washed and unwashed spinach, attempts to reduce the number of microorganisms did not bring the desired effect. The decrease in bacteria count in the lettuce mixes depended on the composition of the autochthonous saprophytic bacteria species. Both phage cocktail application methods effectively improved the microbiological quality of minimally processed products. Whole-spectral phage cocktail application may constitute an alternative food microbiological quality improvement method without affecting food properties.

Bacterial Pathogens in the Food Industry: Antibiotic Resistance and Virulence Factors of Salmonella enterica Strains Isolated from Food Chain Links.

Salmonella is one of the most important foodborne pathogens. Fifty-three strains of Salmonella deposited in the Culture Collection of Industrial Microorganisms-Microbiological Resources Center (IAFB) were identified using molecular and proteomic analyses. Moreover, the genetic similarity of the tested strains was determined using the PFGE method. Main virulence genes were identified, and phenotypical antibiotic susceptibility profiles and prevalence of resistance genes were analyzed. Subsequently, the occurrence of the main mechanisms of ß-lactam resistance was determined. Virulence genes, invA, fimA, and stn were identified in all tested strains. Phenotypic tests, including 28 antibiotics, showed that 50.9% of the strains were MDR. The tet genes associated with tetracyclines resistance were the most frequently identified genes. Concerning the genes associated with ESBL-producing Salmonella, no resistance to the TEM and CTX-M type was identified, and only two strains (KKP 1597 and KKP 1610) showed resistance to SHV. No strains exhibited AmpC-type resistance but for six Salmonella strains, the efflux-related resistance of PSE-1 was presented. The high number of resistant strains in combination with multiple ARGs in Salmonella indicates the possible overuse of antibiotics. Our results showed that it is necessary to monitor antimicrobial resistance profiles in all food chain links constantly and to implement a policy of proper antibiotic stewardship to contain or at least significantly limit the further acquisition of antibiotic resistance among Salmonella strains.

Role of Lactic Acid Bacteria in Food Preservation and Safety.

Fermentation of various food stuffs by lactic acid bacteria is one of the oldest forms of food biopreservation. Bacterial antagonism has been recognized for over a century, but in recent years, this phenomenon has received more scientific attention, particularly in the use of various strains of lactic acid bacteria (LAB). Certain strains of LAB demonstrated antimicrobial activity against foodborne pathogens, including bacteria, yeast and filamentous fungi. Furthermore, in recent years, many authors proved that lactic acid bacteria have the ability to neutralize mycotoxin produced by the last group. Antimicrobial activity of lactic acid bacteria is mainly based on the production of metabolites such as lactic acid, organic acids, hydroperoxide and bacteriocins. In addition, some research suggests other mechanisms of antimicrobial activity of LAB against pathogens as well as their toxic metabolites. These properties are very important because of the future possibility to exchange chemical and physical methods of preservation with a biological method based on the lactic acid bacteria and their metabolites. Biopreservation is defined as the extension of shelf life and the increase in food safety by use of controlled microorganisms or their metabolites. This biological method may determine the alternative for the usage of chemical preservatives. In this study, the possibilities of the use of lactic acid bacteria against foodborne pathogens is provided. Our aim is to yield knowledge about lactic acid fermentation and the activity of lactic acid bacteria against pathogenic microorganisms. In addition, we would like to introduce actual information about health aspects associated with the consumption of fermented products, including probiotics.

Application of Lytic Bacteriophages and Their Enzymes to Reduce Saprophytic Bacteria Isolated from Minimally Processed Plant-Based Food Products-In Vitro Studies.

The aim of this study was to isolate phage enzymes and apply them in vitro for eradication of the dominant saprophytic bacteria isolated from minimally processed food. Four bacteriophages-two Enterobacter-specific and two Serratia-specific, which produce lytic enzymes-were used in this research. Two methods of phage enzyme isolation were tested, namely precipitation with acetone and ultracentrifugation. It was found that the number of virions could be increased almost 100 times due to the extension of the cultivation time (72 h). The amplification of phage particles and lytic proteins was dependent on the time of cultivation. Considering the influence of isolated enzymes on the growth kinetics of bacterial hosts, proteins isolated with acetone after 72-hour phage propagation exhibited the highest inhibitory effect. The reduction of bacteria count was dependent on the concentration of enzymes in the lysates. The obtained results indicate that phages and their lytic enzymes could be used in further research aiming at the improvement of microbiological quality and safety of minimally processed food products.

High-Pressure-Induced Sublethal Injuries of Food Pathogens-Microscopic Assessment.

High Hydrostatic Pressure (HHP) technology is considered an alternative method of food preservation. Nevertheless, the current dogma is that HHP might be insufficient to preserve food lastingly against some pathogens. Incompletely damaged cells can resuscitate under favorable conditions, and they may proliferate in food during storage. This study was undertaken to characterize the extent of sublethal injuries induced by HHP (300-500 MPa) on Escherichia coli and Listeria inncua strains. The morphological changes were evaluated using microscopy methods such as Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Epifluorescence Microscopy (EFM). The overall assessment of the physiological state of tested bacteria through TEM and SEM showed that the action of pressure on the structure of the bacterial membrane was almost minor or unnoticeable, beyond the L. innocua wild-type strain. However, alterations were observed in subcellular structures such as the cytoplasm and nucleoid for both L. innocua and E. coli strains. More significant changes after the HHP of internal structures were reported in the case of wild-type strains isolated from raw juice. Extreme condensation of the cytoplasm was observed, while the outline of cells was intact. The percentage ratio between alive and injured cells in the population was assessed by fluorescent microscopy. The results of HHP-treated samples showed a heterogeneous population, and red cell aggregates were observed. The percentage ratio of live and dead cells (L/D) in the L. innocua collection strain population was higher than in the case of the wild-type strain (69%/31% and 55%/45%, respectively). In turn, E. coli populations were characterized with a similar L/D ratio. Half of the cells in the populations were distinguished as visibly fluorescing red. The results obtained in this study confirmed sublethal HHP reaction on pathogens cells.

Effect of High Hydrostatic Pressure on Stress-Related dnaK, hrcA, and ctsR Expression Patterns in Selected Lactobacilli Strains.

Lactic acid bacteria (LAB) in the natural environment meet multiple stressors such as pH and temperature variations, increased nutrition and metabolite concentrations, harmful chemicals, acidic/oxidative conditions, osmotic pressure, and starvation. However, LAB strains are not subjected to high hydrostatic pressure (HHP) which currently is the most common non-thermal decontamination technology in the food industry. In this context, the LAB response to HHP is more difficult to identify compared to other stress-induced responses, and dnaK, ctsR, and hrcA can serve as essential regulators in this reaction. In the present study, the expression level of dnaK, ctsR, and hrcA mRNAs in 15 LAB strains after the HHP (300 MPa/5') exposure was evaluated. As a result, the HHP-treatment affected the up-regulation of dnaK, ctsR, and hrcA in L. backii KKP 3565, L. backii KKP 3566, L. rhamnosus KKP 3570, L. brevis KKP 3575 strains, whereas, in L. plantarum KKP 3569, L. rhamnosus KKP 3571, L. brevis KKP 3573 all genes were lower expressed. The relative expression level of the dnaK, ctsR, and hrcA either before or after the pressure treatment for L. brevis DSM 6235, L. rhamnosus KKP 3572, L. brevis KKP 3574, L. brevis KKP 3576, L. rossiae KKP 3577, L. curvatus KKP 3578 strains were undetectable. Significant differences in the expression levels were observed, between the control and the HHP treatment strains for dnaK in L. backii KKP 3565, L. backii KKP 3566, L. plantarum KKP 3569, L. rhamnosus KKP 3570, L. rhamnosus KKP 3571, ctsR in, L. backii KKP 3565, L. rhamnosus KKP 3570, L. rhamnosus KKP 3571, and hrcA in L. plantarum KKP 3569, L. rhamnosus KKP 3571. Overall, the studied genes, dnaK, ctsR, and hrcA can be useful markers to indicate the LAB cellular response to HHP. These molecular parameters can help to optimize the desirable LAB growing conditions in industrial processes and to understand the complexity of the stress-related mechanism.

Effects of Pressure Level and Time Treatment of High Hydrostatic Pressure (HHP) on Inulin Gelation and Properties of Obtained Hydrogels.

The aim of this study was the evaluation of the influence of different HHP levels (150 and 300 MPa) and time treatment (5, 10, 20 min) on the gelation and properties of hydrogels with different inulin concentration (15, 20, 25 g/100 g). High-pressure treatment, in tested ranges, induces inulin gels and allows obtaining gel structures even at a lowest tested inulin content (i.e., 15 g/100 g). Selecting the pressure parameters, it is possible to modify the characteristics of the created hydrogels. The use of higher pressure (i.e., 300 MPa) allows to increase the stability of the hydrogels and change their structure to more compressed, which results in higher yield stress, lower spreadability, harder and more adhesive structure. For example, increasing the inulin gelling induction pressure (concentration 20 g/100 g) from 150 to 300 MPa with a time treatment of 10 min resulted in an increase in yield stress from 38.1 to 711.7 Pa, spreadability force from 0.59 to 4.59 N, firmness from 0.11 to 1.46 N, and adhesiveness from -0.06 to -0.65 N. Extending the time treatment of HHP increases this effect, but mainly when higher pressure and a higher concentration of inulin are being used. For example, extension of time treatment at 300 MPa pressure from 5 to 20 min resulted in an increase in yield stress from 774.8 to 1273.8 Pa, spreadability force from 6.28 to 8.43 N, firmness from 1.87 to 2.98 N, and adhesiveness from -0.94 to -1.27 N. The obtained results indicate the possibility of using HHP to create inulin hydrogels tailored to the characteristics in a specific food product.

Characterization and Genome Study of Novel Lytic Bacteriophages against Prevailing Saprophytic Bacterial Microflora of Minimally Processed Plant-Based Food Products.

The food industry is still searching for novel solutions to effectively ensure the microbiological safety of food, especially fresh and minimally processed food products. Nowadays, the use of bacteriophages as potential biological control agents in microbiological food safety and preservation is a promising strategy. The aim of the study was the isolation and comprehensive characterization of novel bacteriophages with lytic activity against saprophytic bacterial microflora of minimally processed plant-based food products, such as mixed leaf salads. From 43 phages isolated from municipal sewage, four phages, namely Enterobacter phage KKP 3263, Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 have lytic activity against Enterobacter ludwigii KKP 3083, Citrobacter freundii KKP 3655, Enterobacter cloacae KKP 3082, and Serratia fonticola KKP 3084 bacterial strains, respectively. Transmission electron microscopy (TEM) and whole-genome sequencing (WGS) identified Enterobacter phage KKP 3263 as an Autographiviridae, and Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 as members of the Myoviridae family. Genome sequencing revealed that these phages have linear double-stranded DNA (dsDNA) with sizes of 39,418 bp (KKP 3263), 61,608 bp (KKP 3664), 84,075 bp (KKP 3262), and 148,182 bp (KKP 3264). No antibiotic resistance genes, virulence factors, integrase, recombinase, or repressors, which are the main markers of lysogenic viruses, were annotated in phage genomes. Serratia phage KKP 3264 showed the greatest growth inhibition of Serratia fonticola KKP 3084 strain. The use of MOI 1.0 caused an almost 5-fold decrease in the value of the specific growth rate coefficient. The phages retained their lytic activity in a wide range of temperatures (from -20 °C to 50 °C) and active acidity values (pH from 4 to 11). All phages retained at least 70% of lytic activity at 60 °C. At 80 °C, no lytic activity against tested bacterial strains was observed. Serratia phage KKP 3264 was the most resistant to chemical factors, by maintaining high lytic activity across a broader range of pH from 3 to 11. The results indicated that these phages could be a potential biological control agent against saprophytic bacterial microflora of minimally processed plant-based food products.

Transcriptional Regulation of the Multiple Resistance Mechanisms in Salmonella-A Review.

The widespread use of antibiotics, especially those with a broad spectrum of activity, has resulted in the development of multidrug resistance in many strains of bacteria, including Salmonella. Salmonella is among the most prevalent causes of intoxication due to the consumption of contaminated food and water. Salmonellosis caused by this pathogen is pharmacologically treated using antibiotics such as fluoroquinolones, ceftriaxone, and azithromycin. This foodborne pathogen developed several molecular mechanisms of resistance both on the level of global and local transcription modulators. The increasing rate of antibiotic resistance in Salmonella poses a significant global concern, and an improved understanding of the multidrug resistance mechanisms in Salmonella is essential for choosing the suitable antibiotic for the treatment of infections. In this review, we summarized the current knowledge of molecular mechanisms that control gene expression related to antibiotic resistance of Salmonella strains. We characterized regulators acting as transcription activators and repressors, as well as two-component signal transduction systems. We also discuss the background of the molecular mechanisms of the resistance to metals, regulators of multidrug resistance to antibiotics, global regulators of the LysR family, as well as regulators of histone-like proteins.

Drug-Releasing Antibacterial Coating Made from Nano-Hydroxyapatite Using the Sonocoating Method.

Medical implant use is associated with a risk of infection caused by bacteria on their surface. Implants with a surface that has both bone growth-promoting properties and antibacterial properties are of interest in orthopedics. In the current study, we fabricated a bioactive coating of hydroxyapatite nanoparticles on polyether ether ketone (PEEK) using the sonocoating method. The sonocoating method creates a layer by immersing the object in a suspension of nanoparticles in water and applying a high-power ultrasound. We show that the simple layer fabrication method results in a well-adhering layer with a thickness of 219 nm to 764 nm. Dropping cefuroxime sodium salt (Cef) antibiotic on the coated substrate creates a layer with a drug release effect and antibacterial activity against Staphylococcus aureus. We achieved a concentration of up to 1 mg of drug per cm2 of the coated substrate. In drug release tests, an initial burst was observed within 24 h, accompanied by a linear stable release effect. The drug-loaded implants exhibited sufficient activity against S. aureus for 24 and 168 h. Thus, the simple method we present here produces a biocompatible coating that can be soaked with antibiotics for antibacterial properties and can be used for a range of medical implants.

Isolation and identification of guaiacol producing Alicyclobacillus fastidiosus strains from orchards in Poland.

The genus Alicyclobacillus comprises a group of Gram-positive, thermo-acidophilic bacteria that are capable of producing highly resistant endospores during unfavorable environmental conditions. The members of this genus inhabit natural environments, including hot springs and soils. The main reason behind the spoilage of final commercial fruit products by Alicyclobacillus is the contamination of fruits with soil at the time of harvesting. Some of the Alicyclobacillus species, including Alicyclobacillus acidoterrestris, are categorized as spoilage bacteria due to their ability to produce off-flavor compounds (e.g., guaiacol and halophenols) that adversely affect the taste and aroma of beverages. In our study, Alicyclobacillus species were isolated from Polish orchard soils and fruits and were subjected to 16S rDNA sequencing. The results of the analysis showed that the isolated strains belonged to A. acidoterrestris and Alicyclobacillus fastidiosus species. All the three isolated strains of A. fastidiosus (f1, f2, f3) exhibited similar morphological and biochemical properties as the strain described in the literature. However, these isolated strains were able to produce guaiacol at temperatures of 20°C, 25°C, and 45°C. Thus, the strains of A. fastidiosus discovered in the present study can be included in the group of spoilage species as they possessed the gene responsible for the production of guaiacol.

Influence of High Hydrostatic Pressure on the Identification of Lactobacillus by MALDI-TOF MS- Preliminary Study.

In the present study, we assessed the ability of MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) to identify microbial strains subjected to high hydrostatic pressure (HHP) as a stress factor. Protein changes induced by HHP can affect the identification of microorganisms when the identification technique is based on the protein profile. We evaluated two methods, namely MALDI-TOF MS and 16S rDNA sequencing, as a valuable tool to identify Lactobacillus species isolated from spoiled food, juices and beers. The data obtained from the protein mass fingerprint analysis of some of the lactobacilli strains showed differences in unpressured and pressured mass spectrum profiles (MSPs), which influenced the results of the identification. Four out of 13 strains (30%) showed different MSP results for unpressured and pressured samples and these results did not overlap with the 16S rDNA identification results. The 16S rDNA sequencing method revealed that five unpressured strains (38%) and four pressured strains (40%) were identified correctly by MALDI-TOF MS. Both methods showed compatible results in 38% of unpressured strains and in 30% of pressured strains. Stress factors, cultivation methods or the natural environment from which the bacteria were derived can affect their protein profile and thus change the mass spectrum. It is necessary to expand the database with a wide range of mass spectra dedicated to a high-throughput study of the microorganisms derived from different environments.

Bacteriophages-a new hope or a huge problem in the food industry.

Bacteriophages are viruses that are ubiquitous in nature and infect only bacterial cells. These organisms are characterized by high specificity, an important feature that enables their use in the food industry. Phages are applied in three sectors in the food industry: primary production, biosanitization, and biopreservation. In biosanitization, phages or the enzymes that they produce are mainly used to prevent the formation of biofilms on the surface of equipment used in the production facilities. In the case of biopreservation, phages are used to extend the shelf life of products by combating pathogenic bacteria that spoil the food. Although phages are beneficial in controlling the food quality, they also have negative effects. For instance, the natural ability of phages that are specific to lactic acid bacteria to destroy the starter cultures in dairy production incurs huge financial losses to the dairy industry. In this paper, we discuss how bacteriophages can be either an effective weapon in the fight against bacteria or a bane negatively affecting the quality of food products depending on the type of industry they are used.

Genetic Heterogeneity of Alicyclobacillus Strains Revealed by RFLP Analysis of vdc Region and rpoB Gene.

PCR-RFLP targeting of the 16S rDNA and rpoB genes, as well as the vdc region, was applied to identify and differentiate between the spoilage and non-spoilage Alicyclobacillus species. Eight reference strains and 75 strains isolated from spoiled juices, juice concentrates, drinks, its intermediates, and fresh apples were subject to study. Hin6I restriction patterns of the 16S rDNA gene enabled distinguishing between all the species analyzed, while the rpoB gene and vdc gene cluster analysis also revealed that there were two major types among the A. acidoterrestris isolates, one similar to the reference strain A. acidoterrestris DSM 2498, and the other similar to the reference strain A. acidoterrestris ATCC 49025. Heterogeneity was also observed among the A. acidocaldarius isolates. RFLP analysis of the 16S rDNA and rpoB genes, as well as vdc region, can be used successfully in the identification and research of intraspecies heterogeneity of the Alicyclobacillus species.

Long-Term Storage of Vegetable Juices Treated by High Hydrostatic Pressure: Assurance of the Microbial Safety.

Food business operators search for new, mild technologies, which extend the shelf life of product without changing the sensory and nutritional properties. High hydrostatic pressure (HHP) meets these requirements; however it also triggers sublethal injury of bacterial cells. Sublethal injuries could spoil the product during storage and potentially pose major public health concerns. This study aims to examine the changes of sublethally injured pathogens cells in two vegetable juices: carrot juice (pH 6.0-6.7) and beetroot juice (pH 4.0-4.2) that are induced by HHP (300-500 MPa). The possibilities of recovery of bacterial cells during 28 days of juices storage at two different temperatures (5°C and 25°C) were determined using plate count methods. During the entire period of storage of carrot juice at refrigerated temperature, the propagation and regeneration of L. innocua strains were observed. Storage at 25°C showed that the number of these bacteria drastically decreased between 14 and 21 days. The above phenomenon was not detected in E. coli case. There was no cells recovery during long-term refrigerated storage for all strains in beetroot juice. However, in some cases spoiling of this product intermittently occurred at 25°C storage temperature. This work demonstrates that carrot juice supports growth and regeneration of HHP-sublethally injured L. innocua, while beetroot juice can be classified as a safe product.