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.

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.

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.

MALDI-TOF MS - application in food microbiology.

Microbiological purity control of food products is of great importance in the food industry. Contaminated food is often characterized by a deteriorated taste, smell, and appearance, and when consumed, it can pose a threat to human health and life. Also, contamination incurs huge financial losses to the food industry. Different methods are used for identification of the microorganisms isolated from food, which are based on phenotypic, immunologic, genetic, and spectroscopic techniques. Unfortunately, these methods have the following disadvantages: laborious, time-consuming, requiring a well-trained spectrometer operator with specialist knowledge, or very accurate, but complicated, and extremely expensive. In recent years, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has been gaining increasing importance in the field of food microbiology. Unlike other techniques used for microorganisms identification, MALDI-TOF MS is more rapid, accurate and cost-efficient, and easy to perform. Thus, this method can be applied in the food industry to quickly and accurately identify microorganisms, which is crucial for controlling the quality of food products. The present review aims to discuss the selected applications of MALDI-TOF MS in food microbiology. It mainly focuses on the characteristics of this method and its potential use in the identification and typing of microorganisms including filamentous fungi, yeasts, and bacteria in fermented beverages (beer and wine), honey, dairy products like yogurt and pasteurized milk, pork, and seafood.

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.

Lactic acid bacteria stress response to preservation processes in the beverage and juice industry.

In this review we summarize stress factors that affect the lactic acid bacteria (LAB) and cause different molecular stress responses. LAB belong to a group of bacteria that is very widespread in food and beverages. They are present, and desired, in fermented products like yogurts, cheese, vegetables, meat or wine. In most of them, LAB are providing positive sensory and nutritive features. However, as harmless and desired microbes in one product, LAB can cause spoilage and a bad taste of others, especially in juices and beverages. LAB are resistant to many stress factors which allows them to survive in harsh environments. The most common stress factors they have to deal with are: heat, cold, acidity, NaCl and high hydrostatic pressure (HHP). Their ability to survive depends on their skills to cope with stress factors. Under stress conditions, LAB activate mechanisms that allow them to adjust to the new conditions, which can influence their viability and technological properties. This ability to adapt to different stress conditions may come from the cross-protection systems they have, as resistance to one factor may help them to deal with the other stress effectors. LAB are highly valuable for the food industry and that is why it is important to understand their stress response mechanisms.