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

Abstract

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.

Development of a Multiplex PCR Assay for Efficient Detection of Two Potential Probiotic Strains Using Whole Genome-Based Primers

“…Detection at the strain level can give definite evidence for the presence of spoilage strains [16] and discriminate against members of the same species with no harmful activity while also providing hints at appropriate decontamination methods. In this vein, we have previously shown that contaminant Loigolactobacillus backii strains are resistant to heat stress but may be sensitive to pressure treatment, resulting in more efficient decontamination approaches in the brewing industry [46]. Importantly, the novel method developed does not require a high level of technical expertise or sophisticated equipment; it can, therefore, be easily employed in the setting of the fermented food industry.…”

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

“…To determine the resistance to hop, the growth kinetics of the KKP 3573 strain was estimated with the automated growth curve analysis system Bioscreen C Pro (Oy AB Ltd., Growth Curves, Finland), as described by Kiousi et al, 2023 [ 25 ]. London Ale beer was used to formulate the starting concentration of 40 IBU, from which other IBU concentrations—5, 10, 20, and 30—were prepared to perform hop resistance analysis on the Bioscreen.…”

“…Genomic DNA from the KKP 3573 strain was isolated using DNeasy PowerFood Microbial Kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer’s protocol and as described by Kiousi et al, 2023 [ 25 ]. Briefly, DNA purity was determined with Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Watertown, MA, USA), and DNA concentration was measured with Qubit 4.0 Fluorometer (Qubit dsDNA BR Assay Kit, Invitrogen, Carlsbad, CA, USA).…”

“…Figure 1 illustrates the hop-related mechanisms of bacterial inhibition in beer. Research is being undertaken to determine which genes in lactobacillus are responsible for the bacteria’s ability to spoil beer [ 5 , 23 , 24 , 25 ]. Detection of marker sequences is essential for better risk assessment in the brewing industry [ 19 ].…”

Molecular Methods for Detecting Microorganisms in Beverages

“…The genome of bacterial strains contained inserts from other contagious species. These genetic constructs contribute to the adaptation of representatives of lactic acid bacteria to the harsh environment of the brewery, including carrying out vital activities at high concentrations of hops [55]. A similar study, in which lactic acid bacteria were identified in beer, was carried out using the PCR method with primers for the hop resistance genes horA and horC.…”