Koji Suzuki

To determine factors affecting the growth of the genus Pectinatus in beer, P. frisingensis ABBC437 was inoculated into 15 different beer samples. Although the pH, alcohol content, and bitterness unit of the 15 beer samples were adjusted to similar values within tolerance, growth kinetics as expressed by OD600 (optical density measured at a wavelength of 600 nm) were considerably different among the beer samples, indicating the presence of “as yet” unidentified factor(s) affecting the growth of P. frisingensis in beer. Chemical compositions of the beer samples were determined, and multiple regression analysis was conducted to predict the OD600 value. As a result, iron was suggested as one of the factors that affects the growth of P. frisingensis in beer because the additions of iron to the beer samples showed enhanced growth of P. frisingensis in a dose-dependent manner. Stimulatory effects of growth by the addition of iron were also observed for P. cerevisiiphilus and P. haikarae. Additionally, effects of diatomaceous earth treatment on the iron concentrations of the beer samples were evaluated. Iron concentrations varied, depending on the brand of diatomaceous earth, and the growth of P. frisingensis in beer showed a high correlation coefficient (0.924, p < 0.01) with the iron concentration in the beer samples. From these results, iron can be considered as an important factor for the growth of the genus Pectinatus in beer and selecting a low iron concentration diatomaceous earth is an effective way to improve the stability of beer against the risk of spoilage by the genus Pectinatus.

In microbiological quality control of final beer products, 100–400 ml beer samples are traditionally filtered on a membrane filter, followed by a lengthy culturing process in/on a laboratory medium that usually requires 3–14 days. This study aims to detect beer spoilage lactic acid bacteria (LAB) in a culture-independent fashion, using a direct polymerase chain reaction (PCR) approach. In order to detect a trace level of spoilage LAB in beer products, pressure cycling technology was evaluated to determine if this technology could improve the direct recovery of DNA from membrane filters for PCR detection. In this study, a mixed cellulose ester filter was adopted to allow a larger volume of beer to be filtrated. As a result of the optimization of the DNA extraction process that minimizes the loss of DNA and the effect of PCR inhibitors, 100 (1–10) cells/300 ml beer were successfully detected for beer spoilage LAB. Furthermore, a challenge test using 3,000 ml beer showed that 100 cells of Levilactobacillus (Lactobacillus) brevis were detectable without the negative effects of beer-originated PCR inhibitors, indicating that a trace level of contamination, corresponding to 1 cell in 300 ml of beer products, is detectable. It was also shown that the test was completed in approximately 8 hours. Therefore, this newly developed method is considered to be useful for the culture-independent direct detection of an extremely low level of beer spoilage LAB without a traditional culturing process. Supplemental data for this article is available online at at.

To expand the applicability of the third-generation DNA sequencing technologies for the brewing industry, the new experimental protocols for quality control (QC) brewery laboratories have been developed, using the MinION system. Firstly, the hop tolerance genes, horA and horC, were amplified with multiplex PCR, and the PCR amplicons were sequenced by the MinION system. As a result, all of the beer spoilage lactic acid bacteria (LAB) strains carrying horA and/or horC were positively identified. Secondly, STA1, SbKEX2 and GEX1, were evaluated as diagnostic marker genes (DMGs) for discriminating Saccharomyces cerevisiae and S. pastorianus strains that possess essentially indistinguishable nucleotide sequences of the rDNA and internal transcribed spacer (ITS) regions. As a consequence of multiplex PCR specific to STA1, SbKEX2 and GEX1, these DMGs were shown to separate these hard-to-discriminate strains into four groups, namely top-fermenting ale yeast strains (S. cerevisiae), bottom-fermenting lager yeast strains (S. pastorianus), non-brewing S. cerevisiae wild yeast strains, and S. cerevisiae var. diastaticus. This study also demonstrated that the multiplexed PCR amplicons are independently recognized and correctly identified by the MinION system. Taken together, the newly developed methods using the MinION system are applicable to the identification and discrimination of important brewery microorganisms for microbiological QC. It was also shown that the concurrent applications of the rDNA-sequencing-based species identification and DMG approaches by the MinION system are useful for the breweries that now manufacture both traditional beers and non-traditional beverages. Supplemental data for this article is available online at https://doi.org/10.1080/03610470.2021.1939606 .

When a beer is contaminated with unknown microorganisms, rapid and accurate identification of the contaminants and risk assessment of their spoilage capability are of critical importance for proper corrective actions. Molecular-based identification techniques have been used for years to identify beer-spoilage bacteria in the breweries. However, these techniques require expensive, advanced analytical instruments (e.g., DNA sequencer, MALDI-TOF MS, etc.), and advanced skills, hindering their practical use for routine microbiological testing in the field. Thus, the evaluation and development of alternative methods for microbial identification that are low-cost, but rapid and provide the same level of accuracy, are a necessity. One particular technology is the nanopore-based sequencer, MinION, developed by Oxford Nanopore Technologies (ONT). MinION is a low cost, portable real-time device for DNA and RNA sequencing. We developed and evaluated a MinION sequencer-based platform for beer-spoilage bacterial identification. The near full length 16S rRNA gene of a wide variety of beer-spoilage bacteria and those commonly found in the brewery environment, were sequenced using three different sequencing kits and analyzed using the EPI2ME cloud-based analysis platform and a consensus sequence-based approach. The methods successfully rapidly identified test-microorganisms at a species level, (approx. 3.5 h from DNA extraction to data analysis) and with a high accuracy, similar to Sanger sequencing (average per. identity > 98.5%). The MinION sequencer-based microbial identification platform was shown to be a powerful tool for on-field rapid and accurate beer-spoilage bacterial identification, which could revolutionize the way microbial identification is performed in a brewery QC laboratory.

Beer spoilage lactic acid bacteria (LAB) often display hard-to-culture characteristics and fail to grow on conventional quality control (QC) laboratory media, while retaining growth capability in beer. This phenomenon is at least partly due to profound adaptation of spoilage LAB to the brewing environments, where multifactorial antibacterial hurdles and low temperature stress are encountered. In this study, changeable culturability of Secundilactobacillus (Lactobacillus) paracollinoides strains, JCM 11969^T and JCM 15729, has been evaluated by comparing the variants obtained under various preceding subculture conditions. Sodium acetate substantially inhibited the growth of hard-to-culture S. paracollinoides variants, with smaller negative impacts for growth observed with yeast extract, peptone, and manganese sulfate. Growth inhibition was also observed at values higher than pH5.3 for hard-to-culture JCM 11969^T variants and higher than pH5.0 for hard-to-culture JCM 15729 variants, respectively. In contrast, culturable S. paracollinoides variants exhibited sensitivities to low pH and grew well at pH 5.6 or even higher. Strikingly, the addition of sodium acetate led to increased colony forming units for culturable JCM 15729 strains, suggesting that sodium acetate exerts both positive and negative effects on the growth of S. paracollinoides strains. It has been also demonstrated that hard-to-culture and culturable states are reversible for S. paracollinoides JCM 11969^T and JCM 15729, depending on the preceding subculture conditions. Taken together, it has been suggested that culturability of S. paracollinoides strains is easily changeable, and brewing microbiologists must ensure their culturability is maintained as observed upon primary isolation.

Beer is a hostile environment for microorganisms to survive. However, some strains of lactic acid bacteria (LAB) have chosen this harsh environment for their ecological niche and evolved by incrementally acquiring various genetic elements to overcome a range of growth hurdles posed by beer. In fact, over 100 genes appear to be involved for one beer spoilage LAB strain to grow in and spoil beer. Although many of them are chromosomally encoded genes, an increasing number of brewery-specific genetic clusters have been also identified on plasmids, which are often horizontally shared by a variety of beer spoilage LAB species/strains. These plasmid-localized genetic factors were once represented by hop tolerance genes, such as horA, horC, and hitA, that were uncovered in the early days of hop tolerance research. But the recent evidence increasingly indicates that beer spoilage potential of LAB is conferred by multifactorial and complex adaptive responses that are related to a large number of defense mechanisms, including divalent cation homeostasis, cell envelope modification, oxidative stress response, and pH homeostasis. Many of these defense systems have been shown to be encoded on plasmids that are carried by beer spoilage LAB. In the past decade, new plasmid-encoded diagnostic marker genes (DMGs), such as gtf_D15 and fabZ, have been additionally reported for the discrimination of beer spoilage potential of LAB strains. This review summarizes the recent progress in this area of research and provides some insights into the microbiological quality control (QC) tests adopted in brewery laboratories.

Until 30 years ago, only a limited number of bacterial and wild yeast species had been recognized as beer spoilers. However, the number of spoilage species have been rapidly increasing since then, reaching approximately 30 species for traditional beer categories. One trend is the continual emergence of new beer spoilage lactic acid bacteria. Many of these novel beer spoilage species are almost exclusively found in the brewing environments and are considered as beer-associated microorganisms. The second trend is the rise of strictly anaerobic beer spoilage bacteria, including Pectinatus and Megasphaera. Currently, close to 10 strictly anaerobic beer spoilage species have been reported. As a countermeasure, species-specific PCR systems have been adopted in breweries, and continually modified to keep up with the increasing number of beer spoilage species. Nevertheless, the circumstances are changing because of the rising trend of non-traditional beverages, as well as the rising use of non-traditional ingredients, in the brewing industry. These non-traditional beverages include non-alcoholic beers and flavored low alcoholic beverages, and spoilage species for these microbiologically unstable products are dramatically increasing in number. The conventional species-specific PCR can only handle the identification of 20–30 species of microorganisms, while novel methods that allow for a much broader microbial identification, such as DNA sequencing, are becoming more necessary. A few brewers are already evaluating the potential quality control applications of inexpensive third generation DNA sequencing technologies with the brewing industry.

Prior investigations by our research group focused on the method development for the simultaneous analysis of sulfated and phosphorylated glycans. Herein, the developed method was applied to analyze minor acidic N-glycans including sulfated and phosphorylated N-glycans in human serum. First, 2-aminobenzoic acid-labeled minor acidic N-glycans were enriched from the serum using a serotonin-immobilized column, which were then separated into groups using hydrophilic interaction liquid chromatography, and analyzed by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. Phosphorylated hybrid-type and sulfated bi-antennary N-glycans were detected in the serum. In addition, we observed that multiple types of glucuronidated N-glycans were present. These results indicate that the developed method is applicable to the analysis of glucuronidated, as well as sulfated and phosphorylated N-glycans. It was also applied to the sera obtained from 17 healthy subjects and 15 pancreatic cancer patients, and the profiles of sulfated, phosphorylated, and glucuronidated N-glycans were compared. The expressed amount of glucuronidated N-glycans was significantly decreased in some pancreatic cancer patients. Numerous examples for the N-glycan analysis in human serum were reported but phosphorylated and glucuronidated glycans were not investigated. The methods described herein allowed the analysis of minor acidic glycans that are typically difficult to detect.

Beer is a microbiologically stable beverage due to several inhibitory factors for microbial growth. In fact, only a restricted number of species can grow in and spoil beer. The vast majority of beer spoilage microorganisms are lactic acid bacteria belonging to the genera Lactobacillus and Pediococcus. However, many beer spoilage microorganisms exhibit extremely poor culturability, and they are hard to detect by the conventional laboratory media adopted for the quality control of beer. This often leads to quality incidents without the detection of the causative agents in breweries. In addition, the continual emergence of new beer spoilage species has been problematic for brewers that traditionally depend on species identification methods for quality control in the brewing processes. To overcome these difficulties, the brewing industry has strived to develop new microbiological media and species-independent methods for comprehensively detecting and identifying beer spoilage microorganisms. Some brewers are now evaluating new techniques, including inexpensive third-generation DNA sequencing, for the comprehensive identification of beer spoilage microorganisms and hygiene indicators. In this review, the recent progress of microbiological quality control methods in unpasteurized beer production is summarized.

There have been many beer-spoilage incidents caused by wild yeasts. Saccharomyces cerevisiae, Dekkera anomala and D. bruxellensis have been recognized as beer-spoilage yeasts in the brewing industry. In contrast, the beer spoilage ability of Brettanomyces custersianus has not been well characterized, although this species was isolated from beer. In this study, the beer-spoilage ability of currently described Dekkera/Brettanomyces yeast species was investigated. As a consequence, D. anomala, D. bruxellensis and B. custersianus were shown to grow in commercial beers. On the other hand, the remaining two Brettanomyces species, B. naardenensis and B. nanus, did not grow in beer. These results indicate that B. custersianus should be recognized as a beer-spoilage species, in addition to S. cerevisiae, D. anomala, and D. bruxellensis. Therefore we developed multiplex polymerase chain reaction (PCR) for the simultaneous detection and identification of B. custersianus and the other beer-spoilage yeast species. For this purpose, PCR primers were designed in the internal transcribed spacer region or 26S rDNA, and each PCR product was made in different sizes to easily discriminate the species from electrophoretic results. Specificity, reactivity and sensitivity of the designed primers were evaluated. As a result, the developed multiplex PCR method was shown to have high specificity and reactivity, and therefore was considered as an effective tool to identify beer-spoilage yeast species. This tool can contribute to microbiological quality assurance in breweries. Copyright © 2015 The Institute of Brewing & Distilling