Accumulation of intracellular S-adenosylmethionine increases the fermentation rate of bottom-fermenting brewer's yeast during high-gravity brewing.

High-gravity brewing has been used to reduce costs and energy, as well as to produce new types of beer with high alcohol content. To identify the key metabolic pathways underlying efficient high-gravity brewing, we explored metabolites that were highly accumulated during alcoholic fermentation under high-maltose conditions using bottom-fermenting brewer's yeast, Saccharomyces pastorianus. Based on metabolomic data, we focused on S-adenosylmethionine (SAM), which may be involved in glycolysis and alcoholic fermentation in the closely related yeast species Saccharomyces cerevisiae. Exogenous SAM led to an increase in fermentation rate in both high-maltose synthetic medium and high-gravity wort. Although SAM is composed of methionine and the adenosine moiety of ATP, neither methionine nor adenosine significantly increased the fermentation rate. These results suggest that SAM is specifically associated with the fermentation rate of bottom-fermenting brewer's yeast. Deletion of the adenosine kinase gene ADO1, which leads to an accumulation of SAM in S. cerevisiae cells, elevated the fermentation rate in high-glucose synthetic medium at 15°C; however, this ado1Δ effect became less significant at higher temperatures. Similarly, a SAM-accumulating S. pastorianus mutant strain, with enhanced resistance to the adenosine analog cordycepin, exhibited a higher fermentation rate in both high-maltose synthetic medium and high-gravity wort. Taken together, our study demonstrates that SAM acts as a positive regulator in high-gravity brewing at low temperatures and that cordycepin resistance could serve as a useful indicator for breeding S. pastorianus strains with high fermentation performance.

Lactobacillus curtus sp. nov., isolated from beer in Finland.

A Gram-stain-positive, catalase-negative and short-rod-shaped organism, designated VTT E-94560, was isolated from beer in Finland and deposited in the VTT culture collection as a strain of Lactobacillus rossiae. However, the results of 16S rRNA gene sequence analysis showed that VTT E-94560 was only related to Lactobacillus rossiae JCM 16176T with 97.0 % sequence similarity, lower than the 98.7 % regarded as the boundary for the species differentiation. Additional phylogenetic studies on the pheS gene, rpoA gene and 16S-23S rRNA internally transcribed spacer region further reinforced the taxonomically independent status of VTT E-94560 and its related Lactobacillus species including L. rossiae and Lactobacillus siliginis. Strain VTT E-94560 also exhibited several differences in its carbohydrate fermentation profiles from those related Lactobacillus species. In addition, DNA-DNA relatedness between VTT E-94560 and these two type strains was 4 % (L. rossiae JCM 16176T) and 12 % (L. siliginins JCM 16155T), respectively, which were lower than the 70 % cut-off for general species delineation, indicating that these three strains are not taxonomically identical at the species level. These studies revealed that VTT E-94560 represents a novel species, for which the name Lactobacillus curtus sp. nov. is proposed. The type strain is VTT E-94560T (=JCM 31185T).

Defective quiescence entry promotes the fermentation performance of bottom-fermenting brewer's yeast.

One of the key processes in making beer is fermentation. In the fermentation process, brewer's yeast plays an essential role in both the production of ethanol and the flavor profile of beer. Therefore, the mechanism of ethanol fermentation by of brewer's yeast is attracting much attention. The high ethanol productivity of sake yeast has provided a good basis from which to investigate the factors that regulate the fermentation rates of brewer's yeast. Recent studies found that the elevated fermentation rate of sake Saccharomyces cerevisiae species is closely related to a defective transition from vegetative growth to the quiescent (G0) state. In the present study, to clarify the relationship between the fermentation rate of brewer's yeast and entry into G0, we constructed two types of mutant of the bottom-fermenting brewer's yeast Saccharomyces pastorianus Weihenstephan 34/70: a RIM15 gene disruptant that was defective in entry into G0; and a CLN3ΔPEST mutant, in which the G1 cyclin Cln3p accumulated at high levels. Both strains exhibited higher fermentation rates under high-maltose medium or high-gravity wort conditions (20° Plato) as compared with the wild-type strain. Furthermore, G1 arrest and/or G0 entry were defective in both the RIM15 disruptant and the CLN3ΔPEST mutant as compared with the wild-type strain. Taken together, these results indicate that regulation of the G0/G1 transition might govern the fermentation rate of bottom-fermenting brewer's yeast in high-gravity wort.

Rapid detection and identification of beer-spoilage lactic acid bacteria by microcolony method.

We evaluated a microcolony method for the detection and identification of beer-spoilage lactic acid bacteria (LAB). In this approach, bacterial cells were trapped on a polycarbonate membrane filter and cultured on ABD medium, a medium that allows highly specific detection of beer-spoilage LAB strains. After short-time incubation, viable cells forming microcolonies were stained with carboxyfluorescein diacetate (CFDA) and counted with muFinder Inspection System. In our study, we first investigated the growth behavior of various beer-spoilage LAB by traditional culture method, and Lactobacillus lindneri and several L. paracollinoides strains were selected as slow growers on ABD medium. Then the detection speeds were evaluated by microcolony method, using these slowly growing strains. As a result, all of the slowly growing beer-spoilage LAB strains were detected within 3 days of incubation. The specificity of this method was found to be exceptionally high and even discriminated intra-species differences in beer-spoilage ability of LAB strains upon detection. These results indicate that our microcolony approach allows rapid and specific detection of beer-spoilage LAB strains with inexpensive CFDA staining. For further confirmation of species status of detected strains, subsequent treatment with species-specific fluorescence in situ hybridization (FISH) probes was shown as effective for identifying the CFDA-detected microcolonies to the species level. In addition, no false-positive results arising from noise signals were recognized for CFDA staining and FISH methods. Taken together, the developed microcolony method was demonstrated as a rapid and highly specific countermeasure against beer-spoilage LAB, and compared favorably with the conventional culture methods.

Rapid and sensitive detection of viable bacteria in contaminated platelet concentrates using a newly developed bioimaging system.

BACKGROUND: Rapid and sensitive methods for the detection of bacteria in platelet concentrates (PCs) are required as well as inactivation techniques to decrease the transfusion-associated risk of infection from bacterially contaminated PCs. In this study, a rapid microbiologic method for the sensitive counting of viable bacteria in PCs was developed by combining a fluorescent staining technique and a bioimaging system. STUDY DESIGN AND METHODS: An esterase indicator, carboxyfluorescein diacetate, was used to detect physiologically active bacteria. Treatment was optimized to selectively remove platelets (PLTs). Bacterial cells trapped on a filter were automatically discriminated from other particles or PLT debris and counted by a bioimaging system. The sensitivity, rapidity, and recovery rates were evaluated using PCs spiked with 14 reference bacterial strains and clinical isolates. RESULTS: Lysis treatment with enzyme and detergent was effective to remove PLTs and white blood cells. Two buffers for fluorescent vital staining were needed for highly sensitive detection of pathogenic bacteria. Fewer than 100 cells spiked in 5-mL PCs were detected by the bioimaging system after treatment and fluorescent staining, and this result shows that PLTs are selectively digested by the treatment. Bacterial cells spiked in 25-mL PCs were detected within 45 minutes (treatment, 15 min; filtration and fluorescent staining, 15 min; automated counting and precise image analysis, 10-15 min). CONCLUSION: The microbiologic method described here is rapid and sensitive, and this method has potential for the screening of PCs contaminated with bacterial cells. Furthermore, this method could contribute to further evaluation of inactivation techniques.

Effects of morphological changes in beer-spoilage lactic acid bacteria on membrane filtration in breweries.

Membrane filter performance was investigated using beer-spoilage lactic acid bacteria (LAB). As a result, beer-adapted LAB strains showed considerably increased penetration rate through filters, as compared with non-adapted strains. Further statistical analyses demonstrated the significant shifts in cell size distribution towards shorter rods, when Lactobacillus brevis and L. lindneri strains were precultured in beer. These results indicate that diminished cell size is responsible for the deteriorated filter performance and, therefore, beer-adapted lactic acid bacteria are regarded as a serious threat to the production of unpasteurized beers. In addition, the selection of test strains and preculture conditions are suggested to be important for the rigorous and standardized evaluation of membrane filter performance in the brewing industry.