Akihiro Mizuno

Effect of Soy Peptide on Brewing Beer

Here, we examined the effect of soy peptides (SPs) on the fermentation and growth of Yeast Bank Weihenstephan 34/70 (W34/70), a bottom-fermenting yeast. We compared fermentation for SP with that for a free amino acid (FAA) mixture having the same amino acid composition as SP, as a nitrogen source. Maltose syrup was used as a carbon source, and the medium contained excess amounts of essential minerals and vitamins. We observed that SP was better than FAA mixture at promoting fermentation and growth and that much more beta-phenylethyl alcohol was produced during fermentation with SP than with FAA mixture. Subsequently, we compared fermentations with the FAA mixture and selected mixtures containing various dipeptides of Phe as a nitrogen source. We found that the rates of Phe metabolism and beta-phenylethyl alcohol generation were much higher when Phe was presented as a dipeptide (Phe-Asp, Phe-Leu, or Phe-Phe) than when presented as FAA. These results show that amino acids such as Phe are absorbed more rapidly when presented as a peptide than as FAA, resulting in a more rapid production of beta-phenylethyl alcohol.

Comprehensive analysis of dipeptides in alcoholic beverages by tag-based separation and determination using liquid chromatography/electrospray ionization tandem mass spectrometry and quadrupole-time-of-flight mass spectrometry.

Fermented foods and beverages contain several different types of dipeptides, which are believed to be important components for taste. To date, however, a method for the comprehensive analysis of dipeptides in these products has not yet been established. In this study, comprehensive analysis of dipeptides in alcoholic beverages was performed by a high-resolution separation method based on the structural characteristics of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized dipeptides as well as dipeptide quantification and structural estimation using ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS), respectively. Dipeptide content was found to differ considerably among Japanese sake, beer, and wine; UHPLC-MS/MS analysis revealed that many types of dipeptides are present in sake. Dipeptide quantification analysis identified 32 types of dipeptides within the concentration range of 1.1-97.2 μM in sake. The analysis was validated by dipeptide recovery of 64.0-107.2% (2.5 μM of standard) with a relative standard deviation of ≤33.2% from an actual alcoholic sample. Furthermore, UHPLC-Q-TOFMS analysis suggested the existence of more than 35 types of dipeptides in sake. Thus, by the combined analysis methods, we discovered that more than 60 dipeptides are present in sake. This research is the first report of dipeptide profiling of fermented alcoholic beverages by comprehensive analysis.

Simple metabolite extraction method for metabolic profiling of the solid-state fermentation of Aspergillus oryzae

Solid-state culture of microorganisms is an important style of culture both in the traditional food industry and in the modern fermentation industry. We propose here a simple method for metabolite extraction from the solid-state fermentation of a filamentous fungus, Aspergillus oryzae, which is known as rice-koji. To evaluate the efficiency of metabolite extraction, liquid chromatography-mass spectrometry (LC/MS) was used for simultaneous detection of a wide range of metabolites including amino acids, organic acids and vitamins, which are of interest in rice-koji making. Among eleven different metabolite extraction methods tested, we found that extraction using acetonitrile/water (50:50), coupled with boiling at 70 °C for 5 min, was advantageous in terms of both extraction yield and metabolism quenching. We consider that our method provides a technical basis for metabolome analysis of a solid-state fermentation.

Evaluation of microbial diversity in sulfite-added and sulfite-free wine by culture-dependent and -independent methods.

The difference in microbiota including non-lactic acid bacteria, non-acetic acid bacteria, and wild yeast during winemaking and in the end-products between sulfite-added and sulfite-free wine, was investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and a culture-dependent method. There were differences between the microorganisms detected by PCR-DGGE and those detected by the culture-dependent method, probably because of the selectivity of culture medium and the characteristics of PCR-based method. In both the red wine and white wine, the microbial diversity of the sulfite-added wine was lower than that of the sulfite-free wine during fermentation. Tatumella terrea was detected from the fermenting must by PCR-DGGE and by the culture-dependent method, even though sulfite inhibited its growth to some extent. We confirmed that the addition of sulfite plays an important role in winemaking by inhibiting the growth of unexpected microorganisms, but on the other hand, it was revealed that some microorganisms can survive and grow in sulfite-added fermenting must. We also analyzed 15 samples of commercial wines by the PCR-DGGE method and detected various microorganisms. Among them, Sphingomonas sp., Pseudozyma sp., Ochromonas sp. and Methylophilus sp. were found for the first time in wine as far as we know. We did not identify a specific microorganism that was detected only from wines without sulfite addition. Thus, the microbiota of end-products seemed to be influenced by other factors, such as filtration before bottling, the production equipment and the storage environment.

Modified COLD-PCR for detection of minor microorganisms in wine samples during the fermentation.

The detection of low-abundant microorganism is difficult when in a sample in which a specific microorganism represents an overwhelming majority using polymerase chain reaction (PCR)-based methods. A modified CO-amplification at Lower Denaturation temperature PCR (mCOLD-PCR) method was developed to detect low-abundant microorganisms using a double-strand RNA probe to inhibit the amplification of the sequence of a major microorganism. Combining the mCOLD-PCR and downstream application (e.g., denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS)), low-abundant microorganisms were detected more efficiently, even when a specific microorganism represents an overwhelming majority of the sample. We demonstrated that mCOLD-PCR-DGGE enabled us to detect Schizosaccharomyces pombe in a model sample coexisting with 10,000 times as many Saccharomyces cerevisiae. When mCOLD-PCR-DGGE was applied in the microbiota analysis of a fermenting white wine, Candida sp. and Cladosporium sp., which were not detected by conventional PCR, were detected. According to the NGS analysis after mCOLD-PCR of a fermenting red wine, the detection ratio of Saccharomyces was decreased dramatically, and the detection ratios of other microorganisms and the numbers of genera detected were increased compared with the conventional PCR. Thus, the application of mCOLD-PCR will reveal comprehensive microbiota of fermented foods, beverages, and so on.

Evaluation of method bias for determining bacterial populations in bacterial community analyses

Various methods are used for analyzing a bacterial community. We recently developed a method for quantifying each bacterium constituting the microbiota by combining a next-generation sequencing (NGS) analysis with a quantitative polymerase chain reaction (NGS-qPCR) assay. Our NGS-qPCR method is useful for analyzing a comprehensive bacterial community because it is enables the easy calculation of the amounts of each bacterium constituting the microbiota. However, it has not been confirmed whether the estimated bacterial community obtained using this NGS-qPCR method corresponds to the results obtained using conventional methods. Accordingly, we prepared model bacterial community samples and analyzed them by several methods (NGS-qPCR, species-specific qPCR, flow cytometry, total direct counting by epifluorescent microscopy [TDC], and plate count). The total bacterial cell densities determined by the PCR-based methods were largely consistent with those determined by the TDC method. There was a difference between the amounts of each bacterium analyzed by NGS-qPCR and species-specific qPCR, although the same trend was shown by both species-specific qPCR and NGS-qPCR. Our findings also demonstrated that there is a strong positive correlation between the cell densities of a specific bacterial group in craft beer samples determined by group-specific qPCR and NGS-qPCR, and there were no significant differences among quantification methods (we tested two bacterial groups: lactic acid bacteria and acetic acid bacteria). Thus, the NGS-qPCR method is a practical method for analyzing a comprehensive bacterial community based on a bacterial cell density.