Effect of kuratsuki Kocuria on sake’s taste varies depending on the combination with different sake yeast strains

doi:10.21203/rs.3.rs-2965635/v1

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Effect of kuratsuki Kocuria on sake’s taste varies depending on the combination with different sake yeast strains

https://doi.org/10.21203/rs.3.rs-2965635/v1

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published 19 Jul, 2023

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Sake brewery-inhabiting (kuratsuki) bacteria affect the flavor of sake and sake yeast mainly produces the flavor of sake. Thus, kuratsuki bacteria may alter the metabolism of sake yeast through interactions between kuratsuki bacteria and sake yeast. In this study, we used the kuratsuki Kocuria strain TGY1127_2 and three sake yeast strains, AK25, K901, and K1801. Although the Brix and acidity during sake production using AK25 differed between sake with and without kuratsuki Kocuria, those using K901 and K1801 did not differ. Thus, sake yeast AK25 interacted with kuratsuki Kocuria and changed its metabolic characteristics. Based on the Brix and acidity changes, it is uncertain whether the K901 and K1801 strains affect sake yeast metabolism. In contrast, the effect of kuratsuki Kocuria on sake’s taste varied depending on the combination of different sake yeast strains. Thus, each sake yeast strain interacted with the kuratsuki bacterium and changed its metabolic characteristics, resulting in a change in the taste of sake. The findings of this study can lead to the brewing of sake using different types of kuratsuki bacteria which can affect the sake’s taste.

sake yeast

kuratsuki bacteria

sake brewing

Kocuria

microbial interaction

flavor and taste

Bacteria other than ethanol-tolerant lactobacilli stop growing or die at the late stages of sake production because of the high ethanol concentration (approximately 20%) produced by sake yeast. Several bacterial species have been isolated during the early stages of sake production at low ethanol concentrations (Terasaki and Nishida 2020, Kanamoto et al. 2021). However, the number of bacterial species isolated from sake production is lower than that based on the DNA sequence of sake (Terasaki et al. 2017, Terasaki et al. 2018, Terasaki and Nishida 2020). Thus, sake yeasts likely interact with different bacteria during sake production. We hypothesized that this interaction between sake yeast and bacteria affects the flavor and taste of sake.

It is well known that yeasts inhabit each sake brewery and have been used in sake brewing, which are called kuratsuki yeasts (Nishida 2021). Japanese words "kura" and "tsuki" respectively correspond to the terms "sake brewery" and "inhabiting." Several kuratsuki yeast strains have been selected as Kyokai strains and are maintained and distributed by the Brewing Society of Japan (Ohya and Kashima 2019). Our previous studies have shown that specific bacteria, called kuratsuki bacteria, inhabit sake breweries and enter the sake production process (Terasaki and Nishida 2020, Nishida 2021, Terasaki et al. 2021a, Terasaki et al. 2021b, Kanamoto et al. 2021). Bacteria entering the sake production process have been strongly suggested to affect the flavor and taste of sake through interactions between sake yeast and the bacteria (Bokulich et al. 2014, Koyanagi et al. 2016, Tsuji et al. 2018, Akaike et al. 2020, Nishida 2021, Ito et al. 2023, Yazaki and Nishida 2023).

The kuratsuki bacteria belonging to the genus Kocuria were isolated from sake production at the Narimasa Sake Brewery, Toyama, Japan (Terasaki et al. 2021a, Terasaki et al. 2021b). The genus Kocuria belongs to the phylum Actinobacteria, which are not lactic acid bacteria. The kuratsuki bacterial isolates affect the flavor of sake (Terasaki et al. 2021a, Nishida et al. 2022, Yazaki and Nishida 2023). For example, kuratsuki Kocuria has different effects on the taste of sake among different koji (Yazaki and Nishida 2023). Koji is made using steamed rice and a koji mold, which converts rice starch into sugar. We used the sake yeast Kyokai strain K1401 and two strains TGY1120_3 and TGY1127_2 of the kuratsuki Kocuria in our experiments (Terasaki et al. 2021a, Yazaki and Nishida 2023). Sake yeasts used in sake production play the greatest role in the flavor and taste of sake. Thus, sakes made using different sake yeasts generally have different flavors and tastes. We believe that the difference in flavor and taste of sake due to the difference in sake yeast is greater than the difference in the taste of sake due to the difference in kuratsuki bacteria. We strongly suggest that sake yeasts interact with kuratsuki bacteria and alter their metabolic characteristics. In this study, we investigated the effects of the combination of sake yeast and kuratsuki bacterium on the taste of sake.

It has been reported that some lactic acid bacteria are also associated with sake brewing (Suzuki et al. 2008, Taniguchi et al. 2010, Bokulich et al. 2014, Koyanagi et al. 2016). Co-culture studies on sake yeast and lactic acid bacteria have been undertaken (Kawarai et al. 2007, Watanabe et al. 2018). During sugarcane ethanol fermentation, interactions between yeast and lactic acid bacteria affect ethanol yield (Senne de Oliveira Lino et al. 2021). Lactic acid bacteria are believed to be closely associated with sake brewing (Nishida 2021). To our knowledge, no kuratsuki bacteria other than lactic acid bacteria have been reported to date. Kuratsuki bacteria have not been investigated because it was considered that bacteria do not have any effect on the sake brewing process, (Nishida 2021).

To date, different varieties of sake rice, sake koji, and sake yeast have been used to create different flavors and tastes of sake. In future, the variety of sake will be further expanded by considering the types of kuratsuki bacteria. This study aimed to confirm the effects of the combination of kuratsuki Kocuria TGY1127_2 and different sake yeasts on the taste of sake.

Cultivation of microorganisms

Sake yeast strains AK25, K901, and K1801 were used and the kuratsuki bacterium Kocuria sp. TGY1127_2 was used in this study. Each strain was grown in TGY medium (5 g/L tryptone, 1 g/L glucose, and 3 g/L yeast extract) and incubated at 25°C for 12 h prior to cultivation. As a control, 200 mL of water was added to 20 g of koji (Miyako koji, Isesou, Tokyo, Japan) and 1 mL of pre-cultivated AK1401 (1.3 × 10⁷ cells), K901 (1.0 × 10⁶ cells), or K1801 (6.0 × 10⁶ cells). The following ingredients were added to 200 mL of water: 20 g of koji, 1 mL of sake yeast precultivated solution, and 1 mL of precultivated TGY1127 2 solution (6.7 × 10⁸ cells). Each mixed solution was statically incubated at 14°C for 13 days. Brix and acidity were measured using a digital refractometer PAL-BX/ACID (ATAGO, Tokyo, Japan) at 0, 1, 2, 3, 6, 7, 8, 10, and 13 days.

Estimation of the taste of the sake

Sake taste was assessed using a taste sensor TS-5000Z (Intelligent Sensor Technology, Inc., Atsugi, Japan). Each taste sensor of TS-5000Z has a different lipid membrane that can detect each taste. The strength of each taste was represented by the magnitude of its current value. In this study, the taste intensity of sake with Kocuria TGY1127_2 minus that of sake without Kocuria TGY1127_2 was calculated. The initial taste in terms of astringent stimulation, bitter miscellaneous taste, saltiness, sourness, and umami were measured using the sensors AE1, CO0, CT0, CA0, and AAE, respectively. Astringency, bitterness, and umami richness were measured using the intensity in the second measurement. Each measurement was repeated four times.

Statistical analysis

Statistical analyses were performed using R software (The R Project for Statistical Computing, http://www.R-project.org/). Analysis of variance (ANOVA) was performed using Bartlett’s test. Pairwise t tests were performed using the Bonferroni technique of p value correction when the ANOVA showed a p value of less than 0.05.

Brix and acidity changes differed among different sake yeast strains

The peak Brix change occurred on the sixth day of incubation during sake production using sake yeast strain K901 (Fig. 1A). In contrast, this occurred on the third day of incubation during sake production using strains AK25 and K1801 (Fig. 1BC). These patterns were similar to those observed with the addition of kuratsuki Kocuria TGY1127_2 using sake yeast strains K901 and K1801 (Fig. 1). However, the patterns were different after the peak in the addition of kuratsuki Kocuria using sake yeast AK25 (Fig. 1C), suggesting that the metabolism of sake yeast AK25 may change after the peak of Brix change by kuratsuki Kocuria addition.

The acidity change patterns were similar among sake production processes using different sake yeast strains (Fig. 2). It increased monotonically during sake production (Fig. 2). However, the effects of kuratsuki Kocuria differed among different sake yeast strains. The effect of sake production using AK25 strain was greater than that of K901 and K1801 (Fig. 2C). The acidity decreased with the addition of kuratsuki Kocuria during sake production using AK25 (Fig. 2C).

The results of Brix and acidity patterns suggest that the kuratsuki Kocuria does not adversely affect the fermentation activity of the sake yeast K1401 in the production process using K901 and K1801, which was found in the sake production process using K1401 (Yazaki and Nishida 2023). However, this adversely affected the fermentation activity of sake yeast AK25 (Figs. 1 and 2). Our findings show that it is necessary to consider the compatibility of sake yeast strains used in sake brewing.

Effects of kuratsuki Kocuria on sake’s taste are different among different sake yeast strains

According to the TS-5000Z taste estimation results, there was a significant difference (p < 0.05) among sake using sake yeasts AK25, K901, and K1801. Using AK25, the taste signal intensities of astringent stimulation, bitter miscellaneous taste, saltiness, sourness, and umami were significantly decreased with the addition of kuratsuki Kocuria (Fig. 3). This was consistent with the changes in Brix and acidity (Figs. 1C and 2C), indicating that Brix and acidity monitoring are important for sake brewing. In sake using K901, the taste signal intensities for astringent stimulation, saltiness, and sourness decreased with the addition of kuratsuki Kocuria (Fig. 3). In contrast, the intensity of bitter miscellaneous increased significantly with the addition of kuratsuki Kocuria (Fig. 3). Using K1801, the taste signal intensities of astringent stimulation, bitter miscellaneous, and saltiness decreased with the addition of kuratsuki Kocuria (Fig. 3). In contrast, the sourness intensity increased with the addition of kuratsuki Kocuria (Fig. 3).

ANOVA results showed that seven tastes other than bitterness were significantly different (p < 0.05) among the different sakes prepared using the three different sake yeasts (Fig. 3). Astringency had different patterns (p < 0.05) of intensity between sakes using the K901 and K1801 strains and between those using the K901 and AK25 strains (Fig. 3). Astringent stimulation, bitter miscellaneous taste, saltiness, sourness, and umami showed different patterns of intensity between sakes using the K901 and K1801 strains, between those using the K901 and AK25 strains, and between those using the K1801 and AK25 strains (Fig. 3). Umami richness showed different patterns of intensity between the sakes prepared using the K1801 and AK25 strains (Fig. 3). Thus, the effects of adding kuratsuki Kocuria to sake on the taste vary among different sake yeasts.

Sake yeast produces chemical components that influence the flavor and taste of sake (Hashiguchi et al. 1999, Katou et al. 2009, Ferdouse et al. 2018, Akaike et al. 2020, Nishida 2021). Therefore, the production of these chemical components may be influenced by the presence of kuratsuki bacteria around the sake yeast.

We showed that the effect of kuratsuki Kocuria strain TGY1127_2 on the taste of sake varies depending on the combination of different sake yeast strains: AK25, K901, and K1801. Although the Brix and acidity during sake production using AK25 differed between sake with and without kuratsuki Kocuria, those using K901 and K1801 did not differ. Thus, sake yeast AK25 interacted with kuratsuki Kocuria and changed its metabolic characteristics. Based on the Brix and acidity changes, it is uncertain whether the K901 and K1801 strains affect sake yeast metabolism. In contrast, the effect of kuratsuki Kocuria on sake taste varied depending on the combination of different sake yeast strains. Thus, each sake yeast strain interacts with the kuratsuki bacterium and changes its metabolic characteristics, resulting in a change in the taste of sake. The effects of bacteria on the sake brewing process have been previously unknown and findings from our study can lead to further expansion of sake brewing by considering different types of kuratsuki bacteria.

Funding

This work was supported by JSPS KAKENHI Grant Number 21H02109 (to H.N.).

Competing Interests

The authors declare no conflict of interest.

Author contributions

M.S. performed the experiments. H.N. designed the study, analyzed the data and wrote the manuscript.

Data availability statement

The data sets in this study are available from the corresponding author on request.

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No competing interests reported.

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Journal Publication

published 19 Jul, 2023

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You are reading this latest preprint version

Effect of kuratsuki Kocuria on sake’s taste varies depending on the combination with different sake yeast strains

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