Antimicrobial activity and productivity of γ-aminobutyric acid in lactic acid bacteria isolated from traditional Gifu ayu-narezushi

Mitsuyo Hori; Yusuke Kawai; Koichiro Noguchi; Kohei Nakamura; Masaya Shimada; Hitoshi Iwahashi; Tomoyuki Nakagawa

doi:10.3136/fstr.FSTR-D-23-00098

Abstract

From traditional Gifu ayu-narezushi, Leuconostoc, Latilactobacillus, and Enterococcus were isolated as dominant genera. Isolated strains of Leuconostoc mesenteroides showed high growth inhibition activity against Staphylococcus aureus and Escherichia coli. The isolated strain, Enterococcus lactis EC52, could produce γ-aminobutyric acid (GABA). These results indicated that Enterococcus lactis and Leuconostoc mesenteroides were lactic acid bacteria, which have GABA-producing and antimicrobial activities, respectively, in traditional Gifu ayu-narezushi.

Introduction

In Japan, salted fish fermented with cooked rice is called “narezushi,” which is a popular traditional dish distributed in each region, and considered to be the progenitor of modern “sushi.” Narezushi is made with the different fish available in each region of the country; for example, funa (Carassius cuvieri; Fujii et al., 2011; Isobe et al., 2002; Tanabe et al., 2022; Tsuda et al., 2012), aji (Japanese horse mackerel, Trachurus japonicus; An et al., 2010; Kiyohara et al., 2012; Koyanagi et al., 2011), saba (chub mackerel, Scomber japonicus; Doi et al., 2021; Kubo et al., 2022; Matsui et al., 2013; Nakagawa et al., 2016), samma (Pacific saury, Cololabis saira; Matsui et al., 2008), yellowtail (Japanese amberjack, Seriola quinqueradiata; Koyanagi et al., 2013), ayu (sweetfish, Plecoglossus altivelis; Matsui et al., 2010; Momose et al., 2005; Nagano et al., 2020; Nomura et al., 2015), and other local fishes.

Ayu-narezushi is a typical narezushi made with sweetfish, and several types of ayu-narezushi are available in various regions of Japan; for example, in Mie (Matsui et al., 2010), Toyama (Nomura et al., 2015), Tochigi (Momose et al., 2005), and Gifu prefectures (Nagano et al., 2020), as well as other local regions. Among the regions producing ayu-narezushi, in Gifu City there is a traditional ayu-narezushi that has been manufactured by cormorant master fishermen, who are called usho. Cormorant fishing (ukai) for ayu has been practiced for over 1 300 years on the Nagara River. Usho have inherited traditional methods for ukai based on a hereditary system. Usho masters also handed down traditional methods for manufacturing Gifu ayu-narezushi, and they prepare Gifu ayu-narezushi for gifts every December. In a previous report, we showed the bacterial biota in Gifu ayu-narezushi using 16S rRNA amplicon sequencing and the transitions in the microbial populations during the fermentation step in detail (Hori et al., 2022). In the early stage of fermentation for Gifu ayu-narezushi, Leuconostoc was the dominant lactic acid bacteria genus, and then the genus Latilactobacillus became the main genus (Hori et al., 2022).

On the other hand, many studies have focused on the functions of lactic acid bacteria strains isolated from several kinds of narezushi, such as the probiotic functions of lactic acid bacteria (Kuda et al., 2013; Yokota et al., 2018; Nishida et al., 2021), production of antibacterial substances (Doi et al., 2020), and others. With respect to the probiotic functions of lactic acid bacteria, it has been reported that some lactic acid bacteria strains produce γ-aminobutyric acid (GABA) (Dahiya et al., 2021; Dhakal et al., 2012). GABA is an inhibitory neurotransmitter (Awapara et al., 1950) that shows some functional effects such as a hypotensive effect on blood pressure (Hayakawa et al., 2004). Therefore, in lactic acid bacteria, the ability to produce GABA is one of their beneficial probiotic abilities. Indeed, Nagano et al. (2020) reported that ayu-narezishi contained high levels of GABA, although the GABA-producing bacterial strain in ayu-narezushi was not identified.

Meanwhile, production of bacteriocins, which are antibacterial peptides produced by bacteria strains, has been considered as a highly desirable trait in several industrial applications (Zendo et al., 2022). Especially, in the food industry, bacteriocins produced by lactic acid bacteria have been widely studied for their potential use in food preservation (Zendo et al., 2022). We have already reported that Lactococcus lactis in Kishu-saba narezushi produce the bacteriocin nisin Z, and that these inhibit the growth of grampositive bacteria, such as Staphylococcus aureus (Doi et al., 2021). Therefore, there is a possibility that lactic acid bacteria strains isolated from Gifu ayu-narezushi have the ability to produce some bacterocins.

In this study, we aimed to isolate the lactic acid bacteria participating in its fermentation of traditional Gifu ayu-narezushi, and to evaluate growth inhibition activity for Gram-positive and -negative bacteria and the GABA-producing ability of lactic acid bacteria isolated from traditional Gifu ayu-narezushi.

Materials and Methods

Sample preparation of Gifu ayu-narezushi Traditional Gifu ayu-narezushi was fermented for 26 days (late stage of the fermentation) in December 2020 by a manufacturer in Gifu City (Fig. 1).

Fig. 1

Image of traditional Gifu ayu-narezushi.

pH measurement of the Gifu ayu-narezushi sample The pH of the ayu-narezushi sample was measured in triplicate using a Piercing pH meter SPH70 (AS ONE Co., Osaka, Japan).

Isolation and identification of lactic acid bacteria In total, 10 g of Gifu ayu-narezushi sample, containing approximately equal amounts of both fish and rice parts, were homogenized with 90 mL of sterile water for 60 s using a Stomacher Lab-Blender 400 (Organo, Tokyo, Japan), and the sample suspensions were diluted with sterile saline solution. The appropriate dilution samples were used for the microbial colony count using the pour plate culture method and nutrient plates (Nissui Pharmaceutical Co., Tokyo, Japan). The plates were incubated at 37 °C for 48 h.

Isolated bacteria strains on the plate were identified using their 16S rRNA sequence. The 16S rRNA genes were amplified from the colonies of the isolated lactic acid bacteria strains using the colony PCR method and primers 20F and 1500R (Nakagawa et al., 2003). The accession numbers for the 16S rRNA sequences for the isolated strains were LC771062–LC771068.

Bacteria growth inhibition test by isolated strains Growth inhibition test by isolated strains was performed using the agar plug diffusion method (Balouiri et al., 2016). Isolated lactic acid bacteria were cultivated on MRS medium under static conditions overnight. The agar piece from the plates on which the isolated lactic acid bacteria were grown was placed on the nutrient plates inoculated with Staphylococcus aureus NBRC 12732, as a standard strain in Gram-positive bacteria, or Escherichia coli NBRC 14237, as a standard strain in Gram-negative bacteria. After overnight incubation at 37 °C, clear zones were observed indicating bacteria growth inhibition.

Lactococcus lactis strain LC41, which had been isolated from Kishu-saba narezushi, was used as a positive control (Doi et al., 2021). Strain LC41 produces nisin Z, which exhibits antimicrobial activity against Gram-positive bacteria, and the strain did not show antimicrobial activity against E. coli (Doi et al., 2021).

Quantitative analysis of GABA Isolated strains were inoculated in MRS medium containing 10 mg/mL monosodium glutamate at OD₆₆₀ = 0.1, and were incubated at 37 °C for 48 h. After cultivation, supernatants were prepared by centrifugation (13 000 rpm, for 3 min).

Quantitative analysis of GABA in the supernatants was performed by AccQ-Tag amino acid analysis according to the instructions of the ACQUITY UPLC H-class system (Waters Co., Milford, MA, USA) (Kubo et al., 2022).

Results

pH values and colony forming units (CFUs) of traditional Gifu ayu-narezushi We first analyzed the pH values of the rice and fish portions and the general viable bacteria counts in the traditional Gifu ayu-narezushi. The pH values of the rice and fish portions were 4.48 ± 0.26 and 4.39 ± 0.29, respectively. The viable bacteria counts in the samples were 5.03 × 10⁷ CFU/g.

Isolation and identification of lactic acid bacteria in traditional Gifu ayu-narezushi Next, we isolated lactic acid bacteria in traditional Gifu ayu-narezushi. The appropriate plate was selected from the plates onto which diluted traditional Gifu ayu-narezushi was inoculated, and all colonies (71 colonies) on the plate were identified by species using their 16S rRNA sequence. All strains (total 71 strains) were identified as belonging to only three genera, Leuconostoc (31 strains [43.7 %]), Latilactobacillus (25 strains [35.2 %]), and Enterococcus (15 strains [21.1 %]), with 16S rRNA sequencing from all strains showing high homology (> 97.0 %) with each strain type. Strains in genera Leuconostoc and Latilactobacillus belonged to the Leuconostoc mesenteroides and Latilactobacillus sakei groups, respectively. In contrast, strains in genus Enterococcus belonged to the two species of Enterococcus lactis and Enterococcus faecium.

Therefore, the main species found in traditional Gifu ayu-nazushi, such as Leuconostoc mesenteroides group and Latilactobacillus sakei, were consistent with our previous work. On the other hand, Enterobacteriaceae was not isolated from traditional Gifu ayu-nazushi in this work, although it was identified by amplicon sequence in previous work (Hori et al., 2022).

Growth inhibition activity of isolated strains to Grampositive and Gram-negative bacteria strains Some lactic acid bacteria possess growth inhibition activity toward other bacteria. Thus, we speculated that lactic acid bacteria strains isolated from Gifu ayu-nazushi would also possess growth inhibition activity against Gram-positive bacteria such as Staphylococcus aureus or Gram-negative bacteria such as Escherichia coli. We found that isolated strains from traditional Gifu ayu-nazushi, such as Enterococcus lactis EC51 and Enterococcus faecium EC52, Leuconostoc mesenteroides LS48 and LS49, and Latilactobacillus sakei LB63 and LB64, showed growth inhibition activity against Staphylococcus aureus, as well as the positive control strain, Lactococcus lactis LC41, which produced the bacteriocin nisin Z (Fig. 2). In particular, Leuconostoc mesenteroides strains exhibited strong growth inhibition activity against not only Staphylococcus aureus but also Escherichia coli compared with other species.

Fig. 2

Growth inhibition test of isolated strains from Gifu ayu-narezushi against S. aureus and E. coli. Lactococcus lactis LC41 was used as control strains.

A white area around the agar plug indicates growth inhibition of S. aureus and E. coli.

GABA-producing ability of lactic acid bacteria isolated from traditional Gifu ayu-narezushi We found that Enterococcus lactis strain EC52 possessed GABA-producing ability, but Leuconostoc mesenteroides and Latilactobacillus sakei strains did not. The growth and GABA content of strain EC52 in MRS medium was OD₆₆₀ = 7.27 ± 0.24 and 1.55 ± 0.064 mM at pH 7.0 and OD₆₆₀ = 2.93 ± 0.24 and 0.931 ± 0.000 mM at pH 5.0. In MRS medium, which was not inoculated with any strains, GABA was not detected.

Discussion

In this study, we isolated lactic acid bacteria from traditional Gifu ayu-narezushi, and also evaluated their growth inhibition activity against Gram-positive and Gram-negative bacteria and the GABA-producing ability.

In traditional Gifu ayu-narezushi, Leuconostoc mesenteroides, Latilactobacillus sakei and Enterococcus species were the main lactic acid bacteria. In a previous study, it was reported that Leuconostoc mesenteroides and Latilactobacillus sakei were the main lactic acid bacteria species in the microbial biota of Gifu ayu-narezushi as revealed by amplicon sequencing (Hori et al., 2022). Thus, it seems that the isolated strains from Gifu ayu-narezushi in this study were largely consistent with the microbial biota in that previous report, and it was suggested that Latilactobacillus sakei and Leuconostoc mesenteroides were influential lactic acid bacteria for the fermentation of traditional Gifu ayu-narezushi. On the other hand, Enterococcus strains, which were not detected by amplicon sequencing in that previous study, were specifically identified in the present study. It is not clear whether the difference in the constituent bacteria strains between traditional Gifu ayu-narezushi samples in the present versus the previous studies was due to differences in their manufacture, in the sample used, or in the identification methods for bacterial biota between the culture method and the amplicon sequencing method.

Moreover, we evaluated growth inhibition activity of isolated strains against Gram-positive bacteria, S. aureus, and Gram-negative bacteria, Escherichia coli. As a result, Leuconostoc mesenteroides strains isolated in this study exhibited high growth inhibition activity against not only S. aureus but also Escherichia coli, compared with other isolates. It was reported that strains of genus Leuconostoc produced several types of bacteriocins (Stiles, 1994; Masuda et al., 2011; Chen et al., 2018). In a case of Leuconostoc mesenteroides isolated from traditional Gifu ayu-narezushi, it seems that the strains, which showed growth inhibition activity against S. aureus and Escherichia coli. In this study, however, we could not identify their growth inhibition compounds. For application of growth inhibition activity of the isolated strains to the food industry, their growth inhibition compounds should be identified. Therefore, the identification of the growth inhibition compounds in Leuconostoc mesenteroides isolated from traditional Gifu ayu-narezushi remains an issue for future work.

Finally, we showed that Enterococcus lactis EC52 isolated from traditional Gifu ayu-narezushi possessed GABA-producing ability, although other isolated strains did not show any such ability (data not shown). This fact indicates the possibility that the traditional Gifu ayu-narezushi used in this study contains GABA. To date, there have been several reports about GABA-production by Enterococcus strains (Tamura et al., 2010; Divyashri and Prapulla, 2015; Jo et al., 2019; Sabna et al., 2021; Sakkaa et al., 2022). GABA productivity of strain EC52 (1.55 ± 0.06 mM) was very low compared with already reported Enterococcus strains, e.g., Enterococcus avium JS-N6B4 (120.9 ± 0.003 mM) (Jo et al., 2019). We expect that the GABA productivity of strain EC52 can be greatly improved by optimization of the GABA producing conditions, such as medium contents, pH, temperature, and other factors. Therefore, our next endeavor is to explore such optimization of the conditions for GABA productivity of strain EC52 and to quantify GABA contents in traditional Gifu ayu-narezushi in future research.

Acknowledgements This research was funded in part by a grant from the Toyo Suisan Foundation to TN and in part by a grant from the Koshiyama Science and Technology Foundation to MH. We would like to express our deepest gratitude to the late Professor Tohru Suzuki for his valuable advice for the study.

Conflict of interest There are no conflicts of interest to declare.

References

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