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. 2025 Feb 10;13(2):388.
doi: 10.3390/microorganisms13020388.

Genomic Insights into Probiotic Lactococcus lactis T-21, a Wild Plant-Associated Lactic Acid Bacterium, and Its Preliminary Clinical Safety for Human Application

Masanori Fukao  1 , Keisuke Tagawa  1 , Yosuke Sunada  2 , Kazuya Uehara  2 , Takuya Sugimoto  2 , Takeshi Zendo  3 , Jiro Nakayama  3 , Shuichi Segawa  1
Affiliations

Genomic Insights into Probiotic Lactococcus lactis T-21, a Wild Plant-Associated Lactic Acid Bacterium, and Its Preliminary Clinical Safety for Human Application

Masanori Fukao et al. Microorganisms. .

Abstract

Lactococcus lactis T-21 is a lactic acid bacterium isolated from wild cranberries in Japan that demonstrates significant immunomodulatory properties and has been incorporated into commercial health products. However, probiogenomic analyses specific to T-21 have remained largely unexplored. This study performed a thorough genomic characterisation of T-21 and evaluated its safety in initial clinical trials. Genomic analysis revealed substantial genetic diversity and metabolic capabilities, including enhanced fermentative potential demonstrated by its ability to metabolise a wide range of plant-derived carbohydrates, and genetic determinants associated with exopolysaccharide biosynthesis and nisin production, distinguishing T-21 from domesticated dairy strains. These attributes, reflective of its wild plant origin, may contribute to its metabolic versatility and unique probiotic functionalities. A preliminary clinical trial assessing the safety of T-21-fermented milk in healthy Japanese adults indicated no significant adverse outcomes, corroborating its safety for human consumption. Together, these findings support the feasibility of utilising non-dairy, wild plant-origin strains in dairy fermentation processes as probiotics. This study expands our understanding of the genomic basis for T-21's probiotic potential and lays the groundwork for further investigations into its functional mechanisms and potential applications in promoting human health.

Keywords: Lactococcus lactis; clinical safety; comparative genomics; human health application; immunomodulatory properties; plant-associated LAB; probiotics.

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Conflict of interest statement

Authors Masanori Fukao, Keisuke Tagawa and Shuichi Segawa were employed by the company Nissin York Co., Ltd. Authors Yosuke Sunada, Kazuya Uehara, Takuya Sugimoto were employed by the company Nissin Foods Holdings Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Nissin York Co., Ltd. and Nissin Foods Holdings. The funder had the following involvement with the study: Takeshi Zendo and Jiro Nakayama benefitted from a research grant from Nissin York. The test products used in this study were supplied by Nissin York for the clinical trials. Nissin York delegated the execution of this research to M&I Science, which conducted the study in collaboration with AMC Nishi-Umeda Clinic.

Figures

Figure 1
Figure 1
Phylogenetic analysis of T-21 and the related Lactococcus lactis subsp. lactis and cremoris strains. Heatmap showing the OrthoANI.
Figure 2
Figure 2
Local collinear blocks (LCBs) between chromosomal sequences of the four Lactococcus lactis strains. Each contiguously coloured region represents an LCB, defined as a region without rearrangement of homologous backbone sequences. The LCBs placed under the vertical bars represent the reverse complement of the reference DNA sequence. The connecting lines between genomes identify the locations of each orthologous LCB in the genome. The white areas inside each LCB represent regions with low similarities. Unmatched regions within an LCB indicate the presence of strain-specific sequences.
Figure 3
Figure 3
Comparative analysis of orthologous genes in L. lactis strains. (a) Venn diagram representing the number of distributions of shared and unique orthologous gene clusters. (b) Bar plot enumerating the quantity of genes under 26 different COG categories among L. lactis subsp. lactis strains listed in Table 1. Error bars represent the standard error. Asterisks represent a statistically significant difference between isolates from plant and dairy (t-test, *, p < 0.05; **, p < 0.01).
Figure 4
Figure 4
Distribution of enzyme families related to metabolic capabilities (a) and putative eps gene clusters (b) in L. lactis strains. (a) Bar plot enumerating the quantity of the genes categorised into the GH and GT families of L. lactis subsp. lactis strains listed in Table 1. Error bars represent the standard error. Asterisks represent a statistically significant difference between isolates from plant and dairy (t-test, *, p < 0.05; **, p < 0.01). (b) T-21 eps gene clusters ranging 12 kb of homology (% amino acid identity) are joined by blocks of different colours as indicated in the figure. Genes were categorised into groups based on the putative or established functions of their products.
Figure 5
Figure 5
Nisin Z gene cluster and specific detection. (a) A comparison of nisin Z gene clusters of selected L. lactis strains. Open reading frames with a known function indicated by gene identification and colour. (b) The mass chromatogram extracted the ions corresponding to [M+3H]3+ of nisin Z (m/z 1111.7–1111.8) from the total ion chromatogram. The inset shows the antimicrobial activity via a spot-on-lawn assay against Listeria innocua ATCC 33090T.
Figure 5
Figure 5
Nisin Z gene cluster and specific detection. (a) A comparison of nisin Z gene clusters of selected L. lactis strains. Open reading frames with a known function indicated by gene identification and colour. (b) The mass chromatogram extracted the ions corresponding to [M+3H]3+ of nisin Z (m/z 1111.7–1111.8) from the total ion chromatogram. The inset shows the antimicrobial activity via a spot-on-lawn assay against Listeria innocua ATCC 33090T.
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