Separation and characterization of the immunostimulatory components in unpolished rice black vinegar (kurozu)

J Biosci Bioeng. 2013 Dec;116(6):688-96. doi: 10.1016/j.jbiosc.2013.05.029. Epub 2013 Jun 27.


Unpolished rice black vinegar (kurozu), a traditional Japanese vinegar, is considered to have beneficial health effects. Kurozu is produced via a static fermentation process involving the saccharification of rice by Aspergillus oryzae, alcohol fermentation by Saccharomyces cerevisiae, and the oxidation of ethanol to acetic acid by acetic acid bacteria such as Acetobacter pasteurianus. Since this process requires about 6 months' fermentation and then over a year of aging, most of these organisms die during the production process and so microbial components, which might stimulate the innate immune system, are expected to be present in the vinegar. In this study, we investigated whether microbial components are present in kurozu, and after confirming this we characterized their immunostimulatory activities. Lyophilized kurozu stimulated murine spleen cells to produce tumor necrosis factor (TNF)-α, at least in part, via Toll-like receptor (TLR) 2 and the Nod-like receptors NOD1 and 2. The active components associated with TLR2 activation were concentrated by Triton X-114-water phase partitioning and hydrophobic interaction chromatography on Octyl Sepharose. TLR4-activating components were also enriched by these methods. The concentrated preparation stimulated murine spleen cells to produce TNF-α and interferon (IFN)-γ. These results indicate that long-term fermented kurozu contains immunostimulatory components and that the TLR2 and TLR4-activating immunostimulatory components of kurozu are hydrophobic. These components might be responsible for the beneficial health effects of kurozu.

Keywords: Acetic acid bacteria; Interferon-γ; Lipopolysaccharide; Lipoprotein; Toll-like receptor; Tumor necrosis factor-α.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetic Acid / chemistry*
  • Acetobacter / metabolism
  • Adjuvants, Immunologic / biosynthesis*
  • Adjuvants, Immunologic / isolation & purification
  • Adjuvants, Immunologic / pharmacology
  • Animals
  • Aspergillus oryzae / metabolism*
  • Ethanol
  • Fermentation
  • HEK293 Cells
  • Humans
  • Immune System / drug effects*
  • Interferon-gamma / metabolism
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Nod1 Signaling Adaptor Protein / metabolism
  • Nod2 Signaling Adaptor Protein / metabolism
  • Octoxynol
  • Oryza / chemistry*
  • Oryza / microbiology
  • Polyethylene Glycols
  • Saccharomyces cerevisiae / metabolism*
  • Toll-Like Receptor 2 / metabolism
  • Toll-Like Receptor 4 / metabolism
  • Tumor Necrosis Factor-alpha / metabolism


  • Adjuvants, Immunologic
  • Nod1 Signaling Adaptor Protein
  • Nod1 protein, mouse
  • Nod2 Signaling Adaptor Protein
  • Nod2 protein, mouse
  • Tlr2 protein, mouse
  • Tlr4 protein, mouse
  • Toll-Like Receptor 2
  • Toll-Like Receptor 4
  • Tumor Necrosis Factor-alpha
  • Ethanol
  • Polyethylene Glycols
  • Interferon-gamma
  • Octoxynol
  • Nonidet P-40
  • Acetic Acid