Large-scale production of HYA-50 and the
launch of CUMECProducing HYA for functional supplement applications
necessitates that all the materials used must themselves be
composed of foods. ìGiven the strict regulations for such products,
our goal was to produce at least 50%-pure HYA in large quantities,î
explains Kitao. ìWe used high-purity sources of linoleic acid, a form
of seed oil, and cultivated lactic acid bacteria in-house for high-
efficiency conversion of this source oil into HYA, which is produced
by gut microbial metabolism in the human digestive tract. It was a
difficult task, but we managed to complete it successfully
through a collaboration with Kyoto
Univer sit y.î
For the mass production of
HYA, the Noster R&D team
decided to use highly pure
(>70%) safflower oil as
the source for the linoleic
acid. Their next task was
devising a procedure that
would yield a high conversion
rate of the linoleic acid into HYA.
Their experiments revealed that it
would be critical to protect the safflower oil
from oxidation and optimize the emulsion of ingredients to
enhance contact between the lactic acid bacteria and linoleic acid.
ìOur process was anaerobic, with a highly efficient reaction in
the emulsified state,î says Kitao. ìThe know-how for cultivating
anaerobic bacteria in the laboratory is one of Nosterís key
technologies. It is not a straightforward process, necessitating
a deep understanding of the behavior of gut microorganisms.
We can now extract many kinds of metabolites directly from gut
mic roor ganisms c ult iv ate d in - house.î
The final step was to recover HYA from the reaction solution
with a purity of more than 50%. Since it had emulsified in the
reaction process, it could not be separated cleanly using normal
centrifugation. The Noster team careful analyzed the effect of
temperature on the physical characteristics of the fatty acidñwater
emulsion, and determined an optimal temperature that led to high-
efficiency separation of 50% pure HYA from the emulsion.
ìUsing this process, we went from being able to produce several
grams of HYA-50 with a purity of 50% to synthesizing kilogram
amounts,î says Kitao. ìThis production technology is at the heart of
our H YA -50 C UMEC pos t biot ic produc t .îBoosting purity for clinical research
The purity of HYA-50 was adequate in a consumer product,
but much higher purityócloser to 99%ówas needed to perform
advanced clinical studies. The most difficult step in producingultrapure HYA was developing a highly efficient refining process.
The conversion of linoleic acid to HYA is a reversible enzymatic
reaction and is therefore never 100% complete. To address this
problem, the Noster team attempted to remove residual linoleic
acid using a fractionation column after the reaction had run its
course. However, this conventional approach did not yield complete
separation, leaving too much linoleic acid residue in the refinement
product.
Nosterís solution was to find lactic acid bacteria strains that
increased the conversion rate, thereby minimizing the amount of
linoleic acid remaining after the reaction. Notably, even
the best lactic acid bacteria strains selected
from Nosterís librar y of about 400
strains for mass production of
high-purity HYA yielded a
maximum conversion rate
of only 80%, leaving 20%
linoleic acid remaining.
To solve this problem,
the project team turned to
Escherichia coli, creating a
strain of this bacteria that carried
the conjugated linoleic acid
hydrase (CLA-HY) enzyme,
which converts linoleic acid
to HYA. After testing numerous
E. coli strains, transfection vectors, and culture conditions, the
research team succeeded in generating a strain capable of
converting close to 100% of linoleic acid to HYA.
Another challenge was that conventional purification processes
rely on diethyl ether. Production of large volumes of HYA would
require large amounts of this ether, which is extremely flammable
and can only be used safely in small amounts, severely limiting the
production potential of pure HYA.
Kitao explains the Noster teamís approach to this problem: ìWe
experimentally found a solvent with low flammability that could
efficiently separate HYA and linoleic acid. But then we hit another
wall. This solvent led to the rapid deterioration of the fractionation
column, and it was not a practical solution for a continuous
purification process. So we still had work to do!î
Eventually, the Noster team developed a customized fractionation
column to efficiently separate HYA and linoleic acid using the new
solvent, but with significantly less deterioration. ìThe combination
of the new solvent and our new column enabled us to scale up the
production of our ultrapure HYA product.îProduction of other lipid metabolites
Over the last decade, Noster researchers have established
PHOTOS: PROVIDED BY NOSTER procedures for producing other types of lipid metabolites inProduced by the Science
0709Product.indd 137 6/30/21 7:28 AM