Page 8 - Shimadzu Journal vol.7 Issue2

Food Development

Development of a High Sensitivity Analytical Method for
Quantitative Analysis of Trace Amounts of D-Amino Acids

Yosuke Nakano and Eiichiro Fukusaki, Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University

Abstract produce (Brückner and Westhauser 1994; Gandolfi et al. 1994), in
marine products (Abe et al. 2005), and in fermented foods. Reports on
When the steric structure of amino acids was devised, it revealed that
most amino acids could exist as mirror-image isotopes (enantiomers) the presence of D-amino acids in fermented foods have also covered a
that are differentiated by an L-/D- notation into L-amino acids (L-form) wide variety of foods such as wine, beer, sake, yogurt, cheese, vinegar,
soy sauce, and Japanese mirin, and microorganism involvement in
and D-amino acids (D-form). Almost all amino acids that occur in nature fermentation and biochemical changes associated with ripening are
are L-amino acids, and D-amino acid function is an area that has not
attracted much interest. More recently, it is starting to appear as if understood to be part of the reason why various D-amino acids are
some foods and particularly microorganism-associated fermented produced (Friedman 1999; Mutaguchi et al. 2013; Inoue et al. 2016).
Food function is broadly divided into a primary nutritional function, a
foods contain D-amino acids, and the function of these foods is gaining
interest. In this context, we have applied analytical technology that secondary sensory (taste fulfillment) function, and a tertiary health
was developed by metabolomics to develop a new analytical method function. The function of D-amino acids in food is often mentioned in
terms of properties related to the secondary taste function and the taste
for D-amino acids that uses a high-sensitivity triple quadrupole mass
spectrometer. In this article, we intend to explain this method and qualities of each D-amino acid. The flavor enhancer L-glutamic acid
describe an example application of this method in fermented food monosodium is a good example of how amino acids play a major role as
analysis. taste modifiers in food production and preparation, though the flavor
and threshold levels of amino acids differ between L- and D-forms
(Schiffman et al. 1981). So far, sensory evaluation and taste receptor
1-1. Current Research into D-Amino Acids analysis has demonstrated the L-form of almost all protein-constituent
Of the 20 different constituent amino acids in proteins, all apart from amino acids produces a bitter taste, while the D-form often produces a
glycine have one or two chiral centers consisting of four different sweet taste (Kawai et al. 2012; Bassoli et al. 2014). Although only L-form
functional groups or atoms bound to a single carbon atom, and a D- amino acids are used as food additives, there is a move towards using
and L-form enantiomer for each of these chiral centers. By convention, some D-form amino acids as new taste substances, though in accordance
the D-/L- notation is commonly used to distinguish between these with the Japanese Ministry of Health, Labour and Welfare regulations
steric arrangements, where the D-form and L-form of an amino acid currently only allows the use of the D-form of alanine, threonine,
are mirror images (enantiomers) of one another with atom arrangements methionine, and tryptophan. Furthermore, permission to use the D-form
that cannot be superimposed (Fig. 1). of these amino acids only extends to their use in a racemic mixture,
hence the D-form can only be used as a food additive together with an
equal amount of the L-form; this has led to a situation in which the
- BNJOP BDJE .JSSPS % BNJOP BDJE unique taste of D-amino acids cannot be utilized to develop original
foods.
As mentioned above, D-amino acids exhibit unique biochemical functions
associated with a variety of aspects of vital phenomena that are not
exhibited by L-amino acids and have therefore been the subject of
broad- ranging research including both fundamental and applied studies.
Novel insights in medicine, food science, and other fields arise largely
.JSSPS JNBHF
thanks to recent remarkable advances in analytical techniques, and the
development of these techniques is still ongoing. There remains room for
Fig. 1 "NJOP "DJE &OBOUJPNFST 3 SFQSFTFOUT B TJEF DIBJO wide-ranging improvements in qualitative and quantitative aspects of
analysis performance, and we look forward to even greater technological
L-form amino acids account for the vast majority of amino acids in the
L-form amino acids account for the vast majority of amino acids in the developments in the future.
natural world, and for many years, all living things on Earth were thought
natural world, and for many years, all living things on Earth were thought
the
from
acids.
However,
mid
to
to be made up of only L-amino acids. However, from the mid-20th century, 1-2. Optical Resolution Technology for Amino Acids
century,
-
20th
up
of
be
made
-
amino
only
L
when the peptidoglycan cell wall layer of bacteria was reported to contain
D-alanine and D-glutamic acid (Stevens et al. 1951), the realization grew Recently, our research laboratory developed a new analytical method
that D-amino acids also existed alongside L-amino acids in the natural for D-amino acid analysis (Konya et al. 2016). This method uses HPLC
world. The relationship between D-amino acids and multiple different to separate chiral amino acids and time-of-flight mass spectrometry
diseases has been a subject of recent discussion, with D-serine thought (TOFMS) for detection. Separation involves using a CROWNPAK CR-I
to affect the pathophysiology of several neurological diseases: Alzhei- (+) column containing a crown ether with a binaphthyl skeleton as the
mer’s disease, schizophrenia, and amyotrophic lateral sclerosis (ALS) chiral stationary phase. At present, the detailed separation mechanism
(Nishikawa 2011; Sasabe et al. 2007). A close relationship between has been unclear, but the presumed mechanism is shown below (Fig. 2).
D-serine and renal dysfunction has also been reported, and D-serine is The amino group on a target amino acid is protonated by a very low
expected to be used as a prognostic predictor in renal disease and as a pH mobile phase used for liquid chromatography (LC). An electrostatic
new biomarker (Sasabe et al. 2014; Kimura et al. 2016). attractive force acts between this protonated amino group and the
crown ether of the stationary phase that retains the amino acid.
As shown above, major developments in D-amino acid research have
occurred in the field of medicine from a physiological perspective, Simultaneously, the crown ether is bound to a binaphthyl with an
though more recent D-amino acid research has been more expansive extremely bulky structure that causes hydrophobic retention but also
and even includes the field of food research. Reports have already varying steric hindrance depending on whether the amino acid is a
shown the presence of D-amino acids in fruit and vegetable agricultural D-form or L-form enantiomer. Electrostatic repulsion acting on the
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