J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Original Research

Volume 12, December 2020, pages 129-139

Identification of short-chain pyroglutamyl peptides in Japanese salted fermented soy paste (miso) and their anti-obesity effect

Figures

Figure 1. Effects of water extracts of miso and synthetic pyroGlu-Leu on weight gain in 45% high fat diet-fed rats. The dose of pyroglutamyl peptides in crude water extract of miso (CE), non-absorbed fraction (N-A), and pooled mixture of 10% and 30% ACN fractions (ACN) (a). Rats were fed on normal (open column) and 45% high fat diet (closed column). Effect of miso extracts on body weight gain (b) and total calorie intake (c) in 45% high fat (HF) diet-fed rats. Effect of synthetic pyroGlu-Leu (0.1 mg/kg body weight) on body weight gain (d) and total calorie intake (e) in the rats. One-letter codes are used for all amino acid residues in panel (a). For b–e, data are shown as mean ± SD (n = 4). * represents significant difference with p < 0.05, as estimated by Dunnett’s test vs HF.

Figure 2. Mass spectrometry chromatograms of pyroglutamyl peptide fraction of soybean miso acquired in the total (upper) and precursor (lower) ion scan modes. Total ion intensity was monitored in the positive mode across the mass to charge ratio (m/z) range of 50–150 (a), 150–200 (b), 200–250 (c), 250–300 (d), and 300–500 (e) (total ion scan). In the precursor ion mode, precursor ions, which generated immonium ion of pyroglutamyl residue (m/z = 84.1), were detected in the positive mode across the same range as the total ion scans. Peaks marked with alphabets (peak a–m and x–y) were further subjected to MS/MS analysis (product ion scan).

Figure 3. Amount of pyroglutamyl di-peptides in 4 types of miso. Short aging and long aging types of rice miso (A, B, C, and D), barley miso, and soybean miso were analyzed by LC-MS/MS in the MRM mode. Data are shown as the average (n = 3–6). The one-letter codes are used for all amino acid residues. pE represents pyroglutamyl (pE) residue.

Figure 4. Effect of synthetic pyroglutamyl peptides on weight gain in 60% very high fat diet-fed rats. Rats were fed on normal (open column) and 60% very high fat diet (closed column). Effect of a mixture of hydrophobic synthetic pyroglutamyl peptides (pEP, pEV, pEI, and pEL) at doses corresponding to those in ACN (×1 ACN-pep) and 10 times higher (×10 ACN-pep) on body weight gain (a) and total calorie intake (b) in rats fed on 60% very HF diet (VHF). Effect of synthetic pyroGlu-Leu (1 mg/kg body weight) on body weight gain (c) and total calorie intake (d). Data are shown as mean ± SD (n = 4). For the mixture of hydrophobic pyroglutamyl peptides, * represents significant difference with p < 0.05, as estimated by Dunnett’s test vs VHF. For pyroGlu-Leu, * represents significant difference with p < 0.05, as estimated by t-test between each diet group.

Figure 5. The effect of pyroglutamyl peptides on weight of epididymal adipose tissue and liver in 60% very high fat diet-fed rats. Rats were fed on normal (open column) and 60% very high fat diet (closed column). Refer the legends for Figure 4 for abbreviation. The effect of mixture of hydrophobic pyroglutamyl peptides (pEP, pEV, pEI, and pEL) on weight of liver (a) and epididymal adipose tissue (b). The effect of synthetic pyroGlu-Leu (1 mg/kg body weight) on weight of liver (c) and epididymal adipose tissue (d). Data are shown as mean ± SD (n = 4). For mixture of hydrophobic pyroglutamyl peptides, * represents significant difference with p < 0.05, as estimated by Dunnett’s test vs VHF. For pyroGlu-Leu, * represents significant differences with p < 0.05, as estimated by t-test between each diet group.

Table

**Table 1.** Sequences of pyroglutamyl peptides and derivatives in miso
Peak	Sequence	Precursor ions (m/z)	Product ions (m/z)
Refer Figure 2 for peak name. The pyroglutamyl residue is represented as pyroGlu. Adduct ions with acetonitrile and proton are marked with . Adduct ions consisting of two molecules and a proton are marked with *. ^a represents immonium ion and related ions of amino acid.
a	pyroGlu-Gly	187.1	28 (pyroGlu^a), 30 (Gly^a), 41, 56 (pyroGlu^a), 76 (y1), 84 (pyroGlu^a)
b	pyroGlu-Ser	217.1	28, 41, 56 (pyroGlu^a), 60 (Ser^a),30 (Gly^a), 84 (pyroGlu^a), 88 (z1), 106 (y1)
c	pyroGlu-Thr	231.2	41, 56 (pyroGlu^a), 74 (Thr^a), 84 (pyroGlu^a), 102 (z1), 120 (y1)
d	pyroGlu-Asp	245.3	70 (Asp^a)
e	pyroGlu-Ala	201.2	41 (pyroGlu^a), 44 (Ala^a), 56, 84 (pyroGlu^a), 90 (y1)
f	pyroGlu-Pro	227.2	41, 56 (pyroGlu^a), 70 (Pro^a), 84 (pyroGlu^a), 116 (y1), 181 (a2)
g	pyroGlu-Val	229.2	41, 56 (pyroGlu^a), 72 (Val^a), 84 (pyroGlu^a), 118 (y1)
h	pyroGlu-Ile	243.2	28, 41, 56, 84 (pyroGlu^a), 86 (Ile^a), 132 (y1)
i	pyroGlu-Leu	243.2	28, 41, 56, 84 (pyroGlu^a), 86 (Leu^a), 132 (y1)
j	pyroGlu-Glu	259.2	41, 84 (pyroGlu^a), 130 (z1)
k	pyroGlu-Gln	258.2	84 (pyroGlu^a), 129 (Gln^a), 130 (z1)
l	pyroGlu-Gly-Ser	274.2	41, 84 (pyroGlu^a), 256 (b3)
m	pyroGlu-Phe	277.2	41, 56, 84 (pyroGlu^a), 120 (Phe^a), 166 (y1)
x	pyroglutamic acid	130.1	28, 41, 56, 84 (pyroGlu^a)
y	pyroglutamic acid methyl ester	144.1	28, 41, 56, 84 (pyroGlu^a)
y*	pyroglutamic acid methyl ester+CH₃CN	185.1	28, 41, 56, 84 (pyroGlu^a), 144 (peak y)
z	pyroglutamic acid ethyl ester	158.1	28, 41, 56, 84 (pyroGlu^a)
z*	pyroglutamic acid ethyl ester+CH₃CN	199.1	28, 41, 56, 84 (pyroGlu^a), 158 (peak z)
x**	Two pyroglutamic acid	259.2	41, 56, 84 (pyroGlu^a), 130 (peak x)
y**	Two pyroglutamic acid methyl ester	287.3	41, 56, 84 (pyroGlu^a), 144 (peak y)
h*	pyroGlu-Ile+CH₃CN	284.2	41, 56, 84 (pyroGlu^a), 132 (Ile y1), 243 (peak h)
i*	pyroGlu-Leu+CH₃CN	284.2	41, 56, 84 (pyroGlu^a), 132 (Leu y1), 243 (peak i)
f**	Two pyroGlu-Pro	453.3	70 (Pro^a), 84 (pyroGlu^a), 116 (Pro y1), 227 (peak f)
z**	Two pyroglutamic acid ethyl ester	315.2	84 (pyroGlu^a), 130 (peak x), 158 (peak z)