sake bitter peptides from rice glutelin

Scope: The acidic subunit of glutelin, a protein in steamed rice, is digested in sake mash by koji-mold-derived enzymes, generating a characteristic oligopeptide (a bitter peptide) bearing a pyroglutamyl group at its N-terminus; this has been shown to possibly be involved in sake's taste.

Note: An account by Hashizume Katsumi (2008). Based on a digestion test of steamed rice using 4 rice-protein mutant lines (esp1-4) and their parent strains (Nipponbare and Yamada Nishiki), and on molecular structural analysis and sensory evaluation of a bitter-peptide preparation isolated from commercial junmai sake.

Note: Hashizume (2008), Table 3 and Figure 3. Purified standards fractionated by reverse-phase HPLC from junmai sake (Yamada Nishiki, 70% rice polishing ratio).

Note: Hashizume (2008), Table 5. Evaluation of a purified preparation dissolved in a 15% ethanol solution.

Scope: In koji-fermented seasoned foods (miso, soy sauce, sake, etc.), glutamine (Gln) freed by enzymatic breakdown by koji mold, or a Gln residue at a peptide's N-terminus, spontaneously cyclizes into pyroglutamic acid (pyroGlu) during heating or ripening. Short-chain pyroglutamyl peptides such as pyroGlu-Leu and pyroGlu-Gln have been identified in sake (Table 1).

Note: Zheng et al. (2022) review, pp. 6–8. Since the sake bitter peptides isolated by Hashizume (2008) likewise carry a pyroglutamyl group at their N-terminus, this is cited as general background for the mechanism. However, no statement calling the short-chain peptides listed by Zheng et al. (2022), such as pyroGlu-Leu, themselves 『苦味』 (in translation: "bitterness") could be found in that review, so this is not used as direct evidence for bitterness (the evidence's relation is set to contextualizes).

Scope: The more the amount of activated-carbon treatment (200/500/1000 mg/l) was increased, the more the peptide concentration in sake (peaks 13,17,18,20) fell, and with 1000 mg/l treatment most fell below the detection limit. In untreated daiginjo (2001–2004 vintage, 40% rice polishing ratio), peak 13 was 10.5–12.1 mM, compared with a lower 0.8–3.5 mM in commercial futsū-shu (regular sake) A–C (consistent with the tendency for futsū-shu using more activated carbon to be lower).

Note: Hashizume (2008), Table 4. Mean ± standard deviation (3 measurements).

Scope: In a steamed-rice digestion test on 3 varieties prepared with rice polishing ratios stepped down from 90% to 40%, Nipponbare showed a tendency for the amount of high-molecular-weight peptide to decrease as the rice polishing ratio fell (i.e., as milling became more thorough), but Yamada Nishiki and Gohyakumangoku showed no such tendency.

Note: Hashizume (2008), Figure 4. Measured data showing that the relationship between rice polishing ratio and bitter-peptide generation depends on the raw-rice variety and is not uniform. Based on a digestion test with koji mold (strain RIB128) (pH 4.3, 37°C, left standing for 1–2 days).

Scope: According to an abstract by Hashizume et al. (publication year not confirmed, original not read in full) introduced in "Research Achievements on Alcoholic Beverages in Reiwa 4" (Journal of the Brewing Society of Japan, vol. 118, no. 4, p.220), adding a peptidase preparation with high ACP activity to sake reduced bitter peptides and increased (pGlu)L-ethyl. The source reports that, on the other hand, adding an enzyme preparation with high (pGlu)-peptide ethyl ester-forming activity and low ACP activity increased (pGlu)LF-ethyl and (pGlu)LFNP-ethyl in sake (without increasing (pGlu)L-ethyl), and it was suggested that this increase in oligopeptide ethyl esters gives a good effect on sake's taste. A separate abstract by Hashizume et al. in the same journal, p.233, states explicitly that an enzyme showing carboxypeptidase-type activity "contributes to the reduction of bitter peptides while also being involved in the generation of dipeptides with poor taste," suggesting that reducing bitterness can carry a quality trade-off as well.

Note: Original not read in full. A secondary citation from an abstracting journal (a summary by the editorial staff). Even for enzyme treatment of the same peptide lineage, the treatment "reduction of bitter peptides = desirable" coexists with the treatment "increase in the esterified derivative = a good effect on taste"; this is included as an example showing this two-sidedness in how bitterness is treated. The addition of the enzyme preparation with high ACP activity itself also carries a two-sided effect, reducing bitter peptides while increasing (pGlu)L-ethyl (see also the separate Hashizume et al. abstract on p.233 of the same journal, vol.118 no.4).

Scope: The author discusses that part of the reason for the previously vaguely understood empirical rule 『タンパク質レベルが高い原料米で造った清酒には雑味が多い』 (in translation: "sake made from rice with a high protein level has more zatsumi") could be captured in terms of protein level, namely the generation of glutelin-derived bitter peptides.

Note: A statement in the closing section (p.579) of Hashizume (2008). This is the author's own interpretive summary, treated as an account that explains an empirical rule long discussed in the industry through the mechanism of bitter-peptide generation. Note that this is a statement in the discussion section, not an experiment that established causation statistically.