The contribution of aromatic components in Katsuobushi to preference formation and reinforcement effect

Figures & data

Fig. 1. Fractionation scheme of arabushi sCO₂ extract.

Notes: Arabushi sCO₂ extract was fractionated by solvent polarity. First, sCO₂ was separated to water-soluble fraction (water layer) and hexane-soluble fraction (hexane layer). The hexane layer was subjected to dehydration, evaporation, and vacuum distillation to yield volatile fraction (Fr. Hex). The Fr. Hex was subjected to silica gel column chromatography and fractionated with different polarity solvents (n-Pentane, n-Pentane with 5% Diethyl ether, Diethyl ether, Methanol). Obtained solution was subjected to evaporation to yield Fr. 1 (n-Pentane), Fr. 2 (n-Pentane with 5% Diethyl ether), Fr. 3 (Diethyl ether), and Fr. 4 (Methanol), respectively.

Table 1. Aromatic components of 125 reconstructed aromatic flavors (125re) and 28 reconstructed aromatic flavors (28re).

No.	Compound	No.	Compound
	Hydrocarbon (9.99%)		Ketone (10.38%)
1	Octane	61	2-Heptanone
2	Decane	62	2-Octanone
3	Dodecane	63^*	1-Octen-3-one
4	Tridecane	64	3-Octen-2-one
5	Tetradecane	65	(E, E)-3,5-Octadien-2-one
6	Pentadecane	66	6-Methyl-5-hepten-2-one
7	Heptadecane	67	2-Hydroxy-3-pentanone
8	Biphenyl	68	2-Methyl-2-cyclopentenone
9	4-Methylbiphenyl	69^*	2-Hydroxy-3-methyl-2-cyclopentenone (Cyclotene)
10	Anthracene	70	2-Decanone
11	β-Pinene	71	3-Nonen-2-one
12	δ-Limonene	72	2-Undecanone
13	γ-Terpinene	73	Acetophenone
	Alcohol (9.28%)	74	β-Ionone
14	Ethyl alcohol	75^*	Maltol
15	1-Pentanol		Acid (12.84%)
16	(E)-2-Penten-1-ol	76	Acetic acid
17	(Z)-2-Penten-1-ol	77	Propanoic acid
18	1-Penten-3-ol	78^*	Butanoic acid
19	1-Hexanol	79	2-Methylpropanoic acid
20	1-Heptanol	80	Pentanoic acid
21	1-Octanol	81^*	3-Methylbutanoic acid
22^*	1-Octen-3-ol	82	2-Methylbutanoic acid
23	(E)-2-Octen-1-ol	83	(E)-2-Methyl-2-butenoic acid
24	(Z)-2-Octen-1-ol	84^*	Hexanoic acid
25^*	(Z)-1,5-Octadien-3-ol	85	(E)-3-Hexenoic acid
26	1-Nonanol	86^*	Octanoic acid
27	Furfuryl alcohol	87	Decanoic acid
28	(Z, Z)-1,5,8-Undecatrien-3-ol	88	Lauric acid
29	Geraniol	89	Myristic acid
30	Benzyl alcohol	90	Palmitic acid
	Aldehyde (6.85%)	91	Benzoic acid
31	Pentanal		Phenol (47.02%)
32	(E)-2-Pentenal	92	Phenol
33	Hexanal	93	o-Cresol
34	(E)-2-Hexenal	94	m-Cresol
35	Heptanal	95^*	p-Cresol
36	(E)-2-Heptenal	96	2-Ethylphenol
37^*	(Z)-4-Heptenal	97^*	3-Ethylphenol
38	(E, E)-2,4-Heptadienal	98^*	4-Ethylphenol
39	Octanal	99	2,5-Dimethylphenol
40	(E)-2-Octenal	100^*	2,6-Dimethylphenol
41	(E, E)-2,4-Octadienal	101	3,4-Dimethylphenol
42	Nonanal	102^*	Guaiacol
43^*	(E)-2-Nonenal	103^*	2-Methoxy-4-methylphenol
44^*	(E, Z)-2,6-Nonadienal	104^*	2-Methoxy-5-methylphenol
45	(E)-2-Decenal	105^*	2,6-Dimethoxyphenol
46^*	(E, E)-2,4-Decadienal	106	3-iso-Propylphenol
47	Benzaldehyde	107	4-Vinylphenol
48	Salicylaldehyde	108	2,4,6-Trimethylphenol
49	Citral	109^*	4-Ethylguaiacol
50^*	Vanillin	110^*	Eugenol
	Ether (0.40%)	111	iso-Eugenol
51	2-Ethylfuran	112^*	4-Propylguaiacol (Dihydroeugenol)
52	2-Pentylfuran		4-Vinylguaiacol
53	2,3-Benzofuran	114	2,6-Dimethoxy-4-methylphenol
54	2-Acetylfuran	115	4-Allyl-2,6-dimethoxyphenol
	Ester (0.14%)	116	Acetovanillon
55	Ethyl formate		N-Compound (2.28%)
56	Ethyl acetate	117	2-Ethylpyridine
	Lactone (0.81%)	118	3-Ethylpyridine
57	γ-Butyrolactone	119	3-Methoxypyridine
58	γ-2-Hexenolactone	120^*	2,5-Dimethylpyrazine
59^*	Coumarin	121	2,6-Dimethylpyrazine
60	6-Methylcoumarin	122	2,3,5-Trimethylpyrazine
		123	2,6-Diethylpyrazine
		124	Quinoline
		125	2-Acetylpyrrole

^* Component detected with GC−O.

Fig. 2. Two-bottle choice test with solvent fraction of arabushi sCO₂ extract.

Notes: Mice were administered dashi-taste solution and dashi-taste solution with aroma extract or aromatic flavor, and tested their preferences for the test solutions for 30 min. (A) A water layer (Water layer) or hexane extract separated from arabushi sCO₂ (Hexane layer) was added to dashi-taste solution and (B) volatile fraction of the hexane layer (Fr. Hex) or further solvent fractionated volatile fractions (Fr. 1–4) added to dashi-taste solution. Values given in the figures are means ± SEM. *p < 0.05, ***p < 0.001 (paired t-test).

Fig. 3. Two-bottle choice test with GC–MS analysis of reconstructed aromatic flavor.

Notes: Mice were administered dashi-taste solution and dashi-taste solution with aroma extract or aromatic flavor, and tested their preferences for the test solutions for 30 min. Reconstructed aromatic flavors (125re, 28re), citral, or arabushi sCO₂ extract (sCO2) was added to dashi-taste solution, respectively. (n = 8, each) Values given in the figures are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (paired t-test).

Fig. 4. CPP test flavored with arabushi sCO₂ extract.

Notes: Mice (n = 12) were conditioned with dashi-taste dextrin solution arabushi sCO₂ extract (sCO2). Values given in the figures are time spent increasing rate (%) of the test solution (means ± SEM). *p < 0.05 (one-sample t-test).

Fig. 5. CPP test flavored with 125 reconstructed aromatic flavor.

Notes: Mice (n = 12) were conditioned with dashi-taste dextrin solution flavored with 125 reconstructed aromatic flavors (125re). Values given in the figures are time spent increasing rate (%) of the test solution (means ± SEM). **p < 0.01 (one-sample t-test).