Revealing the flavor profile of citrus Pu-erh tea through GC-MS-O and untargeted metabolomics

Figures (7) Tables (3)

Figure 1.
Sensory spider plot of three CPT infusion samples, (a) sensory evaluation of three CPT samples based on ten aroma attributes, (b) taste profiles of three CPT samples by E-tongue. Note: *, ** and *** significant at p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001.
Figure 2.
Distribution map of volatile aroma substances, (a) species distribution profile of volatile compounds, (b) concentration distribution of each volatile compounds in three CPT infusion samples.
Figure 3.
Descriptive sensory analysis radar diagram of recombination model and corresponding CPT samples. Note: The sensorial parameters indicated with * are significantly different between samples (p ≤ 0.05).
Figure 4.
Correlation loadings plot for aroma-active compounds (X-matrix) and sensory attributes (Y-matrix) of three CPT samples.
Figure 5.
KEGG enrichment analysis of TOP20 metabolic pathways in untargeted metabolomics.
Figure 6.
Non-volatile compounds that significantly contributed to taste of three CPT samples.
Figure 7.
Flavor wheel of key flavor compounds of CPT infusion samples. Compounds marked with molecular structure in frames (a)−(g) were specific to the IT, OF or both IT and OF.

No.	Compounds	RI^a				Aroma description^b	FD^c			IM^d
		HP-INNOWAX		HP-5MS			FD^c
		cal.	ref.	cal.	ref.		IT	OF	CP
Alkenes
A1	styrene	1295	1272	−	890	sweet, balsam, floral(acacia)	2	1	−	MS, RI, S, O
A2	β-pinene	1135	1115	−	978	woody, pine, hay, green	2	−	−	MS, RI, S, O
A3	(+)-limonene	1232	−	1001	−	citrus, herbal, sweet	4	4	4	MS, RI, S, O
A4	γ-terpinene	1277	1255	−	1064	oily, woody, lime, herbal	−	1	−	MS, RI, S, O
A5	p-cymene	1305	1280	1103	1026	musty, woody, spice	1024	1024	1024	MS, RI, S, O
A6	2,4-dimethyl styrene	1459	1433	1076	1078	phenolic, spicy, soil, plastic	2048	64	2	MS, RI, S, O
Phenols
B1	phenol	2037	2028	−	981	phenolic, plastic, rubber	2	−	4	MS, RI, S, O
B2	o-cresol	1998	2010		1060	musty, phenolic, herbal, leathery	1	−	1	MS, RI, S, O
B3	p-cresol	2950	2079	1097	1098	phenolic, floral(narcissus)	−	−	8	MS, RI, S, O
B4	carvacrol	2226	2225	1296	1307	spice, woody, phenolic	2048	1024	1024	MS, RI, S, O
B5	thymol	2239	2172	1292	1297	herbal, phenolic, roasted	1024	1024	1024	MS, RI, S, O
B6	2,4-di-tert-butylphenol	2323	2330	1512	1513	phenolic	−	2048	2048	MS, RI, S, O
Alcohols
C1	prenol	1340	1323	−	778	fruity, green, floral(lavender)	2	4	−	MS, RI, S, O
C2	linalool	1553	1549	1101	1104	herbal, green, floral(rose),	16	8	8	MS, RI, S, O
C3	α-terpineol	1714	1170	1190	1191	pine, citrus, woody, floral(lilac)	1024	512	32	MS, RI, S, O
C4	Z-carveol	1842	1869	1220	1220	vegetable,green, caraway	16	8	4	MS, RI, S, O
C5	p-cymenol	1868	1851	1185	1188	sweet, fruity(cherry), camphor	128	8	4	MS, RI, S, O
C6	(-)-carveol	1884	1846	1231	1225	minty, green, herbal, spicy	−	4	4	MS, RI, S, O
C7	limonene glycol	2298	2325	1345	1342	minty, roasted,	2	1	1	MS, RI, S, O
C8	perillalcohol	2018	2021	−	1300	spicy(cardamom), floral(violet)	1	2	1	MS, RI, S, O
C9	benzyl alcohol	1903	1885	−	1034	floral(rose), phenolic	−	2048	2	MS, RI, S, O
C10	2-ethylhexanol	1496	1490	−	1026	citrus, fresh, floral, oil, sweet	2048	4	−	MS, RI, S, O
C11	terpinen-4-ol	1602	1636		1177	herbal, woody, earthy, musty	4	32	64	MS, RI, S, O
C12	(-)-pinocarveol	1679	1666	1137	1140	warm, woody, fennel, cereal	−	4	2	MS, RI, S, O
C13	phenylethyl alcohol	1940	1923	−	1121	floral(rose)	16	−	−	MS, RI, S, O
C14	(E)-p-mentha-2,8-dien-1-ol	1645	1641	1120	1121	fatty, popcorn, minty	4	4	1024	MS, RI, S, O
Aldehydes
D1	vanillin	2615	2550	1397	1394	sweet(chocolate), creamy	−	1	4	MS, RI, S, O
D2	nonanal	1410	1396	−	1102	waxy, fatty, orange	−	64	−	MS, RI, S, O
D3	decanal	1513	1504	−	1195	sweet, waxy, citrus(orange), floral	4	−	−	MS, RI, S, O
D4	citronellal	2023	1488	−	1158	sweet, floral, herbal, waxy, citrus	−	4	−	MS, RI, S, O
D5	benzaldehyde	1556	1529	−	961	almond, fruity(cherry)	16	8	−	MS, RI, S, O
D6	perillaldehyde	1814	1807	1276	1279	woody, pine, sweet(balsam), minty	−	32	16	MS, RI, S, O
D7	2-pyrrolaldehyde	2065	2048	1013	1015	musty, beefy, burnt, roasted, smoky	1024	−	1024	MS, RI, S, O
Ketones
E1	dihydrocarvone	1636	1645	1198	1200	herbal, minty, rubber, rice	−	16	8	MS, RI, S, O
E2	β-ionone	1964	1953	1485	1490	powdery, floral(orris), woody	−	8	−	MS, RI, S, O
E3	piperitone	1758	1743	1266	1268	woody, minty, camphor	−	8	4	MS, RI, S, O
E4	(+)-carvone	1764	1744	1244	1245	minty, fruity, spice	1024	2048	1024	MS, RI, S, O
E5	benzophenone	2533	2505	−	1625	floral (rose, geranium)	4	4	2	MS, RI, S, O
E6	4-methoxyacetophenone	2106	2120	1341	1345	fatty, sweet, anisic	2	−	2	MS, RI, S, O
Esters
F1	methyl methanthranilate	2100	2068	1408	1402	fruity, musty, sweet	2048	2048	2048	MS, RI, S, O
F2	methyl anthranilate	2283	2257	−	1338	floral (orange flower), fruity(grape)	1024	2048	1024	MS, RI, S, O
Acids
G1	octanoic acid	2033	2070		1191	fatty, waxy, rancid, oily, green, cheesy	2	1	4	MS, RI, S, O
G2	lauric acid	2489	2502		1570	fatty, fruity(coconut), oily	2	−	2	MS, RI, S, O
G3	palmitic acid	2512	2890	1968	1964	phenolic, waxy, fatty	−	2	4	MS, RI, S, O
Others
H1	dimethyl sulfone	1944	1912	−	915	roasted, sulfurous, burnt	2	2	2	MS, RI, S, O
H2	2,3-dimethyl pyrazine	1373	1352	−	911	nutty, butter, coffee, caramel, roasted	4	−	−	MS, RI, S, O
H3	1,2-dimethoxybenzene	1743	1740	1145	1143	musty, creamy, phenolic, sweet	2	−	1	MS, RI, S, O
^a Retention index of compounds on an HP-INNOWAX column and HP-5MS column. Cal means the RI value calculated by the formula. Ref means the RI value confirmed by comparison retention index to reference standards in the same condition (https://webbook.nist.gov/). ^b Aroma description. The aroma description vocabulary was generated by the GC-O evaluation team by comparing the aroma characteristics at actual concentrations with the literature and spectral library descriptions. ^c FD factor, flavor dilution factor determined on a HP-INNOWAX column. '−' means not being detected. ^d Identification method: MS means identified by comparison with the NIST mass spectral library 11 Vision database; RI means confirmed by comparison retention index; S means confirmed by authentic standard chemicals; O means confirmed by aroma descriptor.

Table 1.

Identification analysis of volatile compounds in citrus Pu-erh tea samples.

No.	Compounds	OT (mg/kg)^A	Concentration (mg/kg)^B			OAV^C			ACI%^D
No.	Compounds	OT (mg/kg)^A	IT	OF	CP	IT	OF	CP	IT	OF	CP
A1	styrene	0.065	26.54^a	8.29^b	−	408.26	127.49	−	0.0526	0.0041	−
A2	β-pinene	0.14	4.09^a	−	−	29.24	−	−	0.0038	−	−
A3	(+)-limonene	0.034	233.62^a	159.49^b	120.07^c	6871.15	4691.00	3531.55	0.8852	0.1513	0.9724
A4	γ-terpinene	1	−	37.35^a	−	−	37.35	−	−	0.0012	−
A5	p-cymene	7.2	94.59^a	80.77^b	76.74^c	13.14	11.22	10.66	0.0017	0.0004	0.0029
A6	2,4-dimethyl styrene	0.085	16.71^c	30.69^b	56.52^a	196.62	361.01	664.97	0.0253	0.0116	0.1831
B1	phenol	5	179.14^a	−	150.89^b	35.83	−	30.18	0.0046	−	0.0083
B2	o-cresol	1.4	19.59^b	−	20.07^a	14.00	−	14.34	0.0018	−	0.0039
B3	p-cresol	0.0039	−	−	83.12^a	−	−	21311.63	−	−	5.7137
B4	carvacrol	2.29	1070.68^c	1413.74^b	2900.15^a	467.54	617.35	1266.44	0.0602	0.0199	0.3487
B5	thymol	1.7	1215.72^a	590.69^c	1011.75^b	715.13	347.46	595.14	0.0921	0.0112	0.1542
B6	2,4-di-tert-butylphenol	0.5	−	120.60^b	955.01^a	−	241.20	1910.02	−	0.0078	0.5259
C1	prenol	0.25	1.78^b	2.88^a	−	7.12	11.54	−	0.0009	0.0004	−
C2	linalool	0.00022	150.37^a	69.68^b	58.20^c	683488.63	316718.51	264532.69	88.0482	10.2151	72.8359
C3	α-terpineol	1.2	1139.07^a	883.02^b	694.31^c	949.23	735.85	578.59	0.1223	0.0237	0.1593
C4	Z-carveol	0.25	1732.26^c	2991.85^a	2243.32^b	6929.04	11967.40	8973.28	0.8926	0.3860	2.4707
C5	p-cymenol	ND	607.73^b	713.37^a	445.52^c	−	−	−	−	−	−
C6	(-)-carveol	0.25	−	285.04^a	274.93^b	−	1140.16	1099.72	−	0.0368	0.3028
C7	limonene glycol	ND	610.42^a	148.96^c	474.19^b	−	−	−	−	−	−
C8	perillalcohol	1.1	169.07^b	279.38^a	147.65^c	153.70	253.98	134.23	0.0198	0.0082	0.0370
C9	benzyl alcohol	2.54	−	69.75^a	48.40^b	−	27.46	19.05	−	0.0009	0.0052
C10	2-ethylhexanol	0.3	333.98^a	42.32^b	−	1113.25	141.07	−	0.1434	0.0045	−
C11	terpinen-4-ol	1.2	282.27^a	220.39^b	155.74^c	235.22	183.66	129.78	0.0303	0.0059	0.0357
C12	(-)-pinocarveol	ND	−	93.72^a	78.28^b	−	−	−	−	−	−
C13	phenylethyl alcohol	0.086	177.55^a	−	−	2064.56	−	−	0.2660	−	−
C14	(E)-p-mentha-2,8-dien-1-ol	ND	1658.63^b	1349.01^c	1829.85^a	−	−	−	−	−	−
D1	vanillin	0.053	−	402.51^a	356.40^b	−	7594.58	6724.47	−	0.2449	1.8515
D2	nonanal	0.0011	−	28.40^a	−	−	25817.10	−	−	0.8327	−
D3	decanal	0.003	54.60^a	−	−	18200.57	−	−	2.3446	−	−
D4	citronellal	0.006	−	58.38^a	−	−	9729.28	−	−	0.3138	−
D5	benzaldehyde	0.75	98.19^a	23.93^b	−	130.92	31.91	−	0.0169	0.0010	−
D6	perillaldehyde	0.03	−	134.70^a	75.84^b	−	4490.11	2528.15	−	0.1448	0.6961
D7	2-pyrrolaldehyde	65	225.12^a	−	147.44^b	3.46	−	2.27	0.0004	−	0.0006
E1	dihydrocarvone	3.25	−	82.27^b	128.87^a	−	25.31	39.65	−	0.0008	0.0109
E2	β-ionone	0.000007	−	18.17^a	−	−	2595440.13	−	−	83.7102	−
E3	piperitone	0.68	−	55.93^a	28.08^b	−	82.25	41.29	−	0.0027	0.0114
E4	(+)-carvone	0.16	1285.06^b	1406.29^a	1215.37^c	8031.61	8789.32	7596.06	1.0346	0.2835	2.0915
E5	benzophenone	ND	149.77^c	591.43^a	157.09^b	−	−	−	−	−	−
E6	4-methoxyacetophenone	ND	76.68^b	−	94.61^a	−	−	−	−	−	−
F1	methyl methanthranilate	0.349	2611.54^b	2902.11^a	2352.80^c	7482.93	8315.50	6741.56	0.9640	0.2682	1.8562
F2	methyl anthranilate	0.003	116.02^b	307.63^a	105.30^c	38672.96	102541.93	35101.20	4.9819	3.3073	9.6647
G1	octanoic acid	3	27.78^b	4.16^c	64.19^a	9.26	1.39	21.40	0.0012	0.0000	0.0059
G2	lauric acid	10	175.08^b	−	431.46^a	17.51	−	43.15	0.0023	−	0.0119
G3	palmitic acid	10	−	337.30^b	1437.30^a	−	33.73	143.73	−	0.0011	0.0396
H1	dimethyl sulfone	ND	243.11^b	145.86^c	492.16^a	−	−	−	−	−	−
H2	2,3-dimethyl pyrazine	0.8	20.18^a	−	−	25.23	−	−	0.0033	−	−
H3	1,2-dimethoxybenzene	ND	389.68^a	−	92.04^b	−	−	−	−	−	−
^A The odor detection thresholds in water were obtained from previous studies^[12,45] and online database (www.vcf-online.nl/VcfHome.cfm). ^B Concentration (mg/kg), The concentration of each volatile compound was calculated based on the calibration equation in Supplemental Table S3. ^C OAV (Odor activity value). ^D ACI (Aroma character impact value). All results were expressed as mean value (n = 3). Values bearing different lowercase letters (a, b, c) were significantly different (p ≤ 0.05).

Table 2.

Quantitative analysis of volatile compounds in citrus Pu-erh tea samples.

No.	Odorants omitted from the complete recombinant	Number^a			Significance^b
No.	Odorants omitted from the complete recombinant	IT	OF	CP	IT	OF	CP
1	octanoic acid, lauric acid, palmitic acid	6	9	11	*	**	**
1−1	octanoic acid	5	6	5	*	*	*
1−2	lauric acid	5	2	5	*	−	*
1−3	palmitic acid	2	6	7	−	*	*
2	thymol, carvacrol, phenol, o-cresol, p-cresol, 2,4-di-tert-butylphenol	8	7	9	**	*	**
2−1	thymol	9	8	8	**	**	**
2−2	carvacrol	6	7	7	*	*	*
2−3	phenol	4	1	5	*	−	*
2−4	o-cresol	5	3	4	*	−	*
2−5	p-cresol	2	4	8	−	*	*
2−6	2,4-di-tert-butylphenol	8	7	8	**	*	**
3	linalool, perillalcohol, p-cymenol, limonene glycol, terpinen-4-ol, prenol, 2-ethylhexanol	15	15	15	***	***	***
3−1	linalool	14	15	14	***	***	***
3-2	perillalcohol	5	6	5	*	*	*
3−3	p-cymenol	2	3	2	−	−	−
3−4	limonene glycol	1	2	3	−	−	−
3−5	terpinen-4-ol	8	9	8	**	**	**
3−6	prenol	5	4	1	*	*	−
3−7	2-ethylhexanol	6	5	3	*	*	−
4	phenylethyl alcohol, α-terpineol, (E)-p-mentha-2,8-dien-1-ol, (−)-pinocarveol, benzyl alcohol, (−)-carveol, Z-carveol	15	14	15	***	***	***
4−1	phenylethyl alcohol	6	1	2	*	−	−
4−2	α-terpineol	8	7	7	**	*	*
4−3	(E)-p-mentha-2,8-dien-1-ol	1	2	2	−	−	−
4−4	(−)-pinocarveol	2	2	3	−	−	−
4−5	benzyl alcohol	3	7	6	−	*	*
4−6	(−)-carveol	3	7	8	−	*	**
4−7	Z-carveol	14	13	13	***	***	***
5	p-cymene, β-pinene, styrene, 2,4-dimethyl styrene, (+)-limonene, γ-terpinene, 1,2-dimethoxybenzene	13	11	11	***	**	**
5−1	p-cymene	6	6	5	*	*	*
5−2	β-pinene	6	3	3	*	−	−
5−3	styrene	11	3	3	**	−	−
5−4	2,4-dimethyl styrene	10	9	11	**	**	**
5−5	(+)-limonene	15	10	9	***	**	**
5−6	γ-terpinene	3	7	2	−	*	−
5−7	1,2-dimethoxybenzene	3	2	1	−	−	−
6	benzaldehyde, 2-pyrrolaldehyde, perillaldehyde, decanal, nonanal, citronellal, vanillin	9	12	13	**	***	***
6−1	benzaldehyde	8	4	1	**	*	−
6−2	2-pyrrolaldehyde	6	3	7	*	−	*
6−3	perillaldehyde	2	7	6	−	*	*
6−4	decanal	9	2	2	**	−	−
6−5	nonanal	3	8	4	−	**	*
6−6	citronellal	4	12	5	*	***	*
6−7	vanillin	5	12	13	*	***	***
7	2,3-dimethyl pyrazine, methyl methanthranilate, methyl anthranilate, dimethyl sulfone	14	15	15	***	***	***
7−1	2,3-dimethyl pyrazine	6	3	4	*	−	*
7−2	methyl methanthranilate	14	15	15	***	***	***
7−3	methyl anthranilate	13	14	15	***	***	***
7−4	dimethyl sulfone	5	3	3	*	−	−
8	(+)-carvone, dihydrocarvone, piperitone, benzophenone, 4-methoxyacetophenone, β-ionone	13	14	15	***	***	***
8−1	(+)-carvone	12	13	13	***	***	***
8−2	dihydrocarvone	3	6	7	−	*	*
8−3	piperitone	4	7	8	*	*	**
8−4	benzophenone	4	5	4	*	*	*
8−5	4-methoxyacetophenone	2	3	4	−	−	*
8−6	β-ionone	4	13	5	*	***	*
^a The number of panelists who perceived the aroma difference by means of a triangle test. Fifteen panelists were invited for aroma omission experiment. ^b Levels of significance, defined based on the number of panelists who were able to determine the difference in aroma omission. −, not significant (0−3, p > 0.05); , significant (4−7, p ≤ 0.05); , highly significant (8-11, p ≤ 0.01); **, very highly significant (12−15, p ≤ 0.001).

Table 3.

Omission tests of three citrus Pu-erh tea based on aroma recombination model.