J Food Bioact

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

Journal website www.isnff-jfb.com

Original Research

Volume 23, September 2023, pages 46-57

Comprehensive profiling of amino and carbonyl compounds in the Hong Yao sour soup, a type of fermented sour soup, in Huangluo Yao Village of Guangxi, China

Figure 1. Size exclusion chromatogram of Hong Yao sour soup monitored at 214 and 254 nm. Fractions eluted from the Superdex Peptide 10/300 GL column were collected every 1 min from 27 min to 44 min. Four peaks eluted after 45 min were named A, B, C, and D, respectively.

Figure 2. LC-MS chromatograms of SEC fractions of Hong Yao sour soup. (a): LC-MS precursor ion scan chromatograms targeting m/z 171 of AccQ-derivatives in positive ion mode in SEC fractions 36–45. (b): LC-MS full scan chromatograms of non-AccQ derivatives in SEC fractions A-D with scanning ranges m/z 50–300. Compounds in peaks with numbers were further analyzed by product ion scan for estimation of structure. Peaks of proteinogenic amino acids were directly indicated with one-letter abbreviations of amino acids on peaks. Peaks that could not be detected in the product ion scan analysis are marked with an asterisk.

Figure 3. LC-MS full scan chromatograms obtained from the cation exchanger unabsorbed fraction at a scanning range of m/z 120–200 (a), 200–250 (b), 250–300 (c), and 300–500 (d) in positive ion mode. Compounds in peaks with numbers were further analyzed by product ion scan for estimation of structure.

Figure 4. Precursor ion scan chromatograms targeting m/z 137 in negative ion mode of Hong Yao sour soup. Peaks identification: 1, lactic acid; 2, acetic acid; 3, pyruvic acid; 4, 2-ketobutyric acid; 5, 3-methyl-2-oxobutyric acid; 6, 3-methyl-2-oxovaleric acid and/or 4-methyl-2-oxovaleric acid.

**Table 1.** Concentrations of amino acids in Hong Yao sour soup and its HCl hydrolysate (mM)
Amino acids	non-hydrolysate	hydrolysate
The data from triplicate analyses are given as mean ± SD. *p< 0.05 represent significant difference between non-hydrolysate and hydrolysate group.
Asp	0.53 ± 0.02	1.26 ± 0.05*
Glu	0.114 ± 0.001	1.01 ± 0.04*
Ser	0.051 ± 0.003	0.59 ± 0.05*
Gly	0.94 ± 0.01	2.01 ± 0.09*
His	0.20 ± 0.01	0.36 ± 0.02*
Arg	0.03 ± 0.01	0.33 ± 0.02*
Thr/GABA	2.35 ± 0.02	2.47 ± 0.10
Ala	1.20 ± 0.01	1.82 ± 0.07*
Pro	0.52 ± 0.01	0.83 ± 0.03*
Tyr	0.06 ± 0.01	0.17 ± 0.02*
Val	0.72 ± 0.02	0.97 ± 0.04*
Met	0.25 ± 0.01	0.28 ± 0.02*
Ile	0.306 ± 0.001	0.45 ± 0.02*
Leu	0.84 ± 0.15	0.86 ± 0.02
Phe	0.369 ± 0.003	0.43 ± 0.02*
Lys	0.34 ± 0.01	1.08 ± 0.03*
Asn	0.317 ± 0.004	–
Gln	0.121 ± 0.004	–

Table 1. Concentrations of amino acids in Hong Yao sour soup and its HCl hydrolysate (mM)

Amino acids

non-hydrolysate

hydrolysate

The data from triplicate analyses are given as mean ± SD. *p< 0.05 represent significant difference between non-hydrolysate and hydrolysate group.

Asp

0.53 ± 0.02

1.26 ± 0.05*

Glu

0.114 ± 0.001

1.01 ± 0.04*

Ser

0.051 ± 0.003

0.59 ± 0.05*

Gly

0.94 ± 0.01

2.01 ± 0.09*

His

0.20 ± 0.01

0.36 ± 0.02*

Arg

0.03 ± 0.01

0.33 ± 0.02*

Thr/GABA

2.35 ± 0.02

2.47 ± 0.10

Ala

1.20 ± 0.01

1.82 ± 0.07*

Pro

0.52 ± 0.01

0.83 ± 0.03*

Tyr

0.06 ± 0.01

0.17 ± 0.02*

Val

0.72 ± 0.02

0.97 ± 0.04*

Met

0.25 ± 0.01

0.28 ± 0.02*

Ile

0.306 ± 0.001

0.45 ± 0.02*

Leu

0.84 ± 0.15

0.86 ± 0.02

Phe

0.369 ± 0.003

0.43 ± 0.02*

Lys

0.34 ± 0.01

1.08 ± 0.03*

Asn

0.317 ± 0.004

–

Gln

0.121 ± 0.004

–

**Table 2.** List of the identified compounds by LC-MS/MS analysis in the positive mode of AccQ-derivatives in SEC fractions 36–45 and non-AccQ derivatives in SEC fractions A and B from Hong Yao sour soup
Peak No.	Retention time (min)	precursor ion (m/z)	Main product ions^a (m/z)	Estimated compound	SEC Fr.
^aInterpretation of a peptide CID spectrum relies mainly on the sequence-specific ions present (a, b, and c ion series and x, y, and z ion series and immonium ions (*).
1	12.1	[242.1]⁺	116, 130, 171	Pyrrolidine	36
2	10.8	[336.1]⁺	56, 74, 100, 145, 166, 272	Methionine sulfoxide	36
3	9.6	[232.1]⁺	116, 128, 143, 145, 171	Ethanolamine	39–40
4	12.5	[244.2]²⁺	84, 116, 128, 145, 171, 172	Lysine	37–45
5	13.2	[368.1]⁺	144, 171, 186, 214	Dopa	39
6	11.0	[274.1]⁺	116, 128, 145, 171	GABA	38–44
7	13.5	[222.2]²⁺	86, 116, 128, 145, 171, 172	Cadaverine	37–39
8	12.5	[215.2]²⁺	72, 89, 116, 128, 145, 171	Putrescine	39–42
9	12.5	[429.3]⁺	72, 98, 115, 145, 171, 285	Putrescine	38–41
10	15.4	[358.1]⁺	171, 188, 358	N-Acetylspermidine	44
11	13.2	[357.2]⁺	116(y1), 171, 187(y2), 242(b2)	Ala-Pro	37
12	16.2	[385.3]⁺	72(V*), 116(y1), 145, 171, 215(y2), 270(b2)	Val-Pro	37–38
13	18.2	[399.2]⁺	86(L*), 116(y1), 171, 229(y2), 256(a2), 284(b2)	Leu-Pro	37–38
14	15.0	[359.1]⁺	72(V*), 145, 171, 189(y2), 242(a2), 270(b2)	Val-Ala	38
15	15.3	[373.2]⁺	86(L*), 132.6, 145, 171, 203(y2), 256(a2), 284(b2)	Leu-Ala	38
16	16.2	[387.3]⁺	72(V*), 145, 171, 217(y2), 242(a2), 270(b2)	Val-Val	38
17	12.5	[487.2]⁺	84(K*), 130, 145, 147(y1)	Lysine	37–38
18	4.7	[138.2]⁺	51, 65, 77, 91, 103	Tyramine	A
19	12.0	[205.1]⁺	29, 91, 115, 118.2, 132, 146, 159, 170, 188	Tryptophan	B
20	12.7	[217.1]⁺	106, 130, 144, 145, 146, 154, 156, 158, 171, 173, 184, 199	L-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid	B
21	4.7	[275.2]⁺	77, 91, 93, 103, 121	2×Tyramine	A

Table 2. List of the identified compounds by LC-MS/MS analysis in the positive mode of AccQ-derivatives in SEC fractions 36–45 and non-AccQ derivatives in SEC fractions A and B from Hong Yao sour soup

Peak No.

Retention time (min)

precursor ion (m/z)

Main product ions^a (m/z)

Estimated compound

SEC Fr.

^aInterpretation of a peptide CID spectrum relies mainly on the sequence-specific ions present (a, b, and c ion series and x, y, and z ion series and immonium ions (*).

12.1

[242.1]⁺

116, 130, 171

Pyrrolidine

10.8

[336.1]⁺

56, 74, 100, 145, 166, 272

Methionine sulfoxide

9.6

[232.1]⁺

116, 128, 143, 145, 171

Ethanolamine

39–40

12.5

[244.2]²⁺

84, 116, 128, 145, 171, 172

Lysine

37–45

13.2

[368.1]⁺

144, 171, 186, 214

Dopa

11.0

[274.1]⁺

116, 128, 145, 171

GABA

38–44

13.5

[222.2]²⁺

86, 116, 128, 145, 171, 172

Cadaverine

37–39

12.5

[215.2]²⁺

72, 89, 116, 128, 145, 171

Putrescine

39–42

12.5

[429.3]⁺

72, 98, 115, 145, 171, 285

Putrescine

38–41

15.4

[358.1]⁺

171, 188, 358

N-Acetylspermidine

13.2

[357.2]⁺

116(y1), 171, 187(y2), 242(b2)

Ala-Pro

16.2

[385.3]⁺

72(V*), 116(y1), 145, 171, 215(y2), 270(b2)

Val-Pro

37–38

18.2

[399.2]⁺

86(L*), 116(y1), 171, 229(y2), 256(a2), 284(b2)

Leu-Pro

37–38

15.0

[359.1]⁺

72(V*), 145, 171, 189(y2), 242(a2), 270(b2)

Val-Ala

15.3

[373.2]⁺

86(L*), 132.6, 145, 171, 203(y2), 256(a2), 284(b2)

Leu-Ala

16.2

[387.3]⁺

72(V*), 145, 171, 217(y2), 242(a2), 270(b2)

Val-Val

12.5

[487.2]⁺

84(K*), 130, 145, 147(y1)

Lysine

37–38

4.7

[138.2]⁺

51, 65, 77, 91, 103

Tyramine

12.0

[205.1]⁺

29, 91, 115, 118.2, 132, 146, 159, 170, 188

Tryptophan

12.7

[217.1]⁺

106, 130, 144, 145, 146, 154, 156, 158, 171, 173, 184, 199

L-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid

4.7

[275.2]⁺

77, 91, 93, 103, 121

2×Tyramine

**Table 3.** Identified compounds by LC-MS/MS analysis of the cation exchanger unabsorbed fraction in positive mode from Hong Yao sour soup
No.	Retention time (min)	precursor ion (m/z)	Main product ion (m/z)	Estimated compounds^a
^aAbbreviations are: Lac-, Ac-, ACN: lactoyl-, acetyl-, acetonitrile, respectively.
1	4.5	[197.0]⁺	81,99	Ortophosphate
2	4.5	[140.1]⁺	63,81,99	Ortophosphate+ACN
3	5.6	[148.1]⁺	43, 60, 70, 88, 106, 130, 148	N-Ac-Ser
4	6	[132.1]⁺	45, 91	Lactic acid+ACN
5	6	[181.1]⁺	45, 91	2×Lactic acid
6	7.3	[127.1]⁺	53, 63, 81, 99, 109	Ethylphosphate
7	9.5	[130.1]⁺	28, 41, 56, 84	pyroGlu
8	10.2	[190.1]⁺	43, 56, 84, 102, 130, 148, 172	N-Ac-Glu
9	8.8	[219.1]⁺	56, 84, 90, 130, 184	N-Lac-Gln
10	16	[220.1]⁺	30.5, 43, 72, 85, 90, 98, 124, 142, 184, 202	Pantothenic acid
11	27.0	[243.1]⁺	43, 56, 84, 86(L*), 132(y1), 197, 226	pyrGlu-Leu
12	30.5	[204.1]⁺	30, 41, 45, 69, 86(I*), 132(y1), 158	N-Lac-Ile
13	31.0	[204.1]⁺	30, 44(L), 45, 86(L), 132(y1), 158	N-Lac-Leu
14	4.7	[261.0]⁺	30, 43, 57, 81, 99, 109, 127, 145	Fructose 1-phosphate
15	9.3	[259.1]⁺	84, 130	2×pyroGlu
16	7.0	[341.0]⁺	43, 53, 81, 99, 109, 225, 243, 323	Fructose 1,6-bisphosphate
17	9.0	[341.0]⁺	43, 53, 81, 99, 109, 225, 243, 323	Glucose 1,6-bisphosphate

Table 3. Identified compounds by LC-MS/MS analysis of the cation exchanger unabsorbed fraction in positive mode from Hong Yao sour soup

No.

Retention time (min)

precursor ion (m/z)

Main product ion (m/z)

Estimated compounds^a

^aAbbreviations are: Lac-, Ac-, ACN: lactoyl-, acetyl-, acetonitrile, respectively.

4.5

[197.0]⁺

81,99

Ortophosphate

4.5

[140.1]⁺

63,81,99

Ortophosphate+ACN

5.6

[148.1]⁺

43, 60, 70, 88, 106, 130, 148

N-Ac-Ser

[132.1]⁺

45, 91

Lactic acid+ACN

[181.1]⁺

45, 91

2×Lactic acid

7.3

[127.1]⁺

53, 63, 81, 99, 109

Ethylphosphate

9.5

[130.1]⁺

28, 41, 56, 84

pyroGlu

10.2

[190.1]⁺

43, 56, 84, 102, 130, 148, 172

N-Ac-Glu

8.8

[219.1]⁺

56, 84, 90, 130, 184

N-Lac-Gln

[220.1]⁺

30.5, 43, 72, 85, 90, 98, 124, 142, 184, 202

Pantothenic acid

27.0

[243.1]⁺

43, 56, 84, 86(L*), 132(y1), 197, 226

pyrGlu-Leu

30.5

[204.1]⁺

30, 41, 45, 69, 86(I*), 132(y1), 158

N-Lac-Ile

31.0

[204.1]⁺

30, 44(L*), 45, 86(L*), 132(y1), 158

N-Lac-Leu

4.7

[261.0]⁺

30, 43, 57, 81, 99, 109, 127, 145

Fructose 1-phosphate

9.3

[259.1]⁺

84, 130

2×pyroGlu

7.0

[341.0]⁺

43, 53, 81, 99, 109, 225, 243, 323

Fructose 1,6-bisphosphate

9.0

[341.0]⁺

43, 53, 81, 99, 109, 225, 243, 323

Glucose 1,6-bisphosphate

**Table 4.** Contents of amino acid metabolites and carboxylic acids in Hong Yao sour soup
Compound^a	Concentration^b	Calibration range	Linearity (r²)	Units
^aAbbreviations are: Lac-, Pyr-, Ac-: lactoyl-, pyroglutamyl-, acetyl-, respectively. ^bThe data from triplicate analysis are given as mean ± SD. ^cSample was diluted for quantification.
N-Lac-Gln	1.43 ± 0.01	0.2–5	0.9983	μM
N-Lac-Leu	0.51 ± 0.03	0.01–2	0.9997	μM
N-Lac-Ile	0.34 ± 0.03	0.01–2	0.9996	μM
N-Pyr-Leu	0.30 ± 0.03	0.1–10	0.9993	μM
N-Ac-Ser	3.63 ± 0.44	0.05–50	0.9980	μM
N-Ac-Glu	187.24 ± 10.03^c	0.05–50	0.9998	μM
Pyruvic acid	0.52 ± 0.03	0.005–0.5	0.9999	mg/L
2-Ketobutyric acid	0.017 ± 0.002	0.0005–0.05	0.9998	mg/L
3-Methyl-2-oxobutyric acid	0.006 ± 0.001	0.0005–0.05	0.9994	mg/L
3-Methyl-2-oxovaleric acid and 4-Methyl-2-oxovaleric acid	0.10 ± 0.01	0.001–0.5	0.9999	mg/L
Succinic acid	87.91 ± 8.18^c	0.001–5	0.9975	mg/L
Glyceric acid	1.68 ± 0.15	0.005–5	0.9918	mg/L
Malic acid	0.027 ± 0.003	0.001–2	0.9902	mg/L
Lactic acid	4,761.21 ± 115.28^c	0.5–10	0.9976	mg/L
n-Caproic acid	0.56 ± 0.01	0.005–5	0.9982	mg/L
n-Butyric acid	0.05 ± 0.01	0.05–5	0.9986	mg/L
Propionic acid	14.16 ± 3.02^c	0.005–2	0.9988	mg/L
Acetic acid	616.33 ± 63.20^c	0.05–5	0.9980	mg/L
Cadaverine	5.43 ± 0.32^c	0.001–0.1	0.9994	mg/L
Putrescine	18.52 ± 1.73^c	0.001–0.1	0.9991	mg/L
Spermidine	0.47 ± 0.001^c	0.001–0.1	0.9987	mg/L
Spermine	0.87 ± 0.03^c	0.001–0.1	0.9972	mg/L
Ethanolamine	8.01 ± 0.14	0.05–10	0.9995	mg/L
Pyrrolidine	0.018 ± 0.001	0.001–0.5	0.9985	mg/L
Methionine sulfoxide	1.77 ± 0.08	0.05–5	0.9975	mg/L
GABA	166.39 ± 10.00^c	0.05–5	0.9996	mg/L
Tyramine	44.09 ± 2.09^c	0.005–0.5	0.9994	mg/L
Phenyethylamine	2.15 ± 0.03	0.05–5	0.9995	mg/L
Tryptamine	7.59 ± 0.58^c	0.005–0.5	0.9998	mg/L
Histamine	1.14 ± 0.01	0.05–5	0.9999	mg/L

Table 4. Contents of amino acid metabolites and carboxylic acids in Hong Yao sour soup

Compound^a

Concentration^b

Calibration range

Linearity (r²)

Units

^aAbbreviations are: Lac-, Pyr-, Ac-: lactoyl-, pyroglutamyl-, acetyl-, respectively. ^bThe data from triplicate analysis are given as mean ± SD. ^cSample was diluted for quantification.

N-Lac-Gln

1.43 ± 0.01

0.2–5

0.9983

μM

N-Lac-Leu

0.51 ± 0.03

0.01–2

0.9997

μM

N-Lac-Ile

0.34 ± 0.03

0.01–2

0.9996

μM

N-Pyr-Leu

0.30 ± 0.03

0.1–10

0.9993

μM

N-Ac-Ser

3.63 ± 0.44

0.05–50

0.9980

μM

N-Ac-Glu

187.24 ± 10.03^c

0.05–50

0.9998

μM

Pyruvic acid

0.52 ± 0.03

0.005–0.5

0.9999

mg/L

2-Ketobutyric acid

0.017 ± 0.002

0.0005–0.05

0.9998

mg/L

3-Methyl-2-oxobutyric acid

0.006 ± 0.001

0.0005–0.05

0.9994

mg/L

3-Methyl-2-oxovaleric acid and 4-Methyl-2-oxovaleric acid

0.10 ± 0.01

0.001–0.5

0.9999

mg/L

Succinic acid

87.91 ± 8.18^c

0.001–5

0.9975

mg/L

Glyceric acid

1.68 ± 0.15

0.005–5

0.9918

mg/L

Malic acid

0.027 ± 0.003

0.001–2

0.9902

mg/L

Lactic acid

4,761.21 ± 115.28^c

0.5–10

0.9976

mg/L

n-Caproic acid

0.56 ± 0.01

0.005–5

0.9982

mg/L

n-Butyric acid

0.05 ± 0.01

0.05–5

0.9986

mg/L

Propionic acid

14.16 ± 3.02^c

0.005–2

0.9988

mg/L

Acetic acid

616.33 ± 63.20^c

0.05–5

0.9980

mg/L

Cadaverine

5.43 ± 0.32^c

0.001–0.1

0.9994

mg/L

Putrescine

18.52 ± 1.73^c

0.001–0.1

0.9991

mg/L

Spermidine

0.47 ± 0.001^c

0.001–0.1

0.9987

mg/L

Spermine

0.87 ± 0.03^c

0.001–0.1

0.9972

mg/L

Ethanolamine

8.01 ± 0.14

0.05–10

0.9995

mg/L

Pyrrolidine

0.018 ± 0.001

0.001–0.5

0.9985

mg/L

Methionine sulfoxide

1.77 ± 0.08

0.05–5

0.9975

mg/L

GABA

166.39 ± 10.00^c

0.05–5

0.9996

mg/L

Tyramine

44.09 ± 2.09^c

0.005–0.5

0.9994

mg/L

Phenyethylamine

2.15 ± 0.03

0.05–5

0.9995

mg/L

Tryptamine

7.59 ± 0.58^c

0.005–0.5

0.9998

mg/L

Histamine

1.14 ± 0.01

0.05–5

0.9999

mg/L