两种养殖模式金背鲤肠道微生物菌群和主体风味差异分析

于美娟; 杨慧; 余长生; 蔡教明; 补子龙; 曾宪文

doi:10.12131/20220270

两种养殖模式金背鲤肠道微生物菌群和主体风味差异分析

doi: 10.12131/20220270

于美娟^{1, 2,},
杨慧¹,
余长生^{2, 3},
蔡教明³,
补子龙³,
曾宪文³

1.
湖南省农业科学院农产品加工研究所，湖南长沙 410125
2.
湖南省水产产业技术体系，湖南长沙 410125
3.
湖南省怀化市畜牧水产事务中心，湖南怀化 418000

基金项目: 湖南省现代农业水产产业技术体系岗位专家专项资金 (湘农发〔2019〕105号)；湖南省自然科学基金 (2020JJ4392)

详细信息

作者简介:
于美娟 (1976—)，女，研究员，硕士，研究方向为水产品加工与综合利用。E-mail: 82923686@qq.com

中图分类号: TS 254.2
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出版历程
- 收稿日期: 2022-10-12
- 修回日期: 2022-12-30
- 录用日期: 2023-01-07
- 网络出版日期: 2023-03-27

Analysis on differences of microbial community structure and main flavor substances of Cyprinus carpio var. Jinbei cultured in paddy fields and ponds

1.
Agricultural Products Processing Institue, Hunan Academy of Agricultural Sciences, Changsha 410125, China
2.
Technology System of Aquatic Industry in Hunan Province, Changsha 410128, China
3.
Huaihua Animal Husbandry and Fishery Affairs Center, Huaihua 418000, China

摘要

摘要: 探讨不同养殖模式下金背鲤 (Cyprinus carpio var. Jinbei) 肠道菌群和鱼肉风味品质差异，对金背鲤的繁殖优化具有重要意义。采用高通量测序技术对稻田放养和池塘养殖模式下金背鲤的肠道微生物进行测序，利用液相色谱和顶空固相微萃取-气相色谱-离子迁移谱联用 (HS-SPME-GC-IMS) 技术测定其滋味物质、挥发性风味化合物，结合感觉阈值计算滋味活性值和相对气味活度值。结果显示：两种养殖模式的金背鲤肠道细菌群落结构差异显著，稻田放养 (FGF) 组以弧菌 (Vibrio)、拟杆菌 (Bacteroides)、交替单胞菌 (Alteromonadales)、希瓦氏菌 (Shewanella)、嗜冷假单胞菌 (Pseudomonas psychrophile) 和Brevinema属为主，池塘养殖 (FGP) 组以莫拉克斯氏菌 (Moraxella) 和克雷伯菌属 (Klebsiella) 为主；FGF组中鲜味肌苷酸含量及其滋味活性值 (1.676 g·kg⁻¹, 6.705) 远高于FGP组 (0.246 g·kg⁻¹, 0.985)，FGF组鲜味氨基酸和甜味氨基酸含量 (0.143和2.052 g·kg⁻¹) 高于FGP组 (0.109和2.001 g·kg⁻¹)，而其苦味氨基酸 (3.193 g·kg⁻¹) 却低于FGP组 (3.836 g·kg⁻¹)；金背鲤的挥发性化合物组分复杂，其关键气味化合物 (ROAV≥1) 和对整体风味有修饰作用的化合物 (0.1≤ROAV<1) 的种类存在差异。菌属与风味物质的相关性分析显示弧菌属、拟杆菌属、克雷伯菌属和摩根菌属 (Morganella) 与风味物质呈显著相关 (0.01≤P<0.05 & 0.001≤P<0.01 & P<0.001)。研究表明，养殖模式影响了金背鲤的肠道微生物，并间接影响了其风味品质。
- 金背鲤 /
- 稻田放养 /
- 池塘养殖 /
- 肠道菌群 /
- 主体风味物质
Abstract: It is important to study the differences of intestinal flora and fish flavor quality of Cyprinus carpio var. Jinbei under different culture modes for its breeding optimization. We applied illumina high-throughput sequencing technology to analyze intestinal microflora of C. carpio var. Jinbei cultured in paddy fields (FGF) and ponds (FGP), respectively. Besides, we used liquid chromatography (LC) to determine the flavor substances, and used headspace solid phase microextraction combining with gas chromatography-ion mobility spectrometry (HS-SPME-GC-IMS) to determine the volatile flavor substances. Then we calculated the taste activity value and relative odor activity by combining the sensory threshold. Significant difference analysis shows that Vibrio, Bacteroides, Alteromonadales, Shewanella, Pseudomonas psychrophila and Brevinema were the main bacteria in FGF group, while Moraxella and Klebsiella were the main bacteria in FGP group. The contents of IMP and flavor activity in FGF group (1.676 g·kg⁻¹ and 6.705) were significantly higher than those in FGP group (0.246 g·kg⁻¹ and 0.985). The contents of umami amino acids and sweet amino acids in FGF group (0.143 and 2.052 g·kg⁻¹) were higher than those in FGP group (0.109 and 2.001 g·kg⁻¹). However, the contents of bitter amino acids in FGF group (3.193 g·kg⁻¹) were lower than those in FGP group (3.836 g·kg⁻¹). Moreover, the components of volatile compounds were complex, and the types of key odor compounds (ROAV≥1) and compounds that could modify the overall flavor (0.1≤ROAV<1) were different. The correlation analysis between bacteria genera and flavor substances shows that Vibrio, Bacteroides, Klebsiella and Morganella were significantly correlated with flavor substances (0.01≤P<0.05 & 0.001≤P<0.01 & P<0.001). The results indicate that cultivation mode affects the intestinal microbes of fish, and affects the flavor quality of fish indirectly.
- Cyprinus carpio var. Jinbei /
- Paddy field culture /
- Pond culture /
- Intestinal bacterial flora /
- Main flavor substances

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图 1 两种养殖模式的金背鲤肠道菌的特征韦恩图

Figure 1. Venn diagram of features of intestinal bacteria in C. carpio var. Jinbei of FGF and FGP groups

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图 2 两种养殖模式的金背鲤肠道微生物Alpha多样性指数

Figure 2. Alpha diversity of intestinal microorganisms in C. carpio var. Jinbei of FGF and FGP groups

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图 3 门水平上的物种相对丰度及差异分析

注：*. 差异显著 (P<0.05)。图4同此。

Figure 3. Relative abundance and differences of species at phylum level

Note: *. Significant differences (P<0.05). The same case in Fig. 4.

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图 4 属水平上的物种相对丰度及差异分析

Figure 4. Relative abundance and differences of species at genus level

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图 5 两种养殖模式的金背鲤肠道微生物菌群的差异性分析

Figure 5. Difference analysis of microbial flora in intestine of C. carpio var. Jinbei of FGF and FGP groups

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图 6 两种养殖模式的金背鲤肌肉中挥发性化合物GC-IMS 指纹图谱

Figure 6. GC-IMS gallrey plot of volatile compounds in muscle of C. carpio var. Jinbei of FGF and FGP groups

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图 7 肠道菌群结构与风味物质的相关性热图分析

Figure 7. Correlation heat map analysis of intestinal flora structure and flavor substances

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表 1 两种养殖模式的金背鲤肌肉中核苷酸及其关联产物含量及K值 (N=3)

Table 1. Nucleotides contents, their related products and K value in muscle of C. carpio var. Jinbei of FGF and FGP groups (N=3)

核苷酸种类 Nucleotide	阈值^[17] Threshold/(g·kg⁻¹)	呈味特征 Taste characteristics	核苷酸含量 Nucleotide content/(g·kg⁻¹)		滋味活性值 TAV
核苷酸种类 Nucleotide	阈值^[17] Threshold/(g·kg⁻¹)	呈味特征 Taste characteristics	稻田放养 FGF	池塘养殖 FGP	稻田放养 FGF	池塘养殖 FGP
腺苷三磷酸 ATP	—	—	0.051±0.0066^a	0.07686±0.0012^b
腺苷二磷酸 ADP	—	—	0.885±0.0271^b	1.935±0.0163^a
腺苷酸 AMP	0.50	鲜甜味	0.1747±0.0173^a	0.146±0.00026^b	0.349	0.291
肌苷酸 IMP	0.25	鲜味	1.676±0.1109^a	0.246±0.0055^b	6.705	0.985
次黄嘌呤核苷 HXR	—	苦味	0.0162±0.0021^b	0.0342±0.0017^a
次黄嘌呤 HX	—	苦味	0.0826±0.0062^b	0.193±0.0035^a
Σ呈味核苷酸 ΣFlavoring nucleotide			2.885±0.17^a	2.63±0.028^b
K值 K value/%			8.805	21.305
注：“—”表示未查到相关阈值和呈味特征；同行中不同字母间存在显著性差异 (P<0.05)；下同。 Note: "—" indicates that the relevant threshold and flavor characteristics are not found; values with different letters within the same line are signifi-cantly different (P<0.05). The same case in the following tables.

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表 2 两种养殖模式下金背鲤肌肉中游离氨基酸与滋味活性值

Table 2. Amino acid and taste activity value in muscle of C. carpio var. Jinbei of FGF and FGP groups

氨基酸Amino acid	阈值^[18-19]Threshold/(g·kg⁻¹)	呈味特征^[20-22]Taste characteristics	游离氨基酸含量 Free amino acid content/(g·kg⁻¹)		滋味活性值TAV
氨基酸Amino acid	阈值^[18-19]Threshold/(g·kg⁻¹)	呈味特征^[20-22]Taste characteristics	稻田放养 FGF	池塘养殖FGP	稻田放养FGF	池塘养殖FGP
苏氨酸 Thr^★	2.60	甜 (+)	0.312±0.0042^b	0.362±0.0041^a	0.119	0.138
赖氨酸 Lys^★	0.50	甜/苦 (−)	0.254±0.0081^b	0.495±0.011^a	0.495	0.991
缬氨酸 Val^▲	0.40	甜/苦 (−)	0.297±0.0031^b	0.357±0.0023^a	0.737	0.888
蛋氨酸 Met^▲	0.30	苦/甜/硫 (−)	0.031±0.00002^b	0.0459±0.0002^a	0.103	0.153
色氨酸 Trp^◆	0.90	苦/芳香 (−)	0.0284±0.0004^a	0.0256±0.0002^b	0.035	0.031
苯丙氨酸 Phe^◆	0.90	苦/芳香 (−)	0.091±0.0004^b	0.119±0.0027^a	0.101	0.13
异亮氨酸 Ile^▲	0.90	苦 (−)	0.0501±0.0006^b	0.819±0.0003^a	0.055	0.091
亮氨酸 Leu^▲	1.90	苦 (−)	0.0982±0.0026^b	0.137±0.00007^a	0.051	0.072
Σ必需氨基酸 ΣEAA			1.162±0.019^b	1.623±0.021^a	1.69	2.49
组氨酸 His^▲	0.20	酸/苦 (−)	1.825±0.002^b	2.139±0.016^a	9.117	10.64
精氨酸 Arg^▲	0.50	甜/苦 (−)	0.0816±0.0018^b	0.181±0.001^a	0.161	0.36
Σ半必需氨基酸 ΣSEAA			1.907±0.0038^b	2.32±0.017^a	9.28	11.00
天冬氨酸 Asp^●	0.03	鲜/酸 (+)	0.0281±0.0005^a	0.0143±0.0003^b	0.923	0.483
谷氨酸 Glu^●	0.05	鲜/酸 (+)	0.114±0.0014^a	0.0949±0.004^b	2.268	1.954
丙氨酸 Ala^★	0.60	鲜/甜 (+)	0.315±0.015^a	0.251±0.014^b	0.542	0.401
丝氨酸 Ser^★	1.50	甜 (+)	0.131±0.0027^b	0.159±0.0001^a	0.086	0.106
甘氨酸 Gly^★	1.30	甜 (+)	1.088±0.017^a	0.905±0.0018^b	0.827	0.697
脯氨酸 Pro^★	3.00	甜/苦 (+)	0.124±0.0016^b	0.174±0.012^a	0.041	0.055
肌氨酸 Sar	—	—	0.125±0.012^a	0.122±0.001^a
酪氨酸 Tyr^◆	0.91	苦/芳香 (−)	0.0693±0.0015^b	0.0869±0.001^a	0.075	0.095
半胱氨酸 Cys^▲	—	苦/甜/硫 (−)	0.639±0.027^a	0.550±0.007^b
Σ非必需氨基酸 ΣNEAA			2.689±0.083^a	2.357±0.041^b	4.279	2.868
羟脯氨酸 Hyp	—	—	0.575±0.01^a	0.604±0.02^a
谷氨酰胺 Gln	—	—	0.08±0.0019^b	0.102±0.0009^a
天冬酰胺 Asn	—	—	0.0031±0.0001^a	0.0034±0.0004^a
瓜氨酸 Cit	—	—	0.0357±0.0006^b	0.0654±0.0001^a
游离氨基酸总量 Total free amino acid			6.361±0.12^b	7.137±0.098^a
^● 鲜味氨基酸总量/占比 Total delicious amino acid/Proportion			0.1425±0.0019^a/2.24%		0.109±0.0042^b/1.53%
^★ 甜味氨基酸总量/占比 Total Sweet amino acid/Proportion			2.052±0.013^a/32.26%		2.031±0.033^a/28.46%
^▲ 苦味氨基酸总量/占比 Total bitter amino acid/Proportion			3.193±0.01^b/50.19%		3.836±0.0071^a/53.74%
^◆ 芳香氨基酸总量/占比 Total aromatic amino acids/Proportion			0.189±0.0021^b/2.96%		0.232±0.0039^a/3.25%

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表 3 两组养殖模式的金背鲤肌肉中挥发性化合物相对含量及其ROVA值

Table 3. Volatile compounds and relative odor activity value in muscle of C. carpio var. Jinbei of FGF and FGP groups　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　μg·kg⁻¹

化合物Compound	阈值^[28-29]Threshold/(μg·kg⁻¹)	气味特征^[28]Odour characteristics	稻田放养 FGF		池塘养殖 FGP
化合物Compound	阈值^[28-29]Threshold/(μg·kg⁻¹)	气味特征^[28]Odour characteristics	相对含量 Relative content	相对气味活度值ROAV	相对含量 Relative content	相对气味活度值ROAV
醛类 Aldehydes			21.42%		9.54%
壬醛 Nonanal	1	脂香、青草味	12.07±2.45^a	100	4.384±0.63^b	100
辛醛 Octanal	0.7	果香叶	6.32±1.1^a	74.80	3.633±0.49^b	43.01
庚醛 Heptanal	3	鱼腥、烤鱼、哈喇味	9.67±1.43^a	26.71	5.641±1.34^b	15.58
E-2-己烯醛 (E)-2-Hexenal	17	青香、脂香、果香味	3.42±1.48^a	1.67	2.54±0.26^b	1.24
己醛-M Hexanal-M	4.5	鱼腥、青草味	44.35±3.09^a	81.56	30.11±4.64^b	55.44
己醛-D Hexanal-D	4.5	鱼腥、青草味	31.66±3.85^a	58.29	11.60±4.72^b	21.36
戊醛 Pentanal	20	果香味	13.1±0.77^a	5.43	6.623±0.85^b	2.74
2-甲基丁醛2-Methylbutanal	1	果香味	4.08±0.29^b	33.8	6.80±1.2^a	56.34
3-甲基丁醛 3-Methylbutanal	1.1	巧克力味、腐臭味	2.71±0.14^b	20.41	4.84±1.11^a	36.45
丁醛 Butanal	1.3	花香、水果香味	5.07±0.72^a	32.31	5.66±0.53^a	36.07
苯甲醛 Benzaldehyde	350	苦杏仁味	2.6±0.44^a	0.062	1.63±0.42^b	0.038
醇类 Alcohols			12.79%		19.19%
1-庚醇 1-Heptanol	330	脂味、酒香	2.07±0.33^a	0.052	1.35±0.31^b	0.034
1-辛烯-3-醇 1-Octen-3-ol	10	蘑菇味、泥土味	3.64±0.54^b	3.16	5.80±2.38^a	4.81
1-己醇 1-Hexanol	5.6	青草味	3.38±0.54^b	5.00	6.22±2.01^a	9.20
正戊醇 1-Pentanol	150.2	面包酒香、果香	3.29±0.19^b	0.18	5.33±0.3^a	0.29
1-戊烯-3-醇 1-Penten-3-ol	358.1	肉香味、鱼腥味	10.68±1.09^a	0.25	9.03±2.05^b	0.021
1-丁醇 1-Butanol	5 000	温和的杂醇油气息	1.21±0.08^a	<0.01	1.04±0.07^a	<0.01
乙醇 Ethanol	100 000	酒味	29.21±6.39^a	<0.01	26.75±2.58^a	<0.01
异丙醇 2-Propanol	—	—	2.85±0.09^b		21.92±3.37^a
3-甲基-1-丁醇 3-Methyl-1-butanol	250	发酵味、油脂味、	1.26±0.12^b	0.04	3.34±0.69^a	0.11
正丙醇 1-Propanol	8 505	—	18.04±1.36^b	0.018	80.69±4.78^a	0.079
异丁醇 Isobutanol	7 000	—	1.26±0.09^b	<0.01	2.16±0.27^a	<0.01
Z-3-己烯-1-醇 (Z)-3-Hexen-1-ol	250	—	0.39±0.06^b	0.013	1.15±0.04^a	0.038
芳樟醇 Linalool	30 000	热带香辛料味	1.96±0.14^a	<0.01	1.46±0.36^a	<0.01
2-甲基-1-丁醇 2-Methyl-1-butanol	1 200	—	1.4±0.04^a	<0.01	1.65±0.25^a	0.01
酮类 Ketones			55.46%		58.01%
3-辛酮 3-Octanone	28	酮香、青香	2.35±0.15^a	0.70	1.76±0.31^b	0.52
3-羟基-2-丁酮 3-Hydroxy-2-butanone	800	脂香味	24.67±1.85^a	0.26	16.73±0.34^b	0.17
1-戊烯-3-酮 1-Penten-3-one	1	蘑菇味、烤焦味	4.54±1.32^a	37.61	1.72±0.16^b	14.25
2-戊酮 2-Pentanone	13 800	果味	4.7±0.2^a	<0.01	4.27±0.42^b	<0.01
2-丁酮-D 2-Butanone-D	50 000	醚香、果香、樟脑香	18.42±1.83^b	<0.01	72.65±3.42^a	0.012
2-丁酮-M 2-Butanone-M	50 000	醚香、果香、樟脑香	35.87±0.82^b	<0.01	40.29±1.69^a	<0.01
2-丙酮 2-Propanone	—	青草味	259.14±7.81^b		370.14±8.67^a
酯类 Esters			2.06%		0.94%
甲酸丁酯 Butyl formate	—		2.95±0.45^a		1.841±0.11^b
乙酸乙酯-M Ethyl Acetate-M	5	醚香、果香	7.65±0.9^a	12.68	4.70±0.37^b	7.79
乙酸乙酯-D Ethyl Acetate-D	5	醚香、果香	2.36±0.5^a	3.91	1.72±0.29^a	2.85
酸类 Acids			1.25%		3.36%
2-甲基丙酸 2-Methylpropanoic acid	—	—	2±0.12^a		2.14±0.15^a
3-甲基丁酸 3-Methylbutanoic acid	—	—	0.57±0.07^b		1.44±0.12^a
丙酸 Propanoic acid	—	—	5.29±0.75^b		25.86±0.47^a
烃类 Hydrocarbon			0.77%		0.19%
β-罗勒烯 beta-Ocimene	34	脂香、甜味	4.85±0.69^a	1.18	1.65±0.77^b	0.40
醚类 Ethers			5.47%		1.86%
二甲基硫醚 Dimethyl sulfide	300	果香、青香味	34.46±2.78^a	0.95	16.28±0.45^b	0.045
其他类 Others			0.78%		6.89%
未鉴定物质1 Unidentified substance 1	—	—	2.49±0.34^b		7.76±0.33^a
未鉴定物质2 Unidentified substance 2	—	—	2.04±0.04^b		50.80±4.06^a
未鉴定物质3 Unidentified substance 3	—	—	0.41±0.03^b		1.71±0.06^a

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