Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 9: 6241 - 6253

DOI: https://doi.org/10.12418/CJAN2025.506

RESEARCH PAPER

Effects of Fermented Mulberry Leaves on Plasma and Liver Metabonomics of Finishing Pigs

  • CUI Yiyan ,
  • YU Miao ,
  • SONG Min ,
  • DENG Dun ,
  • TIAN Zhimei ,
  • LIU Zhichang , * ,
  • MA Xianyong , *
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  • Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition, Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture and Rural Affairs, State Key Laboratory of Swine and Poultry Breeding Industry, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
* LIU Zhichang, associate professor, E-mail: ;
MA Xianyong, professor, E-mail:

Received date: 2025-01-16

  Online published: 2025-09-12

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Abstract

This experiment aimed to investigate the effects of fermented mulberry leaves (FML) on plasma and liver metabolites of finishing pigs. A total of 18 Duroc×Landrace×Large White barrow pigs (78.19±2.05) kg were randomly assigned to 2 groups with 9 replicates per group and 1 pig per replicate. Pigs in control group were fed a basal diet, and pigs in FML group were fed the basal diet supplemented with 10% FML. The test period was 69 days. The results showed as follows: 1) compared with control group, 27 metabolites were down-regulated and 46 metabolites were up-regulated in plasma of FML group, and mainly enriched in metabolic pathways such as histidine metabolism, β-alanine metabolism, glycine, serine and threonine metabolism, phenylalanine metabolism, and linoleic acid metabolism. 2) Compared with control group, 23 metabolites were down-regulated and 24 metabolites were up-regulated in liver of FML group, and mainly enriched in metabolic pathways such as pantothenic acid and coenzyme A biosynthesis, β-alanine metabolism, nicotinate and nicotinamide metabolism, alanine, aspartate, and glutamate metabolism. 3) There were 10 differential metabolites with the same changes in plasma and liver. N-acetyltryptophan, N-methylisoleucine, 2-hydroxyhippuric acid, tetradecanedioic acid, FA 17∶0, kaempferol, and (R)-equol were upregulated, and D-fructose, 16-hydroxyhexadecanoic acid, and 3-hydroxy-3-methylglutaric acid were down-regulated. In conclusion, dietary supplemented with FML can alter the plasma and liver metabonomics of finishing pigs.

Key words： fermented mulberry leaves; pig; metabolomics analysis; liver

Cite this article

CUI Yiyan , YU Miao , SONG Min , DENG Dun , TIAN Zhimei , LIU Zhichang , MA Xianyong . Effects of Fermented Mulberry Leaves on Plasma and Liver Metabonomics of Finishing Pigs[J]. Chinese Journal of Animal Nutrition, 2025 , 37(9) : 6241 -6253 . DOI: 10.12418/CJAN2025.506

随着人们对动物蛋白质需求的持续增加,如何在提高动物生产力的同时保持养殖业的可持续发展十分重要。有效利用非常规饲料资源、探索廉价合适的替代饲料成分是满足以上需求的关键,同时也为环境友好型养殖业提供了可期待的方向。
桑叶通常富含蛋白质、脂类和碳水化合物,是一种良好的饲料原料资源[1]。研究表明,微生物发酵可降低桑叶的单宁含量,提高桑叶营养价值[2]。发酵桑叶(fermented mulberry leaves,FML)对猪生长性能、胴体性能、肉品质和抗氧化能力没有负面影响,且能改善肠道健康,增加营养物质的表观总消化率,降低粪便臭气物质含量[3-4],可作为潜在的饲料原料。
动物对食物的消化和吸收是一个复杂过程。猪摄入的FML在胃肠道中被消化并吸收,产生大量的代谢物,并为机体提供营养。这些代谢物在肝脏首次代谢后进入机体循环系统,在确保生长的同时,对猪产生不同的生理影响。肝脏是最重要的器官之一,在营养物质代谢以及外源性物质的解毒中起着关键作用[5]。血液和肝脏代谢组学分析可以了解动物的生理状态,评估不同营养素、喂养策略、病理状况的影响,以及可能改变动物代谢概况的许多其他因素[6]。在之前的研究中,饲喂FML会提高血浆和尿液部分代谢物含量[7]。但是,目前尚不清楚FML对肥育猪血浆和肝脏代谢组的影响。因此,本试验采用了非靶向代谢组学方法,以探究饲喂FML是否引起肥育猪血浆和肝脏代谢物的显著差异。

1 材料与方法

1.1 试验材料

桑叶由广东省农业科学院蚕业与农产品加工研究所提供。FML制备如下:将桑叶切短(1~2 cm),桑叶与麸皮按质量比(9∶1)混合均匀,加入酒窖片球菌(Pediococcus cellicola)和地衣芽孢杆菌(Bacillus licheniformis)密封发酵[2]。

1.2 试验动物和试验饲粮

动物试验由广东省农业科学院动物科学研究所实验动物伦理委员会批准,编号为GAASIAS-2021-0909。
选取18头132日龄、体重(78.19±2.05) kg的杜×长×大阉公猪,按体重随机分为2组,每组9个重复,每个重复1头猪。对照组(CON组)饲喂基础饲粮,FML组在基础饲粮中添加10% FML。FML营养成分(以干物质计)为:26.88%粗蛋白质,2.14%粗脂肪,14.65%粗纤维,31.43%中性洗涤纤维,14.95%酸性洗涤纤维,12.87%粗灰分,2.67%钙,0.61%总磷。饲粮组成及营养水平见表1。试验期69 d。试验期间猪自由采食和饮水。
表1 饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of diets (air-dry basis) %

项目Items 对照组
CON group		 发酵桑叶组
FML group
原料Ingredients
玉米Corn 69.42 65.23
豆粕Soybean meal 16.49 11.67
麦麸Wheat bran 8.00 7.30
大豆油Soybean oil 2.60 2.60
L-赖氨酸L-Lys 0.38 0.41
L-蛋氨酸L-Met 0.05 0.12
L-苏氨酸L-Thr 0.06 0.08
L-色氨酸L-Trp 0.01 0.05
磷酸氢钙CaHPO4 0.91 0.93
石粉Limestone 0.78 0.31
食盐NaCl 0.30 0.30
发酵桑叶
Fermented mulberry leaves		 10.00
预混料Premix1) 1.00 1.00
合计Total 100.00 100.00
营养水平Nutrient levels2)
消化能DE/(MJ/kg) 14.21 14.49
粗蛋白质CP 15.21 15.18
粗纤维CF 3.21 4.59
钙Ca 0.65 0.71
总磷TP 0.52 0.55
赖氨酸Lys 1.07 1.14
蛋氨酸+胱氨酸Met+Cys 0.56 0.58
苏氨酸Thr 0.60 0.62
色氨酸Trp 0.18 0.19

1)预混料为每千克饲粮提供 Premix provided the following per kg of diets:VA 6 500 IU,VD3 2 000 IU,VE 150 mg,VK3 3 mg,VB12 0.03 mg,VB1 3 mg,VB2 6 mg,VB6 5 mg,烟酸 nicotinic acid 45 mg,D-泛酸 D-pantothenic acid 9 mg,叶酸 folic acid 1 mg,生物素 biotin 0.3 mg,Fe 72 mg,Cu 10 mg,Mn 42 mg,Zn 72 mg,I 0.42 mg,Se 0.2 mg,Mg 34 mg。

2)消化能为计算值,其余为实测值。DE was a calculated value, while the others were measured values.

1.3 FML和饲粮营养成分测定

粗蛋白质含量参照GB/T 6432—2018中方法测定,粗纤维、中性洗涤纤维、酸性洗涤纤维含量参照Van Soest等[8]的方法测定,粗脂肪含量参照GB/T 6433—2006的方法测定,氨基酸含量参照GB/T 18246—2019的方法测定,钙和总磷含量分别参照GB/T 6436—2018和GB/T 6437—2018的方法测定,粗灰分含量参照GB/T 6438—2007的方法测定。消化能参考《猪营养需要量》(GB/T 39235—2020)计算。

1.4 饲养管理与采样

试验在广东省农业科学院动物科学研究所猪场进行。按照猪场常规程序管理。试验第69天晚上进行12 h禁食,此后每组选取6头肥育猪进行耳静脉采血(肝素钠抗凝管),经离心后收集血浆,-80 ℃保存。屠宰后分离出肝脏并收集10 g肝脏,-80 ℃保存。

1.5 代谢组学分析

血浆和肝脏样品的预处理、LC-MS代谢检测以及数据处理由上海敏心生物科技有限公司完成。

1.6 统计分析

使用多元变量统计分析方法对数据进行分析,包括正交偏最小二乘法-判别分析(OPLS-DA)。差异代谢物的筛选条件为变量投影重要度(VIP)>1且P<0.05。将筛选的差异代谢物对KEGG代谢物数据库进行映射匹配,并进一步分析。最后,将差异倍数(FC)前20的差异代谢物通过SPSS 25.0软件进行Spearman相关性分析。

2 结果与分析

2.1 FML对肥育猪血浆代谢组学的影响

2.1.1 血浆差异代谢物的筛选

如图1-A所示,FML组和CON组血浆代谢物明显区分。由表2可知,2组血浆有73个差异代谢物显著不同(VIP>1和P<0.05)。与CON组相比,FML组血浆中有27个下调差异代谢物和46个上调差异代谢物。其中脂肪酸类代谢物占差异代谢物的41%,上调的有19个(壬二酸、亚油酸、十五烷酸等),下调的有11个(反油酸、柠康酸、3-甲基戊二酸等)。氨基酸类代谢物占差异代谢物的15%,上调的有9个(苯乙酰天冬氨酸、2-羟基苯丙氨酸、D-谷氨酰胺等),下调的有2个(苯乙酰甘氨酸、N-乙酰亮氨酸)。
图1 血浆代谢物OPLS-DA和代谢通路分析图

Histidine metabolism:组氨酸代谢;Riboflavin metabolism:核黄素代谢;Sphingolipid metabolism:鞘脂代谢;Beta-alanine metabolism:β-丙氨酸代谢;Glycine, serine and threonine metabolism:甘氨酸、丝氨酸和苏氨酸代谢;Steroid hormone biosynthesis:类固醇激素生物合成;Phenylalanine metabolism:苯丙氨酸代谢;Linoleic acid metabolism:亚油酸代谢;Caffeine metabolism:咖啡因代谢;Glycerolipid metabolism:甘油脂代谢;Pyruvate metabolism:丙酮酸代谢;Glycolysis/Gluconeogenesis:糖酵解/糖异生。

Fig.1 Analysis diagram of plasma metabolite OPLS-DA and metabolic pathways

A:OPLS-DA图;B:代谢通路图。A: plot of OPLS-DA; B: metabolic pathways.

表2 FML对肥育猪血浆代谢物的影响

Table 2 Effects of FML on plasma metabolites in finishing pigs

序号
No.		 代谢物
Metabolites		 变量投影重要度
VIP		 P值
P-value		 log2(差异倍数)
log2(fold change)
1 山柰酚Kaempferol 2.24 0.006 5.31
2 鞘磷脂Sphingomyelin 2.31 <0.001 3.67
3 十四烷二酸Tetradecanedioic acid 2.39 <0.001 3.32
4 13,14-二氢前列腺素F1α 13,14-dihydro prostaglandin F1α 2.23 0.022 2.73
5 3-异丙基苹果酸3-isopropylmalic acid 2.35 0.006 2.73
6 N-乙酰色氨酸N-acetyltryptophan 2.13 0.011 1.97
7 苯乙酰天冬氨酸Phenylacetylaspartic acid 2.04 0.002 1.89
8 3-乙酸吲哚甲酯Methyl indole-3-acetate 1.93 0.040 1.84
9 庚二酸Pimelic acid 2.38 0.001 1.82
10 2-羟基马尿酸2-hydroxyhippuric acid 1.67 0.029 1.51
11 肾上腺素Epinephrine 1.90 0.019 1.50
12 丙三羧酸Tricarballylic acid 1.96 0.015 1.48
13 2-羟基-4-甲基硫代丁酸2-hydroxy-4-methylthiobutanoic acid 2.14 <0.001 1.48
14 (R)-雌马酚(R)-equol 1.93 0.005 1.28
15 柠苹酸Citramalic acid 1.80 0.017 0.93
16 3-苯基乳酸3-phenyllactic acid 1.58 0.026 0.91
17 L-β-高酪氨酸L-beta-homotyrosine 1.57 0.007 0.91
18 13(S)-HOTrE 1.72 0.025 0.86
19 N-甲基异亮氨酸N-methylisoleucine 1.82 0.010 0.79
20 γ-谷氨酰亮氨酸Gamma-glutamylleucine 1.80 0.006 0.75
21 3,5-二羟基癸酸3,5-dihydroxydecanoic acid 1.69 0.022 0.71
22 雄甾酮Androsterone 1.89 0.003 0.70
23 反式10-十七碳烯酸Trans-10-heptadecenoic acid 1.96 0.007 0.67
24 十七烷酸Heptadecanoic acid 1.65 0.019 0.63
25 壬二酸Azelaic acid 1.69 0.034 0.62
26 2-羟基苯丙氨酸2-hydroxyphenylalanine 1.88 0.039 0.60
27 羟基癸二酸Hydroxysebacic acid 2.08 0.001 0.60
28 L-酪氨酸甲酯L-tyrosine methyl ester 2.41 <0.001 0.59
29 亚油酸Linoleic acid 1.70 0.019 0.54
30 马尿酸Hippuric acid 1.60 0.025 0.53
31 十五烷酸Pentadecanoic acid 1.88 0.010 0.52
32 D-谷氨酰胺D-glutamine 1.54 0.031 0.51
33 棕榈油酸Palmitoleic acid 1.63 0.044 0.42
34 棕榈酸Palmitic acid 1.47 0.044 0.39
35 甜菜碱Betaine 1.44 0.042 0.37
36 脱氧核糖Deoxyribose 1.89 0.006 0.36
37 乳清酸Orotic acid 1.54 0.048 0.35
38 6-羟基己酸6-hydroxycaproic acid 1.58 0.039 0.32
39 1-氨基环丙烷-1-羧酸酯1-aminocyclopropane-1-carboxylate 1.74 0.007 0.32
40 2-异丙基苹果酸2-isopropylmalic acid 1.66 0.025 0.29
41 反乌头酸Trans-aconitic acid 1.62 0.026 0.27
42 癸二酸Sebacic acid 1.70 0.026 0.23
43 9-(2,3-二羟基丙氧基)-9-氧代壬酸
9-(2,3-dihydroxypropoxy)-9-oxononanoic acid		 1.69 0.016 0.20
44 辛二酸Suberic acid 1.56 0.045 0.17
45 2-甲基戊二酸2-methylglutaric acid 1.69 0.022 0.11
46 乙酰丙酸Levulinic acid 1.64 0.021 0.11
47 咪唑乙酸Imidazoleacetic acid 1.62 0.049 -1.67
48 反油酸Elaidic acid 2.06 0.009 -1.40
49 肌肽Carnosine 1.66 0.036 -1.20
50 油酸乙酯Ethyl oleate 1.82 0.006 -0.96
51 16-羟基十六烷酸16-hydroxyhexadecanoic acid 1.47 0.046 -0.88
52 脂肪酸16∶3 Fatty acid 16∶3 1.74 0.020 -0.81
53 3-羟基-3-甲基戊二酸3-hydroxy-3-methylglutaric acid 1.45 0.042 -0.60
54 D-果糖D-fructose 1.82 0.010 -0.50
55 L-三烯酸-1,4-内酯L-threonic acid-1,4-lactone 1.27 0.042 -0.50
56 月桂酸Lauric acid 2.24 <0.001 -0.49
57 核黄素Riboflavin 1.54 0.016 -0.46
58 壬酸Perlargonic acid 2.04 <0.001 -0.39
59 10-羟基癸酸10-hydroxydecanoic acid 1.62 0.036 -0.38
60 甘油酸Glyceric acid 1.89 0.018 -0.35
61 溶血磷脂酸LysoPA(i-12∶0/0∶0) 1.89 0.006 -0.34
62 巴豆酸Crotonic acid 2.05 <0.001 -0.30
63 柠康酸Citraconic acid 2.35 <0.001 -0.25
64 胸腺嘧啶Thymine 1.54 0.027 -0.23
65 丙酮酸Pyruvic acid 1.49 0.050 -0.20
66 苯乙酰甘氨酸Phenylacetylglycine 1.90 0.005 -0.20
67 3-甲基戊二酸3-methylglutaric acid 1.47 0.049 -0.16
68 假尿苷Pseudouridine 1.48 0.044 -0.15
69 N-乙酰亮氨酸N-acetylleucine 1.87 0.005 -0.15
70 间苯三酚Phloroglucinol 1.75 0.012 -0.12
71 副黄嘌呤Paraxanthine 1.73 0.014 -0.12
72 2-氧代丁酸2-oxobutyric acid 1.57 0.030 -0.12
73 乙醇酸Glycolic acid 2.15 <0.001 -0.11

2.1.2 血浆差异代谢物的KEGG通路分析

如图1-B所示,血浆差异代谢物主要富集在组氨酸代谢、β-丙氨酸代谢、甘氨酸、丝氨酸和苏氨酸代谢、苯丙氨酸代谢、亚油酸代谢等通路。

2.2 FML对肥育猪肝脏代谢组学的影响

2.2.1 肝脏差异代谢物的筛选

由图2-A可知,FML组和CON组肝脏代谢物明显区分开。如表3所示,2组肝脏有47个差异代谢物显著不同(VIP>1和P<0.05)。与CON组相比,FML组肝脏中有23个下调差异代谢物和24个上调差异代谢物。其中脂肪酸类(9个)和氨基酸类(8个)代谢物分别占差异代谢物的19%和17%。
图2 肝脏代谢物OPLS-DA和代谢通路分析图

Pantothenate and CoA biosynthesis:泛酸和辅酶A生物合成;Beta-alanine metabolism:β-丙氨酸代谢;Nicotinate and nicotinamide metabolism:烟酸和烟酰胺代谢;Glycerophospholipid metabolism:甘油磷脂代谢;Fatty acid degradation:脂肪酸降解;Amino sugar and nucleotide sugar metabolism:氨基糖和核苷酸糖代谢;Alanine, aspartate and glutamate metabolism:丙氨酸、天冬氨酸和谷氨酸代谢;Purine metabolism:嘌呤代谢;Butanoate metabolism:丁酸代谢;Galactose metabolism:半乳糖代谢。

Fig. 2 Analysis diagram of liver metabolite OPLS-DA and metabolic pathways

A:OPLS-DA图;B:代谢通路图。A: plot of OPLS-DA; B: metabolic pathways.

表3 FML对肥育猪肝脏代谢物的影响

Table 3 Effects of FML on liver metabolites in finishing pigs

序号
No.		 代谢物
Metabolites		 变量投影重要度
VIP		 P值
P-value		 log2(差异倍数)
log2(fold change)
1 山奈酚Kaempferol 2.64 0.004 6.74
2 N-乙酰色氨酸N-acetyltryptophan 2.28 0.030 2.13
3 (R)-雌马酚(R)-equol 1.97 0.032 1.55
4 2-羟基马尿酸2-hydroxyhippuric acid 1.88 0.020 1.36
5 胆钙化醇Cholecalciferol 1.78 0.020 1.27
6 十四烷二酸Tetradecanedioic acid 2.11 0.014 1.22
7 乙酰脯氨酸Acetylproline 1.89 0.008 0.84
8 尿苷5'-二磷酸半乳糖Uridine 5'-diphosphogalactose 1.66 0.028 0.78
9 甘氨鹅脱氧胆酸Glycochenodeoxycholic acid 1.72 0.029 0.67
10 N-乙酰氨基苯甲酸N-acetylanthranilic acid 1.64 0.040 0.64
11 N-甲基异亮氨酸N-methylisoleucine 1.96 0.028 0.62
12 尿嘧啶Uracil 1.66 0.028 0.62
13 溶血磷脂酰乙醇胺(20∶3)LysoPE(20∶3) 1.98 0.007 0.59
14 尿苷Uridine 1.57 0.042 0.55
15 十七烷酸Heptadecanoic acid 1.72 0.017 0.54
16 磷酸二羟丙酮Dihydroxyacetone phosphate 1.45 0.049 0.52
17 肉桂酸Cinnamic acid 1.51 0.037 0.45
18 甘氨鹅脱氧胆酸Glycochenodeoxycholic acid 1.80 0.020 0.42
19 2-甲氧基-4-十五烷基苯甲酸2-methoxy-4-pentadecylbenzoic acid 1.51 0.049 0.41
20 SN-甘油-3-磷酸胆碱SN-glycero-3-phosphocholine 1.59 0.042 0.31
21 辅酶A Coenzyme A 1.90 0.008 0.31
22 尿苷二磷酸-N-乙酰葡糖胺UDP-N-acetylglucosamine 2.02 0.005 0.31
23 巴豆酸Crotonic acid 2.07 0.002 0.29
24 烟酸Nicotinic acid 1.70 0.033 0.15
25 亚精胺Spermidine 2.22 0.027 -2.56
26 1-棕榈酰基-2-(9Z-油酰)-sn-甘油-3-磷酰胆碱
PC(16∶0/18∶1(9Z))		 1.23 0.009 -0.96
27 L-蛋氨酸L-methionine 2.23 0.001 -0.92
28 β-丙氨酸β-alanine 1.50 0.037 -0.92
29 16-羟基十六烷酸16-hydroxyhexadecanoic acid 2.03 0.036 -0.90
30 5-甲基四氢叶酸5-methyltetrahydrofolic acid 1.57 0.037 -0.81
31 D-果糖D-fructose 1.52 0.012 -0.76
32 烟酰胺腺嘌呤二核苷酸Nicotinamide adenine dinucleotide 1.63 0.022 -0.74
33 甘氨酰-L-亮氨酸Glycyl-L-leucine 1.85 0.019 -0.68
34 2-苯乙酰胺2-phenylacetamide 1.68 0.044 -0.66
35 泛酸Pantothenic acid 2.21 0.007 -0.64
36 脯氨酰亮氨酸Prolylleucine 1.75 0.027 -0.60
37 泛酸Pantothenic acid 2.04 0.008 -0.49
38 D-景天庚酮糖7-磷酸D-sedoheptulose 7-phosphate 1.87 0.007 -0.48
39 肌酸Creatine 1.65 0.047 -0.43
40 苯乙酸Phenylacetic acid 1.56 0.050 -0.36
41 单磷酸鸟苷Guanosine monophosphate 1.72 0.033 -0.34
42 3-羟基-3-甲基戊二酸3-hydroxy-3-methylglutaric acid 2.02 0.002 -0.33
43 粪臭素Skatole 1.89 0.008 -0.31
44 4-羟基-2-喹啉甲酸4-hydroxy-2-quinolinecarboxylic acid 2.14 0.002 -0.27
45 L-正亮氨酸L-norleucine 1.52 0.042 -0.23
46 正缬氨酸Norvaline 1.76 0.026 -0.20
47 琥珀酸半醛Succinic semialdehyde 2.01 0.003 -0.10

2.2.2 肝脏差异代谢物的KEGG通路分析

如图2-B所示,肝脏差异代谢物主要富集在泛酸和辅酶A生物合成、β-丙氨酸代谢、烟酸和烟酰胺代谢、甘油磷脂代谢、丙氨酸、天冬氨酸和谷氨酸代谢等通路。

2.3 FML对肥育猪血浆和肝脏相同变化差异代谢物的影响

由表4可知,血浆和肝脏中有7个上调差异代谢物和3个下调差异代谢物具有相同变化。这些代谢物主要是氨基酸类、脂肪酸类、黄酮类物质。
表4 FML对肥育猪血浆和肝脏相同变化差异代谢物的影响

Table 4 Effects of FML on differential metabolites with similar changes in plasma and liver in finishing pigs

代谢物
Metabolites		 FML组
FML group		 对照组
CON group		 变量投影
重要度
VIP		 P值
P-value		 log2(差异倍数)
log2 (fold
change)
血浆Plasma
N-乙酰色氨酸N-acetyltryptophan 4.43×10-5 1.13×10-5 2.13 0.011 1.97
N-甲基异亮氨酸N-methylisoleucine 1.04×10-4 5.98×10-5 1.82 0.010 0.79
2-羟基马尿酸2-hydroxyhippuric acid 2.27×10-4 7.98×10-5 1.67 0.029 1.51
十四烷二酸Tetradecanedioic acid 5.25×10-4 5.27×10-5 2.39 0.000 3.32
十七烷酸Heptadecanoic acid 4.40×10-6 2.84×10-6 1.65 0.019 0.63
山奈酚Kaempferol 1.10×10-5 2.77×10-7 2.24 0.006 5.31
(R)-雌马酚(R)-equol 1.55×10-5 6.39×10-6 1.93 0.005 1.28
D-果糖D-fructose 2.97×10-4 4.21×10-4 1.82 0.010 -0.50
16-羟基十六烷酸16-hydroxyhexadecanoic acid 8.72×10-5 1.60×10-4 1.47 0.046 -0.88
3-羟基-3-甲基戊二酸
3-hydroxy-3-methylglutaric acid		 2.53×10-3 3.83×10-3 1.45 0.042 -0.60
肝脏Liver
N-乙酰色氨酸N-acetyltryptophan 1.81×10-5 4.11×10-6 2.28 0.030 2.13
N-甲基异亮氨酸N-methylisoleucine 2.35×10-5 1.53×10-5 1.96 0.028 0.62
2-羟基马尿酸2-hydroxyhippuric acid 5.11×10-5 1.99×10-5 1.88 0.020 1.36
十四烷二酸Tetradecanedioic acid 1.30×10-5 5.55×10-6 2.11 0.014 1.22
十七烷酸Heptadecanoic acid 5.31×10-6 3.64×10-6 1.72 0.017 0.54
山奈酚Kaempferol 1.51×10-5 1.41×10-7 2.64 0.004 6.74
(R)-雌马酚(R)-equol 1.10×10-5 3.77×10-6 1.97 0.032 1.55
D-果糖D-fructose 9.39×10-4 1.59×10-3 1.52 0.012 -0.76
16-羟基十六烷酸16-hydroxyhexadecanoic acid 1.23×10-5 2.30×10-5 2.03 0.036 -0.90
3-羟基-3-甲基戊二酸
3-hydroxy-3-methylglutaric acid		 1.99×10-3 2.51×10-3 2.02 0.002 -0.33

2.4 肥育猪血浆与肝脏差异代谢物的相关性

由图3可知,血浆中下调差异代谢物(咪唑乙酸、油酸、肌肽、油酸乙酯)含量与肝脏中下调差异代谢物(L-蛋氨酸、16-羟基十六烷酸、甘氨酰-L-亮氨酸等)含量呈显著(P<0.05)或极显著(P<0.01)正相关。血浆中上调差异代谢物(山奈酚、鞘磷脂、雌马酚、十四烷二酸等)含量与肝脏下调差异代谢物(L-蛋氨酸、β-丙氨酸、2-苯乙酰胺、泛酸等)含量呈显著(P<0.05)或极显著(P<0.01)负相关,与肝脏上调差异代谢物(山奈酚、雌马酚、乙酰脯氨酸、甘鹅脱氧胆酸等)含量呈显著(P<0.05)或极显著(P<0.01)正相关。肝脏是主要的代谢器官,其改变会影响血浆代谢物的含量。
图3 血浆与肝脏前20差异代谢物相关性

+表示显著相关(P<0.05),++表示极显著相关(P<0.01)。红色代表正相关,蓝色代表负相关。+ mean significant correlation (P<0.05), and ++ mean extremely significant correlation (P<0.01). Red represent positive correlation, blue represent negative correlation.

Imidazoleacetic acid:咪唑乙酸;Elaidic acid:反油酸;Carnosine:肌肽;Ethyl oleate:油酸乙酯;Kaempferol:山奈酚;Sphingomyelin:鞘磷脂;(R)-equol:(R)-雌马酚;Tetradecanedioic acid:十四烷二酸;13,14-dihydro prostaglandin F1α:13,14-二氢前列腺素F1α;3-isopropylmalic acid:3-异丙基苹果酸;N-acetyltryptophan:N-乙酰色氨酸;Phenylacetylaspartic acid:苯乙酰天冬氨酸;Methyl indole-3-acetate:吲哚-3-乙酸甲酯;Pimelic acid:庚二酸;2-hydroxyhippuric acid:2-羟基马尿酸;Epinephrine:肾上腺素;Tricarballylic acid:丙三羧酸;2-hydroxy-4-methylthiobutanoic acid:2-羟基-4-甲硫基丁酸;Citramalic acid:柠苹酸;Phenyllactic acid:3-苯基乳酸;Spermidine:亚精胺;L-methionine:L-蛋氨酸;β-alanine:β-丙氨酸;Nicotinamide adenine dinucleotide:烟酰胺腺嘌呤二核苷酸;2-phenylacetamide:2-苯乙酰胺;Pantothenic acid:泛酸;16-hydroxyhexadecanoic acid:16-羟基十六烷酸;5-methyltetrahydrofolic acid:5-甲基四氢叶酸;D-fructose:D-果糖;Glycyl-L-leucine:甘氨酰-L-亮氨酸;Cholecalciferol:胆钙化醇;Acetylproline:乙酰脯氨酸;Glycochenodeoxycholic acid:甘鹅脱氧胆酸;Uridine 5'-diphosphogalactose:尿苷5'-二磷酸半乳糖。

Fig.3 Correlation between top 20 differential metabolites in plasma and liver

3 讨论

桑叶在肥育猪应用的添加量较低,4%和5%的桑叶降低猪饲料转化率、胴体重、屠宰率,对肥育猪生产性能产生消极影响[9-10],而本课题组前期试验表明,饲喂10% FML对肥育猪生长性能、胴体性能、肉品质均无显著负面影响[3-4,7]。这与FML的摄入改变了肥育猪机体的代谢有关,血浆和肝脏中代谢物的变化改善了猪生理代谢,促进机体健康。动物的生长是一个复杂代谢网络的结果,涉及脂肪酸、氨基酸的利用和蛋白质合成。如下文所述,饲喂FML的猪血浆和肝脏中氨基酸代谢、脂肪酸代谢相关物质显著改变,山奈酚、雌马酚、黄酮类等活性物质含量显著增加,这些变化侧面反映了饲喂FML具有改善机体蛋白质代谢、抗氧化能力、免疫功能的益处[7],也体现了饲喂FML可以抵消直接饲喂桑叶对肥育猪生长性能的负面影响。

3.1 氨基酸相关代谢

氨基酸代谢通路富集在肝脏和血浆中,可能是由于摄入FML改变了蛋白质合成、降解或与生长直接相关的代谢过程。血浆中肌酐和尿酸含量的降低,表明了FML提高膳食氮的利用率,增加蛋白质沉积或减少蛋白质分解[11]。肌酐是动物肌酸的不可逆非酶代谢产物,肌酸是磷酸肌酸的前体,作为人体的能量储存物质[12],猪补充FML后提供了足够的肌酸满足维持所需的能量,减少了肌酐的产生。
饲喂FML提高马尿酸(血浆)和羟基马尿酸(血浆、肝脏)含量。这些酚酸由苯甲酸、羟基苯甲酸与甘氨酸共轭形成,可能由肠道微生物在结肠中分解代谢多酚类物质产生的[13]。补充FML给机体提供了更多的多酚类物质,正如血浆和肝脏中山奈酚、雌马酚含量的提高。哺乳动物中马尿酸合成主要发生在肝脏中,其生物合成过程需要ATP和辅酶A的参与[12],肝脏中辅酶A水平的提高也可能促进了马尿酸的合成。另外,膳食蛋白质的降解是马尿酸形成的主要来源之一[14]。虽然饲粮配方中粗蛋白质含量一致,但FML蛋白质消化率较高[4],猪摄入的蛋白质总量增加,也可导致血浆马尿酸含量增加。这与其他蛋白质相关代谢物、生化指标的改变是一致的[7]。
在血浆苯丙氨酸代谢途径中,差异代谢产物苯乙基甘氨酸的含量显著降低,苯乙基甘氨酸含量的减少有助于保存组织的生长和减少疾病的损害[15]。甜菜碱具有抗炎和抗氧化作用,可以增强肌肉力量和保护肝脏功能[16]。这些代谢物的改变有益于肥育猪的生长。

3.2 脂肪酸相关代谢

血液中脂肪酸类物质含量增加,如壬二酸、亚油酸、十五烷酸、棕榈油酸、癸二酸、辛二酸等,这些代谢物具有很强的抗炎作用[17-18]。亚油酸和棕榈油酸可改善代谢紊乱,促进抗炎因子分泌,减少疾病的发生[19-20]。此外,癸二酸、壬二酸、辛二酸、庚二酸含量显著增加有益于抗疲劳作用[21]。这些脂肪酸含量的增加以及会导致炎症、氧化应激的反油酸含量减少[22],表明了FML摄入能够改善机体健康。
烟酸和烟酰胺代谢在与碳水化合物、脂肪和蛋白质的代谢等各种过程和整体健康中起着重要作用[23]。烟酸含量的增加可显著促进动物的生长[24]。甘油磷脂可以调节活性氧和内源性抗氧化剂的浓度,改变细胞膜的脂肪酸组成,进而影响炎症[25]。肝脏中辅酶A增加与生长促进和饲料效率提高有关[26]。肝脏中这些物质含量的增加有助于机体生长。

3.3 血浆和肝脏中相同变化的差异代谢物

肝脏代谢物和血浆代谢物反映了机体的生理状态,大部分肝脏和血浆下调代谢物含量与上调代谢物含量呈负相关。考虑到肝脏的中心代谢作用,下文主要讨论了在血浆和肝脏中具有相同变化的差异代谢物。
肝脏和血浆中氨基酸衍生物N -乙酰色氨酸和N-甲基异亮氨酸含量上调。N-乙酰色氨酸是一种潜在的抗氧化剂,防止机体蛋白质的热变性和氧化降解,增强机体对自由基的抵抗[27],还参与多胺和色氨酸代谢[28]。N-乙酰色氨酸含量在肝脏和血浆中的提高,表明饲粮中补充FML可能改善机体抗氧化状态。N-甲基异亮氨酸是异亮氨酸的甲基化产物,由肠道微生物菌群对异亮氨酸的酶促生物转化产生[29]。肠道微生物菌群合成的N-甲基异亮氨酸对饲粮敏感,改变饲粮会改变其含量[30]。氨基酸的甲基化代表了一种通过增强被动扩散和增加代谢稳定性来提高肽生物利用度的方法[31],侧面反映了FML中蛋白质的消化利用率更好[4]。
肝脏和血浆中十四烷二酸和十七烷酸含量上调,16-羟基十六烷酸和3-羟基-3-甲基戊二酸含量下调。这些脂肪酸及其衍生物的改变,说明FML具有潜在改变机体脂肪代谢的趋势。十四烷二酸在生物体的能量代谢、细胞膜构成以及信号传导等过程中发挥着重要作用[32]。十七烷酸是部分过氧化物酶体增殖物激活受体δ配体和激动剂,具有线粒体修复功能,减少体内的促炎状态[33],二者的增加有益于机体健康。3-羟基-3-甲基戊二酸促进脂质和蛋白氧化损伤,降低多种抗氧化酶活性[34]。3-羟基-3-甲基戊二酸含量的减少也表明了对动物健康产生不利影响的代谢物减少了,对于动物健康产生积极作用。16-羟基十六烷酸表现出很强的黄嘌呤氧化酶抑制能力[35]。嘌呤类物质在黄嘌呤氧化酶的催化作用下最终转化为尿酸,抑制了尿酸的生成。这与之前的报道结果一致,饲喂FML显著降低了肥育猪血浆和尿液中的尿酸含量[7,36]。
饲粮中的功能性物质会影响动物的健康和新陈代谢。肝脏中观察到的黄酮类物质含量显著提高,这可能与该器官在外源性物质代谢中的作用有关,这意味着FML中黄酮类化合物具有良好的吸收能力[37]。山奈酚减少机体的脂质氧化和肝脏脂质积累,山奈酚和雌马酚具有抗氧化、抗炎、抗菌、肝保护等活性[38-39]。黄酮类化合物在肝脏中发生代谢转化,也可以被结肠微生物菌群代谢[40]。益生菌有助于将山奈酚糖苷生物转化为苷元来提高山奈酚的功能活性和生物利用度[38]。双歧杆菌菌株、乳酸杆菌和片球菌菌株也能产生雌马酚[39]。FML中片球菌丰度显著高于未发酵的,而饲喂FML猪的肠道核心微生物主要富含双歧杆菌[3]。FML丰富的益生菌有利于机体黄酮类物质的吸收转化。另外,血浆中生物活性黄酮类物质含量的增加与其对机体健康益处直接相关,FML的摄入增加了机体黄酮类物质的摄入[41],促进机体健康。

4 结论

补充FML会改变猪肝脏和血浆代谢物。血浆和肝脏中分别筛选出73和47个差异代谢物。其中,肝脏和血浆中山奈酚、(R)-雌马酚、N-乙酰色氨酸、N-甲基异亮氨酸、2-羟基马尿酸、十四烷二酸、十七烷酸的含量显著增加。这些代谢物对猪的健康有显著益处,而那些可能对动物健康不利的代谢物则减少了。

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