丁酸梭菌对肉鸡肠道短链脂肪酸含量和菌群多样性的影响

doi:10.16431/j.cnki.1671-7236.2022.11.014

摘要/Abstract

摘要： 【目的】探究丁酸梭菌在肉鸡消化道内的定植能力以及丁酸梭菌对肉鸡肠道短链脂肪酸含量和菌群多样性的影响。【方法】选择90只健康状况良好的1日龄爱拔益加肉鸡，随机均分为空白组和丁酸梭菌组，每组3个重复，每个重复15只鸡。空白组灌喂生理盐水，丁酸梭菌组灌服荧光标记的丁酸梭菌，3 h及16日龄进行解剖并采集回肠上皮细胞观察荧光情况。随后另外选择30只健康状况良好的1日龄雏鸡，随机分为对照组和试验组，试验组1~6 d饲喂含丁酸梭菌的饲粮，之后饲喂基础饲粮，对照组全程饲喂基础饲粮，试验期21 d。在16和21日龄时，分别采集回肠和盲肠中段内容物，测定乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸、己酸和总短链脂肪酸含量，并进行微生物种类、物种丰度比例、菌属以及蛋白功能丰度等差异分析。【结果】 3 h及16日龄丁酸梭菌组回肠上皮细胞周围均呈现出荧光现象，表明丁酸梭菌能够定植于消化道上皮细胞。短链脂肪酸含量测定结果显示，与对照组相比，在肉鸡回肠内容物中，试验组16日龄乙酸、丙酸、异丁酸、异戊酸和戊酸含量均极显著增加（P<0.01）；21日龄丙酸、异戊酸和戊酸含量均极显著增加（P<0.01），乙酸、异丁酸和总短链脂肪酸含量均显著增加（P<0.05）；在盲肠内容物中，试验组16日龄肉鸡丁酸和戊酸含量均极显著增加（P<0.01），异戊酸和己酸含量显著增加（P<0.05）；21日龄丁酸和异戊酸均极显著增加（P<0.01），乙酸、异丁酸、己酸和总短链脂肪酸均显著增加（P<0.05）。门水平上微生物种类分析发现，16日龄样本中厚壁菌门为优势菌群，丰度最高；21日龄样本中微生物种类增多，其中变形菌门、厚壁菌门和拟杆菌门为优势菌群，丰度较高，16和21日龄试验组的优势菌群均高于对照组。物种丰度比例分析结果显示，与对照组相比，16日龄试验组厌氧菌属的物种丰度比例显著增加（P<0.05），而去氟球菌属、丛毛单胞菌属、肠杆菌属等7种菌属的物种丰度比例显著降低（P<0.05）；21日龄试验组铁杆菌属、毛球菌属等6种菌属的物种丰度比例显著增加（P<0.05），变形菌属、浮霉菌属、装甲菌门GP5的物种丰度属显著降低（P<0.05）。菌属差异分析结果显示，16日龄试验组起重要作用的微生物是布劳特氏菌属，对照组起重要作用的微生物是梭状芽孢杆菌属和未分类毛螺旋菌属；21日龄试验组起重要作用的微生物是未分类拟杆菌、毛球菌属、乳酸杆菌目、乳球菌属等，对照组起重要作用的微生物是诺兰克酸杆菌GP4、丛毛单胞菌科等。菌群蛋白功能丰度分析结果显示，16日龄试验组甲基受体趋化性蛋白、谷胱甘肽S-转移酶、氨基酸转移酶亚基A的丰度均高于对照组，21日龄试验组ECF亚家族RNA聚合酶δ-70因子、丙氨酸的丰度均高于对照组。【结论】丁酸梭菌可以在肉鸡回肠上皮细胞中定植；丁酸梭菌可增加肠道内容物中短链脂肪酸含量、微生物种类及有益菌属，提高物种丰度及功能蛋白丰度，从而促进肠道功能发挥。

关键词: 丁酸梭菌; 定植; 肉鸡; 短链脂肪酸; 肠道菌群

Abstract: 【Objective】 The aim of this study was to investigate the colonization ability of Clostridium butyricum in the digestive tract of broilers and the effects of Clostridium butyricum colonization on intestinal short-chain fatty acid content and microflora diversity of broilers.【Method】 90 one-day-old Abaric broilers with good health were randomly divided into blank group and Clostridium butyricum group with 3 replicates per group and 15 broilers per replicate. The blank group was fed with normal saline, while the Clostridium butyrate group was given fluorescently labeled Clostridium butyrate. At 3 h and 16 days of age, ileum epithelial cells were dissected and collected to observe the fluorescence. Then 30 one-day-old chicks with good health were randomly divided into control and experimental groups. The experimental group was fed the diet containing Clostridium butyricum from 1 to 6 days, followed by the basal diet, while the control group was fed the basal diet throughout the whole period, the period was 21 days. At 16 and 21 days of age, the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid and total short-chain fatty acid in ileum and cecum were determined. The difference analysis of microbial species, species abundance ratio, bacterial species and protein functional abundance were carried out.【Result】 There was fluorescence around the ileum epithelial cells in Clostridium butyricum group at 3 h and 16 days of age, indicating that Clostridium butyricum could colonize gastrointestinal ileum epithelial cells. The results of short-chain fatty acid content determination showed that compared with control group, the contents of acetic acid, propionic acid, isobutyric acid, iso-valeric acid and valeric acid in ileum of broilers at 16 days of age in experimental group were extremely significantly increased (P<0.01). The contents of propionic acid, iso-valeric acid and valeric acid in ileum of broilers at 21 days of age were extremely significantly increased (P<0.01), while the contents of acetic acid、isobutyric acid and total short-chain fatty acid were significantly increased (P<0.05). The contents of butyric acid and valeric acid in cecal of broilers at 16 days of age were extremely significantly increased (P<0.01), while the contents of iso-valeric acid and caproic acid were significantly increased (P<0.05). Compared with control group, the contents of butyric acid and isovaleric acid in cecum of broilers at 21 days of age were extremely significantly increased (P<0.01), while the contents of acetic acid, isobutyric acid, caproic acid and total short-chain fatty acid were significantly increased (P<0.05). The results of microbial species difference analysis at phylum level showed that Firmicutes at 16 days of age were the dominant flora with the highest abundance, and the number of microbial species was increased at 21 days of age samples, and Proteobacteria, Firmicutes and Bacteroidetes were the dominant bacterial groups with high abundance, and the dominant bacteria at 16 and 21 days of age experimental groups were higher than those in control group. The results of species abundance ratio analysis showed that compared with control group, the species abundance ratio of Anaerolineaceae at 16 days of age experimental group was significantly increased (P<0.05), while the proportions of 7 genera, such as Defluviicoccus, Comamonas, Enterobacter were significantly decreased (P<0.05). At 21 days of age in experimental group, the species abundance ratio of 6 genera, such as Ferribacterium, Trichococcus were increased significantly (P<0.05), while Proteobacteria, Plancyomycetes, and Armatimonadetes GP5 were significantly decreased (P<0.05). The results of genus difference analysis showed that the most important microorganisms in experimental group at 16 days of age were Blautia, while the most important microorganisms in control group were Clostridium and unclassified Lachnospiraceae. At 21 days of age, the most important microorganisms in experimental group were unclassified Bacteroidetes, Trichococcus, Lactobacillales and Lactococcus, while the most important microorganisms in control group were norank Acidobacteria GP4 and unclassified Comamonadaceae. The results of functional abundance analysis of flora proteins showed that the abundance of methyl-accepting chemotaxis protein, glutathione S-transferase and amino acidotransferase subunit A at 16 days of age experimental group were higher than those in control group, and the abundance of ECF subfamily RNA polymerases δ-70 factor and alanine at 21 days of age experimental group were higher than those in control group.【Conclusion】 Clostridium butyricum could colonize the ileum epithelial cells of broilers. Clostridium butyricum could increase the content of short-chain fatty acids, microbial species and beneficial bacteria genera in intestinal contents, and improve the abundance of species and functional proteins, so as to promote intestinal function.

Key words: Clostridium butyricum; colonization; broilers; short chain fatty acids; intestinal microflora

中图分类号:

S816.7

单春乔, 翟宏旭, 李娟, 王岩, 于洪敏, 吴怡琦, 刘恩, 刘艳. 丁酸梭菌对肉鸡肠道短链脂肪酸含量和菌群多样性的影响[J]. 中国畜牧兽医, 2022, 49(11): 4239-4251.

SHAN Chunqiao, ZHAI Hongxu, LI Juan, WANG Yan, YU Hongmin, WU Yiqi, LIU En, LIU Yan. Effects of Clostridium butyricum on Intestinal Short-chain Fatty Acid Content and Microflora Diversity of Broilers[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4239-4251.

参考文献

[1] 李修宇, 沈水宝, 韦晓芳, 等.添加丁酸梭菌和复合益生菌对1~35日龄黎村黄鸡肠道形态和菌群的影响[J].饲料研究, 2021, 23:35-40. LI X Y, SHEN S B, WEI X F, et al.Effect of adding Clostridium butyricum and compound probiotics on the intestinal morphology and flora of Licun Yellow chickens from 1-35 d of age[J].Feed Research, 2021, 23:35-40.(in Chinese)
[2] 吴丽媛, 张守明.丁酸梭菌创制技术及在动物养殖中的应用[J].畜牧兽医科技信息, 2021, 12:166-167. WU L Y, ZHANG S M.Production technology of Clostridium butyricum and its application in animal breeding[J].Chinese Journal of Animal Husbandry and Veterinary Medicine, 2021, 12:166-167.(in Chinese)
[3] 简宗辉, 孙帅, 豆腾飞, 等.益生菌在养鸡生产中应用的研究进展[J].畜牧与兽医, 2021, 53(11):129-134. JIAN Z H, SUN S, DOU T F, et al.Advances in the application of probiotics in chicken production[J].Animal Husbandry & Veterinary Medicine, 2021, 53(11):129-134.(in Chinese)
[4] 黄秋连, 王立超, 周昕, 等.丁酸梭菌的来源、功能及其在畜禽生产中的应用[J].黑龙江八一农垦大学学报, 2021, 33(5):53-58. HUANG Q L, WANG L C, ZHOU X, et al.Origin, function of Clostridium butyrate and application in livestock and poultry production[J].Journal of Heilongjiang Bayi Agricultural University, 2021, 33(5):53-58.(in Chinese)
[5] 申文雪, 刘金松, 范小燕, 等.丁酸梭菌替代金霉素对肉鸡腿肌肉品质和抗氧化功能的影响[J].微生物学报, 2021, 62(4):1464-1472. SHEN W X, LIU J S, FAN X Y, et al.Effect of Clostridium butyricum as a replacement of chlortetracycline on quality and antioxidation of broiler leg muscle[J].Acta Microbiologica Sinica, 2021, 62(4):1464-1472.(in Chinese)
[6] 张茜, 潘翠, 张钊, 等.丁酸梭菌联合竹醋液对产蛋后期蛋鸡蛋品质及肠道功能的影响[J].饲料研究, 2021, 44(13):57-60. ZHANG Q, PAN C, ZHANG Z, et al.Effect of Clostridium butyricum combined with bamboo vinegar on egg quality and intestinal function of laying hens in later laying period[J].Feed Research, 2021, 44(13):57-60.(in Chinese)
[7] 张自杰, 芦春莲, 李尚, 等.丁酸梭菌在畜牧业中的研究进展[J].饲料研究, 2021, 44(17):101-103. ZHANG Z J, LU C L, LI S, et al.Research progress of Clostridium butyricum in animal husbandry[J].Feed Research, 2021, 44(17):101-103.(in Chinese)
[8] 何菊, 李筱雯, 崔卫涛, 等.丁酸梭菌CB1制剂对肉鸡生产性能、肠道形态和菌群的影响[J].湖北农业科学, 2021, 60(17):93-97. HE J, LI X W, CUI W T, et al.Effects of Clostridium butyricum CB1 preparations on production performance, intestinal morphology and microflora of broilers[J].Hubei Agricultural Sciences, 2021, 60(17):93-97.(in Chinese)
[9] 易宏波, 唐青松, 侯磊, 等.断奶仔猪肠道健康粉级及其无抗营养策略[J].动物营养学报, 2020, 32(10):4501-4517. YI H B, TANG Q S, HOU L, et al.Intestinal healthy powder levels and anti-nutrition strategies of weaned piglets[J].Chinese Journal of Animal Nutrition, 2020, 32(10):4501-4517.(in Chinese)
[10] 罗贤淑, 孔青.丁酸梭菌在大鼠肠道中的定植及其对肠道菌群的影响[A].中国食品科学技术学会第十八届年会摘要集[C].2022. LUO X S, KONG Q.Colonization of Clostridium butyricum in rat intestine and its effect on intestinal flora[A]. Abstract collection of the 18th Annual Meeting of Chinese Institute of Food Science and Technology[C].2022.(in Chinese)
[11] 郑雨星.鼠李糖乳杆菌和罗伊氏乳杆菌对DSS诱导的小鼠结肠炎的缓解作用及机制分析[D].无锡:江南大学, 2020. ZHENG Y X.Alleviating effects and mechanism analysis of Lactobacillus rhamnosus and Lactobacillus reuteri on DSS induced colitis in mice[D].Wuxi:Jiangnan University, 2020.(in Chinese)
[12] 方志锋.双歧杆菌缓解特应性皮炎的作用及机制研究[D].无锡:江南大学, 2020. FANG Z F.Study on the effect and mechanism of Bifidobacterium in alleviating atopic dermatitis[D].Wuxi:Jiangnan University, 2020.(in Chinese)
[13] 王淑媛, 宋慧.鸡肠道菌群和短链脂肪酸相互关系的研究进展[J].中国微生态学杂志, 2018, 30(7):849-852. WANG S Y, SONG H.Interactions between intestinal microflora and short chain fatty acids in chicken:Research progress[J].Chinese Journal of Microecology, 2018, 30(7):849-852.(in Chinese)
[14] 舒均兰, 徐娥, 桂国弘, 等.丁酸梭菌早期干预对大肠杆菌攻毒肉仔鸡肠道菌群和短链脂肪酸的影响[J].饲料工业, 2021, 42(11):44-52. SHU J L, XU E, GUI G H, et al.Effects of early Clostridium butyricum intervention on short-chain fatty acid and microbial community structure in broiler chickens challenged with Enterotoxigenic Escherichia coli[J].Feed Industry, 2021, 42(11):44-52.(in Chinese)
[15] CISEK A A, BINEK M.Chicken intestinal microbiota function with a special emphasis on the role of probiotic bacteria[J].Polish Journal of Veterinary Sciences, 2014, 17(2):385-394.
[16] ZHANG L, CAO G T, ZENG X F, et al.Effects of Clostridium butyricum on growth performance, immune function, and cecal microflora in broiler chickens challenged with Escherichia coli K88[J].Poultry Science, 2014, 93(1):46-53.
[17] YANG C M, CAO G T, FERKET P R, et al.Effects of probiotic, Clostridium butyricum, on growth performance, immune function, and cecal microflora in broiler chickens[J].Poultry Science, 2012, 91(9):2121-2129.
[18] MERVAT A A, MOHAMED E A, SWELUM A, et al.Single and combined effects of Clostridium butyricum and Saccharomyces cerevisiae on growth indices, intestinal health, and immunity of broilers[J].Animals, 2018, 8(10):184.
[19] PRABHURA J C, CHANDRAKANTH R K.Probiotic potential of Lactobcilli with antagonistic activity against pathogenic strains:An in vitro validation for the production of inhibitory substances[J].Biomedical Journal, 2017, 40(5):270-283.
[20] MU Q, TAVELLA V J, LUO X M.Role of Lactobacillus reuteri in human health and diseases[J].Frontiers in Microbiology, 2018, 9:757.
[21] WU G.Amino acids:Metabolism, functions, and nutrition[J].Amino Acids, 2009, 37(1):1-17.
[22] PONCET N, TAYLOR P M.The role of amino acid transporters in nutrition[J].Current Opinion in Clinical Nutrition and Metabolic Care, 2013, 16(1):57-65.