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Volume 45 Issue 10
Oct.  2021
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Effects of dietary Bacillus subtilis HGcc-1 on gut and liver health, serum complement and gut microbiota of common carp fingerlings (Cyprinus carpio)

  • In order to study the effects of Bacillus subtilis HGcc-1 on the gut and liver health, serum complement and gut microbiota of Cyprinus carpio fingerlings, healthy C. carpio fingerlings with a body weight of (13.10±0.39) g were selected, and randomly divided into HGcc-1 supplementation group and control group, and after 20 weeks of feeding, the growth indicators were measured, and the C. carpio serum endotoxin (LPS), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), total complement and lysozyme were detected, and 16S rRNA sequencing of the gut microbiota was made. At the same time, the gut microbiota induced by HGcc-1 supplement group and the control group were transferred to germ free Danio rerio, and then the levels of endotoxin binding protein (LBP), ALT and AST and the expression levels of C3 and C4 genes of germ free D. rerio were measured; finally, the levels of ALT and AST and the expression levels of C3 and C4 genes in germ free D. rerio were tested when HGcc-1 directly acted on germ free D. rerio. The results showed that HGcc-1 had no effect on the weight gain rate of C. carpio, but adding HGcc-1 significantly reduced the level of serum endotoxin, ALT and AST. The serum total complement level of the HGcc-1 supplement group was significantly increased, compared with control group. The 16S rRNA sequencing results showed that at the phylum level, the HGcc-1 supplement diet group increased the abundance of Fusobacterium by 47.1% compared to control group; the abundance of Proteobacteria was decreased by 70.7% compared with the control group; at the genus level, the HGcc-1 supplement diet group increased the abundance of Cetobacterium by 47.1% compared with the control group; the abundances of Citrobacter and Aeromonas were reduced by 56.6% and 70.9% respectively, compared with the control group. And it was further observed that the intestinal microbiota of HGcc-1 addition group decreased the content of endotoxin binding protein (LBP) and level of AST of the germ free (GF) D. rerio, and significantly increased the gene expression of complement component 3 and the complement component 4 (C3 and C4) of the GF D. rerio. At the same time, the direct interaction between HGcc-1 and GF D. rerio also decreased the ALT and AST levels of the GF D. rerio. This study shows that dietary B. subtilis HGcc-1 can improve the gut and liver health, serum complement and intestinal microbiota homeostasis of C. carpio fingerlings. This study laid a theoretical foundation for the further application of B. subtilis HGcc-1.
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Effects of dietary Bacillus subtilis HGcc-1 on gut and liver health, serum complement and gut microbiota of common carp fingerlings (Cyprinus carpio)

    Corresponding author: YANG Yalin, yangyalin@caas.cn
    Corresponding author: ZHANG Zhen, zhangzhen@caas.cn
  • 1. China-Norway Joint Lab on Fish Gastrointestinal Microbiota, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • 2. Key Laboratory for Feed Biotechnology of Ministry of Agriculture and Rural Affairs, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Abstract: In order to study the effects of Bacillus subtilis HGcc-1 on the gut and liver health, serum complement and gut microbiota of Cyprinus carpio fingerlings, healthy C. carpio fingerlings with a body weight of (13.10±0.39) g were selected, and randomly divided into HGcc-1 supplementation group and control group, and after 20 weeks of feeding, the growth indicators were measured, and the C. carpio serum endotoxin (LPS), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), total complement and lysozyme were detected, and 16S rRNA sequencing of the gut microbiota was made. At the same time, the gut microbiota induced by HGcc-1 supplement group and the control group were transferred to germ free Danio rerio, and then the levels of endotoxin binding protein (LBP), ALT and AST and the expression levels of C3 and C4 genes of germ free D. rerio were measured; finally, the levels of ALT and AST and the expression levels of C3 and C4 genes in germ free D. rerio were tested when HGcc-1 directly acted on germ free D. rerio. The results showed that HGcc-1 had no effect on the weight gain rate of C. carpio, but adding HGcc-1 significantly reduced the level of serum endotoxin, ALT and AST. The serum total complement level of the HGcc-1 supplement group was significantly increased, compared with control group. The 16S rRNA sequencing results showed that at the phylum level, the HGcc-1 supplement diet group increased the abundance of Fusobacterium by 47.1% compared to control group; the abundance of Proteobacteria was decreased by 70.7% compared with the control group; at the genus level, the HGcc-1 supplement diet group increased the abundance of Cetobacterium by 47.1% compared with the control group; the abundances of Citrobacter and Aeromonas were reduced by 56.6% and 70.9% respectively, compared with the control group. And it was further observed that the intestinal microbiota of HGcc-1 addition group decreased the content of endotoxin binding protein (LBP) and level of AST of the germ free (GF) D. rerio, and significantly increased the gene expression of complement component 3 and the complement component 4 (C3 and C4) of the GF D. rerio. At the same time, the direct interaction between HGcc-1 and GF D. rerio also decreased the ALT and AST levels of the GF D. rerio. This study shows that dietary B. subtilis HGcc-1 can improve the gut and liver health, serum complement and intestinal microbiota homeostasis of C. carpio fingerlings. This study laid a theoretical foundation for the further application of B. subtilis HGcc-1.

  • 鲤(Cyprinus carpio)是主要的淡水养殖有鳍鱼类之一,占世界有鳍鱼类总产量的8%[1]。2019年,我国鲤总产量已超过200万t[2]。为了满足市场需求,规模化和集约化养殖模式发展迅速,但同时也存在许多问题需要解决,比如一些植物蛋白代替鱼粉不仅会降低鱼的生长性能,而且导致血清谷丙转氨酶(ALT)和谷草转氨酶(AST)活性水平显著升高[3],造成鱼体肝脏损伤[4]。不合理的养殖方式和饲料配方结构也会导致水产动物肠道菌群失调[5-6],肠道菌群在营养代谢和维持健康过程中发挥着重要作用[7],肠道菌群的紊乱可导致宿主由革兰氏阴性细菌产生的内毒素(LPS)含量显著增加,并且LPS的脂质A成分可能过度刺激巨噬细胞和内皮细胞产生促炎因子[8]。简而言之,不当的养殖方法和饲料会使养殖鱼类更易受到环境中病原菌的侵害[9-11],最终导致产量下降。

    饲料中补充益生菌可能是解决上述问题的方法之一[12],枯草芽孢杆菌 (Bacillus subtilis) 是水产养殖中极具潜力的益生菌菌种,已经广泛应用在水产养殖中,并且有相关研究证明枯草芽孢杆菌作为饲料添加剂时能够提高水产动物的生长性能、抗病能力、免疫功能和抗氧化能力,同时还具有改善肠道菌群、调节脂肪代谢等功能[13-16]。但是,对于肠道菌群与宿主健康的联系关系方面的研究还较少,对其进行深入研究有助于我们对其益生功能的全面理解,从而使其更好地服务于水产养殖。无菌斑马鱼(Danio rerio)模型是研究肠道微生物与鱼类宿主互作的重要模型[17]。一些研究已经使用无菌斑马鱼模型来研究饲料中添加益生菌或益生元诱导的肠道菌群对宿主生长性能、免疫能力和抗病性的影响[18-19]。本研究结合无菌斑马鱼模型探讨了饲料中添加枯草芽孢杆菌HGcc-1对鲤肠肝健康、血清补体以及肠道菌群的影响,为鲤可持续养殖提供指导。

    • 枯草芽孢杆菌HGcc-1由本实验室前期保存(分离自罗非鱼肠道),使用固体发酵:40%麸皮,60%豆粕,加水使其含水量为60%,浸泡2 h,水中添加6%葡萄糖、4%酵母粉和0.1%硫酸锰,调节pH值为7.5,121 °C、高压蒸汽灭菌20 min。将种子菌液接种于固体发酵培养基,接种量5%(体积百分数),30 °C培养96 h。发酵物中HGcc-1含量大于等于1×1010 CFU/g。

      制备了基础饲料和添加HGcc-1的饲料,基础饲料的成分如表1所示,根据前期预实验,添加量为0.3 g/kg的组比添加量为0.2 g/kg的增重率更高,所以选择添加剂量为0.3 g/kg进行正式实验。

      项目
      items
      HGcc-1组
      HGcc-1 group
      对照组
      control
      成分/(g/kg) ingredient
      米糠 rice bran 100 100
      面粉 wheat flour 200 200
      豆粕 soybean meal 200 200
      菜籽粕 rapeseed meal 130 130
      国产鱼粉 domestic fish meal 80 80
      鸡肉粉 Chicken powder 80 80
      猪肉粉 pork powder 40 40
      酒糟 DDGS 100 100
      枯草芽孢杆菌 HGcc-1 0.3 0
      膨润土 bentonite 4.7 5.0
      赖氨酸盐酸盐 lysine 2 2
      蛋氨酸 methionine 0.5 0.5
      氯化胆碱(50%) choline chloride 2 2
      磷酸二氢钙 monocalcium phosphate 20 20
      豆油 bean oil 30 30
      Vc磷酸酯 Vc lecithin 0.5 0.5
      1%鱼预混料 1% fish premix feed[18-19] 10 10
      总和 total 1 000 1 000
      主要营养成分(干物质) main nutrients
      粗蛋白质/% crude protein 36.00 36.63
      粗脂肪/% crude lipid 8.38 9.12
      水分/% moisture 7.07 6.54
      灰分/% ash 8.29 8.41

      Table 1.  Feed ingredient composition and chemical composition

    • 实验鲤购自河北省唐山市鲤鱼种场,并置于90 L的水箱循环系统中。经暂养超过14 d以上,选择体质量为(13.10±0.39) g健康鲤,随机分为HGcc-1组和对照组。HGcc-1组以HGcc-1添加饲料投喂,而对照组以基础饲料投喂,投喂持续20周。每组随机分配3个重复缸,第1周的喂食量为体质量的3%,之后每周在原来的基础上增加2%,每天投喂3次(早上8:00,中午12:00,下午18:00)。在饲养期间,水温平均26.5 °C、溶解氧含量>6.0 mg O/L、pH 7.0~7.2及氨氮含量<0.02 mg/L。养殖实验在中国农业科学院国际农业高新技术产业园中国-挪鱼类消化道微生物联合实验室饲料与养殖评价基地进行。

    • 最后一次喂食结束后,将鱼禁食24 h,测定其体质量,生长指标计算方法:

      存活率(survival rate, SR, %)=Nt/N0×100%

      增重率(weight gain rate, WGR, %)=(WtW0)/W0×100%

      饲料系数(feed conversion ratio, FCR)=Wf /(WtW0)

      日摄食率(daily feeding rate, DFR, %)=Wf /[t×(Wt+W0)/2]

      式中,Nt为终末尾数,N0为初始尾数,Wt为终末质量(g),W0为初始重量(g),Wf为总摄食量,t为实验天数(d)。

    • 对禁食24 h的鱼进行尾静脉取血,取出的血在4 °C下放置10 min,然后4 000 r/min离心10 min,收集上层血清。根据检测试剂盒(南京建成生物科技有限公司)的操作说明检测LPS、ALT、AST、总补体及溶菌酶。

    • 通过16S rRNA测序评估肠道菌群结构,在最后一次进食后4 h取样肠道内容物(n=6),16S rRNA测序在上海美吉生物医药科技有限公司(中国)通过Illumina MiSeq PE300平台完成的,所用引物为338F (5′-ACTCCTACGGGAGGCAGCAG-3′)和806R (5′ -GGACTACHVGGGTWTCTAAT-3′)。使用fastp软件对原始测序序列进行质控,使用FLASH软件进行拼接。使用UPARSE根据97%的相似度对序列进行OTU聚类。利用RDP classifier对每条序列进行物种分类注释,比对Silva 16S rRNA数据库(v138)[18-19]

    • 无菌斑马鱼的准备按照Rawls等[17]和Guo等[20]的操作流程进行。菌群转接:最后一次投喂结束4 h后,取对照组和HGcc-1添加组鲤的肠道内容物,每3尾鱼作为一个样本,磷酸盐缓冲溶液 (PBS) 溶解,均按照106 CFU/mL浓度转接无菌斑马鱼(n=6),并于28 °C培养72 h后取样。单菌直接作用:将活化的HGcc-1菌液加到含有30尾无菌斑马鱼(受精后第4天)的T25细胞培养瓶中,使其终浓度为106 CFU/mL,对照组不加菌,在28 °C处理48 h后取样。

    • 从每个T25细胞培养瓶中收集15尾无菌斑马鱼作为一个样,加入400 μL生理盐水,在冰浴中匀浆,以12 000 r/min离心10 min,收集上清液,然后根据内毒素 (LPS)、体内毒素结合蛋白 (LBP)、ALT及AST试剂盒(南京建成生物技术有限公司)的说明进行测定。

    • 用TRIzol试剂(江苏康为世纪生物技术有限公司)提取无菌斑马鱼全鱼RNA,使用FastKing一步法去除基因组cDNA第一链合成预混试剂[天根生化科技(北京)有限公司]提取第一链互补DNA(cDNA)。使用SYBR Green Supermix[天根生化科技(北京)有限公司]在LightCycler 480(Roche)上进行荧光定量PCR(qPCR引)反应,引物列于表2

      基因
      gene
      正向引物 (5′-3′)
      forward primer (5′-3′)
      反向引物 (5′-3′)
      reverse primer (5′-3′)
      C3 ATGAAGGTGGAGAAGACGGTG CTCACTCGGCAACTGGGAC
      C4 CTGTTGGAGGAGGAGAGGATTC GGTGCTCTCCTGACACGATTG
      β-actin GAAGTGTGGTGTGGACATCCGTAA AGACTCATCGTACTCCTGCTTGCT

      Table 2.  Primer sequences of qPCR

    • 所有统计数据均以均值±标准误(mean±SE)表示。所有结果作图均在GraphPad Prism Version 8软件中进行。T检验用于比较2组数据之间的差异。差异显著水平为P<0.05,考虑到巨大个体差异,针对肠道菌群丰度差异显著水平设为P<0.2;文中 *表示P < 0.05,**表示 P < 0.01,***表示P < 0.001,****表示P<0.0001。

    2.   结果
    • 20周养殖实验结束后,统计了鲤的存活率、增重率、饲料系数和日摄食率。结果显示,添加组和对照组的增重率和饲料系数没有显著差异,同时HGcc-1对存活率没有影响(表3)。

      组别
      groups
      存活率/%
      survival rate
      增重率/%
      weight gain rate
      饲料系数
      feed conversion ratio
      日摄食率/(%/d)
      daily feeding rate
      HGcc-1100954.31±38.701.36±0.041.61±0.04
      CK  100992.54±48.611.34±0.051.59±0.05
      注:值代表6次重复的平均值(mean±SE),下同
      Notes: Data represents the means (mean± SE) of six replicates of each treatment; the same below

      Table 3.  Growth performance and feed utilization of C. carpio

    • HGcc-1添加组的LPS、ALT、AST分别为(0.05±0.01) EU/mL、(10.25±0.93) U/L和(28.97±2.10) U/L,对照组的LPS、ALT、AST分别为(0.16±0.04) EU/mL、(12.60±1.59) U/L和(72.52±20.73) U/L;与对照组相比,HGcc-1添加组显著降低了鲤血清中LBP、ALT和AST水平(P<0.05)(图1)。

      Figure 1.  The effect of HGcc-1 on C. carpio serum LPS (a), ALT (b) and AST (c)

    • 与对照组相比,HGcc-1添加极显著提高了鱼体血清总补体水平(P<0.01),使总补体增加了0.51 IU/mL (图2-a)。对照组和HGcc-1添加组溶菌酶含量分别为(5.45±2.34) μg/mL和(3.09±2.92) μg/mL (图2-b),但没有显著性差异。

      Figure 2.  The effect of HGcc-1 on total complement (a) and lysozyme (b) of C. carpio serum

    • 16S rRNA测序结果显示HGcc-1添加显著改变了鲤肠道菌群结构。在门水平,梭杆菌门(Fusobacteriota)、变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)在2个组中都为优势菌群。HGcc-1添加组中梭杆菌门的丰度比对照组增加了47.1% (P<0.20);变形菌门丰度比对照组减少了70.7% (图3表4P<0.20)。在属水平,鲸杆菌属(Cetobacterium)、柠檬酸杆菌属(Citrobacter)和拟杆菌属(Bacteroides)在2个组中都为优势菌群,HGcc-1添加组中鲸杆菌属的丰度比对照组增加了47.1% (P<0.20);柠檬酸杆菌和气单胞菌属(Aeromonas)的丰度比对照组分别减少了56.6%和70.9% (图4表5P<0.20),拟杆菌属的丰度在2个组之间没有显著差异(图4)。后续对肠道菌群组成进行了PCA分析,结果显示2组肠道菌群组成有显著差异(图5)。

      Figure 3.  The relative abundance of C. carpio gut microbiota at the phylum level

      细菌门
      bacterial phylum
      对照组/%
      control
      HGcc-1添加组/%
      HGcc-1 diet
      梭杆菌门 Fusobacteriota 58.6±29.7 86.2±13.6*
      变形菌门 Proteobacteria 29.7±25.0 8.7±6.6*
      拟杆菌门 Bacteroidetes 6.5±4.8 3.6±6.9
      放线菌门 Actinobacteriota 1.8±2.1 1.0±1.3
      厚壁菌门 Firmicutes 0.7±0.7 0.4±0.3
      注:*. 差异显著 (P<0.05);下同
      Notes: *. signficant differentce, the same below

      Table 4.  The relative abundance of the main phyla in the intestinal microbiota of C. carpio fed with different diets

      Figure 4.  The relative abundance of C. carpio gut microbiota at the genus level

      细菌属
      bacterial genus
      对照组/%
      control
      HGcc-1添加组/%
      HGcc-1 diet
      鲸杆菌属 Cetobacterium58.6±29.786.2±13.6*
      柠檬酸杆菌属 Citrobacter12.2±11.45.3±4.5*
      拟杆菌属 Bacteroides3.5±3.33.4±6.6
      气单胞菌属 Aeromonas3.1±3.30.9±1.6

      Table 5.  The relative abundance of the main genera in the intestinal microbiota of C. carpio fed with different diets

      Figure 5.  PCA analysis of common carp gut microbiota at the phylum level (a) and genus level (b)

    • LBP是体内LPS的转运受体,LBP含量是反应LPS水平的重要指标,无菌斑马鱼实验中,在无法采血的情况下,检测了鱼体内LBP水平。对照组和HGcc-1添加组无菌斑马鱼的LBP含量分别为(126.44±16.02) μmol/g pro和(99.77±6.24) μmol/g pro (图6-aP<0.05);对照组无菌斑马鱼ALT和AST活性分别为(7.05±3.46) U/g pro和(87.77±15.87) U/g prot,HGcc-1添加组分别为(4.22±2.96) U/g pro和(66.22±7.26) U/g pro (图6-b,c),其中AST存在显著性差异(P<0.05)。C3是补体经典激活途径和替代激活途径的重要中心环节,C3激活后才能进行后续补体成分的连锁反应,C3的高低与总补体高低相平行;而C4是补体经典激活途径的重要组成部分,其高表达才能保证补体的有效激活[21];因为在鲤血清检测到总补体显著升高,所以在无菌斑马鱼实验中检测了C3和C4的表达量,本研究结果发现HGcc-1添加组的肠道菌群显著上调了无菌斑马鱼C3和C4的表达量(图6-d,eP<0.05)。

      Figure 6.  The effect of intestinal microbiota altered by feeding C. carpio with HGcc-1 supplement diet on LBP (a), ALT (b), AST (c) and complement (d,e) of GF D. rerio

    • 单菌处理无菌斑马鱼时,鱼体内没有产生LPS的革兰氏阴性菌,因此此处不测LPS和LBP,结果显示,对照组无菌斑马鱼ALT和AST活性分别为(12.42±3.68) U/g pro和(74.54±4.18) U/g pro, HGcc-1添加组分别为(6.63±1.72) U/g pro和(52.77±5.72) U/g pro (图7-a,b)。C3和C4基因的表达量在2组间没有显著差异(图7-c,d)。该结果表明,HGcc-1通过直接作用降低了无菌斑马鱼的ALT和AST水平,但是对C3和C4基因的表达没有影响。

      Figure 7.  The direct effect of HGcc-1 on GF D. rerio ALT (a), AST (b) and complement (c-d)

    3.   讨论
    • 本研究结果表明,HGcc-1虽然没有显著提高鲤的生长性能,但提高血清补体水平和改善肠肝健康的作用较为显著。HGcc-1添加组可显著增加血清中的总补体含量。补体在先天免疫和适应性免疫中发挥着重要作用[22],并且是机体抵御病原体的第一道防线,能够识别外来病原体然后激活补体级联反应以清除病原体[23]。补体是鱼类固有免疫力的重要组成部分[24],补体含量增加表明HGcc-1可增强鱼类抵抗病原体的能力。ALT和AST是肝脏健康的重要指标[25],在本实验的结果中,HGcc-1添加组的血清ALT和AST水平显著降低,显著改善鲤肝脏的健康状态。革兰氏阴性细菌产生的LPS会通过刺激巨噬细胞和内皮细胞产生促炎因子来造成肠道损伤[8],血清LPS的含量是判断肠道健康的重要指标[26],本研究的结果显示,HGcc-1添加组的LPS含量显著下降,表明HGcc-1的添加改善了鲤的肠道健康。

      肠道菌群与鱼类的生长和免疫密切相关。对于水产动物,变形菌门中的许多物种被认为是有害菌,梭杆菌门被认为是鱼类有益菌[27-28]。本研究结果显示,在门水平,梭杆菌门、变形菌门和拟杆菌门在2组中都为优势菌群;在属水平,鲸杆菌属柠檬酸杆菌属和拟杆菌属在2组中都为优势菌群。这与已经报道的研究结果相似[29]。HGcc-1添加组增加了梭杆菌门的丰度,鲸杆菌属是梭杆菌门的重要成员,据报道,从淡水鱼的肠道中分离出的鲸杆菌能够产生维生素B-12[30],维生素B12对红细胞的发育和机体新陈代谢至关重要[31],本研究表明HGcc-1添加组中鲸杆菌的丰度增加了47.1%。同时HGcc-1添加使变形菌门的丰度减少了70.7%,在属水平上,来自变形菌门的柠檬酸杆菌属和气单胞菌属的丰度分别减少了56.6%和70.9%。有研究表明,变形杆菌门的增加是疾病的潜在诊断标志[32]。变形杆菌门中的气单胞菌属是水产养殖中的常见病原体,严重危害水产动物的健康[33-34],柠檬酸杆菌肠炎在中国的集约化水产养殖中已成为日益严重的问题[35]

      利用菌群转接无菌斑马鱼,本研究也揭示了非特异性免疫和肠肝健康指标的变化与添加HGcc-1的饲料投喂鲤后肠道菌群的变化有关。LBP是激活LPS的关键因素,可以显著增强细胞对内毒素的应答[36]。在本研究中,与对照组相比,HGcc-1添加组鲤的肠道菌群显著降低了无菌斑马鱼的LBP含量,表明肠道菌群的变化显著减弱了LPS对鲤肠道的损伤。肠道菌群的间接作用和HGcc-1的直接作用均会降低无菌斑马鱼的ALT和AST水平。有研究表明枯草芽孢杆菌提取物1-Deoxynojirimycin和生物表面活性剂可改善高脂饲料对小鼠 (Mus musculus) 和褐家鼠 (Rattus norvegicus) 肝脏的损伤[37-38]。因此,HGcc-1可能还会分泌一种可以改善肝脏健康的物质。同时HGcc-1所改变的肠道菌群使得无菌斑马鱼C3和C4基因的表达量显著提高,这也证明了HGcc-1通过改变鲤的肠道菌群间接增强了鲤的免疫能力,在之前的报道中也有类似的结果[18-19]

      本研究表明,HGcc-1的益生作用主要体现在几个方面:① HGcc-1对生长指标没有显著影响,也不影响鲤鱼种的存活率;② HGcc-1显著降低鲤血清中LPS含量、ALT和AST水平,显著改善肠肝健康;③ HGcc-1通过激活补体系统增强鲤非特异性免疫;④ 调节肠道菌群方面,HGcc-1增加了梭杆菌门丰度并减少变形菌门丰度;⑤ HGcc-1通过改变鲤的肠道菌群改善补体水平,同时使鲤的肝肠更加健康。综上所述,枯草芽孢杆菌HGcc-1对鲤具有益生作用,可以用于改善鲤肠肝健康及肠道菌群稳态,即有益菌丰度上升。

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