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近年来,随着鱼类需求量增加和水产养殖业不断扩大,鱼类疾病也在威胁着养殖产业,造成严重的经济损失。集约化生产模式和抗生素的滥用使得鱼类疾病难以得到有效的预防和控制,且抗生素的滥用容易产生负面影响,如水产养殖系统中抗生素抗性基因(ARGs)的转移[1]和耐药细菌的出现;它们对大多数水生生物具有一定毒性,易残留且对生态环境造成破坏[2]。寻找和研发抗生素的替代饲料添加剂是目前水产养殖中亟待解决的重大问题。益生菌是一种已被确定的抗生素替代品。联合国粮食及农业组织 (FAO) 和世界卫生组织(WHO)将益生菌定义为当在动物体内定植一定数量后对机体产生有益影响的活性微生物[3]。胃肠道是一个复杂的环境,有不同的微生物群落聚集,益生菌可以影响肠道微生物群落,减少疾病的发生[4],如细菌数量增加、多样性减少会引起肠道微生物生态失调,导致疾病。动物机体的免疫也与微生物的组成密切相关[5]。
国内外都有许多关于益生菌对鱼类肠道菌群影响的报道,益生菌能够调节肠道微生物组成,改善肠道健康[6]。廖庆钊等[7]在罗非鱼饲料中添加乙醇假丝酵母,结果表明,投喂乙醇假丝酵母后,罗非鱼肠道中梭杆菌门、鲸杆菌属和艾克曼菌属等有益菌群的丰度显著上调,蓝细菌门丰度减少。Hongqin 等[8]研究结果表明,在饲料中添加1×105 CFU·g−1的丁酸梭菌增加了罗非鱼肠道菌群的多样性和有益菌(如芽孢杆菌)的相对丰度,降低了条件致病菌(如气单胞菌)的相对丰度。在大黄鱼幼鱼的饲料中添加丁酸梭菌,改变了大黄鱼幼鱼肠道微生物群结构,降低了肠道微生物多样性;添加丁酸梭菌可有效提高大黄鱼幼鱼肠道中丁酸梭菌的丰度,降低大黄鱼幼鱼肠道中一些潜在致病菌的丰度,对大黄鱼的生长起到促进作用。Goncalves 等[9]在虹鳟鱼基础日粮中添加益生菌,饲喂7 d后,其肠道微生物多样性增加,厚壁菌门和梭杆菌门丰度增加,这两个门的成员具有有益的作用,而变形菌门丰度降低。
如何让益生菌在使用过程中长久发挥作用是目前研究的重要关注点。冷冻干燥是生产干燥菌粉的经典方法,干燥过程在真空、低温下进行,从而减少了热降解[10]。该方法通过冷冻含有细胞的水溶液并干燥,通过升华去除水分,但益生菌可能由于冰晶的形成、高渗透压导致的膜损伤、DNA变性和水分蒸发而损坏细胞[11],因此,为了保护益生菌在脱水过程中不被破坏,在冷冻干燥前添加干燥介质作为保护剂[12]。添加保护剂可以提高益生菌在冷冻干燥和储存过程中的稳定性[13]。保护剂在冷冻干燥过程中极为重要,本研究选定脱脂奶粉、海藻糖、乳糖和蔗糖4种常见保护剂进行筛选,旨在获得最适合耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans,YS11)冷冻干燥的保护剂。目前YS11对罗非鱼肠道微生物菌群的影响尚不清楚,因此,本研究探究YS11对罗非鱼幼鱼肠道微生物菌群的影响,为罗非鱼养殖产业益生菌使用和病原菌防治提供新的思路。
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筛选最佳保护剂的浓度(表1),对照组对YS11无法起到保护作用,冻干后存活菌数为(0.03±0.0021)×108 CFU·mL−1,存活率仅有0.19%,远小于其他冻干保护剂的存活率。与对照组相比,4种保护剂均起到了保护细菌免受低温冻伤的情况。不同浓度的保护剂对细菌起到的保护作用也不相同,对比2个蔗糖浓度下的细菌存活率,10%蔗糖组的存活率为9.20%,而5%蔗糖组的存活率为5.86%。对比2个海藻糖浓度下的细菌存活率,10%海藻糖组的存活率为11.48%,而5% 海藻糖组存活率为4.64%。对比2个乳糖浓度下的细菌存活率,10%乳糖组的存活率为9.93%,而20% 乳糖组的存活率为6.84%。对比2个脱脂奶粉浓度下的细菌存活率,10%脱脂奶粉组的存活率为11.16%,而20% 脱脂奶粉组的存活率为2.42%。通过保护剂筛选实验的优化,发现YS11在10%海藻糖的保护下,能够在干燥后保持较高的存活率。
表 1 保护剂对YS11存活率的影响
组别 冻干前/
(109 CFU·mL−1)冻干后/
(108 CFU·mL−1)存活率/% 1%蛋白胨 1.7 ± 0.06 0.03 ± 0.0021 0.19 5% 蔗糖 1.45 ± 0.03 0.85 ± 0.085 5.86 10% 蔗糖 1.45 ± 0.04 1.33 ± 0.071 9.20 5% 海藻糖 1.5 ± 0.1 0.7 ± 0.038 4.64 10% 海藻糖 1.4 ± 0.07 1.61 ± 0.032 11.48 10% 乳糖 1.48 ± 0.06 1.47 ± 0.08 9.93 20% 乳糖 1.37 ± 0.05 0.94 ± 0.081 6.84 10% 脱脂奶粉 1.5 ± 0.08 1.67 ± 0.091 11.16 20% 脱脂奶粉 1.6 ± 0.09 0.39 ± 0.025 2.42 -
从16S rDNA基因的V3 V4 扩增子中产生1 198 583对Reads,双端 Reads 质控、拼接后共产生 1 195 379 条 Clean Reads,每个样品至少产生 79 369 条 Clean Reads ,平均产生79 692 条 Clean Reads。组装的序列平均长度为410 bp。截止97%的操作分类单位聚类产生了整个数据集的总共10 247个操作分类单位。用α多样性和β多样性对样品内部和样品之间的微生物复杂性进行分析。从维恩图中显示(图1-A),Control组、LB组、YS11组、FB组、FLF组共有特征个数680个OTUs,YS11组、FB组、FLF组中特征个数的OTUs数量分别为6、3和4。从稀释性曲线中(图1 -B),笔者发现15个样本中,YS11组、FB组、FLF组微生物多样性有相似的趋势,接近饱和平台,Control组、LB组微生物多样性有相似的趋势。基于整体群落组成的β多样性分析而言,表明添加YS11后,肠道微生物群的聚集相似程度较高。对10 247个OTUs(按97%序列同一性分组)的UniFrac主坐标分析(PCoA)表明(图1-C),对照组和处理组有明显的分离,且对照最相同处理的样品具有较高的分散性,处理组没有显示出与对照的单独聚类,3个处理组出现较高的聚集。通过非加权组平均法(UPGMA)分析进行样品层次聚类(图1-D),YS11、FB、FLF组的肠道微生物群结构相似,聚集在较高的分支内,而Control组和LB组与处理组不同,聚集在较低的分支内。
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为了确定YS11是如何影响罗非鱼肠道微生物群落,在肠道微生物不同的分类尺度上,比较3组不同处理组的微生物群落差异。在门、属、种水平上最丰富的细菌分类群(前10个)出现在所有鱼类肠道样本中。在门水平上(图2-A),罗非鱼肠道内变形杆菌门(Proteobacteria)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)、梭杆菌门(Fusobacteria)构成4个最主要的细菌群落门,其中变形杆菌门在FB组和FLF组的相对丰度高于其他组,在FLF组中厚壁菌门的相对丰度显著高于其他组,其次是拟杆菌门(Bacteroidetes)、酸杆菌门(Acidobacteria)、疣维菌门(Verrucomicrobia)、巴氏杆菌门(Patescibacteria)、软壁菌门(Tenericutes)、绿弯菌门(Chloroflexi)。在属水平上(图2-B),罗非鱼肠道内非培养微杆菌科(uncultured bacterium Microbacteriaceae)、博斯氏菌属(Bosea)、非培养根瘤菌科(uncultured bacterium Rhizobiaceae)、鲸蜡杆菌属(Cetobacterium)为主要细菌属。在种水平上(图2-C),罗非鱼肠道内非培养微杆菌科(uncultured bacterium Microbacteriaceae)、非培养博斯氏菌属(uncultured bacterium Bosea)、非培养根瘤菌科(uncultured bacterium Rhizobiaceae)、非培养鲸蜡杆菌(uncultured bacterium Cetobacterium)为主要细菌种。
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为了检测罗非鱼肠道中病原菌和益生菌的变化,从肠道微生物属水平菌群相对丰度数据中,计算有益菌和病原菌增加或者减少的读数。结果显示杆菌属、梭菌属在FLF组与对照组相比中存在显著差异,数量增加。放线菌属、双歧杆菌属和乳杆菌属无显著差异(表2)。气单胞菌属和爱德华氏菌属在对照组和处理组中均存在显著差异,数量显著减少,其中爱德华氏菌属在处理组中的数据均降为0(表2)。
表 2 YS11对肠道微生物菌群中益生菌和病原菌增加或减少的读数
/条 Control LB YS11 FB FLF 益生菌 Actinomyces 放线菌属 1±2 2±1 27±18 42±50 29±26 Bacillus 杆菌属 17±10 36±10 114±76 161±172 109±33* Bifidobacterium 双歧杆菌属 7±3 8±5 12±13 27±36 14±9 Fusobacterium 梭菌属 20±8 33±18 179±87 105±70 123±38* Lactobacillus 乳杆菌属 101±45 155±63 802±536 605±330 574±222 病原菌 Aeromonas 气单胞菌属 38±58 231±395 1±1*** 12±4** 8±6* Edwardsiella 爱德华氏菌属 182±313 38±61 0*** 0*** 0*** 注:*代表差异显著;**代表差异极显著;***代表差异显著更甚。
Screening of protectants for Lysinibacillus boronitolerans and its effects on intestinal microbiota of tilapia
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摘要: 本研究目的是为了探究耐硼赖氨酸芽孢杆菌(Lysinibacillus boronitolerans,YS11)的保存条件及其对罗非鱼肠道微生物多样性和丰度的影响,为微生物饲料添加剂的研发提供科学依据。选择海藻糖、乳糖、蔗糖和脱脂奶粉4种保护剂,并通过浓度梯度优化筛选最佳保护剂,结果表明,YS11在10%海藻糖中,干燥后保持较高的存活率。罗非鱼幼苗饲喂菌体发酵液,喂养70 d后,通过高通量测序观察YS11对罗非鱼肠道微生物群落的影响,结果表明,添加YS11的处理改变了罗非鱼肠道微生物群及其多样性,提高罗非鱼肠道中有益菌的丰度,降低某些潜在致病菌的丰度。本研究结果证实,10%海藻糖为最佳冷冻干燥保护剂;耐硼赖氨酸芽孢杆菌能够调节肠道微生物群,促进益生菌生长,抑制病原菌生长。Abstract: In order to explore the preservation conditions of Lysinibacillus boronitolerans YS11 and its effect on the diversity and abundance of intestinal microorganisms in tilapia for the research and development of microbial feed additives. Four protectants, i.e. trehalose, lactose, sucrose and skim milk powder, were selected and optimized by concentration gradient. The results confirmed that YS11 maintained a high survival rate after drying in 10% trehalose. Tilapia fingerlings were fed with feeds containing bacterial fermentation broth, and after 70 days of feeding, high-throughput sequencing was performed to observe the effect of the feeds on tilapia intestinal microbial community. The results showed that the feed containing YS11 changed the tilapia gut microbiota and its diversity, increased the abundance of beneficial bacteria in the tilapia gut, and decreased the abundance of some potential pathogenic bacteria. These results confirmed that 10% trehalose was the best freeze-drying protectant. L. boronitolerans can regulate the intestinal microbiota, promote the growth of probiotics, and inhibit the growth of pathogenic bacteria.
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Key words:
- Lysinibacillus boronitoleran /
- gut microbial community /
- tilapia /
- protectant
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表 1 保护剂对YS11存活率的影响
组别 冻干前/
(109 CFU·mL−1)冻干后/
(108 CFU·mL−1)存活率/% 1%蛋白胨 1.7 ± 0.06 0.03 ± 0.0021 0.19 5% 蔗糖 1.45 ± 0.03 0.85 ± 0.085 5.86 10% 蔗糖 1.45 ± 0.04 1.33 ± 0.071 9.20 5% 海藻糖 1.5 ± 0.1 0.7 ± 0.038 4.64 10% 海藻糖 1.4 ± 0.07 1.61 ± 0.032 11.48 10% 乳糖 1.48 ± 0.06 1.47 ± 0.08 9.93 20% 乳糖 1.37 ± 0.05 0.94 ± 0.081 6.84 10% 脱脂奶粉 1.5 ± 0.08 1.67 ± 0.091 11.16 20% 脱脂奶粉 1.6 ± 0.09 0.39 ± 0.025 2.42 表 2 YS11对肠道微生物菌群中益生菌和病原菌增加或减少的读数
/条 Control LB YS11 FB FLF 益生菌 Actinomyces 放线菌属 1±2 2±1 27±18 42±50 29±26 Bacillus 杆菌属 17±10 36±10 114±76 161±172 109±33* Bifidobacterium 双歧杆菌属 7±3 8±5 12±13 27±36 14±9 Fusobacterium 梭菌属 20±8 33±18 179±87 105±70 123±38* Lactobacillus 乳杆菌属 101±45 155±63 802±536 605±330 574±222 病原菌 Aeromonas 气单胞菌属 38±58 231±395 1±1*** 12±4** 8±6* Edwardsiella 爱德华氏菌属 182±313 38±61 0*** 0*** 0*** 注:*代表差异显著;**代表差异极显著;***代表差异显著更甚。 -
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