摘要
目的
解析茶花鸡源罗伊特氏黏液乳杆菌CHF7-2的益生特性和安全性,为开发利用该菌株作为饲用微生态制剂提供理论依据。
方法
通过体外试验检测菌株CHF7-2的黏附性能、产酶性能、抑菌功效和抗氧化活性。利用PacBio Sequel II和Illumina NovaSeq 6000平台对菌株CHF7-2进行全基因组测序,并采用多种生物信息学工具和数据库对其基因组进行注释,从分子层面探究其益生机制和安全性。
结果
体外试验研究表明,菌株CHF7-2展现出良好的益生特性和安全性:具有较强的表面疏水性、自凝聚性和一定的抗氧化能力;能有效抑制大肠杆菌K88、金黄色葡萄球菌ATCC 49521、鸡伤寒沙门氏菌CICC 21510和猪霍乱沙门氏菌CVCC 3383的生长;能够产生蛋白酶和脂肪酶;不产生溶血环,表明其饲用安全性。全基因组测序分析结果显示,菌株CHF7-2的基因组大小为2 116 761 bp,平均G+C含量为38.8%,编码基因数量为2 067个。同时,在CHF7-2基因组中发现了Ⅲ类细菌素EnlA合成基因簇,以及多个与耐酸、耐胆盐、耐热胁迫、耐冷胁迫、黏附、抗氧化和有机酸合成相关的抗应激和益生基因,且基因组中未发现毒力和耐药基因。
结论
罗伊特氏黏液乳杆菌CHF7-2是一株具有潜力的益生菌,有望成为饲用微生态制剂的重要候选菌株。
关键词
茶花鸡,又名傣族鸡,主要分布于云南省西双版纳州的景洪市、勐海县和勐腊县境内,是一种由红原鸡(Gallus gallus)经过长期驯化和选育而成的珍稀热带原始鸡种,具有性早熟、耐粗饲、抗病力强和肉质鲜美等特点,已被列入《国家畜禽遗传资源品种名录》(2021年版
本研究所用的菌株CHF7-2,是从西双版纳勐海县曼贺纳村散养的茶花鸡粪便中分离得到的,通过16S rRNA基因序列分析将其鉴定为罗伊特氏黏液乳杆菌(Limosilactobacillus reuteri),在pH 2.0的MRS肉汤培养基和含有0.3%胆盐的MRS肉汤培养基中分别孵育2 h后,其存活率均超过60%,并且对多种β-内酰胺类和氨基糖苷类抗生素敏感,因此被认为是一株具有潜力的益生菌
然而,随着罗伊特氏黏液乳杆菌在不同物种中被不断鉴定,通过传统方式全面分析其益生、安全等生物学特性变得愈发困难。当前,高通量测序技术的快速发展已成为解决这一问题的关键手段。对益生菌的研究也已深入到基因组学层面,能够检测菌株的耐药和毒力基因,对其安全性进行全面评估,同时快速挖掘益生基因,从而筛选出潜在的益生菌
1 材料与方法
1.1 菌株
罗伊特氏黏液乳杆菌(Limosilactobacillus reuteri) CHF7-2为本课题组邹建华
1.2 主要试剂和仪器
MRS培养基、LB培养基、哥伦比亚血平板,广东环凯微生物科技有限公司;总抗氧化能力(total antioxidant capacity, T-AOC)检测试剂盒、羟自由基清除能力检测试剂盒、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基清除能力检测试剂盒、还原型谷胱甘肽(reduced glutathione, GSH)含量检测试剂盒、超氧化物歧化酶(superoxide dismutase, SOD)活性检测试剂盒,北京索莱宝科技有限公司;土壤/粪便基因组DNA提取试剂盒(磁珠法),天根生化科技(北京)有限公司。
产蛋白酶、脂肪酶和纤维素酶发酵培养基参照Fitri
离心机、NanoDrop,ThermoFisher Scientific公司;Qubit荧光计,Invitrogen公司;PacBio Sequel II测序平台,PacBio公司;Illumina NovaSeq6000测序平台,Illumina公司。
1.3 菌株CHF7-2的黏附性能检测
采用微生物黏附碳氢化合物
疏水性=(1-A1/A0)×100% | (1) |
自聚集性=(1-A2/A0)×100% | (2) |
共聚集性={[(A2+A3)/2-A4]/(A2+A3)/2}×100% | (3) |
式中:A0为初始菌液的OD600值,A1为与二甲苯混匀后菌液的OD600值,A2为初始菌液静置3 h后上清液的OD600值,A3为病原菌液静置3 h后上清液的OD600值,A4为混合静置5 h后上清液的OD600值。
1.4 菌株CHF7-2的产酶性能检测
将活化后的CHF7-2和L. rhamnosus GG菌液分别按1%的体积分数接种于蛋白酶、脂肪酶、淀粉酶和纤维素酶发酵培养基中,培养48 h后检测胞外酶活性,具体方法参照《饲料添加剂酸性、中性蛋白酶活力的测定 分光光度法》(GB/T 28715—2012
1.5 菌株CHF7-2的抑菌作用检测
将活化后的CHF7-2和L. rhamnosus GG菌液分别按1%的体积分数接种于MRS肉汤中,37 ℃静置培养16 h后调整OD600为0.8,2 800×g离心10 min收集上清。分别吸取37 ℃过夜培养的E. coli K88、S. aureus ATCC 49521、S. gallinarum CICC 21510、S. choleraesuis CVCC 3383菌液20 μL,与LB固体培养基(20 mL、50 ℃)剧烈混合后倾注入培养皿中。使用直径为8 mm牛津杯打孔,将CHF7-2与LGG上清液注入琼脂孔中(100 μL/孔),37 ℃培养24 h,使用游标卡尺测量抑菌圈直径。
1.6 菌株CHF7-2的抗氧化活性检测
按照试剂盒说明书的方法,检测菌株CHF7-2的胞外产物、胞内产物的总抗氧化能力(total antioxidant capacity, T-AOC)、羟自由基清除能力、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基清除能力、还原型谷胱甘肽(reduced glutathione, GSH)含量以及超氧化物歧化酶(superoxide dismutase, SOD)活性。
1.7 菌株CHF7-2的溶血试验
采用滤纸片扩散法测定CHF7-2的溶血能力,将无菌滤纸片紧贴于哥伦比亚血琼脂平板,分别吸取20 μL活化后的CHF7-2菌液和强溶血性的金黄色葡萄球菌液(阳性对照)滴于滤纸片上,37 ℃培养24 h,根据菌落周围是否产生溶血圈判断CHF7-2的溶血能力。
1.8 菌株CHF7-2的全基因组测序分析
1.8.1 菌株CHF7-2基因组提取与测序
将活化后的CHF7-2菌液按1%的体积分数接种于MRS肉汤中进行扩大培养(37 ℃、16 h),2 800×g离心10 min收集菌体,并用无菌PBS缓冲液洗涤菌体3次。使用土壤/粪便基因组DNA提取试剂盒(磁珠法)提取菌株CHF7-2的高质量基因组DNA。对提取后的核酸采用NanoDrop、Qubit及琼脂糖凝胶电泳检测其浓度和完整性。DNA浓度和完整性达标后,使用BluePippin全自动核酸片段回收系统回收大片段DNA,构建测序文库。完成DNA文库制备后,使用Qubit及Agilent 2100检测文库浓度和大小以确保质量合格,随后采用PacBio Sequel II和Illumina NovaSeq 6000平台进行测序。
1.8.2 CHF7-2基因组序列分析
使用Hifiasm v0.12软件对PacBio Sequel II三代平台数据(长度≥2 000 bp)进行组装(k-mer值设置为51,其他参数均为默认值),并采用Circlator v1.5.5软件进行环化和调整起始位点。此外,通过Pilon v1.22软件利用Illumina NovaSeq 6000二代测序平台数据进一步纠错,以获得更高准确度的基因组信息。分别利用Rfam v14.1数据库和软件Prodigal v2.6.3、Infernal v1.1.3、tRNAscan-SE v2.0进行编码基因和非编码RNA的预测,并使用Circos v0.66软件绘制CHF7-2的基因组圈图。随后,基于基因本体(gene ontology, GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)数据库,对预测基因进行功能注释。
1.8.3 CHF7-2毒力和抗生素耐药基因检索分析
基于毒力因子数据库(virulence factor database, VFDB) 202
1.9 比较基因组分析
1.9.1 平均核苷酸一致性(average nucleotide identity, ANI)计算
为进一步分析菌株CHF7-2的遗传进化关系,选取27株不同来源且已完成全基因组测序的L. reuteri (
Strains | Genome size (Mb) | G+C content (%) | Origin | GenBank ID |
---|---|---|---|---|
L. reuteri FN041 | 2.4 | 38.5 | Homo sapiens | GCA_024652885.1 |
L. reuteri M2021619 | 2.2 | 39.0 | Homo sapiens | GCA_021459965.1 |
L. reuteri 03 | 2.3 | 39.0 | Homo sapiens | GCA_030517815.1 |
L. reuteri MD IIE-43 | 2.2 | 39.0 | Homo sapiens | GCA_002007085.2 |
L. reuteri subsp. reuteri DSM 20016 | 2.0 | 39.0 | Homo sapiens | GCA_000016825.1 |
L. reuteri ATCC PTA 4659 | 2.1 | 39.0 | Homo sapiens | GCA_030418275.1 |
L. reuteri strain reuteri | 2.0 | 39.0 | Homo sapiens | GCA_009184725.1 |
L. reuteri SD-LRE2-IT | 2.3 | 39.0 | Homo sapiens | GCA_020023775.1 |
L. reuteri DS0384 | 2.2 | 39.0 | Homo sapiens | GCA_021398615.1 |
L. reuteri VHProbi M07 | 2.0 | 39.0 | Homo sapiens | GCA_021228055.1 |
L. reuteri CNEI-KCA3 | 2.1 | 39.5 | Gallus gallus | GCA_013694365.1 |
L. reuteri AM_LB1 | 2.3 | 39.0 | Gallus gallus | GCA_025369755.1 |
L. reuteri 3632 | 2.5 | 38.5 | Gallus gallus | GCA_020978285.1 |
L. reuteri P43 | 2.1 | 39.0 | Gallus gallus | GCA_033570435.1 |
L. reuteri SKKU-OGDONS-01 | 2.3 | 39.0 | Gallus gallus | GCA_003316935.1 |
L. reuteri 19-E-6 | 1.9 | 39.0 | Sus scrofa | GCA_020412465.1 |
L. reuteri ZLR003 | 2.2 | 38.5 | Sus scrofa | GCA_001618905.1 |
L. reuteri AN417 | 2.2 | 39.0 | Sus scrofa | GCA_013348825.1 |
L. reuteri 121 | 2.3 | 39.0 | Sus scrofa | GCA_001889975.1 |
L. reuteri YSJL-12 | 2.2 | 39.0 | Sus scrofa | GCA_006874665.1 |
L. reuteri RE225 | 2.3 | 38.5 | Mus | GCA_030721705.1 |
L. reuteri M1 | 2.3 | 39.0 | Mus | GCA_030345055.1 |
L. reuteri Byun-re-01 | 2.2 | 39.0 | Mus | GCA_003316895.1 |
L. reuteri LL7 | 2.4 | 39.0 | Peromyscus leucopus | GCA_007633215.1 |
L. reuteri YLR001 | 2.4 | 38.5 | Bos mutus | GCA_018884225.1 |
L. reuteri 1B | 2.3 | 39.0 | Equuscaballus | GCA_013487925.1 |
L. reuteri BRD_L17 | 2.0 | 39.0 | Canis lupus familiaris | GCA_026183435.1 |
1.9.2 泛-核心基因集构建
通过Prokka v1.14.
1.9.3 系统发育树构建
通过Roary v3.13.
1.9.4 碳水化合物活性酶(carbohydrate-active enzymes, CAZy)注释
将CHF7-2与
1.10 数据统计及分析
采用SPSS 20.0软件对试验数据进行统计学分析,两组之间的显著性通过t检验进行评估,多组之间使用单因素方差分析(one-way ANOVA),并依据Duncan氏法进行多重比较检验以确定组间差异性。试验数据以平均值±标准差(mean±SD)表示,P<0.05表示差异显著。
2 结果与分析
2.1 菌株CHF7-2的黏附性能
由
Item | Result (%) |
---|---|
Hydrophobicity | 72.46±0.68 |
Self-coagulation | 85.61±0.46 |
Copolymerization with E. coli K88 | 18.95±0.15b |
Copolymerization with S. aureus ATCC 49521 | 52.84±0.16a |
Copolymerization with S. gallinarum CICC 21510 | 15.65±0.16c |
Copolymerization with S. choleraesuis CVCC 3383 | 13.22±0.10d |
Data is presented as mean±SD (n=3) and values with different superscript letters are significantly different (P<0.05).
2.2 菌株CHF7-2的产酶性能
如

图1 菌株CHF7-2的产酶性能
Figure 1 Enzyme production performance of strain CHF7-2. Data is presented as mean±SD (n=3); * indicates P<0.05.
2.3 菌株CHF7-2的抑菌性能
如

图2 菌株CHF7-2的抑菌效果。A:菌株CHF7-2的抑菌效果观察;B:测定菌株CHF7-2抑菌效果时获得的抑菌圈直径(n=3)。
Figure 2 The antibacterial effect of strain CHF7-2. A: Observation of antibacterial effect of strain CHF7-2; B: The diameters of the antibacterial zones obtained when measuring the antibacterial effect of strain CHF7-2 (n=3).
2.4 菌株CHF7-2的抗氧化活性
由

图3 菌株CHF7-2胞外产物及胞内产物的抗氧化活性
Figure 3 Antioxidant activity of extracellular and intracellular products of strain CHF7-2. Data is presented as mean±SD (n=3). A: Total antioxidant activity; B: Superoxide dismutase activity; C: Glutathione content; D: DPPH free-radical scavenging rate; E: Hydroxyl radical scavengingrate. *: P<0.05; **: P<0.01.
2.5 菌株CHF7-2的溶血活性
通过以下标准判断菌株的溶血活性:菌落周边出现草绿色溶血环为α-溶血;菌落周边出现较宽的透明圈为β-溶血;菌落周边不出现溶血环为γ-溶血。如

图4 菌株CHF7-2的溶血活性
Figure 4 Hemolytic property of strain CHF7-2. S. aureus ATCC 49521 was used as a positive control.
2.6 菌株CHF7-2的全基因组基本特征
利用三代PacBio Sequel II和二代Illumina NovaSeq 6000测序平台对菌株CHF7-2进行全基因组测序,结果显示菌株CHF7-2的基因组序列全长为2 116 761 bp,平均G+C含量为38.8%,由1个染色体和2个质粒构成。编码基因数量为2 067个,其中2 045个位于染色体上,12个和10个分别位于质粒1和质粒2上。所有编码基因的总长度为1 840 680 bp,平均长度为890 bp,编码区占据了基因组总长度的86.96%。此外,非编码RNA含有18个rRNA、70个tRNA以及54个其他类型的非编码RNA (other ncRNA)。菌株CHF7-2的基因组圈图如

图5 菌株CHF7-2的基因组圈图。第1圈:基因组大小;第2圈:基因组正链基因;第3圈:基因组负链基因;第4圈:重复序列;第5圈:tRNA和rRNA (蓝色为tRNA,紫色为rRNA);第6圈:G+C含量(浅黄色部分表示该区域G+C含量高于基因组的平均G+C含量,蓝色部分则表示该区域G+C含量低于基因组的平均G+C含量);第7圈:GC-skew (深灰色代表G含量大于C的区域,红色代表C含量大于G的区域)。
Figure 5 Loop diagram of strain CHF7-2 genome. Round 1: Genome size; Round 2: Positive-strand genes in the genome; Round 3: Negative-strand genes in the genome; Round 4: Repeat sequence; Round 5: tRNA and rRNA (Blue represents tRNA, purple represents rRNA); Round 6: G+C content (The light yellow part indicates that the G+C content in this region is higher than the average G+C content of the genome, while the blue part indicates that the G+C content in this region is lower than the average G+C content of the genome); Round 7: GC-skew (Dark gray represents areas with G content greater than C, while red represents areas with C content greater than G).
2.7 基因组功能注释
2.7.1 GO数据库注释结果
基于GO数据库,获得了菌株CHF7-2基因组中编码基因的标准化功能描述信息。注释结果如图6所示,共计1 656个基因得到注释,占全部编码基因的80.12%,与生物学过程(biological process, BP)、细胞组分(cellular component, CC)和分子功能(molecular function, MF)相关的基因数分别为1 210、810和1 365个。其中,在生物学过程中,主要涉及的功能包括代谢过程(metabolic process,917个基因)、细胞过程(cellular process,792个基因)、单有机体过程(single-organism process,562个基因)。在细胞组分方面,主要涉及的功能有膜(membrane,475个基因)、膜组成(membrane part,456个基因)、细胞(cell,437个基因)。在分子功能中,主要的功能单位包括催化活性(catalytic activity,983个基因)、结合活性(binding activity,771个基因)和转运活性(transporter activity,130个基因)。

图6 菌株CHF7-2基因组的GO功能分类
Figure 6 GO functional classification of strain CHF7-2 genome.
2.7.2 KEGG数据库注释结果
KEGG数据库可用于分析物种基因组中各基因表达产物的功能及代谢途径。菌株CHF7-2的KEGG注释结果如

图7 菌株CHF7-2基因组的KEGG代谢通路分类
Figure 7 KEGG metabolic pathway classification of strain CHF7-2 genome.
2.8 细菌素合成基因簇分析
利用在线软件BAGEL4对菌株CHF7-2的抑菌物质信息进行挖掘,其基因组中共鉴定出2个可能与Ⅲ类细菌素Enterolysin A (EnlA)生物合成相关的基因簇(EnlA-1和EnlA-2),核心肽相似率分别为31.58%和39.04%。对相似度较高的EnlA-2基因簇进行注释(图8),该基因簇位于菌株CHF7-2基因组的1 497 513-1 517 945 bp,核心肽Enterolysin A位于1 505 219-1 507 960 bp。orf00001编码聚(核糖醇磷酸酯) β-N-乙酰葡糖胺基转移酶[poly(ribitol-phosphate)beta-N-acetylglucosaminyltransferase];orf00002编码糖基转移酶GlyA (glycosyltransferase GlyA);orf00003编码dTDP-鼠李糖基转移酶RfbG (dTDP-rhamnosyl transferase RfbG);orf00004编码假定O-抗原转运蛋白(putative O-antigen transporter);orf00005编码UDP-吡喃半乳糖变位酶(UDP-galactopyranose mutase);orf00010编码十一异戊烯磷酸半乳糖磷酸转移酶(undecaprenyl-phosphate galactose phosphotransferase);orf00011编码假定糖基转移酶(putative glycosyltransferase) CsbB;orf00013编码调节蛋白(regulatory protein) RecX;orf00018编码假定核糖核酸酶样蛋白(putative ribonuclease-like protein) YfkH;orf00020编码内膜转运蛋白(inner membrane transporter) YjeM,可能与细菌素分泌有关。
(待续)
2.9 益生特性相关基因分析
对菌株CHF7-2的益生特性相关基因进行挖掘,结果显示,其基因组中包含16个耐酸基因、1个耐胆盐基因、3个黏附相关基因、7个抗氧化相关基因、6个核黄素生物合成相关基因、1个乙酸生物合成相关基因、6个耐热胁迫基因和1个耐冷胁迫基因(
Gene ID | Gene name | Gene product |
---|---|---|
Acid resistance | ||
GE000509 | arcA | Arginine deiminase |
GE000477 | arcC | Carbamate kinase |
GE000510 | argR | Transcriptional regulator of arginine metabolism |
GE000785 | argG | Argininosuccinate synthase |
GE000786 | argH | Argininosuccinate lyase |
GE000561 | gadA/B | Glutamate decarboxylase |
GE000562 | gadC | Glutamate: GABA antiporter |
GE000525 | atpB |
F-type |
GE000526 | atpE |
F-type |
GE000527 | atpF |
F-type |
GE000528 | atpH |
F-type |
GE000529 | atpA |
F-type |
GE000530 | atpG |
F-type |
GE000531 | atpD |
F-type |
GE000532 | atpC |
F-type |
GE000191 | nhaC |
N |
Bile salt resistance | ||
GE000784 | cbh | Choloylglycine hydrolase |
Production of adhesion molecules | ||
GE001572 | efg | Elongation factor G |
GE001943 | ltaS | Lipoteichoic acid synthase |
GE001415 | mgs | 1,2-diacylglycerol 3-alpha-glucosyltransferase |
Oxidative resistance | ||
GE000418 | trxR | Thioredoxin reductase (NADPH) |
GE000602 | trxA | Thioredoxin 1 |
2.10 毒力和耐药基因检索分析
随着益生菌资源的不断发掘,其安全性问题日益受到关注。本研究将菌株CHF7-2的基因组通过VFDB和CARD数据库对基因组进行了毒力和抗生素耐药基因的检索分析。结果显示,菌株CHF7-2的基因组中不存在毒力因子和耐药基因,因此可以认为菌株CHF7-2是安全的,适用于畜禽养殖等行业的微生态制剂开发。
2.11 平均核苷酸一致性(ANI)分析
ANI分析是通过比较两两基因组之间的同源序列,在全基因组水平上评估不同物种的亲缘关系,通常认为ANI值大于95%为同

图9 28株罗伊特氏黏液乳杆菌的ANI分析结果
Figure 9 Results of ANI analysis of 28 strains of Limosilactobacillus reuteri.
2.12 泛-核心基因集构建
菌株CHF7-2与

图10 泛基因集和核心基因集曲线
Figure 10 Pan-core gene family curve.
2.13 核心基因构建系统发育树
本研究基于菌株CHF7-2与

图11 基于核心基因构建的系统发育树
Figure 11 Phylogenetic tree constructed based on core genes.
2.14 CAZy注释结果
本研究从基因组层面探究了包括菌株CHF7-2在内的6株鸡源L. reuteri (CHF7-2、CNE1-KCA3、AM_LB1、3632、P43和SKKU-OGDONS-01)对碳水化合物的利用潜力。结果表明,6株L. reuteri共注释到4类碳水化合物活性酶,分别为糖苷水解酶类(glycoside hydrolases, GHs)、糖苷转移酶类(glycosyl transferases, GTs)、辅助模块酶类(auxiliary activities, AAs)和碳水化合物结合模块(carbohydrate-binding modules, CBMs),其中包含15个GHs家族、11个GTs家族、2个CBMs家族和1个AAs家族(

图12 六株鸡源罗伊特氏黏液乳杆菌CAZy数据库注释结果
Figure 12 Annotation results of CAZy database for six chicken-derived Limosilactobacillus reuteri strains.
3 讨论与结论
大量研究表明,生长环境和遗传背景均可影响鸡肠道微生物群落的形成和发育,进而塑造出特定的宿主-微生物共生关
全基因组测序有助于了解目标菌株的基因组结构及基因功
通过对包含CHF7-2在内的28株不同来源的L. reuteri进行ANI分析显示,CHF7-2与罗伊特氏黏液乳杆菌(L. reuteri)模式株DSM 2001
综上所述,罗伊特氏黏液乳杆菌CHF7-2具有良好的黏附性能、抗菌活性和抗氧化活性。同时,其基因组中含有大量潜在的益生基因,且不存在毒力基因和耐药基因。因此,该菌株有望成为饲用微生态制剂的重要候选菌株。
作者贡献声明
徐乐:实验设计、实验操作、数据分析和论文撰写;陈诗宇:实验操作、数据分析和论文撰写;王上:实验操作、数据分析;张志翔:实验操作;董斌:实验操作;林秋叶:实验设计、数据分析、论文审阅和修改;曹振辉:实验设计、数据分析、论文审阅和修改。
利益冲突
作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
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