CN115053935A - 唾液乳杆菌在凡纳滨对虾虾苗培育中的应用 - Google Patents
唾液乳杆菌在凡纳滨对虾虾苗培育中的应用 Download PDFInfo
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Abstract
本发明公开一种唾液乳杆菌在凡纳滨对虾虾苗培育中的应用,属于水产养殖技术领域。本发明为保证唾液乳杆菌对凡纳滨对虾虾苗良好的益生作用,在虾苗的培育过程中,将唾液乳杆菌发酵液拌料放置10min后投喂于虾苗中,有助于促进蛋白质的消化吸收,增加抗氧化代谢物和蜕皮内源性成分的合成,进而显著提高虾苗的体长增长率和体重增长率,促进凡纳滨对虾虾苗生长,更好地满足凡纳滨对虾虾苗培育生产实践的需要。
Description
技术领域
本发明属于水产养殖技术领域,具体涉及一种唾液乳杆菌在凡纳滨对虾虾苗培育过程中提升虾苗生长性能的应用。
背景技术
凡纳滨对虾(Litopenaeus vannamei)属于甲壳类水产动物,分布范围广。耐盐,适宜生长盐度为0.5‰~40‰,适宜生长温度为23~30℃,适宜生长pH为7.5~8.5,水体溶氧量在4.0mg/L以上较好生长。从孵化到成虾为一个养殖周期来算,一般需要100~120天。
凡纳滨对虾是我国重要的海产品之一。2019年,我国凡纳滨对虾年产量已达111.8万吨,占海水养殖对虾总产量的77.1%。然而,随着高密度集约化养殖模式的推广,虾苗在培育过程中易受病毒、弧菌等病原体感染,导致病害频发。虾苗培育中常见的疾病为白斑综合征(白斑综合征病毒所致),致急性肝胰腺坏死病(副溶血性弧菌所致)和生长缓慢(虾肝肠胞虫)等。其中,细菌性疾病以弧菌为主,除了副溶血性弧菌外,还有哈维氏弧菌、鳗弧菌、溶藻弧菌、坎氏弧菌、创伤弧菌和霍乱弧菌等。
目前,应用于虾苗培育中病害防控手段以抗生素等化学药物为主,但其产生的药物残留、抗性基因转移,进而引起食品安全问题和环境污染问题,不利于人和动物的健康。因此,研发一种绿色安全且能有效控制病害的方法十分必要。
益生菌制剂即对动物有益无害的活的微生物,经特定工艺加工制备而成,用于调控养殖水域和水生动物的微生态平衡。它们不产生药物残留,不引起耐药基因转移,适合大面积使用,且不会引起食品安全问题,因此是一种绿色安全环保的可以替代化学药物的有效方法。其中,唾液乳杆菌(Lactobacillus salivarius)作为一种较为新颖的益生菌制剂,凭借其良好的益生作用正越来越受关注。
唾液乳杆菌属于乳酸菌,培养基为可发酵碳水化合物。无鞭毛,不产芽孢,兼性厌氧,***。据报道,唾液乳杆菌可以预防水生动物细菌性疾病,提升水生动物免疫能力,促进水生动物生长存活等。目前,唾液乳杆菌在水产养殖中的研究应用主要集中在鲍鱼和/或鱼如梭鲈、大菱鲆等,而国内关于唾液乳杆菌提高凡纳滨对虾虾苗生长性能的应用研究尚未见报道。
发明内容
为了克服现有技术的缺点与不足,本发明的目的在于提供一种唾液乳杆菌在凡纳滨对虾虾苗培育中的应用。
本实验室从广州市售泡菜中成功分离出唾液乳杆菌(Lactobacillussalivarius)GZPH2,具有广谱抑菌活性,能抑制金黄色葡萄球菌等39株致病菌生长,无溶血现象,不产组胺,耐酸,耐胆盐(CN 104611251 A、一株具有广谱抑菌活性的乳酸菌及其应用)。
本发明的目的通过下述技术方案实现:
本发明提供一种唾液乳杆菌在凡纳滨对虾虾苗培育中的应用。
进一步,所述的唾液乳杆菌在提高凡纳滨对虾生长性能中的应用。
更进一步,所述的唾液乳杆菌在提高凡纳滨对虾虾苗生长性能中的应用。
进一步,所述的唾液乳杆菌在促进凡纳滨对虾虾苗蜕皮和/或生长相关的次级代谢物合成的应用。
优选的,所述的唾液乳杆菌用于上调凡纳滨对虾虾苗代谢物短肽及氨基酸衍生物、倍半萜、类固醇及其衍生物、苯类化合物、类黄酮和/或泛酸。
进一步,所述短肽为二肽;更进一步为Methionyl-Proline、Valyl-Proline、Tyrosyl-Proline和L-phenylalanyl-L-proline中的至少一种;
进一步,所述的氨基酸衍生物为Glutamate,gamma-methyl ester;
进一步,所述倍半萜为法尼基半胱氨酸;
进一步,所述类固醇及其衍生物为3a,21-Dihydroxy-5b-pregnane-11,20-dione、3b-Allotetrahydrocortisol、Convalloside和2-Deoxycastasterone中的至少一种;
进一步,所述苯类化合物为N-[(4-Hydroxy-3-methoxyphenyl)methyl]octanamide、6-Gingerol和Adipostatin A中的至少一种;
进一步,所述类黄酮为Formononetin。
所述的应用是唾液乳杆菌发酵液拌料后投喂于凡纳滨对虾虾苗培育水体中实现。
其中,水体中的唾液乳杆菌浓度为104~106CFU/mL;优选为105CFU/mL。
所述的唾液乳杆菌为唾液乳杆菌(Lactobacillus salivarius)GZPH2,保藏单位:中国典型培养物保藏中心(CCTCC),保藏日期:2014年11月27日,保藏地址:中国武汉市武汉大学,保藏编号:CCTCC NO:M 2014598。该菌株在专利“CN 104611251 A、一株具有广谱抑菌活性的乳酸菌及其应用”中公开。
进一步的,所述唾液乳杆菌发酵液的浓度为8×107~1×108CFU/mL。
所述的唾液乳杆菌发酵液的制备方法,包括以下步骤:
将唾液乳杆菌单菌落接种于MRS液体培养基中,培养用作种子液;然后再以1~5%v/v(优选2%v/v)的接种量接种种子液于MRS液体培养基中,再次培养,即可得到唾液乳杆菌发酵液;其浓度为8×107~1×108CFU/mL。
所述的培养条件为在16~40℃,200rev/min(rpm)的条件下培养12~36h;进一步为在37℃,200rev/min(rpm)的条件下培养24h;
所述的再次培养条件为在16~40℃,静置的条件下培养12~36h;进一步为在37℃,静置的条件下培养24h。
本发明利用唾液乳杆菌GZPH2促进虾苗蜕皮和生长相关的次级代谢物合成,进而强化机体各机能,促进凡纳滨对虾虾苗的生长,通过将唾液乳杆菌发酵液拌料后投喂于凡纳滨对虾虾苗培育水体中实现。
本发明相对于现有技术具有如下的优点及效果:
本发明为保证唾液乳杆菌对凡纳滨对虾虾苗良好的益生作用,在虾苗的培育过程中,将唾液乳杆菌发酵液拌料放置10min后投喂于虾苗中,有助于促进蛋白质的消化吸收,增加抗氧化代谢物和蜕皮内源性成分的合成,进而显著提高虾苗的体长增长率和体重增长率,促进凡纳滨对虾虾苗生长,更好地满足凡纳滨对虾虾苗培育生产实践的需要。
附图说明
图1是实施例2中试验组与空白对照组之间的显著差异代谢物层次聚类热图;其中,横坐标中C7_1、C7_2、C7_3代表空白对照组的三个重复样本;L7_1、L7_2、L7_3代表试验组的三个重复样本。纵坐标代表显著差异代谢物,按代谢物的相对表达量显示颜色,红色越深则该代谢物表达量越高,蓝色越深则越低。
图2是实施例2中空白对照组与试验组之间的KEGG通路富集图;其中,横坐标代表通路名称,纵坐标代表富集率。柱子颜色梯度表示富集的显著性,颜色越深,代表该通路越显著富集,其中p-value<0.05的标记为*。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下列实施例中未注明具体实验条件的试验方法,通常按照常规实验条件或按照制造厂所建议的实验条件。所使用的材料、试剂等,如无特别说明,均为从商业途径得到的试剂和材料。
本发明提供了所述唾液乳杆菌发酵液在凡纳滨对虾虾苗中促进其生长的应用。
以下将通过实施例对本发明进行详细描述。
实施例中涉及到的培养基:
MRS液体培养基(g/L):蛋白胨20;葡萄糖20;牛肉浸粉8;酵母浸膏5;乙酸钠5;磷酸氢二钾2;柠檬酸氢二铵2;七水合硫酸锰0.25;七水合硫酸镁0.1;吐温-80:1;MSG(谷氨酸钠):3;海水晶3;最终pH6.2±0.2;分装后,在121℃、0.1MPa下灭菌处理20min,保存备用。
MRS固体培养基:在MRS液体培养基中加入5g/L碳酸钙,质量比为1.5%的凝胶强度为1300g/cm2的琼脂粉,在121℃、0.1MPa下灭菌处理20min,冷却至60℃时,倾倒于无菌平板,待凝固后翻转,保存备用。
实施例中所用的唾液乳杆菌为唾液乳杆菌(Lactobacillus salivarius)GZPH2。
实施例1唾液乳杆菌发酵液对凡纳滨对虾虾苗(PL 7-8)测试试验
(1)凡纳滨对虾虾苗(PL7-8)准备:PL7-8(postlarvae,PL7-8)是指7-8日龄的凡纳滨对虾仔虾,本次试验的虾苗全部由广东省的一个虾苗孵化场提供,大小一致,活跃健康,无疾病迹象。
(2)唾液乳杆菌发酵液制备:用无菌接种环从MRS平板中挑取单个菌落,接种于50mL MRS液体培养基中,在37℃,200rev/min(rpm)的条件下培养24h,用作种子液。然后,以2%v/v的接种量接种种子液于装有500mL MRS液体培养基的锥形瓶中,在37℃,静置的条件下发酵24h,这样即可得到浓度为8×107~1×108CFU/mL的唾液乳杆菌发酵液。
(3)试验开展:试验共进行7天,在6个水族箱中开展。每缸灌入洁净海水5L,每缸放养65尾虾苗,总共390尾凡纳滨对虾仔虾。具体过程如下所示:
试验分为2组,1个为空白对照组,1个为试验组,每组设置3个平行。试验用水族箱容量为8L,灌水前先用0.01M高锰酸钾溶液消毒,再用无菌水冲洗3遍,随后灌入盐度为15‰的洁净海水,每桶灌入5L。养殖试验前先对水进行曝气处理,采用气泵将养殖水体溶氧量维持在5ppm以上。试验过程中,室内温度维持在28℃左右。
将390尾虾苗随机分成2组,每组3个平行,每个平行含65尾虾苗。所有虾苗在实验实施前先放养于水族箱中培养24h以适应环境。在试验组,将唾液乳杆菌发酵液拌料后静置10min后投喂于虾苗中,最终在水体中的浓度为1×105CFU/mL。在空白对照组,不添加唾液乳杆菌发酵液。每组虾苗皆投喂基础饵料,基础饵料由广东省虾苗孵育场提供的虾苗专用饲料虾片,饲喂量按照每10尾虾苗投喂0.5mg虾片,一天每隔8h饲喂一次,定期清除残余饵料。
养殖采用统一管理方式,连续7天观察虾苗的健康状况,第0天和第7天分别量取虾苗体长和体重。试验结束后,记录活虾数目,计算存活率。
(4)试验结果:
表1空白对照组与试验组之间的生长性能指标
参考指标 | 空白对照组 | 试验组 |
第0天体长 | 11.17±0.17<sup>a</sup> | 10.17±0.25<sup>b</sup> |
第7天体长 | 10.60±0.24<sup>b</sup> | 11.27±0.50<sup>a</sup> |
体长增长量(TLG) | -0.57±0.11<sup>b</sup> | 1.10±0.72<sup>a</sup> |
体长增长率(PTLG) | -5.08±1.11<sup>b</sup> | 10.90±7.40<sup>a</sup> |
第0天体重 | 0.92±0.02<sup>a</sup> | 0.75±0.12<sup>b</sup> |
第7天体重 | 1.00±0.09<sup>a</sup> | 1.04±0.06<sup>a</sup> |
体重增长量(TWG) | 0.08±0.08<sup>b</sup> | 0.29±0.18<sup>a</sup> |
体重增长率(PTWG) | 8.13±5.03<sup>b</sup> | 43.48±19.76<sup>a</sup> |
特定生长率(SGR) | 1.16±0.72<sup>b</sup> | 6.20±2.82<sup>a</sup> |
存活率(SR) | 89.67±5.90<sup>a</sup> | 89.67±2.49<sup>a</sup> |
注:SR(%)=100×N7/N0
TLG(mm)=L7-L0
PTLG(%)=100×(L7-L0)/L0
TWG(mg)=W7-W0
PTWG(%)=100×(W7-W0)/W0
SGR(mg/day)=100×(W7-W0)/7
其中N7为7天试验结束后健康虾苗及试验过程中取样虾苗尾数之和;N0为起始虾苗尾数;L7为最终平均体长;L0为起始平均体长;W7为最终平均体重;W0为起始平均体重。
同一行数值上标字母不同代表差异显著,p<0.05;反之代表无显著性差异,p>0.05。
由表1可知,试验组虾苗的存活率与空白对照组等同(p>0.05)。
在体重增长方面,TWG、PTWG和SGR在试验组虾苗皆显著高于空白对照组(p<0.05);在体长增长方面,TLG和PTLG在试验组虾苗皆显著高于空白对照组(p<0.05)。可知,试验组虾苗表现出明显的生长优势。
综上,本发明的唾液乳杆菌发酵液可以有效提高凡纳滨对虾虾苗(PL7-8)的生长性能,促进体长、体重增长,为凡纳滨对虾虾苗生产养殖业提高经济效益。
实施例2唾液乳杆菌发酵液对凡纳滨对虾虾苗(PL 7-8)代谢组学研究
在实施例1的基础上,取第7天的虾苗进行LC-MS非靶向代谢组学分析,实验过程如下:
(1)样本前处理:精确称量50mg对虾样品,滴加400μL冷甲醇溶液(甲醇:水=4:1);在4℃下,于高通量组织破碎仪下破碎;涡旋混匀后,冰上超声萃取10min,重复3次。随后将样品置于-20℃静置30min。接着在13000g,4℃的条件下离心15min,离心后取上清液,转移至LC-MS带内衬管的进样小瓶中上机分析。为了测评仪器运行时分析***的稳定性,需配备一份质控样品(Quality Control,QC)。QC样品由所有检测样品混合制成。在仪器分析过程中,每6~8个分析样品***一个QC样品。在数据分析时,根据QC样品的聚集程度可用以评价整个分析过程中仪器的稳定性,还可用以捕捉分析***中变异大的变量,保证结果的可靠性。
(3)LC-MS分析:色谱分离:流动相A为水(含0.1%甲酸),流动相B为乙腈/异丙醇(1:1)(含0.1%甲酸);流速为0.40mL/min,进样量为10μL,柱温为40℃。质谱采集:采用离子喷雾电压(ESI)作为质谱的离子源进行检测。质谱源参数:质量范围:70-1050;鞘气:40psi;辅助加热气:10psi;离子源加热温度:400℃;离子化电压(正极):+3500V;离子化电压(负极):-2800V。碰撞能:20-40-60V。
(4)数据预处理及搜库鉴定:上机完成后,将LC-MS原始数据转换格式后导入Progenesis QI(Waters Corporation,Milford,USA)进行数据预处理,流程包括基线过滤、峰识别、峰面积自动积分、保留时间校正和峰对齐等操作。随后通过Progenesis QI软件搜库识别鉴定,将MS和MS/MS质谱信息与公共数据库(如HMDB和Metlin)进行匹配。代谢物的鉴定依据通常为质荷比(m/z)、质量数和保留时间。最终得到一个具有保留时间、质荷比和加合物等参数的数据矩阵。
(5)统计分析:统计分析前,数据矩阵采取总峰面积归一化,并删除QC样本相对标准偏差(RSD)≥30%的变量。利用R软件包ropls工具(version 1.6.2)进行样本主成分分析(PCA)和正交最小偏二乘判别分析(OPLS-DA),并进行200次置换检验来评估模型的准确性。
4.差异代谢物筛选
以VIP(变量权重值)>1,p<0.05为阈值筛选组间显著差异代谢物。同时将配有KEGGID号的代谢物映射到pathway通路进行富集分析,深入挖掘唾液乳杆菌影响虾苗生长的生物学过程。
5.实验结果
(1)差异代谢物筛选及层次聚类分析
符合筛选阈值的显著差异代谢物共33个。如图1和表2所示,相对于空白对照组,试验组上调的代谢物有25个,下调的有8个。其中,显著差异代谢物包括鞘脂、脂酰基化合物、氨基酸、短肽及其衍生物、孕烯醇酮脂类、类固醇及其衍生物、苯类化合物、类黄酮和维生素。
a.氨基酸、短肽及其衍生物:短肽在提高机体抗氧化性能上有重要作用。第7天时,该类化合物在试验组显著上调(p<0.05),说明唾液乳杆菌对短肽的合成有促进作用,提高机体抗氧化性能,保护PUFAs免受脂质过氧化。
b.类固醇及其衍生物:类固醇及其衍生物属于异戊二烯物质,该类化合物与甲壳动物蜕皮和生长调控密切相关。第7天时,类固醇及其衍生物在L组显著上调(p<0.05),说明唾液乳杆菌能促进虾苗类固醇及其衍生物的生成,进而缩短对虾蜕皮周期,这与乳酸菌组对虾体长增加的结果相一致。
c.苯类化合物:苯类化合物通常能抑制超氧化物的产生以及具有良好的抗炎和抗菌性能。Adipostatin A和6-Gingerol结构上含苯酚基,酚类的抗氧化性能可能与其提供电子和通过形成稳定的苯氧基自由基作为自由基清除剂的能力有关。因此,苯类化合物的上调是机体内维持氧自由基(ROS)平衡的代谢表现。第7天时,苯类化合物在L组显著上调(p<0.05),说明唾液乳杆菌对苯类化合物的合成有促进作用。
d.类黄酮:类黄酮具有潜在的生物活性,包括直接抗菌活性、与抗生素的协同作用和抑制细菌毒力。第7天时,类黄酮在L组的含量显著高于C组(p<0.05),说明唾液乳杆菌对类黄酮的合成有促进作用。
表2试验组与空白对照组显著差异代谢物
注:“VIP”指变量权重值,VIP>1代表该代谢物能区分样本差异。VIP值越大,说明代谢物对组间差异的贡献越明显。“pvalue”由独立样本T检验验证试验组与空白对照组代谢物是否具有显著性,pvalue<0.05代表该代谢物在两组间差异显著。“regulated”代表L组代谢物相对于C组的变化情况。
(2)KEGG通路富集分析
KEGG通路富集分析如图2和表3所示。结果显示,显著富集的代谢通路有6条(p<0.05),分别为Terpenoid backbone biosynthesis(萜类骨架的生物合成)、Vitamindigestion and absorption(维生素的消化与吸收)、beta-Alanine metabolism(β-丙氨酸代谢)、Pantothenate and CoA biosynthesis(泛酸和CoA的生物合成)、Sphingolipidmetabolism(鞘脂代谢)以及Sphingolipid signaling pathway(鞘脂信号通路)。其中富集的代谢物分别为Farnesylcysteine(法尼基半胱氨酸)、Pantothenic Acid(泛酸)和Sphinganine(二氢鞘氨醇)。
法尼基半胱氨酸属于倍半萜,为可促进甲壳动物蜕皮的关键内源性成分。萜类骨架的生物合成通路中,法尼基半胱氨酸在该通路上调,说明唾液乳杆菌能使该通路活跃,促进虾苗蜕皮,这与L组虾苗的体长得率显著增加的结果相一致。
泛酸又称维生素B5,在无饮食补充的情况下,由肠道共生细菌合成。在泛酸和CoA的生物合成通路中,泛酸在由泛酸激酶、磷酸泛酸-半胱氨酸连接酶、PPC-脱羧酶、去磷酰-CoA焦磷酸化酶和去磷酸肌酸激酶组成的酶***共同作用下形成乙酰CoA。乙酰CoA是碳分解代谢途径和能量代谢中关键代谢中间体,包括糖酵解、丙酮酸氧化和脂肪酸β氧化。由此,我们认为唾液乳杆菌的施用有助于调节虾苗机体泛酸和CoA的生物合成,促进乙酰CoA合成,参与能量代谢,为机体生长提供足够的能量。
综上可知,从代谢层面来看,唾液乳杆菌可通过调节代谢物上调/下调,进而调节机体生长性状。
表3 KEGG通路代谢物富集情况
KEGG通路 | 代谢物 |
Terpenoid backbone biosynthesis* | Farnesylcysteine(↑) |
Vitamin digestion and absorption* | Pantothenic Acid(↑) |
beta-Alanine metabolism* | Pantothenic Acid(↑) |
Pantothenate and CoA biosynthesis* | Pantothenic Acid(↑) |
Sphingolipid metabolism* | Sphinganine(↑) |
Sphingolipid signaling pathway* | Sphinganine(↑) |
注:“*”代表该通路显著富集,“↑”代表试验组中的含量相对于空白对照组上调。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.唾液乳杆菌在凡纳滨对虾虾苗培育中的应用,其特征在于:
所述的唾液乳杆菌为唾液乳杆菌(Lactobacillus salivarius)GZPH2。
2.权利要求1中所述的唾液乳杆菌在提高凡纳滨对虾生长性能中的应用。
3.根据权利要求2所述的应用,其特征在在于:
所述的唾液乳杆菌在提高凡纳滨对虾虾苗生长性能中的应用。
4.权利要求1中所述的唾液乳杆菌在促进凡纳滨对虾虾苗蜕皮和/或生长相关的次级代谢物合成的应用。
5.根据权利要求4所述的应用,其特征在于:
所述的唾液乳杆菌用于上调凡纳滨对虾虾苗代谢物短肽及氨基酸衍生物、倍半萜、类固醇及其衍生物、苯类化合物、类黄酮和/或泛酸。
6.根据权利要求5所述的应用,其特征在于:
所述短肽为Methionyl-Proline、Valyl-Proline、Tyrosyl-Proline和L-phenylalanyl-L-proline中的至少一种;
所述的氨基酸衍生物为Glutamate,gamma-methyl ester;
所述倍半萜为法尼基半胱氨酸;
所述类固醇及其衍生物为3a,21-Dihydroxy-5b-pregnane-11,20-dione、3b-Allotetrahydrocortisol、Convalloside和2-Deoxycastasterone中的至少一种;
所述苯类化合物为N-[(4-Hydroxy-3-methoxyphenyl)methyl]octanamide、6-Gingerol和Adipostatin A中的至少一种;
所述类黄酮为Formononetin。
7.根据权利要求1~6任一项所述的应用,其特征在于:
所述的应用是唾液乳杆菌发酵液拌料后投喂于凡纳滨对虾虾苗培育水体中实现。
8.根据权利要求7所述的应用,其特征在于:
水体中的唾液乳杆菌浓度为104~106CFU/mL。
9.根据权利要求7所述的应用,其特征在于:
所述的唾液乳杆菌发酵液的制备方法,包括以下步骤:
将唾液乳杆菌单菌落接种于MRS液体培养基中,培养用作种子液;然后接种种子液于MRS液体培养基中,再次培养,即得到唾液乳杆菌发酵液。
10.根据权利要求9所述的应用,其特征在于:
所述种子液的接种量为1~5%v/v;
所述的培养条件为在16~40℃,200rev/min的条件下培养12~36h;
所述的再次培养条件为在16~40℃,静置的条件下培养12~36h。
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