CN106616042A - 一种植物乳杆菌饲料及其应用 - Google Patents
一种植物乳杆菌饲料及其应用 Download PDFInfo
- Publication number
- CN106616042A CN106616042A CN201611203828.2A CN201611203828A CN106616042A CN 106616042 A CN106616042 A CN 106616042A CN 201611203828 A CN201611203828 A CN 201611203828A CN 106616042 A CN106616042 A CN 106616042A
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- CN
- China
- Prior art keywords
- cadmium
- parts
- feedstuff
- lactobacillus plantarum
- tilapia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Fodder In General (AREA)
Abstract
本发明公开了一种植物乳杆菌饲料及其应用,属于微生物技术领域。本发明将植物乳杆菌(Lactobacillus plantarum)CCFM 8610以离心浓缩获得的菌剂以饲料添加剂的形式加入到水生动物饲料预混料中进行制粒,含活菌数大于108CFU/g成品饲料的益生菌颗粒饲料。以该方法获得的饲料能够提高罗非鱼的生长速率;降低镉暴露罗非鱼体内镉含量,尤其是罗非鱼可食用部位的镉含量;改善镉暴露罗非鱼的血细胞指标以及血清生化指标;缓解镉暴露罗非鱼体内氧化应激损伤;调节镉暴露罗非鱼肠道微生物的组成及数量,抑制肠道内致病菌Flavobacterium与Pseudomonas的丰度;非常具有应用前景。
Description
技术领域
本发明涉及一种含植物乳杆菌鱼饲料及其应用,属于微生物技术领域。
背景技术
镉是人体非必需元素,具有很强的毒性以及致癌性。在美国毒物和疾病登记署(ATSDR)于2015年公布的危害物质目录中,镉位列第七位。工业上镉常用于电池、合金、颜料的生产。工业生产中含镉的废弃物若未经过处理,直接排放到自然环境中,会造成区域内环境重金属镉污染。环境中的镉会通过生物吸收以及体内蓄积作用会不断在食物链中积累,进而危害处于食物链顶端的人类的健康。近年来随着工业的不断发展,环境镉污染问题也愈发突出,其中水环境镉污染也在世界范围内广泛报道。水体环境镉超标不仅会对水生生物产生毒害,引发多种急慢性疾病,从而影响水产养殖业。同时,水生生物吸收水体中的镉并在体内逐渐蓄积,不仅威胁水产品的食用安全性,消费者的身体健康也会受到潜在危害。现有对镉的毒性研究中,水体中镉超标会使鱼类产生氧化应激,并对鱼有一定的免疫毒性,从而导致鱼的一些重要器官(如腮、肝、肾等)出现损伤并影响其正常的生理功能,进而抑制鱼类正常生长,并增加鱼类死亡率。
目前处理水生生物镉中毒的可选方法较少,其主要方法集中于水体中镉元素的减除以及镉毒害作用的缓解。水体中镉元素的减除主要依赖于吸附剂的吸附作用,如粉末活性炭、膨润土、海泡石等,但靠吸附剂降低水环境中镉浓度使用性较差且成本太高。缓解镉毒害则主要集中降低镉的生物利用率以及缓解机体氧化应激产生的危害,主要方法是在饲料中添加抗氧化剂以及镉吸附剂。其中镉吸附剂主要有壳聚糖,葡甘露聚糖与膨润土等,抗氧化物质则主要包括维生素C、无机硒等。但吸附剂的摄入会破坏水生生物体内原有的离子平衡,而添加抗氧化物质则成本过高。固而寻找一种高效且低成本的方法缓解镉对水生生物的毒害有着重要的意义。
已有的研究表明,鱼类的肠道微生物在鱼类的免疫力、新陈代谢能力以及抵抗病原菌能力方面有的不可或缺的作用。肠道菌群紊乱会使鱼肠道免疫系统失调并导致鱼类患病率增加。已有研究表明,益生菌可作为饲料添加剂混入饲料,并可有效调节鱼的肠道菌群,改善鱼的生长性能以及维持鱼体内免疫平衡。同时,某些特定的乳酸菌菌株具有高效吸附镉离子的特性,也可作为潜在吸附剂添加进饲料,加之乳酸菌的培养成本较低,可用于大规模的饲料制作。并且益生菌在缓解水生生物镉中毒方面的研究较少,因此,探究一种能够缓解水生生物镉中毒的饲料,进而降低水生生物可食用部位的镉含量,具有很大的研究意义以及应用价值。
发明内容
本发明的第一个目的在于提供一种水生动物饲料,所述饲料含有≥108CFU/g的植物乳杆菌CCFM 8610活菌。
在本发明的一种实施方式中,所述的植物乳杆菌CCFM 8610。该菌株已于2012年5月3日在北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物中心保藏,其保藏号为CGMCC No.6077,该菌株在申请号为201210322124.2的发明专利中公开。
在本发明的一种实施方式中,所述饲料是由植物乳杆菌CCFM 8610的菌剂与食品学上可接受的载体组成的。
在本发明的一种实施方式中,所述食品学上可接受的载体是指水生动物饲料的基础配料。
在本发明的一种实施方式中,所述水生动物饲料含有粗蛋白30~35份,粗脂肪5~10份,粗纤维10~15份,粗灰分10~15份。
在本发明的一种实施方式中,所述水生动物饲料含有大豆粕、次粉、面粉、鱼粉、矿物盐、氯化胆碱、复合维生素、酪蛋白、豆粕、大豆油、VC-磷酸酯钠、微晶纤维素、磷酸二氢钙中的至少一种。
在本发明的一种实施方式中,所述水生动物饲料含有大豆粕20~25份、次粉20~25份、面粉20~25份、鱼粉10~15份、矿物盐1~2份、氯化胆碱0.5~1份、复合维生素1~2份、酪蛋白8~10份、豆粕20~25份、大豆油5~10份、VC-磷酸酯钠0.2~0.5份、微晶纤维素2~4份、磷酸二氢钙1~2份。
本发明的第二个目的是提供一种水生动物饲料的生产方法,是将配料粉碎,与植物乳杆菌CCFM 8610菌液混合,制备成菌体含量≥108CFU/g的饲料。
在本发明的一种实施方式中,所述配料包括大豆粕、次粉、面粉、鱼粉、矿物盐、氯化胆碱、复合维生素、酪蛋白、豆粕、大豆油、VC-磷酸酯钠、微晶纤维素、磷酸二氢钙中的至少一种。
在本发明的一种实施方式中,所述菌液的制备包括以下步骤:将植物乳杆菌CCFM8610按体积比以2-4%的接种量接种到MRS培养基中,37℃下培养16~24h,离心并洗涤菌体,用生理盐水重悬使活菌浓度≥1011CFU/mL。
在本发明的实施方式中所述的饲料制作包括以下步骤:将粒状原料粉碎处理后与粉状原料按比例混合,得到预混饲料,将所述菌液按14mL/kg预混饲料的比例均匀混入预混饲料,以小型颗粒饲料机进行制粒,制粒温度为65℃。
本发明的第三个目的是提供一种饲喂水生动物的方法,所述方法是应用含有植物乳杆菌CCFM 8610的水生动物饲料饲喂水生动物。
在本发明的一种实施方式中,所述水生动物是罗非鱼,所述饲喂是按罗非鱼体重3~5%进行饲喂。
本发明还提供所述饲料在制备低含镉水产品中的应用。
有益效果:本发明的植物乳杆菌CCFM 8610在鱼饲料中的应用能够加快罗非鱼的生长速率,使其相对增长速率提高5%以上,并能有效降低罗非鱼肌肉中镉含量,由0.14μg/g降低至0.057μg/g,达到GB2762-2012中规定的鱼类镉限量标准(0.1μg/g),缓解罗非鱼体内因镉中毒导致的氧化应激损伤,改善镉暴露罗非鱼的血细胞指标以及血清生化指标,对于制备低镉含量、天然无重金属污染的罗非鱼及以罗非鱼肉为原料的食品非常具有应用前景。
附图说明
图1为本发明的饲料制作方法流程图;
图2是罗非鱼血液生理指标的变化:A,红细胞压积;B,血红蛋白浓度;C,白细胞数量;D,红细胞平均容量。注:标有不同字母的组表示有差异显著(P<0.05);
图3是罗非鱼血清生化指标的变化:A,总胆固醇;B,总甘油三酯;C,谷草转氨酶;D,谷丙转氨酶;E,高密度脂蛋白;F,总蛋白。注:标有不同字母的组表示有差异显著(P<0.05)
图4是罗非鱼肝胰脏丙二醛(A)和谷胱甘肽过氧化物酶活性(B)的变化;注:标有不同字母的组表示有差异显著(P<0.05);
图5是罗非鱼脑丙二醛(A)和超氧化物歧化酶活性(B)变化;注:标有不同字母的组表示差异显著(P<0.05);
图6是罗非鱼粪便微生物组成变化(属水平);
图7是罗非鱼粪便中微生物相对丰度的变化:A,Cetobacterium,Plesiomonas和Deefgea的相对丰度变化;B,Flavobacterium,Pseudomonas,Cellvibrio和Acinetobacter的相对丰度变化。
具体实施方式
实施例1水生动物饲料的制备
按表1-2所示的基础配方及营养组成配制饲料配料,按附图1所示的方法制备饲料,具体步骤如下:
将植物乳杆菌CCFM 8610按体积比以2-4%的接种量接种到MRS培养基中,37℃下培养18h,离心并洗涤菌体,用生理盐水重悬浮,使活菌浓度≥1011CFU/mL。将粒状原料经初清、粉碎处理后与粉状原料按表1配方混合,得到预混饲料;将上述菌液按14ml/kg预混饲料的比例均匀混入预混饲料后,以小型颗粒饲料机进行制粒,制粒温度为65℃,得到含该菌的颗粒饲料,所得颗粒饲料的活菌数大于108CFU/g。
表1基础饲料组成及含量
表2基础饲料组成和营养成分含量
实施例2植物乳杆菌CCFM 8610对镉暴露罗非鱼生长性能影响的实验
选取健康的罗非鱼200尾,初始体重平均值±标准差为34.0±1.16g,随机分成四组,每组三个平行,每个平行16-17尾。分组分别是,对照组:正常水体中饲养,饲喂基础日粮;益生菌CCFM 8610组:正常水体中饲养,饲喂CCFM 8610益生菌饲料;Cd暴露组:暴露于1mg/L的Cd2+溶液中,饲喂基础饲料;益生菌CCFM 8610干预组:暴露于1mg/L的Cd2+溶液中,饲喂CCFM 8610益生菌饲料。所使用的CCFM 8610饲料为附图1中以浓缩菌液方式制作的颗粒饲料,饲料的每日饲喂量约为罗非鱼体重的3%。实验为期4周,期间记录罗非鱼的饲喂量、体重和死亡情况,每周收集一次罗非鱼粪便。结束后记录罗非鱼体重,尾静脉取血后处死罗非鱼进行解剖。利用以下公式对罗非鱼相对增长速率、饲料转化率、存活率进行计算。
相对增长速率(%)=[(四周后鱼重(g)-初始鱼重(g))/初始鱼重(g)]×100%
饲料转化率=四周摄入饲料总量(g)/(四周后鱼重(g)-初始鱼重(g))
存活率(%)=(四周后存活鱼数量(尾)/初始鱼数量(尾))×100%"
计算结果如表3所示。由表3可知,对比CCFM 8610组和对照组,发现在相对增长速率和饲料转化率上均有有显著性差异,说明益生菌促进了罗非鱼的生长。
表3罗非鱼的生长指标变化
注:不同字母代表的组别间具有显著性差异(P<0.05)
实施例3植物乳杆菌CCFM 8610对镉暴露罗非鱼血液生理以及血清生化指标改善作用实验
按照实施例1所述以罗非鱼进行动物实验和分组,实验结束后对罗非鱼进行尾部静脉取血,每个处理组取15条,其中一半血液放入添加有抗凝剂(EDTA-2K)的无菌EP管中,使用血细胞分析仪(BC-5300V;Mindray,China)进行血液生理指标分析,另一半经静置1h后离心(3000g,10min)得到血清,使用全自动生化分析仪(BS-400;Mindray,China)进行血清生化分析。
测定结果如附图2和3所示。由附图2可知,镉暴露能显著减少罗非鱼血液中白细胞数量(WBC),同时红细胞压积(HCT)以及红细胞平均容积(MCV)降低,血红蛋白含量(HGB)下降。而通过CCFM 8610饲料干预,白细胞数量(WBC)和血红蛋白含量(HGB)下降趋势受到抑制。由附图3可知,镉暴露会显著改变罗非鱼血清中TC,TG,TP,ALT,AST和HDL含量(P<0.05),而通过益生菌饲料干预,TG,ALT和AST的变化趋势受到抑制。通过对比对照组和CCFM 8610组,可知含饲喂CCFM 8610的饲料会提高鱼血液中HGB含量以及血清中HDL含量,对其他指标并无显著性影响。以上数据表明,CCFM 8610能够改善镉暴露罗非鱼血液生理以及血清生化指标。
实施例4植物乳杆菌CCFM 8610对镉暴露罗非鱼体内镉含量的降低作用实验
按照实施例1所述以罗非鱼进行动物实验和分组,实验结束后处死罗非鱼进行解剖,取肝胰脏、肾脏、脾脏、鳃、脑、肠组织,背部肌肉,放入微波消解炉(MARS;CEM,UK)中消解20min,得到的消解液使用电感耦合等离子体质谱仪(NexIon-300X;PerkinElmer)测定镉含量。实验结果如表4所示。
表4罗非鱼组织镉含量
注:-表示未检测到;标有不同字母的组表示差异显著(P<0.05)
对比CCFM 8610干预组与镉暴露组可以发现,在脾、肾、腮、肠组织中,镉含量呈现显著性差异,而在脑和肝胰脏中差异则不显著,但是仍然表现出CCFM 8610干预组要低于Cd暴露组。同时可以发现,镉暴露组中罗非鱼背部肌肉中的镉含量达到了0.14μg/g,超出了GB2762-2012中规定的鱼类镉限量标准(0.1μg/g)。而通过饲喂益生菌饲料,罗非鱼背部肌肉中镉含量降低了59%,从0.14μg/g降低至0.057μg/g,达到GB2762-2012中规定的鱼类镉限量标准(0.1μg/g)。以上数据表明CCFM 8610能够降低镉暴露罗非鱼体内的镉含量,并显著减少罗非鱼可食用部位(即肌肉)中的镉含量。
实施例5植物乳杆菌CCFM 8610缓解镉暴露罗非鱼氧化损伤实验
以实施例4所述以罗非鱼进行动物实验和分组,实验结束后处死罗非鱼进行解剖,取肝胰脏,肾脏和脑,按照南京建成试剂测试盒的方法分别测定丙二醛(MDA)、还原性谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)的水平。测试结果如附图4和5所示。
测试结果如附图4和5所示,CCFM 8610抑制了罗非鱼因镉暴露造成的MDA含量和GSH-Px活性在肝胰脏中的变化,以及SOD活性和MDA含量在脑中的变化。SOD是生物体内重要的抗氧化酶,是生物体内清除自由基的首要物质,可对抗与阻断因氧自由基对细胞造成的损害,并及时修复受损细胞,复原因自由基造成的对细胞伤害,是生物体内氧化应激损伤的一种标志酶;GSH-Px是体内广泛存在的一种抗氧化酶,能将有毒的过氧化物催化成无毒物质。两者都是重金属暴露的生物标志物;MDA是一种生物体内的自由基与脂质发生过氧化反应的终产物,是生物体内氧化应激损伤的一种标志酶。以上结果说明,CCFM 8610缓解了罗非鱼镉中毒引起的氧化应激和氧化损伤。
实施例6
以实施例1所述以罗非鱼进行动物实验和分组,每周从水体中收集粪便,使用粪便DNA提取试剂盒(Omega Bio-tek,Norcross,GA,U.S.)提取粪便中所含DNA,并使用PCR扩增细菌16S rRNA中的V4V5区,扩增引物为515F(5′-barcode-GTGCCAGCMGCCGCGG-3′),907R(5′-CCGTCAATTCMTTTRAGTTT-3′)。
对扩增子进行电泳纯化,使用胶回收试剂盒(Axygen Biosciences,CA,US)回收纯化产物,并用荧光仪(Promega,US)进行定量。将纯化后的扩增子等量混合后,利用IlluminaMiSeqTM测序服务技术平台对混合物进行双尾测序。测序结果储存于ncbi的Sequence ReadArchive(SRA)数据库中。(收录号:SRP089871)
使用QIIME(版本1.17)对测序结果进行de-multiplex以及quality-filter。使用UPARSE将相似度在97%以上的片段序列聚类成为分类操作单元(OTUs),并利用UCHIME识别并移除嵌合序列。使用DPR分类(http://rdp.cme.msu.edu/)以及SILVA(SSU115)16S rRNA数据库对每个16S rRNA进行比对分类(置信度阈值为70%)。
罗非鱼肠道菌群分类结果如图6、7,镉暴露会降低肠道微生物多样性以及显著改变肠道菌群的结构。在属水平上,镉暴露会显著增加罗非鱼肠道内Flavobacterium,Pseudomonas,Cellvibrio和Acinetobacter的数量,同时显著降低Cetobacterium,Plesiomonas和Deefgea的数量。而通过饲喂含CCFM 8610饲料,镉暴露罗非鱼肠道内的Flavobacterium和Pseudomonas的丰度得到了抑制。由于这两种菌是常见的致病菌,故可认为含CCFM 8610饲料可改善罗非鱼肠道微生物结构,并有效抑制肠道病原菌的生长。
随后利用植物乳杆菌的特异性引物(LP-F,5′-GGAGCCGCTATTAGTATTTTCAT-3′和LP-R,5′-AATACAAGCAAGTCTTGGACCAG-3′)对罗非鱼粪便中该种群进行定量分析。结果如表5,可见镉暴露会降低罗非鱼能显著降低罗非鱼肠道内植物乳杆菌的丰度(第四周),而饲喂含CCFM 8610饲料则能显著增加罗非鱼肠道内该种群的丰度。
表5罗非鱼粪便中植物乳杆菌含量
以上数据表明植物乳杆菌CCFM 8610能调节镉暴露罗非鱼肠道微生物的组成及丰度,并有潜在的抑制肠道病原菌生长的能力。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种植物乳杆菌饲料及其应用
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> 人工序列
<400> 1
ggagccgcta ttagtatttt cat 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列
<400> 2
aatacaagca agtcttggac cag 23
<210> 3
<211> 23
<212> DNA
<213> 人工序列
<400> 3
ggagccgcta ttagtatttt cat 23
<210> 4
<211> 23
<212> DNA
<213> 人工序列
<400> 4
aatacaagca agtcttggac cag 23
Claims (10)
1.一种水生动物饲料,其特征在于,所述饲料含有≥108CFU/g的植物乳杆菌CCFM 8610活性成分。
2.根据权利要求1所述的饲料,其特征在于,所述饲料是由植物乳杆菌CCFM 8610的菌剂与食品学上可接受的载体组成的。
3.根据权利要求2所述的饲料,其特征在于,含有粗蛋白30~35份,粗脂肪5~10份,粗纤维10~15份,粗灰分10~15份。
4.根据权利要求2所述的饲料,其特征在于,含有大豆粕20~25份、次粉20~25份、面粉20~25份、鱼粉10~15份、矿物盐1~2份、氯化胆碱0.5~1份、复合维生素1~2份、酪蛋白8~10份、豆粕20~25份、大豆油5~10份、VC-磷酸酯钠0.2~0.5份、微晶纤维素2~4份、磷酸二氢钙1~2份。
5.一种水生动物饲料的生产方法,其特征在于,将配料粉碎,与植物乳杆菌CCFM 8610菌液混合,制备成菌体含量≥108CFU/g的饲料。
6.根据权利要求5所述的方法,其特征在于,所述菌液按如下步骤制备:将植物乳杆菌CCFM 8610的菌液按体积比以2-4%的接种量接种到MRS培养基中,37℃下培养16~24h,离心并洗涤菌体,用生理盐水重悬使活菌浓度≥1011CFU/mL。
7.一种饲喂水生动物的方法,所述方法是应用权利要求1所述的水生动物饲料饲喂水生动物。
8.根据权利要求7所述的方法,其特征在于,所述水生动物是罗非鱼,所述饲喂是按罗非鱼体重3~5%进行饲喂。
9.权利要求1-4任一所述的饲料在制备低含镉水产品中的应用。
10.权利要求7所述方法在制备低镉含量的水生动物食品中的应用。
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