CN102028128A - 镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂及其在饲料脱毒中的应用 - Google Patents
镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂及其在饲料脱毒中的应用 Download PDFInfo
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- CN102028128A CN102028128A CN2010106013914A CN201010601391A CN102028128A CN 102028128 A CN102028128 A CN 102028128A CN 2010106013914 A CN2010106013914 A CN 2010106013914A CN 201010601391 A CN201010601391 A CN 201010601391A CN 102028128 A CN102028128 A CN 102028128A
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- toxin
- feed
- fusarium
- zearalenone
- biological
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Abstract
本发明公开了一种镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂及其在饲料脱毒中的应用，由消胆安、铝硅酸盐、碳酸铵、酵母细胞壁中的三种或多种(可任意比例)与内酯水解酶混合而成。所述的内酯水解酶质量含量40％-60％。本发明脱毒剂脱毒效果好，含生物脱毒剂的母猪饲料，生产出具有高效降解饲料中的F-2毒素含量的饲料，从而改善了饲料的营养质量，促进了母猪的健康成长，利于其适时发情和提高发情质量，提高母猪的年产胎数和产仔数，达到提高猪场养殖效益的目的。
Description
技术领域
本发明涉及一种脱毒剂，特别涉及一种镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂。
背景技术
真菌毒素是对粮食和饲料危害最大的生物性污染物之一。F-2毒素又名玉米赤霉烯酮(Zearalenone，F-2)为白色晶体，是由多种镰刀菌在代谢过程中产生的一种非类固醇结构产物，其具有雌激素样活性。主要对动物繁殖机能造成影响。F-2毒素是2，4--二羟基苯甲酸内酯类化学物，化学名称为6-(10-羟基-6-氧基碳烯基)-β-雷锁酸-u-内酯，主要由禾谷镰刀菌产生，此外还有三线镰刀菌(Fusarium tricinctum)、木贼镰刀菌(Fusarium equiseti)、雪腐镰刀菌(Fusarium nivale)、粉红镰刀菌(Fusarium roseum)，黄色镰刀菌(Fusarium culmorum)、串珠镰刀菌(Fusarium moniliforme)、茄病镰刀菌(Fusarium solani)、尖孢镰刀菌(Fusarium oxysporum)和表球镰刀菌(Fusarium episphaeria)等。F-2毒素最初于1962年由Stob M(1962)等从污染了禾谷镰刀菌的发霉玉米中分离得到，F-2毒素为白色晶体，分子式C18H22O5，熔点161-163℃，不溶于水，溶于碱性溶液、乙醚、苯及甲醇、乙醇等。其甲醇溶液在紫外光下呈明亮的绿蓝色荧光。Urry W H等(1966)应用经典化学、核磁共振和质谱技术确定了玉米赤霉烯醇的化学结构。1979年Mirocha C J等阐明其生物合成途径。
F-2毒素没有甾体结构，但具有雌激素活性。F-2毒素可由胃肠道持续吸收，肝肠循环可使F-2毒素在胃肠道滞留时间延长，F-2毒素主要随粪便排出，少量F-2毒素可由乳汁排泄。在鼠和禽，氚标记的F-2毒素在体内无蓄积作用。F-2毒素主要有2条代谢途径与葡萄糖醛酸结合，还原为玉米赤霉烯醇，玉米赤霉烯醇有2种异构体，即a-玉米赤霉烯醇(a-F-2ralenone)和β-玉米赤霉烯醇(β-F-2ralenone)。a-玉米赤霉烯醇的雌性激素活性为玉米赤霉烯酮的3倍，β-玉米赤霉烯醇的雌性激素活性小于或等于玉米赤霉烯酮。a-玉米赤霉烯醇转化为β-玉米赤霉烯醇(β-F-2ralenone)也属于解毒。a-玉米赤霉烯醇熔点较低(168--169℃)，而β-玉米赤霉烯醇熔点较高(174-176℃)。F-2毒素及其代谢物均有游离型和结合型，通过β-葡萄糖醛酸的作用，结合型转为游离型。
1.生殖毒性
在多种动物中对猪繁殖性能的影响报道较多，青年母猪对F-2毒素最敏感。F-2毒素可引起未性成熟母猪外阴水肿、子宫增大、乳腺增生，甚至直肠和阴道脱出；镜检表现为子宫内膜和子宫肌层细胞增生引起的水肿。有关F-2毒素对青年母猪性成熟和生产性能影响的研究报道较少，而且结论不尽一致。对于性成熟母猪，F-2毒素可引起多种生殖功能失调。对于怀孕
母猪主要是引起流产、死胎、新生仔猪死亡和胎儿干尸、胚胎吸收、少胎和弱胎。对于泌乳母猪，Chang等(1979)报道，饲喂不同剂量F-2毒素的饲料，可导致发情抑制、卵巢萎缩和子宫角弯曲；断乳--发情间隔时间延长；但不影响断乳后再配种。Christensen等(1972)报道，未成熟公猪采食含F-2毒素的玉米，可使睾丸重减少30％，甚至可中断精子生成；出现性欲降低和血液睾丸酮浓度下降。
牛对F-2毒素不如猪敏感，但与F-2毒素或产生F-2毒素的镰刀菌有关的不孕、产乳下降、雌激素过多症已有报道(Mirocha，1974)。饲喂含14mg/kgF-2毒素的大麦，可引起奶牛不孕；饲料中含25--100mg/kgF-2毒素，可导致荷斯坦牛吞咽障碍和生殖器出血，但发情和排卵正常(Mirocha，1978)，引起青年牛乳腺增大的饲料中分离出F-2毒素；用含385--1925ng/kgF-2毒素的饲料喂牛，产乳量不变，且血、乳、尿及组织中测不出F-2毒素(Shreeve，1979)。Noller等((1979)报道，F-2毒素污染的玉米含500ng/kgF-2毒素，不影响牛奶及乳脂产量；初情牛饲喂含250mg/kg F-2毒素共3个发情周期，受孕率为62％，而对照为87％(Weaver，1986)。Smith等(1986)报道，日25mgF-2毒素，可导致绵羊排卵率和怀孕率下降，Olsen等(1986)用800mg/kgF-2毒素饲喂小火鸡，不影响其采食、增重和血液睾丸酮浓度，但导致行为异常和肉冠增大；饲料中F-2毒素达100mg/kg，可使火鸡产蛋下降(Allen等，1983)。量。Jian Ying等研究了F-2及其代谢产物ZEL对老鼠精液质量、受精能力和睾丸激素浓度的负面影响。
2.致癌性
F-2有致癌性，能导致DNA收敛、染色体失常，从而产生遗传方面的危害。孙剑寒等研究发现F-2能通过活化人甾体激素和外源化学物受体诱导细胞色素P4503A4(CYP3A4)转录表达。严继承等研究发现F-2在1μmol/L，以上就能抑制细胞间隙连接通讯功能，提示它在一定条件下可能有促癌作用。F-2会结合到人的雌激素受体和刺激人乳腺癌细胞生长，免疫学研究表明在人体内有致癌性
细胞毒性
Obremski等(2005)报道：猪采食低F-2(400ugF-2/kg)污染的饲粮，肠粘膜形态结构虽然无显著变化，但杯状细胞的活性增加，囊泡中粘蛋白原浓度提高，淋巴细胞浸润，内分泌细胞和小肠隐窝底部的潘氏细胞活性增加，表明F-2调动了小肠粘膜的局部防御机制。该研究结果提示：F-2的作用不仅仅只局限于对生殖系统的影响。Vaca等(1988)指出：F-2以浓度依赖方式增加丙二醛(malondialdehye，MDA)的生成，通过增强脂质的过氧化反应诱导Vero细胞和Caco-2细胞的氧化损伤。因此，氧化损伤可能也是F-2发挥毒性作用的主要途径之一。
3.肝毒性
给雌性大鼠腹膜内注射F-2即(1.5-5.0mg/BW)，48h后ALT、AST、ALP、血清肌肝、胆红素等生化指标以及红细胞压积、平均红细胞容积(MCV)、血小板和白细胞等血液学参数发生改变(Maaroufi等，1996)，表明F-2有一定的肝毒性，对血凝过程也有一定损害作用。
孙美乐等(1997)在大鼠肝细胞体外培养液中加入0.5-15mgF-2/mL，结果使培养液中白蛋白及细胞内DNA含量下降，提示F-2对体外培养大鼠肝细胞有损伤作用。
4.免疫毒性
F-2能抑制植物血凝素刺激的人外周血淋巴细胞增殖，还能抑制刀豆素A和美洲商陆有丝分裂原刺激的B细胞和T细胞形成，可降低家畜对病原菌的抵抗力，增加对肿瘤的敏感性，改变巨噬细胞功能，降低NK细胞活力，降低迟发性超敏反应，F-2及其衍生物ZEL诱导胸腺萎缩和激活巨噬细胞。摄入10mg/kgF-2两周，降低对李斯特菌属抵抗力但不影响抗体形成细胞对SRBCS应答或迟发性超敏反应(Dean等1994)。对免疫器官的毒性作用报道也较多。5F-2毒素与其它毒素的毒力互作效应
检测霉菌毒素对小鼠免疫功能的影响时发现，F-2可显著降低同时给予DON对小鼠迟发性超敏反应的抑制效应(Kuiper等，1998)。DON与F-2联合呈剂量依赖性降低人和大鼠淋巴细胞增殖，F-2抑制淋巴细胞增殖的IC50是DON的250倍，两者具有加性效应(Additiveeffects)(Atkinson和Miller，1984)。DON和F-2对猪的联合效应表明：F-2不影响采食量、增重和饲料效率，但随日粮DON水平的增加线性降低采食量和增重(P＜0.05)。方差分析表明两种毒素无交互作用(P＝0.35)，但F-2以任何浓度存在时可进一步降低采食量(P＝0.08)。饲粮中DON增加胃、子宫和肾上腺的相对重，F-2不影响这些器官的相对重。赵虎等2009研究表明：日粮中添加1～3mg/kg F-2毒素不影响仔猪的平均日增重和饲料转化效率，可以增强仔猪细胞和体液免疫功能。苏军研究表明：镰刀菌毒素DON、F-2通过抑制细胞增殖内的抗氧化系统使细胞受损，从而导致动物消化道形态结构异常，并通过下调及转运载体的基因表达，降低养分吸收而导致动物生产性能下降，具有加性作用，表现出抗营养效应。
我国的F-2毒素的污染状况是比较广泛和严重的；因此去毒技术的研究尤其是生物去毒技术的研究是非常急迫的。
发明内容
本发明的目的是提供一种镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂及其在饲料脱毒中的应用，该种脱毒剂可彻底把F-2降解为无毒物质。
一种镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂，由消胆安、铝硅酸盐、碳酸铵、酵母细胞壁中的三种或多种(可任意比例)与内酯水解酶混合而成。
所述的内酯水解酶质量含量40％-60％。
所述的镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂，优选由消胆安、铝硅酸盐、碳酸铵、酵母细胞壁与内酯水解酶混合而成，各成分质量含量为消胆安10％-20％、铝硅酸盐10％-20％、碳酸铵10％-20％、酵母细胞壁10％-20％、内酯水解酶40％-60％。
所述的内酯水解酶为Fusarium oxysporum AKU 3702的内酯水解酶或内酯水解酶zhd101或两者任意比例混合物。
Naoko等克隆了F-2内酯水解酶zhd101，并在大肠菌表达，结果重组的大肠杆菌表现出很强的水解内酯酶的能力(NaokoTA，ShuichiO，Takehiko S，et al.Metabolism ofzearalenone by geneticallymodified organisms expressing the detoxification gene from clonostachysrosea[J]Applied andEnvironmental Microbiology，2004，70(6)：3239-3249)；Michihiko等对Fusarium oxysporum AKU3702的内酯水解酶基因进行了分析，根据内酯水解的氨基酸序列设计PCR引物，结果扩增产物长大约1000bp，进一步分析发现它含有5个内含子，6个外显子(Michihiko K，Makoto S，ChigusaS，et al.Lactonering-cleaving enzyme：Genetic analysis，novel RNA editing，and evolutionaryimplications[J]Proc.Nat.1 Acad.Sc.i USA.，1988，95：12787-12792)；Tomoko等把zhd101转化到玉米，结果玉米也表出对F-2的降解能力(Tomoko I，Naoko TA，Noriyuki O，et al.Reducedcontamination by the fusarium mycotoxin in zearalenone in maize kernels through geneticmodification with a detoxification gene[J]Applied and Environmental Microbiology，2004，73(5)：1622-1629)
所述的镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂在饲料脱毒中的应用。
所述的镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂在饲料脱毒中的应用方法为：在被F-2毒素污染的饲料中添加有效量的镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂。
一种添有上述镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂的饲料。
一种添有上述镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂的母猪饲料，由以下按重量份计的组分组成：被镰刀菌毒素玉米赤霉烯酮毒素(F-2毒素)污染的柠檬酸渣100-110份、豆油15-20份、豆粕745-760份、食盐17-19份、石粉51-55份、磷酸氢钙24-28份、赖氨酸24-26份、胆碱4-6份、蛋氨酸3-5份、苏氨酸3-5份、复合微量元素8-10份、复合维生素8-10份、抗氧化剂0.5-0.7份、镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂0.2-0.4份。
所述的添有上述镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂的母猪饲料，还包括以下组分：微生态制剂0.5-1份、复合酶制剂0.2-1份。
所述的复合微量元素是硫酸铜、硫酸铁、硫酸锰、硫酸锌、亚硒酸钠、氯化钴、碘酸钙中的两种或多种组合而成。
所述的复合维生素是维生素A、维生素D3、维生素E、维生素K3、维生素B1、维生素B2、维生素B6、维生素B12、烟酸、泛酸、叶酸、生物素中的两种或多种组合而成。
所述的微生态制剂是乳酸菌、酵母菌中的一种或多种组合而成。
所述的复合酶制剂是蛋白酶、淀粉酶、木聚糖酶、植酸酶等中的三种或数种任意比例混合而成。
复合酶制剂：提高肠道酶的含量，刺激酶的发育，同时通过参与一种或几种催化反应有效降解饲料中的F-2毒素含量，提高猪对饲料的吸收利用率，有效屏蔽F-2毒素带来环境雌激素效应。
抗氧化剂：保证饲料在储存过程中不发生氧化，保证维生素等营养物质的有效含量。
本发明镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂与单独微生物菌---内酯水解酶比较的效果见下表
表1
本发明添有上述镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂的母猪饲料，生产出具有高效降解饲料中的F-2毒素含量的饲料，从而改善了饲料的营养质量，促进了母猪的健康成长，利于其适时发情和提高发情质量，提高母猪的年产胎数和产仔数，达到提高猪场养殖效益的目的。
试验结果
为了验证本发明对猪的饲用效果，公司技术部于2008年9月在养殖户猪场进行了为期160天的饲喂试验。试验采用单因素对比试验设计，60头体重接近、健康的后备猪被随机分为试验组和对照组，每组包括3个重复，每个重复包括10头。试验结果表明：1、配种成功率(％)：试验组比对照组提高了11％；2、产仔数：试验组比对照组提高了17.7％；3、产仔窝重：试验组比对照组提高了24.55％。试验组的配方同实例3；对照组为未添加生物脱毒剂的空白对照。
表2单端孢霉烯族毒素生物脱毒技术对猪生产性能的影响
| 项目 | 对照组 | 试验组 |
| 配种成功率(％) | 76 | 87 |
| 产仔数(头) | 9 | 10.6 |
| 产仔窝重(千克) | 12.34 | 15.37 |
具体实施方式
实施例1：
一种添有上述镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂的母猪饲料，由以下按重量份计的组分组成：被F-2毒素污染的柠檬酸渣100份，豆油15份，豆粕742.3份，食盐17份，石粉51份，磷酸氢钙24份，赖氨酸24份，胆碱4份，蛋氨酸3份，苏氨酸3份，复合微量元素(硫酸铜与硫酸铁1∶1质量比)8份，复合维生素(维生素A、维生素E、维生素B1与维生素B21∶1∶1∶1的质量比)8份，抗氧化剂0.5份，镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂(消胆安10％、铝硅酸盐10％、碳酸铵10％、酵母细胞壁10％、Fusarium oxysporum AKU 3702的内酯水解酶60％)0.2份。
使用方法：体重60千克左右的猪，使用本实施例饲料配合日粮后饲喂(本实施例饲料25％+60％玉米+15％麸皮)，它的主要功能是高效降解饲料中的F-2毒素含量，从而改善了饲料的营养质量，促进了母猪的健康成长，利于其适时发情和提高发情质量，提高母猪的年产胎数和产仔数，达到提高猪场养殖效益的目的。
实施例2：
一种添有上述镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂的母猪饲料，由以下按重量份计的组分组成：被F-2毒素污染的柠檬酸渣85.1份，豆油20份，豆粕745份，食盐18份，石粉52份，磷酸氢钙26份，赖氨酸25份，胆碱5份，蛋氨酸3.5份，苏氨酸3.2份，复合微量元素(硫酸锌与亚硒酸钠质量比1∶1)8份，复合维生素(烟酸、泛酸与叶酸1∶1∶1质量比)8份，抗氧化剂0.5份，微生态制剂(酵母菌)0.5份、复合酶制剂(淀粉酶、木聚糖酶、植酸酶质量比1∶1∶1)0.2份，镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂0.2份(消胆安20％、铝硅酸盐10％、碳酸铵10％、酵母细胞壁20％、Fusarium oxysporum AKU 3702的内酯水解酶20％、内酯水解酶zhd10120％)。
使用方法同实施例1。
实施例3：
一种添有上述镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂的母猪饲料，由以下按重量份计的组分组成，被F-2毒素污染的柠檬酸渣110份，豆油25份，豆粕737.7份，食盐19份，石粉50份，磷酸氢钙27份，赖氨酸26份，胆碱6份，蛋氨酸4份，苏氨酸3.5份，复合微量元素(氯化钴与碘酸钙质量比1∶1)10份，复合维生素(维生素A、维生素D3与维生素E质量比2∶1∶1)10份，抗氧化剂0.6份，微生态制剂(乳酸菌)0.8份、复合酶制剂(蛋白酶、淀粉酶与木聚糖酶质量比1∶1∶1)0.6份，镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂0.4份(消胆安15％、铝硅酸盐15％、碳酸铵15％、酵母细胞壁15％、内酯水解酶zhd10140％)。
使用方法同实施例1。
Claims (10)
1.一种镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂，其特征是，由消胆安、铝硅酸盐、碳酸铵、酵母细胞壁中的三种或多种与内酯水解酶混合而成。
2.根据权利要求1所述的镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂，其特征是，所述的内酯水解酶质量含量40％-60％。
3.根据权利要求1所述的镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂，其特征是，由消胆安、铝硅酸盐、碳酸铵、酵母细胞壁与内酯水解酶混合而成，各成分质量含量为消胆安10％-20％、铝硅酸盐10％-20％、碳酸铵10％-20％、酵母细胞壁10％-20％、内酯水解酶40％-60％。
4.根据权利要求1-3任一项所述的镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂，其特征是，所述的内酯水解酶为Fusarium oxysporum AKU 3702的内酯水解酶或内酯水解酶zhd101或两者任意比例混合物。
5.权利要求1-3任一项所述的镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂在饲料脱毒中的应用。
6.一种添有权利要求1-3任一项所述的镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂的饲料。
7.根据权利要求6所述的饲料，其特征是，由以下按重量份计的组分组成：被镰刀菌毒素玉米赤霉烯酮毒素污染的柠檬酸渣100-110份、豆油15-20份、豆粕745-760份、食盐17-19份、石粉51-55份、磷酸氢钙24-28份、赖氨酸24-26份、胆碱4-6份、蛋氨酸3-5份、苏氨酸3-5份、复合微量元素8-10份、复合维生素8-10份、抗氧化剂0.5-0.7份、镰刀菌毒素玉米赤霉烯酮毒素的生物脱毒剂0.2-0.4份。
8.根据权利要求7所述的饲料，其特征是，还包括以下组分：微生态制剂0.5-1份、复合酶制剂0.1-1份。
9.根据权利要求6或7所述的饲料，其特征是，所述的复合微量元素是硫酸铜、硫酸铁、硫酸锰、硫酸锌、亚硒酸钠、氯化钴、碘酸钙中的两种或多种组合而成；所述的复合维生素是维生素A、维生素D3、维生素E、维生素K3、维生素B1、维生素B2、维生素B6、维生素B12、烟酸、泛酸、叶酸、生物素中的两种或多种组合而成。
10.根据权利要求8所述的饲料，其特征是，所述的微生态制剂是乳酸菌、酵母菌中的一种或多种组合而成；所述的复合酶制剂是蛋白酶、淀粉酶、木聚糖酶、植酸酶中的三种或数种任意比例混合而成。
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| CN106721932A (zh) * | 2016-11-25 | 2017-05-31 | 中州大学 | 一种饲料用畜禽生物脱霉剂及其应用 |
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