CN110236081A - 一种霉菌毒素生物降解剂在仔猪饲料中的应用 - Google Patents
一种霉菌毒素生物降解剂在仔猪饲料中的应用 Download PDFInfo
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- CN110236081A CN110236081A CN201910616937.4A CN201910616937A CN110236081A CN 110236081 A CN110236081 A CN 110236081A CN 201910616937 A CN201910616937 A CN 201910616937A CN 110236081 A CN110236081 A CN 110236081A
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- A—HUMAN NECESSITIES
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
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- A—HUMAN NECESSITIES
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Abstract
本发明实施例公开了一种霉菌毒素生物降解剂在仔猪饲料中的应用;本发明通过将霉菌毒素生物降解剂用于仔猪饲料中,能够有效降低仔猪饲料中AFB1和ZEA在肝脏中的残留,进而能够更好地降低AFB1和ZEA对仔猪肝脏、空肠和子宫的损伤,同时能够解决饲料中AFB1和ZEA诱导的仔猪腹泻率高及仔猪血清中胆固醇、谷丙转氨酶、谷草转氨酶、乳酸脱氢酶含量高的问题。
Description
技术领域
本发明实施例涉及仔猪饲料技术领域,具体涉及一种霉菌毒素生物降解剂的应用。
背景技术
黄曲霉毒素B1(Aflatoxin B1简写为AFB1)是二氢呋喃氧杂萘邻酮的衍生物,含有一个双呋喃环和一个氧杂萘邻酮(香豆素);黄曲霉毒素B1是已知的化学物质中致癌性最强的一种。黄曲霉毒素B1对包括人和若干动物具有强烈的毒性,其毒性作用主要是对肝脏的损害。
玉米赤霉烯酮主要污染玉米、小麦、大米、大麦、小米和燕麦等谷物;其中玉米的阳性检出率为45%,最高含毒量可达到2909mg/kg;小麦的检出率为20%,含毒量为0.364~11.05mg/kg;玉米赤霉烯酮的耐热性较强,110℃下处理1h才被完全破坏;玉米赤霉烯酮具有雌激素样作用,能造成动物急慢性中毒,引起动物繁殖机能异常甚至死亡,可给畜牧场造成巨大经济损失。
黄曲霉毒素B1(AFB1)和玉米赤霉烯酮(ZEA)是谷物和动物饲料中广泛存在的两种霉菌毒素,给动物和人类带来了大量的经济损失和健康问题。为了消除其毒性,近些年来,人们尝试许多物理和化学的方法,但是收效甚微,难以缓解或降低饲料中存在的AFB1和ZEA对动物造成的损伤。
发明内容
为此,本发明实施例提供一种霉菌毒素生物降解剂在仔猪饲料中的应用,以解决现有技术中仔猪饲料中存在的AFB1和ZEA对仔猪组织损伤的问题。
为了实现上述目的,本发明实施例提供如下技术方案:
根据本发明实施例的第一方面提供一种霉菌毒素生物降解剂在仔猪饲料中的应用。
进一步地,所述应用为霉菌毒素生物降解剂在缓解仔猪饲料中黄曲霉毒素B1和玉米赤霉烯酮影响仔猪腹泻中的应用。
进一步地,所述应用为霉菌毒素生物降解剂在降低仔猪饲料中黄曲霉毒素B1和玉米赤霉烯酮在仔猪肝脏残留中的应用。
进一步地,所述应用为霉菌毒素生物降解剂在缓解仔猪饲料中黄曲霉毒素B1和玉米赤霉烯酮对仔猪肝脏、空肠或子宫组织损伤中的应用。
进一步地,所述应用为霉菌毒素生物降解剂在缓解仔猪饲料中黄曲霉毒素B1和玉米赤霉烯酮提高仔猪血清胆固醇含量中的应用。
本发明通过将霉菌毒素生物降解剂用于仔猪饲料中,能够有效降低仔猪饲料中AFB1和ZEA在肝脏中的残留,进而能够更好地降低AFB1和ZEA对肝脏的损伤,同时能够解决饲料中AFB1和ZEA诱导的仔猪腹泻率高及仔猪血清中胆固醇、谷丙转氨酶、谷草转氨酶、乳酸脱氢酶含量高的问题。
进一步地,所述霉菌毒素生物降解剂包括干酪乳杆菌、产朊假丝酵母菌、枯草芽孢杆菌、黄曲霉毒素B1降解酶和玉米赤霉烯酮降解酶;优选地,所述干酪乳杆菌的保藏编号为CGMCC1.2884;所述产朊假丝酵母菌的保藏编号为CGMCC2.0615;枯草芽孢杆菌的保藏编号为CGMCC1.0504。
本发明中对黄曲霉毒素B1降解酶和玉米赤霉烯酮降解酶的来源不做严格限制,优选地,所述黄曲霉毒素B1降解酶和玉米赤霉烯酮降解酶由米曲霉培养得到;具体地通过如下方法培养得到:
选取降解AFB1和ZEA的米曲霉菌株(保藏编号为CGMCC5817),将其涂布于PDA固体培养基中,置于30℃恒温培养箱,培养至产生大量孢子时收获;将适量灭菌生理盐水加入平皿中,用玻璃涂布棒将平皿上的孢子轻轻刮下,用4层纱布过滤除去菌丝残体,最后将米曲霉孢子浓度调整为1×108CFU/mL;然后将12mL米曲霉孢子悬液接种于米曲霉产酶固体发酵培养基(麸皮42g,玉米6g,豆粕12g,加入蒸馏水36mL,搅拌均匀,高压灭菌),置于30℃恒温培养箱中培养5-7d,待发现有大量米黄色孢子产生时收获,按照液固比为10:1的比例将生理盐水与固体发酵培养物混合均匀,30℃摇床振荡2h,静置4h,之后用8层纱布过滤,然后将滤液以10000r/min离心5min,最后用0.22μm滤膜过滤除菌后,保存于4℃备用;得AFB1降解酶活为284.3U/L,ZEA降解酶活为31.0U/L;活定义:在pH为8.0,温度为37℃条件下,1min能降解1ng AFB1或ZEA所需要的酶量,定义为一个酶活单位。
根据本发明实施例的第二方面提供一种仔猪饲料,所述仔猪饲料中干酪乳杆菌、产朊假丝酵母菌或枯草芽孢杆菌含量为(0.5-1.5)×109CFU/kg;所述仔猪饲料中黄曲霉毒素B1降解酶的酶活为280-290U/kg;所述仔猪饲料中玉米赤霉烯酮降解酶的酶活为28-35U/kg。
进一步地,所述仔猪饲料中干酪乳杆菌、产朊假丝酵母菌或枯草芽孢杆菌含量为1×109CFU/kg;所述仔猪饲料中黄曲霉毒素B1降解酶的酶活为284.3U/kg;所述仔猪饲料中玉米赤霉烯酮降解酶的酶活为31U/kg。
本发明通过限定仔猪饲料中所述霉菌毒素生物降解剂的各组分的具体用量,能够更好地降低AFB1和ZEA对仔猪的影响。
本发明实施例具有如下优点:
(1)本发明通过将霉菌毒素生物降解剂用于仔猪饲料中,能够有效降低仔猪饲料中AFB1和ZEA在肝脏中的残留,进而能够更好地降低AFB1和ZEA对肝脏的损伤,同时能够解决饲料中AFB1和ZEA诱导的仔猪腹泻率高及仔猪血清中胆固醇、谷丙转氨酶、谷草转氨酶、乳酸脱氢酶含量高的问题。
(2)本发明通过限定仔猪饲料中所述霉菌毒素生物降解剂的各组分的具体用量,能够更好地降低AFB1和ZEA对仔猪的影响。
附图说明
为了更清楚地说明本发明的实施方式或现有技术中的技术方案,下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍。显而易见地,下面描述中的附图仅仅是示例性的,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图引伸获得其它的实施附图。
图1为本发明实验例中提供的肝脏、空肠和子宫的HE染色图;
图2为本发明实验例中提供的肝脏、空肠和子宫中TNF-α表达免疫组化图;
图3为本发明实验例中提供的肝脏、空肠和子宫中Caspase-3表达免疫组化图;
图4为本发明实验例中提供的肝脏、空肠和子宫中的Nf-κB表达免疫组化图;
图5为本发明实验例中提供的肝脏、空肠和子宫中与细胞凋亡、结构屏障、营养物质转运和炎性因子相关基因表达情况。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
以下实施例采用的各原料如下:
干酪乳杆菌:保藏编号为CGMCC1.2884;购自中国普通微生物菌种保藏管理中心;
产朊假丝酵母菌:保藏编号为CGMCC2.0615;购自中国普通微生物菌种保藏管理中心;
枯草芽孢杆菌:保藏编号为CGMCC1.0504;购自中国普通微生物菌种保藏管理中心;
米曲霉菌:保藏编号为CGMCC5817,购自中国普通微生物菌种保藏管理中心;
黄曲霉毒素B1降解酶和玉米赤霉烯酮降解酶通过如下方法培养得到:
选取降解AFB1和ZEA的米曲霉菌株,将其涂布于PDA固体培养基中,置于30℃恒温培养箱,培养至产生大量孢子时收获;将适量灭菌生理盐水加入平皿中,用玻璃涂布棒将平皿上的孢子轻轻刮下,用4层纱布过滤除去菌丝残体,最后将米曲霉孢子浓度调整为1×108CFU/mL;然后将12mL米曲霉孢子悬液接种于米曲霉产酶固体发酵培养基(麸皮42g,玉米6g,豆粕12g,加入蒸馏水36mL,搅拌均匀,高压灭菌),置于30℃恒温培养箱中培养5-7d,待发现有大量米黄色孢子产生时收获,按照液固比为10:1的比例将生理盐水与固体发酵培养物混合均匀,30℃摇床振荡2h,静置4h,之后用8层纱布过滤,然后将滤液以10000r/min离心5min,最后用0.22μm滤膜过滤除菌后,保存于4℃备用;得AFB1降解酶活为284.3U/L,ZEA降解酶活为31.0U/L。
实施例1
本实施例为一种仔猪饲料,该仔猪饲料中干酪乳杆菌、产朊假丝酵母菌或枯草芽孢杆菌含量为0.5×109CFU/kg;所述仔猪饲料中黄曲霉毒素B1降解酶的酶活为290U/kg;所述仔猪饲料中玉米赤霉烯酮降解酶的酶活为35U/kg。
实施例2
本实施例为一种仔猪饲料,该仔猪饲料中干酪乳杆菌、产朊假丝酵母菌或枯草芽孢杆菌含量为1.5×109CFU/kg;所述仔猪饲料中黄曲霉毒素B1降解酶的酶活为280U/kg;所述仔猪饲料中玉米赤霉烯酮降解酶的酶活为28U/kg。
实施例3
本实施例为一种仔猪饲料,该仔猪饲料中干酪乳杆菌、产朊假丝酵母菌或枯草芽孢杆菌含量为1×109CFU/kg;所述仔猪饲料中黄曲霉毒素B1降解酶的酶活为284.3U/kg;所述仔猪饲料中玉米赤霉烯酮降解酶的酶活为31U/kg。
实验例
试验方法
试验地点河南省某养殖场;按照品种相同、胎次相近,体重相近的原则,选择150头42±2日龄、体重为11.03±0.5kg断奶“杜×长×大”三元杂交仔猪。随机分为3组,每组3个重复,10头/重复,公母各半,每个重复单圈饲养。各处理组间初始体重差异不显著(P>0.05);饲养试验共进行60d;所有仔猪均在同一猪舍内饲养,保持舍内清洁干燥通风,夏季温度23-31℃,相对湿度55-65%;采用自由采食和自由饮水,其它饲养管理均按照猪场统一要求进行。
试验日粮
试验基础日粮的配制及营养水平参照NRC(2012)小猪10-30kg的营养需要标准配置,采用玉米-豆柏型全价日粮,供试各组的营养水平完全相同,试验日粮为粉料;日粮配方及营养水平如表1所示;
表1日粮组成及营养水平(%)
注:预混料中每千克全价日粮提供:维生素B12 1.00μg,维生素E 100IU,维生素B11.00mg,维生素D3 3000IU,维生素K 1.50mg,维生素A 15000IU,维生素B2 6.25mg,泛酸35.00mg,胆碱50.00mg,烟酸61.8mg,叶酸2.00mg,生物素0.075mg;铜20.00mg,锌150.00mg,铁120.00mg,碘0.30mg,锰52.34mg,硒0.30mg。
试验分组设计:
用发霉的玉米100%等量替换玉米-豆粕型基础饲粮中的正常玉米,发霉玉
米中AFB1含量为76.68μg/kg、ZEA含量为42.16μg/kg;额外添加ZEA纯品,按照如下表2中试验分组设计中的AFB1含量为40μg/kg、ZEA含量为500μg/kg的日粮进行配制。
表2试验分组设计
1、试验期间每天记录每圈猪的采食量,及每头猪的腹泻情况,并做好记录。根据公式腹泻率=每组仔猪腹泻头数/试验总头数/试验天数×100%计算腹泻率,计算结果如表3所示;
表3
注:同行中字母相同代表差异不显著(P>0.05),字母不同则代表差异显著(P<0.05)。
由表3可知,C组腹泻率显著低于B组(P<0.05),腹泻率下降了54.30%,与A组相比,腹泻率差异不显著。
2、在试验结束后分别取料样,取仔猪肝脏组织各10g加入10mL预冷的80%甲醇溶液,用组织匀浆仪进行匀浆,然后采用酶联免疫吸附(ELISA)法测定AFB1和ZEA含量;黄曲霉毒素B1定量检测试剂盒:德国拜发ELISA试剂盒FAST Aflatoxin B130/15(R-Biopharm,Germany,货号:R1211);玉米赤霉烯酮定量检测试剂盒:德国拜发ELISA试剂盒FAST Zearalenon SC(R-Biopharm,Germany,货号:R5505);检测结果如表4所示:
表4
注:同行中字母相同代表差异不显著(P>0.05),字母不同则代表差异显著(P<0.05)。
由表4可知,ZEA在肝脏中的残留,A组和C组要显著比B组低31.87%和46.64%(P<0.05);AFB1在肝脏中的残留,C组显著低于B组51.43%(P<0.05),与A组差异不显著(P>0.05)。
3、在A、B和C三个处理组中,选取肝脏、空肠和子宫相同部位各约1cm3,用生理盐水冲洗干净,并用滤纸吸干水分,置于10%的福尔马林固定液中固定,24h之后换液一次,用于组织切片制作。
(一)、采用苏木精-伊红(HE)染色,染色步骤:取10%甲醛溶液中固定好的肝脏、空肠和子宫组织进行流水冲洗,经乙醇逐级脱水,二甲苯透明,石蜡包埋处理后,进行下面的操作:
(1)切片脱蜡:二甲苯脱蜡,梯度酒精冲洗掉二甲苯,蒸馏水洗涤;
(2)苏木素染色3min;
(3)用水冲洗,洗去苏木精液:1min,1%盐酸-乙醇10s(显微镜下观察效果);
(4)水洗1-2s,1%伊红染色10min;
(5)脱水,90%、95%、100%梯度酒精脱水各10min;
(6)二甲苯3-5min,中性树胶封片;
(7)显微镜观察,照相。
(二)、免疫组化(生物素-卵白素法-Sp法)步骤:
(1)切片脱蜡,PBS洗3×5min;
(2)柠檬酸抗原修复10min,自然冷却;
(3)PBS浸泡5min,3次(3×5min);
(4)3%H2O2封闭内源性过氧化物酶,室温20min;
(5)PBS浸泡5min,3次(3×5min);
(6)滴加山羊血清50μL/片,室温封闭内源性生物素20min;
(7)甩除勿洗,滴加一抗(1:100)50μL/片,4℃过夜;
(8)PBS浸泡5min,3次(3×5min);
(9)滴加二抗50μL片,37℃、30min;
(10)PBS浸泡5min,3次(3×5min);
(11)滴加辣根酶标记卵霉链白素50μL/片,37℃、30min;
(12)PBS浸泡5min,3次(3×5min);
(13)3,3-二氨基苯联胺(Diaminobenzidine,DAB)显色,显微镜观察;
(14)苏木素复染,梯度酒精脱水;二甲苯透明,中性树胶封片。
HE染色观察结果如图1所示;
由图1可知,从肝脏HE染色结果看,B组与A相比,肝脏炎症严重,肝小叶消失,肝板结构不明显,肝细胞水肿明显,炎症细胞浸润,个别肝细胞出现坏死,少量出现核溶解和核破裂现象;而C组炎症细胞减少,肝细胞水肿有所缓解。从空肠HE染色结果看,B组肠绒毛出现断裂,黏膜上皮脱落,小肠粘膜结构不完整;而C组空肠绒毛完整,肠粘膜正常。从子宫HE染色结果看,A、B和C三组之间差异不大,A组子宫正常,子宫内膜平整光滑,B组有较轻的炎症,C组较正常。
免疫组化各组织中Caspase-3、Nf-κB及TNF-α表达图如图2-4所示;
由图2-4计算肝脏、空肠及子宫中Caspase-3、Nf-κB及TNF-α表达量,计算结果如表5所示;
表5
注:同行中字母相同代表差异不显著(P>0.05),字母不同则代表差异显著(P<0.05)。
由图2-4和表5可知,Caspase-3蛋白免疫组织化学染色呈现棕黄色阳性颗粒,主要定位在细胞浆内,各组空肠组织Caspase-3蛋白都有不同程度阳性表达,其中A组Caspase-3蛋白呈现弱阳性表达,C组Caspase-3蛋白的表达强度较B组有所减弱。A、B和C组三组的NF-κB、TNF-α和Caspase-3的阳性率见表5,Caspase-3在肝脏和空肠中皆表达,其中B组和C组之间差异不显著,但是C组有降低的趋势,均显著高于A组(P<0.05),C组子宫中Caspase-3阳性表达率显著低于B组29.80%(P<0.05);NF-κB在肝脏和子宫中的表达,C组显著低于B组(P<0.05),阳性表达率分别降低了49.96%和8.71%;在空肠中的表达,C组与B组差异不显著(P>0.05),但有降低的趋势;TNF-α在肝脏、空肠和子宫中的表达,C组显著低于B组(P<0.05),阳性率分别降低了25.59%、44.93%和4.41%。
4、取-80℃的肝脏、空肠和子宫组织样本,加入1mL Trizol用组织匀浆仪匀浆,用Trizol法提取各组织样本RNA,反转录成cDNA,操作按照宝生物工程(大连)有限公司RT reagent Kit反转录试剂盒说明书进行,反转录cDNA于-20℃保存。引物由上海生物工程科技有限公司合成(见表6)。
表6荧光定量PCR引物序列
检测的肝脏、空肠和子宫组织中与细胞凋亡、结构屏障、营养物质转运和炎性因子相关基因表达量的检测结果如图5所示;
由图5可知,与B组相比,C组肝脏、空肠和子宫中细胞促凋亡因子Bax和Caspase-3的mRNA丰度均显著下调(P<0.05),而空肠和子宫中的抑制细胞凋亡因子Bcl-2则显著上调(P<0.05);与肠道上皮屏障功能相关的细胞因子ZO-1和Occludin在空肠和子宫中均显著上调(P<0.05),但在肝脏中ZO-1显著上调(P<0.05),而Occludin显著下调(P>0.05);与营养转运相关的细胞因子在空肠中PepT1和GLUT2显著上调(P<0.05),ASCT2和SGLT1显著下调(P<0.05),在肝脏中ASCT2和GLUT2显著上调(P<0.05),SGLT1显著下调(P<0.05);在子宫中PepT1、ASCT2和GLUT2均显著上调(P<0.05),SGLT1各组差异不显著(P>0.05)。
与B组相比,A组和E组空肠中炎性因子IL10、IL8、IL1β和TNF-α表达量下调(P<0.05),而IL6则上调(P<0.05);E组肝脏中IL10和IL8的表达量上调(P<0.05),A组肝脏中IL6的表达量也出现上调(P<0.05);A组和E组卵巢中IL10的表达量下调(P<0.05),而IL6和IL8则出现则表现为上调(P<0.05),另外A组IL1β出现上调而TNF-α出现下调(P<0.05)。
综上,A组的大部分炎性因子的mRNA丰度的变化与C组趋于一致,说明本发明中霉菌毒素生物降解剂具有缓解霉菌毒素对仔猪中组织损伤的作用,可使空肠、肝脏和子宫的功能恢复正常。
5、采样前空腹12h,每个重复随机选取1头仔猪,前腔静脉采血10mL,倾斜静置3h,取分离血清转移至1.5mL离心管,-20℃冰箱保存。采用全自动生化分析仪(型号:Au480,美国贝克曼库尔特公司)测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)和乳酸脱氢酶(LDH)总胆固醇含量。
测定结果如表7所示;
表7
注:同行中字母相同代表差异不显著(P>0.05),字母不同则代表差异显著(P<0.05)。
由表7可知,C组总胆固醇、谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)和乳酸脱氢酶(LDH)分别比B组降低了59.60%、47.78%、36.32%、46.26%和36.52%(P<0.05)。
虽然,上文中已经用一般性说明及具体实施例对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (9)
1.霉菌毒素生物降解剂在仔猪饲料中的应用。
2.根据权利要求1所述的应用,其特征在于,所述应用为霉菌毒素生物降解剂在缓解仔猪饲料中黄曲霉毒素B1和玉米赤霉烯酮影响仔猪腹泻中的应用。
3.根据权利要求1所述的应用,其特征在于,所述应用为霉菌毒素生物降解剂在降低仔猪饲料中黄曲霉毒素B1和玉米赤霉烯酮在仔猪肝脏残留中的应用。
4.根据权利要求1所述的应用,其特征在于,所述应用为霉菌毒素生物降解剂在缓解仔猪饲料中黄曲霉毒素B1和玉米赤霉烯酮对仔猪肝脏、空肠或子宫组织损伤中的应用。
5.根据权利要求1所述的应用,其特征在于,所述应用为霉菌毒素生物降解剂在缓解仔猪饲料中黄曲霉毒素B1和玉米赤霉烯酮提高仔猪血清胆固醇含量中的应用。
6.根据权利要求1-5任一所述的应用,其特征在于,所述霉菌毒素生物降解剂包括干酪乳杆菌、产朊假丝酵母菌、枯草芽孢杆菌、黄曲霉毒素B1降解酶和玉米赤霉烯酮降解酶。
7.根据权利要求6所述的应用,其特征在于,所述黄曲霉毒素B1降解酶和玉米赤霉烯酮降解酶由米曲霉菌种培养得到。
8.一种仔猪饲料,其特征在于,所述仔猪饲料中干酪乳杆菌、产朊假丝酵母菌或枯草芽孢杆菌含量为(0.5-1.5)×109CFU/kg;所述仔猪饲料中黄曲霉毒素B1降解酶的酶活为280-290U/kg;所述仔猪饲料中玉米赤霉烯酮降解酶的酶活为28-35U/kg。
9.根据权利要求8所述的仔猪饲料,其特征在于,所述仔猪饲料中干酪乳杆菌、产朊假丝酵母菌或枯草芽孢杆菌含量为1×109CFU/kg;所述仔猪饲料中黄曲霉毒素B1降解酶的酶活为284.3U/kg;所述仔猪饲料中玉米赤霉烯酮降解酶的酶活为31U/kg。
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