动物营养学报 >

2024 , Vol. 36 >Issue 7: 4339 - 4349

DOI: https://doi.org/10.12418/CJAN2024.374

研究论文

乳酸菌肽聚糖对雏鸭饲粮中黄曲霉毒素B1的脱毒效果研究

  • 邵怡豪 , 1, 2 ,
  • 王煜琦 1, * ,
  • 张宇豪 1 ,
  • 张广明 1 ,
  • 肖发沂 1 ,
  • 李庆梅 , 1, **
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  • 1 山东畜牧兽医职业学院动物科技学院,潍坊 261061
  • 2 烟台市牟平区动物疫病预防与控制中心,烟台 264100
**李庆梅,副教授,E-mail:

* 同等贡献作者

邵怡豪(1994—),男,山东烟台人,中级兽医师,硕士,研究方向为动物营养代谢病与中毒病。E-mail:

Copy editor: 武海龙

收稿日期: 2023-12-27

  网络出版日期: 2024-07-09

基金资助

山东省现代农业产业技术体系创新团队建设项目(SDAIT-21-03)

山东省新旧动能转换重大产业攻关项目(2021-50)

山东省高等学校低碳绿色养殖新技术研发中心项目(2022)

收起

Study on Detoxification Effects of Lactic Acid Bacteria Peptidoglycan on Aflatoxin B1 of Ducklings' Diet

  • SHAO Yihao , 1, 2 ,
  • WANG Yuqi 1 ,
  • ZHANG Yuhao 1 ,
  • ZHANG Guangming 1 ,
  • XIAO Fayi 1 ,
  • LI Qingmei , 1, **
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  • 1 Department of Animal Science and Technology, Shandong Vocational Animal Science and Veterinary College, Weifang 261061, China
  • 2 Muping Animal Disease Control Center, Yantai 264100, China
**E-mail:

* Contributed equally

Received date: 2023-12-27

  Online published: 2024-07-09

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摘要

本试验旨在探究乳酸菌肽聚糖对黄曲霉毒素B1(AFB1)的吸附作用,为乳酸菌肽聚糖的制备和饲粮中AFB1的脱毒提供科学依据及理论基础。试验以4种乳酸菌(嗜酸乳杆菌、嗜热链球菌、罗伊氏乳杆菌和植物乳杆菌)为原料提取乳酸菌肽聚糖,采用高效液相色谱法比较4种乳酸菌肽聚糖对AFB1的体外吸附率,选取体外吸附效果最好的罗伊氏乳杆菌肽聚糖进行动物试验。选择1日龄健康樱桃谷鸭120只,随机分为5组,每组4个重复,每个重复6只。对照组饲喂基础饲粮,AFB1组在基础饲粮中添加0.10 mg/kg AFB1,试验组在AFB1组饲粮中分别添加0.10%(Ⅰ组)、0.15%(Ⅱ组)和0.20%(Ⅲ组)罗伊氏乳杆菌肽聚糖。预试期3 d,正试期21 d。结果表明:1)乳酸菌肽聚糖种类、添加量及二者的交互作用均能够显著影响AFB1的体外吸附率(P<0.05),其中10.0 mg/mL罗伊氏乳杆菌肽聚糖对AFB1的体外吸附率最高,达75.29%。2)与对照组相比,AFB1组的平均日增重、平均日采食量显著降低(P<0.05),料重比显著升高(P<0.05);血浆谷草转氨酶(AST)、谷丙转氨酶(ALT)、碱性磷酸酶(AKP)活性及丙二醛(MDA)含量显著升高(P<0.05),肝脏、脾脏、胸腺和法氏囊指数显著升高(P<0.05),血浆免疫球蛋白G(IgG)、免疫球蛋白M(IgM)、γ-干扰素(IFN-γ)、白细胞介素-2(IL-2)、白细胞介素-4(IL-4)含量和超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及总抗氧化能力(T-AOC)显著降低(P<0.05)。3)与AFB1组相比,Ⅰ、Ⅱ、Ⅲ组的平均日增重显著升高(P<0.05),料重比显著降低(P<0.05);Ⅰ、Ⅱ、Ⅲ组的血浆IgG、IFN-γ、IL-2、IL-4含量和SOD、GSH-Px活性显著升高(P<0.05),血浆AST、AKP、ALT活性及MDA含量显著降低(P<0.05);Ⅱ、Ⅲ组的胸腺和法氏囊指数显著降低(P<0.05)。由此可见,不同乳酸菌肽聚糖对AFB1体外吸附效果不同,饲粮中添加罗伊氏乳杆菌肽聚糖能够部分消除AFB1对雏鸭造成的生长性能下降,改善AFB1所导致的免疫功能降低以及肝脏毒性。

关键词： 肽聚糖; 乳酸菌; 黄曲霉毒素B1; 生长性能; 脱毒效果

本文引用格式

邵怡豪 , 王煜琦 , 张宇豪 , 张广明 , 肖发沂 , 李庆梅 . 乳酸菌肽聚糖对雏鸭饲粮中黄曲霉毒素B1的脱毒效果研究[J]. 动物营养学报, 2024 , 36(7) : 4339 -4349 . DOI: 10.12418/CJAN2024.374

Abstract

This experiment was conducted to explore the adsorption effect of lactic acid bacteria peptidoglycan on aflatoxin B1 (AFB1), so as to provide a theoretical basis for the preparation of lactic acid bacteria peptidoglycan and the detoxification of AFB1 in diet. The experiment used 4 types of lactic acid bacteria (Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus reuteri and Lactobacillus plantarum) as raw materials to extract lactic acid bacteria peptidoglycan, the in vitro adsorption rates of 4 types of lactic acid bacteria peptidoglycan on AFB1 were compared by high-performance liquid chromatography, and the Lactobacillus reuteri peptidoglycan with the best in vitro adsorption effect was selected for animal experiment. A total of 120 healthy Cherry Valley ducks were allocated into 5 groups with 4 replicates per group and 6 ducks per replicate. The control group was fed a basal diet, the AFB1 group was fed the basal diet supplemented with 0.1 mg/kg AFB1, and the experimental groups were fed the AFB1 group diets supplemented with 0.10% (group Ⅰ), 0.15% (group Ⅱ) and 0.20% (group Ⅲ) Lactobacillus reuteri peptidoglycan, respectively. The pre-experimental period lasted for 3 days, and the experimental period lasted for 21 days. The results showed as follows: 1) the species, addition amount and their interaction of lactic acid bacteria peptidoglycan could significantly affect the in vitro adsorption rates for AFB1 (P<0.05), and the 10.0 mg/mL Lactobacillus reuteri peptidoglycan had the highest in vitro adsorption rates for AFB1, which reached 75.29%. 2) Compared with the control group, the average daily weight gain and average daily feed intake of the AFB1 group were significantly decreased (P<0.05), and the feed to gain ratio was significantly increased (P<0.05); the aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AKP) activities and malondialdehyde (MDA) content in plasma were significantly increased (P<0.05), the liver, spleen, thymus and bursa of Fabricius indexes were significantly increased (P<0.05), and the immunoglobulin G (IgG), immunoglobulin M (IgM), γ-interferon (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4) contents and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and total antioxidant capacity (T-AOC) in plasma were significantly decreased (P<0.05). 3) Compared with the AFB1 group, the average daily weight gain of groups Ⅰ, Ⅱ and Ⅲ were significantly increased (P<0.05), and the feed to gain ratio was significantly decreased (P<0.05); the IgG, IFN-γ, IL-2, IL-4 contents and SOD, GSH-Px activities in plasma of groups Ⅰ, Ⅱ and Ⅲ were significantly increased (P<0.05), and the AST, AKP, ALT activities and MDA content in plasma were significantly decreased (P<0.05); the thymus and bursa of Fabricius indexes of groups Ⅱ and Ⅲ were significantly decreased (P<0.05). In conclusion, the lactic acid bacteria peptidoglycan has different in vitro adsorption capacity on AFB1. Dietary supplemented with Lactobacillus reuteri peptidoglycan has a certain recovery effect on the decline in growth performance of ducklings affected by AFB1, and can improve the reduced immune function and liver toxicity caused by AFB1.

Key words： peptidoglycan; lactic acid bacteria; aflatoxin B1; growth performance; detoxification effects

霉菌毒素是由各种霉菌产生的有毒次级代谢产物,主要是由曲霉菌属(Aspergillus)、青霉菌属(Penicillium)和镰刀菌属(Fusarium)等霉菌产生。在一项为期8年的全球饲粮和饲料原料中霉菌毒素的研究结果显示,72%的样品检测出至少1种霉菌毒素存在,38%的样品发现存在有多种霉菌毒素共同污染的状况[1]。自从1960年发现黄曲霉毒素B1(AFB1)后,AFB1已被确定为影响动物和人类健康的重要毒素[2]。联合国粮农组织(FAO)允许饲粮中的黄曲霉毒素最大残留量为总量(AFB1+黄曲霉毒素B2+黄曲霉毒素G1+黄曲霉毒素G2)小于15 μg/kg,我国《饲料卫生标准》(GB 13078—2017)也对饲粮中AFB1的限量做出了规定。虽然AFB1的理化性质极为稳定,但至今未见有关AFB1长期大量积累的报道,故可以推断自然界中存在可降解或吸附AFB1的微生物。
已有大量的研究表明,乳酸菌可通过细胞壁上的特殊结构,以非共价键相互作用与AFB1结合,降低其在胃肠道中的吸收,保护身体免受毒素侵害[3⇓⇓-6]。肽聚糖(peptidoglycan,PGN)是细菌细胞壁的重要组成成分,与细菌的生长分裂密切相关。Piotrowska[7]通过对3株乳酸菌吸附赭曲霉毒素A效果进行研究发现,热灭活后的细菌表现出的吸附效果比相同浓度的活菌高几倍。有研究确定,霉菌毒素与细菌细胞壁发生物理结合,其结合的主要位点位于肽聚糖[8⇓-10],且与其结构变化密切相关[11]。目前,国内外关于乳酸菌肽聚糖在动物生产中的研究较少,特别是乳酸菌肽聚糖对于AFB1的吸附作用的相关研究。因此,本试验选取4种不同乳酸菌进行细胞壁肽聚糖的提取鉴定,通过定量检测和动物试验深入研究乳酸菌肽聚糖对于AFB1的吸附作用,旨在为乳酸菌肽聚糖的制备及其对AFB1的脱毒提供科学依据及理论基础。

1 材料与方法

1.1 试验材料

嗜酸乳杆菌(Lactobacillus acidophilus)ACCC 11073、嗜热链球菌(Streptococcus thermophilus)ATCC 19258、罗伊氏乳杆菌(Lactobacillus reuteri)ATCC 23272和植物乳杆菌(Lactobacillus plantarum)ACCC 03954为实验室保存;AFB1标准品购自北京百灵威科技有限公司;枯草芽孢杆菌(Bacillus subtilis)肽聚糖标准品购自美国Sigma-Aldrich公司;溶菌酶(200 000 IU)购自上海酶联生物科技有限公司;试验用其他有机溶剂均为国产分析纯。

1.2 乳酸菌肽聚糖的提取鉴定

将活化好的嗜酸乳杆菌、嗜热链球菌、罗伊氏乳杆菌和植物乳杆菌分别接种至MRS培养基中,扩大培养后8 500 r/min离心5 min收集菌体,用无菌去离子水洗涤2次,然后采用三氯乙酸法(TCA法)提取肽聚糖[12]。肽聚糖提取主要步骤为磷壁酸的去除、蛋白质的去除、脂类物质的去除与S-层蛋白的去除,最后收集沉淀并洗涤,经冻干机冷冻干燥保存。采用溶菌酶进行肽聚糖鉴定,发现提取物能够被溶菌酶完全溶解,证明所提取物质为肽聚糖。

1.3 体外吸附试验

上述提取的4种乳酸菌肽聚糖分别设5.0、7.5、10.0 mg/mL 3个添加量,每个添加量3个重复。按照设定的添加量将乳酸菌肽聚糖加入5 mL 10 μg/mL AFB1水溶液中,超声波振荡使乳酸菌肽聚糖与溶液充分混匀,置于37 ℃、150 r/min的恒温摇床反应8 h后取出。反应结束后,6 000 r/min离心5 min,取上清液过3次0.22 μm水系滤膜到2 mL进样小瓶,通过高效液相色谱仪(Agilent 1100,美国)检测AFB1含量,色谱条件为:SB-C18色谱柱(Agilent ZORBAX,美国),流动相为乙腈-水(体积比4∶6),流速为1.0 mL/min,进样体积50 μL,柱温30 ℃。荧光检测器激发波长(EX)为365 nm,发射波长(EM)为430 nm,检测时间18 min。AFB1吸附率计算公式如下:
AFB1吸附率(%)=[(空白样中AFB1含量-上清液中AFB1含量)/空白样中AFB1含量]×100。

1.4 动物试验

1.4.1 试验设计

试验于2023年9—11月在山东畜牧兽医职业学院新旧动能转换智慧农牧示范园(寒亭)进行。试验采用单因素完全随机设计,选取1日龄樱桃谷鸭120只,随机分为5组,每组4个重复,每个重复6只,各重复之间体重接近(P>0.05)。对照组饲喂基础饲粮,AFB1组在基础饲粮中添加0.1 mg/kg AFB1,试验组在AFB1组饲粮中分别添加0.10%(Ⅰ组)、0.15%(Ⅱ组)和0.20%(Ⅲ组)罗伊氏乳杆菌肽聚糖。预试期3 d,正试期21 d。基础饲粮参照NRC(1994)营养需要配制,其组成及营养水平见表1。基础饲粮中粗蛋白质、粗脂肪、钙、总磷含量分别参照国家标准GB/T 6432—2018、GB/T 6433—2006、GB/T 6436—2018和GB/T 6437—2018测定,代谢能参照《山东配合饲料资源应用商务章典》[13]中饲料原料代谢能值计算而得。试验温度为30~32 ℃,相对湿度为35%~50%。试验鸭自由采食和饮水,按正常免疫程序进行免疫接种。
表1 基础饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of the basal diet (air-dry basis)%

项目 Items 含量 Content
原料 Ingredients
玉米 Corn 63.10
小麦麸 Wheat bran 5.00
豆粕 Soybean meal 24.57
鱼粉 Fish meal 4.00
食盐 NaCl 0.30
磷酸氢钙 CaHPO4 1.80
L-赖氨酸 L-Lys 0.06
DL-蛋氨酸 DL-Met 0.17
预混料 Premix1) 1.00
合计 Total 100.00
营养水平 Nutrient levels2)
代谢能 ME/(MJ/kg) 12.49
粗蛋白质 CP 19.00
粗脂肪 EE 3.27
钙 Ca 0.87
总磷 TP 0.70

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:VA 8 500 IU,VB1 4.5 mg,VB2 7.0 mg,VB6 3.5 mg,VB12 0.02 mg,VD 700 IU,VE 20 IU,VK 3 mg,生物素 biotin 0.25 mg,叶酸 folic acid 2.1 mg,泛酸 pantothenic acid 10.5 mg,烟酸 nicotinic acid 65 mg,胆碱 choline 1 750 mg,Cu (as copper sulfate) 12.3 mg,Fe (as ferrous sulfate) 91 mg,Mn (as manganese sulfate) 58 mg,Zn (as zinc sulfate) 80 mg,I (as potassium iodide) 0.50 mg,Se (as sodium selenite) 0.20 mg。

2)代谢能为计算值,其余为实测值。ME was a calculated value, while the others were measured values.

1.4.2 AFB1攻毒饲粮的制备

根据预试验结果选定0.1 mg/kg为动物试验中饲粮中AFB1添加量。取10 mg AFB1标准品溶于5 mL无水乙醇中,配制成2 mg/mL的AFB1无水乙醇溶液,将2 mg/mL的AFB1无水乙醇溶液5 mL倒入100 g基础饲粮中,充分混匀,70 ℃干燥2 h,待饲粮完全干燥后,按比例配制AFB1攻毒饲粮。配制完成后进行四分法取样,采用上转发光免疫分析仪(UPT-3A-1800,北京热景生物技术股份有限公司)测定AFB1含量。

1.4.3 样品采集及指标测定

正试期第1天和最后1天对雏鸭空腹称重,统计耗料量,计算平均日增重(ADG)、平均日采食量(ADFI)和料重比(F/G)。
正试期最后1天对雏鸭进行心脏采血,肝素钠抗凝后3 000 r/min、10 min离心取血浆,使用试剂盒检测血浆免疫球蛋白G(IgG)、免疫球蛋白M(IgM)、白细胞介素-2(IL-2)、白细胞介素-4(IL-4)、白细胞介素-6(IL-6)、γ-干扰素(IFN-γ)含量,血浆总抗氧化能力(T-AOC)和谷丙转氨酶(ALT)、谷草转氨酶(AST)、超氧化物歧化酶(SOD)、碱性磷酸酶(AKP)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量,试剂盒均购自南京建成生物工程研究所,具体步骤按其说明书进行。
试验结束时扑杀雏鸭并立即分离肝脏、肾脏、胸腺、脾脏和法氏囊,生理盐水漂洗,滤纸吸干,分别称重,计算脏器指数:
脏器指数(%)=(脏器重量/体重)×100。

1.5 数据统计分析

采用SPSS 26.0统计软件对体外吸附结果进行一般线性模型(GLM)分析,统计模型中主效应包括乳酸菌肽聚糖种类、添加量及二者的交互作用。动物试验组间比较使用单因素方差分析(one-way ANOVA),并用Duncan氏法多重比较检验差异显著性,结果以平均值和均值标准误(SEM)表示,P<0.05表示差异显著。

2 结果

2.1 不同乳酸菌肽聚糖对AFB1体外吸附率的影响

如表2所示,乳酸菌肽聚糖种类、添加量及二者的交互作用均能够显著影响AFB1的体外吸附率(P<0.05)。主效应分析表明,罗伊氏乳杆菌肽聚糖对AFB1的体外吸附率显著高于嗜热链球菌肽聚糖、嗜酸乳杆菌肽聚糖和植物乳杆菌肽聚糖(P<0.05);乳酸菌肽聚糖添加量为7.5和10.0 mg/mL时对AFB1的体外吸附率显著高于添加量为5.0 mg/mL时(P<0.05)。在乳酸菌肽聚糖添加量为10 mg/mL时,不同菌种来源乳酸菌肽聚糖对于AFB1的体外吸附率由高到低依次为罗伊氏乳杆菌肽聚糖、植物乳杆菌肽聚糖、嗜热链球菌肽聚糖、嗜酸乳杆菌肽聚糖。以上结果表明,添加量为10 mg/mL的罗伊氏乳杆菌肽聚糖能够更好地吸附AFB1,因此选取罗伊氏乳杆菌肽聚糖进行后续动物试验。
表2 不同乳酸菌肽聚糖对AFB1体外吸附率的影响

Table 2 Effects of different lactic acid bacteria PGN on adsorption rate of AFB1 in vitro

项目
Items		 添加量
Addition amount/(mg/mL)		 吸附率
Adsorption rate/%


嗜热链球菌肽聚糖
Streptococcus thermophilus PGN		 5.0 51.77ef
7.5 68.13bc
10.0 65.51c


罗伊氏乳杆菌肽聚糖
Lactobacillus reuteri PGN		 5.0 66.51bc
7.5 73.67a
10.0 75.29a


嗜酸乳杆菌肽聚糖
Lactobacillus acidophilus PGN		 5.0 54.76de
7.5 58.28d
10.0 65.00c


植物乳杆菌肽聚糖
Lactobacillus plantarum PGN		 5.0 47.79f
7.5 56.99de
10.0 70.99ab
SEM 1.49
主效应 Main effects



种类 Species		 嗜热链球菌肽聚糖 Streptococcus thermophilus PGN 61.80b
罗伊氏乳杆菌肽聚糖 Lactobacillus. reuteri PGN 71.82a
嗜酸乳杆菌肽聚糖 Lactobacillus acidophilus PGN 59.34b
植物乳杆菌肽聚糖 Lactobacillus plantarum PGN 58.59b


添加量 Addition amount		 5.0 55.21b
7.5 64.27a
10.0 69.20a


P值 P-value		 种类 Species <0.01
添加量 Addition amount <0.01
种类×添加量 Species×addition amount <0.01

同列数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。

In the same column, values with different small letter superscripts mean significant difference (P<0.05), while with the same or no letter superscripts mean no significant difference (P>0.05).

2.2 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭生长性能的影响

如表3所示,与对照组相比,AFB1组的平均日增重、平均日采食量显著降低(P<0.05),料重比显著升高(P<0.05)。与AFB1组相比,Ⅰ、Ⅱ、Ⅲ组的平均日增重显著升高(P<0.05),料重比显著降低(P<0.05),平均日采食量无显著差异(P>0.05);但Ⅰ、Ⅱ、Ⅲ组的平均日增重、平均日采食量显著低于对照组(P<0.05),料重比显著高于对照组(P<0.05)。
表3 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭生长性能的影响

Table 3 Effects of AFB1 challenged diet supplemented with Lactobacillus reuteri PGN on growth performance of ducklings

项目
Items		 组别 Groups SEM P值
P-value
对照 Control AFB1 Ⅰ Ⅱ Ⅲ
平均日增重 ADG/g 38.05a 26.32c 30.66b 30.46b 32.39b 0.54 <0.01
平均日采食量 ADFI/g 98.61a 82.25b 88.10b 84.41b 87.34b 1.17 <0.01
料重比 F/G 2.59c 3.12a 2.87b 2.77bc 2.70bc 0.36 <0.01

同行数据肩标不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05)。下表同。

In the same row, values with different small letter superscripts mean significant difference (P<0.05), while with the same or no letter superscripts mean no significant difference (P>0.05). The same as below.

2.3 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭血浆免疫指标的影响

如表4所示,与对照组相比,AFB1组的血浆IgG、IgM、IFN-γ、IL-2和IL-4含量显著降低(P<0.05)。与AFB1组相比,Ⅰ、Ⅱ、Ⅲ组的血浆IgG、IFN-γ、IL-2和IL-4含量显著升高(P<0.05),Ⅱ、Ⅲ组的血浆中IgM含量显著升高(P<0.05);且Ⅰ、Ⅱ、Ⅲ组的血浆IgG、IgM、IFN-γ和IL-4含量与对照组无显著差异(P>0.05),Ⅰ、Ⅱ组的血浆IL-2含量与对照组无显著差异(P>0.05),Ⅲ组的血浆IL-2含量显著高于对照组(P<0.05)。
表4 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭血浆免疫指标的影响

Table 4 Effects of AFB1 challenged diet supplemented with Lactobacillus reuteri PGN on plasma immune indexes of ducklings

项目
Items		 组别 Groups SEM P值
P-value
对照 Control AFB1 Ⅰ Ⅱ Ⅲ
免疫球蛋白G IgG/(g/L) 13.69a 9.16b 11.70a 11.81a 12.21a 0.44 <0.01
免疫球蛋白M IgM/(g/L) 1.72a 1.27b 1.58ab 1.70a 1.76a 0.06 0.04
γ-干扰素 IFN-γ/(pg/mL) 85.18a 60.47b 83.43a 80.26a 86.90a 2.66 <0.01
白细胞介素-2 IL-2/(pg/mL) 60.50b 50.12c 61.36b 64.40ab 73.00a 2.04 <0.01
白细胞介素-4 IL-4/(pg/mL) 93.35a 62.19b 78.04a 83.41a 90.09a 3.13 <0.01
白细胞介素-6 IL-6/(pg/mL) 40.87 41.38 44.66 39.61 42.75 1.24 0.78

2.4 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭血浆抗氧化和肝脏损伤指标的影响

如表5所示,与对照组相比,AFB1组的血浆AST、ALT、AKP活性及MDA含量显著升高(P<0.05),血浆T-AOC及SOD、GSH-Px活性显著降低(P<0.05)。与AFB1组相比,Ⅰ组的血浆AST、AKP、ALT活性及MDA含量显著降低(P<0.05),血浆SOD、GSH-Px活性显著升高(P<0.05);且Ⅰ组的血浆AKP、SOD、GSH-Px活性和MDA含量及T-AOC与对照组无显著差异(P>0.05)。与AFB1组相比,Ⅱ组的血浆AST、AKP、ALT活性及MDA含量显著降低(P<0.05),血浆SOD、GSH-Px活性显著升高(P<0.05);且Ⅱ组的血浆AST、SOD、GSH-Px活性和MDA含量及T-AOC与对照组无显著差异(P>0.05)。与AFB1组相比,Ⅲ组的血浆AST、AKP、ALT活性和MDA含量显著降低(P<0.05),血浆SOD、GSH-Px活性及T-AOC显著升高(P<0.05);且Ⅲ组的血浆AST、AKP、ALT活性和MDA含量及T-AOC与对照组无显著差异(P>0.05),血浆SOD、GSH-Px活性显著高于对照组(P<0.05)。
表5 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭血浆抗氧化和肝脏损伤指标的影响

Table 5 Effects of AFB1 challenged diet supplemented with Lactobacillus reuteri PGN on plasma antioxidant and liver injury indexes of ducklings

项目
Items		 组别 Groups SEM P值
P-value
对照 Control AFB1 Ⅰ Ⅱ Ⅲ
谷草转氨酶 AST/(U/L) 7.63c 15.02a 10.66b 9.71bc 8.46c 0.63 <0.01
碱性磷酸酶 AKP/(U/L) 276.63c 413.21a 316.17bc 330.18b 319.90bc 11.76 <0.01
谷丙转氨酶 ALT/(U/L) 15.05c 21.69a 18.63b 18.21b 17.52bc 0.59 <0.01
丙二醛 MDA/(nmol/mL) 10.93c 15.02a 14.11bc 12.19bc 11.54bc 0.56 0.01
总抗氧化能力 T-AOC/(U/mL) 6.44a 4.01b 5.34ab 6.02ab 7.02a 0.36 0.04
超氧化物歧化酶 SOD/(U/mL) 101.21b 93.73c 101.30b 99.27b 106.76a 1.16 <0.01
谷胱甘肽过氧化物酶 GSH-Px/(U/mL) 488.36b 336.13c 459.58b 456.81b 602.93a 22.74 <0.01

2.5 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭脏器指数的影响

如表6所示,与对照组相比,AFB1组肝脏、脾脏、胸腺和法氏囊器官指数显著升高(P<0.05)。与AFB1组相比,Ⅱ组的肝脏指数显著降低(P<0.05),Ⅱ、Ⅲ组的胸腺指数和法氏囊指数显著降低(P<0.05);且Ⅱ、Ⅲ组的肝脏、脾脏和胸腺与对照组无显著差异(P>0.05),Ⅰ组的肝脏、脾脏和胸腺指数显著高于对照组(P<0.05),Ⅰ、Ⅱ、Ⅲ组的法氏囊指数显著高于对照组(P<0.05)。各组之间肾脏指数无显著差异(P>0.05)。
表6 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭脏器指数的影响

Table 6 Effects of AFB1 challenged diet supplemented with Lactobacillus reuteri PGN on organ indexes of ducklings%

项目
Items		 组别 Groups SEM P值
P-value
对照Control AFB1 Ⅰ Ⅱ Ⅲ
肝脏指数 Liver index 2.89c 3.70a 3.58ab 3.22bc 3.25abc 0.07 <0.01
肾脏指数 Kidney index 1.24 1.45 1.37 1.37 1.38 0.03 0.52
脾脏指数 Spleen index 0.12b 0.16a 0.15a 0.14ab 0.14ab 0.01 0.04
胸腺指数 Thymus index 0.36c 0.48a 0.43ab 0.39bc 0.41bc 0.01 <0.01
法氏囊指数 Bursa of Fabricius index 0.14c 0.21a 0.19ab 0.18b 0.18b 0.01 <0.01

3 讨论

3.1 乳酸菌肽聚糖对AFB1的体外吸附效果评价

目前乳酸菌对霉菌毒素脱毒机制主要依靠细胞壁的吸附作用,加热与酸处理可导致细胞壁结构发生改变,使肽聚糖交联减少,表面通透性增加,暴露大量的吸附位点,有利于吸附外来物质[14]。Mogahed Fahim等[15]研究表明,植物乳杆菌RM1和副干酪乳杆菌KC39的无细胞上清液降低了婴儿配方奶粉中的AFM1含量。Zou等[16]对乳酸菌进行热、酸处理后发现,其上清液中脱氧雪腐镰刀菌烯醇和T-2毒素含量显著减少。本试验对4种乳酸菌肽聚糖体外吸附AFB1的效果检测表明,不同肽聚糖对AFB1的体外吸附率表现出菌株特异性[17]。
肽聚糖之间的差异主要体现在肽尾和肽桥的部分,其免疫效应的发挥与肽尾和肽桥的交联程度相关[11]。Hernandez-Mendoza等[17]对乳酸菌-AFB1复合物进行反复洗涤,发现有不同程度的AFB1重新释放回溶液中,因此判断AFB1通过微弱的相互作用附着在细菌壁肽聚糖上。肽聚糖中较高的天冬氨酸含量反映了肽聚糖含有较多带正电的游离氨基,与肽尾未端D-丙氨酸(D-Ala)中含有带负电的游离羧基、糖链上的带负电羟基,共同通过静电作用等方式促进肽聚糖对AFB1吸附[18]。本试验中,10.0 mg/mL的罗伊氏乳杆菌肽聚糖对AFB1具有较高的吸附效果,吸附率达75.29%。物理吸附所形成的复合物极易被有机溶剂、机械振荡等因素分离破坏,致使结合的霉菌毒素被再度释放。本试验所采取的体外吸附方法虽然去除了有机溶剂的影响,但在样品检测过程中必经的离心步骤必然会破坏部分肽聚糖-AFB1复合物的复合状态。因此推测,各乳酸菌肽聚糖对AFB1的实际吸附效果理论上应高于本试验所得结果。

3.2 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭生长性能的影响

ŚliZewska等[19]研究发现,随着饲粮中AFB1水平提高,肉鸡平均日采食量、平均日增重显著下降。Chen等[20]通过饲粮中添加不同水平的AFB1发现,试验组的北京鸭平均日增重显著下降;Yang等[21]同样发现AFB1能够显著降低肉鸭对饲粮的利用效率。本试验结果表明,雏鸭饲粮中添加0.1 mg/kg的AFB1显著降低了平均日增重,显著提高了料重比,该结果与Han等[22]的研究结果一致。目前公认的是,饲粮中添加乳酸菌可以提高育雏期家禽的生长性能[23-24]。Chen等[25]在饲粮中添加唾液乳杆菌,发现其不仅可以有效降解AFB1,还能够提高生长性能,改善肉品质,增强特异性抗体产生。陈晓宇等[26]在蛋雏鸡饲粮中添加1 000 mg/kg罗伊氏乳杆菌肽聚糖,发现雏鸡平均日增重提高,料重比降低。本试验进一步证明了罗伊氏乳杆菌肽聚糖可以降低AFB1对雏鸭的危害,随着攻毒饲粮中罗伊氏乳杆菌肽聚糖的添加,雏鸭的平均日增重和料重比均得到明显恢复。

3.3 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭血浆免疫指标的影响

AFB1能够抑制体液免疫和细胞免疫机能,降低巨噬细胞的吞噬能力,并影响其炎性因子的分泌,从而影响机体的免疫水平[27]。本试验中,与对照组相比,AFB1组的血浆IgG、IgM、IFN-γ、IL-2和IL-4含量显著降低,与谢庆[28]的研究结果一致。推测其原因为AFB1通过与DNA、RNA发生特异性结合,抑制蛋白质合成,进而抑制机体免疫因子的产生,引发免疫抑制[29]。张桂枝等[30]试验结果表明,饲喂含AFB1的饲粮后雏鸡血清免疫球蛋白A(IgA)、IgG、IgM含量显著降低;但在饲粮中添加乳酸菌发酵制剂后,其免疫器官发育和免疫球蛋白含量均恢复正常。还有研究发现,使用乳酸菌与甘草提取物混合饲养,可改善AFB1中毒肉鸡的血液指标[31]。本试验中,在AFB1饲粮中添加罗伊氏乳杆菌肽聚糖后,雏鸭血浆中各项指标含量均有所提高,证明罗伊氏乳杆菌肽聚糖能够缓解AFB1对雏鸭免疫系统的损伤。

3.4 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭血浆抗氧化和肝脏损伤指标的影响

AFB1在动物体内主要表现出强烈的肝脏毒性,AKP、ALT和AST活性是临床上用于判断肝脏损伤的重要指标[28]。AKP紧密结合于肝细胞膜内,当肝脏损伤时由肝血窦进入血液循环[32];ALT主要存在于细胞质中,而AST主要存在于肝脏细胞线粒体内。当肝脏细胞受到损伤时,AKP、ALT和AST会进入血液,造成血液中的活性增加。有研究表明,AFB1能够显著提高雏鸡血清ALT、AST和ALP活性[29],并且通过下调核因子E2相关因子2(Nrf2)通路,增加脂质过氧化产物水平,降低抗氧化酶活性和抗氧化成分水平,从而诱导氧化应激[33]。本试验发现,饲喂AFB1攻毒饲粮可以使雏鸭血浆AST、ALT和AKP活性显著上升,而脂质氧化产物MDA含量显著增加,表明AFB1提高了雏鸭体内氧自由基水平,细胞损伤加剧,血浆SOD、GSH-Px活性降低,T-AOC减弱,表明血浆中的抗氧化酶减少,雏鸭的抗氧化能力下降。饲粮中添加乳酸菌复合添加剂,可以减轻黄曲霉毒素引起的毒性损伤,提高抗氧化能力[34],并降低AST、ALT和AKP活性,减轻肝脏病理损害[31]。本试验中,AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖可显著降低雏鸭血浆AST、ALT和AKP活性,证明罗伊氏乳杆菌肽聚糖可以降低饲粮中AFB1含量,起到保护雏鸭肝脏的效果;雏鸭血浆MDA含量降低,血浆SOD、GSH-Px活性及T-AOC升高,证明罗伊氏乳杆菌肽聚糖能够减轻AFB1对雏鸭氧化损伤程度[35]。

3.5 AFB1攻毒饲粮中添加罗伊氏乳杆菌肽聚糖对雏鸭脏器指数的影响

研究表明,AFB1能够显著增加肉鸭肝脏和脾脏指数[36]。同样,王晓琼[37]在番鸭饲粮中添加不同水平的AFB1,发现随饲粮中AFB1水平的提高,脾脏和法氏囊指数均呈升高趋势。本试验结果同样表明,AFB1能够显著提高胸腺、脾脏和法氏囊指数,其原因可能是由AFB1引起的机体免疫系统抑制或损伤,免疫器官代偿增重,而肝脏指数的上升同样代表肝脏为AFB1的主要作用靶器官[38]。研究发现,乳酸菌和酿酒酵母组合添加能够减少饲粮中AFB1残留,减轻肝脏和肾脏的组织学病变,改善肉品质[19]。本试验中,添加罗伊氏乳杆菌肽聚糖组的雏鸭脏器指数均随其添加量的增加呈下降趋势,说明罗伊氏乳杆菌肽聚糖对内脏器官肿大的损伤具有一定程度的减轻效果。

4 结论

① 4种乳酸菌肽聚糖对AFB1均具有良好的体外吸附效果,其中以10.0 mg/mL罗伊氏乳杆菌肽聚糖的吸附效果最佳,吸附率为75.29%。
② 在本试验条件下,饲粮中添加罗伊氏乳杆菌肽聚糖能够部分消除AFB1对雏鸭造成的生长性能下降,改善AFB1所导致的免疫功能降低以及肝脏毒性,其中添加量为0.20%的罗伊氏乳杆菌肽聚糖能够使各指标基本恢复至正常水平。

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