动物营养学报  2014, Vol. 26 Issue (9): 2797-2804   PDF (1170 KB)    
引用本文
盛清凯, 成建国, 赵红波, 窦红艳, 宣玉娟, 武英. 外源L-色氨酸、果聚糖、酪蛋白对猪粪发酵液粪臭素浓度的影响[J]. 动物营养学报, 2014, 26(9): 2797-2804.  
SHENG Qingkai, CHENG Jianguo, ZHAO Hongbo, DOU Hongyan, XUAN Yujuan, WU Ying. Effects of Additional L-Tryptophan, Fructan and Casein on Skatole Concentration in Pig Manure Fermentation Broths[J]. Chinese Journal of Animal Nutrition, 2014, 26(9): 2797-2804.  
外源L-色氨酸、果聚糖、酪蛋白对猪粪发酵液粪臭素浓度的影响
盛清凯, 成建国, 赵红波, 窦红艳, 宣玉娟, 武英    
山东省农科院畜牧兽医研究所, 山东省畜禽疫病防治与繁育重点实验室, 济南 250100
收稿日期：2014-4-3
基金项目：国家自然科学基金项目（31172245）
作者简介：盛清凯（1971-），男，山东陵县人，研究员，博士，研究方向为单胃动物营养与环境科学。E-mail：qksheng@163.com
摘要：为了减少粪臭素在猪体外的污染，本试验旨在研究不同剂量的外源L-色氨酸、果聚糖和酪蛋白对猪粪发酵液中粪臭素及相关合成物浓度的影响。将80头体重为（50.0±0.5） kg的“杜×长×大”三元杂交猪随机分为对照组和试验组，每组4个重复，每重复10头猪，分别饲喂基础饲粮和添加0.2%枯草芽孢杆菌的试验饲粮。6周后，每组随机选取8头猪，采集其新鲜粪便，配制粪水厌氧发酵液用于发酵试验。发酵试验采用3×3正交设计，发酵液中外源L-色氨酸、酪蛋白、果聚糖的添加剂量分别为0、0.05%、0.10%，0、0.125%、0.250%，0、0.75%、1.50%。色谱法测定发酵液中色氨酸、吲哚、吲哚-3-乙酸、粪臭素浓度，荧光标记单链构象多态性及片段长度多态性技术与毛细管电泳技术结合测定空白对照组和空白试验组发酵液中微生物的种群结构。结果表明：1）对照组色氨酸、吲哚、吲哚-3-乙酸、粪臭素浓度显著高于试验组（P<0.05）。2）外源L-色氨酸为发酵液中色氨酸、粪臭素的主要影响因素，酪蛋白为对照组中吲哚的主要影响因素，果聚糖为发酵液中吲哚-3-乙酸的主要影响因素。3）外源L-色氨酸、果聚糖与饲粮中的枯草芽孢杆菌无互作（P>0.05）。4）空白对照组猪粪发酵液优势菌以芽孢杆菌（CWBIB1434）为主，含量为26%；空白试验组发酵液以非解乳链球菌（AF201899）为主，含量为36%。 由此得出，降低猪粪中色氨酸的含量及在饲粮中添加枯草芽孢杆菌有助于减少猪粪发酵液中的粪臭素浓度。
关键词： 猪粪     色氨酸     果聚糖     酪蛋白     粪臭素    
Effects of Additional L-Tryptophan, Fructan and Casein on Skatole Concentration in Pig Manure Fermentation Broths
SHENG Qingkai, CHENG Jianguo, ZHAO Hongbo, DOU Hongyan, XUAN Yujuan, WU Ying    
Shandong Provincial Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China
Abstract: In order to reduce the pollution of pig skatole in vitro, this study investigated the effects of different doses of additional L-tryptophan (L-Trp), fructan and casein on skatole and its related compounds concentration in pig manure fermentation broths. Eighty Duroc×Landrace×Yorkshire pigs with (50.0±0.5) kg body weight were randomly allotted into control group and test group with 4 replicates per group and 10 pigs per replicate. Pigs were accordingly fed a basal diet and an adding 0.2% Bacillus subtilis test diet. After 6 weeks, 8 pigs in each group were randomly selected; their fresh manures were collected and prepared manure anaerobic broths for fermentation experiment. A 3×3 orthogonal design was used in the fermentation experiment. The additional doses of L-Trp, casein, fructan in fermentation broths were 0, 0.05% and 0.10%; 0, 0.125% and 0.250%; 0, 0.75% and 1.50%, respectively. The concentrations of Trp, indole-3-acetic acid (IAA), indole and skatole were detected by high performance liquid chromatography (HPLC) and microbial communities in fermented broths in blank control and blank test groups were detected by fluorescnce-based PCR-SSCP-FLP (F-PCR-SSCP-FLP) and capillary electrophoresis. The results showed as follows: 1) the concentrations of Trp, indole, IAA and skatole in test group were significantly decreased compared with the control group (P<0.05). 2) L-Trp was the main influence factor on Trp and skatole concentration in fermentation broths, respectively, casein was the main influence factor on indole concentration in the control group, and fructan was the main factor on IAA concentration in fermentation broths. 3) Additional L-Trp or fructan and Bacillus subtilis did not interact (P>0.05). 4) The bacteria Bacillus (CWBIB1434) was predominant and its content was 26% in the blank control group, and the Lactose Streptococcus (AF201899) was predominant and its content was 36% in the blank test group. The conclusion is that the reduction of Trp in pig manure and additional Bacillus subtilis in diet are beneficial to the abatement of skatole pollution in vitro.
Key words: pig manure     tryptophan     fructan     casein     skatole    

粪尿污染已成为严重的社会公害，养殖污染更加引起人们的注意。粪臭素是一种重要的粪便恶臭物质，不但污染环境，而且降低猪肉的品质，并且易导致疾病的发生。为了减少猪体内粪臭素等臭味物质的产生，人们常在饲粮中添加有益菌、果聚糖、酶制剂或配制氨基酸平衡饲粮等[1, 2]，以及给猪去势及注射疫苗。饲粮中添加有益菌可以调节肠道菌群，减少氨等臭味的产生[3]。果聚糖作为双歧杆菌益生因子，可能通过改变肠道微生物区系和发酵模式来影响粪臭素的产生[4]。配制氨基酸平衡饲粮可以降低饲粮粗蛋白质含量，有利于减少粪氮的排泄。色氨酸为猪的必需氨基酸，氨基酸平衡饲粮中常常添加。去势则降低猪的生产性能。饲粮中添加有益菌或果聚糖、饲喂氨基酸平衡饲粮、生猪去势以及注射疫苗只是减少生猪体内粪臭素的产生，对于猪粪便中粪臭素在猪体外的影响尚未见报道。本文研究外源L-色氨酸、果聚糖、酪蛋白添加对猪粪发酵液中粪臭素浓度的影响，为有效减少猪体外粪臭素的污染提供数据参考。 1 材料与方法 1.1 试验动物

80头体重为(50.0±0.5) kg的“杜×长×大”三元杂交商品猪。 1.2 试验设计

将80头猪随机分为对照组和试验组，每组4个重复，每重复10头猪。2组分别饲喂基础饲粮及添加0.2%枯草芽孢杆菌的试验饲粮，均为水泥地面饲养。枯草芽孢杆菌含量为109 CFU/g。6周后，对照组和试验组每组随机选取8头猪，每头猪采集新鲜粪便280 g，分别将其悬浮在2.8 L无菌厌氧的矿物质介质[每升含NaHCO3 5.0 g，NaCl 0.9 g，(NH4)2SO4 0.9 g，KH2PO4 0.45 g，K2HPO4·3H2O 0.45 g，CaC12·2H2O 0.03 g， MgCl2 0.02 g，MnSO4·4H2O 0.01 g，CoCl2·6H2O 0.01 g， FeSO4·7H2O 0.01 g，浓度为10%的半胱氨 酸溶液1.0 mL]中。将悬浮液随即转移到无菌充 满CO2的塑料袋中，拍打5 min，后用6层灭菌纱布过滤以除去粗微粒物质，配制成10%(m/v)粪水液[5]。从每头猪的10%粪水液中各取2.7 L，将其分别厌氧分装。每头猪的2.7 L粪水液都各自分装于27个150 mL无菌瓶中，每瓶100 mL，每3瓶为1组，共分为9组。将9组瓶按3×3正交试验设计，分别加入不同剂量的L-色氨酸、果聚糖和酪蛋白(表1)，38 ℃厌氧下分别培养24 h，然后从各瓶中分别取出20 mL溶液，进行粪臭素及相关合成产物浓度的测定。从对照组8头猪的空白发酵液(空白对照组)和试验组8头猪空白发酵液(空白试验组，即表1中试验1号)中各取1 mL各自混匀，分别液氮冷冻保存，进行菌群分析。

表1 猪粪发酵液中L-色氨酸、果聚糖和酪蛋白添加量 Table 1 The supplemental doses of L-Try,fructan and casein in pig manure fermentation broths
1.3 粪臭素浓度的测定

参考夏枚生等[5]高效液相色谱方法测定粪臭素及相关合成物浓度。测定设备及梯度洗脱程序调整为：美国Waters W2475高效液相色谱仪，色谱柱为Attlantisdc18、5 μm、4.6 mm×250 mm不锈 钢柱；柱箱温度30 ℃，荧光检测波长为激发波长 Ex280 nm、发射波长Em360 nm，流动相流速1.0 mL/min，进样量10 μL，具体见表2。L-色氨酸和吲哚-3-乙酸(IAA)标准品为Sigma公司产品,粪臭素(3-甲基吲哚)和吲哚标准品为Fluka公司产品。

表2 样品梯度洗脱程序 Table 2 Sample gradient elution program
1.4 猪粪发酵液中菌群分析

提取DNA，利用通用引物V3-340F、V3-532R扩增细菌16S rDNA V3区，利用荧光标记单链构象多态性及片段长度多态性(F-SSCP-FLP)技术进行PCR扩增，然后3730xl全自动分析仪进行毛细管电泳分离[6]，NCBI数据库进行Blast比对，确定发酵液中优势菌群的种类及含量。 1.5 数据处理

采用SAS 9.1软件进行分析，对照组数据和试验组数据平均值比较采用单因素方差分析(one-way ANOVA)及t检验,数据结果用平均值±标准误(n=8)表示。极差值及互作采用多因素方差分析(proc GLM)及Duncan氏法多重比较,极差值以最小二乘均值±标准误表示。P＜0.05表示差异显著，P＜0.01表示差异极显著。 2 结 果 2.1 不同处理对猪粪发酵液中粪臭素和相关合成产物浓度的影响

由表3可知，试验组中色氨酸、吲哚、吲哚-3-乙酸及粪臭素浓度显著或极显著低于对照组(P＜0.05或P＜0.01)，表明试验饲粮中添加枯草芽孢杆菌可以有效减少猪粪发酵液中色氨酸、吲哚、吲哚-3-乙酸及粪臭素的浓度。

表3 不同处理对猪粪发酵液中粪臭素和相关合成产物浓度的影响 Table 3 Effects of different treatments on skatole and its related compounds concentration in pig manure fermentation broths mg/L
2.2 不同外源物质对发酵液中粪臭素和相关合成产物浓度影响的极差分析

由表4可知，对于色氨酸，对照组和试验组中外源L-色氨酸的极差值大于酪蛋白和果聚糖，均极显著大于酪蛋白(P＜0.01),表明外源L-色氨酸为主要影响因素。对于吲哚，对照组中酪蛋白极差值最大，酪蛋白为主要影响因素，试验组果聚糖为主要影响因素。对于吲哚-3-乙酸，果聚糖为主要影响因素，极差值均极显著高于L-色氨酸和酪蛋白(P＜0.01)。对于粪臭素，对照组和试验组中的外源L-色氨酸极差值最大，表明外源L-色氨酸为粪臭素的主要影响因素。

表4 不同外源物质对发酵液中粪臭素和相关合成产物浓度影响的极差分析 Table 4 Analysis of range for effects of different additional substances on skatole and its related compounds concentration in pig manure fermentation broths
2.3 不同外源物质与饲粮中枯草芽孢杆菌的交互作用

由表5可知，L-色氨酸、酪蛋白、果聚糖与枯草芽孢杆菌对发酵液中色氨酸、吲哚、吲哚-3-乙酸、粪臭素浓度的交互作用P值皆大于0.05，皆无交互作用。

表5 不同外源物质与枯草芽孢杆菌的互作(P值) Table 5 Interaction of different additional substances and Bacillus subtilis (P-value)
2.4 空白对照组和空白试验组发酵液菌群结构

由表6可知，空白对照组中优势菌为芽孢杆菌(CWBIB1434)，含量为26%。空白试验组中优 势菌为非解乳链球菌(AF201899)，含量为36%； 其次为噬淀粉乳杆菌，含量为16%。空白对照组和试验对照组优势菌种类及组成皆不相同，表明发酵液中的菌群受饲粮枯草芽孢杆菌影响。

表6 空白发酵液中的优势菌群 Table 6 The dominant bacteria in blank fermentation broths

3 讨 论

养猪过程中产生的粪臭素，作为一种恶臭物质，不仅污染生猪及人类生活环境，并且也严重降低了猪肉品质。对于粪臭素，人们注重其对猪肉品质的影响，忽略了对环境的影响。粪臭素具有致癌的可能性[7]，并可导致牛羊肺气肿或肺水肿，对于粪臭素应给予高度重视。

粪臭素是猪体内色氨酸降解后产生的一种芳香族物质，色氨酸生成吲哚、吲哚-3-乙酸，后者脱羧转化为粪臭素。粪臭素代谢途径目前已经基本清楚，其在大肠中产生后，一部分通过粪便排出体外，另一部分通过门静脉到达肝脏代谢，代谢产物由尿排出；没有被代谢的粪臭素沉积在脂肪和肌肉组织中，影响猪肉质量。猪粪中分离出的产乙酸梭菌(Clostridium drakei、Clostridium scatologenes)[8]、梭菌C.disporicum[9]、梭菌(ATCC25775)[10]、乳酸菌(11201)[11]参与了色氨酸向粪臭素的转化。

本试验中发酵液补充外源L-色氨酸，其为猪粪发酵液中色氨酸及粪臭素浓度的主要影响因素，该结论与给猪盲肠中注射外源色氨酸后增加血液中粪臭素浓度的结论近似[12]，与饲粮中色氨酸对粪臭素影响不明显的结论[13]不同，不同原因可能在于外源色氨酸添加方式不同。Raffael等[14]认为，饲粮中色氨酸对粪臭素影响不明显的原因可能在于外源色氨酸在小肠中被吸收，不能被结肠中的微生物所利用。

理论上认为酪蛋白、饲粮中的蛋白质可以分解为色氨酸、酪蛋白或饲粮中的蛋白质间接影响粪臭素和吲哚的合成。本试验中，酪蛋白并非为粪臭素浓度的主要影响因素，该结论与Lin等[15]饲粮蛋白质对猪粪臭素几乎无影响的报道结论近似。Lundstrma等[16]报道粪臭素的浓度并不随着蛋白质的增加而增加。本试验中酪蛋白为对照组中吲哚浓度的主要影响因素，并非为试验组中酪蛋白浓度的主要影响因素，推断酪蛋白对粪臭素的作用可能与枯草芽孢杆菌相关。酪蛋白、饲粮蛋白质导致肠道内环境的改变与肠道内微生物发酵有关[17, 18]。

果聚糖作为一种益生元，通过促进肠道内有益菌的增殖而发挥肠道菌群的调节功能，从而提高动物的生产性能。本试验中果聚糖为试验组吲哚浓度的主要影响因素，与Xu等[19]结论一致。Xu等[19]认为0.5%~1.0%果聚糖增加吲哚的含量，而非粪臭素的含量，可能的原因为通过改变发酵环境的pH而影响菌群的生态系统。本试验中试验组和对照组的菌群生态系统确实改变，但是否通过pH尚无法判定。本试验中果聚糖也是吲哚-3-乙酸浓度的主要影响因素，推断也可能与菌群生态系统改变有关。本试验中果聚糖与枯草芽孢杆菌无交互作用，推断果聚糖可能通过其他菌发挥作用。夏枚生等[5]报道添加1.0%和1.5%的果寡糖显著降低猪粪发酵液色氨酸降解率和粪臭素相对产率,显著提高吲哚相对产率,显著降低梭菌和大肠杆菌数量,显著增加双歧杆菌和总厌氧菌数量。

猪体内色氨酸在微生物及相关酶的作用下转化为粪臭素，调控肠道中粪臭素转化过程中的菌群可以降低粪臭素的浓度。Vhile等[20]用洋姜饲喂未去势的公猪，结肠和直肠中的产气荚膜梭菌(C.perfringens)的数量降低，脂肪组织中的粪臭素含量显著下降。Li等[21]发现金华猪和外来长白猪的粪臭素相比，基因型的差异对于猪肠道菌群的影响起了重要作用。本试验中空白试验组猪粪发酵液的优势菌为非解乳链球菌(AF201899)和噬淀粉乳杆菌，空白对照组中的优势菌为芽孢杆菌(CWBIB1434)，空白对照组和试验对照组优势菌种类及组成皆不相同，表明发酵液中的菌群受饲粮枯草芽孢杆菌影响。

本试验中空白试验组发酵液中非解乳链球菌(AF201899)为优势菌，含量为36%。由于试验样品为猪粪，因此确定AF201899来源于猪肠道，该结论与国外从猪粪及猪肠道中检出非解乳链球菌(AF201899)的结论[22, 23]一致。关于AF201899的功能，目前尚未见报道。Rinkinen等[24]报道非解乳链球菌为狗空肠和粪便中产乳酸的优势菌，Vandanme等[25]报道非解乳链球菌分泌物具有β-糖苷酶、α-牛乳糖苷酶、淀粉酶、脲酶的活性，酸来源于半乳糖、牛乳糖等物质。饲粮中的乳酸可以降低肠道中粪臭素的含量[26, 27]。李凤[28]报道粪臭素可以在体外被乳酸链球菌6020厌氧降解。AF201899与粪臭素之间的关系还需进一步研究。本试验空白试验组中还检出优势菌噬淀粉乳杆菌，与噬淀粉乳杆菌存在于猪肠道中的报道一致[29]。噬淀粉乳杆菌为一种有益菌[30, 31]，可以抑制大肠杆菌导致的Toll样受体4(TLR4)感染信号[32]，分泌乳酸及淀粉酶[33]，猪结肠中在半乳低聚糖和双歧杆菌的作用下促进生长[34]。目前尚未发现噬淀粉乳杆菌直接降解粪臭素的报道。Meng等[35]报道乳酸杆菌(1.12)分泌的蛋白酶可以降解粪臭素。 4 结 论

① 降低粪便中色氨酸的含量有助于减少猪体外粪臭素的产生。

② 饲粮中添加枯草芽孢杆菌有助于降低猪粪发酵液中粪臭素的浓度。

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