全 文 ：第 15 卷 第 5 期
Vol. 15 No. 5
草 地 学 报
ACTA AGRESTIA SINICA
2007 年 9 月
Sep. 2007
文章编号: 10070435( 2007) 05050603 博士论文摘要
青贮中苜蓿蛋白的降解特性及化学添加剂的影响
博士生: 郭旭生 导师: 周 禾 玉 柱
(中国农业大学草地研究所, 北京 100094)
苜蓿 (M edicago sativa L. )在青贮时, 其
中的蛋白被大量降解形成非蛋白氮,而非蛋白
氮不能被反刍家畜有效地利用, 导致苜蓿青贮
料中氮的严重流失。本研究对青贮过程中苜蓿
蛋白的降解以及单宁酸、甲酸和甲醛等对蛋白
降解的抑制机理做了探讨,并分析了苜蓿青贮
后粗蛋白、氨基酸组成的变化、蛋白降解所形成
肽的种类及不同添加剂对其的影响。在此基础
上, 利用体外法对苜蓿青贮料的营养价值做了
初步评价。
1 苜蓿青贮过程中氮组分和蛋白降解酶的变
化及添加剂对其的影响
研究了苜蓿青贮过程中甲酸、甲醛及单宁
酸对苜蓿青贮料中氮的组分及植物蛋白降解酶
活性的变化的影响。苜蓿在青贮开始到第 7 d
时,甲酸能够有效地抑制蛋白的降解。单宁酸
没有甲酸和甲醛抑制蛋白降解的效果好, 但从
青贮开始到青贮 35 d的过程中,添加单宁酸处
理组苜蓿青贮料中非蛋白氮和游离氨基酸氮的
增加量要低于对照组和其它添加剂处理组。苜
蓿青贮过程中,蛋白降解酶羧基肽酶和酸性蛋
白降解酶随青贮时间的延长其活性降低的较
慢,而氨基肽酶则在青贮第 7 d 时就失活。因
此, 苜蓿青贮过程中对蛋白降解起主要作用的
是羧基肽酶和酸性蛋白降解酶。
2 苜蓿青贮过程中蛋白分子质量分布的变化
采用 SDSPAGE 不连续电泳方法研究了
苜蓿青贮不同时间,即 0, 1, 3, 5, 7, 14, 21, 35 d
时青贮料中蛋白分子质量分布的变化。结果表
明,苜蓿青贮料中分子量在 43 kDa~ 97. 4 kDa
范围内的蛋白从青贮青贮的第 1 d至青贮的第
35 d之间变化不是很明显, 而且在青贮的第
35 d时有些蛋白才被降解。而分子量在 20. 1
kDa~ 43 kDa 范围内的蛋白在青贮第 5 d 后被
大部分降解。上述结果说明,苜蓿在青贮时其中
的酶蛋白和细胞质及叶绿体可溶性蛋白被大部
分降解,而剩余的蛋白大部分为叶绿体膜蛋白。
3 苜蓿青贮料肽分子质量分布及不同添加剂
对其的影响
采用凝胶( SephadexG25, 中级)层析过滤
法分析了苜蓿青贮料中蛋白降解所形成肽的大
小及不同添加剂对其的影响。试验结果表明,
对照组苜蓿青贮料浸提液中的肽多为小肽,即
二肽和三肽。而甲酸处理的苜蓿浸提液中多为
3~ 7个氨基酸残基的寡肽, 而小肽的含量较
少。甲醛和单宁酸处理的苜蓿浸提液中的肽链
多为含有 3~ 4个氨基酸残基的寡肽。同时,甲
醛处理的苜蓿浸提液中也含有一定量的 7个氨
基酸残基以下的寡肽。
4 化学添加剂对苜蓿青贮粗蛋白及氨基酸组
成的影响
根据康乃尔净碳水化合物和蛋白质体系,
研究了甲酸,甲醛,单宁酸及这三种添加剂两两
混合处理对苜蓿青贮料中粗蛋白、氨基酸组成
的影响。苜蓿在青贮 40 d后,其中的大多数真
蛋白转变成了Fract ion A即非蛋白氮。而所有
添加剂处理,特别时甲酸/甲醛混合处理组显著
降低了青贮料中 Fraction A的含量( P< 0. 05)。
真蛋白组分 Fraction B1在添加剂特别是甲醛处
理组苜蓿青贮料中的含量显著高于其在对照组
苜蓿青贮料中的含量( P< 0. 05)。而在甲酸/甲
醛和单宁酸/甲醛混合处理的苜蓿青贮料中大部
分真蛋白分别为 Fract ion B3和 Fract ion B2。甲
醛和单宁酸处理后,增加了苜蓿青贮料中不能被
收稿日期: 20070622; 修回日期: 20070626
作者简介: 郭旭生( 1978) ,男,博士,研究方向为草产品加工与利用的研究, Email: guoxush eng_ 78@ 163. com
导师简介: 周 禾( 1955) ,男,教授,主要从事草坪、草产品加工与利用的研究, Email: zhouh e@ cau. edu . cn
玉 柱( 1963) ,男,教授,主要从事草产品加工与利用的研究, Email: yuzhu 3@ sohu. com
第 5期 郭旭生:青贮中苜蓿蛋白的降解特性及化学添加剂的影响
家畜消化利用的真蛋白组分 Fract ion C 的含
量。甲酸处理的苜蓿青贮料中总的氨基酸含量
高于对照组和其它添加剂处理的苜蓿青贮料中
的含量。从单个氨基酸的变化来看, 苜蓿青贮
过程中一些支链氨基酸和蛋氨酸能够较好的保
存,而酸性和碱性氨基酸则大部分被降解。
5 苜蓿青贮料体外消化特性及不同添加剂对
其的影响
采用活体外两阶段法研究了苜蓿干草及苜
蓿青贮料 DM, CP, NDF 及 ADF 的体外消失
率。苜蓿青贮料分别为青贮时添加甲酸,甲醛,
单宁酸,甲酸+ 单宁酸,甲醛+ 单宁酸,甲酸+
甲醛及对照组苜蓿青贮料。与苜蓿干草相比,
对照组苜蓿青贮料的 DM, CP 和 NDF 的体外
消失率均较高。各添加剂处理降低了苜蓿青贮
料中 DM 的体外消化率。对照组 CP 的体外消
化率显著高于其它添加剂处理组 ( P< 0. 05)。
除了甲酸和单宁酸混合处理组外,对照组苜蓿
青贮料其 ADF 的体外消化率与其它各添加剂
处理组之间差异不显著。但对照组苜蓿青贮料
其 NDF 的体外消化率显著高于添加剂处理组
( P< 0. 05)。
关键词: 苜蓿青贮; 蛋白降解; 蛋白组成; 肽; 氨基酸; 化学添加剂
中图分类号: S816. 53 文献标识码: A
完成日期: 2007年 5月
Study on the Characteristics of Proteolysis in Ensiled Alfalfa Treated
with Different Chemical Additives
Candidate: GUO Xusheng A dvisors: ZHOU He and YU Zhu
( In st itu te of Grass lan d S cien ce, China Agricultu ral University, Beijing 100094, China)
Ensiling alfalfa ( Medicago sativa L. ) forag e is an alternative w ay to avoid damage by w eather and
loss o f leaf by shat tering in the process o f haymaking . How ever, the problem is that the ex tensive prote
o lysis is usual ly occurred during ensiling r esult ing in the nit rog en of the ensiled alfalfa can not be ef ficiently
ut ilized by ruminant . T his experiment w as conducted to invest igate the protein proteolysis during alfalfa
fo rag e ensiling and the inhibit ing mechanism o f different addit iv es on it . Meanwhile, the changes of crude
pro tein, components o f amino acids, and pept ides in the ensiled forag e w ith dif ferent additives w ere ana
lyzed. M oreover, the deg radat ion characterist ics of the ensiled forage w ere also invest ig ated using in vi tr o
method.
Experiment 1. Changes in the N components and plant enzymatic activity during ensi lage of alfalfa treated
with different additives
Effects of formic acid, formaldehyde and tw o lev els of tannic acid on changes in the N components and
plant enzymatic act ivity during ensilage of alfalfa w ere studied. Inhibit ion o f proteolysis by form ic acid w as
more ef fective than the other addit ives during the first 7 d of ensiling. T annic acid w as not as effect ive at
inhibit ing production of nonpr otein N, ammoniaN and free am inoacid N as formic acid and formalde
hyde. How ever, increment of nonpr otein N and free am inoacid N in silag e f rom day 1 to day 35 of ensi
l ing w ere less w ith the higher level of tannic acid than that in the contro l and other additivetr eated silages.
Carboxypept idase and acid pr otease lo st it s act ivit ies slow ly w ith increasing t ime of ensiling. Aminopeptid
ase act ivity in all silages declined rapidly af ter ensiling.
Experiment 2. Changes in protein molecular weight distributions in ensi led alfalfa during ensilage
Study w as conducted to investig ate the changes o f protein mo lecular w eight in alfalfa silages ensiled 0,
1, 3, 5, 7, 14, 21, and 35 d, respect ively, using the SDSPA GE system contained discont inuous gel. The
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草 地 学 报 第 15卷
results show that changes in proteins w ith molecular w eight between 43. 0 kDa to 97. 4 kDa were no t dis
t inct f rom 1 to 35 days of ensiling . Proteins w ith molecular w eight betw een 20. 1 kDa to 43 kDa w ere dis
appeared at day 5 of ensiling. It w as concluded that the enzyme pro teins, cytoplasmic, and chlor oplastic
soluble proteins w ere larg ely degraded during ensiling, w hile the membranous pro teins w ere r elat ively w ell
preserv ed.
Experiment 3 Characterization of peptides in ensiled alfalfa treated with different additives
Effects of dif ferent additives on pept ides size w ere invest igated using gel f iltr at ion. T he addit iv es of
fo rmic acid, formaldehyde, and tannic acid w ere added to the alfalfa forag e at ensiling fo r 35 days. The re
sults show that the pept ides in the contr ol silage mainly w ere t ri or dipept ides. M ost of pept ides in the
fo rmic acid t reated silag e contained 3- 7 amino acid r esiduals, and most of pept ides in the formaldehyde o r
tannic acid treated silag es contained 3- 4 amino acid r esiduals. Furthermore, there w er e some peptides
contained 7 amino acid residuals in formaldehyde t reatedsilag e.
Experiment 4. Characterization of crud protein and amino acid components in ensi led alfalfa treated with dif
ferent additives
Effects of formic acid, formaldehyde, tannic acid, and the m ix tur e of each tw o of the addit ives on crud
pro tein and amino acid components w ere studied based on the Co rnell Net Carbohydrate and Protein Sys
tem in ensiled alfalfa. After 40 d of ensiling, most of forage t rue pro teins w ere converted to fr act ion A
( nonprotein nit rog en) and all o f the added addit ives signif icantly reduced fraction A content in the ensiled
fo rag es ( P< 0. 05) . The content of f ract ion B1 ( Water So luble True Protein) in all of the additivetreated
silages w as significant ly higher than that in control silag e, especially in fo rmaldehydetr eated silage ( P< 0.
05) . Large amounts of t rue protein in the fo rmic acid/ formaldehyde t reated and tannic acid/ formaldehyde
tr eatedsilages w er e f ract ions B3 ( Neutr al deterg ent insoluble nitr ogen minus acid detergent inso luble nit ro
gen) and fract ion B2 ( Water insoluble protein minus neutral deterg ent insoluble nit rog en) , respectively.
Formaldehyde or tannic acid increased the f ract ion C ( Acid Detergent Inso luble Nitr ogen) pr otein in the en
siled alfalfa. Amino acids w er e w ell preserved in additivetr eated silag es in compassion w ith the control si
lag e. Concentr at ion of to tal amino acid w as higher in formic acidt reated silages than that in the control and
the o ther addit iv et reated silages. T he change pat terns in individual amino acid in all of the silages indica
ted that branched chain am ino acids and methionine w ere r elat ively w ell preserved during fermentat ion, but
no t fo r the basic and acidic amino acids.
Experiment 5. Characteristics of in vi tro digestion in ensiled alfalfa treated with different additives
The dig est iv e rates of DM, CP, NDF and ADF of alfalfa hay and ensiled alfalfa forage w ere studied u
sing in vi tro method. T he ensiled fo rages w ere t reated with fo rmic acid, formaldehyde, tannic acid, and
the m ixture of each tw o above addit iv es or w ithout any additiv es at ensiling. The digestive rates of DM,
CP and ADF in the control silag e w ere higher than that of alfalfa hay. All of the addit ives decreased the di
gest ive rate of DM of ensiled forages. The CP dig est ive rate o f the contro l silage w as significant ly higher
than that of addit ivet reated silag e ( P< 0. 05) . No differ ence w as detected betw een the silage t reated w ith
fo rmic acid or formaldehyde alone and silages t reated with the mix ture o f fo rmic acid and formaldehyde o r
fo rmic acid and tannic acid. No difference o f digest ive rates of ADF was also observed betw een the contro l
silage and silages t reated w ith addit ives except for silag es t reated w ith formic acid and tannic acid m ixture.
How ever , the DNF digest ive rate of the contr ol silag e w as significant ly higher than that of additivetreated
silages ( P< 0. 05) .
Key words: Alfalfa silage; Proteolysis; Protein components; Pept ides; Am ino acids; Chemical addi
t ives
(责任编辑 才 杰 梁艳萍)
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