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Capillary electrophoresis ( CE ) is a newly developed
separation technique that enable rapid separation with high
resolution and high efficiency, and only need small amounts of
buffer solution and samples. Racemization, cross-linking
reactions and degradation may occur during food processing,
especially when protein-containing foods were treated with
alkali. In this study, Dns-DL-amino acids were separated by
micellar electrokinetic capillary chromatography (MEKC).
Effects of the pH values of backgrou
electrolyte (BGE)、surfactant concentrations、the types of
chiral selectors、capillary length and applied voltage on
separation were investigated in detail. In addition, effects of
the types and concentrations of organic modifiers on separation
were also discussed herein. In order to establish the optimun
operation conditions for enantiomeric separation of the racemic
amino acids, apply the results in a model system to recemic
mixtures of Dns-DL-amino acids in the alkali-treated duck
pidan. Besides, the ch
es of content of lysinoalanine in duck pidan during pickling
periods were investigated. In addition, the pepsin
digestibilities in alkali-treated protein were also determined.
Finally, summarized the favorite and adverse results in the
alkali-treated protein preducts; an optimun processing method
will be suggested for these products processed. Results
obtained from various stage of this study were presented as
following:
Increasing in the pH values and SDS concentration of the
background electrolyte (BGE) buffer solution were found to
improve the resolution (Rs) and to prolong the migration time
of Dns-DL-amino acids. Rs of each DL-amino acid was less than
1.0, when 100 mM SDS、75 mM b-CD and 250 mM borate (pH8.3) were
utilized as BGE buffer solution. However, as the pH value of
BGE buffer was increased to 9.5, all Dns-DL-amino acids, except
for Dns-DL-Ser and Dns-DL-Ala, were completely separated into
two individual pe
p and almost reached baseline separation. The separation was
fruther enhanced when the SDS concentration was raised to
150mM, all DL-amino acids, except for Dns-DL-Ser、Dus-DL-Ala
and Dns-DL-Met, were completely reached baseline separation,
and the efficiency expressed as theoretical plates number
exceeded 105. Dns-D-amino acids were found to be
eluted first when compared to the corresponding Dns-L-amino
acids, indicating that the cyclodextrin forms stronger
complexes with Dns-D-amino acids enantionmers.
The species of cyclodextrin affect enantiomeric
separation of DL-amino acids, with b-CD as a chiral selector
when 150 mM SDS、pH 9.5 was utilized as BGE buffer, the elution
peaks associated with Dns-DL-Leu, Dns-DL-Phe and Dns-DL-Glu
were be partially overlapped. By replacing b-CD with g-CD as a
chiral selector , the corresponding DL-enantiomers were
completely separated, g-CD has been found to be more effective
in imporving Rs、separation efficiency and shortening the
migration times . Besides, increa
ang the length of capillary and applied voltage were found to
be beneficial for increasing Rs and separation efficiency.
Addition of organic modifier, such as 10% acetonitrile, to the
background electrolyte solution (BGE) helped remarkably improve
the enantioselectivity and separation of D-Met and D-Leu.
Addition of organic modifiers can improve or worsen chiral
resolution, depending on the types and concentrations of
organic modifiers and the properties of analytes. The addition
of 30% methanol to the BGE improve the separation of Dns-DL-Ser
and Dns-DL-Thr, which were poorly separated in the absensence
of 30% methanol, while the addition of acetonitrile was proven
to be ineffective. For the separation of basic amino acids,
the composition and the pH value of BGE showed be different
from that used in
e neutral and acidic amino acids, because of the variations of
structure and electric charges.
The racemization of amino acids in alkali-treated duck
pidan during pickling periods have been studied by micellar
electrokinetic capillary chromatography (MEKC). The decreasing
order of racemization was Ser＞Asp＞Glu＞Phe＞Leu＞Val＞Thr＝
Ile, for duck albumen; while the order for duck yolk was Asp＞
Glu＞Phe＞Len＞Val. Based on the results of amino acid analysis
Cys、Ser、Thr、Lys and Arg showed major losses during the
alkali treatment and the remaining ratio was 35.5%、73.2%、88.1%
、70.8% and 67.9%, r
pectively. Therefore, alkali treatment can cause recemization
and severe destruction of
essential amino acids.
Study on LAL formation and factors inhibiting
lysinoalanine formation revealed that the LAL contents of duck
albumen and yolk increased with the time of pickling period.
The rapid increasing rate of LAL formation in duck albumen
could be due to the instant increase pH; in contrast, the LAL
content in duck yolk as less than l m mole / 100g protein
during the first 9 day pickling period. But at the storage
period between 12 and 20 days, the rapid increasing rate of
LAL formation was considered to be du
a
to the pH higher than 9 in yolk. Besides, addition of metal
salts and reductants were found available for preventing the
LAL formation.
b Pepsin digestibility tests revealed that the
digestibility of both duck albumen and yolk decreased with the
increasing period of pickling time, which could be close
related to the racemization of amino acids and the formation of
LAL.
於１。然而，當背景電解質之pH提高到9.5，除了Dns-DL-Ser及Dns -DL- - Ala外，其餘D
ns-DL-amino acids均可完全分離出二個獨立的波峰，且幾近於基準線的解析。當SDS濃度
提高為150 mM時，可加強其分離效果，除了Dns-DL-Ser、Dns-DL-Ala及Dns-DL-Met外，其
餘胺基酸完全可以達到基準之解析，且分離效率之理論板數高達數十萬。此外，Dns-D-胺
基酸對掌體均比Dns-L-胺基酸對掌體遷移較快，顯示Ｄ胺基酸對掌體與環狀糊精的結合較
強。
環狀糊
精的種類也會影響胺基酸對掌體之解析，於150mM SDS，pH 9.5之分離條件下，b-CD對Dns
-DL-Leu,Dns-DL-Phe及Dns-DL-Glu僅能部分解析，而使用g-CD則可將三者完全分離，且g-
CD與b-CD比較，發淚g-CD可提高解析度、分離效率及縮短遷移時間。此外，延長毛細管長
度及提高操作電壓，可提高解析度及分離效率。添加有機修飾劑如10％之氰甲烷於背景電
解質中，可促進D-Met及D-Leu之分離。
添加有機修飾劑可提高或降低光學異構物之解
析度，此與有機修飾劑的種類、濃度及分析物的性質有關。對於較難解析的Dns-DL-Ser及
Dns-DL-Thr而言，添加30％之甲醇可加強其分離效果，而氰甲烷則無此效果。鹼性胺基酸
由於結?
c及電荷異於其餘胺基酸，因此必須改變緩衝溶液的種類及pH值才能將它們分離。
以微膠體毛細管電泳層析進行鴨蛋
皮蛋製琵中胺基酸消旋化反應之探討，發淚鴨蛋在鹼處理過琵中，蛋白中胺基酸消旋化之
快慢次序依序為Ser＞Asp＞Glu＞Phe＞Leu＞Val＞Thr＝Ile；而蛋黃中其胺基酸消旋化之
快慢順序依序為Asp＞Glu＞Phe＞Leu＞Val＞Ile＝Ser＝Thr，因此，各胺基酸消旋琵度之
差異，除了與側鏈取代基的性質有關外，也與蛋白質之結構有關。而成品皮蛋中胺基酸成
分分析結果顯示Cys、Ser、Thr、Lys及Arg之含量減少較多，其殘留量百分比分別為35.5
％、73.2％、88.1％、70.8％及67.9％。可知鹼處理除消旋化反應外，必需胺基酸之鹼珮
壞也十分嚴重
在鴨蛋皮蛋製琵中，
LAL生成量之變化情形及抑制劑添加效應之探討中，結果顯示蛋白及蛋黃中LAL之生成量皆
隨著處理時間之增加而增加。蛋白在浸暝初期LAL快速生成，此乃因pH急速上升之結果；
蛋黃方面，由於浸暝初期pH值較低，浸暝９天後LAL生成量仍少於1m mole/100g protein
，至浸暝第12天由於pH值大於９，因此LAL有較快之生成速率。此外，添加金屬鹽類及還
原劑有明顯抑制LAL之生成。
胃蛋白酉每消化率之測試結果顯示消化率隨處理時間之
增加而降低，此消化率之降低與胺基酸之消旋化反應及LAL之生成有關。
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