臺灣博碩士論文加值系統

大豆粕(soybean meal；SBM)是大豆經過抽油加工後主要的副產物，因富含蛋白質(40%)，故常被當作家畜飼料中蛋白質的主要來源。唯豆粕中含有抗營養因子，降低其利用率。本研究旨在探討利用米麴菌(Aspergillus oryzae)在固態發酵模式下去除大豆粕之抗營養因子：包括脹氣因子(寡醣類)與抗原蛋白質(β-conglycinin和glycinin)之可行性。
首先進行發酵基質前處理，發現豆粕吸水力極佳，其與水比例為1:2.5以上時吸水之程度可達飽和，而加熱蒸煮過之豆粕其水分含量值偏高因此可省略此步驟。利用三角瓶(250 ml)進行A. oryzae之豆粕固態發酵培養最適培養條件為：豆粕與水比例1:1、接種量5%以及培養溫度為25℃，α-半乳糖苷酶(4.52 U/g)和酸性蛋白酶(109.85 U/g)的活性最高，且粗蛋白質含量增加10.73%。其次利用麴盤式進行豆粕之固態發酵培養，其最適培養條件為：豆粕與水比例2:1、接種量1%、培養溫度30℃及相對溼度90%下，培養時間30~35小時，此時有較佳活性的α-半乳糖苷酶(4.83 U/g)和酸性蛋白酶(43.05 U/g)，且能完全消除豆粕中的寡醣以及抗原蛋白質β-conglycinin，並降低glycinin含量。粗蛋白質和胺基酸態氮含量也分別增加為51.43% 和0.45%。
經固態發酵24小時後之豆粕的粗酵素液，其α-半乳糖苷酶最適作用溫度和pH值分別為47℃和4.5，而酸性蛋白酶最適作用溫度和pH值分別為52℃和2.6，且兩酵素在37~52℃下活性均甚為穩定。利用上述條件，將發酵24小時之豆粕，加緩衝液促進其酵素水解作用時之最適作用條件為：豆粕與水比1:2，pH值3.0，於42℃下作用一小時，對於原本豆粕中的抗營養因子寡醣水蘇四糖以及過敏蛋白質β-conglycinin可去除，而對棉籽糖和glycinin蛋白質則有部份降低的效果。

Soybean meals have been used extensively as protein sources in animal feedstuffs because of their high protein content (around 44%). However, the thermo-stable anti-nutritional factors, i.e., allergenic proteins (mainly β-conglycinin and glycinin) and flatulence-producing factors (mainly stachyose and raffinose) that commonly exist in these soybean meals will affect the growth and health of animals and have limited their use in young animal diets. The purpose of this study was to investigate the factors affecting the solid-state fermentation condition of soybean meal by koji-mold Aspergillus oryzae in order to reduce anti-nutritional factors and improve its nutritional value.
Pre-treatment of raw material before fermentation was investigated. The water absorption ability was good for soybean meal. The moisture content reached equilibrium when ratio of soybean meal to water was at 1: 2.5, and which was independent of soaking time. Cooking methods had no effect on moisture content and could be omitted. The optimum fermentation conditions performed in Erlenmeyer flasks (capacity 250 ml) were established. The optimal ratio of soybean meals to water was 1:1 (moisture content 58.82%), and the fermentation was performed at 25℃ for 6~7 days with inoculum size at 5% (v/w). Peak activities of α-galactosidase and acid protease obtained were 4.52 and 109.85 U/g, respectively.
The optimum solid-state fermentation conditions by tray method were established. The optimal ratio of soybean meals to boiled water was 2:1 (moisture content 41.09%), and the fermentation was performed at 30℃ under 90% RH for 30~35 hr with inoculum size at 1% (v/w). Maximumα-galactosidase activity and acid protease activity obtained were 4.83 and 43.05 U/g, respectively. The oligosaccharides (stachyose and raffinose) of soybean meal were removed after fermented for 42 hr. Theβ-conglycinin of allergenic protein was also removed and the amount of glycinin decreased. Moreover, crude protein content and the amino nitrogen content of soybean meal increased after fermentation for 30~35 hr, and the maximum values obtained were 51.43% and 0.453%, respectively.
The optimum temperature and pH for both α-glactosidase and acid protease activity from crude enzyme solution of a fermented soybean meal (24 hr) were determined. The optimum temperature of α-galactosidase and acid protease activity were 47℃and 52℃, respectively. The optimum pH of α-galactosidase and acid protease activity were 4.5 and 2.6 U/g, respectively. These two enzymes were stable in the temperature range of 37-52℃.
The optimum conditions of limited enzymatic hydrolysis of 24 h fermentation were determined. The ratio of fermented soybean meal to water was 1:2, and the reaction was carried out at pH 3.0 and 42℃ for 1 hr. Under this condition, all stachyose and part of raffinose in the fermented soybean meal were removed. The β-conglycinin of allergenic protein was degraded and the amount of glycinin also decreased considerably.
It is concluded that solid-state fermentation of soybean meal by Aspergillus oryzae produced high levels of α-galactosidase and acid protease, and thereafter increased crude protein and amino nitrogen content. Contents of both flatulence-producing factors and allergenic proteins were largely removed. Nutritional value of soybean meals by such solid-state fermentation will be considerably upgraded.

中文摘要...................................... II
英文摘要………………………………………………………… III
壹、前言………………………………………………………… 1
貳、文獻整理…………………………………………………… 3
【壹】大豆……………………………………………………… 3
一、大豆的介紹與特性………………………………………… 3
二、大豆的用途………………………………………………… 3
【貳】大豆粕………………………………………………… 4
一、大豆粕的介紹與特性……………………………………… 4
二、大豆粕之製法……………………………………………… 4
三、大豆粕之製造工程………………………………………… 6
四、大豆粕於豬飼料中之應用………………………………… 8
【參】大豆中之抗營養因子……………………………………… 9
一、胰蛋白酶抑制因子………………………………………… 9
二、寡醣………………………………………………………… 11
三、抗原蛋白質………………………………………………… 13
【肆】固態發酵………………………………………………… 21
一、簡介………………………………………………………… 21
二、固態發酵的特性…………………………………………… 23
三、固態發酵之優缺點………………………………………… 25
【伍】關於Aspergillus屬與Aspergillus oryzae……… 26
一、Aspergillus屬的特性………………………………… 26
二、Aspergillus oryzae的特性………………………… 27
三、Aspergillus oryzae所產生之酵素………………… 28
參、材料與方法…………………………………………………… 31
【壹】實驗材料…………………………………………………… 31
一、原料………………………………………………………… 31
二、實驗菌株…………………………………………………… 31
三、培養基……………………………………………………… 31
四、化學藥劑…………………………………………………… 31
【貳】儀器設備…………………………………………………… 32
【參】電腦套裝軟體……………………………………………… 34
【肆】實驗方法…………………………………………………… 34
一、實驗大綱…………………………………………………… 34
二、實驗方法…………………………………………………… 35
(一) 孢子懸浮液的製備……………………………………… 35
(二) 黃豆粕固態發酵前處理………………………………… 35
(三) 黃豆粕三角瓶固態發酵培養…………………………… 36
(四) 黃豆粕麴盤式固態發酵培養…………………………… 37
(五) 發酵黃豆粕加緩衝液促進麴菌酵素作用最適條件之探討：粗酵素液之分析………………………………………37
(六) 發酵黃豆粕加緩衝液促進麴菌酵素作用最適條件之探討……38
三、分析方法……………………………………………………… 39
四、統計分析及圖形繪製………………………………………… 44
肆、結果與討論…………………………………………………… 45
【壹】黃豆粕固態發酵前處理…………………………………… 45
一、黃豆粕最適浸漬與蒸煮條件之探求……………………… 45
(一) 不同水分比例添加量對黃豆粕水分含量之影響……… 45
(二) 浸漬時間對黃豆粕水分含量之影………………………… 47
(三) 蒸煮方法和時間對黃豆粕水分含量之影響……………… 47
【貳】黃豆粕三角瓶固態發酵培養……………………………… 52
一、最適固態發酵培養條件之探討……………………………… 52
(一) 水分含量…………………………………………………… 52
(二) 接種量……………………………………………………… 58
(三) 不同培養溫度之探求……………………………………… 61
【參】黃豆粕麴盤式固態發酵培養……………………………… 70
一、最適固態發酵培養條件之探討……………………………… 70
最適培養時間…………………………………………………… 70
【肆】發酵黃豆粕加緩衝液促進麴菌酵素作用最適條件之探討 88
一、 粗酵素液之分析…………………………………………… 88
(一) 最適反應溫度和pH值之探求……………………………… 88
(二) 熱穩定性之探求…………………………………………… 92
二、發酵黃豆粕加緩衝液作用最適條件之探討………………… 97
(一) 作用pH值與溫度之影響…………………………………… 97
(二) 添加緩衝液量與作用時間之影響………………………… 101
伍、結論…………………………………………………………… 109
陸、參考文獻……………………………………………………… 111
圖表附錄…………………………………………………………… 126

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