第二章 加工前處理條件對紅豆中γ-胺基丁酸含量之影響
十、 紅豆組成分分析
紅豆原料組成分分析結果如表2.5.，粗蛋白質、粗脂肪、灰分及總膳食纖 維含量以乾重表示。產自萬丹鄉之紅豆水分含量為13.85％，與產自巴西南部 的紅豆(水分含量約為13.07％)差異不大，高於產自泰國的紅豆(水份含量11.57
％)。紅豆主要成分以碳水化合物為主，含量約為52.64％，較產自巴西的紅豆 低(碳水化合物含量約62.26％)，與產自泰國的紅豆相去不遠(59.08 ％)；粗蛋 白質含量19.58 g/100 g，高於產自巴西(粗蛋白質含量20.30 g/100 g)與泰國(粗 蛋白質含量19.91 g/100 g)的紅豆，灰分含量4.86 g/100 g，較產自泰國及巴西的 紅豆略低(Gohara et al., 2016; Sai-Ut et al., 2009)，本研究與前人文獻中豆類中 組成分的差異應為栽培品種與生長條件不同所致(Sai-Ut et al., 2009)。
紅豆經於37℃浸泡8小時後，除了水分含量顯著提高(p＜0.05)、粗蛋白質 含量顯著減少(p＞0.05)外，其餘粗脂肪、灰分、膳食纖維及碳水化合物含量與 未經浸泡之紅豆相比，均無顯著差異(p＞0.05)。浸泡會促進豆類種子內儲藏蛋 白分解，以利提供發芽作用所需營養源(Barampama and Simard, 1995)，本研究 中紅豆經過37℃、浸泡8小時，粗蛋白質含量顯著下降之原因，應為在水分存 在下，微生物繁殖及酵素系統活化，使蛋白質降解為游離胺基酸造成。
肆、 結論
一、 紅豆經清洗後添加 5 倍重量自來水，於 4℃、25℃、37℃浸泡 2~60 小時以篩 選 GABA 生成量最高之前處理條件，其中，於 37℃浸泡 8 小時之紅豆 GABA 含量由 6.81 mg/100 g 增加至 90.27 mg/100 g，較完全未經浸漬處理紅豆提高 約 13 倍，GAD 活性由 4.4 U g-1 DW 提昇至 59.6 U g-1 DW，水份含量由 13.8
％增加至 47.3％，生菌數由 1.30 log cfu/g 增加至 5.44 log cfu/g，紅豆浸泡液 pH 值由 7.3 下降至 5.8，花青素含量由 1.13 μmole/g 下降至 0.85 μmole/g，總 酚含量由 14.85 mg GAE/g DW 提高至 18.04 mg GAE/g DW，顯示在高於室溫 條件下浸泡，有助活化紅豆中的 GAD，進而提高紅豆之 GABA 含量，浸泡 過程中種子的代謝作用，也促進酚類化合物形成，而提高總酚含量，惟浸泡 過程，並不利於花青素的穩定作用，造成經過浸泡之紅豆，花青素含量下降。
二、 於 37℃浸泡 8 小時之紅豆，再經-20℃低溫冷凍 48 小時並於室溫解凍 24 小 時，GABA 含量可增加至 154.51 mg/100 g，較完全未經任何處理之新鮮紅豆 高出約 22.7 倍，與浸泡處理後未經冷凍解凍之紅豆相比，僅提高 1.7 倍，冷 凍解凍過程使紅豆中花青素含量及總酚含量大幅下降。
三、 經浸泡過之紅豆再經冷凍處理，製程中需消耗大量能源，且冷凍、解凍處理 尚需延長加工時間達 72 小時，卻僅能將紅豆中 GABA 含量提高 1.7 倍，實 不符大量生產之經濟效益，本研究選擇將紅豆於 37℃浸泡 8 小時後，不經冷 凍及解凍流程，直接製備成紅豆乳，接種不同益生菌，以探討益生菌發酵作 用對提升紅豆乳γ-胺基丁酸之影響。
表 2. 1. 用於高壓液相層析法及毛細管電泳分析之主要胺基酸衍生劑 Table 2.1. Major amino acid derivative reagents for HPLC and CZE separations
Reagent Availability of commercial kit
Detection Detection Pre-/post-separation
aIt was also been used for the quantitation of total amino acids without separation.
bAlso susceptible to electrochemical detection.
PITC: Phenylisothiocyanate
Dabsyl-Cl:4-dimethylaminoazobenzene-4'-sulfonyl chloride Dansyl-Cl:1-dimethylaminonaphthalene-5-sulfonyl chloride F-MOC: 9-Fluorenylmethyl chloroformate
OPA: o-phthaldialdehyde
AQC: 6-aminoquinolyl-N-hdroxysuccinimicly crbamate BQCA: 3-benzoyl-2-quinolinecarboxaldehyde
RP-HPLC: Reversed-phase high-performance liquid chromatography CEC: cation-exchange chromatography
CZE: capillary zone electrophoresis TLC: thin-layer chromatography IP: ion paired
Fl: fluorescence detection Vis: visible detection UV: ultraviolet detection
LIF:laser induced fluorescence detection
(Aristory and Toldrá, 2004)
表 2.2. 浸泡溫度及時間對紅豆 GABA 含量之影響
Table 2.2. Effect of soaking temperature and time on GABA content of adzuki beans Soaking
time(hr)
GABA content(mg/100g adzuki bean)1
4℃ 25℃ 37℃
2 16.67±1.05B,f2 20.52±1.17A,f 21.85±1.52A,g 4 18.52±0.60C,f 27.01±1.48B,e 62.11±1.83A,f 8 24.57±1.13C,e 43.25±0.46B,d 90.27±0.78A,b 12 34.45±3.57C,d 63.98±2.25B,c 91.34±1.07A,ab 24 58.48±0.85C,c 67.16±1.55B,bc 93.35±0.97A,a 30 64.65±1.54C,b 68.80±1.50B,b 88.67±1.04A,b 36 64.85±1.45C,b 73.90±2.60B,a 83.40±1.57A,d 48 68.17±1.41B,a 68.10±1.64B,b 85.81±1.58A,c 60 69.60±0.50A,a 66.85±2.68A,bc 70.92±3.00A,e
1. GABA content of unsoaking adzuki beans:6.81±0.83 mg/100 g adzuki bean.
2. Values are expressed as mean ± SD (n = 3). Means with different uppercase letters within the same row are significantly different (p ＜ 0.05). Means with different lowercase letters within the same column are significantly different (p＜0.05).
表 2.3. 浸泡溫度及時間對紅豆麩胺酸脫羧酶活性之影響
Table 2.3. Effect of soaking temperature and time on GAD activity of adzuki beans Soaking
time(hr)
GAD activity(U g-1 DW)1
4℃ 25℃ 37℃
2 11.12±1.11B,f2 13.32±0.76Af 14.19±0.99Ag 4 12.87±1.03C,f 17.54±1.15Be 39.66±1.37Af 8 17.22±1.14C,e 28.77±0.93B,d 59.48±1.22A,ab 12 22.23±1.54C,d 41.97±1.42B,c 59.73±1.39A,ab 24 37.98±1.46C,c 44.39±1.08B,b 61.49±1.06A,a 30 41.71±1.54C,b 44.64±1.02B,b 57.79±1.02A,bc 36 42.78±1.01C,ab 47.98±1.69B,a 53.92±1.02A,d 48 44.60±1.27B,a 44.19±1.06B,bc 55.98±1.59A,cd 60 37.27±0.93B,c 43.59±2.02A,bc 46.06±1.94A,e
1. GAD activity of unsoaking adzuki beans：4.34±0.08 U g-1 DW.
2. Values are expressed as mean ± SD (n = 3). Means with different uppercase letters within the same row are significantly different (p ＜ 0.05). Means with different lowercase letters within the same column are significantly different (p＜0.05).
表 2.4. 冷凍及解凍處理對紅豆 GABA 含量之影響
Table 2.4. Effect of freezing and thawing conditions on GABA content of adzuki beans
Freezing temperature
Freezing time
(h)
GABA content (mg/100g adzuki bean) No thawing Thawing for 12
hours
Thawing for 24 hours -4℃ 12 96.56±1.60B,e 99.26±5.93B,d 106.98±3.94A,e
24 104.59±2.88C,d 117.11±5.07Bc 132.41±5.69A,c 48 113.06±1.33C,b 119.34±4.93B,bc 138.39±2.64A,c -20℃ 12 101.16±2.84B,d 119.79±8.26A,bc 124.98±5.33A,d 24 106.47±3.25C,c 127.19±3.18B,ab 146.74±4.90A,b 48 118.92±2.50C,a 135.19±4.78B,a 154.51±4.15A,a Liquid
nitrogen
12 89.99±1.42B,f 90.99±2.22B,d 107.91±4.12A,e 24 90.70±0.97B,f 92.76±2.58B,d 108.31±1.42A,e 48 90.17±1.27B,f 91.60±1.67B,d 114.95±4.80A,e
1. GABA content of soaking adzuki beans without freezing and thawing treatment:90.27±
0.78 mg/100 g adzuki bean.
2. Values are expressed as mean ± SD (n = 3). Means with different uppercase letters within the same row are significantly different (p ＜ 0.05). Means with different lowercase letters within the same column are significantly different (p＜0.05).
表 2.5. 紅豆組成分分析
Table 2.5. Proximate composition of adzuki beans
Constituent Raw beans1 After soaking treatment2 Crude protein(g/100g DW) 19.58±0.52 B 18.46±0.38 A
Crude fat(g/100g DW) 1.16±0.15 A 1.14±0.13 A Crude ash(g/100g DW) 4.86±0.37 A 4.94±0.42 A Total dietary fiber(g/100g DW) 21.75±0.81 A 21.51±0.36 A Soluble dietary fiber(g/100g DW) 19.82±0.74 A 19.64±0.46 A Insoluble dietary fiber(g/100g DW) 1.93±0.11 A 1.86±0.10 A Carbohydrate(g/100g DW) 52.64±1.40 A 53.96±0.64 A
1Each value represents mean ± SD (n = 3). Means with different uppercase letters within the same row are significantly different (p＜0.05).
2Soaking treatment:adzuki beans were soaked in five-fold weight of water (w/w) at 37°C for 8 h.
圖 2.1. 胺基酸 PITC 衍生反應圖。
Fig. 2.1. Amino acid derived with phenylisocyanate.
(Sherwood, 2000)
＋
Alkaline pH
Amino acid
PTC- amino acid
Phenylisothiocyanate, PITC
圖 2.2. 實驗架構。
Fig. 2.2. The framework of the study.
生 鮮 紅 豆 高雄 8 號
清 洗
添加 5 倍水(w/w,自來水)
紅 豆 浸 漬 試 驗
4℃ 25℃ 37℃
第 2、4、8、12、24、30、36、48 及 60 小時測定紅豆水份含量、pH、生菌 數、花青素含量、總酚含量、GABA 含量及 GAD 活性，篩選最適浸漬條件
最適浸漬條件浸漬後之紅豆
瀝 乾
-4℃ -20℃ 液態氮
冷 凍
第 12、24、48 小時取出，分別在室溫下解凍 12 及 24 小時，測 定紅豆花青素含量、總酚含量、GABA 含量、GAD 活性，篩選 最適前處理條件
圖 2.3. 浸泡溫度及時間對紅豆水分含量之影響。
Fig. 2.3. Effects of soaking temperature and time on moisture content in adzuki beans.
Each value represents mean ± SD (n = 3).
0 10 20 30 40 50 60 70 80
0 2 4 8 12 24 30 36 48 60
4℃
25℃
37℃
Soaking time (h)
Moisture content (％)
圖 2.4. 浸泡溫度及時間對紅豆生菌數含量之影響。
Fig. 2.4. Effects of soaking temperature and time on total counts of adzuki beans. Each value represents mean ± SD (n = 3).
0 2 4 6 8 10 12 14
0 2 4 8 12 24 30 36 48 60
Total counts (log cfu/g)
Soaking time(h)
4℃
25℃
37℃
圖 2.5. 紅豆於不同溫度浸泡之浸泡液 pH 值變化。
Fig. 2.5. Effects of different soaking temperature and time for adzuki beans on pH values of soaking solution. Each value represents mean ± SD (n = 3).
3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8
0 2 4 8 12 24 30 36 48 60
pH value
Soaking time (h) 4℃
25℃
37℃
圖 2.6.浸泡溫度及時間對紅豆花青素含量之影響。
Fig. 2.6. Effects of soaking temperature and time on anthocyanin content in adzuki beans.
Each value represents mean ± SD (n = 3).
0.00 0.20 0.40 0.60 0.80 1.00 1.20
0 2 4 8 12 24 30 36 48 60
Anthocyanin content (μmole/g)
Soakimg time (h)
4℃
25℃
37℃
圖 2.7. 浸泡溫度及時間對紅豆總酚含量之影響。
Fig. 2.7. Effect of soaking temperature and time on total phenolic content in adzuki beans. Each value represents mean ± SD (n = 3).
12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00
0 2 4 8 12 24 30 36 48 60
Total phenolic content (mg GAE/ g DW)
soakimg time (h)
4℃
25℃
37℃
圖 2.8. 冷凍及解凍處理對紅豆花青素含量之影響。
Fig 2.8. Effects of freezing and thawing conditions on anthocyanin content of adzuki beans.
*4F12H：freezing temperature at -4℃ for 12 h; 4F24H: freezing temperature at -4℃ for 24 h; 4F48H：freezing temperature at -4℃ for 48 h; 20F12H：freezing temperature at -20℃ for 12h; 20F24H：freezing temperature at -20℃ for 24 h; 20F48H:freezing temperature at -20℃ for 48 h; NF12H: freezing in liquid nitrogen for 12 h; NF24H:
freezing in liquid nitrogen for 24 h; NF48H: freezing in liquid nitrogen for 48 h.
*Anthocyanin content of soaking adzuki beans without freezing and thawing treatment:
0.85 ± 0.03 μmole/g.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
4F12H 4F24H 4F48H 20F12H 20F24H 20F48H NF12H NF24H NF48H
Anthocyanin content (μmole/g)
Treatment condition
0hr thawing 12hr thawing 24hr thawing
圖 2.9. 冷凍及解凍處理對紅豆總酚含量之影響。
Fig 2.9. Effect of freezing and thawing conditions on total phenol content of adzuki beans.
*4F12H：freezing temperature at -4℃ for 12 h; 4F24H: freezing temperature at -4℃ for 24 h; 4F48H：freezing temperature at -4℃ for 48 h; 20F12H：freezing temperature at -20℃ for 12h; 20F24H：freezing temperature at -20℃ for 24 h; 20F48H:freezing temperature at -20℃ for 48 h; NF12H: freezing in liquid nitrogen for 12 h; NF24H:
freezing in liquid nitrogen for 24 h; NF48H: freezing in liquid nitrogen for 48 h.
*Total phenol content of soaking adzuki beans without freezing and thawing treatment：
18.04 ± 0.14 μmole/ g.
0 2 4 6 8 10 12 14 16 18 20
4F12H 4F24H 4F48H 20F12H 20F24H 20F48H NF12H NF24H NF48H
Total phenol content (GAE/ g DW)
Treatment condition
0hr thawing 12hr thawing 24hr thawing
圖 2.10. 冷凍及解凍處理對紅豆麩胺酸脫羧酶活性之影響。
Table 2.10. Effect of freezing and thawing conditions on glutamate carboxylase of adzuki beans.
*4F12H：freezing temperature at -4℃ for 12 h; 4F24H: freezing temperature at -4℃ for 24 h; 4F48H：freezing temperature at -4℃ for 48 h; 20F12H：freezing temperature at -20℃ for 12h; 20F24H：freezing temperature at -20℃ for 24 h; 20F48H:freezing temperature at -20℃ for 48 h; NF12H: freezing in liquid nitrogen for 12 h; NF24H:
freezing in liquid nitrogen for 24 h; NF48H: freezing in liquid nitrogen for 48 h.
*GAD activity of soaking adzuki beans without freezing and thawing treatment：59.48
± 1.22 U g-1 DW.
0 10 20 30 40 50 60 70 80 90 100 110
4F12H 4F24H 4F48H 20F12H 20F24H 20F48H NF12H NF24H NF48H
Glutamate decarboxylase activity (U g-1 FW)
Treatment condition
0hr thawing 12hr thawing 24hr thawing
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