水酶法提取花生油和水解蛋白的中试工艺及花生ACE抑制肽的研究
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摘要
花生是世界上最重要的油料作物之一,花生仁中的脂肪含量约为50%左右,蛋白质含量也很高,约为24%～36%。传统的制油工艺包括压榨法和溶剂浸出法,压榨法劳动强度大,动力消耗也大,而浸出法采用有机溶剂制油,其生产安全性及食用安全性都不够高,花生蛋白质资源也未能得到充分的利用。水酶法提取花生油和水解蛋白的实验室工艺已经比较成熟,为了将其应用于工业化生产,本论文在前人实验室工艺的基础上,进行了水酶法提取花生油和水解蛋白的中试扩大实验,对水酶法提油的机理进行了初步探讨,并对中试工艺所得的花生水解蛋白进行了生物活性研究,主要研究内容及结果如下。
论文首先进行了水酶法提取花生油和水解蛋白的中试扩大实验,比较了两种中试工艺。在中试工艺1中引入三相离心机同时分离游离油、油水混合物和不溶残渣,并使用碟片离心机将油水混合物中的油和水进一步分离得到游离油、乳状液和水解液。水解液经喷雾干燥后得到花生水解蛋白粉。最终,总游离油得率为73.77%,水解蛋白得率为55.10%。中试工艺2仅采用三相离心机分离油、油水混合物和不溶残渣,游离油得率分别达到79.68%(带皮花生)和81.95%(脱皮花生),水解蛋白得率分别达到73.05%(带皮花生)和72.20%(脱皮花生),达到了实验室小试的游离油和水解蛋白的得率。中试工艺得到的花生油其色泽、过氧化值、酸值等各项品质基本达到国家三级花生油的标准,因此水酶法提取的花生油经过简单精炼即可食用。
采用扫描电子显微镜和透射电子显微镜对酶解不同时间的花生细胞和乳状液的超微结构进行了观察,并测定了界面及水相蛋白质的圆二色性和表面疏水性,初步探讨了水酶法提取花生油和水解蛋白的碱提及酶解机理。花生经干法破碎后,细胞中的大部分细胞内容物在碱提过程中被溶出,然后其中的蛋白质在溶液中进一步被蛋白酶酶解,释放出包裹的油滴,同时乳状液界面上的蛋白质在酶解过程中也受到蛋白酶的作用被降解,其二级结构发生了明显的变化,界面膜变薄并发生破裂,乳状液破乳,大大提高了油和水解蛋白的得率。
采用DA201-C大孔吸附树脂对花生水解蛋白进行了脱盐和初步纯化,动态吸附和解吸试验表明:花生水解蛋白以50mg/mL的浓度上样,流速为60mL/h,用去离子水洗涤层析柱,当电导率降至与去离子水相当时,采用75%的乙醇进行解吸,收集洗脱液,真空浓缩蒸去乙醇,冷冻干燥后即得花生粗肽,脱盐率为91.02%,蛋白含量提高到近80%。随后测定了花生水解蛋白和脱盐花生肽的还原能力、清除DPPH·、·OH和O2—·的能力,脱盐花生肽抗氧化性强于花生水解蛋白。同时还测定了花生水解蛋白和脱盐花生肽的ACE抑制活性,花生肽的ACE抑制活性略优于花生水解蛋白。
以ACE抑制率为指标,采用葡聚糖凝胶Sephadex G-15、半制备和分析型RP-HPLC进一步分离纯化花生肽,得到G3-R2-r3和G3-R4-r2这两个具有最高ACE抑制活性的组分,然后利用基质辅助激光解吸电离-飞行时间-飞行时间( MALDI-TOF-TOF )串联质谱对这两个组分进行结构鉴定,得到Tyr-Thr-Thr-Pro-Th(rYT-5)、Tyr-Thr-Thr-Pro-Val-Th(rYT-6)、Arg-Arg-Met-Leu-Tyr(RY-5)和Pro-Gly-Arg-Val-Tyr(PY-5)4种具有ACE抑制活性的花生肽的氨基酸序列。按照这4种肽的氨基酸结构合成得到4种花生纯肽,选择其中ACE抑制率最高的花生肽PY-5与Sephadex G-15分离得到的具有最高ACE抑制活性的G3组分一起进行SHR大鼠体内降血压功能实验,证实了花生ACE抑制肽在大鼠体内确实具有降血压功能。
Peanut is one of the most important oilseeds in the world. The oil content of peanut is about 50% and the protein content is 24% to 36%. Conventional industrial oil processing involves mechanical pressing and solvent extraction. Mechanical pressing is labor intensive and energy consuming, while solvent extraction has some problems of production security and edible safety, and the protein would not be used completely. The laboratory technique of aqueous enzymatic extraction of peanut oil and protein hydralysates has matured. In order to apply the technique into industrial production, pilot-scale process of aqueous enzymatic extraction of peanut oil and protein hydrolysates was carried out based on the previous results in this dissertation. The mechanism of aqueous enzymatic extraction of oil and the peanut protein hydrolysates obtained from pilot-scale process were studied. The results are as follows:
First the pilot-scale process of aqueous enzymatic extraction of peanut oil and protein hydrolysates was carried out and two processes were compared. The three-phase separator was chosen to separate oil, oil-water mixture and insoluble residue simultaneously in pilot-scale process 1. Then oil-water mixture was treated by the disk separator to obtain free oil, emulsion and hydrolysate solution. After spray drying, peanut protein hydrolysates powder was obtained from hydrolysate solution. As a result, total free oil and protein hydrolysates yields were 73.77% and 55.10% respectively. In pilot-scale process 2, only three-phase separator was used. The free oil yields were 79.68% (with hull) and 81.95% (dehulled), while protein hydrolysates yields were 73.05% (with hull) and 72.20% (dehulled). The yields have achieved to the level of laboratory technique. The color, peroxide value, acid value and other qualities of peanut oil from pilot-scale processes reached the national standard of third class peanut oil, so the peanut oil obtained from aqueous enzymatic extraction process can be edible after simple refining.
The peanut cells and emulsion at different enzymatic time were observed by SEM and TEM. The circular dichroism and surface hydrophobicity of protein on the emulsion interface and in aqueous phase were measured to learn the mechanism of aqueous enzymatic extraction of peanut oil and protein hydrolysates. After dry grinding, most cell content came out into the solution during alkaline extraction process, then the protein was hydrolyzed by protease and oil was released. At the same time, protein on the emulsion interface was also degraded by protease. As a result, its secondary structure has obviously changed, protein film on the interface broke, and the emulsion was demulsified. Thus, free oil and protein hydrolysates yields were greatly improved.
The macroporous adsorption resins (type: DA201-C) were used to treat the peanut protein hydrolysates to remove salt. The results of dynamic adsorption and desorption show that 50mg/mL peanut protein hydrolysates were adsorbed onto mcroporous at 60mL/h rate. Then the chromatographic column was washed with deionized water. When the conductivity of the eluent decreased until it was the same with deionized water, desorption was carried out with 75% ethanol. After the eluent was collected, evaporated and freeze-dried, we got crude peanut peptides, the protein content of which reached about 80% and the desalination ratio is 91.02%. Both peanut protein hydrolysates and desalted peanut peptides were made reducing power, scavenging abilities on free radicals including DPPH·,·OH and O2—·testing and ACE inhibitory activity testing. The results shown that desalted peanut peptides has stronger antioxidant activity and ACE inhibitory activity than peanut protein hydrolysates.
Peanut peptides were separated and purified by Sephadex G-15, semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC) and analytical RP-HPLC. After these, two active fraction G3-R2-r3 and G3-R4-r2 were obtained, which were tested to have better ACE inhibitory activities. The amino acids sequences of these peptides were gained using MALDI-TOF-TOF tandem mass spectrometry, which were Tyr-Thr-Thr-Pro-Thr(YT-5), Tyr-Thr-Thr-Pro-Val-Thr(YT-6), Arg-Arg-Met-Leu-Tyr(RY-5) and Pro-Gly-Arg-Val-Tyr(PY-5).We synthesized the four peanut ACE inhibitory peptides according to the sequences. Then G3 which was separated by Sephadex-15 and the peanut ACE inhibitory peptide which has the highest ACE inhibitory activity were chosen to test the effects on blood pressure in spontaneously hypertensive rats (SHR). And it’s proved that peanut ACE inhibitory peptides can control blood pressure effectively in SHR.
论文首先进行了水酶法提取花生油和水解蛋白的中试扩大实验,比较了两种中试工艺。在中试工艺1中引入三相离心机同时分离游离油、油水混合物和不溶残渣,并使用碟片离心机将油水混合物中的油和水进一步分离得到游离油、乳状液和水解液。水解液经喷雾干燥后得到花生水解蛋白粉。最终,总游离油得率为73.77%,水解蛋白得率为55.10%。中试工艺2仅采用三相离心机分离油、油水混合物和不溶残渣,游离油得率分别达到79.68%(带皮花生)和81.95%(脱皮花生),水解蛋白得率分别达到73.05%(带皮花生)和72.20%(脱皮花生),达到了实验室小试的游离油和水解蛋白的得率。中试工艺得到的花生油其色泽、过氧化值、酸值等各项品质基本达到国家三级花生油的标准,因此水酶法提取的花生油经过简单精炼即可食用。
采用扫描电子显微镜和透射电子显微镜对酶解不同时间的花生细胞和乳状液的超微结构进行了观察,并测定了界面及水相蛋白质的圆二色性和表面疏水性,初步探讨了水酶法提取花生油和水解蛋白的碱提及酶解机理。花生经干法破碎后,细胞中的大部分细胞内容物在碱提过程中被溶出,然后其中的蛋白质在溶液中进一步被蛋白酶酶解,释放出包裹的油滴,同时乳状液界面上的蛋白质在酶解过程中也受到蛋白酶的作用被降解,其二级结构发生了明显的变化,界面膜变薄并发生破裂,乳状液破乳,大大提高了油和水解蛋白的得率。
采用DA201-C大孔吸附树脂对花生水解蛋白进行了脱盐和初步纯化,动态吸附和解吸试验表明:花生水解蛋白以50mg/mL的浓度上样,流速为60mL/h,用去离子水洗涤层析柱,当电导率降至与去离子水相当时,采用75%的乙醇进行解吸,收集洗脱液,真空浓缩蒸去乙醇,冷冻干燥后即得花生粗肽,脱盐率为91.02%,蛋白含量提高到近80%。随后测定了花生水解蛋白和脱盐花生肽的还原能力、清除DPPH·、·OH和O2—·的能力,脱盐花生肽抗氧化性强于花生水解蛋白。同时还测定了花生水解蛋白和脱盐花生肽的ACE抑制活性,花生肽的ACE抑制活性略优于花生水解蛋白。
以ACE抑制率为指标,采用葡聚糖凝胶Sephadex G-15、半制备和分析型RP-HPLC进一步分离纯化花生肽,得到G3-R2-r3和G3-R4-r2这两个具有最高ACE抑制活性的组分,然后利用基质辅助激光解吸电离-飞行时间-飞行时间( MALDI-TOF-TOF )串联质谱对这两个组分进行结构鉴定,得到Tyr-Thr-Thr-Pro-Th(rYT-5)、Tyr-Thr-Thr-Pro-Val-Th(rYT-6)、Arg-Arg-Met-Leu-Tyr(RY-5)和Pro-Gly-Arg-Val-Tyr(PY-5)4种具有ACE抑制活性的花生肽的氨基酸序列。按照这4种肽的氨基酸结构合成得到4种花生纯肽,选择其中ACE抑制率最高的花生肽PY-5与Sephadex G-15分离得到的具有最高ACE抑制活性的G3组分一起进行SHR大鼠体内降血压功能实验,证实了花生ACE抑制肽在大鼠体内确实具有降血压功能。
Peanut is one of the most important oilseeds in the world. The oil content of peanut is about 50% and the protein content is 24% to 36%. Conventional industrial oil processing involves mechanical pressing and solvent extraction. Mechanical pressing is labor intensive and energy consuming, while solvent extraction has some problems of production security and edible safety, and the protein would not be used completely. The laboratory technique of aqueous enzymatic extraction of peanut oil and protein hydralysates has matured. In order to apply the technique into industrial production, pilot-scale process of aqueous enzymatic extraction of peanut oil and protein hydrolysates was carried out based on the previous results in this dissertation. The mechanism of aqueous enzymatic extraction of oil and the peanut protein hydrolysates obtained from pilot-scale process were studied. The results are as follows:
First the pilot-scale process of aqueous enzymatic extraction of peanut oil and protein hydrolysates was carried out and two processes were compared. The three-phase separator was chosen to separate oil, oil-water mixture and insoluble residue simultaneously in pilot-scale process 1. Then oil-water mixture was treated by the disk separator to obtain free oil, emulsion and hydrolysate solution. After spray drying, peanut protein hydrolysates powder was obtained from hydrolysate solution. As a result, total free oil and protein hydrolysates yields were 73.77% and 55.10% respectively. In pilot-scale process 2, only three-phase separator was used. The free oil yields were 79.68% (with hull) and 81.95% (dehulled), while protein hydrolysates yields were 73.05% (with hull) and 72.20% (dehulled). The yields have achieved to the level of laboratory technique. The color, peroxide value, acid value and other qualities of peanut oil from pilot-scale processes reached the national standard of third class peanut oil, so the peanut oil obtained from aqueous enzymatic extraction process can be edible after simple refining.
The peanut cells and emulsion at different enzymatic time were observed by SEM and TEM. The circular dichroism and surface hydrophobicity of protein on the emulsion interface and in aqueous phase were measured to learn the mechanism of aqueous enzymatic extraction of peanut oil and protein hydrolysates. After dry grinding, most cell content came out into the solution during alkaline extraction process, then the protein was hydrolyzed by protease and oil was released. At the same time, protein on the emulsion interface was also degraded by protease. As a result, its secondary structure has obviously changed, protein film on the interface broke, and the emulsion was demulsified. Thus, free oil and protein hydrolysates yields were greatly improved.
The macroporous adsorption resins (type: DA201-C) were used to treat the peanut protein hydrolysates to remove salt. The results of dynamic adsorption and desorption show that 50mg/mL peanut protein hydrolysates were adsorbed onto mcroporous at 60mL/h rate. Then the chromatographic column was washed with deionized water. When the conductivity of the eluent decreased until it was the same with deionized water, desorption was carried out with 75% ethanol. After the eluent was collected, evaporated and freeze-dried, we got crude peanut peptides, the protein content of which reached about 80% and the desalination ratio is 91.02%. Both peanut protein hydrolysates and desalted peanut peptides were made reducing power, scavenging abilities on free radicals including DPPH·,·OH and O2—·testing and ACE inhibitory activity testing. The results shown that desalted peanut peptides has stronger antioxidant activity and ACE inhibitory activity than peanut protein hydrolysates.
Peanut peptides were separated and purified by Sephadex G-15, semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC) and analytical RP-HPLC. After these, two active fraction G3-R2-r3 and G3-R4-r2 were obtained, which were tested to have better ACE inhibitory activities. The amino acids sequences of these peptides were gained using MALDI-TOF-TOF tandem mass spectrometry, which were Tyr-Thr-Thr-Pro-Thr(YT-5), Tyr-Thr-Thr-Pro-Val-Thr(YT-6), Arg-Arg-Met-Leu-Tyr(RY-5) and Pro-Gly-Arg-Val-Tyr(PY-5).We synthesized the four peanut ACE inhibitory peptides according to the sequences. Then G3 which was separated by Sephadex-15 and the peanut ACE inhibitory peptide which has the highest ACE inhibitory activity were chosen to test the effects on blood pressure in spontaneously hypertensive rats (SHR). And it’s proved that peanut ACE inhibitory peptides can control blood pressure effectively in SHR.
引文
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