摘要
葵花籽是一种营养价值较高的油料作物,但对其开发利用还不够。本文以高油葵花籽为对象,开展了水酶法提取葵花籽油工艺以及脱脂葵花粕的利用研究,对促进葵花籽的综合开发利用具有重要的理论和应用意义。
首先研究了水酶法提取葵花籽油技术。引入连续搅拌罐膜生物反应器,大大降低产物抑制作用,提高了反应效率。考察了水酶法提取葵花籽油的工艺条件,如缓冲液pH值、热处理温度及热处理时间对游离油提取率的影响。结果表明:缓冲液pH值为4.8、热处理温度110℃及时间60min时可获得较高的游离油提取率。在确定热处理工艺的基础上,通过单因素试验及响应面试验,得到了水酶法提取葵花籽油最适条件为:固液比1:8,加酶量(纤维素酶:果胶酶=2:1)2%,酶解pH值为4.7,酶解温度为50℃,酶解时间为5.5h,游离油提取率为89.8%。
采用番红染色显微摄影法研究了水酶法提油过程中热处理及酶解过程对细胞结构的破坏。结果表明葵花籽经上述加工过程,细胞结构基本被破坏。以苏丹Ⅲ染色显微摄影法研究了水酶法提油过程中油脂释放情况,结果显示酶解有利于油脂的释放,从微观角度证明了水酶法提油工艺的可行性。
研究了水酶法提油工艺对葵花籽油质量的影响。结果表明:热处理时缓冲液pH值、热处理温度及热处理时间对葵花籽油的色泽没有影响;缓冲液pH值在3-7之间随pH值的增大,油脂酸值由2.17降为1.98mgKOH/g,变化不明显,但过氧化物值却从1.36增至2.53meq/kg,影响较大;热处理温度及热处理时间对葵花油酸值、过氧化物值影响较大。尽管酶解时间长达5h,但对葵花油色泽几乎没有影响,对酸值和过氧化物值影响也很小。
以水酶法提取葵花籽油后的脱脂葵花粕为原料,通过盐提酸沉法提取葵花籽中的分离蛋白,盐溶盐析法分离其主要蛋白成分球蛋白,盐溶有机溶剂沉淀分离其蛋白成分清蛋白。与大豆分离蛋白进行比较,研究了葵花蛋白的结构、功能及理化性质。溶解度曲线表明葵花蛋白主要成分球蛋白在pH4左右溶解度最小,而葵花浓缩蛋白和分离蛋白在pH5左右显示出最小的溶解度;葵花浓缩蛋白、分离蛋白和球蛋白的功能性质除吸水性低于大豆分离蛋白外,其吸油性、起泡性、起泡稳定性、乳化活力和乳化稳定性稍高于大豆分离蛋白,葵花球蛋白的乳化性特别高,葵花蛋白的乳化性主要在于其主要成分11S球蛋白。
凝胶层析表明葵花分离蛋白有5个组分,其中3个主要组分为11S球蛋白、7S球蛋白和2S清蛋白,相对分子质量分别为380,000,100,000和27,000,11S球蛋白为葵花分离蛋白的主要组分。葵花蛋白SDS-PAGE显示葵花11S球蛋白由10条主带和两条微带组成,10条主带的相对分子质量分别为53,500、42,600、39,500、33,200、30,800、26,500、24,000、22,500、20,800和19,200;葵花2S清蛋白由4条主带和一些微带组成;而葵花分离蛋白的谱带比11S球蛋白和2S清蛋白总和还多,因为葵花分离蛋白中还有7S球蛋白和其他一些变性蛋白。圆二色性(CD)分析葵花球蛋白和葵花清蛋白的主要二级结构为β-折叠和不规则卷曲,葵花蛋白属于β-折叠型蛋白质。差示扫描量热(DSC)研究显示葵花11S球蛋白和葵花2S清蛋白干粉热变性温度分别为123.5℃和122.8℃,葵花蛋白具有较高的变性温度。荧光发射光谱研究显示葵花11S球蛋白和2S清蛋白荧光发射光谱最大荧光强度分别在345nm和340nm左右,色氨酸对荧光的贡献较大。
研究了Alcalase、Protamex和Flavourzyme蛋白酶对葵花蛋白的酶水解过程,以水解度、水解物中可溶性蛋白含量和抗氧化活力为指标,得出了各酶水解葵花蛋白的较佳条件。采用凝胶层析法,对各酶不同时间水解产物中多肽相对分子质量分布进行了测定。结果表明:Alcalase碱性蛋白酶水解反应进行1h,水解物中多肽的相对分子质量集中分布在590-2,975之间;Protamex复合蛋白酶水解葵花蛋白1h,水解液中多肽的相对分子质量主要分布在370-4,226范围内;Flavourzyme风味蛋白酶水解葵花蛋白1h,水解液中86.70%的多肽相对分子质量集中在370-5,117。
采用超滤、离子交换层析、凝胶层析和反相色谱对Flavourzyme水解脱脂葵花粕1h的水解产物进行分离纯化,得到一个抗氧化活性肽。质谱测定的抗氧化活性肽的氨基酸序列为:Ala-Cys- Ala-His-Asp-Lys-Val,抗氧化活力为79.6U/mL,该活性肽未见报道。开展了好食脉孢霉发酵脱脂葵花粕产生抗氧化活性肽的研究。对好食脉孢霉发酵脱脂葵花粕产生抗氧化活性肽的培养基组成、发酵条件进行了优化,同时对发酵产物浸提液中蛋白的电泳和肽的相对分子质量分布等内容进行了研究,在发酵产物中发现大量具有抗氧化活性的物质,其相对分子质量主要分布在3,396-166之间。
sunflower seed was an oil corn with high nutritional value,but this resource was not sufficiently developed. The aqueous enzymatic extraction oil and the utilization of defatted sunflower meal were studied with the high oil sunflower seed as raw material, which to provide theoretical and practical foundation of comprehensive utilization of sunflower seed. At first, the aqueous enzymatic extraction technology was studied. a continuous stirred tank membrane reactor(CSTMR) system was applied to the aqueous enzymatic extraction of sunflower seed oil, which decreases the production inhibition in the enzymatic hydrolysis process and increase reaction rate. The operation conditions were established, and influences of buffer pH, the temperature, time of hydrothermal treatment on yield of free oil were studied. The results showed that a high yield of free oil was obtained when sunflower seed was treated by hydrothermal treatment at 110℃for 60min in pH4.8 citrate buffer. The optimum conditions of extraction sunflower seed oil were determined by condition test and response surface analysis test. 89.8% free oil was obtained when sunflower seed was hydrolyzed at following conditions: enzyme amount: 2% (W/W), temperature: 50℃, time: 5.5h, dilution ratio: 1/8 (W/W).
The influences of hydrothermal treatment and enzymatic hydrolysis on the damage of cell structure during the process of aqueous enzymatic extraction were studied with microphotograph of safranin stained technology. The results exhibited that the seed cell structure was destroyed during the mentioned process. Microphotograph of sudanⅢstained technology was used to research of the oil releasing process, which showed that it was very easy to release oil from enzymatic hydrolysis seed. All the research results mentioned above, in a microstructure standard, suggested that the aqueous enzymatic extraction technology of sunflower seed oil was feasible.
The influence of aqueous enzymatic extraction on the quality of sunflower seed oil was also studied. The results showed that the temperature, time of hydrothermal treatment and pH of buffer had no influence on the color. The value of acid decreased from 2.17 to 1.98 mgKOH/g, while peroxide value varied from 1.36 to 2.53 meq/kg, according to the pH value increased from 3 to 7. The temperature and time of hydrothermal treatment had a great effect on the value of acid and peroxide value. Although hydrolysis process last 5h, it had no influence on the color, and the value of acid, peroxide and fatty acid content had rarely been affected by hydrolysis process.
Sunflower isolated proteins, sunflower globular proteins and sunflower albumins were prepared from the defatted sunflower meal. Compared with soybean isolated proteins, the structure, function and physiochemical properties of sunflower proteins were investigated. The results showed that sunflower concentrated protein and isolated proteins have lowest solubility when pH value was 5, while sunflower globular proteins have lowest solubility when pH value was 4. Functional properties of sunflower isolated proteins were better or close to those of soybean isolated proteins, apart from the water absorption capacity of sunflower isolated protein was lower than that of soybean isolated proteins. The fat absorption capacity, the foam formation and foam stability, the emulsification activity and emulsification stability of sunflower isolated proteins were all higher than that of soybean isolated proteins, especially emulsification activity of sunflower globular proteins was very good.
Sunflower isolated proteins showed five fractions by gel filtration and three major fractions having relatively molecular weights of 380,000,100,000 and 27,000. 11S globular proteins were the major fractions of sunflower isolated proteins. It was found that 10 major bands and 2 minor bands in sunflower globulins while sunflower albumins have 4 major bands and some minor bands by gel electrophoresis. The relatively molecular weight of the major bands of sunflower globular proteins were 53,500、42,600、39,500、33,200、30,800、26,500、24,000、22,500、20,8kDa和19.200. SDS-PAGE of sunflower isolated proteins found that there more bands than total bands of 11S globulins and 2S albumins. It indicated that sunflower isolated proteins had 7S globular proteins and other proteins apart from the 11S globular proteins and 2S albumin proteins. CD showed that the major structures of sunflower globular proteins and sunflower albumin proteins wereβ-sheet structure and random coil. Sunflower protein isβ- sheet type protein. DSC showed that the denaturation temperature of 11S sunflower globulins powder and 2S sunflower albumins powder were 123.5℃and 122.8℃respectively, sunflower proteins had high denaturation temperature. The fluorescence emission spectrum of the 11S sunflower globulins and 2S sunflower albumins gave a maximum at 345nm and 340nm. It indicated a greater contribution of tryptophan residues.
Hydrolyzation of sunflower proteins with Alcalase, Protamex and Flavourzyme were studied. Taking DH, the soluble protein content and antioxidative activity of the hydrolysate as criteria, suitable conditions of sunflower proteins hydrolyzed with each enzyme were obtained. The molecular weight distribution of polypeptides in the hydrolysate processed with the three kinds of enzymes at different hydrolysis time was determined by gel chromatography. The results showed that relatively molecular weight mainly distributed from 590 to 2,975 when sunflower proteins hydrolysate was prepared with alcalase for 1h.
Accordingly, the relatively molecular weight mainly distributed from 370 to 4,226 when it was prepared with protamex. However, when sunflower proteins was treated with flavourzyme for 1h, the relatively molecular weight mainly distributed from 370 to 5,117.
One antioxidative peptide was obtained from the hydrolysate prepared with flavourzyme for 1h by ultrafiltration, ion exchange chromatography, gel chromatography and reversed phase chromatograpy methods. Amino acid sequences was determined by mass spectrograph analysis, which was Ala-Cys-Ala-His-Asp-Lys-Val.Antioxodative activity of this polypeptides was 79.6U/mL, which was a new polypeptide.
Neurospora sitophilo was applied to the fermentation of the defatted sunflower meal to produce the antioxidative peptide. The cultivation base content and the fermentation operation condition were optimized. The gel electrophoresis of the fermentation product soaping liquid and the molecular weight distribution of the peptide were also studied. A lot of antioxidative products were found in the soaping liquid, which relatively molecular weight mainly distributed from 3,396 to 166 .
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