鸡腿菇子实体蛋白提取、特性及酶解研究
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摘要
鸡腿菇(Coprinus comatus)味道鲜美,营养丰富,富含多种生物活性物质且生产成本低,周期短,抗逆性强,单产高,经济效益显著,具有广阔的发展前景。但其研究基础尚显薄弱,尤其在其蛋白质的研究方面。
本文以鸡腿菇子实体为原料,优化了其蛋白的碱溶酸沉法最适提取工艺,研究了其蛋白的特性,寻求到了其蛋白酶解的最适酶制剂,并得到酶解最佳工艺条件。试验内容与结果如下：
(1)以鸡腿菇子实体为原料,以提取液pH值、提取温度、料液比、提取时间为其蛋白提取的影响因素,采用正交试验优化碱溶酸沉法提取蛋白的工艺条件,结果表明：鸡腿菇子实体蛋白提取的最佳工艺参数为提取液pH 11.5,提取温度50℃,料液比1：15,提取时间50min,蛋白提取率为24.3%,在pH 3.5条件下沉淀蛋白效果最佳。
(2)对制备所得的鸡腿菇子实体蛋白特性分析表明：其分子量主要集中在50-90kDa、20-35kDa和13-17kDa等区间内；必需氨基酸含量占氨基酸总量的40.57%,必需氨基酸含量与非必需氨基酸含量的比值为0.68,为理想蛋白质；在碱性条件下,鸡腿菇子实体蛋白显示了良好的溶解性、乳化性、起泡能力。
(3)以鸡腿菇子实体蛋白为原料,分别用木瓜蛋白酶、Alcalase 2.4L、Neutrase 0.8L、Protamex、Flavorzyme 1000L在各自适宜的条件下进行酶解,比较酶解物的水解度(DH)、蛋白质回收率(PR)、感官品质、抗氧化活性等。结果表明鸡腿菇子实体蛋白在Alcalase 2.4L的作用下,其水解度、蛋白质回收率最高,感官品质较为优良,抗氧化活性相对较高。研究了酶活底物比、底物浓度、酶解温度、pH值、时间对Alcalase 2.4L酶解鸡腿菇子实体蛋白的影响,并采用响应面法(RSM)优化得到了最佳工艺条件为酶解pH 9.0、温度54.8℃,酶活底物比6271 U/g、底物浓度1.0%、酶解时间240min,在此条件下,得到水解度为24.05%。
The basidiomycete, Coprinus comatus, is a kind of edible fungus reputed for its delicious taste and high nutrition. It contains a variety of functional active compounds. The production cost is low, with short production cycle, strong stress resistance and high yield. So it has broad propect for development. But the research on it remains rather weak, especially its protein.
In this paper, the extraction process of the fruit body protein of Coprinus comatus was optimized and its functional properties were studied. The best protease for enzymolysis of the fruit body protein of Coprinus comatus was determinded and the technical parameters were optimized. The main results were presented as follows:
(1) The effects of leaching solution pH value, temperature, ratio of solid to liquid, time on the extraction of the fruit body protein of Coprinus comatus were investigated. The optimum conditions of alkaline extraction were determined by orthogonal test plan and the isoelectric point was explored. In addition, the molecular weight distribution, amino acid composition and functional properties of the protein were studied. The results showed that the optimum extraction conditions were:pH at 11.5, temperature at 50℃, solid:liquid ratio(w/v) at 1:15, time at 50min. The isoelectric point was pH 3.8. At these conditions, the protein extraction rate was 24.3%, purity 80.7%.
(2) The molecular weight of the extracted protein mainly distributed in 50-90kDa, 20-35kDa and 13～17kDa areas and contained relatively complete amino acid composition. The ratio of essential amino acid to total amino acid is 40.57%, essential amino acid to nonessential amino acids at 0.68, which determinded it a good protein. The protein had good solubility, emulsibility and foaming properties at high alkaline condition.
(3) Five proteases (Papain, Alcalase 2.4L, Neutrase 0.8 L, Protamex, Flavorzyme 1000L) were used to hydrolyze the fruit body protein of Coprinus comatus at their suitable conditions. Based on the comparision of degree of hydrolysis (DH), protein recovery (PR), sensory evaluation and antioxidant activities of hydrolysates, Alcalase 2.4L was the most suitable protease for the protein, for its highest DH and PR, better sensory quality and antioxidant activities. The effects of enzymolysis temperature, pH, time, ratio of enzyme activity to substrate ratio ([E]/[S]) and substrate concentration on DH were studied. Response surface methodology was used to optimise the hydrolysis parameters of the fruit body protein of Coprinus comatus by Alcalase 2.4L. The results showed that the optimum conditions were pH at 9.0, temperature at 54.8℃, [E]/[S] at 6271U/g, time at 240min, substrate concentration at 1%. At these conditions, the DH was 24.05%.
本文以鸡腿菇子实体为原料,优化了其蛋白的碱溶酸沉法最适提取工艺,研究了其蛋白的特性,寻求到了其蛋白酶解的最适酶制剂,并得到酶解最佳工艺条件。试验内容与结果如下：
(1)以鸡腿菇子实体为原料,以提取液pH值、提取温度、料液比、提取时间为其蛋白提取的影响因素,采用正交试验优化碱溶酸沉法提取蛋白的工艺条件,结果表明：鸡腿菇子实体蛋白提取的最佳工艺参数为提取液pH 11.5,提取温度50℃,料液比1：15,提取时间50min,蛋白提取率为24.3%,在pH 3.5条件下沉淀蛋白效果最佳。
(2)对制备所得的鸡腿菇子实体蛋白特性分析表明：其分子量主要集中在50-90kDa、20-35kDa和13-17kDa等区间内；必需氨基酸含量占氨基酸总量的40.57%,必需氨基酸含量与非必需氨基酸含量的比值为0.68,为理想蛋白质；在碱性条件下,鸡腿菇子实体蛋白显示了良好的溶解性、乳化性、起泡能力。
(3)以鸡腿菇子实体蛋白为原料,分别用木瓜蛋白酶、Alcalase 2.4L、Neutrase 0.8L、Protamex、Flavorzyme 1000L在各自适宜的条件下进行酶解,比较酶解物的水解度(DH)、蛋白质回收率(PR)、感官品质、抗氧化活性等。结果表明鸡腿菇子实体蛋白在Alcalase 2.4L的作用下,其水解度、蛋白质回收率最高,感官品质较为优良,抗氧化活性相对较高。研究了酶活底物比、底物浓度、酶解温度、pH值、时间对Alcalase 2.4L酶解鸡腿菇子实体蛋白的影响,并采用响应面法(RSM)优化得到了最佳工艺条件为酶解pH 9.0、温度54.8℃,酶活底物比6271 U/g、底物浓度1.0%、酶解时间240min,在此条件下,得到水解度为24.05%。
The basidiomycete, Coprinus comatus, is a kind of edible fungus reputed for its delicious taste and high nutrition. It contains a variety of functional active compounds. The production cost is low, with short production cycle, strong stress resistance and high yield. So it has broad propect for development. But the research on it remains rather weak, especially its protein.
In this paper, the extraction process of the fruit body protein of Coprinus comatus was optimized and its functional properties were studied. The best protease for enzymolysis of the fruit body protein of Coprinus comatus was determinded and the technical parameters were optimized. The main results were presented as follows:
(1) The effects of leaching solution pH value, temperature, ratio of solid to liquid, time on the extraction of the fruit body protein of Coprinus comatus were investigated. The optimum conditions of alkaline extraction were determined by orthogonal test plan and the isoelectric point was explored. In addition, the molecular weight distribution, amino acid composition and functional properties of the protein were studied. The results showed that the optimum extraction conditions were:pH at 11.5, temperature at 50℃, solid:liquid ratio(w/v) at 1:15, time at 50min. The isoelectric point was pH 3.8. At these conditions, the protein extraction rate was 24.3%, purity 80.7%.
(2) The molecular weight of the extracted protein mainly distributed in 50-90kDa, 20-35kDa and 13～17kDa areas and contained relatively complete amino acid composition. The ratio of essential amino acid to total amino acid is 40.57%, essential amino acid to nonessential amino acids at 0.68, which determinded it a good protein. The protein had good solubility, emulsibility and foaming properties at high alkaline condition.
(3) Five proteases (Papain, Alcalase 2.4L, Neutrase 0.8 L, Protamex, Flavorzyme 1000L) were used to hydrolyze the fruit body protein of Coprinus comatus at their suitable conditions. Based on the comparision of degree of hydrolysis (DH), protein recovery (PR), sensory evaluation and antioxidant activities of hydrolysates, Alcalase 2.4L was the most suitable protease for the protein, for its highest DH and PR, better sensory quality and antioxidant activities. The effects of enzymolysis temperature, pH, time, ratio of enzyme activity to substrate ratio ([E]/[S]) and substrate concentration on DH were studied. Response surface methodology was used to optimise the hydrolysis parameters of the fruit body protein of Coprinus comatus by Alcalase 2.4L. The results showed that the optimum conditions were pH at 9.0, temperature at 54.8℃, [E]/[S] at 6271U/g, time at 240min, substrate concentration at 1%. At these conditions, the DH was 24.05%.
引文
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