Enzymatic extraction and physicochemical properties of Porphyra haitanensis protein
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摘要: 鲍鱼内脏中含有丰富的可分解藻类多糖的水解酶。为实现坛紫菜 (Porphyra haitanensis) 蛋白的高效提取和产业化制备，采用鲍鱼内脏酶对坛紫菜进行酶法破壁提取紫菜蛋白，并比较冷冻干燥与喷雾干燥对蛋白理化性质的影响。结果显示，鲍鱼内脏酶酶解坛紫菜提取蛋白的最佳条件为：加酶量7.6%、酶解时间2.8 h、酶解温度35 ℃、料液比质量体积比为1∶25，在该条件下获得的蛋白得率为 (238.65±2.13) mg∙g−1。观察坛紫菜的外观及细胞形态，发现鲍鱼内脏酶能显著破坏坛紫菜细胞壁。冷冻干燥坛紫菜蛋白 (Freeze-dried P. haitanensis protein, FPP) 在不同pH下的溶解性和乳化性能均优于喷雾干燥坛紫菜蛋白 (Spray-dried P. haitanensis protein, SPP) (P<0.01)，而SPP的表面疏水性与接触角均高于FPP (P<0.01)。扫描电镜结果显示， FPP为表面光滑的片状结构，而SPP呈大小较为均一、表面有凹槽的球状颗粒。综上，鲍鱼内脏酶能有效破坏坛紫菜细胞壁，使水溶性蛋白溶出。制备得到的蛋白均具有良好的理化特性，其中冷冻干燥所制备蛋白的理化性质更佳。Abstract: Abalone viscera is rich in hydrolytic enzymes that can decompose algal polysaccharides. In order to realize the highly efficient extraction and industrial production of Porphyra haitanensis protein, we used abalone visceral enzymes to break the cell wall of P. haitanensis to extract porphyra protein, and compared the physicochemical properties in proteins prepared by freeze drying and air drying. The results show that the optimal enzymatic conditions were obtained as follows: enzyme dosage of 7.6%, enzymatic hydrolysis time of 2.8 h, enzymatic hydrolysis temperature of 35 ℃, and material-to-liquid ratio of 1:25. The protein yield under above conditions was (238.65±2.13) mg∙g−1. The results of appearance morphology and cell morphology of Porphyra haitanensis indicate that abalone viscera enzymatic digestion could break down the cell wall of P. haitanensis significantly. Freeze-dried P. haitanensis protein (FPP) showed better solubility and emulsification properties than spray-dried P. haitanensis protein (SPP) at different pHs (P<0.01), while the surface hydrophobicity and contact angle of SPP were higher than those of FPP (P<0.01). Scanning electron microscopy shows that FPP had a smooth lamellar surface, while SPP had a more uniform spherical particle size with grooves on the surface. In conclusion, the abalone viscera enzyme was effective in breaking down the cell wall of P. haitanensis and leaching out the water-soluble proteins. All the prepared proteins had good physicochemical properties, while the freeze-dried proteins are better than air-dried proteins.
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图 1 加酶量、温度、时间和料液比对鲍鱼内脏酶酶解紫菜的影响
注：同一序列中不同小写字母表示显著差异 (P<0.05)；图6、图7同此。
Figure 1. Effects of enzyme addition, temperature, time and material-to-liquid ratio on enzymatic digestion of P. haitanensis by abalone viscera enzyme
Note: Different lowercase letters in the same series indicate significant differences (P<0.05). The same case in Fig. 6 and Fig. 7.
图 2 加酶量、时间和温度交互作用响应面图和等高线图
Figure 2. Response surface and contour plots of enzyme addition, time and temperature interactions
图 3 不同破壁方式紫菜蛋白得率和固形物得率对比
注：不同大、小写字母表示显著差异 (P<0.05)。
Figure 3. Comparison of protein yield and solids yield of P. haitanensis by different cell wall breaking methods
Note: Different uppercase and lowercase letters in the same series indicate significant differences (P<0.05).
图 4 不同破壁方式紫菜外观形态及光学显微镜图片 (10×40)
注：从左到右依次为未处理、溶胀、冻融、超声、鲍鱼内脏酶酶解；a—e为外观形态，f—j为光学显微镜图片。
Figure 4. Appearance morphologies and optical microscope images of P. haitanensis by different cell wall breaking methods (10×40)
Note: From left to right: untreated, lysed, freeze-thawed, sonicated and abalone visceral enzymatic digestion; a–e. appearance morphologies, f–j. optical microscope images.
图 6 pH 对冻干坛紫菜蛋白和喷干坛紫菜蛋白的溶解度和表面疏水性的影响
Figure 6. Effect of pH on solubility and surface hydrophobicity of FPP and SPP
图 7 pH 对冻干坛紫菜蛋白和喷干坛紫菜蛋白乳化性和乳化稳定性的影响
Figure 7. Effect of pH on emulsification and emulsion stability of FPP and SPP
表 1 Box-Behnken Design 设计方案
Table 1. Box-Behnken Design solution
因素 Factor 水平 Level −1 0 1 A 加酶量 Enzyme dosage/% 6 7 8 B 酶解时间 Enzymolysis time/h 1 2 3 C 酶解温度 Enzymolysis temperature/℃ 30 35 40 
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表 2 鲍鱼内脏酶酶解紫菜响应面实验设计及结果
Table 2. Response surface experimental design and results of enzymatic digestion of P. haitanensis by abalone viscera enzyme
序号
No.A：加酶量
Enzyme
dosage/%B：时间
t/hC：温度
Temperature/
℃Y：蛋白得率
Protein yield/
(mg∙g−1)1 6 1 35 158.46 2 8 1 35 189.04 3 6 3 35 183.47 4 8 3 35 242.13 5 6 2 30 174.96 6 8 2 30 215.56 7 6 2 40 168.66 8 8 2 40 209.65 9 7 1 30 185.99 10 7 3 30 219.76 11 7 1 40 178.40 12 7 3 40 217.07 13 7 2 35 229.99 14 7 2 35 232.14 15 7 2 35 231.90 16 7 2 35 226.36 17 7 2 35 228.45
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表 3 回归模型方差分析
Table 3. Regression model analysis of variance
来源 Source 平方和SS 自由度df 均方MS F P 显著性Sig. 模型 Model 11 105.40 9 1 233.91 244.51 <0.000 1 ** A 3 648.83 1 3 648.83 723.04 <0.000 1 ** B 2 832.76 1 2 832.76 561.33 <0.000 1 ** C 63.27 1 63.27 12.54 0.009 5 ** AB 197.04 1 197.04 39.04 0.000 4 ** AC 0.038 1 0.038 0.007 554 0.933 2 BC 5.99 1 5.99 1.19 0.312 1 A2 2 092.93 1 2 092.93 414.93 <0.000 1 ** B2 849.05 1 849.05 168.24 <0.000 1 ** C2 981.24 1 981.24 194.44 <0.000 1 ** 残差 Residual 35.33 7 5.05 失拟差 Lack of fit 11.75 3 3.92 0.66 0.616 3 不显著 纯误差 Pure error 23.58 4 5.90 总和 Total 11 140.72 16 注：*. 差异显著 (P<0.05)；**. 差异极显著 (P<0.01)。 Note: *. Significant differences (P<0.05); **. Extremely significant differences (P<0.01).
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