Optimization of Preparing Technology of Clam Peptide and Its Enhanced Immunomodulatory Effect
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摘要: 目的：以菲律宾蛤仔（Ruditapes philippinarum）为原料，研究蛋白肽制备工艺及其增强免疫活性。方法：以蛋白水解度为评价指标，筛选最适蛋白酶，采用单因素实验和响应面试验确定最佳酶解条件；氨基酸分析仪分析蛤蜊肽氨基酸组成；通过小鼠器官/体重比值、小鼠脾淋巴细胞转化实验、血清溶血素实验、小鼠腹腔巨噬细胞吞噬鸡红细胞实验、NK细胞活性实验评价蛤蜊肽的增强免疫活性。结果：菲律宾蛤仔蛋白肽制备的最适蛋白酶为胰蛋白酶，最佳酶解条件为温度48.4 ℃，pH8.0，加酶量3795 U/g，料液比1:2，水解时间4 h，该工艺下蛋白水解度达到15.33%，蛋白肽重均分子量为418 Da；其氨基酸组成合理，必需氨基酸占比达到41.48%；经口给予小鼠不同剂量的蛤蜊肽30 d，与空白对照组比较，小鼠的脏器比值无显著影响（P>0.05），低剂量（700 mg/（kg·d））与高剂量（2800 mg/（kg·d））下能显著提高血清溶血素水平（P<0.05），低剂量（700 mg/（kg·d））与中剂量（1400 mg/（kg·d））下能显著提高小鼠腹腔巨噬细胞吞噬鸡红细胞吞噬率（P<0.01），具有较好的增强免疫活性。结论：该方法下制备的蛤蜊肽水解程度较高、分子量低且分布集中，并具有一定增强免疫活性，具有开发成保健食品或特殊膳食食品的潜能。Abstract: Objective: Ruditapes philippinarum was used as raw material to investigate the preparation process of peptide and its enhanced immunomodulatory effect. Methods: The hydrolysis degree of protein was used as an evaluation index to screen the optimal protease. The single factor experiment and response surface test were used to determine the best enzymatic hydrolysis condition. The amino acid composition was analyzed using amino acid analyzer. And organ/body weight ratio, spleen lymphocyte transformation test, serum hemolysin experiment, peritoneal macrophage swallowing chicken red blood cell test, and NK cell activation experiment were used to assess the clam peptide enhanced immune activity. Results: Trypsin was the most suitable protease in the preparation of protein peptides from Ruditapes philippinarum. The optimal enzymatic conditions were temperature 48.4 ℃, pH8.0, enzyme concentration 3795 U/g, solid-liquid ratio 1:2, and hydrolysis time 4 hours. The average molecular weight of protein-peptide was 418 Da with 15.33% hydrolysis degree under optimal enzymatic conditions. The amino acid composition of the clam peptide was reasonable and the proportion of essential amino acids reached 41.48%. The organ ratio of BALB/c mice did not change significantly as compared to the control group after receiving various doses of oral clam peptides for 30 days. Furthermore, the serum hemolysin levels were significantly (P<0.05) increased of low dose (700 mg/(kg·d)) and high dose (2800 mg/(kg·d)) groups and the phagocytic rate of peritoneal macrophage swallowing chicken red blood cells was significantly (P<0.01) increased of low dose (700 mg/(kg·d)) and middle dose (1400 mg/(kg·d)) groups, which indicated the enhanced immunomodulatory effect of the clam peptide. Conclusions: The clam peptide produced using this process shows a high degree of hydrolysis, a low molecular weight, and a concentrated distribution with enhanced immune function. It has the potential to produce healthy foods or foods for special dietary uses.
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图 2 不同蛋白酶酶解后分子量分布图谱
Figure 2. The distribution plots of molecular weight after enzymatic hydrolysis by varies proteases
图 3 酶解温度对蛤蜊蛋白水解度的影响
Figure 3. Effect of enzymatic temperatures on the degree of hydrolysis of clam protein
图 5 加酶量对蛤蜊蛋白水解度的影响
Figure 5. Effect of enzyme concentration on the degree of hydrolysis of clam protein
图 6 料液比对蛤蜊蛋白水解度的影响
Figure 6. Effect of solid-liquid ratio on the degree of hydrolysis of clam protein
图 7 酶解时间对蛤蜊蛋白水解度的影响
Figure 7. Effect of enzymatic time on the degree of hydrolysis of clam protein
图 8 双因素交互作用对蛤蜊蛋白水解度的响应面
Figure 8. Response of two-factor interaction to the degree of hydrolysis of clam protein
图 10 蛤蜊肽对小鼠免疫功能的影响
Figure 10. Effect of clam peptide on immune function in mice
注：与NC组比较，*P<0.05、**P<0.01。
表 1 六种蛋白酶酶解参数
Table 1. Enzymatic parameters of six proteases
酶种类 pH 温度（℃） 时间（h） 加酶量（U/g） 料液比 碱性蛋白酶 10 45 4 3000 1:3 中性蛋白酶 7 45 4 3000 1:3 胰酶 8 50 4 3000 1:3 复合蛋白酶 7 50 4 3000 1:3 风味蛋白酶 7 50 4 3000 1:3 木瓜蛋白酶 6.5 45 4 3000 1:3 
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表 2 响应面设计因素水平表
Table 2. Response surface design factor level table
水平 因素 A pH B 温度（℃） C 加酶量（U/g） ?1 7 40 2000 0 8 50 3000 +1 9 60 4000 
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表 3 响应面试验结果
Table 3. Results of response surface experiment
试验号 A pH B 温度（℃） C 加酶量（U/g） D 水解度DH（%） 1 9 50 2000 9.41 2 7 50 2000 11.04 3 8 40 2000 6.64 4 8 50 3000 12.67 5 7 40 3000 9.41 6 8 60 4000 8.2 7 8 50 3000 12.79 8 8 50 3000 12.3 9 8 50 3000 14.6 10 8 60 2000 7.54 11 9 50 4000 12.79 12 7 50 4000 13.95 13 8 50 3000 14.11 14 9 40 3000 9.35 15 9 60 3000 8.57 16 7 60 3000 6.27 17 8 40 4000 13.93 
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表 4 回归模型的方差分析
Table 4. Analysis of variance of regression equation
来源 平方和 自由度 均方 F值 P值 显著 模型 133.26 9 14.81 5.35 0.0189 * A-pH 1.05 1 1.05 0.38 0.557 B-温度 21.62 1 21.62 7.82 0.0267 * C-加酶量 38.75 1 38.76 14.02 0.0072 ** AB 1.04 1 1.04 0.38 0.559 AC 0.058 1 0.058 0.021 0.8893 BC 10.99 1 10.99 3.97 0.0865 A2 0.056 1 0.056 0.02 0.8905 B2 56.97 1 56.97 20.6 0.0027 ** C2 1.22 1 1.22 0.44 0.5279 残差项 19.36 7 2.77 -- -- 失拟项 15.36 3 5.12 5.11 0.0744 纯误差 4.00 4 1.00 -- -- 总离差 152.62 16 -- -- -- 注：*表示显著P<0.05；**表示极显著P<0.01。 
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表 5 蛤蜊肽氨基酸分析
Table 5. The amino acid analysis of clam peptide
氨基酸种类 含量（%） 天门冬氨酸Asp 3.97 谷氨酸Glu 7.27 丙氨酸Ala# 3.88 甘氨酸Gly 4.73 胱氨酸Cys 0.74 脯氨酸Pro# 2.26 丝氨酸Ser 2.16 酪氨酸Tyr# 2.52 非必需氨基酸总量（NEAA） 27.53 组氨酸His* 1.28 精氨酸Arg* 4.15 半必需氨基酸总量（SEAA） 5.43 缬氨酸Val# 3.08 蛋氨酸Met# 1.32 苯丙氨酸Phe# 4.93 异亮氨酸Ile# 2.57 亮氨酸Leu# 4.21 赖氨酸Lys* 4.86 苏氨酸Thr 2.40 色氨酸Trp# 0.00 必需氨基酸总量（EAA） 23.37 疏水氨基酸总量（HAA） 24.77 正电荷氨基酸总量（PCAA） 10.29 氨基酸总量（TAA） 56.33 EAA/TAA 41.48 HAA/TAA 43.98 PCAA/TAA 18.26 注：#为疏水氨基酸，*为正电荷氨基酸。 
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表 6 蛤蜊肽对小鼠脏器指数的影响
Table 6. Effect of clam peptide on organ indices in mice
组别 脏器指数（%） 肝脏 脾 左肾 右肾 NC 4.36±0.33 0.44±0.05 0.76±0.05 0.78±0.05 PC 4.31±0.35 0.46±0.09 0.79±0.07 0.84±0.07 GL 4.08±0.29 0.46±0.04 0.80±0.05 0.81±0.05 GM 4.33±0.29 0.49±0.07 0.85±0.06 0.84±0.05 GH 4.41±0.38 0.43±0.06 0.85±0.07 0.85±0.08 
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