Optimization of Sturgeon Cartilage Products and Its Regulation on Human Chondrocyte Inflammation
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摘要: 本文旨在以鲟鱼软骨为原料制得一种鲟鱼软骨制品，并探究其对人软骨细胞炎症的影响。通过高温高压技术对鲟鱼软骨和龙筋进行热液化处理并加工制得了一种鲟鱼软骨制品，通过单因素及正交试验确定了其最佳制备条件，通过制备含药血清来培养人软骨细胞，以MTT、阿尔新兰染色、ELISA等方法考察了鲟鱼软骨制品对白细胞介素1β（Interleukin 1β，IL-1β）诱导细胞炎症模型的影响。结果表明，鲟鱼软骨制品的最佳制备条件为热液化温度120 ℃、热液化时间20 min、熬煮温度80 ℃、熬煮时间30 min，此条件下该产品的感官评分为59.69分，且鲟鱼软骨制品组的含药血清不具备细胞毒性，能够刺激软骨细胞分泌胶原蛋白及蛋白聚糖等细胞外基质。同时，该产品可以显著提升炎症细胞的增殖活力达156%（P<0.05），且可以显著抑制炎症状态下软骨细胞NO、INOS、IL-6、COX-2和PGE2的表达水平（P<0.05）。Abstract: The purpose of this paper was to prepare a sturgeon cartilage product from sturgeon cartilage and explore its effect on inflammation of human chondrocytes. A kind of sturgeon cartilage product was prepared by thermal liquefaction of sturgeon cartilage and dragon tendon at high temperature and high pressure. The optimum preparation conditions were determined by single factor and orthogonal test. Human chondrocytes were cultured by preparing drug-containing serum and the effects of sturgeon cartilage products on Interleukin 1β (IL-1β)-induced cell inflammatory model were investigated by MTT, Alsinland staining and ELISA. The results showed that the best preparation conditions of sturgeon cartilage products were as follows: Hot liquefaction temperature was 120 ℃, hot liquefaction time was 20 min, boiling temperature was 80 ℃, boiling time was 30 min. Under this condition, the sensory score of the product was 59.69. The drug-containing serum of sturgeon cartilage products group had no cytotoxicity and could stimulate chondrocytes to secrete collagen and proteoglycan and other extracellular matrix. Meanwhile, the product could significantly increase the proliferation activity of inflammatory cells by 156% (P<0.05), and significantly inhibited the expression levels of NO, INOS, IL-6, COX-2 and PGE2 in the inflammatory chondrocytes (P<0.05).
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Key words:
- sturgeon /
- cartilage /
- osteoarthritis /
- chondrocyte /
- inflammatory factor /
- process optimization
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图 2 不同热液化条件对鲟鱼软骨制品感官品质的影响
Figure 2. Effects of different thermal liquefaction conditions on sensory quality of sturgeon cartilage product
注：（a）为热液化时间；（b）为热液化温度。
图 3 不同熬煮条件对鲟鱼软骨制品感官品质的影响
Figure 3. Effects of different boiling conditions on sensory quality of sturgeon cartilage product
注：（a）为熬煮时间；（b）为熬煮温度。
图 4 鲟鱼软骨制品外观图
Figure 4. Appearance of sturgeon cartilage product
注：（a）、（b）为鲟鱼软骨制品外观侧视图和俯视图。
图 6 软骨细胞阿尔新蓝染色
Figure 6. Alcian blue staining of chondrocytes
注：1：未造模组；2：空白对照组；3：空白血清组; 4: 阳性对照组；5~7：鲟鱼软骨制品血清低、中、高浓度组。
图 7 不同组含药血清对软骨细胞活力的影响
Figure 7. Effect of different groups of drug-containing serum on chondrocyte viability
图 8 不同组含药血清对软骨细胞培养液中细胞因子表达水平的影响
Figure 8. Effect of different groups of drug-containing serum on cytokine expression in chondrocyte culture medium
注：（A）、（B）、（C）分别为INOS和NO、IL-8和IL-6、 COX-2和PGE2的表达水平。
表 1 正交试验因素水平设计
Table 1. Factor and level of orthogonal experimental
水平 因素 热液化温度（℃） 热液化时间（min） 熬煮温度（℃） 熬煮时间（min） 1 110 15 70 20 2 120 20 80 30 3 130 25 90 40 
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表 2 感官评定标准
Table 2. Sensory evaluation standard
项目 总分（分） 色泽 性状 气味、滋味 浅棕黄色，色泽透亮均匀 质地均匀透明，无分层 香味浓郁协调，无腥味，无苦涩感，细腻顺滑 46~60 浅棕黄色，色泽略暗均匀 质地均匀，略透明，无分层 香味稍淡，无腥味，略涩，口感顺滑 31~45 棕黄色，色泽暗淡 质地略不均匀，有絮状物漂浮 无香味，略带腥味，偏甜或偏酸，略涩，口感略微下降 16~30 深棕色，色泽较黑不均匀 质地不均匀，有絮状沉淀 难以接受的臭味，腥味重，过甜或过酸，有涩味，口感难以接受 0~15
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表 3 各单糖成分的线性回归方程和范围
Table 3. The linear equations and linear ranges of each monosaccharide
单糖成分 线性回归方程 决定系数（R2） 线性范围（μg/mL） 甘露糖 y=26550x−15442 0.9996 1.50~650.00 氨基葡萄糖 y=18445x−15004 0.9991 1.50~650.00 葡萄糖醛酸 y=32008x+123354 0.9994 1.50~650.00 氨基半乳糖 y=18664x+12116 0.9997 1.50~650.00 葡萄糖 y=18008x−487660 0.9992 1.50~650.00 半乳糖 y=38562x−6624 0.9994 1.50~650.00
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表 4 正交试验结果
Table 4. Results of orthogonal experiment
实验号 A热液化温度 B热液化时间 C熬煮温度 D熬煮时间 感官评分（分） 1 1 1 1 1 52.86 2 1 2 2 2 58.11 3 1 3 3 3 55.72 4 2 1 2 3 59.19 5 2 2 3 1 59.17 6 2 3 1 2 58.51 7 3 1 3 2 58.33 8 3 2 1 3 57.78 9 3 3 2 1 57.12 K1 166.69 170.38 169.15 169.15 K2 176.87 175.06 174.42 174.95 K3 173.23 171.35 173.22 172.69 k1 55.56 56.79 56.38 56.38 k2 58.96 58.35 58.14 58.32 k3 57.74 57.12 57.74 57.56 R 3.40 1.56 1.76 1.94
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表 5 方差分析结果
Table 5. Results of variance analysis
方差来源 自由度 F比 显著性 热液化温度 2 3.58 * 热液化时间 2 0.43 熬煮温度 2 0.56 熬煮时间 2 0.64 注：*表示显著，P<0.05。
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表 6 鲟鱼软骨制品理化指标检测结果
Table 6. Results of physical and chemical indexes of sturgeon cartilage product
项目 结果 定量限 限值 说明 甲基汞（mg/kg） 未检出 0.025 ≤1.0 符合 多氯联苯（PCBs）（mg/kg） 未检出 − ≤0.5 符合 N-二甲基硝胺（μg/kg） 未检出 1.0 ≤4.0 符合 铅（Pb）（mg/kg） 未检出 0.04 ≤1.0 符合 铬（Cr）（mg/kg） 未检出 0.1 ≤2.0 符合 镉（Cd）（mg/kg） 未检出 0.003 ≤0.1 符合 苯甲酸（g/kg） 未检出 0.010 不得检出 符合 无机砷（mg/kg） 未检出 0.08 ≤0.1 符合
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表 7 鲟鱼软骨制品微生物指标检测结果
Table 7. Results of microbiological index detection of sturgeon cartilage product
项目 结果 n c m M 说明 沙门氏菌/25 g 未检出 5 0 0 − 符合 金黄色葡萄糖球菌（CFU/g） <10 5 1 100 1000 符合 副溶血性弧菌（MPN/g） <0.3 5 1 100 1000 符合 注：n：同一批次产品应采集的样品件数；c：最大可允许超出m值的样品数；m：微生物指标可接受水平的限量值；M：微生物指标的最高安全限量值。
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表 8 鲟鱼软骨制品主要营养成分检测结果
Table 8. Results of main nutrients of sturgeon cartilage product
项目（g/100 g） 方法 结果 检出限 胶原蛋白 羟脯氨酸含量换算 2.20 0.1 硫酸软骨素 GB/T 20365-2006 2.82 0.1 氨基葡萄糖 GB/T 20365-2006 1.17 0.1 注：羟脯氨酸含量换算方法，参考陆剑锋等[20]的方法。
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表 9 含药血清中单糖成分分析
Table 9. Analysis of monosaccharide composition in drug-containing serum
组别 摩尔浓度比率（%） 甘露糖（%） 氨基葡萄糖（%） 葡萄糖醛酸（%） 氨基半乳糖（%） 葡萄糖（%） 半乳糖（%） 空白对照组 27.56±0.12a 27.66±0.04c 0.99±0.16a 0.58±0.04b 28.40±0.74c 14.80±1.42b 阳性对照组 24.44±0.24bc 31.83±1.24a 0.16±0.02c 0.62±0.14a 31.05±0.86b 11.90±0.24cd 产品低浓度组 26.30±0.09ab 30.04±0.84b 0.08±0.06d 0.57±0.27b 27.34±1.24d 15.67±0.06ab 产品中浓度组 25.15±0.14b 32.26±1.66a 0.08±0.08d 0.59±0.12b 31.08±1.08b 10.84±1.04d 产品高浓度组 23.43±0.60c 30.01±0.57b 0.22±0.04b 0.64±0.02a 33.05±0.26a 12.65±0.84c 注：同列不同小写字母表示组间差异显著（P<0.05）。
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