海参自溶过程中生化变化及抗氧化活性寡肽的研究
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摘要
海参是海洋中最常见的无脊椎动物,具有极高的营养价值。海参具有极强的自溶能力,在受到外界物理因素、化学因素和生理因素等刺激后,会出现吐肠和体壁软化的现象,严重影响了海参的品质,给其运输和加工等带来极大不便。对海参自溶进行系统研究,对促进海参深加工产业的发展具有重要意义。
本文针对海参体壁和海参肠的自溶特性,研究其在自溶过程中所发生的一系列化学成分的变化和鲜度的变化,明确海参自溶过程的影响因素。同时,利用海参自溶技术制备海参肠自溶寡肽,并对其进行抗氧化活性的评价和氨基酸序列的鉴定。论文所得主要结论如下：
(1)在海参体壁和海参肠自溶过程中,蛋白质和糖类两种主要化学成分均发生不同程度的变化。考察温度、pH、NaCl浓度和液料比对海参体壁和肠自溶过程的影响,发现温度和pH是两个最主要的影响因素。根据不同条件下海参体壁和肠自溶过程中主要化学成分溶出情况的变化规律,确定TCA可溶性寡肽溶出量为海参体壁自溶过程的评价指标,同时,还可采用还原糖溶出量作为海参肠自溶过程的评价指标。
(2)海参肠在室温自溶和50℃自溶前后,组织蛋白酶B和组织蛋白酶L的活性均明显增强。室温自溶时,分别提高了11%和6%,50℃自溶时,分别提高了11%和17%。半胱氨酸蛋白酶抑制剂E-64、抗痛素及碘乙酸可显著抑制海参肠自溶,在浓度为1mmol/L条件下,抑制率分别达到48.97%、47.24%和12.24%。同时,半胱氨酸蛋白酶的激活剂DTT对海参自溶过程有明显的促进作用。上述结果表明半胱氨酸蛋白酶可能在海参肠自溶过程中起重要作用。
(3)控制海参自溶时,应限制在较低的温度,pH可以控制在偏碱性范围内,而且要尽量避免阳光的照射。在加工即食海参等制品时,温度要超过60℃,以抑制海参自溶酶活性,进而保证产品具有良好的形态。利用自溶时,温度应控制在37～60℃,pH应控制在4～7之间：
(4)采用响应面法研究不同影响因素对海参体壁自溶过程的交互作用。结果表明,分别以温度、pH、NaCl浓度和温度、pH、底物浓度为变量因素所建立的响应面模型均可以准确的反映不同因素对海参体壁自溶过程的交互作用。pH和底物浓度对海参体壁自溶过程的交互作用显著,表现为在较低底物浓度下,pH越低,TCA可溶性寡肽含量下降越快,对海参体壁自溶越不利。
(5)研究海参体壁在25℃和46℃自溶过程中的鲜度变化。结果显示,pH随自溶时间延长呈先下降后上升趋势,在自溶20 h时最低。挥发性盐基氮(TVBN)的含量随着自溶时间的延长而逐渐增加,在25℃和46℃条件下分别自溶44 h和32 h时,TVBN超过20mg/100g。46℃自溶32h时,TVC超过106 cfu/g。上述结果表明,本研究所采用的4h自溶条件下的海参体壁自溶与微生物作用无关。
(6)以TCA-可溶性寡肽含量(以干基计)为响应值,采用响应面法优化得到海参肠自溶的最佳条件为温度48.30℃、pH 4.43、料液比1：3和自溶时间4 h。
(7)海参肠自溶水解物主要由分子质量小于3 kDa的组分组成,含有17种氨基酸,具有一定的DPPH自由基清除能力、Fe2+螯合能力和还原能力,其SC50、CC50和ACo.5值分别为4.04、5.91和8.06 mg/mL。
(8)海参肠自溶水解物经Sephadex G-15分离纯化后得到4个组分,分别为Ⅰ、Ⅱ、Ⅲ和Ⅳ,其中组分Ⅳ的DPPH自由基清除能力、Fe2+螯合能力和对DNA氧化损伤的保护作用最强,这可能与其Ile和Leu等含量较高有关。
(9)组分Ⅳ经ESI-MS/MS进行氨基酸序列分析,得到三种具有抗氧化活性的寡肽,分别为两种四肽Val-Thr-Pro-Tyr (479 Da)和Val-Leu-Leu-Tyr(507 Da)和一种六肽Val-Gly-Thr-Val-Glu-Met (635 Da)。在浓度为2mg/mL时,三种寡肽均对DNA氧化损伤具有明显的保护作用。说明利用自溶技术获得的海参肠抗氧化自溶寡肽作为功能性基料具有潜在的应用价值。
Sea cucumber is the most common invertebrate in the ocean with high nutritive value. It is susceptive to the environmental changes caused by physical, chemical and physiological factors and easy to undergo autolysis. The subsequent phenomena of gut vomitting and body wall softening will happened, which lead to severe deterioration in sea cucumber quality and bring great difficulties to the transportation and processing of sea cucumber. It is significant and necessary for the development of sea cucumber deep-processing industries to study the autolysis of sea cucumber systematicly.
In the present study, the biochemical changes including main chemical composition changes and freshness changes were investigated and the influencing factors of sea cucumber autolysis process were determined according to the autolysis characteristic of sea cucumber body wall and guts. Meanwhile, the autolysis method was adopted to prepare oligopeptides from sea cucumber guts. The antioxidant activities of oligopeptides were evaluated and their amino acid sequences were identified. The main conclusions were as followed:
(1) Both protein and sugar were changed during the autolysis process of sea cucumber body wall and guts. pH and temperature were found to be the two main factors by investigating the effects of pH, temperature, NaCl concentration and liquid to sample ratio on the autolysis process of sea cucumber body wall and guts. TCA-soluble oligopeptide was determined to be the evaluation indicator for the autolysis process of sea cucumber body wall and guts. Meanwhile, the reducing sugar content could also be used as the evaluation indicator for the autolysis of sea cucumber guts.
(2) The activities of cathepsin B and cathepsin L from sea cucumber guts were enhanced by 6%～17% after autolysis under different temperatures (25℃and 50℃). The inhibitors of cathepsin B and cathepsin L including E-64, antipain and iodoacetic acid significantly inhibited the autolysis of sea cucumber guts. Their suppressing rates of TCA-soluble oligopeptides contents reached to 48.97%,47.24% and 12.24%, respectively. Meanwhile, dithiothreitol as a activitor of cathepsin B and cathepsin L obviously promoted the autolysis process of sea cucumber guts. These results suggest that cathepsin B and cathepsin L may play an important role in the sea cucumber autolysis process.
(3) In order to control the autolysis the temperature should be low and the pH should be controlled in the alkaline range in the transportation and processing of sea cucumber. In addition, sea cucumber should be sheltered from direct sunlight to avoid the inductive effects of ultraviolet irradiation on its autolysis. When the ready-to-eat sea cucumber was produced, the heating treatment should be performed at the temperature higher than 60℃in order to inhibit the activities of endogenous enzyme and maintain good appearance. Ultraviolet irradiation could be used in utilization of sea cucumber autolysis, and the temperature and pH should be in the range of 37～60℃and 4-7, respectively.
(4) The combined interaction of different influencing factors on the sea cucumber autolysis was studied by response surface methodology. The results showed that the two models which were established on the basis of temperature, pH, NaCl concentration and temperature, pH and substrate concentration, respectively, could reflect the interaction accurately. The combined effect of pH and substrate concentration on the autolysis process of sea cucumber was significant. The TCA-soluble oligopeptides content decreased more quickly as the pH got more lower when the substrate concentration was relatively low, which was worse for the autolysis of sea cucumber body wall.
(5) The freshness changes during the autolysis process of sea cucumber body wall were investigated at both 25℃and 46℃. The results showed that pH reached to minimum at 20 h and then increased gradually. The contents of TVBN were increased gradually with the autolysis period prolonged, and reached higher than 20mg/100g when the body wall was autolyzed for 44 h and 32 h at 25℃and 46℃, respectively. When the body wall was autolyzed for higher than 8h, the TVC were increased gradually and reached to 106 cfu/g at 32 h. These results suggest that microorganisms were not involved in the autolysis process of sea cucumber body wall under the experimental conditions in the present study.
(6) The optimum autolytic conditions of sea cucumber guts were obtained by response surface methodology. Temperature at 48.30℃, pH at 4.43, sample to ratio 1:3 and time 4 h were found to be the optimal conditions to obtain autolysis hydrolysates from the guts of sea cucumber.
(7) The autolysis hydrolysates were mainly composed of components below 3 kDa and it contained 17 kinds of amino acids. The autolysis hydrolysates had DPPH radical scavenging capacity, Fe2+-chelating ability and reducing power. The values of SC50, CC50 and AC0.5 were 4.04,5.91 and 8.06 mg/mL, respectively.
(8) Four different fractions, namelyⅠ,Ⅱ,Ⅲand IV, were obtained after the autolysis hydrolysates were separated by Sephadex G-15. Fraction IV exhibited the highest DPPH radical scavenging capacity, Fe2+-chelating ability and protective effect against hydroxyl radicals-induced DNA damage, which may be related with the high content of Ile, Leu and Val.
(9) The fraction IV was analyzed by ESI-MS for molecular mass determination and ESI-MS/MS for the characterization of peptides. Three kinds of antioxidative oligopeptides, including two tetrapeptides Val-Thr-Pro-Tyr (479 Da), Val-Leu-Leu-Tyr (507 Da) and a hexapeptide Val-Gly-Thr-Val-Glu-Met (635 Da), were identified. All of them showed obvious protective effects against hydroxyl radicals-induced DNA damage. protective effect against DNA damage. These results suggest that antioxidant oligopeptides derived from the guts of sea cucumber by autolysis method could be utilized for functional foods.
本文针对海参体壁和海参肠的自溶特性,研究其在自溶过程中所发生的一系列化学成分的变化和鲜度的变化,明确海参自溶过程的影响因素。同时,利用海参自溶技术制备海参肠自溶寡肽,并对其进行抗氧化活性的评价和氨基酸序列的鉴定。论文所得主要结论如下：
(1)在海参体壁和海参肠自溶过程中,蛋白质和糖类两种主要化学成分均发生不同程度的变化。考察温度、pH、NaCl浓度和液料比对海参体壁和肠自溶过程的影响,发现温度和pH是两个最主要的影响因素。根据不同条件下海参体壁和肠自溶过程中主要化学成分溶出情况的变化规律,确定TCA可溶性寡肽溶出量为海参体壁自溶过程的评价指标,同时,还可采用还原糖溶出量作为海参肠自溶过程的评价指标。
(2)海参肠在室温自溶和50℃自溶前后,组织蛋白酶B和组织蛋白酶L的活性均明显增强。室温自溶时,分别提高了11%和6%,50℃自溶时,分别提高了11%和17%。半胱氨酸蛋白酶抑制剂E-64、抗痛素及碘乙酸可显著抑制海参肠自溶,在浓度为1mmol/L条件下,抑制率分别达到48.97%、47.24%和12.24%。同时,半胱氨酸蛋白酶的激活剂DTT对海参自溶过程有明显的促进作用。上述结果表明半胱氨酸蛋白酶可能在海参肠自溶过程中起重要作用。
(3)控制海参自溶时,应限制在较低的温度,pH可以控制在偏碱性范围内,而且要尽量避免阳光的照射。在加工即食海参等制品时,温度要超过60℃,以抑制海参自溶酶活性,进而保证产品具有良好的形态。利用自溶时,温度应控制在37～60℃,pH应控制在4～7之间：
(4)采用响应面法研究不同影响因素对海参体壁自溶过程的交互作用。结果表明,分别以温度、pH、NaCl浓度和温度、pH、底物浓度为变量因素所建立的响应面模型均可以准确的反映不同因素对海参体壁自溶过程的交互作用。pH和底物浓度对海参体壁自溶过程的交互作用显著,表现为在较低底物浓度下,pH越低,TCA可溶性寡肽含量下降越快,对海参体壁自溶越不利。
(5)研究海参体壁在25℃和46℃自溶过程中的鲜度变化。结果显示,pH随自溶时间延长呈先下降后上升趋势,在自溶20 h时最低。挥发性盐基氮(TVBN)的含量随着自溶时间的延长而逐渐增加,在25℃和46℃条件下分别自溶44 h和32 h时,TVBN超过20mg/100g。46℃自溶32h时,TVC超过106 cfu/g。上述结果表明,本研究所采用的4h自溶条件下的海参体壁自溶与微生物作用无关。
(6)以TCA-可溶性寡肽含量(以干基计)为响应值,采用响应面法优化得到海参肠自溶的最佳条件为温度48.30℃、pH 4.43、料液比1：3和自溶时间4 h。
(7)海参肠自溶水解物主要由分子质量小于3 kDa的组分组成,含有17种氨基酸,具有一定的DPPH自由基清除能力、Fe2+螯合能力和还原能力,其SC50、CC50和ACo.5值分别为4.04、5.91和8.06 mg/mL。
(8)海参肠自溶水解物经Sephadex G-15分离纯化后得到4个组分,分别为Ⅰ、Ⅱ、Ⅲ和Ⅳ,其中组分Ⅳ的DPPH自由基清除能力、Fe2+螯合能力和对DNA氧化损伤的保护作用最强,这可能与其Ile和Leu等含量较高有关。
(9)组分Ⅳ经ESI-MS/MS进行氨基酸序列分析,得到三种具有抗氧化活性的寡肽,分别为两种四肽Val-Thr-Pro-Tyr (479 Da)和Val-Leu-Leu-Tyr(507 Da)和一种六肽Val-Gly-Thr-Val-Glu-Met (635 Da)。在浓度为2mg/mL时,三种寡肽均对DNA氧化损伤具有明显的保护作用。说明利用自溶技术获得的海参肠抗氧化自溶寡肽作为功能性基料具有潜在的应用价值。
Sea cucumber is the most common invertebrate in the ocean with high nutritive value. It is susceptive to the environmental changes caused by physical, chemical and physiological factors and easy to undergo autolysis. The subsequent phenomena of gut vomitting and body wall softening will happened, which lead to severe deterioration in sea cucumber quality and bring great difficulties to the transportation and processing of sea cucumber. It is significant and necessary for the development of sea cucumber deep-processing industries to study the autolysis of sea cucumber systematicly.
In the present study, the biochemical changes including main chemical composition changes and freshness changes were investigated and the influencing factors of sea cucumber autolysis process were determined according to the autolysis characteristic of sea cucumber body wall and guts. Meanwhile, the autolysis method was adopted to prepare oligopeptides from sea cucumber guts. The antioxidant activities of oligopeptides were evaluated and their amino acid sequences were identified. The main conclusions were as followed:
(1) Both protein and sugar were changed during the autolysis process of sea cucumber body wall and guts. pH and temperature were found to be the two main factors by investigating the effects of pH, temperature, NaCl concentration and liquid to sample ratio on the autolysis process of sea cucumber body wall and guts. TCA-soluble oligopeptide was determined to be the evaluation indicator for the autolysis process of sea cucumber body wall and guts. Meanwhile, the reducing sugar content could also be used as the evaluation indicator for the autolysis of sea cucumber guts.
(2) The activities of cathepsin B and cathepsin L from sea cucumber guts were enhanced by 6%～17% after autolysis under different temperatures (25℃and 50℃). The inhibitors of cathepsin B and cathepsin L including E-64, antipain and iodoacetic acid significantly inhibited the autolysis of sea cucumber guts. Their suppressing rates of TCA-soluble oligopeptides contents reached to 48.97%,47.24% and 12.24%, respectively. Meanwhile, dithiothreitol as a activitor of cathepsin B and cathepsin L obviously promoted the autolysis process of sea cucumber guts. These results suggest that cathepsin B and cathepsin L may play an important role in the sea cucumber autolysis process.
(3) In order to control the autolysis the temperature should be low and the pH should be controlled in the alkaline range in the transportation and processing of sea cucumber. In addition, sea cucumber should be sheltered from direct sunlight to avoid the inductive effects of ultraviolet irradiation on its autolysis. When the ready-to-eat sea cucumber was produced, the heating treatment should be performed at the temperature higher than 60℃in order to inhibit the activities of endogenous enzyme and maintain good appearance. Ultraviolet irradiation could be used in utilization of sea cucumber autolysis, and the temperature and pH should be in the range of 37～60℃and 4-7, respectively.
(4) The combined interaction of different influencing factors on the sea cucumber autolysis was studied by response surface methodology. The results showed that the two models which were established on the basis of temperature, pH, NaCl concentration and temperature, pH and substrate concentration, respectively, could reflect the interaction accurately. The combined effect of pH and substrate concentration on the autolysis process of sea cucumber was significant. The TCA-soluble oligopeptides content decreased more quickly as the pH got more lower when the substrate concentration was relatively low, which was worse for the autolysis of sea cucumber body wall.
(5) The freshness changes during the autolysis process of sea cucumber body wall were investigated at both 25℃and 46℃. The results showed that pH reached to minimum at 20 h and then increased gradually. The contents of TVBN were increased gradually with the autolysis period prolonged, and reached higher than 20mg/100g when the body wall was autolyzed for 44 h and 32 h at 25℃and 46℃, respectively. When the body wall was autolyzed for higher than 8h, the TVC were increased gradually and reached to 106 cfu/g at 32 h. These results suggest that microorganisms were not involved in the autolysis process of sea cucumber body wall under the experimental conditions in the present study.
(6) The optimum autolytic conditions of sea cucumber guts were obtained by response surface methodology. Temperature at 48.30℃, pH at 4.43, sample to ratio 1:3 and time 4 h were found to be the optimal conditions to obtain autolysis hydrolysates from the guts of sea cucumber.
(7) The autolysis hydrolysates were mainly composed of components below 3 kDa and it contained 17 kinds of amino acids. The autolysis hydrolysates had DPPH radical scavenging capacity, Fe2+-chelating ability and reducing power. The values of SC50, CC50 and AC0.5 were 4.04,5.91 and 8.06 mg/mL, respectively.
(8) Four different fractions, namelyⅠ,Ⅱ,Ⅲand IV, were obtained after the autolysis hydrolysates were separated by Sephadex G-15. Fraction IV exhibited the highest DPPH radical scavenging capacity, Fe2+-chelating ability and protective effect against hydroxyl radicals-induced DNA damage, which may be related with the high content of Ile, Leu and Val.
(9) The fraction IV was analyzed by ESI-MS for molecular mass determination and ESI-MS/MS for the characterization of peptides. Three kinds of antioxidative oligopeptides, including two tetrapeptides Val-Thr-Pro-Tyr (479 Da), Val-Leu-Leu-Tyr (507 Da) and a hexapeptide Val-Gly-Thr-Val-Glu-Met (635 Da), were identified. All of them showed obvious protective effects against hydroxyl radicals-induced DNA damage. protective effect against DNA damage. These results suggest that antioxidant oligopeptides derived from the guts of sea cucumber by autolysis method could be utilized for functional foods.
引文
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