鲤鱼卵钙离子结合活性肽的制备及钙结合机制的研究
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摘要
本论文以鲤鱼鱼卵为研究对象，优化酶解工艺制备鱼卵钙离子结合活性肽（FEP），优化活性肽与钙离子结合条件，制备鱼卵肽钙复合物（FEP-Ca），通过构建缺钙大鼠模型研究FEP-Ca促钙吸收活性。从FEP中分离纯化高活性钙离子结合肽并鉴定其结构，对活性肽与钙结合的机制进行探讨。研究结果如下：
1.鲤鱼卵酶解制备FEP条件优化
鲤鱼卵脱脂后含有1.09%的磷，氮磷摩尔比为31.31，蛋白质中丝氨酸摩尔百分数为6.81%。以水解度和酶解液钙离子结合活性为指标，优化了鱼卵酶解工艺。结果表明胰蛋白酶对鱼卵的酶解效果最好，当鱼卵脱磷率为30.39%时，在49℃，加酶量3000U/g，底物浓度2%，pH9.0条件下，水解度为31.15%，酶解液结合钙活性最好，500mg/ml酶解液能结合0.67mmol/g的钙。
2. FEP-Ca复合物制备条件优化及其稳定性
以FEP的单位蛋白钙结合量和钙结合率为评价指标，对肽钙结合反应条件进行优化。结果表明在FEP浓度为5g/L，钙离子浓度为5mM，pH8.0，45℃，1h条件下，FEP单位蛋白钙结合量为0.86mmol/g，钙结合率为86%。此条件下制备的FEP-Ca复合物磷含量为3.78%，氮含量为11.15%，钙含量为7.27%，氮磷摩尔比为6.53。
FEP-Ca复合物具有较好的酸碱稳定性，在pH5-8范围内钙保留率高达90%以上。一定的加热处理会导致FEP-Ca复合物钙保留率下降，经过高温杀菌后钙保留率为71.33%。FEP-Ca复合物具有较高的抗消化性，经过胃蛋白酶、胰蛋白酶共同作用后，钙保留率为89.69%。磷酸盐能与FEP竞争钙离子，随着磷酸盐浓度的上升，FEP-Ca的钙保留率显著下降（p <0.05）。当磷酸盐浓度为30mM时，钙保留率降至37.05%。
3. FEP-Ca促钙吸收活性研究
缺钙大鼠饲喂FEP-Ca后，可显著增加体重、促进钙吸收、维持血钙和ALP水平稳定，增加骨重、骨密度和骨钙含量，增强骨生物力学性能，使上述指标达到或优于正常对照组水平。FEP-Ca对缺钙大鼠的补钙效果显著优于CaCO3(p <0.05)，而与CPP-Ca无显著性差异。FEP-Ca有望成为新型的补钙制剂。
4. FEP中高活性组分的分离纯化及结构鉴定
FEP经超滤分离后，将分子量小于5kDa的组分依次经羟基磷灰石层析、分子排阻层析及RP-HPLC分离纯化得到高活性组分，经ESI-QTOF-MS/MS分析，鉴定其氨基酸序列为(pS)S(pS)AF(pS)(pS)ELAR，分子量为1461Da，含有5个丝氨酸，其中有4个被磷酸化修饰，是一种典型的磷酸肽。
5.活性肽钙结合机制研究
人工合成高活性组分（IPP），经质谱和红外光谱分析表明，生理条件下磷酸基团是钙离子的优先结合部位，1分子IPP能结合4个钙离子，羧酸基团未参与钙离子的结合。红外光谱和圆二色谱分析表明，IPP生理条件下完全以无序状态存在。当同时升高pH（10以上）和钙离子浓度（3mM以上）后，羧基解离，钙离子对磷酸基团电荷产生屏蔽效应，使IPP在疏水作用和氢键作用下形成β折叠结构，并在两个羧酸根之间形成盐桥，进而构建网状结构，形成IPP-Ca纳米颗粒。而当体系中含有混合肽时，则可在pH6-8范围与钙离子结合形成纳米颗粒。
IPP在过饱和磷酸钙溶液中与磷酸根竞争与钙的结合，并吸附到Ca-P簌和无定形纳米粒（ACP）表面，抑制晶核形成和ACP定向聚集结晶，还能吸附到结晶表面抑制结晶长大产生沉淀。
In this thesis, fish egg peptides (FEP) with calcium-binding activity wereprepared from carp eggs through enzymatic technology and the parameters of FEPbinding calcium reaction were optimized to prepare peptide-calcium complex(FEP-Ca). The effects of FEP-Ca on increasing calcium bioavailability were furtherstudied in calcium-deficiency rats. A novel peptide was purified and identified toexplore the mechanism of the peptide binding calcium. The details of the work wouldbe shown as follows:
1. Preparation of FEP through enzymatic technology from carp eggs
Calcium-binding activity and degree of hydrolysis (DH) were adopted as indexto optimize the enzymatic technology. The trypsin showed the highest ability toprepare peptides with calcium-binding activity from carp eggs. Dephosphorizationtreatment of carp eggs could significantly increase the degree of hydrolysis. However,excessive dephosphorization was disadvantageous to calcium-binding activity. Theoptimization of enzymatic conditions for preparation calcium-binding peptide was asfollows: the degree of dephosphorization of carp eggs30.39%, trypsin dosage3000U/g, substrate concentration2%, pH9.0, temperature49℃and hydrolysis time12h.The DH and calcium-binding activity of the hydrolysate reached31.15%and0.67mmol/g.
2. Preparation of FEP-Ca complex and its stability
Based on binding rate, The conditions of FEP binding calcium were optimized asfollows: FEP concentration5g/L, calcium concentration5mM, temperature45℃, pH8.0and reaction time1h. In these conditions the binding rate of FEP was86%. Thecontents of phosphorus, nitrogen and calcium were3.78,11.15,7.27%respectivelyand the molar ratio of phosphorus to nitrogen was6.53.
FEP-Ca complex owned good ability to resist pH with more than90%calciumretention rate at the pH range of5-8. After heated at121.1℃for15min, the calcium retention rate was as high as71.33%. FEP-Ca complex also could resist digestion bypepsin and trypsin with89.69%calcium retention rate after treated with the twoenzymes. However, the calcium retention rate significantly decreased as the additionof phosphate because phosphate can compete with FEP to bind calcium.
3. Effect of FEP-Ca on enhancing calcium bioavailability in vivo
In vivo the effect of FEP-Ca on increasing calcium bioavailability were studiedin calcium-deficiency rats. During the experimental period, calcium absorption and itsaccumulation in bone was significantly increased by FEP-Ca supplementation. Thelevels of serum calcium, bone mineral density, bone calcium content andbiomechanical properties of the FEP-Ca group were significantly higher than those ofCaCO3group (p <0.05), but similar to the CPP-Ca group (p>0.05). FEP-Ca isexpected to become a novel calcium nutraceutical additive in food industry due toenhancing Ca bioavailability by its intake.
4. Purification and identification of calcium-binding peptideAfter ultrafiltration, the fraction with molecular weight <5kDa (U) was collectedand then purified with hydroxylapatite chromatography (HAC), H3eluted with themaximum concentration of phosphate buffer (400mmol/L) exhibited the highestcalcium binding ability of5.03mmol/g. Amino acid content analysis showed that theSer content of H3is about3times more than that of U but the contents of Thr andTyr are almost identical. H3was further purified using size exclusionchromatography (SEC) and reverse phase high-performance liquid chromatography(RP-HPLC), an oligophosphopeptide with the highest calcium binding ability (7.62mmol/g) was obtained. Its sequence was identified as (pS)S(pS)AF(pS)(pS)ELARthrough ESI-QTOF tandem mass analysis.
5. Mechanism of peptide binding calcium
The peptide with the sequence (pS)S(pS)AF(pS)(pS)ELAR (IPP) wassynthesized for further study. MS and FTIR spectrum of IPP and IPP-Ca showed thatphosphate had the priority to binding calcium. Under physiological conditions onemole of IPP could bind four moles of calcium and carboxyl group could not bindcalcium. FTIR and CD spectrum further revealed that regardless of the presence calcium IPP was present in the state of unorded structure in solution underphysiological conditions, without any secondary structures such as α-helix or β-sheet.When pH reached to10with calcium concentration up to3mM, Ca2+boundphosphate and shielded negative charges of it, which made the formation of β-sheet inIPP via hydrophobic interactions and hydrogen bond. Then Ca2+served as asalt-bridge between carboxyl group and induce the formation of nanoparticles.However in the presence of other peptides, Ca2+could induce the peptides solution tofomate nanoparticles at the pH range of6-8.
In the supersaturated solution of hydroxylapatite (HAP), IPP could compete withphosphate to bind calcium and adsorb to the surfaces of Ca-P nanoclusters andamorphous calcium phosphate granules (ACP), which inhibited the nuleation ofcalcium phosphate and the aggregation of ACP to crystal. IPP also could adsorb to thesurfaces of calcium phosphate crystals, thus inhibited the growth and precipitation ofcrystals.
1.鲤鱼卵酶解制备FEP条件优化
鲤鱼卵脱脂后含有1.09%的磷，氮磷摩尔比为31.31，蛋白质中丝氨酸摩尔百分数为6.81%。以水解度和酶解液钙离子结合活性为指标，优化了鱼卵酶解工艺。结果表明胰蛋白酶对鱼卵的酶解效果最好，当鱼卵脱磷率为30.39%时，在49℃，加酶量3000U/g，底物浓度2%，pH9.0条件下，水解度为31.15%，酶解液结合钙活性最好，500mg/ml酶解液能结合0.67mmol/g的钙。
2. FEP-Ca复合物制备条件优化及其稳定性
以FEP的单位蛋白钙结合量和钙结合率为评价指标，对肽钙结合反应条件进行优化。结果表明在FEP浓度为5g/L，钙离子浓度为5mM，pH8.0，45℃，1h条件下，FEP单位蛋白钙结合量为0.86mmol/g，钙结合率为86%。此条件下制备的FEP-Ca复合物磷含量为3.78%，氮含量为11.15%，钙含量为7.27%，氮磷摩尔比为6.53。
FEP-Ca复合物具有较好的酸碱稳定性，在pH5-8范围内钙保留率高达90%以上。一定的加热处理会导致FEP-Ca复合物钙保留率下降，经过高温杀菌后钙保留率为71.33%。FEP-Ca复合物具有较高的抗消化性，经过胃蛋白酶、胰蛋白酶共同作用后，钙保留率为89.69%。磷酸盐能与FEP竞争钙离子，随着磷酸盐浓度的上升，FEP-Ca的钙保留率显著下降（p <0.05）。当磷酸盐浓度为30mM时，钙保留率降至37.05%。
3. FEP-Ca促钙吸收活性研究
缺钙大鼠饲喂FEP-Ca后，可显著增加体重、促进钙吸收、维持血钙和ALP水平稳定，增加骨重、骨密度和骨钙含量，增强骨生物力学性能，使上述指标达到或优于正常对照组水平。FEP-Ca对缺钙大鼠的补钙效果显著优于CaCO3(p <0.05)，而与CPP-Ca无显著性差异。FEP-Ca有望成为新型的补钙制剂。
4. FEP中高活性组分的分离纯化及结构鉴定
FEP经超滤分离后，将分子量小于5kDa的组分依次经羟基磷灰石层析、分子排阻层析及RP-HPLC分离纯化得到高活性组分，经ESI-QTOF-MS/MS分析，鉴定其氨基酸序列为(pS)S(pS)AF(pS)(pS)ELAR，分子量为1461Da，含有5个丝氨酸，其中有4个被磷酸化修饰，是一种典型的磷酸肽。
5.活性肽钙结合机制研究
人工合成高活性组分（IPP），经质谱和红外光谱分析表明，生理条件下磷酸基团是钙离子的优先结合部位，1分子IPP能结合4个钙离子，羧酸基团未参与钙离子的结合。红外光谱和圆二色谱分析表明，IPP生理条件下完全以无序状态存在。当同时升高pH（10以上）和钙离子浓度（3mM以上）后，羧基解离，钙离子对磷酸基团电荷产生屏蔽效应，使IPP在疏水作用和氢键作用下形成β折叠结构，并在两个羧酸根之间形成盐桥，进而构建网状结构，形成IPP-Ca纳米颗粒。而当体系中含有混合肽时，则可在pH6-8范围与钙离子结合形成纳米颗粒。
IPP在过饱和磷酸钙溶液中与磷酸根竞争与钙的结合，并吸附到Ca-P簌和无定形纳米粒（ACP）表面，抑制晶核形成和ACP定向聚集结晶，还能吸附到结晶表面抑制结晶长大产生沉淀。
In this thesis, fish egg peptides (FEP) with calcium-binding activity wereprepared from carp eggs through enzymatic technology and the parameters of FEPbinding calcium reaction were optimized to prepare peptide-calcium complex(FEP-Ca). The effects of FEP-Ca on increasing calcium bioavailability were furtherstudied in calcium-deficiency rats. A novel peptide was purified and identified toexplore the mechanism of the peptide binding calcium. The details of the work wouldbe shown as follows:
1. Preparation of FEP through enzymatic technology from carp eggs
Calcium-binding activity and degree of hydrolysis (DH) were adopted as indexto optimize the enzymatic technology. The trypsin showed the highest ability toprepare peptides with calcium-binding activity from carp eggs. Dephosphorizationtreatment of carp eggs could significantly increase the degree of hydrolysis. However,excessive dephosphorization was disadvantageous to calcium-binding activity. Theoptimization of enzymatic conditions for preparation calcium-binding peptide was asfollows: the degree of dephosphorization of carp eggs30.39%, trypsin dosage3000U/g, substrate concentration2%, pH9.0, temperature49℃and hydrolysis time12h.The DH and calcium-binding activity of the hydrolysate reached31.15%and0.67mmol/g.
2. Preparation of FEP-Ca complex and its stability
Based on binding rate, The conditions of FEP binding calcium were optimized asfollows: FEP concentration5g/L, calcium concentration5mM, temperature45℃, pH8.0and reaction time1h. In these conditions the binding rate of FEP was86%. Thecontents of phosphorus, nitrogen and calcium were3.78,11.15,7.27%respectivelyand the molar ratio of phosphorus to nitrogen was6.53.
FEP-Ca complex owned good ability to resist pH with more than90%calciumretention rate at the pH range of5-8. After heated at121.1℃for15min, the calcium retention rate was as high as71.33%. FEP-Ca complex also could resist digestion bypepsin and trypsin with89.69%calcium retention rate after treated with the twoenzymes. However, the calcium retention rate significantly decreased as the additionof phosphate because phosphate can compete with FEP to bind calcium.
3. Effect of FEP-Ca on enhancing calcium bioavailability in vivo
In vivo the effect of FEP-Ca on increasing calcium bioavailability were studiedin calcium-deficiency rats. During the experimental period, calcium absorption and itsaccumulation in bone was significantly increased by FEP-Ca supplementation. Thelevels of serum calcium, bone mineral density, bone calcium content andbiomechanical properties of the FEP-Ca group were significantly higher than those ofCaCO3group (p <0.05), but similar to the CPP-Ca group (p>0.05). FEP-Ca isexpected to become a novel calcium nutraceutical additive in food industry due toenhancing Ca bioavailability by its intake.
4. Purification and identification of calcium-binding peptideAfter ultrafiltration, the fraction with molecular weight <5kDa (U) was collectedand then purified with hydroxylapatite chromatography (HAC), H3eluted with themaximum concentration of phosphate buffer (400mmol/L) exhibited the highestcalcium binding ability of5.03mmol/g. Amino acid content analysis showed that theSer content of H3is about3times more than that of U but the contents of Thr andTyr are almost identical. H3was further purified using size exclusionchromatography (SEC) and reverse phase high-performance liquid chromatography(RP-HPLC), an oligophosphopeptide with the highest calcium binding ability (7.62mmol/g) was obtained. Its sequence was identified as (pS)S(pS)AF(pS)(pS)ELARthrough ESI-QTOF tandem mass analysis.
5. Mechanism of peptide binding calcium
The peptide with the sequence (pS)S(pS)AF(pS)(pS)ELAR (IPP) wassynthesized for further study. MS and FTIR spectrum of IPP and IPP-Ca showed thatphosphate had the priority to binding calcium. Under physiological conditions onemole of IPP could bind four moles of calcium and carboxyl group could not bindcalcium. FTIR and CD spectrum further revealed that regardless of the presence calcium IPP was present in the state of unorded structure in solution underphysiological conditions, without any secondary structures such as α-helix or β-sheet.When pH reached to10with calcium concentration up to3mM, Ca2+boundphosphate and shielded negative charges of it, which made the formation of β-sheet inIPP via hydrophobic interactions and hydrogen bond. Then Ca2+served as asalt-bridge between carboxyl group and induce the formation of nanoparticles.However in the presence of other peptides, Ca2+could induce the peptides solution tofomate nanoparticles at the pH range of6-8.
In the supersaturated solution of hydroxylapatite (HAP), IPP could compete withphosphate to bind calcium and adsorb to the surfaces of Ca-P nanoclusters andamorphous calcium phosphate granules (ACP), which inhibited the nuleation ofcalcium phosphate and the aggregation of ACP to crystal. IPP also could adsorb to thesurfaces of calcium phosphate crystals, thus inhibited the growth and precipitation ofcrystals.
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