【作者】 金文刚；

【导师】 朱蓓薇；

【作者基本信息】 西北农林科技大学 ， 水产品加工及贮藏工程， 2013， 博士

【摘要】 近年来，我国沿海扇贝养殖规模逐渐扩大，扇贝加工量也随之增加。扇贝柱产品在生产和加工过程中，产生了大量低值副产物（包括贝壳、裙边、中肠腺、生殖腺），约占扇贝湿重的70%，尚未得到有效开发利用。扇贝生殖腺蛋白质含量较高，是制备蛋白水解物或活性肽的良好资源。本文以虾夷扇贝生殖腺为研究对象，通过分析雌、雄生殖腺的化学组成，初步研究了中性蛋白酶对雌、雄生殖腺的水解效果及产物功能特性。一方面考察生殖腺水解物的抗氧化活性和理化功能特性，并从水解物中进行抗氧化肽的分离纯化和氨基酸序列鉴定；另一方面，通过美拉德反应修饰和钙螯合修饰制备生殖腺水解物的衍生物，考察其功能特性。主要研究内容及结果如下：（1）化学组成分析表明，虾夷扇贝生殖腺中粗蛋白的质量分数分别达到干重的62.89±0.87%（雌性生殖腺，SFG）和81.66±0.03%（雄性生殖腺，SMG）。将4%底物浓度的变性处理（100℃，10min）和未变性处理（对照组）的虾夷扇贝生殖腺匀浆，利用中性蛋白酶在50℃条件下恒温水解180min后，制备得到虾夷扇贝雌性生殖腺水解物（SFGHs）和雄性生殖腺水解物（SMGHs）。结果表明，SFG经中性蛋白酶水解后，水解效果较好，而中性蛋白酶水解SMG后， SMGHs呈弱凝胶特性。虾夷扇贝雌性生殖腺和雄性生殖腺经变性处理后进行酶解，肽得率和水解度明显高于对照组。与对照组相比，虾夷扇贝雌性生殖腺多肽经变性处理后，肽得率和水解度分别提高了94.78%和36.16%。利用Sephadex G-25凝胶过滤层析对SFGHs进行分离，发现变性处理组的组分较对照组相比更为集中，而且具有一定的还原能力。经高效液相色谱进行分子量分布测定，结果表明变性处理的SFGHs分子量主要分布在250Da～5000Da范围内，约占整个水解液的74.78%，符合生物活性肽的分子量分布范围。因此，对SFG进行热变性处理后，采用中性蛋白酶进行酶解，可以从水解产物中分离纯化得到抗氧化肽组分。（2）针对SMGHs呈弱凝胶性质，制备不同水解度的SMGHs对其理化功能特性进行研究。结果表明，SMGHs的理化功能特性较SMG有所改善。深度水解能提高SMGHs的溶解性和凝胶性，而有限水解后，持水/油性和表面疏水性较好。SMGHs的乳化性与水解度无关，但是受pH环境的制约。SMG的水解导致SMGHs起泡性和泡沫稳定性下降，水解度越大，起泡性和泡沫稳定性越差。氨基酸分析表明，SMG和SMGHs含有丰富的甘氨酸、赖氨酸、丙氨酸、谷氨酸和天冬氨酸，其中必需氨基酸占总氨基酸的41.63%～42.90%，具有较高的营养价值，可作为多功能蛋白基料应用于食品工业中。（3）扫描电镜观察显示，SMGHs呈现多孔、三维网状结构。SMGHs中肽段的分子量主要集中在小于1000Da和大于10000Da的组分，所占比例分别为46.86%和30.30%。采用质构仪研究了SMGHs的凝胶特性，并与几种商品亲水胶体进行比较。结果表明，SMGHs的硬度和稠度分别为40.49±1.96g和479.02±37.04g·sec，和1.5%的瓜尔豆胶和卡拉胶比较接近（p>0.05），并和1.0～2.0%的黄原胶和0.5～1.0%的明胶相当。SMGHs的粘度为23.69±1.92g，接近1.0%的瓜尔豆胶和黄原胶、1.0～1.5%的明胶和1.5%的卡拉胶。SMGHs具有良好的凝胶性质，在某些条件下可替代瓜尔豆胶、明胶、黄原胶和卡拉胶，在食品体系中可能会起到增稠和胶凝作用。（4）为了进一步阐明SMGHs的凝胶机制，通过向SMGHs添加一些能破坏蛋白质分子间作用力的化学试剂，来研究其凝胶性质的变化。结果表明，添加尿素和PG后，SMGHs凝胶强度下降，并且与添加浓度呈正相关。添加8M尿素后，SMGHs凝胶特别弱。添加0.3M的NaCl、KCl、CH₃COONa和NaSCN后，SMGH凝胶性质得到显著增强（p<0.05），但是随着添加浓度的增加，SMGHs凝胶性质下降，特别是3M NaSCN对SMGHs凝胶形成的抑制作用。另外，添加DTT、2-ME和NEM也可以降低凝胶性质（p<0.05），但是整体凝胶状态未发生变化。这说明维持SMGHs凝胶形成的分子间作用力主要是疏水相互作用和静电作用。（5）以DPPH自由基清除能力、Fe²⁺螯合能力和还原能力为指标，对SFGHs的抗氧化活性进行了研究。结果表明，SFGHs对DPPH自由基清除能力的IC₅₀值（DPPH自由基清除率达到50%时的样品浓度）为9.44mg/mL，对Fe²⁺螯合能力的IC₅₀值（螯合率达到50%时的样品浓度）为0.94mg/mL，还原能力的AC_0.5值（吸光度为0.5时的样品浓度）为5.88mg/mL。氨基酸分析表明，SFGHs中Gly、Glu、Asp、Val和Lys含量相对较高，其中必需氨基酸占总氨基酸的40.51%。SFGHs经Sephadex G-25凝胶过滤层析分离后得到6个组分，其中4号组分（F4）具有较强的DPPH自由基清除能力。进一步对F4组分进行质谱分析，得到两个寡肽被鉴定为His-Met-Ser-Tyr（P1）和Pro-Glu-Ala-Ser-Tyr（P2）。P1和P2经人工合成后，对DPPH自由基清除能力呈现一定量效关系，其中30mM的P1和P2对DPPH自由基的清除能力分别达到54.03±3.95%和50.27±1.36%，弱于对照BHA（20mM）。另外，P1和P2对羟基自由基诱导的DNA氧化损伤具有较好的保护作用。（6）在SFGHs与还原糖模拟体系下，研究了其美拉德反应产物的抗氧化活性。结果表明，SFGHs美拉德反应的适宜单糖为核糖、反应pH为8.0，肽糖质量比为1:2、适宜反应时间4⁶h、适宜温度80¹00℃。在此条件下，制备虾夷扇贝雌生殖腺水解物-核糖美拉德反应产物（SFGHs-MRPs），发现SFGHs-MRPs对DPPH自由基清除能力的IC₅₀值为49.55μg/mL，还原能力的AC_0.5值为34.34μg/mL，分别是SFGHs的190.51和171.23倍。另外，SFGH-MRPs在浓度为32μg/mL以上时，对羟自由基诱导DNA氧化损伤具有较强的保护能力。SFGHs与核糖美拉德反应过程中，反应体系的pH呈下降趋势，并且温度越高或反应时间越长，pH下降越明显；反应体系的紫外吸收、褐变程度、荧光强度和抗氧化活性呈上升趋势，并且温度越高或反应时间越长，增幅越明显。相关分析表明，虾夷扇贝雌生殖腺水解物-核糖美拉德反应体系pH与OD₂₉₄、OD₄₂₀、荧光强度、DPPH自由基清除能力和还原能力均呈现负相关，而其它各指标间表现出显著正相关（p<0.01）。（7）为了优化虾夷扇贝雌性生殖腺多肽-Ca²⁺螯合物的制备工艺，在单因素试验的基础上，采用响应面二次回归正交旋转组合试验设计，分析了反应pH、时间、温度和多肽/氯化钙质量比对螯合物得率的影响。结果表明，虾夷扇贝雌生殖腺多肽-Ca²⁺螯合物最优制备工艺为：pH8.0、温度64.8℃、反应时间为22min、多肽与氯化钙质量比为3.4:1，螯合物得率预测值为6.78%，实际得率为6.81%，响应面预测模型较为可靠，可用于指导实际生产。虾夷扇贝雌性生殖腺多肽-Ca²⁺螯合物中氮和钙质量分数分别为6.67±0.11%和9.75±0.17%。多肽中的氨基和羧基可能参与了Ca²⁺的螯合反应。虾夷扇贝生殖腺多肽-Ca²⁺螯合物经体外模拟胃肠道消化后，钙透析率为41.86±2.33%，该螯合物具有较好的钙生物利用度。更多还原

【Abstract】 In recent years, the amount of processed scallop products increased with the enlargementof coastal acquculture in China. During the processing of adductor muscle product, a largequantity of byproducts such as shells, mid-gut gland, mantle lobe, gonad （ovary and testis）,accounting for about70%of the scallop body, are produced and underutilized. Scallop gonadcontained protein at a high level, which can be used as good resources for preparation ofprotein hydrolysate or bioactive peptides.Taking scallop Patinopecten yessoensis gonad as research object, this dissertationassayed the chemical composition of scallop Patinopecten yessoensis gonad （male and female,respectively）, and a pilot study on hydrolysis effects and functional properties of proteinhydrolysate derived from scallop Patinopecten yessoensis gonad （male and female,respectively） was conducted. On one hand, antioxidant activity and functional properties ofscallop Patinopecten yessoensis gonad hydrolysates were investigated, and antioxidantpeptides was also isolated and identified from the gonad hydrolysate. On the other hand, thederivatives of Patinopecten yessoensis gonad hydrolysates through modification of maillardreaction and calcium chelating were prepared and its functional characterizations wereinvestigated. The main research contents and results were as follows:（1） The proximate composition indicated that the crude protein contents （on dry basis） ofscallop Patinopecten yessoensis gonad were62.89±0.87%（female gonad） and81.66±0.03%（male gonad）, respectively. The scallop Patinopecten yessoensis gonad homogenates atprotein concentration of4%were treated with the denaturated（100℃,10min） andnon-denaturated （control） groups, then subjected to enzymatic hydrolysis with commercialneutrase at50℃for180min, respectively. After this, scallop female gonad hydrolysates（SFGHs） and male gonad hydrolysates （SMGHs） were obtained. The results showed thatscallop female gonad（SFG） can be effectively hydrolyzed by neutrase, while scallop malegonad（SMG） was insensitive to neutrase, for the SMGHs exhibited gelation-like properties.The yield of peptides and degree of hydrolysis were higher than control for SFG and SMGafter neutrase hydrolysis by the denaturing pretreatment. Compared with control, the yield of peptides and degree of hydrolysis of SFG after neutrase hydrolysis were increased by94.78%and36.16%, respectively, by denaturing treatment. SFGHs were separated by Sephadex G-25gel filtration chromatography, and the results suggested that the fractions in SFGHs wereconcentrated by denaturing treatment, and with reducing power. The molecular massdistribution of SFGHs with denaturing pretreatment ranged from250to5000Da, accountingfor74.78%of the total, by HPLC. Therefore, SFG could be effectively hydrolyzed usingneutrase in combination with denaturing treatment, and peptides with antioxidant activitycould be isolated through SFGHs.（2） As for the gelation-like protein hydrolysate from neutrase-hydrolyzed scallop malegonad （SMGHs）, the functional properties of SMGHs with different degree of hydrolysis （DH）were investigated. The results showed that hydrolysis with neutrase improved the gelationproperty, solubility, water holding capacity （WHC）, oil holding capacity （OHC） and surfacehydrophobicity （SH）, but not foaming capacity （FC） of SMG. The SMGHs at high DH（11.86%） showed better gelation property and solubility than that at low DH （4.94⁷.53%）.However, the maximum values of WHC, OHC and SH of SMGHs were found at DH of4.94%, significantly higher than （p<0.05） or equivalent to （p>0.05） that of soy protein isolate（SPI） for WHC and OHC. Emulsifying capacity of SMGH is independent of DH, butrestricted by pH environment. The emulsifying activity index （EAI） of all SMGH wassignificantly higher than that of SPI in pH5（p<0.05）, and slightly higher than or equivalent tothat of SPI in pH7. Meanwhile, SMG and SMGHs were abundant in glycine, lysine, alanine,glutamic acid and aspartic acid, containing all the essential amino acids （41.63%⁴2.90%ofthe total amino acids）. These results imply that SMGHs might be utilized as multifunctionaland nutritive ingredients in food industry.（3） SMGHs chiefly consisted of peptides both below1,000Da （46.86%） and above10,000Da （30.30%）, and showed a porous, three-dimensional network observed by scanningelectron microscopy. The gelation properties of SMGHs were studied by texture analyzer, andcompared with several commercial hydrocolloids. The results showed that SMGHs exhibitedfirmness of40.49±1.96g, consistency of479.02±37.04g·sec and cohesiveness of23.69±1.92g, which were similar with that of1.0%¹.5%guar gum,1.5%carrageenan,1.0%².0%xanthan gum and0.5%¹.5%gelatin. These results suggest that the fine gelation properties ofSMGH could be used as replacement of guar, gelatin, xanthan gum and carrageenan undercertain conditions, which may act as thickening and gelation agents in food systems.（4） To further understand the gelation mechanism of SMGHs, we investigated the effectof various chemicals on molecular forces of the gel network of SMGHs, by monitoringchanges of gel properties. The results showed that addition of urea, and propylene glycol （PG） decreased the gel strength of SMGHs with the increase of concentration, and only a veryweak gelation was observed when the concentration of urea reached8M. The overall gelproperties of SMGHs were improved in the presence of0.3M NaCl, KCl, CH₃COONa andNaSCN （p<0.05）. However, elevated salt concentration led to decreased gel properties ofSMGHs, especially the inhibiting effect of NaSCN at3M. In addition, inclusion ofdithiothreitol （DTT）,2-mercaptoethanol （2-ME）, and N-ethylmaleimide （NEM） alsodiminished the gel properties at high concentration （p<0.05）, but not severely, and theiraddition did not change the gelation-like profiles of SMGHs. These results suggest that the gelnetwork of SMGHs was primarily maintained by hydrophobic and electrostatic interactions.（5） Scallop Patinopecten yessoensis female gonad hydrolysates （SFGHs） possessed highantioxidant activity. The IC₅₀value for DPPH radical scavenging activity was9.44mg/mL,IC₅₀value for Fe²⁺-chelating activity was0.94mg/mL and AC_0.5value for reducing powerwas5.88mg/mL. SFGHs was fractionated into six fractions by Sephadex G-25gel filtrationchromatography, Number4（F4） fraction exhibited highest DPPH radical scavenging activity.The F4fraction was analyzed by mass spectrography, two oligopeptides were identified asHis-Met-Ser-Tyr （P1） and Pro-Glu-Ala-Ser-Tyr （P2）. After synthesis, both P1and P2showedcertain dose-DPPH radical scavenging activity relationships（30mM:54.03±3.95%and50.27±1.36%, respectively）, but weaker than contro（20mM BHA）. Besides, both P1and P2exibited strong protective effects against hydroxyl radical induced DNA oxidative damage.（6） Antioxidant activity of maillard reaction products （MRPs） of scallop Patinopectenyessoensis female gonad hydrolysate（SFGHs）-sugar model system were investigated. Theresults showed that the optimum conditions for SFGHs maillard reaction were ribose, pH8.0,and peptide to sugar mass ratio1:2, reaction time4⁶h, temperature80¹00℃. The MRPswere prepared under these conditions at95℃. The IC₅₀value of SFGHs-MRPs for DPPHradical scavenging activity was49.55μg/mL, and The AC_0.5for reducing power was34.34μg/mL, which were190.51and171.23times as for SFGHs, respectively. SFGHs-MRPs （32μg/mL or above） also showed good protective effect against hydroxyl radical induced DNAoxidative damage.During the process of SFGHs-ribose maillard reaction, the pH value of the model systemdeclined, and pH reduction was more obvious when the temperature was higher or thereaction time was longer. The UV absorbance, browning extent, fluorescence intensity, andantioxidant activity of the system were growing. The increasing seemed obvious when thetemperature was higher or the reaction time was longer. Correlation analysis showed thatnegative correlation between UV absorbance, browning extent, fluorescence intensity, DPPHradical scavenging activity, and reducing power of the system against pH were observed, while the correlation of other indexes were significantly positive（p<0.01）.（7） In order to optimize the preparation conditions of scallop （Patinopecten yessoensis）gonad hydrolysate-Ca²⁺complex, on the basis of single factor experiments, a quadratic centralrotary combination design of response surface methodology was used to analyze the influenceof pH, time, temperature, and mass ratio of peptide to calcium on the yield of peptide-Ca²⁺complex. The results showed that the optimum parameters for peptide-Ca²⁺chelating were pH8.0,22min,64.8℃, and peptide to calcium ratio of3.4:1. Under these conditions, thepredicted yield was6.75%, the actual yield was6.81%, and the complex contained6.67±0.11%nitrogen and9.75±0.17%calcium. Infrared spectroscopic study showed that bothamino and carboxyl groups are involved in the complex. Calcium dialyzability of the complexafter in vitro digestion was41.86±2.33%, but showed no difference with control （p>0.05）.Therefore, scallop （Patinopecten yessoensis） gonad hydrolysate-Ca²⁺complex might havegood calcium bioavailability.更多还原