【作者】 陆应林；

【导师】 周光宏；

【作者基本信息】 南京农业大学 ， 食品科学， 2012， 博士

【摘要】 南京板鸭是我国著名的腌腊制品,有着500多年的历史,以其独特的风味深受广大消费者喜爱。然而,落后的作坊式生产方式在一定程度上限制了它的可持续发展,主要原因是有关南京板鸭传统加工方面理论研究较少,工艺参数不明确。本研究是863项目“中国传统及特色食品和畜产品生产技术与产品开发”(2002AA248031)的一部分。研究的目的是在南京板鸭传统加工工艺基础上,通过对肌肉蛋白的降解规律、组织蛋白酶的活性变化及影响因素和南京板鸭风味成分的分析研究,探讨南京板鸭风味形成的机理,为南京板鸭传统工艺的现代化改造提供理论依据。具体研究内容和结果如下：1.鸭肉中组织蛋白酶B、L和H的特性研究从樱桃谷鸭腿肌中提取组织蛋白酶B、L和H,分别研究了pH值、温度、盐、蛋白酶激活剂和抑制剂对三种组织蛋白酶活力的影响。结果表明：组织蛋白酶B、L和H的适宜反应pH值分别是5.0～5.5、4.5～5.5和6.5～7.0；组织蛋白酶B和L的耐酸性强于H。组织蛋白酶B、L和H的适宜反应温度别是35℃～40℃、40℃～45℃和45℃～50℃;组织蛋白酶B和L的耐热性强于组织蛋白酶H。食盐的添加量影响组三种织蛋白酶的活力,随着食盐含量的增加,三种组织蛋白酶的活力降低。激活剂L-cysteine和DTT对三种组织蛋白酶有一定的激活作用；而巯基抑制剂E-64对三种组织蛋白酶有明显抑制作用,抑制剂EDTA和PMSF对组织蛋白酶B和L基本上无抑制作用,对组织蛋白酶H的抑制作用也较弱。用响应曲面法研究了板鸭加工过程中的主要因素(温度、盐含量和pH值)对组织蛋白酶B、L和H活力的影响。建立响应曲面回归方程用来预测南京板鸭加工过程中组织蛋白酶B、L和H的实际活力。结果显示：温度和盐含量显著影响三种组织蛋白酶的活力(P<0.0001),并且温度和盐之间的交互作用对组织蛋白酶B、L和H的活力产生显著影响(P<0.05)。温度和pH值的交互作用对组织蛋白酶L的活力产生显著影响(P<0.05)。2.南京板鸭加工过程中组织蛋白酶B、L和H的活力变化研究选取100只樱桃谷肉鸭为原料,按传统工艺生产南京板鸭,分别在原料、干腌、湿腌、排坯、风干5天、风干10天和风干15天7个工艺点随机抽取6只板鸭,分割腿肌和胸肌作为待测样品。测定了腿肌肉样中组织蛋白酶B、L和H的活力及相关指标在加工过程中的变化。结果显示：原料鸭腿肌中组织蛋白酶B活力最高,组织蛋白酶H活力最低,三种组织蛋白酶活力在加工过程中均发生下降。干腌结束后三种组织蛋白酶活力均发生显著下降(p<0.05),干腌结束到风干5天,三种组织蛋白酶活力下降幅度趋缓,而在风干阶段,三种组织蛋白酶活力变化明显变慢。组织蛋白酶B的活力从原料鸭腿肌中4.87U/g下降到成品板鸭(风干15天)的1.02U/g,成品板鸭中残余活力为原料鸭的20.84%；组织蛋白酶L活力由3.72U/g下降到1.11U/g,残余活力为29.76%；组织蛋白酶H活力由1.75U/g下降到0.40U/g,残余活力为22.61%。加工过程中腿肌水分含量下降,由原料鸭中的78.67%下降到成品鸭的60.97%,盐含量由原料鸭中的0.15%上升到14.42%,湿腌后pH值在加工过程中的无显著变化。响应曲面回归方程预测结果显示,除干腌阶段外,组织蛋白酶B、L种H的实际活力在其它加工阶段相对稳定,织蛋白酶B、L和H的预测实际活力低于其潜在活力。3.南京板鸭加工过程中肌肉蛋白降解的研究对板鸭腿肌肉样中总氮、非蛋白态氮、蛋白质小肽和游离氨基酸等相关指标进行分析,结果表明：南京板鸭加工过程中,胸肌和腿肌的水分含量下降,盐含量上升,总氮的含量呈下降趋势；经干腌和湿腌后非蛋白态氮的含量显著下降(P<0.05)。传统工艺加工的南京板鸭成品率为63.83%。成品板鸭中胸肌和腿肌中的水分含量分别为60.12%和60.97,盐分含量分别为14.62%和14.42%。南京板鸭加工过程中蛋白质的降解指数为6.62-8.41。采用RP-HPLC分析研究板鸭加工过程中肌肉中的小肽,在14分钟内共获得6个比较显著的多肽洗脱峰,说明板鸭中多肽的极性大多比较强,干腌后洗脱峰的总面积呈现显著增加(P<0.05)。小肽总量和氨基酸总量呈正相关(R2=0.82)。各种游离氨基酸的浓度随着板鸭加工进行都有不同程度的增加,加过程中,G1u增加最多,是原料鸭的2.19倍,其次是lys、Pro、Gly和Ala等。加工结束后,板鸭肌肉中含量较高的游离氨基酸主要有Arg、Glu、Thr和His等。试验最后还提取加工过程中各阶段板鸭胸肌中全肌蛋白、肌原纤维蛋白和肌浆蛋白,进行SDS-PAGE电泳实验,结果发现：板鸭在加工过程中,胸肌中全肌蛋白、肌浆蛋白和肌原纤维蛋白都发生不同程度的降解。4.南京板鸭加工过程中风味成分的变化研究采用固相微萃取和GC/MS技术提取和测定了南京板鸭传统工艺加工过程中各工艺点的挥发性风味成分,研究南京板鸭加工各阶段挥发性物质种类以及各类挥发性物质相对含量和绝对含量的变化。结果显示：在南京板鸭加工过程中共有90种化合物被鉴定出来,这些化合物包括烃类化合物26种,醛类化合物18种,醇类13种,酮类化合物10种,酸类8种,酯类化合物5种,硫化合物2种,胺类化合物1种,醚类化合物1种,含氮化合物2种,呋喃类化合物4种。其中原料鸭腿肉中分离鉴定出34种挥发性化合物,成品板鸭(风干15天)腿肉中有53种。在板鸭不同的加工阶段,板鸭腿肌中鉴定的化合物数量、出峰面积和比例都在不断变化,干腌使得鸭肉氧化程度增加,挥发性化合物种类和总峰面积迅速增加。但在湿腌阶段,部分风味物质流入到腌制液中,板鸭风味成分总峰面积下降。从湿腌到风干15天,风味成分总峰面积逐渐增大,风味物质逐渐增加。原料鸭中含量较高的风味化合分别是醇类、醛类、酸类和烃类,成品南京板鸭中醛、醇、烃和酮类化合物含量较高,分别为38.00%、26.72%、18.61%和11.61%,它们占总风味成份的94.94%。在板鸭加工过程中,醇含量呈现先上升后下降趋势；烃类含量先下降后上升；醛类和酮类化合物的变化趋势非常相似,风干前变化不大,风干过程中含量增加较快,其中己醛在板鸭风干15天达到23.91%。更多还原

【Abstract】 Nanjing dry-salted duck is one of the most famous dry-salted meats of china with a long production history of over500years; It has a very strong flavor characteristic and is enjoyed by consumers. However, the laggard processing technology adopted in small-scale workshops has limited the sustainable development of Nanjing dry-salted. The main reason is the deficiency in basic research during traditional processing of Nanjing dry-salted duck, even some processing parameters are still not concrete. This research is a part of the items entitled "Producing technology and products exploitation for Chinese traditional&character food and animal products "(2002AA248031) under the high technology and development program of P. R. The aim of current project was to investigate the rule of muscle protein degradation, the changes of muscle cathepsin activities and their influencing factors, and analyzing the flavor substances of Nanjing dry-salted duck during traditional processing. This could be beneficial to provide a basic heory in improving the processing technology and realizing the industrial production of Nanjing dry-salted duck. The contents and results are as follows.1. Study on properties of Cathepsin B, L and H in duck muscleCathepsin B, L and H were extracted from muscle of cherry valley duck by biochemical techniques. The influence of temperature, pH, salt content, activators and inhibitors on the activities of Cathepsin B, L and H was studied. The activities of Cathepsin B, L and H with optimum pH were5.0-5.5,4.5-5.5, and6.5-7.0respectively. The optimum temperatures were35℃-40℃,40℃-45℃and45℃-50℃respectively. The activities of Cathepsin B, L and H were significantly influenced by salt concentration, which decreased along with increase of salt concentration. L-cysteine and DTT could increase the activity of cathepsin. On the contrary, the activities of Cathepsin B, L and H were completely inhibited by E-64. Cathepsin B and L were not inhibited by EDTA and PMSF.The effects of main influencing factors (temperature, salt content and pH) on the activities of cathepsin B, L and H were firstly studied using response surface methodology. The actual activities of cathepsin B, L and H were estimated with the gained response surface regression equations. The results showed that the activities of three cathepsins were significantly influenced by temperature and salt content (P<0.0001). The activities of cathepsin B, L and H were interaction effects between temperature and salt content (P<0.05), The activities of cathepsin L were interaction effects between temperature and pH value(P<0.05).2. Studies on changes of the activities of cathepsin B, L and H during processing of Nanjing dry-salted duckNanjing dry-salted duck were processed according to typical traditional processing technology with100cherry valley ducks. Muscle samples were taken from six ducks, raw ducks, cured, salted, piled, and then drying for5,10and15days stage. Leg muscle Samples were used to analyze the changes of the activities of cathepsin B, L, H and relative indices during the processing of Nanjing dry-salted duck. Results showed that the activity of cathepsin B was the highest; activity of cathepsin H was the lowest. While cathepsin B, L and H displayed similarly potential activities which all all dropped gradually during processing. The potential activity of three cathepsin significantly decreased in dry-cured finished (p<0.05), which was not different in drying stage. The activity of cathepsin B of leg muscle decreased from4.87U/g at raw duck stage to1.02U/g at ripened duck (drying15days). The residue activity in ripened duck was about20.84%.The activity of cathepsin L from3.72U/g to1.11U/g. The residue activity was about29.76%. The activity of cathepsin H was changed from1.75U/g to0.40U/g, and the residue activity was about22.61%. Moisture contents of leg muscle decreased from78.67%in raw ducks to60.97%in ripened duck. Salt contents of leg muscle increased from0.15%in raw duck to14.42%. The changes of pH were insignificant after salted stage.The response surface regression equations estimated that the actual activities of cathepsin B, L and H were lower than their corresponding potential activities except for cured stage during processing of Nanjing dry-salted duck.3. Studies on changes of muscle protein degradation during the processing of Nanjing dry-salted duckMuscle samples indices was studied by analyzing moisture content, salt content, total nitrogen, non-nitrogen, peptides and free amino acids during processing of Nanjing dry-salted duck.The results indicated that the moisture content decreased while the salt content increased in breast and leg muscles. The total nitrogen content decreased, and the non-protein nitrogen content increased by dry-curing and salting. The ratio of final product of traditional Nanjing dry-salted duck was63.83%.The moisture content and salt content in breast and leg muscle of ripened duck were60.12%and60.97,14.62%and14.42%respectively. The proteolytic index (P.I.%) of Nanjing dry-salted duck was6.62-8.41.Peptides of muscle were studied by RP-HPLC during the processing of Nanjing dry-salted duck. Six remarkable elution peaks were discovered in14min. Total elution area increased significantly by curing (P<0.05). The amount of peptides were positively correlated with FAA contents (R2=0.82)All kinds of FAA increased in concentration along with the progressing of Nanjing dry-salted duck. The glutamic acid content, which is the main palatable compound of the Nanjing dry-salted duck, increased2.19folds. followed by Lys、Pro、Gly and Ala. FAA concentration of the Arg, Glu, Thr and His were high in the leg muscle of Nanjing dry-salted duck.The experiment studied the changes of the myofibrillar and sarcoplasmic protein of breast muscles during the processing of Nanjing dry-salted duck. Electrophortic studies of the muscle myofibrilar and sarcoplasmic proteins showed the disappearance of protein chain. Protein of Nanjing dry-salted duck was degradated in different levels.4. Studies on the changes of flavor compounds in Nanjing dry-salted duck by typical traditional processingChanges in class, absolute and comparative amount of volatile flavor compounds in Nanjing dry-salted duck during typical traditional processing were extracted and identified by solid phase microextraction (SPME) and Gas Chromatography-Mass Spectrometry (GC/MS) technology using the samples mentioned above. The results showed that total90kinds of volatile compounds were identified during Nanjing dry-salted duck processing. These compounds including26kinds of hydrocarbons,18kinds of aldehydes,13kinds of alcohols,10kinds of ketones,8kinds of carboxylic acids,5kinds of esters,2kins sulfur compounds,1kind of amines, lkind of aethers,2kinds nitrogen,4kinds of furans.34kinds of volatile compounds were identified in leg muscle of raw duck. Ripened Nanjing dry-salted duck (drying15days) were53kinds of volatile compounds. During different processing stages of Nanjing dry-salted duck, the amount of volatile flavor compounds, total Peak area and proportion were constantly changing in leg muscle. Dry-curing accelerated the oxidation, kinds and total Peak area of volatiles compounds increased rapidly. However, total Peak area of volatiles compounds decreased in salted stages, which may be part of flavor substances into the salted liquid. From salted to drying15days,total peak area of volatiles compounds increased gradually with the increase of flavor substances. The contents of alcohols, aldehydes, carboxylic acids and ydrocarbons in leg muscle were higher than others volatile compounds in raw duck. The contents of aldehydes, alcohols, hydrocarbons and ketones in leg muscle were38.00%,26.72%,18.61and11.61%in ripened Nanjing dry-salted duck, respectively. The change trends of aldehydes and ketones compounds were very similar. Contents of aldehydes and ketones increased rapidly during drying stage. While hexanal content was23.91%in drying15days.更多还原