肌肉非蛋白氮和游离氨基酸在金华火腿加工过程中的变化

以60只浙江兰溪当地杂交猪后腿为原料,按传统工艺加工金华火腿,分析了股二头肌中非蛋白氮(Non-proteinnitrogen,NPN)和游离氨基酸(Freeaminoacids,FAA)浓度随加工时间而变化的规律。结果表明,肌肉中的NPN和FAA含量都随金华火腿加工进程逐渐升高;NPN在后熟期和晒腿期间升高速度最快,而FAA在成熟过程中升高最快,成品火腿的蛋白质降解指数在14～20之间;成品火腿中的FAA占NPN的70%以上,其中以Arg、Glu、Leu、Lys、Ala和Val等含量较高。FAA总含量比腌制前提高13.8倍,火腿中大部分FAA浓度为腌制前的10～20倍,其中增加比例较大的FAA有Lys、Asp、Ser、Tyr和Ile等。火腿中大部分游离氨基酸的浓度多倍于其感觉阈值,因此可能对火腿风味的形成有重要贡献。     

金华火腿工艺过程蛋白质水解及其相关性研究

本文通过对金华火腿工艺过程总氮、非蛋白氮及游离氨基酸的分析研究,来确定其蛋白质分解与工艺过程产品水分、NaCl含量及温湿度条件的相关性;结果表明:工艺过程中非蛋白氮和游离氨基酸的变化与工艺过程湿度和产品水分的降低、温度和NaCl含量的提高呈正相关,且蛋白质在工艺过程中发生了持续降解。     

The Role of Muscle Proteases and Lipases in Flavor Development During the Processing of Dry-Cured Ham

The processing of dry-cured ham is very complex and involves numerous biochemical reactions that are reviewed in this article. Muscle proteins undergo an intense proteolysis, resulting in a great number of small peptides and high amounts of free amino acids. The enzymes responsible of these changes are proteinases (cathepsins B, D, H, and L and, to a less extent, calpains) and exopeptidases (peptidases and aminopeptidases). Muscle and adipose tissue lipids are also subject to intense lipolysis, generating free fatty acids by the action of lipases that, in a second stage, are transformed to volatiles as a result of oxidation. Sensory profiles of dry-cured ham are strongly affected by these enzymatic reactions. In addition, the activity levels of the muscle enzymes significantly depend on the properties of raw ham, such as age and crossbreeding as well as the process conditions such as temperature, time, water activity, redox potential, and salt content. Thus, the control of the muscle enzyme systems, mainly proteases and lipases, is essential for the standardization of the processing and/or enhancement of flavor quality of dry-cured ham.     

Biochemical changes during processing of traditional Jinhua ham

Jinhua ham is the most famous traditional meat product of China and one of the most famed dry-cured hams in the world. Its processing consists of six stages: green ham preparation, salting, washing and sun-drying and shaping, ripening, and post-ripening. Intense proteolysis and lipolysis occur during processing period. As a result, the content of free amino acids in final ham products is 14-16 times that of green ham, and 191 volatile compounds have been identified during processing, which make a major contribution to the flavor of Jinhua ham.     

Proteolysis,protein oxidation and protease activity in dry‐cured Xuanwei ham during the salting stages

Twenty‐four experimental dry‐cured Xuanwei hams were salted using a standard method for 90 days. The proteolysis, protein oxidation and protease activities in biceps femoris (BF) and semimembranoesus (SM) muscles of dry‐cured Xuanwei ham were investigated during the salting phase. At the end of salting, the salt content increased to 35.2 g kg^?1 muscle in BF and 54.2 g kg^?1 muscle in SM. During the salting stage, salt soluble proteins were degraded mainly into water soluble proteins that were further broken down to peptides with molecular weights mostly greater than 1 kDa. Although large amounts of smaller peptides and free amino acids were generated, especially when the hams were aged. The carbonyl contents were increased but lower than 1.57 nmol mg^?1 proteins in muscles during the salting stage. The cathepsin B, dipeptidyl peptidase I (DPP I), alanyl (AAP), arginyl (RAP) and leucyl (LAP) aminopeptidase all remained active while salt content strongly inhibited cathepsin L and DPP IV in the first 90 days. The results suggested that the salting process promoted the hydrolysis of proteins, and increased the muscle protein oxidation at a slower rate.     

Characterization of proteolysis during the ripening of semi-dry fermented sausages

The respective contribution of indigenous enzymes and enzymes from starter bacteria to proteolysis in fermented sausages were determined by comparing the proteolytic changes occurring in sausages resulting from the presence of a proteolytic strain of Staphylococcus carnosus, i.e. S. carnosus MC 1 to the proteolytic changes occurring in control sausages containing glucono-δ-lactone (GDL) and an antibiotic mixture. Proteolysis was quantified by assaying for non-protein nitrogen (NPN) and free amino acids. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed phase high performance liquid chromatography (RP-HPLC) were used to qualitatively assess the proteolytic changes in the sarcoplasmic and myofibrillar proteins as ripening progressed. The concentration of NPN and free amino acids increased in both sausages initially, but subsequently decreased towards the end of ripening in sausages inoculated with the starter culture. SDS-PAGE showed a similar pattern of proteolysis of sarcoplasmic proteins in both sausages, while of the two sausage types; the S. carnosus MC 1 inoculated sausages exhibited the most intense degradation of myofibrillar proteins, especially myosin and actin. RP-HPLC profiles of 2% trichloroacetic acid (TCA)-soluble peptides for the two sausage types were similar, with the production of numerous hydrophilic peptides. N-Terminal amino acid sequence analysis and sequence homology with proteins of known primary structure showed that six of the TCA-soluble peptides were released from the sarcoplasmic (myoglobin and creatine kinase) and myofibrillar (troponin-I, troponin-T and myosin light chain-2) proteins. In addition, the initial degradation of sarcoplasmic proteins was due to the activity of indigenous proteinases, while both indigenous and bacterial enzymes contributed to the initial degradation of myofibrillar proteins. Furthermore, indigenous enzymes were responsible for the release of TCA-soluble peptides, which, were further hydrolysed by bacterial enzymes.     

不同地域典型干腌火腿肌原纤维蛋白的氧化特性及体外消化性对比

为比较几种典型干腌火腿的可消化吸收性,以巴马火腿、金华火腿、宣威火腿、帕尔玛火腿为原料,研究4 种干腌火腿的肌原纤维蛋白表面疏水性、巯基和二硫键含量、超微结构、蛋白降解程度、消化前后粒径及体外消化率。结果表明：金华火腿肌原纤维蛋白的表面疏水性显著高于巴马、宣威、帕尔玛火腿（P＜0.05）。微观结构分析显示金华火腿肌原纤维蛋白交联和聚集程度最低,肌原纤维排布更为伸展;帕尔玛火腿聚集程度最大,呈现明显聚集状。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳谱图显示金华火腿肌原纤维蛋白在体外消化过程中大幅降解,促进了肌原纤维蛋白粒径的降低。体外模拟消化结果表明,经胃蛋白酶、胰蛋白酶及α-凝乳蛋白酶作用后,金华火腿肌原纤维蛋白水解速率最高（P＜0.05）;帕尔玛火腿肌原纤维蛋白水解速率最低（P＜0.05）。这表明疏水基团的暴露和蛋白结构的伸展为消化酶提供了更多的识别位点,促进了金华火腿中肌原纤维蛋白的水解。综上,肌原纤维蛋白的氧化程度显著影响蛋白的体外消化率。     

金华火腿加工过程中肌肉肌内脂的水解变化研究

研究了金华火腿股二头肌 (bicepsfemoris)肌内脂肪在加工过程中不同时期的水解变化情况。肌内脂使用氯仿 甲醇溶液提取 ,采用固相萃取法将中性脂 (主要为甘油脂 )、游离脂肪酸和磷脂分离 ,用毛细管气相色谱分别分析甘油脂、游离脂肪酸和磷脂脂肪酸的种类与比例。通过比较不同时期样品的色谱结果 ,发现在金华火腿原料中磷脂脂肪酸的不饱和脂肪酸百分含量较高 ,尤其是多不饱和脂肪酸的含量高达 4 9 2 2 %。且在金华火腿成熟过程中 ,磷脂水解作用显著 ,而甘油脂脂肪酸百分比例比较稳定 ,说明甘油脂水解作用相对较弱。     

Effect of prefreezing hams on endogenous enzyme activity during the processing of Iberian dry-cured hams

The use of frozen/thawed raw material in the processing of Iberian dry-cured ham has been studied to determine its effect on the sensory quality of the final product. The proteolysis and lipolysis processes were measured by the proteolytic and lipolytic enzyme activities and free amino acids and free fatty acids. The thawed Iberian hams had lower salt contents throughout the process. The use of thawing raw material did not affect the proteolytic enzymes, cathepsins, aminopeptidases and dipeptidylpeptidases, only the activity of dipeptidylpeptidase III was reduced due to thawing. Moreover, there were no differences in the content of free amino acids between fresh and thawed hams during the whole process. However, the use of thawing hams affected the lipolytic activity. The activity of phospholipase and neutral lipase were significantly higher in the thawed hams and also the content of free fatty acids, at all the stages analyzed. Consumer sensory analysis showed thawed Iberian hams had the lowest hardness, probably due to an intense proteolysis. The acceptability of the Iberian hams was similar between fresh and thawed hams.     

Development of a rapid, specific and efficient procedure for the determination of proteolytic activity in dry-cured ham: definition of a new proteolysis index

A method was adapted to determine proteolytic activity in dry-cured ham using fluorescamine-specific labelling of N-terminal α-amino groups of peptides and amino acids. Fluorescence of the complex was measured using a microplate procedure and optimum excitation and emission wavelengths of 375nm and 475nm, respectively. A new proteolysis index (PI) was defined as the percentage ratio of the N-terminal α-amino group content to the total protein content of the ham extract. The robustness of the method was evaluated by measuring PI in pork meat samples subjected to standardized processing conditions and in samples extracted from industrial hams taken at different stages of processing. For the industrial samples, a comparison with the classic nitrogen procedure of PI determination was performed and a formula relating the two PIs was established. The rapidity, sensitivity and specificity of the procedure make it a good candidate for a screening test to evaluate ham quality in industry.     

Generation of non-protein nitrogen and volatile compounds by Penicillium chrysogenum Pg222 activity on pork myofibrillar proteins

A non-toxigenic strain of proteolytic Penicillium chrysogenum Pg222 isolated from dry-cured ham was tested for its ability to generate non-protein nitrogen (NPN) and volatile compounds from muscle myofibrillar proteins. The activity of mold led to higher accumulation of non-protein and amino acidic nitrogens than control samples. Volatile compound analysis revealed the presence of branched compounds, such as 3-methylbutanal and 3-methylbutanol only in samples inoculated with the mold. Similarly, compounds such as ethanol, propanol and 2-methoxy ethanol were detected only in inoculated samples at all sampling time. 3- and 2-methylpentane, benzoic and acetic acids, 2-butanone and 2-ethylhexanol, pyridine and 3-carene were detected occasionally, but only in the Pg222 batch. The proteolytic activity of P. chrysogenum Pg222 lead to accumulation of soluble NPN compounds, in addition to the generation of volatile compounds of great interest for dry-cured meat products. Therefore, this mold could be appropriate to be used as a non-toxigenic starter culture during the ripening of dry-cured meat products to stimulate proteolysis and flavour development.     

Effect of intensifying high‐temperature ripening on proteolysis,lipolysis and flavor of Jinhua ham

BACKGROUND: The flavor quality of dry‐cured ham comes from proteolysis, lipolysis and lipid oxidation, Maillard reaction and Strecker amino acid degradation. Intense proteolysis, lipolysis and lipid oxidation make major contributions to flavor development of dry‐cured ham. Increasing the temperature in fermenting and ripening could promote these reactions and accelerate flavor development in dry‐cured hams. The specific aroma flavor of Jinhua ham is developed only during long‐time high‐temperature ripening in July and August. Our objective was to effectively shorten the process time by intense high‐temperature ripening based on the flavor and quality features of traditional Jinhua ham. RESULTS: Muscle dehydration rate of 80‐day ripened hams (29.43 ± 1.16%) was higher than that of the traditional process (P < 0.05). The total free fatty acids in ripened hams of 45–80 days were all higher than that of traditional hams (P < 0.05) and the level of TBARS was significantly lower (P < 0.01). The flavor profile of modern‐processed hams was different from that of the traditional Jinhua ham. The contents of carboxylic acids and aldehydes were obviously higher than those of the traditional products (P < 0.05). The results of organoleptic evaluation for flavor and quality showed that 80‐day ripened hams reached the first‐grade level of traditional Jinhua ham. CONCLUSION: Long‐time (25–30 days) intensifying high‐temperature ripening (35–37 °C) could accelerate the proteolysis, lipolysis, lipids oxidation, flavor development and effectively shorten the process time based on the traditional flavor and quality features of dry‐cured ham. Copyright © 2009 Society of Chemical Industry     

Changes of alanyl aminopeptidase activity and free amino acid contents in biceps femoris during processing of Jinhua ham

Sixty experimental Jinhua hams were processed by a traditional method. The potential alanyl aminopeptidase (AAP) activity in biceps femoris was determined. The effects of temperature, salt content, sodium nitrate content and pH on muscle AAP were evaluated using response surface methodology. Porcine muscle was found to possess very strong potential AAP activity that decreased gradually during processing from 201,635 U g⁻¹ before salting to 6147 U g⁻¹ after aging. Temperature, pH and salt content had significant exponential effects on AAP activity (P < 0.001). Both temperature and salt content interacted with pH in their effects on AAP activity (P < 0.01). However, 0–50 mg L⁻¹ sodium nitrate had no detectable effect on AAP activity (P > 0.05). The regression model showed muscle AAP maintaining its activity all through Jinhua ham processing, indicating that muscle AAP may generate free amino acids during the processing and storage of Jinhua ham. The concentrations of free amino acids increased significantly (P < 0.05) during Jinhua ham processing, except for arginine and cystine. The concentrations of most free amino acids were 5–20 times higher in the final product than in hams before salting. Final concentrations exceeded thresholds for sensory detection, thus implicating an important role of free amino acids in the determination of Jinhua ham flavor.     

生姜蛋白酶和猕猴桃蛋白酶对干腌羊火腿蛋白质降解的影响

为了促进干腌羊火腿肌肉蛋白质降解,加快其风干成熟进程,缩短加工周期,提高羊火腿的品质,在干腌羊火腿中添加生姜蛋白酶和猕猴桃蛋白酶。以带骨鲜羊后腿肉为试验材料,分别设计一个对照组和两个试验组;对照组不采取处理,而试验组分别添加0.05%的生姜蛋白酶和猕猴桃蛋白酶,并在相应条件下进行风干成熟,检测干腌羊火腿的总氮(Total nitrogen,TN)含量、非蛋白氮(Non-protein nitrogen,NPN)含量和蛋白质降解指数(Proteolysis index,PI)等蛋白质降解指标,并通过SDS-PAGE(聚丙烯酰氨凝胶电泳)分析了干腌羊火腿肌肉蛋白质的降解情况。结果表明,与对照组相比,猕猴桃蛋白酶处理组和生姜蛋白酶处理组的TN含量风干成熟后分别增加了1.2倍和1.3倍、NPN含量分别增加1.5倍和1.7倍、PI分别上升1.2倍和1.3倍;SDS-PAGE分析结果表明,生姜蛋白酶降解肌肉蛋白的效率较猕猴桃蛋白酶强。通过蛋白质将指数和SDS-PAGE电泳结果可知,生姜蛋白酶对肌肉蛋白的降解程度比猕猴桃蛋白酶大。     

PROTEOLYTIC CHANGES DURING PROCESSING IN TURKISH DRY‐CURED MEAT PRODUCT (PASTIRMA) WITH DIFFERENT SALT LEVELS

ABSTRACT

The effects of different salt (NaCl) contents on proteolytic changes during processing in the dry‐cured meat product past?rma were investigated. Past?rma samples were produced with three different NaCl levels (3, 6 and 9%) and proteolytic changes were monitored. A progressive increase in nonprotein nitrogen (NPN) and free amino acids (FAAs) was detected during processing. High‐salt past?rma samples had the highest FAAs. Drying and pressing stages before pasting with çemen resulted in a decrease in water‐soluble nitrogen (WSN) and a slight increase in NPN and FAA. High salt level and processing time resulted in the accumulation of FAAs. The increase in WSN, NPN and FAA contents after çemen treatment indicated that the çemen used in the pasting process had an impact on accumulation of these components in past?rma. However, no significant effect of salt level was observed on WSN and NPN. Sarcoplasmic proteins decreased considerably after salting due to drainage. Myofibrillar proteins degraded during processing, particularly in samples with 6 and 9% NaCl.

PRACTICAL APPLICATIONS

Proteolysis is one of the most important biochemical changes occurring during the processing of dry‐cured meat products such as ham or past?rma. The effect of additional salt concentrations and processing stages on proteolysis in Turkish dry‐cured meat product, past?rma, was studied during processing. Processing stages (salting, drying, pressing and pasting with çemen mixture) resulted in increases in NPN and FAA contents. High salt level (9% NaCl) and processing stages, particularly pasting with çemen mixture, facilitated the accumulation of FAA. Salting resulted in denaturation or insolubilization of proteins. Insolubilization of sarcoplasmic proteins was clearly seen in samples with 6 and 9% NaCl, depending on increased salt concentration during processing. In contrast, myofibrillar proteins in samples with 6 and 9% NaCl degraded during processing. From these results, processing conditions (salt concentration of past?rma and pasting with çemen) can determine the compounds released from protein breakdown during processing.

     

Evaluation of protein degradation and flavor compounds during the processing of Xuan'en ham

Protein degradation occurs during the processing of dry-cured ham, which has important influences on the flavor and quality of products. The aim of this work was to study the degradation kinetics of myofibrillar proteins (MPs) and sarcoplasmic proteins (SPs) extracted from the biceps femoris muscle during the processing of Xuan'en ham. A relationship between protein degradation and the flavor formation was found. During the processing of Xuan'en ham, MPs and SPs were mainly degraded in the salting stage and incipient fermentation. Accompanied by protein degradation, the content of carbonyl group in SPs increased gradually, but in MPs, it first increased and then decreased. Interconversion between sulfhydryl and disulfide bonds was investigated during this processing. Oxidation, degradation, and thermal effects significantly affected the surface hydrophobicity of proteins. More than one hundred volatile compounds have been identified at each stage of ham preparation. Among them, organic acids were the predominant group, followed by hydrocarbons, aldehydes, alcohols, ketones, and esters.     

Time course of peptide fingerprints in semimembranosus and biceps femoris muscles during Bayonne ham processing

The aim of this work was to define reliable markers of muscle and processing time in dry-cured ham using a rapid, precise semi quantitative method for the protein fraction soluble in low ionic strength buffer. For this purpose protein labchip Agilent was used to separate proteins and peptides and accurately determine their molecular weights and concentrations electrophoretically. In this way the protein fingerprinting of dry-cured ham at different process times was characterised, together with targets and products of proteolysis. In addition, the comparison of all the electrophoregrams indicated muscle and dry-curing process markers.     

金华火腿加工过程中的理化特性

以传统金华火腿为原料 ,跟踪测定了火腿加工全过程中半膜肌和股二头肌的水分、NaCl、非蛋白态氮 ,水溶性氮和游离氨基酸含量的变化情况。结果表明 ,半膜肌和股二头肌的水分含量逐渐降低 ;NaCl含量逐步上升 ,水溶性氮的含量在加工过程中先降低后升高 ,非蛋白态氮的含量也逐步上升。各种游离氨基酸随着加工的进行 ,含量逐渐增加 ,尤其是谷氨酸 ,金华火腿主要的鲜味物质 ,在加工过程中含量显著增加。     

金华火腿粗肽液的体外抗氧化活性

在金华火腿长期加工过程中,蛋白质在内源酶作用下产生了大量不同长短的肽段.提取金华火腿粗肽液,通过测定不同质量浓度粗肽液清除自由基、螯合金属离子、抑制脂质过氧化和蛋白质氧化的能力,并与相应质量浓度的丁基羟基甲苯(BHT)和谷胱甘肽(GSH)作比较,衡量金华火腿粗肽液的抗氧化能力.结果表明:随着质量浓度的增加,金华火腿粗肽液的抗氧化能力显著增加.在质量浓度为1.5mg/mL时,金华火腿粗肽液清除羟基自由基能力达到90％;质量浓度为5mg/mL时,金华火腿粗肽液清除1,1-二苯基-2-三硝基苯肼(DPPH)自由基能力与BHT和GSH相当,高达95％;金华火腿粗肽液螯合金属离子的能力显著高于BHT和GSH;当质量浓度为4mg/mL时,金华火腿粗肽液具有和BHT和GSH相当的抑制脂质过氧化能力,并能一定程度抑制蛋白质氧化.结论:金华火腿粗肽液的抗氧化能力与BHT与GSH相当.     

Influence of pre-cure freezing of Iberian ham on proteolytic changes throughout the ripening process

This work aimed to investigate the effect of pre-cure freezing Iberian hams on proteolysis phenomena throughout the ripening process. Non-protein nitrogen (NPN), peptide nitrogen (PN) and amino acid nitrogen (AN) as well as amino acid and dipeptide evolution followed the same trend in both refrigerated (R) and pre-cure frozen (F) Iberian hams during processing. At the different stages of ripening, there were no differences in the content of NPN and AN while F dry-cured hams had higher levels of PN than R hams at the final step. This seemed to be more related to the salt content (lower in F than in R hams) than to the pre-cure freezing treatment. Most amino acids and dipeptides detected showed higher concentrations in F than in R Iberian hams at the green stage, being rather similar at the intermediate phases. At the final stage, the effects of pre-cure freezing of Iberian hams were not well defined, higher levels of some amino acids and dipeptides were found in R than in F Iberian hams whereas other amino acids were lower in R than in F hams.