Muscle and Adipose Tissue Aminopeptidase Activities in Raw and Dry-Cured Ham.

Free amino acids have been analyzed in biceps femoris muscle and adipose tissue from raw and dry-cured ham. A high increase was observed for all amino acids except glutamine and taurine. Major increases were in glutamic acid, arginine, alanine, valine, leucine, and lysine. A survey of five aminopeptidase activities of muscle and adipose tissue from raw and dry-cured ham was performed by using 7-amino-4-methylcoumarin derivatives of five amino acids (Leu, Arg, Ala, Tyr and pGlu) as substrates. Optimum activity was found at neutral pH and around 37°C, except the leucyl hydrolyzing activity which was 45°C. High recoveries of activity (25–75%) were obtained in the dry-cured ham. These enzymes might be responsible for free amino acids increasing during dry-curing.     

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.     

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.     

Nutritional pork meat compounds as affected by ham dry-curing

This work is focused on the determination of compounds of nutritional interest that are naturally present in pork meat and how they are affected during the processing of dry-cured ham. Such compounds are creatine, creatinine, coenzyme Q₁₀, glutathione, carnosine, anserine, carnitine, taurine, cystine, cysteine and the essential amino acids. Their antioxidant and antyhipertensive functions were evaluated. Of all the assayed substances, only glutathione decreased totally during processing. Carnosine, creatinine, anserine and glutathione showed antioxidant, while cysteine, glutathione and carnosine showed antyhipertensive activity. So, dry-cured ham constitutes an excellent source of essential amino acids (all essential amino acids exhibited a large increase during processing) and other nutritionally interesting compounds such as cystine, cysteine, carnosine, anserine, taurine, carnitine and coenzyme Q₁₀.     

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.     

Identification and significance of the N-terminal part of swine pyruvate kinase in aged Parma hams

Within a project aimed at studying the peptide fraction in dry-cured Parma hams, a peptide was purified by means of reversed phase-high performance liquid chromatography (RP-HPLC) and identified by its molecular mass and amino acid sequence analysis. The peptide showed a very high degree of homology with the N-terminal part of different mammalian pyruvate kinases reported in databases and was accordingly identified as the N-terminal part of swine pyruvate kinase, whose sequence had never been reported before. The peptide was determined quantitatively by comparison with a suitable internal standard (Phe-Phe) in commercial ham samples with different age degrees. The peptide was found to be ubiquitous in Parma ham and its amount to increase during ageing even if a large variability was found within each assayed maturing time. The correlation found with the ham proteolysis degree (P<0.01) suggests that this peptide is related to the endopeptidasic activity. The peptide did not show a significant relationship with bitterness perception in assayed dry-cured ham, in agreement with the hypothesis that this taste is more related to free amino acids and low molecular weight peptides.     

SUBCUTANEOUS ADIPOSE TISSUE LIPOLYSIS IN THE PROCESSING OF DRY-CURED HAM

ABSTRACT The activities of subcutaneous adipose tissue lipases and esterases were assayed at different stages (0 to 15 months) in the processing of dry-cured ham. The formation of free fatty acids during the process was also determined. Maximal generation of free fatty acids occurred during the first 10 months. Simultaneously, the triglyceride content decreased while the diglycerides increased during the aging period. Neutral and basic lipases showed maximal activity at the beginning of the process but only neutral lipase remained as the main enzyme responsible for the reported lipolysis during the drying ripening stages. Adipose tissue esterases showed excellent stability but the generation of volatile free fatty acids was negligible, suggesting a minor role of these enzymes.     

Changes of arginyl and leucyl aminopeptidase activities in biceps femoris along Jinhua ham processing

Sixty experimental Jinhua hams were processed by a traditional method. The potential arginyl (RAP) and leucyl (LAP) aminopeptidase activities in biceps femoris were determined. The effects of temperature, salt content, sodium nitrate content and pH value on muscle RAP and LAP activities were evaluated using response surface methodology. Porcine muscle was found to possess very strong potential RAP and LAP activities that decreased gradually during processing from 165,980.97Ug(-1) and 89,201.52Ug(-1) before salting to 3164.70Ug(-1) and 5373.66Ug(-1) after aging, respectively. Temperature, pH value and salt content had significant effects on RAP and LAP activities (P<0.0001). They interacted with each other in their effects on RAP activity (P<0.0001) and influenced LAP activity in exponential way (P<0.05). However, 0-50mgL(-1) sodium nitrate had no detectable effect on either RAP or LAP activity (P>0.05). The regression models showed that muscle RAP and LAP maintained their activities all through Jinhua ham processing. LAP could display very strong actual activity during aging and post-aging, indicating that muscle LAP may play the most important role in generating free amino acids during the processing of Jinhua ham.     

Generation of volatile flavour compounds as affected by the chemical composition of different dry-cured ham sections

The composition of different dry-cured ham sections has been determined in order to study their contribution to flavour generation. The proteins, free amino acids, free fatty acids and volatile compounds were analysed in different sections of dry-cured hams with 7 and 12 months of processing. The main differences among sections were detected in free amino acids and volatile compounds. To sum up, there was a higher concentration of free amino acids in the centre section than in the butt and shank sections. However, after 12 m of ripening the difference among sections was lower and only the butt was the one with the lowest concentration. Similar results were obtained in the content of volatile compounds, the centre part of the hams contained a higher proportion of volatile compounds after 7 m of ripening than the shank and butt sections. Nevertheless, in 12 months dry-cured hams there are changes in the proportions of volatile compounds being the centre section the one with the highest proportion of volatile compounds due not only to the higher concentration of free amino acids but also to the oxidation of unsaturated fatty acids.     

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.     

Contribution of a selected fungal population to proteolysis on dry-cured ham

The proteolytic changes taking place in dry-cured hams lead to increases in free amino acids. Such free amino acids not only contribute to flavour, but also serve as precursors of volatile compounds. Several months of ripening time are required to allow the particular flavour to develop. The fungal population allowed to grow on the surface of some types of dry-cured could play a key role on proteolysis, as it has been shown for dry-cured sausages. The purpose of this work was to study the possible contribution of fungi to proteolysis in dry-cured ham. For this, a strain each of non-toxigenic Penicillium chrysogenum (Pg222) and Debaryomyces hansenii (Dh345), selected for their proteolytic activity on myofibrillar proteins, were inoculated as starter cultures. Changes in the high ionic strength-soluble proteins of an external muscle (adductor) revealed in only 6 months higher proteolysis in the inoculated hams when compared to non-inoculated control hams. Proteolytic strains among the wild fungal population on non-inoculated control hams prevented from obtaining similar differences at the end of processing. However, inoculation with Pg222 and Dh345 led to higher levels for most free amino acids at the external muscle in fully dry-cured hams. In addition, the concentration for some of the more polar free amino acids (i.e. Asp, Glu, Ser and Gln) in inoculated hams was higher at external than at internal (biceps femoris) muscles. These promising results deserve further studies to know the impact of a selected fungal population on the volatile compounds and sensory properties of dry-cured ham.     

Effect of high pressure treatment on colour, microbial and chemical characteristics of dry cured loin

The effect of high pressure on sliced and vacuum packaged commercial dry-cured pork loin was determined by analysing the induced colour changes, the residual enzymatic activities of cathepsins, aminopeptidases and dipeptidilpeptidases and the changes in the content of free amino acids and also its effect on lipid oxidation, as TBARS (Thiobarbituric reactives substances) and volatile flavour compounds. High pressure treatments above 300 MPa affected the colour of dry cured pork loins producing an increase in lightness and decreased redness and these differences were detected during all vacuum storage. High pressure produced a reduction in the activity of aminopeptidases and dipeptidylpeptidases. The untreated samples showed an increase in free amino acid content during vacuum storage while the pressurised samples showed no significant increases probably due to the reduction in aminopeptidase activity. The oxidative stability of the pressurised dry-cured loins was not affected as observed by the absence of differences in TBARS values and in the abundance of volatile compounds from the lipid oxidation. However, the pressurised treatment produced a reduction of several flavour compounds, particularly those derived from Maillard reactions, although they are regenerated during vacuum storage. In summary, high pressure treatment after the ripening of dry-cured loin affects its quality but the differences can be minimised by vacuum storage.     

不同来源和年份诺邓火腿的理化和呈味性质

为研究市售常见诺邓火腿的差异,对具有地域特色的诺邓火腿进行综合评价。以洋三元白猪、诺邓黑猪为原材料,按照诺邓火腿工艺加工1、3 年火腿,测定其理化指标、质构特性和游离氨基酸含量,并用电子舌分析其滋味特性差异。结果表明：4 组火腿质地存在明显差异;4 组火腿中均分离出18 种游离氨基酸,精氨酸含量最丰富,亮氨酸、赖氨酸和谷氨酸次之;洋三元白猪火腿（W组）的游离氨基酸含量高于诺邓黑猪火腿（B组）,B组的鲜味氨基酸相对含量高于W组,氨基酸的释放量可明显将1 年和3 年的火腿区分开;电子舌结果表明,B1组的滋味丰富性最大,B3组的咸味最大但鲜味最小,W1组的鲜味较大但丰富性较小,W3组的咸鲜味和丰富性适中。综上,鲜味、丰富性和咸味是诺邓火腿重要的味觉指标,诺邓黑猪火腿滋味较为丰富,B3组硬度、咸味最大,加工过程中要控温控湿。     

Relations between compositional traits and sensory qualities of French dry-cured ham

The left hams of 20 pig carcasses were processed by dry-curing for either 179 (n = 10) or 273 days (n = 10). The Biceps femoris was taken at the end of the processing and submitted to chemical and sensory analyses. The following chemical traits were determined: pH, moisture, salt content, fatty acid content and composition of lipid classes (triglycerides, phospholipids, free fatty acids), nitrogen fractions (soluble and insoluble protein, non-protein nitrogen, free amino acids) and volatile compounds. Sensory analysis was performed by a panel of 12 trained members, who scored texture traits (firmness, dryness, fibrousness, mellowness), taste (salty, acid), intensity of aromas (typical of dry ham, fresh meat, fat, cured meat, rancid, metal), persistence of dry ham aroma, persistence of taste after deglutition, persistence of astringency, persistence of metallic taste. The sensory qualities of dry-cured ham showed noticeable changes between days 179 and 273. Firmness, dryness and intensities of aromas typical of dry-cured ham and cured meat were enhanced with time. Neither pH, moisture nor salt content played a noticeable role in determining the sensory quality of dry-cured ham. Among the volatile compounds, the levels of several ketones and 1-butanol were significantly correlated with the aromas of dry ham and cured meat, while rancid aroma was related to aldehydes, ethylacetate, 2,3-pentanedione and nonane. Therefore, the aromas of dry ham and cured meat appeared to be determined mainly by the products of lipid oxidation. However, they were related also to unidentified volatile compounds, whose identification would be of particular interest.     

A comprehensive review on molecular mechanism of defective dry-cured ham with excessive pastiness,adhesiveness, and bitterness by proteomics insights

Excessive bitterness, pastiness, and adhesiveness are the main organoleptic and textural defects of dry-cured ham, which often cause a lot of financial losses to manufacturers and seriously damage the quality of the product. These sensory and textural defects are related to the protein degradation of dry-cured ham. Proteomics shows great potential to improve our understanding of the molecular mechanism of sensory and textural defects and identify biomarkers for monitoring their quality traits. This review presents some of the major achievements and considerations in organoleptic and textural defects of dry-cured ham by proteomics analysis in the recent decades and gives an overview about how to correct sensory and textural defects of dry-cured ham. Proteomics reveals that muscle proteins derived from myofibril and cytoskeleton and involved in metabolic enzymes and oxygen transport have been identified as potential biomarkers in defective dry-cured ham. Relatively high residual activities of cathepsin B and L are responsible for the excessive degradation of these protein biomarkers in defective dry-cured ham. Ultrasound-assisted mild thermal or high-pressure treatment shows a good correction for the organoleptic and textural defects of dry-cured ham by changing microstructure and conformation of muscle proteins by accelerating degradation of proteins and polypeptides into free amino acids.     

Effect of brine thawing/salting for time reduction in Spanish dry-cured ham manufacturing on proteolysis and lipolysis during salting and post-salting periods

The simultaneous brine thawing/salting operation has been proposed as an effective alternative for the accelerated processing of frozen cured hams. The aim of the present study was to study the effect of this new technology on the lipolytic and proteolytic enzymes responsible for the generation of flavour precursors during the salting and post-salting stages of the manufacturing of Spanish dry-cured ham. The effect of the frozen and thawed process produced a higher proteolytic and lipolytic activity than in the fresh traditional salted hams (FPS) that was detected by a higher concentration in free amino acids and free fatty acids. On the other hand, the brine thawed/salted treatments, at atmospheric pressure (BTS) and with vacuum impregnation (BTS-TP), produced an acceleration of the myofibrillar degradation that occurred in the BTS and BTS-TP hams in comparison to FPS. However, the lipolysis was affected by the frozen treatment but not by the brine thawed treatments, as few differences in free fatty acids and lipase activity were detected among the frozen and brine thawed hams at the end of post-salting stage.     

EFFECT OF CARNOSINE, ANSERINE AND OTHER ENDOGENOUS SKELETAL PEPTIDES ON THE ACTIVITY OF PORCINE MUSCLE ALANYL AND ARGINYL AMINOPEPTIDASES

The effect of endogenous dipeptides (carnosine and anserine) and peptidic fractions from meat extracts on aminopeptidase activity, alanyl and arginyl aminopeptidases purified from porcine skeletal muscle, was studied. Carnosine inhibited the activity of porcine arginyl aminopeptidase (RAP); the inhibition being stronger with the purified form of the enzyme than in the muscle extract. The RAP inhibition was competitive. One of the peptidic fractions isolated from meat extracts inhibited RAP activity But the degree of inhibition depended on the extent of purification. The major aminopeptidase present in muscle, alanyl aminopeptidase (AAP), was neither inhibited by natural dipeptides nor arty of the peptidic fractions. The study of meat inhibitory peptides provides a better understanding of the action of proteolytic enzymes during meat processing.     

Dipeptidyl peptidase activities along the processing of Serrano dry-cured ham

The activity of pork muscle dipeptidyl peptidases I, II, III, and IV was followed during the processing of Serrano dry-cured ham. The effects of NaCl, pH, and temperature on these enzymes have also been studied for a better understanding of their importance in ham processing. The lysosomal enzymes DPP I and DPP II showed maximal activity at pH around 5.5-6.0, i.e., close to the pH in ham. At low temperatures, only DPP I and DPP IV showed relevant activity. NaCl considerably inhibited DPP activities except DPP I which was only slightly affected. Dipeptidyl peptidases remained active during the whole process except DPP II whose activity disappeared after 240 days of dry-curing.     

Purification and properties of an arginyl aminopeptidase from Debaryomyces hansenii

A metallo arginyl aminopeptidase (EC 3.4.11.6) activated by Co(2+) was isolated from Debaryomyces hansenii CECT 12487. The enzyme was purified after precipitation with protamine sulphate, followed by a weak anion exchange chromatography, gel filtration chromatography and a strong anion exchange chromatography. The arginyl aminopeptidase (AAP) was purified 337 folds, with a 18% recovery. The AAP appeared to be a dimer with a molecular mass of 101 kDa. The enzyme was active in the pH range from 6 to 9. The optimal activity was detected at pH 7.0 and at 37 degrees C. AAP activity was inhibited by typical aminopeptidase inhibitors (puromycin and bestatin), reducing agents (DTT), chelating agents (EDTA, EGTA and phenantroline) and sulphydryl groups reagents (iodoacetate). Ca(2+), Mn(2+) and Co(2+) activated the enzyme, while Cu(2+), Cd(2+), Hg(2+) and Mg(2+) inhibited it. The K(m) values calculated for Arg-AMC (7-amido-4-methylcoumarin) and Leu-AMC were 0.071 and 0.094 mM, respectively. The enzyme showed maximum specificity for basic amino acids (Arg and Lys), but was also able to hydrolyze non-charged amino acids (Leu, Met and Ala) and, at a minor rate, aromatic amino acids (Phe and Tyr). AAP showed higher activity when an acid residue was located at the C-terminal position of dipeptides.The described purification of an arginyl aminopeptidase from the yeast D. hansenii can contribute to the lack of knowledge about the exopeptidase activity in one of the yeasts more frequently isolated in sausage and to understand its role during the ripening of a fermented sausage.