Proteolytic activity of Penicillium chrysogenum and Debaryomyces hansenii during controlled ripening of pork loins

The role of micro-organisms on the ripening process of dry-cured ham, particularly with respect to proteolysis, is not clear. This is partially due to the lack of an adequate system to study changes on a sterile control meat product for long ripening times. Using a meat system based on sterile pork loins ripened under aseptic conditions for 106 days, the contribution to the proteolysis of two micro-organisms isolated from dry-cured ham has been established. Changes were studied by SDS-PAGE of sarcoplasmic and myofibrillar proteins, capillary zone electrophoresis (CZE) of low ionic strength-soluble nitrogen compounds, and HPLC of free amino acids. Debaryomyces hansenii Dh345 did not show any significant proteolytic activity. However, Penicillium chrysogenum Pg222 showed high proteolytic activity on myofibrillar proteins resulting in an increase in soluble nitrogen compounds. For this, P. chrysogenum Pg222 should be considered to be used as starter culture in meat products made using long ripening times.     

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.     

Contribution of a selected fungal population to the volatile compounds on dry-cured ham

Dry-cured ham is obtained after several months of ripening. Different fungi strive on the surface, including toxigenic molds. Proteolysis and lipolysis by the endogenous and microbial enzymes seem to play a decisive role in the generation of flavor precursors in dry-cured meat products. In addition, fungi show a positive impact on the volatile compounds of ripened pork loins. However, the contribution of the fungal population to flavor formation in dry-cured ham remains unclear. One selected strain each of Penicillium chrysogenum and Debaryomyces hansenii was inoculated as starter cultures on dry-cured ham. Volatile compounds extracted by solid phase micro-extraction technique were analyzed by gas chromatography/mass spectrometry. A trained panel evaluated flavor and texture of fully ripened hams. The wild fungal population on non-inoculated control hams correlates with higher levels of short chain aliphatic carboxylic acids and their esters, branched carbonyls, branched alcohols, and some sulfur compounds, particularly at the outer muscle. Conversely, P. chrysogenum and D. hansenii seem to be responsible for higher levels of long chain aliphatic and branched hydrocarbons, furanones, long chain carboxylic acids and their esters. The very limited impact of P. chrysogenum on pyrazines in inoculated hams can be due to the activity of the yeast. Lower levels for some of the more volatile linear carbonyls at the ham surface suggest an anti-oxidant effect by micro-organisms. The differences in volatile compounds did not show a neat impact on flavor in the sensorial analysis. Nonetheless, inoculated hams got a better overall acceptability, which has to be attributed to their improved texture. The lower toughness of inoculated hams is a direct consequence of an early settling of a highly proteolytic mold. Thus, the use of selected fungi as starter cultures may be useful to obtain high-quality and safe dry-cured 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.     

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.     

Effect of Penicillium chrysogenum and Debaryomyces hansenii on the volatile compounds during controlled ripening of pork loins

During ripening of meat products such as dry-cured ham, the moulds and yeasts that proliferate on the surface may contribute to flavour development. However, their contribution to volatile components of dry-cured meat products is not known. One strain each of Penicillium chrysogenum and Debaryomyces hansenii, selected from dry-cured ham by their proteolytic activity, were tested to determine their effect on the volatile compounds during ripening. Sterile pork loins were inoculated and ripened for 106 days. Volatile compounds collected with a Solid Phase Micro-Extraction (SPME) fibre were analysed by GC/MS. Inoculation of pork loins with P. chrysogenum lead to a decrease in compounds attributed to lipid oxidation and to an increase of compounds derived from free amino acids. Inoculation with D. hansenii seemed to favour the formation of complex alcohols.     

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.     

Lipolytic and proteolytic properties of dry-cured boneless hams ripened in modified atmospheres

We studied proteolytic and lipolytic properties of dry-cured boneless ham (porcine quadriceps femoris) made with chilled (10°C, 48 h) or frozen/thawed meat (frozen at -20°C frozen for 90 days and followed by thawing at 10°C for 48 h) were determined. Dry-cured meats were stored in modified atmosphere packages (100% N(2) and a mixture of 75% N(2)+25% CO(2)) at 15°C with the intention of reducing ripening space. Results showed that dry-cured hams made with frozen/thawed raw meat had more salt, volatile fatty acids and free fatty acid content after salting and smoking. Whereas, samples prepared with chilled meats contained more nitrogenous compounds (water-soluble nitrogen, non-protein nitrogen, and free amino acids). Volatile and free fatty acid contents in all samples significantly increased with storage. Acetic acid was the predominant volatile fatty acid. To confirm lipolytic activity in dry-cured ham stored in modified atmospheres, we calculated the lipolytic coefficient. The lipolytic coefficients of all samples were positive values and significantly (P<0.05) increased with storage indicating lipolysis in samples were still active. Furthermore, nitrogenous compounds in dry-cured ham significantly (P<0.05) increased with storage indicating proteolysis in samples were not affected by modified atmosphere storage. Aerobic, anaerobic and lactic acid bacteria counts in dry-cured meats were stable to modified atmospheres storage for 20 weeks at 15°C. Flavor, texture and color score in sensory evaluation for dry-cured ham made with chilled meat were significantly higher than that made with frozen/thawed meat. All samples had high overall acceptance scores in sensory evaluation. Results in this study suggested that dry-cured boneless ham stored in modified atmospheres for 20 weeks at 15°C was another feasibility to ripen the meat without affecting lipolysis, proteolysis, microbiology and sensory quality.     

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.     

Hydrolytic activity of Penicillium chrysogenum Pg222 on pork myofibrillar proteins

Moulds grow on many different dry-cured meat products and are able to hydrolyse muscle proteins. However, their contribution to proteolysis in these products is not well known. Only recently, the ability of just a few strains of Penicillium spp. to increase proteolysis in dry-cured meat products has been shown. For these strains to be used as starter cultures, their hydrolytic activity under standard conditions should be characterised. With this purpose, the effect of Penicillium chrysogenum Pg222 on pork myofibrillar proteins has been assayed in a culture medium containing 5% (w/v) NaCl. SDS-PAGE revealed that Pg222 was responsible for extensive hydrolysis of the main myofibrillar proteins except alpha-actinin. The proteolysis led to increases in free amino acids, reaching peak values at 84 h. Ala, Tyr and Lys were present in the greatest amount. These results suggest that P. chrysogenum Pg222 would contribute to development of desired texture and flavours in dry-cured meat products.     

Proteolysis in biceps femoris during Jinhua ham processing

Sixty experimental Jinhua hams were processed by a traditional method. The nitrogen fractions and free amino acids in biceps femoris were analyzed. Intense proteolysis was found in ham muscle and totally more than 10% of muscle proteins were degraded during the course of Jinhua ham processing. The proteolytic index of Jinhua ham was between 14 and 20. Both insoluble and soluble proteins were degraded to some degree and the later showed more intense degradation. In the soluble fraction, the percentage of non-protein nitrogen (NPN) increased gradually whereas that of protein nitrogen decreased during processing (P<0.05). However, very small amount of peptides larger than 1kDa was accumulated during the whole course of processing, which proved that intense degradation reactions were also happened to them, especially at the post ripening stage when dramatic decrease of nitrogen fraction from all the peptides larger than 1kDa was found. As a result, more than 90% of the NPN products from muscle proteolysis were free amino acids and peptides of MW less than 1kDa that might make important contribution to Jinhua ham taste and provide precursors for the generation of volatile flavor compounds in ham muscle. The pattern of muscle proteolysis coincided with the reported changes of muscle proteolytic enzymes during processing, seemingly indicating that the enzymes could have played important roles in ham muscle proteolysis.     

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。     

Effect of the inoculation of a starter culture and vacuum packaging (during resting stage) on the appearance and some microbiological and physicochemical parameters of dry-cured ham

The effect of the inoculation of a starter culture and vacuum packaging (during the resting stage) on dry-cured ham appearance, microbiological and physicochemical parameters was studied. Half of the 36 processed hams were inoculated, after salting, with a commercial starter culture containing lactic-acid bacteria, Gram-positive catalase-positive cocci and yeasts. 18 hams per group (inoculated and non-inoculated) remained vacuum packaged during resting. Microbiological analyses were carried out on the lean surface during processing, on subcutaneous fat tissue at the drying stage and on lean tissue in the finished product. Appearance was evaluated during processing. Physicochemical analyses (NaCl, H(2)O, proteolysis index, a(w)) were done on Semimembranosus and Biceps femoris in the final product. Inoculation caused a reduction of mould growth and oil drip. Vacuum packaging induced increased proteolysis and increases in all microbial counts and a reduction of oil drip, mould growth and weight loss during processing.     

Oligopeptides and free amino acids in Parma hams of known cathepsin B activity

Sixteen Parma hams were manufactured following regular standard procedures up to the 13th month of processing and then divided into two groups according to established levels of cathepsin B activity in fresh hams. Each group was selected in order to include the same number of hams with low or high cathepsin B activity. The two groups were further split to be kept for an additional month at two different temperatures (18 and 26 °C) and then analysed for proximate composition, free amino acids and oligopeptides (MW<500 Da). All hams were analysed by sensory analysis (panel test) to be scored for saltiness, bitterness and the flavour of “ageing”. Fresh hams with higher cathepsin B activity were shown by means of two-way ANOVA to yield dry-cured hams more proteolysed (P<0.01), while the higher temperature of the final ageing period seemed to play a major role in lowering ham dryness. Bitterness was particularly well perceived in most proteolysed hams and it was significantly related to the higher amounts of lipophilic amino acids and lipophilic oligopeptides (identified by mass spectrometry), in agreement with the literature. Thus, the endopeptidase activity in fresh hams, together with the presence of specific lipophilic amino acids and oligopeptides in dry-cured hams, can be regarded as a molecular marker of bitter taste development.     

Factors affecting dry-cured ham consumer acceptability

The objectives of the present study were (1) to compare the relative importance of price, processing time, texture and intramuscular fat in purchase intention of dry-cured ham through conjoint analysis, (2) to evaluate the effect of dry-cured ham appearance on consumer expectations, and (3) to describe the consumer sensory preferences of dry-cured ham using external preference mapping. Texture and processing time influenced the consumer preferences in conjoint analysis. Red colour intensity, colour uniformity, external fat and white film presence/absence influenced consumer expectations. The consumer disliked hams with bitter and metallic flavour and with excessive saltiness and piquantness. Differences between expected and experienced acceptability were found, which indicates that the visual preference of consumers does not allow them to select a dry-cured ham that satisfies their sensory preferences of flavour and texture.     

金华火腿特征风味物质研究进展

金华火腿是我国传统的干腌火腿,以其特殊的风味而著称。通过对金华火腿及其它干腌火腿主要风味物质、形成机理、提取及鉴定方法等研究现状的综述,表明对金华火腿特征风味物质的深入研究有利于了解风味物质形成规律,为金华火腿加工工艺控制及火腿调味料开发应用提供理论基础。     

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.     

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.     

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.