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.     

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.     

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.     

Proteolytic activity of lactic acid bacteria strains and fungal biota for potential use as starter cultures in dry-cured ham

During the processing of dry-cured meat products, sarcoplasmic and myofibrillar proteins undergo proteolysis, which has a marked effect on product flavor. Microbial proteolytic activity is due to the action of mostly lactic acid bacteria (LAB) and to a lesser extent micrococci. The proteolytic capacity of molds in various meat products is of interest to meat processors in the Mediterranean area. Eleven LAB and mold strains from different commercial origins were tested for proteolytic activity against pork myosin, with a view to possible use of these strains as starter cultures for Iberian dry-cured ham. Proteolytic activity was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LAB strains with the highest proteolytic activity were Lactobacillus plantarum (L115), Pediococcus pentosaceus (Saga P TM), and Lactobacillus acidophilus (FARGO 606 TM). The best fungal candidate was Penicillium nalgiovense LEM 50I followed by Penicillium digitatum, Debaryomyces hansenii, and Penicillium chrysogenum.     

传统腌腊肉制品挥发性风味物质的研究进展

本文以干腌火腿、腊肉、风鸭、发酵香肠和风干鱼制品为代表,对中国传统腌腊肉制品的挥发性风味物质进行了探讨。从总体上阐述了我国传统腌腊肉制品挥发性风味的形成机理,详尽列表总结分析了不同制品的特征风味化合物,分析它们之间的共性与异性,并从原料特性及加工工艺两个主要方面分析了差异性形成的可能原因。一方面,丰富了传统腌腊肉制品的形成机制,另一方面,为生产调控肉制品的风味提供理论依据。     

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.     

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.     

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.     

贵式肉品中乳酸菌的选育及其对发酵里脊火腿风味品质的影响

以锦屏和榕江酸肉、凯里和遵义香肠、威宁和盘县火腿以及凯里、遵义和毕节风肉9 种贵州发酵肉为对象,对其中乳酸菌分离鉴定并进行蛋白质降解、生理生化特性、生产适应性、安全性研究。根据初筛结果选取1 株蛋白降解能力强且具有肉类发酵剂特性的乳酸菌SJ-4,接种发酵里脊火腿,以未接种火腿为对照,分析该菌株对发酵里脊火腿色泽、质构、电子舌响应值及风味的影响。结果表明：菌株SJ-4能耐受6 g/100 mL NaCl、150 mg/kg NaNO2,不产NH3、H2S、H2O2,无氨基酸脱羧酶活性,具有分解蛋白质的能力但无分解脂肪活性,最适温度37 ℃,最适pH 6.0,培养4 h进入对数生长期,培养18 h pH值可降至3.80,已达到肉类发酵剂条件。经16S rDNA测序鉴定为植物乳杆菌（Lactobacillus plantarum）。菌株SJ-4接种对里脊火腿色泽、硬度、黏聚性均影响显著,添加该菌后有助于产品滋味形成,且关键风味物质如壬醛、3-羟基-2-丁酮等含量增加。植物乳杆菌SJ-4具有作为高活性肉制品发酵剂的潜力。     

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.     

Proteomic analysis of water soluble and myofibrillar protein changes occurring in dry-cured hams

The myofibrillar fraction of raw ham muscles and dry-cured hams with different ripening times was extracted in denaturing and reducing conditions and subjected to two-dimensional gel electrophoresis. The two-dimensional maps gave overall pictures of the already noted progressive disappearance of actin, tropomyosin and myosin light chains during ripening. In addition, two fragments from Myosin Heavy Chain proteolysis, marked as myosin chain fragments MCF1 and MCF2, were identified by immunodetection and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Furthermore, a new form of actin on two-dimensional gel was identified by MALDI-TOF peptide mapping. In 12-month-old dry-cured ham, most myofibrillar proteins were completely hydrolyzed. At this stage of ripening, in fact, in some Parma and S. Daniele dry-cured ham samples, myosin heavy chain fragments and other unidentified neo-formed spots were found. Some of the sarcoplasmic proteins in water extracts from pork meat markedly decreased in amount or disappeared totally, during ripening. Surprisingly, two-dimensional gel electrophoresis maps of the water soluble protein fraction from dry-cured ham showed the presence of two spots identified as tropomyosin α- and β-chain. This result suggests that some of the saline soluble myofibrillar proteins can disappear from this fraction because of salt solubilization and not due to complete enzyme action. Two-dimensional gel electrophoresis (2-DGE) has proved a powerful tool to evaluate the enzymatic susceptibility of meat proteins and the evolution of protein map fragmentation throughout ripening process as well as a means of obtaining a standard fingerprinting map characterizing the final product.     

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.     

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.     

Analysis of volatile compounds of Iberian dry-cured loins with different intramuscular fat contents using SPME-DED

In order to study the effect of both, ripening time and IMF content on the volatile profile of Iberian dry-cured loin, volatile compounds from the headspace of 10 Iberian dry-cured loins with different intramuscular fat contents (IMF), low (average IMF content of 2.3%) and high (average IMF content of 6.7%), at days 40 and 55 of the ripening process were analysed using SPME coupled to a direct extraction device (DED) and subsequent gas chromatography/mass spectrometry (GC/MS). Chromatographic areas of the main chemical families detected (hydrocarbons, aldehydes, alcohols, ketones and acids) increased with ripening time. A higher total chromatographic area was detected in the headspace of high IMF loins compared to low IMF ones. Several volatile compounds derived from lipid oxidative reactions, such as hexanol, octanal, (E,E)-2,4-heptadienal or (E)-2-decenal, and others from the degradation of certain amino acids, such as dimethylsulfide, 3-methylbutanal or phenylacetaldehyde, showed higher chromatographic areas in the headspace of high IMF loins than in low IMF ones. Thus, IMF content could influence both the generation of volatile compounds and the transfer of such compounds from the product matrix to the headspace.     

Differentiation of yeasts growing on dry-cured Iberian ham by mitochondrial DNA restriction analysis, RAPD-PCR and their volatile compounds production

The efficiency of mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR and volatile compounds analysis to differentiate yeast biotypes involved in flavour development of dry-cured Iberian ham throughout the ripening process is evaluated. For this purpose, 86 yeasts isolated from Iberian hams in the main ripening stages at different industries of the four Protected Designations of Origin of this product, were used. The combination of mtDNA restriction analysis and RAPD-PCR using the primer (GACA)₄ showed a higher variability in the yeast species detected than obtained using only mtDNA restriction analysis. Only two species, Debaryomyces hansenii and Candida zeylanoides, were identified throughout the whole ripening process and a wide diversity of biotypes was found in these two species, with those of D. hansenii predominating. Clear differences between biotypes were detected in the generation of volatile compounds, with the biotype C2-2 of D. hansenii showing the highest concentrations of volatiles. The combined use of mtDNA restriction analysis and RAPD-PCR distinguishes yeast biotypes with different production of volatile compounds. In addition, analysis of the production profile of volatile compounds is needed to differentiate yeast strains of the same biotype recovered at different stages of ripening. Thus, the combination of these three methods could be very useful to select or monitor yeasts as starter cultures in dry-cured meat products.     

不同微生物菌株对干腌肉块挥发性风味化合物的影响

以干腌猪肉块为原料,分别接种表皮葡萄球菌(Staphylococcus epidermidis)、木糖葡萄球菌(Staphylococcus xylosus)和耳氏葡萄球菌(Staphylococcus auricularis)后按火腿加工工艺进行发酵生产干腌肉块。采用固相微萃取(SPME)和气相色谱- 质谱联用(GC-MS)进行挥发性风味化合物的测定,研究不同干腌发酵肉块中挥发性风味化合物的种类和相对含量,以探讨不同微生物发酵剂对干腌发酵肉块中挥发性风味化合物的影响。本实验共鉴定出149 种挥发性风味化合物,包括17 种醛、32 种醇、44 种烷烃、28 种酯、10 种酮、10 种酸、5 种杂环化合物、1 种酚和1种醚。不同微生物菌株对干腌发酵肉块挥发性风味化合物的种类、数量和相对含量都产生了非常明显的影响。对照处理组的发酵肉块中烃类物质的相对含量较高,木糖葡萄球菌处理的发酵肉块中杂环类化合物相对含量较高,木糖葡萄球菌处理的发酵肉块中醛类物质相对含量较高、耳氏葡萄球菌处理的发酵肉块中酯类物质的相对含量较高。     

金华火腿质量与原料选择的关系研究

中式火腿如金华火腿与西方地中海地区的干腋火腿都属于高档的肉类制品。金华火腿质量取决于两个主要方面：一是独特的加工方法；二是严格的原料选择。本文从选料着手，分析在金华火腿加工过程中原料的质量对火腿成熟的作用，以及最终对产品质量的影响。     

Lipids in muscles and adipose tissues, changes during processing and sensory properties of meat products

Dry-cured meat products represent a large part of the meat products on the European market. The technologies develop for these products lead to the production of a large scale of meat products with typical sensory traits. Numerous studies have been devoted to optimise the quality traits of these products which are considered as traditional products by the consumer and provide a high added value to the producer. Among the components of the raw material, lipids play a key role in the final quality of these products. Many sensory traits of dry-cured meat products depend on lipid traits of muscle and adipose tissues of fresh meat and on their degradation through a complex set of lipolytic and oxidative reactions during processing. Lipid traits of both muscle and adipose tissues of fresh meat are strongly related to pig rearing conditions, mainly genotype and feeding strategy. During processing, lipids undergo intense lipid hydrolysis controlled by both lipases and phospholipases, which remain active all along the process. Lipids are also subjected to oxidation, which generates numerous volatile compounds. These volatiles contribute to some typical aroma notes of dry-cured meat products such as rancid, aged ham and dry-cured odours. This paper reviews the recent knowledge on the influence of lipid traits of fresh meat, lipid hydrolysis and oxidation on the development of sensory traits of dry-cured meat products.     

Irradiated Chinese Rugao ham: Changes in volatile N-nitrosamine, biogenic amine and residual nitrite during ripening and post-ripening

N-nitrosamines, biogenic amines and residual nitrite are harmful substances and often present in cured meat. The effects of gamma-irradiation (γ-irradiation) on these chemicals in dry-cured Chinese Rugao ham during ripening and post-ripening were investigated. Rugao hams were irradiated at a dose of 5kGy before ripening and were then ripened in an aging loft. Although γ-irradiation degraded tyramine, putrescine and spermine, on the other hand, it promoted the formation of spermidine, phenylethylamine, cadaverine and tryptamine. Residual nitrite was significantly reduced by γ-irradiation. N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-nitrosopyrrolidine (NPYR) were found in Chinese Rugao ham during ripening and post-ripening but could be degraded with γ-irradiation. The results suggest that γ-irradiation may be a potential decontamination measure for certain chemical compounds found in dry-cured meat.     

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.