Enhancement of the flavour development of dry fermented sausages by using a protease (Pronase E) and a cell‐free extract of Penicillium camemberti

The present work was an attempt to improve the organoleptic properties of dry fermented sausages by enhancing the release of amino acids and their further transformation into ammonia, ketoacids and some volatile compounds responsible for the ripened flavour. For this purpose a protease (Pronase E from Streptomyces griseus) and a cell‐free extract (CFE) of a Penicillium camemberti strain selected for its L ‐amino‐oxidase activity were used. The addition of Pronase E alone led to a large increase in the content of free amino acids. The CFE showed deaminative activity, which gave rise to the accumulation of some important volatile compounds such as 2‐methylpropanal and 2‐ and 3‐methylbutanal. Organoleptic analysis reflected an improvement in the quality of the sausages, especially when the two treatments were combined. © 2002 Society of Chemical Industry     

Microbial and physico-chemical changes during the ripening of dry fermented sausages superficially inoculated with or having added an intracellular cell-free extract of Penicillium aurantiogriseum

The effect of inoculation of the "salchichón" (dry fermented sausage) surface with an atoxygenic, proteolytic and lipolytic strain of Penicillium aurantiogriseum and/or the addition of an intracellular cell-free-extract (ICFE) of the same mould on the ripening process was studied. Four batches of salchichón were manufactured: control, superficially inoculated, added with the intracellular cell free extract and combination of both treatments. Superficial mould modified Micrococcaceae counts, pH, free amino acids, lipid fractions, TBARS and some organic acids. The ICFE degraded free amino acids, producing a rise in ammonia. Both treatments combined accelerate the proteolysis and lipolysis of the sausages and also the further amino acid degradation.     

Use of bacterial extracts to enhance amino acid breakdown in dry fermented sausages

The effect of the intracellular cell-free extracts (ICFEs) of two bacterial strains (Lactobacillus sakei GO and Bacillus pumilus) on the amino acid catabolism and the sensory properties of dry fermented sausages, was investigated. Extracts were added to sausages alone or in combination with a protease, papain. Amino acid breakdown was monitored by the changes in free amino acids, ammonia and amine content during the ripening process. A 15% decrease in the content of free amino acids was observed in sausages added with the ICFE from L. sakei GO. Furthermore, the extract of L. sakei GO significantly reduced (54-68%) the content of the amino acids considered as precursors of the typical ripened flavour, i.e., valine, leucine and isoleucine. Chemical changes were not reflected in a significant improvement of the sensory quality of sausages added with the ICFEs. The potential use of the bacterial ICFEs studied in the present work for the manufacture of dry fermented sausages, and its comparison with the use of fungal extracts, are discussed.     

The contribution of Penicillium aurantiogriseum to the volatile composition and sensory quality of dry fermented sausages

An atoxygenic, proteolytic and lipolytic strain of Penicillium aurantiogriseum was tested for its ability to accelerate the production of volatile compounds and to improve the sensory properties of dry fermented sausages. The following batches of sausages were prepared: control; superficially inoculated with a spore suspension; added with an intracellular cell free extract; and superficially inoculated and added with the intracellular cell free extract. Higher levels of lipid oxidation products were found in the aroma extracts of sausages without a mould cover. In contrast, branched aldehydes and alcohols presented higher concentrations in superficially inoculated and extract added sausages, while esters only showed higher concentration in the first ones. The sensory analysis showed that sausages prepared with both treatments received the highest scores in all the properties evaluated, which demonstrated both the potential of this mould as producer of volatile compounds and the effectiveness of combining both treatments.     

Effect of the fungal protease EPg222 on proteolysis and texture in the dry fermented sausage ‘salchichón’

Proteolytic activity of the fungal protease EPg222 and its effect on the texture of the dry fermented sausage ‘salchichón’, which has a long ripening process, was assayed. Samples with enzyme added showed a significant reduction of myofibrillar protein concentration during the ripening period, compared with a control, and proteolytic activity of the enzyme led to a higher accumulation of non‐protein and ‐amino acid nitrogen. Analysis of volatile compounds in ripened dry fermented sausages revealed that only in EPg222 batches were branched compounds derived from amino acids catabolism detected that are associated with the flavour of dry cured meat products. The texture profile analysis showed reduction in hardness, gumminess and chewiness values in treated compared with control sausages. The effect of this enzyme could be of great interest in stimulating proteolysis, in flavour development and in reducing the hardness of dry fermented sausages produced by a long ripening process. Copyright © 2004 Society of Chemical Industry     

Changes in selected biochemical and sensory parameters as affected by the superficial inoculation of Penicillium camemberti on dry fermented sausages

An atoxigenic strain of Penicillium camemberti was superficially inoculated on fermented sausages in an attempt to improve their sensory properties. The growth of this mould on the surface of the sausages resulted in an intense proteolysis and lipolysis, which caused an increase in the concentration of free amino acids, free fatty acids (FFA) and volatile compounds. Many of these were derived from amino acid catabolism and were responsible for the "ripened flavour", i.e. branched aldehydes and the corresponding alcohols, acids and esters. The development of the fungal mycelia on the surface of the sausages also protected lipids from oxidation, resulting in both lower 2-thiobarbituric acid (TBARS) values and lipid oxidation-derived compounds, such as aliphatic aldehydes and alcohols. The sensory analysis of superficially inoculated sausages showed clear improvements in odour and flavour and, as a consequence, in the overall quality of the sausages. Therefore, this strain is proposed as a potential starter culture for dry fermented sausage production.     

Effects of muscle shortening and proteolysis on Warner-Bratzler shear force in beef longissimus and semitendinosus

The effect of the addition of the fungal protease EPg222 on the sensory characteristics of dry fermented sausage "salchichón" ripened with commercial starter cultures was investigated. Sausages were prepared with purified EPg222 and Staphylococcus carnosus, Staphylococcus xylosus, and Lactobacillus sakei as starter cultures, ripened for 145 days and compared with a control batch only inoculated with the starter cultures. Dry fermented sausages ripened with EPg222 and starter cultures showed higher amount of NPN and volatile compounds derived from amino acid catabolism, than control ripened only with starter cultures. Several branched aldehydes, acids and alcohols such as 2- and 3-methylbutanoic acid and 2-methylpropanol were detected only in enzyme treated samples. Sensory analysis reflected higher values for aroma intensity of sausages treated with EPg222 and lower values of hardness than control. The effect of EPg222 may be of great interest to improve sensory characteristics of dry fermented sausages ripened with starter cultures.     

Proteolysis in dry fermented sausages: The effect of selected exogenous proteases

The effect of three commercial proteases (pronase E from Streptomyces griseus, aspartyl proteinase from Aspergillus oryzae and papain) on protein breakdown and the sensory characteristics of dry fermented sausages was investigated. Water soluble, non-protein, 5% phosphotungstic acid soluble, 5% sulphosalicylic acid soluble and total volatile basic nitrogen contents increased during fermentation, stabilizing later until the end of ripening (26th day). Nitrogen values were always greater in the aspartyl proteinase added batch in comparison with the other protease added batches. Total free amino acid changes showed a similar pattern to those observed for the 5% sulphosalicylic acid soluble nitrogen. The electrophoretic studies demonstrated that proteolysis of high molecular weight myofibrillar and sarcoplasmic proteins was more prominent in protease added batches. It was especially intensive in papain one. The dominant amino acids at the end of ripening were similar in all batches. Tyramine and histamine increased throughout ripening. No significant differences in sensory properties were found between control and pronase E and papain added batches, but they were significantly different (p < 0.01) from the sausages containing aspartyl proteinase, due to an excessive softening. The effect of exogenous enzyme addition on the flavour potentiation of dry fermented sausage is discussed.     

Effect of Debaryomyces spp. on the proteolysis of dry-fermented sausages

The effect of the addition of Debaryomyces spp. on the microbial, chemical properties and degradation of meat proteins in dry fermented sausages was investigated. The manufacture of dry fermented sausages with Debaryomyces spp. produced a slow decline in pH during early drying stage. However, the final product had lower ammonia, and higher acetic and d-lactic acids without producing any effect on the final pH. Sarcoplasmic proteins were not affected by Debaryomyces spp. but the degradation of myofibrillar proteins was accelerated at the beginning of the drying stage even though the final sausage, inoculated with Debaryomyces spp., had lower myofibrillar proteolysis. The content of free amino acids was similar at the beginning of the drying stage for all the studied batches. However, the high differences in the content of free amino acids at the end of the process could be attributed to the Debaryomyces spp. activity. The addition of a higher amount of Debaryomyces spp. did not contribute to a major proteolysis degree.     

Improvement of the Sensory Properties of Dry Fermented Sausages by the Superficial Inoculation and/or the Addition of Intracellular Extracts of Mucor racemosus

ABSTRACT: The effect of inoculation of the "salchichón" (dry fermented sausage) surface with an atoxigenic, proteolytic, and lipolytic strain of Mucor racemosus and/or the addition of a intracellular extract of the same mold on the ripening process was studied. Four batches of salchichón were manufactured: control, superficially inoculated, added with intracellular extract, and a combination of both treatments. Superficial mold modified the amino and free fatty acid contents, volatile compounds, and sensory properties. Intracellular extract degraded free amino acids, producing a rise in ammonia and volatile compounds. Both treatments combined improved significantly the sensory properties, which were given scores higher than control sausages at the end of ripening.     

Volatile compound generation in dry fermented sausages by the surface inoculation of selected mould species

The effect of the inoculation of dry fermented sausage surface with an atoxigenic, proteolytic and lipolytic strain of Mucor racemosus, Penicillium aurantiogriseum and Penicillium camemberti on the volatile composition was studied. The analysis of the headspace volatile compounds using gas chromatography/mass spectrometry enabled the identification of 55 volatiles. The study showed that every mould species produced a different volatile profile which was also different from that of the control sausages. Compounds derived from amino acid catabolism, i.e. branched aldehydes and the corresponding alcohols, were produced in higher amounts in sausages inoculated with Penicillium spp. On the other hand, volatiles coming from the microbial esterification were related to sausages inoculated with M. racemosus. The development of the fungal mycelia on the sausage surface protected lipids from oxidation, thus giving rise to fewer lipid oxidation products in the inoculated sausages.     

Use of Lactococcus lactis subsp. cremoris NCDO 763 and α-ketoglutarate to improve the sensory quality of dry fermented sausages

The aim of the present work was to enhance the degradation of free amino acids in dry fermented sausages as precursors of volatile compounds responsible for the ripened flavour. For this purpose, Lactococcus lactis subsp. cremoris NCDO 763, its intracellular cell free extract (ICFE) and α-ketoglutarate were added to sausages. Papain was also used to increase the amount of free amino acids. When L. lactis was inoculated in sausages, an increase in the proteolytic phenomena was observed. The addition of α-ketoglutarate increased transamination phenomena in batches where it was added. The enhancement of these phenomena determined a noticeable rise in the content of glutamic acid (the main final product in transamination reactions) and a decrease, among other amino acids, of valine and leucine, with the formation of high amounts of their derivatives 2-methylpropanal and 3-methylbutanal. These aldehydes are responsible for the ripened flavour of dry fermented sausages. Sensory analysis showed an improvement of odour and flavour when L. lactis and α-ketoglutarate were combined. On the other hand, the intracellular cell free extract of L. lactis did not show any important activity in relation to amino acid breakdown even when used together with α-ketoglutarate and/or papain.     

Nonprotein Nitrogen (NPN) and Free amino acid contents of dry, fermented and nonfermented sausages

Fermented and nonfermented sausages were analyzed for concentration changes in total and nonprotein nitrogen (NPN) and in free amino acid profiles during processing. Sausages fermented by Pediococcus pentaceous at 38°C and heated to 60°C had increases (p < 0·05) in total N and NPN contents and increases in concentration (≥5 mg per 100 g dry sample) for 14 of 20 free amino acids. Nonfermented sausages had no increase (p > 0·05) of NPN content and increases in only 6 of 20 free amino acids after heating to 60°C. When the effects of three starter cultures were compared, NPN concentrations after fermentation and heating and after drying were highest to lowest in the following order: P. pentaceous >P. acidilactici with M. varians >P. acidilactici. Variations of free amino acid patterns in the fermented, dried sausages were found which indicated differences in metabolic activity between the cultures. These variations did not (p > 0·05) affect sensory scores for the dried sausages. Overall, these results for rapidly fermented and mildly heated dry sausages were similar to results previously reported for 'European-style' dry sausages processed with prolonged low temperature fermentations.     

传统与强化发酵羊肉香肠微生物多样性及代谢物差异分析

为探究发酵方式对羊肉香肠中微生物群落和代谢物影响，采用高通量测序和代谢组学技术分析传统（CT）和强化（QH）发酵羊肉香肠中的微生物群落及代谢物差异，及微生物与差异显著代谢物之间的相关性。结果表明，强化发酵对羊肉香肠微生物群落有明显的影响。属水平上，CT以明串珠菌属（Leuconostoc）、拉恩氏菌属（Rahnella）、环丝菌属（Brochothrix）、德巴利氏酵母属（Debaryomyces）占优势；QH以明串珠菌属、环丝菌属、嗜冷杆菌属（Psychrobacter）、片球菌属（Pediococcus）、德巴利氏酵母属、青霉属（Penicillium）占优势。代谢物方面，强化发酵增加了风味和滋味物质的种类和含量，尤其是酯类和氨基酸类。基于多元统计分析，共鉴定出29 个挥发性和16 个非挥发性显著差异代谢物（|差异倍数|＞2且变量重要性投影＞1，P＜0.05）；相关性结果表明，7 个细菌属（明串珠菌属、环丝菌属、片球菌属、嗜冷杆菌属、肠球菌属（Enterococcus）、拉恩氏菌属和葡萄球菌属（Staphylococcus））和7 个真菌属（德巴利氏酵母属、青霉属、曲霉属（Aspergillus）、unspecified_Aspergillaceae、unspecified_Capnodiales、根霉属（Rhizopus）和unspecified_Basidiomycota）被视为核心微生物（|r|＞0.8，P＜0.05）。     

Metabolism of amino acids, dipeptides and tetrapeptides by Lactobacillus sakei

The microbial degradation of proteins, peptides and amino acids generates volatiles involved in the typical flavor of dry fermented sausage. The ability of three Lactobacillus sakei strains to form aroma compounds was investigated. Whole resting cells were fermented in phosphate buffer with equimolar amounts of substrates consisting of dipeptides, tetrapeptides and free amino acids, respectively.Dipeptides disappeared quickly from the solutions whereas tetrapeptides were only partially degraded. In both approaches the concentration of free amino acids increased in the reaction mixture but did not reach the equimolar amount of the initial substrates. When free amino acids were fed to the bacteria their levels decreased only slightly. Although peptides were more rapidly degraded and/or transported into the cells, free amino acids produced higher amounts of volatiles.It is suggested, that after transport into the cell peptides are only partially hydrolyzed to their amino acids, while the rest is metabolized via alternative metabolic pathways. The three L. sakei strains differed to some extend in their ability to metabolize the substrates to volatile compounds. In a few cases this was due to the position of the amino acids within the peptides. Compared to other starter cultures used for the production of dry fermented sausages, the metabolic impact of the L. sakei strains on the formation of volatiles was very low.     

Release of aroma compounds from dry-fermented sausages as affected by antioxidant and saliva addition

The effect of the addition of an antioxidant and saliva on the release of aroma compounds from dry fermented sausages was studied by extracting the headspace at different times using solid phase micro-extraction technique. The compounds were analysed by gas chromatography using a FID detector and identified by mass spectrometry. The addition of butylated hydroxytoluene to dry fermented sausages produced a significant reduction of the release of most of the volatile compounds indicating an oxidation process during sampling. The addition of water and saliva to the dry fermented sausages produced a higher release of pentanal, hexanal and octanal, whilst pentane, 1-octanol, pentanoic acid, and propanal showed a lower rate of release. Moreover, phenylethyl alcohol, 2-butanone, 2,3-pentanedione and 2-nonanone, derived from bacterial metabolism, presented a lower release rate when water and saliva were added. The rate of release of the compounds from dry fermented sausages was not related with the compound hydrophobicity value. Further research is needed to elucidate the effect of fermented sausage matrix on the partitioning of the compounds and its effect on aroma perception.     

Dry fermented sausages made with a protease from Aspergillus oryzae and/or a starter culture

Sausages with a starter culture (Lactobacillus plantarum + Staphylococcus carnosus), a protease (Fungal Protease from Solvay Enzymes) and both enzyme/starter were produced from the same raw matter under the same conditions for 15 days of ripening. The lowest pH values were found in Fungal/starter sausages. Significantly higher amounts of free amino acids (FAA) were seen in sausages containing enzyme from the 3rd day of ripening, whereas amino acids from peptides (PAA) were significantly higher only after 15 days of ripening. Enzyme addition gave rise to changes in 10 of the 15 FAA analyzed. Histidine was the main amino acid from the peptide fraction that increased in both sausages containing added enzyme. Although trained panelists detected some sensorial benefits in the sausages with added enzyme, the effects were not as marked as might have been expected.     

Use of Fourier transform infrared spectroscopy to evaluate the proteolytic activity of Yarrowia lipolytica and its contribution to cheese ripening

The interaction between tyrosine-decarboxylase and proteolytic activities of a Lactobacillus curvatus and Staphylococcus xylosus, respectively, on biogenic amine production during the ripening and the storage of dry fermented sausages was investigated. Water content, pH, proteolysis parameters, microbial counts, and biogenic amine contents were monitored in spontaneously and starter fermented sausages. The use of proteolytic staphylococci as starter resulted in a higher content of non-protein nitrogen and total free amino acids. Tyramine was the main amine produced in all batches. However, tyrosine-decarboxylase activity of the L. curvatus starter strain was weak and yielded lower amounts of tyramine than those produced by the wild mioroflora in the control batch. Association between tyramine production and proteolysis could only be established in a defectively dried batch. Putrescine and cadaverine accumulation was efficiently reduced in the starter-mediated fermentation, in agreement with the lower development of enterobacteria. Phenylethylamine and tryptamine were only detected in the spontaneously fermented sausages, while histamine, spermine and spermidine did not vary during the ripening. Biogenic amine levels and related parameters showed significant changes during the storage of dry sausages depending on the temperature and the batch. As a general rule, changes in the pH, proteolysis, microbial counts, and biogenic amine contents were stronger at 19 degrees C than at 4 degrees C. The results suggest that refrigeration would be advisable for preventing further accumulation of biogenic amines during the storage of dry fermented sausages.     

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.     

黄酒机械成型麦曲制曲过程中真菌动态变化的研究

采用传统分离培养和免培养的方法研究了机械化黄酒麦曲堆放成型过程中真菌群落的动态变化情况。研究结果显示,机械化黄酒麦曲堆放成型过程中演替存在的真菌大致分13个属,其中犁头霉属、曲霉属、毛霉属真菌存在于整个麦曲成型堆放过程中。在麦曲成型堆放的前期,麦曲中主要存在犁头霉属(Absidia corymbifera)、曲霉属(Aspergillus oryzae)、毛霉属(Rhizomucor Pusillus)、青霉属(Penicilliumaurantiogriseum)、毕赤酵母属(Pichia burtonii)等真菌。在麦曲成型堆放的中后期,除前期存在的真菌外,还存在裸胞壳属(Emericellanidulans)、散囊菌属(Eurotiumamstelodami)、青霉属(Penicillium chrysogenum)、棒孢酵母属(Clavispora lus-itaniae)、毕赤酵母属(Pichia anomala)、伊萨酵母属(Issatchenkia orientali)、平脐蠕孢属(Bipolaris spicifera)和复膜孢酵母属(Saccharomycopsis fibuligera)。反映出机械化黄酒麦曲在成型堆放过程中真菌多样性丰富,这些真菌微生物在麦曲品质和黄酒大罐酿造中均有重要的影响。