Ripening Of Dry Fermented Sausages Research Articles

Influence of mixed starters on the degradation of proteins and the formation of peptides with antioxidant activities in dry fermented sausages

This study aimed to investigate the influence of mixed starters (Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201) with a high protease activity on the degradation of proteins and the formation of peptides with antioxidant activities during the fermentation and ripening of dry fermented sausages. The starters mainly affected the proteolytic characteristics during the fermentation stage, and their beneficial effects persisted until the end of ripening. Electrophoresis analysis indicated that the starters could more rapidly and severely hydrolyze proteins on days 1 and 4 than on other days. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and ferric ion reducing antioxidant power (FRAP) value of the peptides extracted from the starter-inoculated group significantly increased (P < 0.05) compared with those from the control group. After 23 days of ripening, the free amino acid (FAA) content also increased significantly (P < 0.05) because of the addition of starters. Thus, mixed starters could help delay oxidative changes in dry fermented sausages.

Actin proteolysis during ripening of dry fermented sausages at different pH values.

In dry fermented sausages, myofibrillar proteins undergo intense proteolysis generating small peptides and free amino acids that play a role in flavour generation. This study aimed to identify small peptides arising from actin proteolysis, as influenced by the type of processing. Two acidification profiles were imposed, in order to mimic the pH normally obtained in southern-type and northern-type dry fermented sausages. The identification of peptides was done by liquid chromatography coupled to mass spectrometry in a data-independent positive mode of acquisition (LC-MSE). During manufacturing of the dry fermented sausages, actin was highly proteolysed, especially in nine regions of the sequence. After fermentation, 52 and 42 actin-derived peptides were identified at high and low pH, respectively, which further increased to 66 and 144 peptides, respectively, at the end of ripening. Most peptides were released at the cleavage sites of cathepsins B and D, which thus play an important role.

Impact of Casing Damaging on Aflatoxin B1 Concentration During the Ripening of Dry-Fermented Meat Sausages

The aim of this article is to investigate the impact of casing damaging on the formation of aflatoxin B1 (AFB1) during the ripening of dry-fermented meat sausages. The level of AFB1 contamination was determined in 24 samples using the ELISA immunoassay throughout a six-month production period. While with intact casing samples no contamination was observed throughout the whole production process, in damaged casing samples AFB1 was detected in the ripening end-stages in the range of 1.62–4.49 μg/kg. The results showed that casing damaging occurring during long-term ripening of dry-fermented sausages can cause AFB1 contamination, possibly arising on the grounds of diffusion of this mycotoxin from the product surface to its interior.

English

The aim of this work was to study the effect of Staphylococcus xylosus and Lactobacillus plantarum on microbial and physicochemical characteristics of a dry fermented poultry meat sausage. The microbial results show that lactic acid bacteria (LAB) and coagulase negative staphylococci (CNS) represent the two predominant microflora in all stages of ripening. The number of enterobacteria decreased significantly in sausages previously inoculated respectively with L. plantarum and with a mixed starter cultures, due to the acidifying and antibacterial activities of lactic acid bacteria. The moisture content of control and inoculated sausages decreased in all stages of ripening which allows a good preservation and consequently, improved their shelf life. Moreover, total free amino acids content increased during ripening of dry fermented sausages. Their concentrations were higher in sausages previously inoculated with starter cultures than in control ones. The main amino acids present in all stages of ripening were aspartic and glutamic acids, arginine, glycine, threonine, alanine, tyrosine, phenylalaline, leucine and isoleucine. &nbsp; Key words: Staphylococcus xylosus, Lactobacillus plantarum, ripening, dry fermented sausage. &nbsp

Effect of Starter Cultures Combinations on Lipolytic Activity and Ripening of Dry Fermented Sausages

The effect of starter culture combinations on the quality of Turkish type dry fermented sausage (sucuk) were evaluated during ripening and storage periods. Sucuk formulations were produced without (control) and with three different starter culture combinations; i) Staphylococcus carnosus+Pediococcus pentosaceus, ii) Staphylococcus carnosus+ Lactobacillus sakei, and iii) Staphylococcus carnosus+Pediococcus pentosaceus+ Lactobacillus sakei. Analysis of microbiological, physico-chemical and lipase enzyme levels of samples were conducted until the 60th day. Interactions among the presence of lipolytic starter cultures, lipase enzyme levels and thiobarbituric acid reactive substances were also evaluated both in ripening and drying periods. There were apparent differences on microbiological and chemical properties between samples prepared with starters and control. It has been concluded that the use of lipolytic starter cultures in suitable combination would have positive effect on the acceleration of ripening and improvement of the quality of dry fermented sausages.

Meat-model system development for proteolytic activity determination.

Many technological parameters that affect the nature and functional properties of proteins are involved in the preparation of meat products. The dry-curing process is quite complex because of the coexistence of enzymes from endogenous and bacterial origins. The protein breakdown that takes place during the ripening of dry fermented sausages leads to an increase in the concentration of peptides and free amino acids. The proteolytic events have been thoroughly investigated not only because of their physiological significance but also for their technological connotations in terms of texture and flavor development. Lactic acid bacteria and Staphylococcus or Kocuria are used as starter cultures in fermented meat products. In recent years, the proteolytic system of lactobacilli involved in meat fermentation became the focus of an increasing number of studies because of the technological roles of these organisms. Although results obtained from broth systems show proteolytic activity, studies involving food systems must be done to confirm their effectiveness. A simplified soluble muscle extract to determine the capacity to degrade meat proteins by lactic acid bacteria was developed using a spectrophotometric method based on the reaction of the alpha-amino groups released by hydrolysis with o-phtaldialdehyde and beta-mercaptoethanol to form an adduct (1-thioalkyl-2-alkylisoindoles) that is enhanced at basic pH and absorbs strongly at 360 nm.

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.

Accelerated ripening of dry fermented sausages

Research on accelerated ripening of dry fermented sausages started in the early 1990s. Fermented sausages manufacture is a very important part of meat industry in many countries and the acceleration of ripening would result in a reduction of the storage time and would increase the profit margin and the competitiveness of the end product. The different strategies that have been assayed with this purpose include ripening at elevated temperature, use of genetically modified starter bacteria, addition of enzymes and addition of slurry systems. Over the last decade, numerous studies have been carried out, especially on the addition of enzymes. The aim of this paper is to review and update the knowledge on this topic. The more recent approaches in this field, such as the use of microbial extracts, are also presented.

Changes in the Components of Dry-Fermented Sausages during Ripening

Several chemical changes occur during the ripening of dry-fermented sausages that determine the flavor and odor of the end product. The phenomena that take place during fermentation, that is, both acidification of the sugars by lactic acid bacteria and reduction of nitrates and nitrites to nitric oxide by micrococci have been known for several years. However, the chemical changes involved in this process, and, particularly, the agents responsible have not yet been established, although they have been attributed to changes in the majority components (proteins and lipids) and to the ingredients added (spices and condiments) in the preparation of the original mixture.The typical flavor and odor of dry-fermented sausages cannot be attributed to volatile substances alone, but to a large number of volatile and nonvolatile compounds present in the product in suitable proportions. Microbial growth in the sausage together with activity of the meat endogenous enzymes are undoubtedly partially responsible for the development of a number of aromatic and sapid compounds. However, lipid autooxidation reactions are also an important source of these substances, and it is not yet known which of these processes is more important in sausage ripening. Much research has focused on the break up of triglycerides into free fatty acids, diglycerides, and monoglycerides during ripening and the progressive increase in the amounts of different carbonyl oxidation products. Carbonyl compounds probably play a significant role in determining the flavor because, in general, these have very low perception thresholds, in the ppm and ppb range. Similarly, the protein breakdown to yield peptides and amino acids has been studied extensively, the latter being substrates of several microbial and chemical reactions that generate many flavor compounds.

Effect of proteolytic starter cultures of Staphylococcus spp. on biogenic amine formation during the ripening of dry fermented sausages

The effect of proteolytic starter cultures of Staphylococcus carnosus and Staphylococcus xylosus on biogenic amine production was examined during the fermentation process of dry sausages. Microbial counts (lactic acid bacteria, Micrococcaceae and Enterobactenaceae), pH, moisture and proteolysis-related parameters were also studied. The polyamines spermine and spermidine were the main amines found in the raw material and they only showed slight fluctuations during the fermentation. The four elaborated batches presented a significant ( P<0.001) formation of tyramine and putrescine. The main rate of amine production was during the first three days, when a sharp pH decrease and the development of lactic acid bacteria occurred. Sausages fermented with starters had lower amounts of tyramine than naturally fermented sausages (control), but differences in the Micrococcaceae counts were only significant during the first week of the ripening process. A slight formation of diaminopropane, cadaverine, agmatine, tryptamine and phenylethylamine was observed. The amounts of histamine were constant and remained below 0.5 mg/kg of dry matter, while serotonin, octopamine and dopamine were not detected. The sausages with Staphylococcus as starter culture showed strong proteolysis that was correlated with higher pH values than those of the control sausages. However, no positive correlation was found between the proteolysis index and biogenic amine production. Since proteolysis was stronger during the second half of the ripening process, the release of free amino acids as amine precursors occurred later than the early amine production.

Improved ion chromatography–integrated pulsed amperometric detection method for the evaluation of biogenic amines in food of vegetable or animal origin and in fermented foods

An improved method for the simultaneous determination of underivatized biogenic amines, cadaverine, putrescine, spermidine, histamine, tyramine and some amino acids precursors, histidine and tyrosine, in food products, based on ion-exchange chromatography (IC) with integrated pulsed amperometric detection (IPAD) has been developed. The method was successfully used for the analysis of biogenic amines and amino acids in food both of vegetable (kiwi, Actinidia chinensis) and animal origin, (fish, pilchard), as well as in fermented foods, such as cheese (Emmenthal) and dry sausages (salami). The method was also successfully used to study the changes in biogenic amines during the ripening of dry fermented sausages (salami). The analytes were extracted from foods with perchloric acid and the extracts were purified by liquid–liquid partition using n-hexane. Determination of biogenic amines was performed through cation-exchange chromatography with isocratic elution and IPAD. The detection limits for the analytes under investigation were found to range from 1.25 to 2.50 ng, at a signal-to-noise ratio of 3:1. Average recoveries ranged from 85.5 to 97.4% and R.S.D. values ranged from 3.4 to 8.8. The proposed method offers a number of advantages over our previous IPAD method, such as the application to a larger number of analytes and matrices, a simpler extraction procedure and clean-up, isocratic elution using low acid and base concentrations, an improved chromatographic separation and a lower detection limit.

Effect of selected mould strains on proteolysis in dry fermented sausages

In order to contribute to the knowledge of the actual role played by moulds in the proteolytic process occurring during the ripening of dry fermented sausages, a total of six different species of Penicillium and Mucor genera, previously isolated from Spanish dry fermented sausages and selected because of their proteolytic activity against meat proteins, were inoculated on the surface of dry sausages (manufactured in a local factory) with natural and artificial casings. In all batches, water-soluble nitrogen, non-protein nitrogen, phosphotungstic-acid-soluble nitrogen, free amino acids and total volatile nitrogen were studied. The results indicated that the greater difference between control and inoculated batches corresponded to water-soluble and non-protein nitrogen fractions in those inoculated with Penicillium spp. The batch inoculated with Mucor showed an increase in low-molecular-weight-fractions. The proteolytic processes were more intense in batches prepared with natural casings due to the most intense colonization by moulds. It was concluded that the influence of moulds depends on the mould strain and on the type of casing used.

Development of mathematical model to describe the acidification occurring during the ripening of dry fermented sausage

To describe the evolution of pH values during the ripening process of fermented sausages, the use of a mathematical model is proposed. The model is both easy to use and statistically adequate. It can be employed to distinguish the different stages in the acidification process associated with the fermentation of sausages. It can be successfully used in the control of the manufacture of sausages when this is carried out in temperature-controlled chambers equipped with a microprocessor to continuously record pH values. The mathematical development of the function shows that the most rapid decline in pH occurs on the second day of ripening, coinciding with the highest levels of microbial development; on the 9th hour of the third day a stationary phase begins, which undergoes a slight increase from the 14th day. In order to better interpret the process, various parameters involved in the proton concentration of the sausage mixture were determined: water activity, fermentative microbial population ( Lactobacillaceae, Micrococcaceae) and metabolites derived from fermentative enzyme activity (lactic acid, acetic acid, total free fatty acids, total free amino acids).

Accelerated production of dry fermented sausage

The scope of this paper is to review work connected with accelerated ripening of dry fermented sausages by addition of proteolytic enzymes. An overview of the following topics is given: practical sausage experiments with addition of various proteinases of bacterial origin, including data from sensory, biochemical and gc/ms analyses; biochemical and genetic characterization of the enzyme shown to be most useful in these experiments, the serine proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151; experiments to transform starter cultures with the genes for production of this proteinase and proposals for future work in this field.

Potential technological interest of a Mucor strain to be used in dry fermented sausage production

The effect of several parameters on the proteolytic activity of a non-toxigenic Mucor strain previously isolated from Spanish dry fermented sausages was studied. For this, pH and NaCl concentration of a culture medium and temperature and incubation time were modified to reach values near to those used for ripening of dry fermented sausages. The optimum conditions for the development of proteolytic activity were a NaCl concentration of 30 g litre −1, pH of 5.5 and a temperature of 18 °C. The results, however, obtained at 14 °C (temperature of the final phase of ripening), indicated that the strain studied would be able to produce proteases at this temperature and, therefore, contribute to the proteolytic phenomena occurring during ripening of dry fermented sausages and to the development of the flavour and the odour characteristics of these products.

Effect of the addition of pancreatic lipase on the ripening of dry-fermented sausages — Part 1. Microbial, physico-chemical and lipolytic changes

The effect of the addition of nine different amounts (3–500 units) of pancreatic lipase on the microbial and physico-chemical parameters and lipid fractions during the ripening of dry fermented sausages has been studied. No differences between conventional and lipase-added sausages were found for pH, dry matter and water activity. The addition of lipase caused a greater accumulation of products resulting from the triglyceride breakdown, mainly diglycerides and free fatty acids (FFA). The maximum rate of lipolysis was observed during the first week of the ripening process, specially in the fermentation phase. The greater the pancreatic lipase added, the higher lipolysis observed. At the end of the ripening, the levels of total FFA were clearly higher (1·5 to 5-fold) in all lipase-added batches than in the controls. This fact gave rise to the accumulation of a great amount of FFA, which can contribute either by themselves to the flavour of the sausage or can be available as substrates for further transformations which may generate other flavour compounds.

Effect of the addition of pancreatic lipase on the ripening of dry-fermented sausages — Part 2. Free fatty acids, short-chain fatty acids, carbonyls and sensory quality

The effect of the addition of nine different amounts ( 3–500 units) of pancreatic lipase on the composition of free fatty acids (FFA), short-chain fatty acids and carbonyls, as well as the sensory quality, has been studied on dry fermented sausages. The lipase produced a greater release of all fatty acids in relation to the control sausages. The greater the pancreatic lipase content, the higher the release of these fatty acids, the most important ones being myristic, palmitoleic and oleic acids. A lower release of linoleic acid was observed, probably because of its oxidative degradation. At the end of the ripening in all lipase-added batches, a clear increase of the carbonyl content was noticed in relation to conventional sausages. No consistent changes were observed in the short-chain fatty acid fraction. In the sensory evaluation, the highest significant differences ( P < 0·01) between control sausages and lipase-added batches were observed when 60 and 90 lipase units were used. According to the chemical and sensory analyses, it can be concluded that the addition of 60 and 90 units to sausages seems to be useful to enhanced the flavour of these products.

Accelerated ripening of dry fermented sausage by addition of a Lactobacillus proteinase

Accelerated ripening of dry sausage was investigated by adding two different concentrations of proteinase isolated from Lactobacillus paracasei ssp. paracasei NCD0151 to sausage mixtures. Sausages with proteinase showed a greater pH decrease and a greater increase in lactic acid formation, water loss, protein degradation and peptide formation than control, no enzyme, sausages. Changes were most pronounced in the sausages containing 12 Ug‐1 proteinase. Addition of proteinase caused a specific increase in glutamate, serine and lysine content. Sensory evaluation after 14 days of ripening showed a significant increase in flavour intensity, maturity flavour, acidity, bitter taste and hardness in the proteinase‐containing sausages. Addition of proteinase induced changes earlier in the fermentation and ripening period than in the control, thereby indicating an acceleration of maturation.

Effect of the addition of pronase E on the proteolysis in dry fermented sausages

The effect of the addition of pronase E at two different concentrations on protein breakdown during the ripening of dry fermented sausage was studied. In all batches, water-soluble, non-protein and 5% phosphotungstic acid soluble nitrogens increased sharply during the first days of ripening, then became stabilized until the end of the process (26th day), and the total volatile nitrogen consistently increased during ripening. The greater the pronase E added the higher were the values reached for all these fractions. The changes in total free amino acids showed a similar pattern to that observed for the phosphotungstic acid soluble nitrogen. Histamine and tyramine progressively increased throughout the ripening. By sensory evaluation, no significant differences between the control batch and the batch with the lowest amount of added pronase E were found, but both batches were significantly different (P < 0·1) from the batch manufactured with the highest concentration of pronase E, which was classed as objectionable by the panellists because of its excessive softness.

Microorganisms and lipolysis in the ripening of dry fermented sausages

SummaryThe actual roles of two strains of microorganisms (Micrococcus‐12 and Lactobacillus‐56) isolated from Spanish dry sausages have been studied by using an aseptic method for making experimental sausages. The fermentation of sugars by lactobacilli is clearly demonstrated by the pH changes and the nitrate and nitrite reduction by micrococci is inferred by the changes in the sausage colour. The lipolysis occurring during ripening of experimental sausages seems to be mainly due to the lipases from meat rather than those elaborated by the organisms used.