Fermented sausages are a heterogeneous group of products differentiated in relation to raw material (type of lean meat and fat), the mincing degree of meat batter, ingredients (salt concentration, nitrate/nitrite, spices and herbs, other additives), size (diameter and weight), type of casing, starters, and ripening conditions (temperature, relative humidity, use of moulds, and/or smoke) [1
]. After casing, several modifications occur during fermentation and ripening, which bring to the acquisition of the organoleptic and textural properties desired for each type of sausage. These modifications are influenced by physico-chemical factors due to the action of salt on the formation of a gel structure and autoxidation reactions and biochemical activity due to the endogenous enzymes of the meat and microbial activities [3
]. The solubilization and gelification of myofibrillar proteins exert an essential role in the formation and development of fermented sausages’ structure. In detail, this process is widely influenced by the salt concentration within the meat batter and the decrease of pH following lactic fermentation [4
]. In this context, in fermented sausages, proteolysis is one of the main phenomena taking place during ripening [6
]. Meat proteins are the target of enzymes such as peptidases and proteases such as calpains and cathepsins (in particular cathepsin D), which catalyse the hydrolysis of the myofibrillar proteins first to polypeptides and, subsequently, to smaller peptides leading in the final step to the formation of free amino acids [7
]. Moreover, sausage microbiota actively contributes to protein hydrolysis and peptide formation especially during ripening. This proteolytic activity has been widely studied in staphylococci and moulds even though a role of lactic acid bacteria (LAB) has been demonstrated [9
]. Afterward, free amino acids and small oligopeptides can be further metabolized by microorganisms resulting in energy production (i.e., arginine metabolism), development of flavor compounds (for branched chain or aromatic amino acids) and accumulation of biogenic amines (BAs) [12
]. These latter compounds can be present in food, which causes several adverse reactions in the consumers. In fact, ingestion of food containing high amounts of biogenic amines is implicated in the headache, heart palpitations, vomiting, and diarrhea since histamine and tyramine are the most dangerous and are responsible for symptomatology known as “scombroid fish poisoning” and “cheese reaction,” respectively [15
]. The presence of BAs in food is dependent both on food contamination by decarboxylating microorganisms on the precursor availability (proteins and/or free amino acids) and on different intrinsic, environmental, and technological factors. In fermented sausages, the presence of high precursor concentrations and of decarboxylase positive non-starter microflora during the ripening period can favor a high accumulation of these compounds (tyramine and putrescine) [16
]. In these products, since BA reduction is often limited by the fermentation and ripening conditions, the main tool to counteract their accumulation is the choice of appropriate starter cultures able to rapidly and persistently colonize the meat batter and have the capacity to inhibit or reduce the growth of aminobiogenetic wild microorganisms (i.e., with bacteriocin producing strains) [15
Alongside proteolysis, lipolysis plays a fundamental role during ripening. Endogenous lipase and esterase produce free fatty acids, precursors for oxidative transformations bringing the formation of compounds (alkanes, alkenes, aldehydes, alcohols, ketones) with a strong impact on the aroma profile of sausages. In addition, several authors underlined a possible role of microbial lipases (and esterases) produced by molds and staphylococci [17
]. All these transformations contribute to the formation of the “typical” characteristics, which are associated to each sausage typology and concern physico-chemical, textural, and sensorial features as well as product safety [18
]. It has been reported that sausage diameter plays a relevant role in the formation of the desired characteristics. In fact, it affects many important events during ripening such as the removal of water and the presence of oxygen inside the sausage, which, in turn, influence the biochemical activities of this crucial period [19
]. Recently, some authors applied a linear discriminant analysis to highlight the role of diameter on the aroma profile and chemico-physical characteristics of different Italian industrial fermented sausages typologies [20
Among the main drivers of the complex phenomena, which take place during ripening, the use of starter cultures together with rigorous temperature and relative humidity conditions are the main tools adopted by the fermented sausage industry to improve the quality and safety of its products [22
]. In Italy, the use of selected starter cultures [consisting in lactic acid bacteria (LAB), staphylococci, and molds] is nowadays widespread in industrial products [23
]. However, in many cases, the introduction of this technological practice has not been accompanied by deeper studies of starter fermentation impact on the sausage characteristics and health features in relation to the type of process and product.
In this work, two different diameter Milano-type dry fermented sausages were industrially produced to evaluate the effects of fermentation performed by two different LAB starter cultures (Lactobacillus sakei
and Pediococcus pentosaceus
) on the presence of BAs during the production and the ripening. Moreover, lipolytic and proteolytic activities during fermentation, maturation, and in the final products were assessed to understand the role of LAB in ripening patterns. For the same production, microbiota evolution (both by culture-dependent and culture-independent methods), physico-chemical parameters and aroma profile were evaluated. The results have been recently published [25
Four batches of Milano-type dry fermented sausages were industrially produced with the aim to evaluate the effects of two different LAB starter cultures and diameter on BA content and sample ripening patterns during the production and at the end of maturation. Montanari et al. [25
] evaluated the same samples as far as physico-chemical, microbiological, and aroma characteristics during processing and in the final products. It was found that results were influenced by the diameter in the staphylococcal population and the formation of volatile organic compounds. Nevertheless, the same authors reported that the choice of L. sakei
or P. pentosaceus
as starter cultures had a direct effect on the fermentation and the acidification rate. In fact, the sausages inoculated with L. sakei
showed a slower pH decrease during the fermentation and small sausages fermented by L. sakei
presented significant higher pH values (p <
0.05) with respect to those inoculated with P. pentosaceus
starter cultures at the end of ripening.
BA analysis revealed that tyramine and putrescine were the most important ones, as reported for these kinds of products [16
]. The production of putrescine in food is often associated with the activity of Gram negative bacteria (pseudomonads and enterobacteria), but, in this case, the higher pseudomonads survival found in sausages inoculated with P. pentosaceus
] can only partially explain this diversity among samples. On the other hand, the production of putrescine by some Gram positive LAB has been demonstrated and these bacteria are considered as the main producers of BAs in fermented meat products [40
]. Putrescine can directly derive from the decarboxylation of ornithine, which, in turn, is obtained by the metabolism of arginine (via the enzyme arginase). Alternatively, it can be obtained through the activity of a specific deiminase acting on agmatine (AgDI), which derives from arginine decarboxylation. This latter pathway has been demonstrated in several LAB including in P. pentosaceus
and L. sakei
]. Since starters are selected on the basis of their lack of aminobiogenic potential, the high putrescine content in a large sausage inoculated with P. pentosaceus
could be ascribed to the presence of some indigenous L. sakei
, which has been previously highlighted by Montanari et al. [25
]. These wild strains could produce putrescine from agmatine through the AgDI pathway, which produces ammonia toward acid stress due to the strong pediococci fermentation.
The tyramine presence in dry fermented sausages has been mostly related to the tyrosine decarboxylase activity of LAB [16
]. Van Ba et al. [44
] compared five different commercial starter cultures containing different LAB strains and their effects on the quality characteristics and BA contents in sausages. In agreement with our findings, these authors found that starter containing P. pentosaceus
led to a higher sausage tyramine content in comparison with L. sakei
in which the latter starter was more suitable for the production of high-quality products with lowered BA concentration.
BA content in samples differed in relation to the diameter as it has already been observed by several authors. Miguélez-Arrizado et al. [45
] demonstrated that the tyramine accumulation in Spanish sausages was higher in salchichon (diameter 5–12 cm) rather than in the fuet (diameter < 3 cm) and tyramine was mainly accumulated in the central part of sausages. Similar results were reported by Komprda et al. [46
] in typical Czech sausages and by Latorre-Moratalla [47
] in fuet and llonganissa. In Italian salami, a similar trend was observed by Anastasio et al. [19
] and, recently, Tabanelli et al. [21
] found that BA amounts could be related to the size of sausages with the larger diameter including a higher concentration. The lower BA level in small size sausages can be attributed to the shorter ripening time and, consequently, to the time needed to reach aw
values able to inhibit the metabolism of decarboxylating microorganisms and/or the action of decarboxylases. Moreover, it has been demonstrated that the ability to produce BAs is negatively influenced by increasing concentration of NaCl [48
]. In addition, the lower small size product acidity (which reduces the role of pH cell protective mechanism of decarboxylases) can limit BA accumulation. Some authors found higher tyramine production by Lactococcus lactis
under anaerobic conditions [51
]. This could be a supplementary reason for explaining the higher BA content in the sausages with a larger diameter, which caused a lower oxygen diffusion inside the product.
In the present study, the electrophoretic profiles of the myofibrillar proteins were investigated. On the other hand, being the main target for endogenous muscle peptidases [52
], the sarcoplasmic protein fraction was not considered within this study.
Overall, the relative abundance of contractile (myosin and actin) and cytoskeletal proteins as well as of those polypeptide chains composing the tropomyosin complex profoundly changed during fermentation and ripening. In detail, the significant reduction at the end of ripening in the relative intensities of the bands ascribed to the main myofibrillar proteins (MHC, desmin, actin, tropomyosin, and troponin C) agreed with previous studies performed on dry sausages. A relevant decrease and even a complete degradation of MHC was observed during ripening [11
]. In particular, since MHC had two regions target different proteases, the enzymatic hydrolysis of this protein led to several breakdown products with a wide range of molecular weight (50–150 kDa). In this context, MHC degradation was associated with a simultaneous appearance of a degradation product that has a molecular weight of 110 kDa. Similarly, the broad degradation of actin (42 kDa) agreed with previous investigations carried out to assess proteolysis during the processing of beef, pork, and horse-made fermented sausages [53
]. Actin breakdown during ripening was widely demonstrated by electrophoresis [29
] as well as more deeply investigated, resulting in the identification of its peptides by proteomic analysis through LC-MSE [7
]. After ripening, the increased staining intensities of the electrophoretic bands ascribed to troponin T, troponin C, and tropomyosin might be the result of the co-migration of peptides derived from MHC and other high-molecular weight proteins [11
Aside from the starter cultures, a diameter-effect on the amount of troponin C and proteolytic fragments was observed in salami inoculated with L. sakei
. This might be partially attributed to the different pH increase observed during ripening, which is mainly attributable to the faster respiration of lactic acid by molds. In fact, mold hyphae
requires a higher time in large size sausages to penetrate inside the product due to the reduced oxygen availability within the mass [25
]. The presence of two distinctive fragments (with molecular weight of 95.8 and 36.1 kDa) identified in salami inoculated with L. sakei
might be attributed to the exopeptidases activity and to the wide set of intracellular peptidases (such as endopeptidases, aminopeptidases, dipeptidases, and tripeptidases) found in L. sakei.
These enzymes can be released as a consequence of cell lysis during fermentation, which leads to the formation of several polypeptides [52
]. In the same time, the distinctive electrophoretic bands of 191, 174, and 150 kDa and the faster degradation of the polypeptide chains observed in salami inoculated with P. pentosaceus
likely resulted from hydrolysis of high-molecular weight proteins such as MHC, nebulin, vinculin, and titin. In addition, the accumulation of a 173 kDa degradation product was previously observed after nine days of processing in dry-fermented sausages [57
Lipids are the major fraction of fermented sausages and they play a key role in their nutritional and sensory quality. Thus, the changes that occur to lipids due to lipolytic and oxidative phenomena during the ripening process were studied for these samples.
Diacylglycerols (DAGs) are a useful parameter to evaluate the extent of lipolysis and they are considered a more reliable parameter with respect to free fatty acids, which can easily react with other analytes of the system [58
]. The increase in D32, D34, and D36 series during ripening in all samples could be due to the fact that pork fat is mainly constituted by fatty acids (FAs) with 16 and 18 carbon atoms [59
] such as oleic (C18:1c9, ~50% of the total FAs), palmitic (C16:0, ~23%), stearic (C18:0, ~13%), and linoleic (C18:2, ~11%) acids (data not shown). The increase of the total DAG content and the decrease of MUFA class at the end of maturation in the sausages inoculated with L. sakei
could be related. Therefore, the starter culture with L. sakei
seems to have a higher effect on lipolysis in fermented sausages than P. pentosaceus
and these results reveal that the principal FAs losses for lipolysis were MUFA (oleic acid, as principal one), which was already evaluated in previous investigations [60
Lipid oxidation was also analyzed in order to better describe the lipidic evolution in dry fermented sausages and evaluate their final quality. Peroxide index (PV) and TBARS values were respectively used as indices of primary and secondary products of oxidation and they were relatively high in all samples. Other studies already reported high values for oxidative parameters in fermented sausages [61
], which might be because of the product manufacture: grinding and mixing of the meat increase the surface exposed to oxygen and oxidation catalysts [63
]. However, these peroxide values were lower than 25 meq of O2
/kg of fat, which is the limit of acceptability for fatty foods and did not show differences (p <
0.05) among the samples.
Moreover, this TBARS behavior was already observed in other research studies [61
] because the peroxides, which are the primary oxidative products, degrade in malonaldehyde (MDA) and other reactive compounds. MDA reacts with amino acids, sugars, and nitrite during ripening and storage, which brings a decrease in TBARS.