Preliminary Assessment of Microbiological and Physicochemical Properties of Artisanal “Chouriço de Carne” Fermented Sausages, Manufactured in Northern Portugal †

: “Chouriço de carne” is a Portuguese ready-to-eat dry fermented sausage which relies on effective spontaneous fermentation to ensure its microbial safety and desired organoleptic properties. This study aimed to assess selected microbiological and physicochemical characteristics of artisanal chouriço produced by 14 different regional producers. Aerobic mesophilic bacteria, lactic acid bacteria, Staphylococcus aureus, Clostridium spp., Listeria spp. and Salmonella spp., in addition to pH, water activity and moisture, were evaluated for each of 70 samples. Principal Component Analysis of all these attributes was performed to build quality maps of the analyzed lots. The results showed great variability between sausages of different producers within the same geographic region, and S. aureus , Clostridium spp., Salmonella spp. and Listeria spp. were identiﬁed in sausages of several producers, highlighting the need for stricter microbiological control and standardization of production processes among artisanal producers of chouriço.


Introduction
"Chouriço de carne" is a Portuguese ready-to-eat dry fermented sausage, manufactured predominantly in the Trás-os-Montes and Alentejo regions, where it is a staple food in the daily diet of rural populations.Generally, this type of sausage is produced with roughly chopped pork meat and fat, seasoned/marinated with salt, wine, water, garlic, sweet and/or spicy paprika and bay leaf.This mixture is left to rest at low temperatures for up to 4 days; it is then filled in dry natural casings (pre-rehydrated) and hung to naturally ferment (without the aid of starter cultures) and dry by means of an intermittent smoking process for several weeks."Chouriço de carne" is still produced in an artisanal manner in certain regions of the interior of Northern Portugal and can present extensive nutritional, Biol.Life Sci.Forum 2023, 26, 56 2 of 6 organoleptic, physicochemical and microbiological diversity, depending on the raw materials and the production processes of each producer, as well their standardization and hygiene practices [1][2][3].
This study aimed (i) to assess the variability of selected microbiological and physicochemical attributes of chouriço manufactured by different artisanal producers representative of the northeastern Portugal region and (ii) to comprehend the associations between the studied properties through quality maps generated by principal component analysis.

Materials and Methods
Five samples of finished "chouriço de carne" from the same lot of production were purchased from 14 artisanal producers situated in the counties of Bragança, Carrazeda de Ansiães, Mirandela, Mogadouro, Miranda do Douro, Vila Flor, Vimioso and Vinhais, located in the Bragança district, northeastern Portugal.Samples were transported to and stored in refrigeration at the laboratory until analysis.Under aseptic conditions, the surface of the sausage was cleaned, the casing removed and its contents portioned for physicochemical and microbiological analysis.

Physicochemical Analysis
The physicochemical properties measured were pH, water activity (a w ) and moisture.For pH measurement, 10 g of sample was homogenized (Interscience Bag Mixer 400, France) in 90 mL of Buffered Peptone Water (611014 Liofilchem, Italy) and the homogenate's pH was measured in triplicate using a pH meter (FiveGo F2, Mettler-Toledo, Switzerland) equipped with an LE438-IP67 electrode.To measure a w , a portion of the ground sample was pressed to fit into a disposable cuvette into an Aqualab 4TE water activity meter (4TE Decagon, Pullman, WA, USA, sourced in Portugal) and the values recorded in duplicate.Moisture was determined according to ISO standards [4].

Statistical Analysis
To synthesize the information contained in the 10 physicochemical and microbiological parameters analyzed, and to understand their interrelationships, the data were submitted to Principal Component Analysis (PCA) using the R software (version 4.3.1,R Foundation for Statistical Computing, Austria).The analysis was conducted using the function prcomp() and the library factoextra, resulting in a varimax-rotated solution for three factors.Using the three-dimensional PCA, quality maps of the microbiological and physicochemical attributes of chouriço were generated by projecting the scores of the samples onto the length of the principal components, and the scores were grouped by producer (i.e., county origin).

Results and Discussion
The microbiological and physicochemical quality of "chouriço de carne" sausages produced in an artisanal manner in northeastern Portugal exhibited substantial variability, as observed from the producer-specific mean values for counts of total mesophiles (5.301-9.013log CFU/g), LAB counts (7.151-11.405log CFU/g), presumptive C. perfringens (<0.699-2.504log CFU/g), S. aureus counts (<1.699-4.000log CFU/g), pH (4.707-6.230),a w (0.7717-0.972) and moisture (11.86-50.23%).While Listeria spp. was never enumerated, it was detected from one of the fourteen sampled producers at an incidence of 1 (five positive samples out of five tested from producer "MirandaDoDouro") and Salmonella spp. was detected in one producer at an incidence of 0.40 (two positive samples out of five tested samples from producer "VilaFlor").
The contribution of the sausages' quality variables to the principal components can be assessed through their correlations to each of the extracted components (Table 1).Three components were extracted, which account for 63% of the variability observed in the 10 quality attributes studied.The first component (PC1), which explains 26% of the data variability, presents a highly positive correlation to a w (R = 0.87) and moisture (R = 0.84) and a highly negative association with pH (R = −0.85)(Table 1).For this reason, PC1 explains that slower drying is required for efficient fermentation.Moisture and water activity (a w ) are measurements related to the availability of water in foods, with the first pertaining to total water in a food, both in free and bound forms, while a w is an expression of water available in a food in its free form only.This free water is essential to bacterial biological functions, and a rapid reduction in its levels results in an abrupt drop in microbial growth.In fermented foods, this hinders the development of essential fermentative bacterial communities, contributing to a higher pH of the product and a rise in spoilage and pathogenic bacterial populations [12].In accordance with the quality maps depicted in Figure 1, the producers "Mirandela1" and "VilaFlor" apply slower/longer drying in their production process, while producers "Braganza2", "MirandaDoDouro", "Vimioso" and "Vinhais3" seem to employ a quicker drying period.
populations [12].In accordance with the quality maps depicted in Figure 1, the producers "Mirandela1" and "VilaFlor" apply slower/longer drying in their production process, while producers "Braganza2", "MirandaDoDouro", "Vimioso" and "Vinhais3" seem to employ a quicker drying period.As for the second component (PC2), explaining 24% of data variation, it is highly and positively correlated with LAB (isolated from MRS, R = 0.87; and from M17, R = 0.84) and mesophilic bacteria (R = 0.79) and characterizes the extent of fermentation or the length of production (Table 1).Longer fermentation/maturation times are necessary for a sustained increase in mesophilic lactic acid bacteria.In turn, greater LAB populations should lead to faster and stronger pH drop, which is required for microbial safety, and LAB metabolic by-products also contribute to flavor development in the finished product [12].The quality maps (Figure 1) show that sausages from the producers "MirandaDoDouro" and "VilaFlor" went through a longer or more efficient fermentation, while the producers "Mogadouro", "Carrazeda" and "Braganza2" employed a shorter fermentation period in the production of their sausages, or their fermentation was less successful.
The third component (PC3), although having a moderate weight (12% of the data variability), is positively associated with S. aureus (R = 0.66) and Clostridium spp.(R = 0.69), describing sausages of low microbiological safety (Table 1 and Figure 2).S. aureus and Clostridium spp.were detected in ten and six factories each and simultaneously in five ("Braganza2", "Mirandela2", "MirandaDoDouro", "Vinhais1" and "Vinhais2").Knowing that S. aureus is a normal commensal bacterium known to asymptomatically colonize the human skin and mucous membranes, and C. perfringens is found widely in environmental soil as well as in the intestinal tract of humans and animals, and adding to the fact that contamination of foods generally occurs due to poor hygiene of food handlers and/or high initial loadings in raw materials, it is clear to see a link between poor manufacturing practices and poor hygiene conditions [12].As for the second component (PC2), explaining 24% of data variation, it is highly and positively correlated with LAB (isolated from MRS, R = 0.87; and from M17, R = 0.84) and mesophilic bacteria (R = 0.79) and characterizes the extent of fermentation or the length of production (Table 1).Longer fermentation/maturation times are necessary for a sustained increase in mesophilic lactic acid bacteria.In turn, greater LAB populations should lead to faster and stronger pH drop, which is required for microbial safety, and LAB metabolic by-products also contribute to flavor development in the finished product [12].The quality maps (Figure 1) show that sausages from the producers "MirandaDoDouro" and "VilaFlor" went through a longer or more efficient fermentation, while the producers "Mogadouro", "Carrazeda" and "Braganza2" employed a shorter fermentation period in the production of their sausages, or their fermentation was less successful.
The third component (PC3), although having a moderate weight (12% of the data variability), is positively associated with S. aureus (R = 0.66) and Clostridium spp.(R = 0.69), describing sausages of low microbiological safety (Table 1 and Figure 2).S. aureus and Clostridium spp.were detected in ten and six factories each and simultaneously in five ("Braganza2", "Mirandela2", "MirandaDoDouro", "Vinhais1" and "Vinhais2").Knowing that S. aureus is a normal commensal bacterium known to asymptomatically colonize the human skin and mucous membranes, and C. perfringens is found widely in environmental soil as well as in the intestinal tract of humans and animals, and adding to the fact that contamination of foods generally occurs due to poor hygiene of food handlers and/or high initial loadings in raw materials, it is clear to see a link between poor manufacturing practices and poor hygiene conditions [12].
The occurrence of Salmonella spp. or Listeria spp. was not correlated with any of the three principal components as these variables had no significant loading.
The detection of such pathogenic agents in these meat products emphasizes the urgent need to reinforce food safety knowledge and practices among Portuguese artisanal producers.The occurrence of Salmonella spp. or Listeria spp. was not correlated with any of the three principal components as these variables had no significant loading.
The detection of such pathogenic agents in these meat products emphasizes the urgent need to reinforce food safety knowledge and practices among Portuguese artisanal producers.

Figure 1 .
Figure 1.Quality maps of the principal components one and two of the physicochemical and microbiological attributes of "chouriço de carne" sausage, showing projections of the individual samples from each of the fourteen producers and variable loadings.

Figure 1 .
Figure 1.Quality maps of the principal components one and two of the physicochemical and microbiological attributes of "chouriço de carne" sausage, showing projections of the individual samples from each of the fourteen producers and variable loadings.

Figure 2 .
Figure 2. Quality maps of the principal components one and three of the physicochemical and microbiological attributes of "chouriço de carne" sausage, showing projections of the individual samples from each of the fourteen producers and variable loadings.

Table 1 .
Correlation coefficients of the tested physicochemical and microbiological attributes of "Chouriço de carne" sausage, with the three Varimax-rotated factors (PC1, PC2 and PC3), alongside communalities and their explained variances.