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Article

Evaluation of the Nutritional Quality and Shelf Life of Fermented Processed Sheep Salami Inoculated with Lactobacillus casei and Lactobacillus paracasei

by
Natália Martins dos Santos do Vale
1,
Michelle Rayssa Pereira de Melo
1,
Adelmo Cavalcante Pascoal Filho
2 and
Jenyffer Medeiros Campos Guerra
2,*
1
Universidade Federal Rural de Pernambuco, Rua Dom Manuel de Medeiros, s/n-Dois Irmãos, Recife 52171-900, PE, Brazil
2
Departamento de Engenharia Química, Universidade Federal de Pernambuco, 1235-Cidade Universitária, Av. Prof. Moraes Rego, Recife 50670-901, PE, Brazil
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(13), 5509; https://doi.org/10.3390/app14135509
Submission received: 29 May 2024 / Revised: 19 June 2024 / Accepted: 20 June 2024 / Published: 25 June 2024
(This article belongs to the Section Applied Microbiology)
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Abstract

:
The aim of this study was to produce two fermented processed foods made from sheep meat, one of them inoculated with the probiotic Lactobacillus casei and the other with Lactobacillus paracasei, evaluating their microbial growth, viability, and the efficiency of these microorganisms as bioconservants. To that effect, physicochemical, physical, and microbiological analyses were carried out, as well as a count of lactic acid bacteria (LAB). The results regarding the physicochemical and physical characteristics were in accordance with Brazilian legislation, except for the humidity and carbohydrate analysis and water activity, the values of which were not in accordance with the legislation. However, the microbiological results regarding the pathogenic microorganisms’ growth were within the standard established in the legislation until the end of the time of analysis. Regarding the presence of probiotics, both fermented processed foods had values higher than 107 CFU/g relative to LAB count until the limit of 84 days. From these results, it can be concluded that the probiotics are viable, showing bioconservative activity and proper consumption conditions, since they are in conformity with the legislation.

1. Introduction

It is known that changes in the lifestyle and eating habits of the population have been improving because of the search for health and the increase in life expectancy. Currently, one of the priorities of the world’s population in the maintenance of quality of life is directly associated with the type of food every human being consumes. Healthy food is characterized as something that can meet all of the body’s requirements, including the presence of nutrients and the lack of excesses that cause long-term harm [1]. This factor has contributed to the search for alternatives for the consumption and production of healthier foods that can improve wellbeing and benefit health. In this context, the products that are known as functional foods are found and have been assuming a big space in the life of consumers [2].
The functional food market is composed of products that positively influence intestinal health. This type of food shows many positive activities when it comes to health, including its potential to stimulate the immune system, reduce the risk of cardiovascular problems, osteoporosis, obesity, and some types of cancer, as well as improve memory and physical condition [3]. In this context, compounds with added probiotic cultures that can turn food functional by being inoculated in the formulations during processing are included, such as lactic acid bacteria (LAB) [4,5].
There are many genres of bacteria with evidence of being probiotic. For example, Lactobacillus and Bifidobacteria are associated with a lower risk of developing food allergies, such as to gluten and other types of proteins [6]. These lactic acid bacteria contribute to the prevention of infections, in addition to having anticarcinogenic effects [7]. They also improve digestion and nutrient absorption by the consumer’s intestinal cells and act in bioconservation, which shows that they highly influence food [8].
The meat industry has been interested in the development of products in this field, seeking higher competitiveness in the market. Sheep meat and its derivatives constitute a group of food with high nutritional value, given that they are rich in protein and contain a number of vitamins and minerals. Moreover, they show many benefits for the human body, including strengthening the immune system, which is directly linked to the reduction in the occurrence of diseases such as cancer and anemia [9,10].
One of the most commonly consumed meat products are salami-type fermented sausages, which display optimal characteristics for the multiplication of lactic acid bacteria. LAB can be inoculated during the formulation, aiming to turn the product into a type of functional food, increasing the nutritional quality of the meat used in its production [11].
Some commonly known conservation methods, such as using fermentation during processing, are characterized by many biochemical, biophysical, and microbiological changes [12]. The practice of making salami-type fermented sausages uses fermentation as a basic conservation principle; however, combined methods of preservation can be used, such as probiotic inoculation, allowing the achievement of a high-quality and stable final product [13]. Furthermore, the use of probiotics makes it possible to reduce the use of curing salts (nitrites and nitrates), which decreases the possibility of nitrosamine (potentially carcinogenic substance) formation, a potent carcinogenic substance that causes damage to DNA, base mutations, and induces cancer, mainly the colorectal type [14,15].
Therefore, there is an increasing need to understand the incorporation of these bacteria in food products and their effect throughout their growth. The field of predictive microbiology is one of the main tools responsible for measuring food safety and it studies the microbial growth in food through environmental conditions and their storage conditions or functional capacity [16].
In view of the above, the aim of this study was to make fermented processed sheep salami and to evaluate the inoculation and development of Lactobacillus casei and Lactobacillus paracasei in regard to its nutritional quality, probiotic strain viability, physical, physicochemical and microbiological characteristics, as well as its shelf life.

2. Materials and Methods

The experiment was carried out through to the following steps: preparation of the sheep salami; physical, physicochemical, and microbiological analyses; shelf life analysis; and evaluation of microbial growth through bacteria count. These steps were executed in the Animal Products Technology Laboratory (Meat) of the Chemical Engineering Department (DEQ) of the Federal University of Pernambuco (UFPE) (Latitude: 8°03′03.46970″ S; Longitude: 34°57′05.45910″ W; Altitude: 4.217 m).

2.1. Production of the Salami-Type Fermented Sausages

Three formulations of fermented processed sheep meat were made and named S1, S2, and S3, which correspond to the following: fermented processed sheep meat with curing salts, fermented processed sheep meat with the addition of Lactobacillus casei, and fermented processed sheep meat with the addition of Lactobacillus paracasei, respectively. The first refers to the standard product used as reference for the results, having curing salts added to maintain shelf life, while the second and third ones refer to the products of the study. They were used to understand the growth curve with viable cells from both probiotic strains throughout the production process until the final product, as well as which of them will maintain no pathogenic growth until the end of the product’s 84-day shelf life.
The lamb shank samples were purchased from the local market in the city of Recife, PE. The company Frigomalta in the city of Igarassu, PE provided the bacon samples. The formulations of the fermented processed food that was produced are described in Table 1, evidencing, for each assay, the Lactobacillus strains that was used.
After the ingredients, a starter culture (Staphylococcus xylosus and Pediococcus pentosaceus) and probiotic strains (L. casei LC03 and L. paracasei LCP00, both with 1011 viable cell), were mixed in a cutter (MBI-98P model Becker, Santa Catarina, Brazil), the salami were processed with the help of a mechanical stuffing machine (CAF Machines, São Paulo, Brazil) in cellulose casings, tied, and taken to the Biochemical Oxygen Demand (B.O.D.) incubator for fermentation for seven days and maturation for twenty-one days. The humidity and temperature were controlled in different ways according to the maturation process for twenty-eight days.
At the end of the maturation process, the processed fermented foods were submitted to storage in a cold chamber ranging from 4 to 7 °C and analyzed every 14 days until their expiration date of 84 days. During that time, the microbiological analyses established in the legislation were carried out and the growth of Lactobacillus casei and Lactobacillus paracasei was evaluated. Also, physical and physicochemical analyses were executed at 0 days after the maturation process, counting from the product’s shelf life. The processing of the fermented processed meat was according to the following flowchart (Figure 1):

2.2. Microbiological Analysis

Microbiological analyses were carried out according to the methodology described in the American Public Health Association (APHA). In accordance with the resolution RDC no. 60 from the National Health Surveillance Agency (ANVISA) [17], which makes the Official Analytical Methods for Microbiological Analyses for Control of Animal Origin Products official, the fermented processed sheep meat formulations were submitted to microbiological analyses of the following microorganisms: Staphylococcus aureus, Salmonella sp., Clostridium perfringens, and Eschericha coli [18].

2.3. Physical and Physicochemical Analyses

The physicochemical analyses carried out were the following: pH, titratable acidity, humidity, lipids, proteins, ash, carbohydrate, caloric value, and water activity; the physical analyses carried out were the following: texturometer and colorimetry. All the analyses were performed according to the official analytical procedures for animal origin products according to the normative instruction no. 22 [19]. The same analyses were performed after the production of the fermented processed meat, inoculation of the probiotics, and the fermentation and maturation period, some of these analyses being conducted at 0 days after maturation and some at 84 days, the expiration date.

2.4. Statistical Analyses

The physicochemical and microbiological analyses and the probiotic acid lactic bacteria’s growth evaluation were carried out and based on those results, it was necessary to perform statistical analyses that expressed the mean and standard deviation, using the analysis of variance (ANOVA) and the Tukey comparison test to determine significant differences between the means, with a level of significance of p < 0.05. The analyses were executed using the software, Minitab Statistical (https://osbsoftware.com.br/produto/minitab-statistical-software) [20].

2.5. Lactic Acid Bacteria Count

Before inoculation in the salami-type fermented sausages, the probiotic strains were slowly defrosted through the “overnight” process in a refrigerator at temperatures between 4 and 8 °C. After that, the pure lyophilized L. casei and L. paracasei strains each weighed 0.30 g and were inoculated in 300 mL of MRS broth for each strain, in order to pre-activate them. The same samples were incubated for approximately 24 h in a B.O.D. incubator at 36 ± 1 °C. After the Lactobacillus pre-activation, the MRS broth from each strain was placed in Falcon tubes, which were refrigerated and centrifuged. The supernatant was discarded, and the precipitate was added to the mass of processed food after the addition of the other ingredients and the starter culture, which also needed to be activated in water with 2% sucrose added for 30 min.
In parallel to the production of the processed meat, 1 mL of the total volume of the precipitate of each culture was taken out and used for the count of the number of viable initial cells from the inoculates in petri dishes with MRS agar. They were incubated for approximately 48 h in a B.O.D. incubator at 36 ± 1 °C in order to identify the efficiency of the strains at the moment of inoculation. With that, the L. casei cell concentration obtained after centrifugation and added to the S2 Formulation was of 7.5 × 107 CFU. mL−1; whereas the L. paracasei one, added to the S3 Formulation, was of 7.5 × 107 CFU. mL−1.
The control for the probiotics’ growth was achieved from the total count of the viable lactic acid bacteria in petri dishes, using the pour plate technique and an overcoat in an MRS agar medium of 1 mL aliquots from each of the ten dilutions of saline solutions prepared. After the solidification of the medium at 18 °C, the plates were incubated in anaerobic conditions in the B.O.D. incubator at 36 °C ± 1 °C for 72 h and the colonies were expressed in CFU/g. Finally, with the goal of analyzing the probiotics’ growth, the bacteria count was carried out at a 14-day interval, for a total period of 84 days for the S2 and S3 assays, which contained probiotics in their formulations.

3. Results

3.1. pH Values of the Salami-Type Fermented Sausages

During the production of the fermented processed meat, the initial counts for the inoculation of Lactobacillus casei and Lactobacillus paracasei, as well as the sample’s pH, were followed. The early pH analysis was carried out in quintuplicate, and the initial Lactobacillus count was performed before they were inoculated in the S2 and S3 salami assays, a number of viable cells of 107 CFU/mL and 106 CFU/mL were introduced in the formulation, for L. paracasei and L. casei, respectively. The pH is described in Table 2.

3.2. Microbiological Analysis of the Salami-Type Fermented Sausages

The analyses were carried out 0 days after maturation, early in the product’s shelf life, as well as at 84 days, the expiration date These samples showed results according to what was expressed in RDC no. 60 [17], as described in Table 3.
In the microbiological assays that were performed, the presence of Escherichia coli, coagulase-positive Staphylococci and Salmonella was not detected in any of the samples during the evaluation period, which showed that there were no microbial contaminations during all of the product’s shelf life. That perception is related to the product’s pH showing few alterations, with results between 5.0 and 5.3, showing that it is possible to avoid the food deterioration by controlling these factors to enhance its quality and not bring a risk to the consumer’s health [21].
In view of the above, when the starter cultures are added to the fermented processed food, they have the role of inhibiting pathogenic microorganisms and increasing shelf life, because of the reduction in pH, which is why they are named protectors. However, when the probiotic cultures are inoculated as well, in addition to contributing to the nutritional and microbiological quality of the product, they show the capacity of helping in the promotion of consumer health, as well as influencing the inhibition of the pathogenic microorganisms during all of the fermented processed food storage time, guaranteeing safer food for consumption [22].

3.3. Physical and Physicochemical Analyses of the Salami-Type Fermented Sausages

The results obtained from the physical and physicochemical characterization of the fermented salami-type sheep sausages are described in Table 4. They were compared with the values determined by the legislation IN no. 22 [19].
According to Table 4, it is possible to observe that the lipid, protein, carbohydrate, and ash content were not significantly different in the three assays. The lipid and protein amounts were within the limits established by Brazilian law. Similar results were obtained in the study using probiotic sausages [23]. It is worth mentioning that the protein percentage in all the assays was superior to the minimum established by the legislation, showing values higher than 30%. Concerning humidity, the samples inoculated with probiotics did not show significant levels, but they were significantly higher than those of standard salami.

3.4. Humidity and Water Activity Analyses

The results of the humidity and water activity analyses in these periods of time are shown in Table 5.
In evaluating the results of the humidity level on day 0, it was observed that none of the results stayed up to the minimal standards required by legislation [19].

3.5. pH Analysis throughout Shelf Life

The results of pH analyses are demonstrated in Figure 2 and Table 6.
Ideally, the final pH of processed meat should vary between 4.6 and 5.5, which may depend on a number of factors, for example, the speed of acidification in starter cultures and the presence of yeast. Higher pH values can indicate that the fermentation process was not effective in the production of lactic acid and are responsible for forming peptides, amino acids and nitrogen compounds [24]. In view of that, checking the pH in processed meats is a process executed in order to identify one of their quality criteria. For that reason, when the pH values are under 6.2, meat products become more protected against the action of unwanted microorganisms [9].

3.6. Lactic Acid Bacteria Growth Analyses throughout Shelf Life

The results obtained from laboratory analyses of the lactic acid bacteria present in assays S2 and S3 of the salami-type fermented sausages containing probiotics are expressed in Table 7.
It was observed from Table 7 that the S2 and S3 assays had viable and active lactic acid bacteria counts during all 84 days of the shelf life of these products, showing values higher than 107 CFU/g. According to the National Health Surveillance Agency (ANVISA), values must be between 107 and 109 CFU/g, which correspond to the minimum amount of active and viable probiotic microorganisms that have to be present for the product to be classified as functional and to guarantee that the strains effectively promote their benefits.

3.7. Correlation Analysis between the Physical and Growth Parameters of Lactobacillus

The results obtained in the linear correlations between pH, water activity, texture, and acidity analyses with Lactobacillus casei and Lactobacillus paracasei growth are described in Table 8.
R values refer to linear regression, where the closest the result is to 1, the higher the linear interaction between the factors and their correlation. However, it is important to examine p values for a more efficient analysis. They represent the Pearson coefficient, showing that values < 0.05 indicate there was linear interaction between the accessed parameters and the probiotics’ growth. F values refer to the variation between sample means/variation within samples.

4. Discussion

4.1. Processing of the Salami-Type Fermented Sausages

pH values before fermentation remained close, not showing any significant difference. After maturation, an increase in pH values was observed. Those values are in line with the ones shown in a study using sausages inoculated with Lactobacillus plantarum BG 112 [22]. In this stage, the culture starter plays an important role, since it is responsible for fermenting the sources of carbohydrates in the medium. In the case of the formulations in this research, sucrose was used, releasing lactic acid and reducing pH in the medium in the first days. In addition to that, metabolites that help in the development of technological and sensory characteristics and the microbial quality of the product can be produced [23,25].
In the formulations from the S2 and S3 assays, pH values had an even greater increase when compared with the standard assay S1, which only had the starter culture, while the others had Lactobacillus in them. The pH values during the first days of fermentation and maturation guarantee the quality of the salami because of the inhibition of unwanted microorganisms, like E. coli and S. aureus [1].
At the end of maturation, the acidification kept hitting pH levels between 5.0 and 5.3, which coincides with the isoelectric point of meat proteins, causing a faster water release in the product, bringing on a reduction in the capacity of the meat to retain water and facilitating the drying process of the final product [22,26].
Finally, in the storage process a change in the product occurs, which depends mostly on the initial acidity of the product, considering that the pH itself will take a fall in the fermentation and maturation stages. Those changes also depend on storage temperature as well as on the acidifying power of the starter culture and on the cooling process of the product, which is responsible for stopping fermentative activity in inappropriate cases [25].

4.2. Microbiological Analyses

When the coagulase-positive Staphylococci is present in food, it is identified as an indicator of contamination from the manipulator’s skin, mouth, and nasal cavities. In view of that, according to the three assays showing results below what is required in current legislation, it is shown that the manufacture process for these fermented processed meats was carried out according to Good Practices of Manufacturing and Manipulation, maintaining the manipulator’s hygiene, cleanliness, and care.
As an indicator of the sanitary hygienic processing conditions of these fermented processed foods and their pathogenic microbial growth, the Escherichia coli count was calculated, showing results below current legislation, which indicates that the presence of probiotic strains influences the inhibition of this pathogen, resulting in an integrate final product with guaranteed quality and safety for consumption [27].
Regarding Salmonella spp., all of the samples analyzed showed its absence in 25 g, an indicator that there was no contamination. That bacteria is responsible for a potential source of foodborne outbreaks in humans (known as salmonellosis) when present in food, which shows the importance of proper processing and storage [23].

4.3. Physical and Physicochemical Analyses

As described in Table 5, the macronutrient contents were not divergent in the three assays. Out of these nutrients, the lipid content can vary in sheep meat according to some factors, like race, feeding, age, and sex. The main changes in the lipid fraction result from the hydrolysis of triglycerides by lipases, releasing free fatty acids, which are responsible for a contribution in flavor, texture, and appearance of the products [22,28]. Moreover, the meat fat has buffer characteristics, since it protects the probiotic microorganisms in salami from the adverse conditions of the gastrointestinal tract, and can determine shelf life because of the lipid oxidation control if the storage is adequate [22].
Protein values are assigned to sheep meat in natura, since it has a higher protein content when compared to beef or pork. Furthermore, during the production of salami, other ingredients are added, such as bacon, which also contains a protein percentage in its composition. Proteins are the main functional and structural components in processed meats, which are turned into peptides and amino acids when they are degraded during salami processing, which causes beneficial changes, contributing to the formation of the characteristic flavor of the final product [22].
Regarding the amount of carbohydrates, the results obtained did not fall within the limits defined by legislation. That accumulation is mainly due to the addition of sucrose and the inoculation of Lactobacillus, since that is an important parameter due to their transformation into lactic acid by the action of bacteria. Moreover, it also helps guarantee safety and quality of the final products since it has a protective effect against a number of unwanted microorganisms [25]. Similar results to the ones found in this study are shown in fermented processed foods that also contain probiotic strains, which had values between 6.70% and 7.19%. These values, when compared to processed meats that did not have these strains added to them, had values between 4.81% and 6.26%, showing that the presence of probiotics can increase the amount of carbohydrates in the product after maturation [29].
Concerning the ashes, although legislation did not have a standard value for those analyses, the results were compared with the Food Composition Table [30], showing a standard value of at least 5.90%, which matches with the values found in the present study. Like carbohydrates, for ash content, the ingredients added in the salami were also investigated, showing the importance of the impact that these substances bring to the value of ashes obtained in the final product. Other studies that used probiotic strains in the composition of processed meats have average values between 5.48% and 7.49% of ashes, higher than the ones in this work [23,29].
The titratable acidity has the main function of granting aroma and flavor, as well as maintaining the acid feature in the final product. This parameter does not have values established in the legislation; however, it is evident by the three assays performed that the Lactobacillus casei inoculate has higher acidity levels, which indicates a greater production of lactic acid, potentiated by the probiotic during fermentation and maturation. In contrast, the results obtained in the S2 assay (containing Lactobacillus casei) had the lowest acidity levels, showing that these bacteria did not help the starter cultures in the production of lactic acid in the final product. That being said, it is understood that the Lactobacillus casei strain has a higher capacity to develop and multiply in a meat medium.
The shear force is evaluated to measure the texture of salami, an important parameter for the sensory evaluation of the product. After the maturation process, salami tend to have a greater shear force because of the drying process and biochemical reaction that occurs during this stage. The drop in humidity can interfere negatively with the texture of the salami, implanting in a greater proteolysis and consequently a greater softness in the final product, an unwanted characteristic for salami, which means that the higher force applied, the harder the texture of the product will be, which leads to less humidity and present water activity (Aw) [26,31]. The results obtained did not have significant difference between them, as well as the S2 and S3 assays, that had similar results to those of lamb meat for shear force between 3.56 kgf and 5.38 kgf [32].
The determination of color is conducted with the goal of assessing the color changes in salami after the treatments at the end of the storage time. The analysis was carried out at the end of maturation, by measuring the samples directly, in which “L*” stands for brightness, “a*” for the red color index, and “b*” for the yellow color index. The more water present in the product, the greater luminosity it has, because the loss of water can cause its darkening. Because of that, the values found for L. casei show less luminosity and yellowish color when compared to the other two assays [26]. The “b*” values refer to the consumption of oxygen by lactic acid bacteria during the exponential phase, which in a study of salami added to rice had results between 5.45 and 14.96, meaning improved visual characteristics for the fermented processed food [9,23]. The “a*” values were according to what was reported in another study regarding processed meat that had results between 10.62 and 12.09. This parameter is directly related to the curing and maturing of the salami, because during those processes, myoglobin interacts with nitric oxide, producing nitrosomyoglobin, a typical component of curated meat responsible for its redness [23].

4.4. Humidity and Water Activity Analyses of the Salami-Type Fermented Sausages

The values for S2 and S3 treatment containing Lactobacillus kept the humidity percentage even higher, matching the results obtained in studies with salami containing probiotic strains, being present in between 32 and 35% of the finished product [29]. Therefore, it was understood that the main factors that influence humidity levels are the following: the amount of humidity in meat in natura above 70% which impacts derived products, the control of the physical parameters in the maturation and conservation process, and the presence of Lactobacillus.
After 84 days of storage, it was possible to find humidity results showing that only assays 2 and 3 (containing probiotics in their composition) exhibited values lower than the maximum established in the legislation (40%), and close to the ones found in TACO (34.7%) [23,30]. In view of the above, it was shown that humidity values were lower in assays S2 and S3 and higher in assay S1 during storage, which indicates that the presence of probiotic strains diminishes the absorption of relative humidity, since the conservation medium was made properly, with vacuum packaging under refrigeration between 5 and 10 °C. Some studies with different varieties of microbial cultures in the composition of fermented processed sheep meat had humidity values between 35.85 and 46.62% during storage, which agrees with what was found in this research for assays S1 and S2 during 0 and 84 days [22].
Regarding water activity, values on day 0 are above the maximum according to legislation, which agrees with the humidity results found, since the higher the humidity, the higher the free water content, which is shown by water activity levels being high, which also makes the medium more susceptible to microbial growth [11]. Those values must be kept up to 0.92, since that indicates product stability and the end of processing, guaranteeing the product’s quality until its expiration date.
Roselino et al. (2018) also evaluated salami inoculated with Lactobacillus and the results obtained were higher than the standard described in the legislation, which was between 0.93 and 0.97, showing coherence in the results found in the present study and indicating that the presence of probiotic strains can influence an increase in the final product’s water activity. Many factors can affect high water activity levels, like the relation between the decrease in pH, the high humidity, the presence of probiotic strains, and maturation and storage conditions [22,33].
After 84 days of storage, the water activity set in the product changed, making the results from assays S2 and S3 fit the standards required by law (maximum of 0.92). Furthermore, all the assays showed a reduction in water activity during the maturation process when stored in proper conditions, which happened mostly because of the dehydration of salami, which showed similar results to the ones from a study with ripened Italian salami [34]. That result promotes a higher quality and safety guarantee, demonstrating that over time the presence of Lactobacillus reduces the presence of available water in the medium where the pathogenic microorganisms could develop [11].

4.5. pH Analysis over Shelf Life

After the fermentation and maturation of the processed meats, the pH tends to drop because of the production of lactic acid by starter cultures inoculated with probiotics that help that decrease, especially in the first days post-maturation [23].
The results of day 0 between S2 and S3 did not show significant difference; however, both were different from S1. It was noticeable over time that assay S2 showed the lower statistic variation when it came to pH values.
The results referring to the first 28 days of storage are coherent with the values obtained in the acidity analysis. The higher acidity value was found in assay S2, according to the lowest pH value found in the same period. Similar pH values (between 5.36 and 5.5 in the first days of storage) were found in a study with salami inoculated with L. plantarum [23]. That pH follow-up is extremely important to assess product stability, since its decrease can provoke water loss and a reduction in the meat’s water holding capacity, which makes the product dry easier and reduces water activity levels [35].
pH values were compared at the end of the 84-day storage time, the end of the maturation period, and a significant difference in the three assays was observed. Formulations from assays S1 and S3 had the highest values, which gradually increased over time because of the decay of lactic acid production since the first month of storage. That indicates that Lactobacillus paracasei (present in assay S3) did not help fermentative activity in an efficient way for over 28 days. The same thing happened in another study using fermented processed meat which had their pH increasing over the storage period of 120 days [29]. The increase in pH can be assigned to the appearance of basic compounds from the degradation of proteins [36].

4.6. Lactic Acid Bacteria Growth Analyses over Shelf Life

Before maturation, after the filling process, the salami had a count higher than 107 CFU/g which allowed them to be considered probiotics and continue the fermentation and maturation processes. After those stages, the growth of Lactobacillus casei increased considerably, results similar to those found in another study with L. casei-inoculated salami [36], and that of Lactobacillus paracasei diminished, indicating that it did not multiply significantly during fermentation; however, some viable cells were still found.
Over their growth time, both strains had noticeable growth from the bacterial count, showing that storage and the absence of curing salts did not influence the evolution of these Lactobacillus negatively. The decay of these bacteria started on day 56 of storage, although viable strains were maintained above 107 CFU/g even after that time, which reveals that the product is viable for 84 days, guaranteeing a functional probiotic product during its shelf life.
The viable cell count in the probiotics with L. casei and L. paracasei increased gradually post-maturation and after the first 14 days, exhibiting their activity over that time in viable quantities. In the first 14 days, the samples showed a count of 107 and 1010 CFU/g for L. casei and L. paracasei, respectively, during the lag phase, when bacteria are still adapting to the new environment and their growth is mostly affected by pH and acidity over the processed food’s shelf life.
In days 28 through 42 the greatest population growths were observed for both the processed meats, reaching a count in order of 1015 and 1013 CFU/g for L. casei and L. paracasei, respectively, which happened because of the increase in present cells during the lag or exponential phase, when the strains have greater growth in the medium. However, a reduction in that was observed in the samples after 70 days of storage, getting to the stabilization and decline phase with 1.82 × 107 and 6.8 × 107 CFU/g, when the probiotics stopped growing and started the cell death stage. These results over storage periods showed that the most active viable cells are found between the 14th and 70th day of storage.
That fact is noticeable when the parameters of acidity, pH, humidity, and water activity are evaluated to help the population growth of the microbial Lactobacillus strains over storage time, especially during the first 28 days [37]. The survival of probiotic bacteria in fermented products depends on many factors, like the interaction between the species found in the food, culture conditions, chemical composition of the medium, final acidity of the product, salt content, nutrient availability, growth promoters and inhibitors, dissolved oxygen, the amount of probiotic inoculate, incubation time, and storage temperature [38].
Even with the pH variation, Lactobacillus developed over time. When we compare the results of this research with other studies in the literature, we found that processed meat with the addition of Lactobacillus acidophillus at the end of maturation had values of 108 CFU/g and, after 90 days of storage, the formulations show a decrease in the population of Lactobacillus spp. [29]. Another study assessed the growth of L. casei in salami and also obtained values of 108 CFU/g for viable cells [37]. A population of 107 CFU/g viable cells was found in research with L. paracasei in fermented processed meat with reduction in nitrate and nitrite at the end of the storage period. All those studies showed similar results to the ones found at the end of the storage period in the current study [1].
The values observed confirmed a resistance to pH and humidity changes and indicate that the strains used in fermentation are capable of surviving and multiplying in the meat used. Despite the numeric reduction in the microorganism population, a daily intake higher than 10 g of these fermented processed meats would be enough to follow the recommendations of ANVISA and for the possible health benefits to be observed [39].

4.7. Analysis of the Correlation between the Physical Parameters and Lactobacillus Growth

The results found showed that both probiotics (for showing R values close to 1) had linear interactions between the evaluated parameters, except for the pH, which had a significantly different variation over time for both assays, making it clear that it is not possible to associate its variation with the growth of both Lactobacillus directly.
When analyzing p values, it was observed that they corroborated R values, because the analysis for the individual effects of the evaluated parameters, except pH, showed results < 0.05, which made it possible to infer that they had a linear correlation with the growth of both probiotics. From that, as it was discussed over this study and shown in Table 8, it is possible to affirm that the physical and physical–chemical parameters that impact microbial growth in this study are the same for L. casei and L. paracasei.

4.8. Limitations and Future Directions

Throughout the research, limitations were found when it came to the availability of equipment to carry out the proper analyses; it was necessary to schedule and make partnerships with other laboratories in the University. Other limitations were also found when it came to financing the materials and substances used in the research, since the authors did not have a sponsorship.
In light of the results found in this study, it was possible to better understand the behavior of L. casei and L. paracasei in meat products regarding the quality and safety of that food. With that, the interest to understand the use of different Lactobacillus and their dosage arose, aiming to understand the efficiency in conservation and applicability of those microorganisms’ activity in the human body through in vitro simulation.

5. Conclusions

The fermented processed sheep salami have physical and physicochemical features, according to Brazilian legislation, that characterize a product with appropriate qualities for consumption, within the standards of identity and quality. Regarding the carbohydrate and water activity analyses, the results did not follow the required standards by law, especially because of the ingredients present in the product’s formulation and the inoculation of Lactobacillus.
According to the microbiological results, all the assays showed an absence of pathogenic microorganisms, obtaining a high-quality food product that is safe for consumption, and has no signs of contamination that can be a health hazard for humans. Consequently, we were able to make probiotic processed meat containing viable L. casei and L. paracasei cells, with at least 107 CFU/g 84 days post-inoculation, showing functional benefits and bioconservation potential.
Given the results obtained, the efficiency of the use of these microorganisms in meat products and of the development of new products with the inoculation of the analyzed probiotics is noticeable.

Author Contributions

N.M.d.S.d.V.: conceptualization, methodology, investigation, resources, formal analysis, writing—original draft, visualization. M.R.P.d.M.: resources, writing—review and editing, visualization. A.C.P.F.: resources, software. J.M.C.G.: methodology, investigation, resources, writing—review and editing, visualization. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors honestly thank the support received by Universidade Federal Rural de Pernambuco and Universidade Federal de Pernambuco, located in the city of Recife in Pernambuco, in Brazil, institutions where this research was carried out, and the support of the advisors during the trajectory of this work, as well as the company Frigomalta in Igarassu-Pernambuco, that provided the raw materials used in the production of the processed foods.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 05509 g001
Figure 1. Flowchart of the processing of fermented salami-type sheep meat sausage.
Figure 1. Flowchart of the processing of fermented salami-type sheep meat sausage.
Applsci 14 05509 g001
Applsci 14 05509 g002
Figure 2. pH curve during storage time of fermented sausages.
Figure 2. pH curve during storage time of fermented sausages.
Applsci 14 05509 g002
Table 1. Composition of sausage formulations fermented with Lactobacillus inoculation.
Table 1. Composition of sausage formulations fermented with Lactobacillus inoculation.
IngredientsQuantities (%)
S1S2S3
Leg of lamb77.5077.5077.50
Bacon20.0020.0020.00
Curing salts0.25--
Sucrose0.500.700.70
Pepper0.100.100.10
Garlic0.130.130.13
Antioxidant0.250.250.25
Sodium chloride1.251.251.25
Starter Culture0.020.020.02
Lactobacillus casei-0.05-
Lactobacillus paracasei--0.05
S1: Standard salami without probiotic. S2: Salami with Lactobacillus casei. S3: Salami with Lactobacillus paracasei.
Table 2. pH values during salami processing.
Table 2. pH values during salami processing.
RehearsalSalami TypeSalami’s Initial pH before FermentationpH after Maturation
S1Standard5.82 a ± 0.145.19 a ± 0.03
S2L. casei5.85 a ± 0.035.02 a ± 0.02
S3L. paracasei5.75 a ± 0.015.04 a ± 0.01
Means followed by the same lowercase letter in the columns do not differ statistically by Tukey’s test at 5% probability. S1: Standard salami without probiotic. S2: Salami with Lactobacillus casei. S3: Salami with Lactobacillus paracasei.
Table 3. Results of the microbiological evaluation performed on the sample of fermented salami from sheep meat at 0 days after maturation and at 84 days.
Table 3. Results of the microbiological evaluation performed on the sample of fermented salami from sheep meat at 0 days after maturation and at 84 days.
RehearsalAt 0 Days after MaturationAt 84 Days
Escherichia coli (CFU/g) *Coagulase-Positive Staphylococci (CFU/g) *Salmonella in 25 g (CFU/25 g) **Escherichia coli (CFU/g) *Coagulase-
Positive
Staphylococci
(CFU/g) *		Salmonella in 25 g (CFU/25 g) **
S1<1.00 × 101<1.00 × 101Absence ***<1.00 × 101<1.00 × 101Absence ***
S2<1.00 × 101<1.00 × 101Absence ***<1.00 × 101<1.00 × 101Absence ***
S3<1.00 × 101<1.00 × 101Absence ***<1.00 × 101<1.00 × 101Absence ***
Legislation ****<2.00 × 101<1.00 × 101Absence ***<2.00 × 101<1.00 × 101Absence ***
S1: Standard salami without probiotic. S2: Salami with Lactobacillus casei. S3: Salami with Lactobacillus paracasei. * Values are expressed in colony forming units per gram (CFU/g). ** Values are expressed in colony forming units per 25 g (CFU/25 g). *** Expressed result represents absence of growth considering the limit of the method. **** Resolution RDC No. 60 of 2019 [17].
Table 4. Physical and Physicochemical Composition of Salami.
Table 4. Physical and Physicochemical Composition of Salami.
SampleS1S2S3Legislation
Lipids (%)30.69 a ± 0.0033.85 a ± 0.0829.72 a ± 0.03Max. 35.00
Protein (%)35.05 a ± 1.6633.19a ± 1.8931.14 a ± 1.94min. 20.00
Total carbohydrates (%)9.62 a11.59 a8.06 aMax. 4.00
Ash (%)2.98 a ± 0.002.65 a ± 0.002.64 a ± 0.00-
Caloric Value (Kcal)454.89483.76427.27-
ColorimetryL = 41.26 ± 0.53
a* = 12.71 ± 0.95
b* = 10.98 ± 0.66		L = 35.62 ± 0.83
a* = 10.82 ± 0.94
b* = 7.88 ± 0.71		L = 49.97 ± 0.57
a* = 9.04 ± 0.27
b* = 11.53 ± 0.46		-
Texture (KgF)6.53 a ± 1.634.25 a ± 1.419.45 a ± 1.42-
Acidity (g/mL)9.42 a ± 0.0114.14 a ± 0.007.48 a ± 0.01-
Means followed by the same superscript in the columns do not differ statistically by Tukey’s test at 5% probability (p < 0.05). S1: Standard salami without probiotic. S2: Salami with Lactobacillus casei. S3: Salami with Lactobacillus paracasei. All analyses were performed in triplicate, with the average taken and the standard deviation added; finally, the values were compared to the IN legislation no. 22 [19].
Table 5. Moisture analysis and water activity performed on samples of fermented salami sausages.
Table 5. Moisture analysis and water activity performed on samples of fermented salami sausages.
SamplesAw-Day 0Humidity (%)-Day 0Aw-Day 84Humidity (%)-Day 84
S10.95 a ± 0.0040.90 a ± 0.030.93 a ± 0.0043.91 a ± 0.01
S20.94 b ± 0.0041.89 a ± 0.040.90 b ± 0.0032.46 b ± 0.03
S30.96 a ± 0.0044.56 a ± 0.040.92 a ± 0.0038.66 c ± 0.02
LegislationMax. 0.92Max. 40.00Max. 0.92Max. 40.00
Means followed by the same superscript in the columns do not differ statistically by Tukey’s test at 5% probability (p < 0.05). S1: Standard salami without probiotic. S2: Salami with Lactobacillus casei. S3: Salami with Lactobacillus paracasei. All analyses were performed in triplicate, with the average taken and the standard deviation added; finally, the values were compared to the IN legislation no. 22 [11].
Table 6. pH analysis carried out throughout storage time.
Table 6. pH analysis carried out throughout storage time.
pH
Maturation TimeS1S2S3
05.19 ± 0.03 aA5.02 ± 0.02 aB5.04 ± 0.01 aB
145.35 ± 0.01 bA5.13 ± 0.03 bB5.20 ± 0.01 aC
286.04 ± 0.21 cA5.11 ± 0.01 bB6.14 ± 0.05 bA
426.03 ± 0.17 cA5.36 ± 0.05 cB6.44 ± 0.35 cC
566.23 ± 0.10 dA5.28 ± 0.01 dB7.43 ± 0.04 dC
706.47 ± 0.06 eA5.37 ± 0.03 cB7.64 ± 0.18 eC
847.14 ± 0.06 fA5.53 ± 0.03 eB8.09 ± 0.06 fC
Lowercase letters are used for vertical comparisons and capital letters for horizontal comparisons. Equal letters are used for vertical or horizontal comparisons that are not significantly different. Tukey’s test at 5% probability (p < 0.05). S1: Standard salami without probiotic. S2: Salami with Lactobacillus casei. S3: Salami with Lactobacillus paracasei.
Table 7. Count of lactic acid bacteria along the shelf-life of salami-type fermented sausages.
Table 7. Count of lactic acid bacteria along the shelf-life of salami-type fermented sausages.
Lactic Acid Bacterial Count (CFU/g)
Days/RehearsalsS2S3
0–Before1.61 × 10103.37 × 1011
0–After1.64 × 10135.40 × 108
144.00 × 1074.30 × 1010
281.35 × 10154.52 × 1013
424.52 × 10131.12 × 1013
561.20 × 1083.19 × 107
701.82 × 1076.8 × 107
842.3 × 1071.1 × 107
0–Before: Lactic acid bacteria viability at the time of inoculation in the produced mass. 0–After: Viability of lactic acid bacteria soon after salami maturation. S2: Salami with Lactobacillus casei. S3: Salami with Lactobacillus paracasei.
Table 8. Linear correlation coefficients for the parameters of L. casei and L. paracasei count.
Table 8. Linear correlation coefficients for the parameters of L. casei and L. paracasei count.
RFp
S2Acidity0.9998454833.3420.000005
pH0.7295971.7070.319841
Aw0.98882966.0150.003312
Texture0.996225197.5460.000654
S3Acidity0.9996836302.2570.000000
pH0.6761233.3680.140357
Aw0.97786487.3640.000730
Texture0.995705462.6450.000028
Values of p < 0.05 indicate there was significant effect when evaluated with a linear correlation between the microorganism count and the individual effects of the studied parameters. S2: Salami with Lactobacillus casei. S3: Salami with Lactobacillus paracasei.
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Vale, N.M.d.S.d.; Melo, M.R.P.d.; Filho, A.C.P.; Guerra, J.M.C. Evaluation of the Nutritional Quality and Shelf Life of Fermented Processed Sheep Salami Inoculated with Lactobacillus casei and Lactobacillus paracasei. Appl. Sci. 2024, 14, 5509. https://doi.org/10.3390/app14135509

AMA Style

Vale NMdSd, Melo MRPd, Filho ACP, Guerra JMC. Evaluation of the Nutritional Quality and Shelf Life of Fermented Processed Sheep Salami Inoculated with Lactobacillus casei and Lactobacillus paracasei. Applied Sciences. 2024; 14(13):5509. https://doi.org/10.3390/app14135509

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Vale, Natália Martins dos Santos do, Michelle Rayssa Pereira de Melo, Adelmo Cavalcante Pascoal Filho, and Jenyffer Medeiros Campos Guerra. 2024. "Evaluation of the Nutritional Quality and Shelf Life of Fermented Processed Sheep Salami Inoculated with Lactobacillus casei and Lactobacillus paracasei" Applied Sciences 14, no. 13: 5509. https://doi.org/10.3390/app14135509

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