Production of probiotic fermented salami using Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, and Bifidobacterium lactis

Abstract

The objectives of the study were to improve the functionality of fermented salami using probiotics, to evaluate the effects of the addition of probiotics on the physicochemical and microbiological characteristics and sensory acceptance of fermented salami, and to introduce a brand‐new probiotic food to the market for meat products. Fermented salami samples were produced using various formulations, including no probiotic (A), non‐probiotic starter cultures (B) or probiotic cultures [Lacticaseibacillus rhamnosus LR32 200B (C), Lactiplantibacillus plantarum LP115 400B (D), Bifidobacterium lactis BB12 (E), and L. rhamnosus LR32 200B + L. plantarum LP115 400B (F)]. The samples were kept at 4°C for 60 days, and their probiotic viability as well as their chemical, physical, microbiological, and sensory qualities were assessed at intervals of 0, 15, 30, 45, and 60 days. The probiotic addition enhanced the safety and quality of the product while favorably affecting the microbiological, physical, chemical, and sensory properties of the samples. The sample produced with mixed probiotics (F) had the highest moisture and fat content and the lowest pH. Lactic acid bacteria counts were found above 6.0 log CFU/g in the samples produced with probiotic at the end of the storage. Probiotic added products were rated higher than products without probiotics in terms of color, texture, flavor, and overall acceptance during storage. Consequently, a probiotic fermented salami with high probiotic cell counts and meeting the sensory preferences of the consumers was produced.

1. INTRODUCTION

The diet of many people worldwide includes meat and meat products, which are a significant group of highly nutritious foods. Fermented salami and sausages are the most commonly consumed meat products all over the world. In the production of fermented salami, starter cultures are used in order to rapidly lower the pH of the raw meat and to achieve the desired sensory quality of the final product. However, in recent years, the use of functional starter cultures, which have important industrial and nutritional functions, has been investigated. Functional starter cultures offer additional features compared to conventional starter cultures, such as improving and optimizing the fermentation process and producing tastier, safer, and healthier products. Salami is a dried fermented meat product produced with meat, fat, nitrite, and spices. The fermentation process is carried out during the production of salami by microorganisms that are inherent on the raw materials and/or are inoculated as starters. Lactic acid bacteria (LAB) and coagulase‐negative staphylococci are commonly used in the production of fermented salami (Pérez‐Burillo et al., 2020). During fermentation, activity of microorganisms and their enzymes leads to biochemical, microbial, and physical changes. The fermentation and curing processes of meat are critical for safety and shelf life. Although pathogen inhibition is ensured by low pH, low water activity (a_w), and salting (nitrite or nitrate addition), aroma structure and color properties are improved by coagulase‐negative staphylococci and micrococci, especially by LAB (Bis‐Souza et al., 2020; Cenci‐Goga et al., 2018; Pérez‐Burillo et al., 2019). Moreover, LAB were proved highly competitive during the fermentation and ripening process, controlling the growth of undesired microorganisms (Kołożyn‐Krajewska & Dolatowski, 2012). Proteolytic enzymes produced by starter cultures during ripening and fermentation are another reason for the tenderness of the products. Enterococcus, Lactobacillus, Pediococcus, Leuconostoc, and Lactococcus genera, which are facultatively anaerobic, were commonly used in fermented meat products (Bis‐Souza et al., 2020). It has been reported that Lacticaseibacillus rhamnosus is generally found as the dominant flora in the ripening stage of meat products and can be used in the production of fermented salami due to its presence in both the microflora of meat and the microbiota of healthy people (Jofre et al., 2015).

In recent years, there has been a significant increase in consumer demand for meat products containing functional ingredients. Therefore, numerous research has been carried out to develop meat products with functional properties, with an emphasis on ways to raise the amounts of health‐promoting compounds and decrease the amounts of compounds that may be harmful to the consumer's health. When reviewing the studies on probiotic meat products, it is important to note that these studies were mostly carried out in fermented and dried meat products. Because of its high fat and salt content as well as absence of healthy ingredients, salami, a dry‐fermented meat product, is frequently seen as a food with an unbalanced nutritional value (Pérez‐Burillo et al., 2019). One of the ways to improve the functionality of fermented salami is to add probiotics to the product (Martinez et al., 2014). Fermented and dried salami provides suitable conditions for the survival of probiotics because they can be produced and consumed without heating. In addition, bioactive peptides produced during fermentation enrich the meat products nutritionally (Khan et al., 2011). Some studies on the production of probiotic meat products have been shown in the literature (Blaiotta et al., 2018; Cavalheiro et al., 2021; Pérez‐Burillo et al., 2020; Ruiz et al., 2014). These studies mostly investigate the effect of probiotics on product quality and in vitro health and the synbiotic effect of prebiotic and probiotic use (Bis‐Souza et al., 2020; Pérez‐Burillo et al., 2020). A few studies have investigated the effect of probiotics on product safety (de Souza Barbosa et al., 2015; Pidcock et al., 2002). On the other hand, research on the use of probiotic bacteria in various meat products, such as cooked sausages and fresh ground or raw meat, is still rare, mainly because of the processing involved in the production of these products, such as heating and adding additives. This is why the majority of these studies are based on fermented meat products. Cenci‐Goga et al. (2018) assessed the impact of a starter culture (Enterococcus faecium 614, L. lactis 16 and 340, and L. casei 208) on the low‐temperature fermentation and ripening of Italian dry sausage. They found that the use of probiotic starter cultures reduced the growth of L. innocua, Escherichia coli, S. aureus, and Salmonella Derby, possibly producing a safer product without degrading sensory quality. Sidira et al. (2019) conducted a study to investigate the effect of different sugar levels on the shelf life and qualitative characteristics of dry‐fermented sausages containing either free or immobilized L. casei ATCC 393. They stated that L. casei prevented spoilage of the sausages regardless of the sugar concentration tested. The addition of immobilized L. casei ATCC 393 cells also gave the product a unique flavor. The pH, moisture, and salt content of the salami should be taken into consideration when choosing the probiotics that will be used in the studies to impart probiotic qualities to salami. Lactiplantibacillus plantarum, L. rhamnosus, L. pentosus, Ligilactobacillus salivarius, L. casei, L. paracasei, Limosilactobacillus reuteri, L. acidophilus, P. acidilactici, and Bifidobacterium lactis are the probiotic cultures mainly used for the development of probiotic meat products (Bis‐Souza et al., 2020; Coelho et al., 2019; Jofre et al., 2015; Muthukumarasamy & Holley, 2006; Pavli et al., 2020; Rubio et al., 2014; Ruiz et al., 2014; Slima et al., 2018; Zhang et al., 2020). Among these, Lactobacillus and/or Bifidobacterium spp. are commonly used in the production of fermented sausages or salami due to their presumed probiotic properties (Holko et al., 2013). They are also normally present in the human gastrointestinal (GI) tract. Furthermore, L. rhamnosus LR32 was found to be tolerant to nitrate, salt, and low pH (Agüero et al., 2020) and was considered the most promising probiotic strain (Carballo, 2021). L. plantarum LP115 400B is the other probiotic commonly used in meat products. B. lactis BB12 is known for its cholesterol‐lowering, antimutagenic, anticancer, and gut microbiota‐regulating properties (de Campos et al., 2022). It is considered a potential starter for meat fermentation (Libera et al., 2015). It has been reported that when probiotics are consumed together with sausages, their viability in the GI system increases, which is related to the sausage matrix, protecting the bacteria from the acidic environment of the host. In addition, it has been observed that studies performed on probiotic meat products mostly focus on sensory properties and microbiological quality of the product, whereas there are limited studies on the viability of probiotic microorganisms throughout the shelf life of the product (Libera et al., 2020; Sirini et al., 2022). On the other hand, the commercial use of probiotic bacteria in fermented salami is still uncommon in our country. Furthermore, fermented salami is an abundant part of the diet of many people. However, salami is considered to be low in nutrients because it is high in fat and low in bioactive compounds. There is therefore a need to improve the functional properties of salami. It can be consumed without heat treatment, making it an ideal matrix for the delivery of probiotics, thereby increasing the viability of the probiotics. Additionally, it may help probiotic bacteria to survive in the GI tract.

For these reasons, this study aims to produce a new probiotic fermented salami that will be produced and consumed without heat treatment and to offer a novel functional meat product to the food industry. L. rhamnosus, L. plantarum, and B. lactis were selected as probiotics because these cultures are suitable for use in the meat industry as mentioned in publications by other authors.

2. MATERIALS AND METHODS

2.1. Probiotic strains

L. plantarum LP115 400B (Danisco), L. rhamnosus LR32 200B (Danisco), and B. lactis BB12 (Chr. Hansen) were used as probiotic cultures. Skim milk medium (10% w/v) (Pınar Dairy Products Industry Inc. Co.) was combined with 0.1 g of lyophilized culture for the activation and the incubation was performed at 37°C for 30 min (dos Santos Cruxen et al., 2017).

2.2. Probiotic fermented salami production

The research aimed to develop a probiotic fermented salami (Figure 1). Formulations were obtained using beef and fat (Pınar Entegre Et ve Un San. A.Ş.), probiotic strains, starter cultures (Latilactobacillus curvatus, L. sakei, S. carnosus, Meat Cracks), salt (Estuz), nitrite salt (Tunçkaya), antioxidant (sodium ascorbate, CSPC, Luwei), dextrose (Asian Winner), salami flavoring (Firmenich), and permeable salami casing (Kalle). Fermented salami production was carried out in three repetitions with six different recipes, which did not contain starter culture (A), contain a starter culture without probiotic properties (B), and contain only probiotic cultures (C, D, E, and F) (Table 1). Probiotic cultures were added at a level of 12.0 log CFU/g.

FIGURE 1.

Production of probiotic fermented sausage.

TABLE 1.

Recipes used for the probiotic fermented salami production.

Ingredients	A	B	C	D	E	F
Beef (kg)	80.00	80.00	80.00	80.00	80.00	80.00
Fat (kg)	18.00	18.00	18.00	18.00	18.00	18.00
Salt (kg)	1.00	1.00	1.00	1.00	1.00	1.00
Nitrite salt (kg)	0.15	0.15	0.15	0.15	0.15	0.15
Antioxidant (kg)	0.10	0.10	0.10	0.10	0.10	0.10
Dextrose (kg)	0.10	0.10	0.10	0.10	0.10	0.10
Flavoring (kg)	0.65	0.65	0.65	0.65	0.65	0.65
Starter culture (Meat cracks)	X	0.02	X	X	X	X
Lacticaseibacillus rhamnosus LR32 200 B	X	X	0.01	X	X	0.007
Lactiplantibacillus plantarum LP 115 400 B	X	X	X	0.014	X	0.007
Bifidobacterium lactis BB12	X	X	X	X	0.018	X
Total	100.00	100.025	100.014	100.014	100.000	100.014

The fermented salami production (20 cm length) was conducted in three independent replicates, six different recipes (A, B, C, D, E, and F) were prepared per batch and per storage time (0, 15, 30, 45, and 60), and a total of 90 (3 replicates × 6 recipes × 5 storage time) fermented salami were produced during the experiment. Analyses were applied to six different recipes for each storage time and carried out with two parallels and three repetitions.

2.3. Proximate composition

Moisture (TS, 1743 ISO, 1442, 2001), fat (Association of Official Analytical Collaboration [AOAC], 1996a), and protein (AOAC, 1996b) analyses were performed on the 0, 15, 30, 45, and 60 days of the storage.

The moisture content of the samples was measured according to the procedures of AOAC (2000). According to Flynn and Bramblett (1975), the chloroform–methanol extraction method was used to evaluate the fat content of the samples. A protein measurement analyzer (Leco FP‐528, St. Joseph), which is a microprocessor‐based, software‐controlled equipment for determining nitrogen content, was used to determine the protein content of the samples.

2.4. pH

The pH was determined in triplicate using a pH meter (Metrohm 691 brand) (AOAC, 1995).

2.5. Salt

The homogenized sample was titrated with 0.1 M AgNO₃ and the result was expressed as %NaCl (AOAC, 2005).

2.6. Texture analysis

TA‐XT Plus (Stable Micro Systems) was used to conduct texture profile analysis on fermented salami samples (1 × 1 × 1 cm³). Three repetitive measurements were made for each sample. As a result of the measurements, the resistance of the samples to the applied force was recorded (Slima et al., 2018).

2.7. Color measurement

Color properties of fermented salami samples were measured using an 8 mm aperture of the chromatic color difference colorimeter of the Konica Minolta Chroma Meter (Konica Minolta, CR‐400/410, the measuring range: 0.01–160.00) with a D65 light source and a 10° standard observer. The CIE L* (dark–light), a* (red–green), and b* (yellow–blue) color coordinates were recorded on the surface of the samples after 20 min blooming. Prior to color measurements being made on the surfaces of the samples at room temperature, the samples were cut into rounds. Three readings were taken for each sample (Slima et al., 2018). Each sample was subjected to four measurements, and three samples from each sample group were assessed.

2.8. Microbiological analysis

Overall, 0.1% peptone water (PW, pH 7.0 ± 0.2) was used to obtain sample homogenates. A Stomacher (Stomacher Lab‐Blender 400) was used to homogenize the sample (25 g) with PW (225 mL of 0.1%, w/v) for 60 s. The samples were diluted appropriately by tenfold in PW before being duplicate‐plated on/in growth media.

Probiotic counts of fermented salami samples were determined periodically during storage on the 0, 15, 30, 45, and 60 days. In order to selectively enumerate L. rhamnosus LR32 200B and L. plantarum LP115 400B, pH of MRS Agar (Man Ragosa Sharpe Agar, pH 5.6–5.9, Merck) was adjusted to 5.2 (0.5–2 mL of acetic acid/100 mL MRS) and the incubation was carried out anaerobically at 37°C for 72 h (Karimi et al., 2012). TOS‐MUP (TOS Propionate Agar & MUP Selective Supplement, pH 6.7 ± 0.2, Merck) medium was used for B. lactis BB12 enumeration. A volume of 5 mL MUP Supplement was added to 95 mL of TOS Propionate Agar medium at 48 ± 1°C and the plates underwent a 72‐h anaerobic incubation period at 37°C (Bunesova et al., 2015).

In addition, to determine the microbiological quality of fermented salami samples, total mesophilic aerobic bacteria (TMAB), mold and yeast, total coliform, E. coli O157:H7, Salmonella spp., Listeria monocytogenes analyzes were performed.

• (A)TMAB count was determined by pour plating on Plate Count Agar (pH 6.8–7.2, Merck) and incubating the plates at 30°C for 3 days (TS 3834 ISO, 2293, 1996).
• (B)For mold and yeast enumeration, appropriate dilutions of the samples were inoculated on Dichloran Rose Bengal Chloramphenicol Agar (pH 5.6, Merck) and the incubation was carried out at 25°C for 5–7 days (Food and Drug Administration/Bacteriological Analytical Mannual (FDA/BAM), 2001).
• (C)Total coliform count was determined on Violet Red Bile Agar (pH 7.4 ± 0.2, Neogen) according to the double layer plate method and the incubation was conducted at 37°C for 24 h (ISO 4832, 2006).
• (D)After enrichment of samples in PW at 37 ± 1°C for 16–20 h, Salmonella sp. was detected by real‐time PCR (Biorad CFX96, IQ‐Check® AOAC certificate number: 010802) (TS EN ISO 6579‐1, 2017).
• (E)After enrichment of the samples in PW at 30 ± 1°C for 25 ± 1 h, detection of L. monocytogenes was performed by real time PCR (Biorad CFX96) (AOAC, 2006).
• (F)Detection of E. coli O157:H7 in samples was performed by using 3 M molecular detection system (AOAC, 2016).

2.9. Sensory evaluation

Sensory analysis was conducted using a 10‐point hedonic scale as noted by Altug Onogur and Elmaci (2005). The color, flavor, texture, and overall appreciation properties of fermented salami were assessed at the Sensory Evaluation Laboratory of Pınar Et R&D Center. A total of three independent replicates were carried out and two slices of fermented salami were used per recipe in each replicate. A total of 36 fermented salami slices (2 fermented salami slices per recipe × 6 recipes × 3 independent replicates) were used. Prior to evaluation, each sample was cut into a round shape. Each sample was assigned to a panelist in random order and identified by a three‐digit random code. The sensory evaluation was carried out by a panel including seven trained panelists (seven female) aged between 20 and 50 years. During the training sessions, the panelists received an explanation of the rating criteria. All sensory evaluation sessions were performed between 14.00 and 17.00. The same panelists performed all the sensory evaluations and each panelist evaluated all the samples (2 slices of salami per recipe and a total of 12 samples per replicate) during the analysis.

The study was approved by the Science and Engineering Sciences Scientific Research and Publication Ethics Committee, Ege University (Ethics ID: E.806332, approved on 01/08/2022).

2.10. Statistical analysis

Each of the three independent production batches of every determination was carried out in triplicate. Analyses were repeated at least three times for relevant tests in each independent production. Statistical analysis was carried out using the SPSS 21.0 (SPSS Inc.). To evaluate statistical difference between independent groups (proximate composition, pH, salt, texture, color, TMAB, mold‐yeast count and probiotic viability, color properties of fermented meat samples), one‐way analysis of variance and Duncan's multiple comparison tests, where the different formulations and storage periods were set as factors, were used at a significance level of p < 0.05. The mean ± standard error was used to express the results.

3. RESULTS AND DISCUSSION

Fermented salami production was performed by using six different receipts: without starter culture (A), with non‐probiotic starter cultures (B), and probiotic starter cultures including L. rhamnosus (C), L. plantarum (D), and B. lactis (E), which were used separately and also as the mixed culture of L. rhamnosus and L. plantarum (F) in salami formulations. All samples were investigated in terms of chemical (moisture, fat, protein, pH, and salt), physical (texture and color), microbiological (probiotic viability, TMAB, mold‐yeast, total coliform, L. monocytogenes, Salmonella spp., and E. coli O157:H7), and sensory properties during storage.

3.1. Chemical composition of the samples

The moisture content and a_w are factors intrinsically linked. Because a lower a_w value can prevent the growth of pathogens and spoilage microorganisms, it is safer and better for food quality. Conversely, the loss of moisture results in the formation of a cloudy viscose solution, which lowers the L* value (Bis‐Souza et al., 2020; Mafra et al., 2022). The moisture content of the sample during storage was ranged between 38.35% and 42.91% (Table 2). The moisture content of the samples as not significantly changed at the end of fermentation (p > 0.05). However, the highest moisture value (40.9%) was found in F, produced by two different probiotic cultures (L. rhamnosus and L. plantarum), and the lowest moisture content (38.4%) was observed in E, produced by B. lactis at the end of the storage (p < 0.05). However, when the moisture contents of the samples were compared in terms of storage, there was no statistical difference among A, B, and C (p > 0.05), whereas the moisture contents of D, E, and F were significantly reduced at the end of the storage (p < 0.05). Ayyash et al. (2019) observed a decrease in a_w of fermented camel and beef sausages during the storage period. They suggested that the presence of small molecules such as organic acids and short peptides, as well as the decrease in moisture content, could be the reason. Similarly, in this study, the pH of D, E, and F is lower than the other samples, which is related to the organic acids produced and may be explain the low moisture content. The results are also consistent with the study by Roselino et al. (2018).

TABLE 2.

Chemical compositions of fermented salami samples during storage.

		Sample groups
	Days	A	B	C	D	E	F
Moisture (%w/w)	0	41.3 ± 0.7a	40.9 ± 0.9a	41.3 ± 1.0a	42.9 ± 0.8a,B	41.7 ± 0.3a,C	42.4 ± 0.1a,C
	15	40.9 ± 1.0a	40.5 ± 1.0a	42.0 ± 1.3a	42.9 ± 0.8a,B	41.9 ± 0.2a,C	42.0 ± 0.3a,BC
	30	40.7 ± 0.7ab	41.1 ± 0.6ab	41.9 ± 1.2b	42.4 ± 0.6b,B	39.3 ± 0.1a,B	41.3 ± 0.4ab,AB
	45	40.5 ± 0.4bc	40.0 ± 0.5ab	39.9 ± 1.0ab	40.0 ± 0.4ab,A	38.4 ± 0.0a,A	42.1 ± 0.0c,BC
	60	39.9 ± 0.4ab	40.0 ± 0.3ab	39.8 ± 1.1ab	39.6 ± 0.7ab,A	38.4 ± 0.1a,A	40.9 ± 0.3b,A
Fat (%w/w)	0	30.8 ± 0.5a	30.8 ± 1.4a	30.2 ± 1.8a	29.4 ± 0.4a,A	31.0 ± 0.3a,A	33.5 ± 0.4b
	15	30.1 ± 0.1ab	30.5 ± 0.5ab	29.1 ± 1.0a	30.4 ± 0.5ab,AB	31.0 ± 0.1b,A	33.3 ± 0.5c
	30	29.8 ± 0.3a	28.9 ± 1.3a	29.7 ± 0.8a	30.0 ± 0.2a,A	31.3 ± 0.4a,A	34.1 ± 0.9b
	45	30.2 ± 0.4a	32.0 ± 1.6ab	30.4 ± 0.7ab	31.5 ± 0.3ab,B	32.9 ± 0.2b,B	33.0 ± 0.8b
	60	30.6 ± 0.4a	30.7 ± 1.0a	30.7 ± 1.0a	31.7 ± 0.5ab,B	33.3 ± 0.3b,B	33.6 ± 0.5b
Protein (%w/w)	0	22.4 ± 1.3c	23.2 ± 0.7c	22.7 ± 0.5c	21.3 ± 0.4bc,A	19.2 ± 0.5ab,A	18.7 ± 0.1a,A
	15	22.5 ± 0.6c	22.4 ± 0.1c	22.1 ± 0.1c	20.9 ± 0.3b,A	20.3 ± 0.1ab,B	19.5 ± 0.1a,AB
	30	23.0 ± 0.0c	23.5 ± 1.1c	21.8 ± 0.2bc	21.1 ± 0.6b,A	22.6 ± 0.0bc,D	19.2 ± 0.4a,AB
	45	22.8 ± 0.3c	21.6 ± 1.2bc	21.7 ± 0.5bc	21.8 ± 0.4bc,A	20.7 ± 0.3ab,B	19.6 ± 0.3a,B
	60	25.9 ± 3.0b	22.2 ± 1.3ab	21.5 ± 0.8ab	21.7 ± 0.6ab,A	21.6 ± 0.0ab,C	19.6 ± 0.1a,B

Note: Values are the mean ± SD. Values in the same row with different lowercase letters (a, b, and c) show a significant difference (p < 0.05). Values in the same column with different uppercase letters (A, B, and C) show a significant difference (p < 0.05).

The fat content of the samples was found in the range of 28.93%–34.09% (Table 2). The highest fat content was found in F, as parallel in moisture content (p < 0.05). When the fat contents of the samples were compared based on storage times, no significant difference was found between the samples, except D and E (p > 0.05).

The protein content was ranged between 18.71% and 25.92% (Table 2). The lowest protein content was found in F (p < 0.05), as in contrast to the moisture and fat contents. When the protein contents of the samples were compared in relation to the storage times, no significant difference was found between the samples, except for E and F (p > 0.05). The ability to break the protein molecules in food is limited to a few species microorganisms that can produce extracellular proteolytic enzymes (Suryaningsih et al., 2019). Due the synergistic effect between the cultures and the proteolytic enzyme activities, the protein molecules in the salami produced using the combined starter culture (F) were broken down, and the protein content was reduced compared to the control group. This could also be due to differences in the intensity of the breakdown of meat proteins (Cullere et al., 2020). Protein contents in E and F were noticeably greater at the end of storage than they were at beginning (p < 0.05) (Table 2). The main reason for the increase in protein, fat, and ash contents may be the decrease in moisture content (Ayyash et al., 2020). Similarly, Ayyash et al. (2019) found that the protein, fat, and ash contents of fermented camel and beef sausages increased after 21 days of storage (p < 0.05).

As a result, there was no significant difference in the last week of storage between the salami samples produced with the individual use of probiotic cultures and the control groups (except F). Fermented salami produced by mixed culture of L. rhamnosus and L. plantarum (F) showed differences in terms of moisture, fat, and protein contents. Similarly, Slima et al. (2018) observed that the moisture content of fermented sausages produced by probiotics (L. plantarum TN8 and P. acidilactici MA 18/5 M) was higher than the control group, but this situation was also associated with the reduction in meat rate due to addition of fiber to the products containing probiotics. The protein content was found to be lower in the sample produced with the mixed culture (F). It can be seen that the sample with high protein and low fat content is the sample produced with L. rhamnosus LR32 200 B (C). Therefore, it is recommended to use single cultures instead of mixed cultures in the production of probiotic salami.

3.2. pH

Determining the pH is essential for checking the stability of the product. As it affects quality and safety of products, the ability of probiotics to produce rapid and sufficient organic acid is an important characteristic. Lowering the pH also reduces the water‐binding properties of the meat and shortens the drying time of sausages (Holck et al., 2017). However, one of the biggest problems in salami/sausage processing is excessive acid formation, often associated with color abnormalities. Depending on the acid concentration, the sour taste of fermented meat products is preferred in some countries and undesirable in others. Therefore, the acid production capacity of starter cultures plays an important role in their selection, as it determines the quality characteristics and safety of the final product (Agüero et al., 2020).

In this study, the pH of the salami samples was in the range of 5.14–5.56 during storage (Table 3). The highest pH was found in A, produced without any starter culture, whereas the lowest value was found in F, produced by mixed probiotics (p < 0.05). The mixed probiotic cultures (F) produced a higher acidity during the fermentation and drying stages, which persisted throughout the storage period, resulting in a difference in pH values. However, it was found that the pH of the samples did not change statistically during storage, except E (p > 0.05). The pH value of the samples achieved after fermentation also affects the pH stability during storage period. This is because a low pH prevents the growth of pathogens or spoilage microorganisms, thereby preventing the pH rising or dropping. Composition of the salami can interact with the probiotic cultures and affect their activities in different ways, which can affect pH stability. Storage temperature also affects pH stability by affecting the activity of probiotic cultures and other microorganisms in the salami.

TABLE 3.

pH values of fermented salami samples.

	pH
Days	A	B	C	D	E	F
0	5.5 ± 0.0c,A	5.4 ± 0.1b,A	5.4 ± 0.0b,A	5.4 ± 0.0b,A	5.4 ± 0.0b,C	5.1 ± 0.0a,A
15	5.5 ± 0.1b,A	5.4 ± 0.1b,A	5.4 ± 0.0b,A	5.4 ± 0.0b,A	5.4 ± 0.0b,BC	5.2 ± 0.1a,A
30	5.5 ± 0.0d,A	5.4 ± 0.0c,A	5.4 ± 0.0bc,A	5.4 ± 0.0b,A	5.4 ± 0.0bc,C	5.1 ± 0.0a,A
45	5.5 ± 0.1b,A	5.5 ± 0.2b,A	5.4 ± 0.0b,A	5.4 ± 0.0b,A	5.4 ± 0.1b,B	5.2 ± 0.1a,A
60	5.6 ± 0.0d,A	5.5 ± 0.0cd,A	5.4 ± 0.1bc,A	5.4 ± 0.0bc,A	5.3 ± 0.0b,A	5.1 ± 0.0a,A

Note: Values are the mean ± SD. Values in the same row with different lowercase letters (a, b, c, and d) show a significant difference (p < 0.05). Values in the same column with different uppercase letters (A, B, and C) show a significant difference (p < 0.05). The same lowercase letters within a line indicate that there was no significant difference (p > 0.05).

In a study, the pH of fresh fermented sausages produced with probiotic L. plantarum TN8 (8.0 log CFU/g) was found significantly lower than the control group, and this decrease was linked with the organic acids, produced by probiotics (Slima et al., 2017). The pH of fermented sturgeon sausage produced with the use of probiotics (L. paraplantarum S4, L. paraplantarum L‐ZS9, L. pentosus 31‐1, L. rhamnosus LGG ATCC53101, L. plantarum P, L. plantarum Y9, L. plantarum pl2, and L. acidophilus N4) was lower than in the control group (Wang et al., 2015). In another study, the pH of Spanish‐type fermented sausages produced with L. rhamnosus GG and L. plantarum 299 V additions was compared with the control group. The pH of probiotic products was significantly lower than the control group and the lowest pH was observed in the samples containing L. rhamnosus GG (Rubio et al., 2013). These results are in parallel with our results, where the sample produced by the mixed probiotic cultures (L. rhamnosus and L. plantarum) (F) had a lower pH (Table 3). pH value less than 5.2 is considered essential for meat products to be shelf stable. The lower pH values in the sample produced by the mixed probiotic cultures (F, pH 5.1) revealed that the mixed cultures enhanced the fermentation process, most likely by producing more organic acids (Ayyash et al., 2019). This also suggests a synergistic effect between cultures (Ruiz et al., 2014).

Fibrillar proteins require a lower pH to coagulate, which improves the firmness and cohesiveness of the end product and makes it easier to slice. Rapid and sufficient organic acid production, resulting in a pH drop, is also necessary for the inhibition of pathogens and spoilage microorganisms to enhance the safety and quality of salami (Agüero et al., 2020). However, it has been demonstrated that lowering the pH from 5.6 to 4.9 following fermentation has an impact on the viability probiotics (L. rhamnosus GG and E‐97,800) in fermented sausages (Erkkilä et al., 2001).

3.3. Salt concentration of the samples

Salt is typically added to fermented sausages to enhance the flavor and extend the shelf life. Moreover, meat has better texture, tenderness, and juiciness when its myofibrillar proteins are solubilized by salt (NaCl), which increases the proteins’ ability to hydrate and bind water. The sodium content of processed meat products ranges from 1.5 to 2.7 g/100 g of product, according to the USDA National Food Database (Sirini et al., 2021). A higher salt concentration leads to weight loss in salami, resulting in a loss of free water and a decrease in a_w and increase in hardness. Furthermore, the salt concentrations used in fermented sausages inhibit growth of undesired microorganisms and thereby promote growth of more salt‐tolerant LAB. Hence, a high salt concentration inhibits the probiotic growth. It is crucial to select a starter culture that can tolerate salt concentration used (Agüero et al., 2020; Bis‐Souza et al., 2020). In this study, the salt concentration of salami samples varied between 1.66% and 2.25% during storage (Table 4). The lowest salt concentration was in F during storage (p > 0.05). In general, there is no significant difference between the samples. On the other hand, the salt concentration of the samples stored for 30 and 45 days has changed so much. This could be linked with the structure of the product. Due to the manufacturing process of the products, not including an emulsification step, the product may not be homogenized exactly for the whole structure. As a result, the ingredients may not be evenly distributed, resulting in slight variations in salt concentration. Other possible reason could be the uncontrolled moisture loss during storage of the product for 30 and 45 days.

TABLE 4.

Salt concentrations of fermented salami samples.

	Salt (%, w/w)
Days	A	B	C	D	E	F
0	2.1 ± 0.0a,B	2.0 ± 0.0a,A	2.0 ± 0.0a,AB	1.9 ± 0.4a,A	2.0 ± 0.0a,A	1.7 ± 0.0a,A
15	2.1 ± 0.0a,B	2.1 ± 0.1a,A	2.1 ± 0.1a,AB	1.8 ± 0.3a,A	2.1 ± 0.0a,B	1.7 ± 0.1a,A
30	2.2 ± 0.1a,B	2.1 ± 0.1a,A	2.1 ± 0.1a,AB	1.9 ± 0.4a,A	2.1 ± 0.0a,C	1.7 ± 0.0a,A
45	1.8 ± 0.1ab,A	1.9 ± 0.1b,A	1.9 ± 0.0ab,A	1.9 ± 0.1ab,A	1.9 ± 0.0ab,A	1.7 ± 0.2a,A
60	2.2 ± 0.0 cd,B	2.1 ± 0.1c,A	2.3 ± 0.0d,B	2.2 ± 0.1 cd,A	2.0 ± 0.0b,A	1.7 ± 0.0a,A

Note: Values are the mean ± SD. Values in the same row with different lowercase letters (a, b, c, and d) show a significant difference (p < 0.05). Values in the same column with different uppercase letters (A and B) show a significant difference (p < 0.05).

Rubio et al. (2014) investigated the viability of probiotic cultures (L. casei CTC 1677, L. casei CTC 1678, L. plantarum 299v, L. rhamnosus CTC 1679, L. casei Shirota, and L. rhamnosus GG) in fermented sausages having reduced salt and fat contents. The results showed that the salt concentration of fermented sausages was generally 4%, and the probiotic level of the samples, whose salt concentration was reduced by 25%, was around 7.0–8.0 log CFU/g during storage. However, the salt concentrations of the samples produced within the scope of this study are even lower than the salt concentration of the products produced with the reduced salt concentration. In another study performed by Abdallah et al. (2018), L. rhamnosus FERMP‐15,120 showed a higher growth rate (11 log CFU/g) in the sausages with 50% salt (NaCl) reduction with significant differences to control samples (p < 0.05). Considering these results, the low salt concentration provides an advantage for maintaining the viability of probiotics.

Consequently, pathogens and spoilage microorganisms in fermented salami are generally inhibited by a combination of hurdles: high salt, presence of curing salt, low aw, reduction of redox potential, growth of competitive starter culture, and reduction of pH. Among these, salt has many important functions in fermented salami, contributing to taste, texture, quality and overall acceptability, as well as safety. To prevent the growth of pathogens and spoilage microorganisms, it is important to determine the correct salt concentration and to select a salt‐tolerant starter culture.

3.4. Texture values of the samples

Probiotic strains can influence the technological properties of the final product, such as texture. Texture is a key factor in determining whether or not consumers will accept meat products. The texture of the samples was evaluated in terms of hardness values. During storage, the hardness values of the samples produced by probiotics were 1978.4–2249.7 g/cm² in C, 2001.3–2177.3 g/cm² in D, 1906.6–1996.1 g/cm² in E, and 2005.9–2116.4 g/cm² in F, whereas these values were 1623.0–1800.2 and 996.4–1161.8 g/cm² for A and B, respectively (p < 0.05) (Table 5). A significant difference was observed between the values of the samples during storage, and the hardness values of the samples produced by probiotic cultures were found to be significantly higher than the control samples (p < 0.05). A higher concentration of lactic acid produced by probiotics may be the cause of the increase in hardness, as it causes a greater loss of water and, which in turn, increases hardness (Roselino et al., 2018; Sirini et al., 2022). Therefore, the high hardness value in samples containing probiotics is associated with a pH close to the isoelectric point of protein, thereby increasing water loss (Bis‐Souza et al., 2020). On the other side, F, where the pH was not highest, showed higher hardness values. Similarly, Ayyash et al. (2019) observed that an increase in hardness value of fermented camel and beef sausages during storage. They associated this with a decrease in moisture content. However, in a study, the addition of L. plantarum TN8 to fresh fermented sausages reduced the hardness value of the sausage samples. At the end of the storage (10 day), the hardness value of the control group was 1.61, while it was decreased to 0.59–0.77 and 0.67–0.86 for the samples containing 7.0 log CFU/g L. plantarum TN8 and 8.0 log CFU/g L. plantarum TN8, respectively (Slima et al., 2017). These results are not parallel with the results obtained in the current study.

TABLE 5.

Texture of fermented salami samples during storage.

	Texture (hardness, g/cm2)
Days	A	B	C	D	E	F
0	1714.6 ± 7.0b,D	996.4 ± 6.3a,A	2113.7 ± 8.8e,B	2007.2 ± 2.3d,A	1906.6 ± 3.2c,A	2096.5 ± 8.4e,C
15	1623.0 ± 6.2b,A	1161.8 ± 1.8a,D	2135 ± 8.7e,C	2090.6 ± 5.8d,B	1953.6 ± 3.3c,B	2116.4 ± 9.4e,C
30	1800.2 ± 5.5,E	1013.4 ± 3.5a,B	2249.7 ± 4.9f,D	2153.6 ± 3.3e,C	1993.2 ± 7.4c,C	2055.3 ± 4.7d,B
45	1690.6 ± 5.2b,C	1094.2 ± 3.4a,C	1978.4 ± 7.7c,A	2177.3 ± 3.3f,D	1996.1 ± 0.3d,C	2011.1 ± 4.7e,A
60	1670.5 ± 5.8b,B	1096.5 ± 3.0a,C	1996.3 ± 1.2cd,A	2001.3 ± 1.7d,A	1987.8 ± 3.6c,C	2005.9 ± 3.7d,A

Note: Values are the mean ± SD. Values in the same row with different lowercase letters (a, b, c, d, and e) show a significant difference (p < 0.05). Values in the same column with different uppercase letters (A, B, C, D, and E) show a significant difference (p < 0.05).

Consequently, consumer acceptance of meat products is significantly influenced by their texture. In this study, although the hardness values of C and E are higher than those of the control group, the sensory evaluation shows that they are acceptable in terms of texture. Comparing the two groups of samples (C and E), it can be said that L. rhamnosus LR32 200 B has a positive effect on texture when used alone in the production of probiotic salami, but not when used in combination with other cultures.

3.5. Color properties

The color of food plays a crucial role in defining its quality, as well as its retail value and its ability to influence consumers’ opinions about the quality of food and meat (Suryaningsih et al., 2019). It is well known that a drop in pH and a reduction in oxygen are critical factors in the development of color during the fermentation and maturation of meat products. A decrease in yellowness (b*) values, an increase in redness (a*) as a result of the characteristic red‐cured nitrosomyoglobin formation, and a decrease in brightness (L*) as a result of drying are the changes that occur during this process. In the final product (after fermentation and drying), the lowest L* (brightness) value was found in A (42.10), whereas the highest value was observed in F (48.52). At the end of the storage, the L* values of all samples except F were found to be higher than the initial values (p < 0.05) and the highest L* value was in A (49.21) (p < 0.05). Besides, only the L* value of F was not changed during storage (p > 0.05). When the pH value of the food is close to the isoelectric point of the protein, it lowers the water holding capacity. Therefore, variations in L* value may be caused by differences in pH. Furthermore, the decrease in L* value during the storage can be related to the lower moisture content. Sample F had a higher L* value than the control at the beginning of the storage period. The presence of mixed cultures and the resulting production of lactic acid may be the cause of this increased fermentation activity.

The a* value is an important parameter used to determine the product freshness in meat and meat products (Tang et al., 2006). In this study, the a* values of the salami samples varied between 17.40 and 27.46 (Table 6). The lowest a* value was detected in A (control) (p < 0.05) at the end of the storage. When the a* values of the samples were compared at the end of storage, there was no statistical difference between B and F samples (p > 0.05), whereas the other samples showed this difference to be significant (p < 0.05).

TABLE 6.

Color values of fermented salami samples during storage.

		Color values (L*, a*, b*)
	Days	A	B	C	D	E	F
L*	0	42.1 ± 1.5a,A	46.3 ± 0.1b,A	47.2 ± 0.1b,cB	46.7 ± 0.1bc,B	46.5 ± 0.0bc,B	48.5 ± 0.2c
	15	48.0 ± 0.3b,B	46.5 ± 0.1a,A	47.4 ± 0.1b,B	46.4 ± 0.0a,A	46.4 ± 0.0a,B	47.7 ± 0.5b
	30	43.6 ± 0.1a,A	47.4 ± 0.1c,B	47.2 ± 0.1c,B	46.8 ± 0.1c,BC	45.1 ± 0.5b,A	47.3 ± 0.3c
	45	50.5 ± 0.3c,C	48.2 ± 0.2b,C	46.6 ± 0.1a,A	47.1 ± 0.1ab,D	47.4 ± 0.2ab,C	48.1 ± 0.8b
	60	49.2 ± 0.3c,BC	46.6 ± 0.1a,A	47.4 ± 0.1a,b,B	47.0 ± 0.1ab,CD	46.7 ± 0.1a,BC	48.0 ± 0.8b
a*	0	22.4 ± 0.1a,D	26.2 ± 0.1d,C	24.6 ± 0.1bc,A	24.4 ± 0.7b,AB	25.6 ± 0.1 cd,D	24.7 ± 0.4bc,AB
	15	20.0 ± 0.3a,C	27.5 ± 0.1d,D	26.3 ± 1.0 cd,B	23.7 ± 0.5b,A	25.5 ± 0.1c,D	26.4 ± 0.5 cd,C
	30	18.8 ± 0.1a,B	26.0 ± 0.1d,B	24.8 ± 0.2c,A	23.9 ± 0.1b,A	25.0 ± 0.1c,C	24.8 ± 0.5d,AB
	45	18.0 ± 0.3a,AB	21.5 ± 0.1b,A	24.3 ± 0.1d,A	24.8 ± 0.1d,AB	22.4 ± 0.1c,A	23.8 ± 0.3d,A
	60	17.4 ± 0.1a,A	26.1 ± 0.0d,BC	27.0 ± 0.1e,B	25.5 ± 0.1c,B	24.5 ± 0.0b,B	25.9 ± 0.3d,BC
b*	0	7.1 ± 0.1b,C	5.9 ± 0.4a,A	6.1 ± 0.1a,A	6.0 ± 0.0a,A	6.0 ± 0.0a,AB	6.5 ± 0.3ab
	15	6.2 ± 0.0a,B	7.3 ± 0.1b,C	7.8 ± 0.4b,C	6.1 ± 0.1a,A	5.9 ± 0.0a,A	7.0 ± 0.5b
	30	4.9 ± 0.1a,A	6.9 ± 0.1e,B,C	6.8 ± 0.1d,e,B	6.3 ± 0.1bc,B	6.0 ± 0.1b,B	6.5 ± 0.2 cd
	45	7.1 ± 0.1d,C	5.6 ± 0.0a,A	6.1 ± 0.1a,b,A	6.2 ± 0.1bc,B	5.9 ± 0.1ab,A	6.7 ± 0.3c
	60	5.0 ± 0.1a,A	6.7 ± 0.1b,c,B	7.4 ± 0.2d,B,C	6.9 ± 0.1cd,C	6.2 ± 0.1b,C	6.6 ± 0.4bc

Note: Values are the mean ± SD. Values in the same row with different lowercase letters (a, b, c, and d) show a significant difference (p < 0.05). Values in the same column with different uppercase letters (A, B, C, and D) show a significant difference (p < 0.05). Statistical analysis was evaluated separately for L*, a*, and b* values.

At the beginning, there was no significant difference between the b* values of the samples, except A, produced without any culture addition (p > 0.05) (Table 6). However, among the samples, the highest b* value was 7.77 on the 15th day of C, and the lowest value was 4.85 on the 30th day of A. During storage, only b* value of A was significantly decreased (p < 0.05), whereas this value was increased in B, C, D, and E (p < 0.05). Besides, as observed in L* value, the b* value of F was not changed during storage (p < 0.05). Comparing the color values with the sensory analysis results, it can be seen that the samples with the highest L* and a* value among the probiotic salami samples are C and F, and these samples also have a high color score. Similarly, Table 8 shows that these samples have high levels of overall acceptance.

TABLE 8.

Sensory properties of fermented salami during storage.

		Sensory evaluation scores
	Days	A	B	C	D	E	F
Color	0	8.2 ± 0.1a,A,B	8.4 ± 0.1b	9.3 ± 0.1d,B	9.1 ± 0.1d	8.1 ± 0.1a	8.8 ± 0.1c
	15	8.0 ± 0.1a,A	8.4 ± 0.1a,b	9.1 ± 0.2c,A,B	8.9 ± 0.1b,c	8.3 ± 0.3a,b	8.8 ± 0.1b,c
	30	8.3 ± 0.1a,B	8.4 ± 0.1a	9.1 ± 0.1b,A,B	9.0 ± 0.1b	8.4 ± 0.3a	8.8 ± 0.1a,b
	45	8.0 ± 0.1a,A	8.3 ± 0.2a,b	9.0 ± 0.1c,d,A,B	9.1 ± 0.1d	8.1 ± 0.1a	8.7 ± 0.2b,c
	60	8.1 ± 0.1a,A,B	8.3 ± 0.2a	8.9 ± 0.1b,A	9.0 ± 0.1b	8.1 ± 0.1a	8.7 ± 0.2b
Flavor	0	7.6 ± 0.1a,B	8.1 ± 0.1b	9.3 ± 0.2e,B	9.0 ± 0.1d,e	8.5 ± 0.2b,c	8.8 ± 0.2c,d
	15	7.3 ± 0.1a,A,B	8.1 ± 0.1b	9.2 ± 0.1d,B	9.1 ± 0.1d	8.2 ± 0.3b,c	8.7 ± 0.2c,d
	30	7.4 ± 0.1a,A,B	8.1 ± 0.1b	9.0 ± 0.1d,A,B	8.9 ± 0.1c,d	8.4 ± 0.3b,c	8.8 ± 0.3c,d
	45	7.3 ± 0.1a,A,B	8.1 ± 0.1b	8.9 ± 0.1c,d,A	9.1 ± 0.1d	8.5 ± 0.2b,c	8.8 ± 0.3c,d
	60	7.2 ± 0.1a,A	8.1 ± 0.1b	8.9 ± 0.1c,A	8.9 ± 0.1c	8.4 ± 0.3b,c	8.8 ± 0.3c
Texture	0	7.5 ± 0.1a,b	7.9 ± 0.1b	8.3 ± 0.1c,A,B,C	7.4 ± 0.1a,A	7.7 ± 0.2a,b	8.8 ± 0.2d
	15	7.8 ± 0.1a,b	8.1 ± 0.1b	8.6 ± 0.1c,C	7.8 ± 0.1a,b,A,B	7.5 ± 0.1a	8.8 ± 0.2c
	30	7.7 ± 0.2a	8.0 ± 0.2a,b	8.3 ± 0.1b,c,B,C	8.0 ± 0.1a,b,B	7.6 ± 0.2a	8.6 ± 0.2c
	45	7.5 ± 0.1a	7.9 ± 0.1a,b	8.0 ± 0.1b,A	7.7 ± 0.1a,b,A,B	7.7 ± 0.2a,b	8.5 ± 0.1c
	60	7.5 ± 0.1a	7.7 ± 0.1a,b	8.1 ± 0.1b,c,A,B	7.5 ± 0.1a,A	7.7 ± 0.2a	8.6 ± 0.2c
Overall acceptance	0	7.2 ± 0.1a	8.0 ± 0.1b,A,B	8.7 ± 0.1c,A	8.6 ± 0.1c,A,B	8.1 ± 0.2b	9.0 ± 0.1c
	15	7.8 ± 0.2a	8.1 ± 0.1a,b,B	9.0 ± 0.1c,B	8.9 ± 0.1c,B,C	8.2 ± 0.2b	9.0 ± 0.1c
	30	7.5 ± 0.3a	8.1 ± 0.1b,B	8.9 ± 0.1d,A,B	8.9 ± 0.1d,C	8.3 ± 0.3b,c	8.8 ± 0.1c,d
	45	7.4 ± 0.2a	7.9 ± 0.1b,A,B	8.7 ± 0.1c,A	8.5 ± 0.1c,A	7.8 ± 0.2a,b	8.7 ± 0.2c
	60	7.2 ± 0.1a	7.7 ± 0.2b,A	8.8 ± 0.1c,d,A,B	8.3 ± 0.2c,A	8.3 ± 0.2c	8.8 ± 0.1d

Note: Values are the mean ± SD. Values in the same row with different lowercase letters (a, b, c, d, and e) show a significant difference (p < 0.05). Values in the same column with different uppercase letters (A, B, C, and D) show a significant difference (p < 0.05).

In a study performed by Slima et al. (2018), it was determined that the a* and b* values of sausages produced with probiotic L. plantarum remained stable during storage, and this result linked with the high spice content of the product. In the study, it was also reported that the L* value of all samples increased until the 4th day of the storage. This result explained by protein coagulation, which cause increase in the release of water on the surface and thus increase light scattering. Similarly, the L* values of fermented salami samples produced in this study increased during storage, except F. It was reported that fermented sturgeon sausages produced with probiotics showed higher brightness, redness, and yellowness values when compared to the control groups (Wang et al., 2015). In another study, L. plantarum TN8 (8.0 log CFU/g) addition did not affect the L* value of fresh fermented sausage but significantly reduced the a* and b* values when compared to the control group (Slima et al., 2017).

In general, pH and a_w affect the L* value. The presence of free water on the surface of the meat product also affects the L* values; therefore, these values will vary depending on the area or method of measurement. Another reason for the increased redness could be that the inoculated probiotics are able to reduce nitrites, allowing them to continue producing red pigments. On the other hand, the b* value (yellow/blue) is mainly affected from lipid oxidation.

Results indicate that inoculation with L. rhamnosus positively impacted the color, flavor, and overall acceptance of the probiotic fermented salami. Consequently, the use of different strains, the use of mixed cultures, and the different fermentation abilities of the strains have resulted in differences in the color properties of the product.

3.6. Microbiological properties

Pathogen (Salmonella spp., L. monocytogenes, and E. coli O157:H7) detection as well as TMAB, total coliform bacteria, and mold‐yeast counts were applied. Microbiological results are an indicator of product quality. Total coliforms, TMAB, and mold‐yeast counts are influenced by the processing conditions and the hygienic quality of the raw materials. High TMAB, mold‐yeast, and coliform counts indicate poor hygienic conditions during production. Monitoring the counts of these groups of microorganisms in salami helps to ensure the safety and quality of the product. On the other hand, TMAB count greater than 6.00 log CFU/g indicates that the product contains dominating microorganisms. However, if LAB has been used as a processing aid, there are no restrictions. Fermented products are produced by the addition of intentionally added bacterial cultures. These bacteria are the predominant microorganisms in the product and other bacteria are usually present in lower amounts due to the high acidity of the fermentation process (Libera et al., 2015; Trząskowska et al., 2014).

During storage, all salami samples complied with the Turkish Food Codex Microbiological Criteria Regulation, which states that Salmonella spp., L. monocytogenes, and E. coli O:157:H7 should be negative in fermented meat products (Anonymous, 2011). The total coliform bacteria and mold‐yeast counts of the samples were below the detection limit, indicating that good manufacture practices have been applied. On the other hand, it could also be linked with the inhibitory compounds (e.g., bacteriocin, organic acids, and hydrogen peroxide) produced by probiotics, which inactivate or inhibit the microorganisms. Bacteriocin producing strains have been described and used in production of fermented meat products to inactivate Listeria spp. Hugas et al., 1995). According to Gómez et al. (2016), the presence of L. lactis 368 (bac−), L. curvatus MBSa3 (bac+), and L. sakei MBSa1 (bac+) reduced pathogen counts by more than six logs compared to controls. Probiotic colonization of hard surfaces is another extremely promising strategy for controlling pathogen growth and preventing pathogen proliferation. Hashemi et al. (2023) stated that LAB strains (L. pentosus PTCC 1872, L. paraplantarum PTCC 1965, and L. plantarum PTCC 1745) caused a decrease in pH and, as a result, inhibited the growth of pathogens (S. Typhi PTCC 1609 and S. aureus PTCC 1826), which improved consumer health and increased the food safety of fermented meat.

TMAB counts of the samples were in the range of 7.87–14.93 log CFU/g (Table 7). The lowest TMAB count was obtained from the sample produced using two different probiotic cultures (F), whereas C, the sample produced with L. rhamnosus, had the highest count. All sample groups, with the exception of F, had a decrease in TMAB counts during storage, and in C, D, and E, this decline was statistically significant (p < 0.05) (Table 7). At the end of storage, the lowest TMAB count (7.55 log CFU/g) was found in A, which is the sample produced without culture. The sample produced with L. rhamnosus had the highest count (8.36 log CFU/g) (Table 7). These results are in parallel with the findings of Libera et al. (2015), who determined the TMAB counts of the Polish‐specific dried neck meat samples containing B. lactis BB‐12 as a probiotic (6.08–6.11 log CFU/g) were higher than the control group (5.82 log CFU/g). The microbiological properties of Spanish type fermented sausages produced with the addition of L. rhamnosus GG and L. plantarum 299 V were also investigated by Rubio et al. (2013) and Enterobacteriaceae count was found to be 6.43 log CFU/g in the samples produced without probiotic culture, whereas this count was determined below the detection limit in the samples containing probiotic. All these results show that using probiotics in the receipts provide safety of meat products.

TABLE 7.

Microbiological properties of fermented salami samples during storage (log CFU/g).

			Microbiological properties
	Days		A	B	C	D	E	F
TMAB	0	11.1 ± 1.6ab		10.5 ± 2.0ab	14.9 ± 0.4b,C	14.9 ± 0.4b,B	12.4 ± 2.1b,B	7.9 ± 0.0a,A
	15	11.9 ± 2.0a		11.7 ± 2.4a	12.7 ± 2.5a,BC	12.7 ± 2.9a,AB	8.4 ± 0.0a,A	8.2 ± 0.3a,AB
	30	9.8 ± 1.4ab		10.3 ± 0.6ab	12.0 ± 1.2b,ABC	11.1 ± 1.2ab,AB	9.9 ± 0.6ab,AB	8.6 ± 0.2a,B
	45	9.0 ± 0.6a		8.4 ± 0.5a	9.0 ± 0.4a,AB	10.6 ± 0.1b,AB	9.5 ± 0.4ab,AB	8.4 ± 0.1a,B
	60	7.6 ± 0.2a		7.9 ± 0.0a	8.4 ± 0.2a,A	8.3 ± 0.5a,A	8.3 ± 0.3a,A	8.3 ± 0.1a,AB

Note: Values are the mean ± SD. Values in the same row with different lowercase letters (a and b) show a significant difference (p < 0.05). Values in the same column with different uppercase letters (A, B, and C) show a significant difference (p < 0.05).

Abbreviation: TMAB, total mesophilic aerobic bacteria.

Fermented salami is a highly valued traditional food. The high moisture content (70%–80%) and availability of proteins, peptides, amino acids, and minerals make raw meat a suitable medium for the growth of pathogens. Traditional methods of producing fermented salami may not guarantee microbiologically safe products. Dry or semidry products have been linked to several foodborne outbreaks. Therefore, the safety of salami must be guaranteed to protect the health of consumers (Holck et al., 2017).

Physicochemical characteristics (e.g., a_w, pH, and salt content) affect the microbiological quality of salami. The pH drop that occurs during fermentation prevents the growth of undesirable microorganisms that can grow at neutral pH. In addition, maintaining this pH during storage ensures microbial quality and safety. The salt used also has an antimicrobial effect on undesirable microorganisms. Factors, such as low pH, salt, and low moisture content, create a hurdle effect and prevent the growth of these microorganisms.

Probiotics have capable of inhibiting or inactivating the growth of pathogens associated with meat. Therefore, they are mainly used to improve the safety of dry fermented meat products. This is mainly due to their ability to aid acidification and produce antimicrobial metabolites (e.g., bacteriocin). The three main candidates for use as probiotic starter cultures in salami production are L. rhamnosus LR32, L. rhamnosus R0011, and L. paracasei Lpc‐37 (Agüero et al., 2020; Gómez et al., 2016).

3.7. Probiotic viability of the samples

LAB counts of fermented salami samples were detected during storage (Figure 2). At the end of the storage, LAB counts of A (sample produced without starter culture) decreased by 2.17 log CFU/g in comparison with the beginning (p < 0.05). Similarly, the count of B (sample containing non‐probiotic starter culture) was significantly decreased from 6.87 to 5.45 log CFU/g at the end of the storage (p < 0.05) (Figure 2).

FIGURE 2.

Lactic acid bacteria (LAB) counts of fermented salami samples. Values are the mean ± SD. Different lowercase (a, b, and c) indicates the statistical difference between samples (p < 0.05). Different uppercase (A, B, and C) indicates the statistical difference between storage times (p < 0.05).

When L. rhamnosus 32 200B was used in the formulation (C), LAB count of the sample was increased during storage from 7.41 to 9.67 log CFU/g in the first 30 days of the storage. Although LAB counts decreased by 1.0 log CFU/g between 30th and 45th days of the storage, it completed 60 days of storage at 8.26 log CFU/g. According to Jofre et al. (2015), L. rhamnosus CTC 1679 strain in fermented salami product remained viable at 8.0 log level in the final product after 75 days of storage. In another study, the count of LAB in fermented sausages produced with low acidity, reduced sodium, and fat content using probiotic strains (L. casei CTC 1677, L. casei CTC 1678, L. rhamnosus CTC 1679, L. plantarum 299 V, L. rhamnosus GG, and L. casei Shirota) remained constant at around 8.0 log CFU/g during 74 days of storage, where L. rhamnosus CTC1679 was found as the predominant strain (Rubio et al., 2014). Similar results were also obtained by Bis‐Souza et al. (2020), when probiotic L. casei SJRP66 and L. casei SJRP169 strains were used in the production of Italian‐type low‐fat fermented salami. These results show that fermented salami/sausage could be a suitable food product that can be used for probiotic intake with no sensory problems.

LAB counts of D were increased from 6.93 to 10.30 log CFU/g in the first 15 days of storage. After 15th day, LAB counts decreased by 2.0 log CFU/g until the end of 30th day and completed the storage at 8.40 log CFU/g. In a study performed by Pavli et al. (2020), L. plantarum L125 (8.0 log CFU/g) was used as a starter culture during the fermented sausage production. As parallel in our results, the LAB count found in the samples enriched with probiotic was 7.46 log CFU/g, whereas the count of LAB in the control group decreased significantly to 5.01 log CFU/g at the end of 182 days of storage at 4°C. In another study, the microbiological, sensory, and chemical properties of sausages produced with the addition of probiotic L. plantarum TN8 and P. acidilactici MA 18/5 M (at 7.0 log CFU/g) were examined during storage at 4°C for 12 days. The LAB counts of the samples containing probiotics at the beginning of the storage were 2 logarithmic units higher than the control group. At the end of the storage, LAB count was 8.45 log CFU/g in the sample containing probiotic L. plantarum TN8 while this count was 8.22 log CFU/g in the sample containing P. acidilactici MA 18/5 M, and LAB counts of probiotic added products were significantly higher (2.0 log CFU/g) than the control group (Slima et al., 2018). Zhang et al. (2020) investigated the effects of bacteriocin‐producing L. plantarum LPL‐1 strain on the production of salt‐reduced fermented sausage. The results revealed that the use of L. plantarum LPL‐1 changed the bacterial composition of the product, reduced the diversity of microflora, and inhibited spoilage bacteria in the fermentation and maturation stages.

LAB counts of E produced with the addition of B. lactis BB12 were 6.85 log CFU/g at the beginning of the storage, whereas it increased to 8.28 log CFU/g at the end of 45th day and completed the storage at the level of 6.93 log CFU/g. Similarly, the viability of probiotic B. lactis BB12 (6.3 and 6.6 log CFU/g) added in Polish dried neck meat was investigated during maturation at 4°C for 12 months (Libera et al., 2015). At the end of the period, the LAB counts in the samples were determined as 5.32 and 6.18 log CFU/g. L. acidophilus and B. lactis viability in Italian fermented salami was investigated in a study. It was stated that L. acidophilus and B. lactis were present in the product at the level of 9.0 log CFU/g at the end of the fermentation (Ruiz et al., 2014).

When L. rhamnosus 32 200B and L. plantarum 115 400B were used combined as a probiotic mixture (1:1) (F), LAB counts were detected at around 8.0 log CFU/g during storage (p > 0.05). These results showed that LAB values of the samples produced by the addition of probiotics (C, D, E, and F) met 6.0 log CFU/g live bacteria requirement, which is a critical level for the product to be identified as probiotic.

As a result, the initial LAB counts of the fermented salami samples varied between 6.87 and 8.05 log CFU/g, the highest LAB count was observed in F, which produced by different probiotic cultures, and the lowest count was observed in control sample (B, produced by non‐probiotic culture). When the beginning and the end of the storage were compared, it can be observed that the LAB counts of C, D, E, and F were not significantly changed (p > 0.05). However, LAB counts of control samples (A and B) were significantly decreased during storage (p < 0.05) (Figure 2). However, Muthukumarasamy and Holley (2006) added probiotic L. reuteri to sausage dough at 7.0 log CFU/g with and without encapsulation and found that at the end of the fermentation and maturation stages, the count of L. reuteri decreased by 0.5 log CFU/g in the encapsulated cells and 2.0 log CFU/g in the free cells. The count of live free probiotics basically decreased under 6.0 log CFU/g level, whereas encapsulated cell count remained above 6.0 log CFU/g. In another study, L. plantarum MF1291, L. plantarum MF1298, L. plantarum DC13, L. pentosus MF1300, and L. salivarius DC5 were presented to the volunteers as a mixture or added to fermented sausage. The results showed that the probiotic viability in the GI system increased when consumed with sausage, and this was explained as the sausage matrix protecting the bacteria from the acidic environment of the host.

The viability of probiotics in salami is influenced by a wide range of factors such as pH, hydrogen peroxide, organic acids, natural microflora, temperature, oxygen, moisture, salt, sugar, and additives (Rouhi et al., 2013). For example, low pH is one of the most important factors limiting the growth and stability of probiotics. a_w is another critical factor affecting the growth, activity, and even survival of microorganisms. On the other hand, when choosing a suitable starter culture, it is important to consider not only the characteristics of each probiotic strain but also how these strains interact in a mixed culture. The ideal strains should be able to properly ferment the carbohydrates and have an acceptable acidification rate. They should also be resistant to the potential antagonistic effects of other cocultured strains, tolerate certain deleterious characteristics of fermented salami, such as low pH and the presence of salt and nitrate/nitrite, and not adversely affect the sensory qualities of the product. One of the most important factors in the viability of probiotics is the fermentation temperature. Optimum growth occurs at 40–42°C for Lactobacillus and 37–41°C for Bifidobacterium. In this study, L. plantarum and L. rhamnosus were found to be the cultures that were better adapted to environmental conditions and maintained a high level of viability. The high viable cell counts of the L. rhamnosus and L. plantarum, at the end of the storage (60 days), indicate that the probiotic strains proliferated more rapidly than the commercial culture and B. lactis. This is due to differences in the strains used and resistance to environmental conditions.

Product properties, probiotic strains, storage, and fermentation conditions affect cell viability. In the current study, the selected probiotics were found suitable to keep their viability in the selected product receipt without the need for any protection technique such as microencapsulation. However, according to O'Hara and Shanahan (2007), depending on the strain, probiotics have the ability to affect human health at three different levels: (i) by competing with other microorganisms for nutrients, producing antimicrobial agents; (ii) by strengthening mucosal barriers; and/or (iii) by altering the host immune system. Probiotics should be consumed in foods sufficiently (>10⁸–10⁹ CFU/g) to have a beneficial effect. Therefore, future studies should include in vivo studies to investigate the health effects of these products. Besides their contribution to human health, probiotics used in fermented meats are known to have a positive effect on the microbiological quality and sensory properties of the product through the acidification of the final product when appropriate strains are used in sufficient amounts (Muthukumarasamy & Holley, 2006; Pidcock et al., 2002).

Consequently, the fact that these products maintain high levels of probiotic viability during storage indicates their potential for commercialization, especially salami produced with L. plantarum or L. rhamnosus, which can be offered to consumers as a functional meat product.

Some researchers have examined in vitro and in vivo health effect of probiotic fermented meat consumption. In one study, after 7 days of storage, all probiotic (L. plantarum) camel sausages, with the exception of the control, showed up to a 70% increase in cytotoxicity activity against the Caco‐2 cell line (Ayyash et al., 2019). A 4‐week human intervention involving the daily consumption of 30 g of probiotic salami was carried out by Perez‐Burillo et al. (2020) on 24 healthy volunteers. The study investigated whether a salami fermented with the probiotic L. rhamnosus HN001 and added citrus fiber would have any health benefits. A probiotic effect was evident as L. rhamnosus was found in all samples from the intervention group but not in any samples from the control group. They stated that after consumption of the reformulated salami, there was a reduction in inflammation, an increase in butyrate synthesis, and an improvement in plasma antioxidant indicators.

3.8. Sensory properties of the samples

Probiotics influence the aroma and flavor of fermented meat products through their enzymatic activity. The enzymatic activity of probiotics is a useful tool for quickly recreating the distinctive flavor of meat products and can also increase the variety of product flavors. Improving the sensory quality of a product could be achieved by using carefully selected probiotic strains with lipolytic and/or proteolytic activity, which can produce strong aroma compounds (Rouhi et al., 2013). The unique flavor and texture of fermented meats are influenced by a variety of substances, including peptides, amino acids, carbonyls, volatile flavor compounds, and degradation products of lipolysis and proteolysis. Color is one of the factors that visually influences consumer preferences. When the color properties of the salami samples were evaluated, the samples produced with probiotics were more liked than the control group (A). Among the salami samples produced using probiotic cultures, the most liked samples were C and D (p > 0.05), whereas the least liked one was E (p < 0.05) in terms of color properties during storage (Table 2).

The flavors of the samples produced with probiotics were more liked than the control groups (A and B). Among the salami samples produced using probiotic cultures, the most liked samples were C and D (p > 0.05), whereas the least liked one was E (p < 0.05) in terms of flavor properties as observed in color properties during storage (Table 2). These results demonstrated that the use of probiotics improved the flavor properties of the product.

The most liked sample in terms of texture characteristics was F, whereas C was also liked in the second order (Table 8). On the other hand, no statistical difference was found between the texture scores of A, B, E, and F during storage (p > 0.05).

According to the evaluation of the salami samples in terms of overall acceptance, the most admired samples were obtained as C (on the 15th day) and F (on the 0th and 15th days). There was no significant difference between C, D, and F during storage (except for the 60th day evaluation) (p > 0.05), whereas A and B produced without probiotic addition were less acceptable at the end of the storage (p < 0.05) (Table 8). Besides, the general acceptance scores of A, E, and F did not change statistically during storage (p > 0.05).

Similarly, Pavli et al. (2020) reported that fermented sausage samples produced with the use of L. plantarum L125 were more appreciated than other sample groups. In addition, the structural and sensory properties of the products enriched with probiotics (L. plantarum TN8 and P. acidilactici MA 18/5 M) have been improved (Slima et al., 2018). However, in another study, no significant sensory differences were found between the control and probiotic L. reuteri (7.0 log CFU/g) containing sausage samples (Muthukumarasamy & Holley, 2006). Coelho et al. (2019) also reported that the a_w, pH, moisture, acidity, and LAB counts of fermented dried salami samples produced using probiotic L. paracasei (8 log) and prebiotic lactulose (3%) were not affected by the functional ingredients used in the formulation and there was no difference in the sensory properties of the product compared to the control group.

In another study, fermented sturgeon sausages produced by probiotics (L. paraplantarum S4, L. paraplantarum L‐ZS9, L. plantarum P, L. plantarum Y9, L. plantarum pl2, L. pentosus 31‐1, L. rhamnosus LGG ATCC53101, and L. acidophilus N4) were evaluated for sensory properties. The general acceptance scores of all sausage samples were found to be higher than 4 (in 5 scale). It was determined that the sausage sample produced with L. plantarum in terms of texture and color properties, and the sample produced with L. pentosus in terms of aroma properties were the most appreciated samples. As the sausage sample produced with L. pentosus is less appreciated in terms of texture and color properties compared to L. plantarum, it has been reported that the use of these two strains together will give better sensory results (Wang et al., 2015). These results are in parallel with our results, which revealed that the samples containing probiotics were more appreciated in terms of sensory properties, and therefore the use of probiotics in such products could contribute to the sensory properties of the products. This is because probiotics, through their enzyme systems, break down the carbohydrates, proteins, and fats in the product, releasing various compounds that can have a positive or negative effect on the sensory properties of the product. The use of carefully selected strains capable of producing high levels of aroma compounds can improve sensory quality.

4. CONCLUSION

In the study, it is aimed to produce probiotic fermented salami, which is produced without heat treatment and fermented using probiotic cultures. All salami samples complied with the Turkish Food Codex Microbiological Criteria Regulation. The results showed that L. plantarum LP115 400 B, L. rhamnosus LR32 200B, and B. lactis BB12 strains can be used in the production of probiotic fermented salami, which can maintain their viability at high levels (>6 log CFU/g) for at least 60 days at 4°C. The results showed that L. plantarum LP115 400 B, L. rhamnosus LR32 200B, and B. lactis BB12 could be used as potential starter cultures for the production of probiotic fermented salami. It was also shown that, in addition to the natural microflora, a high number of probiotic microorganisms (>6 log CFU/g) can be found in the product during 60 days of refrigerated storage. As probiotics maintain a high level of viability in the products, it is thought that regular consumption of these products may provide the expected health benefits by modulating the gut microbiota. Probiotic‐fermented meats may not only benefit human health. They must also be profitable. In the conducted study, it was revealed that the use of probiotics, especially L. plantarum LP115 400 B and L. rhamnosus LR32 200B, contributed significantly to the sensory properties of salami. It has been concluded that the production of “Probiotic Fermented Salami” can be achieved with the use of L. plantarum LP115 400 B, L. rhamnosus LR32 200B, and B. lactis BB12 strains, as stated in Turkish Food Codex Nutrition and Health Claims Regulation. According to the data obtained, of L. plantarum LP115 400 B and L. rhamnosus LR32 200B strains are the main candidates for use as salami starters because they showed high growth than others. It is thought that these results will make an important contribution to meat industry. The investigation of probiotic strains as starters and the demonstration that the probiotic strain can be used alone, without the need for a commercial culture, were the innovative aspects of this work. The selection of a new starter culture is theoretically supported by considerations of safety and technological properties (such as growth, sensory properties, stability, and viability). It is expected that strains (L. plantarum LP115 400 B and L. rhamnosus LR32 200B) will act as both beneficial microorganisms and starter cultures.

Consequently, the greatest advancement in meat science can be achieved by using probiotic microorganisms as a starter culture. This will increase the safety profile of the product, potentially improve consumer health and possibly change the unfavorable perception of sausages by some consumers, creating the perception of consuming a healthy product. However, further studies should be applied to detect the combined effects of probiotics used in various food products. Moreover, molecular approaches should be used in future studies to prove that these microorganisms are added probiotic strains.

AUTHOR CONTRIBUTIONS

Olcay Tukel: Conceptualization; investigation; writing—original draft; methodology; writing—review and editing. Ilkin Sengun: Conceptualization; investigation; writing—original draft; methodology; validation; writing—review and editing; project administration; supervision.

CONFLICT OF INTEREST STATEMENT

The authors declare no known competing financial interest.