Fermented sausage production using E . faecium as starter culture : Physicochemical and microbiological profile , sensorial acceptance and cellular viability

Fermented sausages are defined as a mixture of lean meat and fat, curing salts, sucrose and spices, stuffed in a natural or artificial casing and submitted to fermentation and air-drying process. Starter culture and ripening process may affect the quality and acceptability of the final product. Current research evaluates the use of Enterococcus faecium as starter culture in fermented sausage production and its physicochemical and microbiological profile during maturation process, coupled to sausage sensory acceptance after ripening. Enterococcus faecium showed 10.9 log CFU g and remained viable after the ripening period with 8.32 log CFU g. Fermented sausage was monitored during the ripening period by physicochemical (pH control, water activity and weight loss) and microbiological (analysis of coagulasepositive Staphylococcus, coliforms and Salmonella spp.) analyses. All tests complied with standards established by Brazilian legislation and did not interfere in final product quality. Results showed that E. faecium was resistant to curing salt and sodium chloride, maintaining its viability during ripening and conferring beneficial effects on fermented sausage technological characteristics. E. faecium also proved to be in vitro resistant to simulate passage through the human digestive tract. Fermented sausage containing E. faecium had better sensory acceptance than commercial sausage evaluated.


Introduction
Although meat is an important source of proteins in human diet and is consumed worldwide, it is also a rich culture medium for microbe development and fast deterioration due to its high nutrient concentration (Font-i-Furnols & Guerrero, 2014).Thus, the development of meat products as embedded meat is a strategy to increase shelf life and, consequently, new and safe products to consumers.
Brazilian legislation describes embedded meat as products elaborated with fresh meat, edible offals and seasonings, embedded in natural or artificial casings and submitted or not to cooking, drying or ripening (Brasil, 1997).Sausage is an example of embedded meat and is produced by microbial fermentation of lean and fat meat, curing salts (nitrite and nitrate), sodium chloride, sugars and spices (garlic, pepper, nutmeg, etc.), stuffed in natural or synthetic casings followed by ripening under controlled conditions of moisture and temperature (Coloretti et al., 2014, Essid & Hassouna, 2013).
Starter culture comprises non-pathogenic microorganisms capable of fermenting raw material which contains nitrogen compounds and sugar (e.g.meat batter), improves sausage technological (color), sensorial characteristics (flavor, odor) and also maintains the traditional product profile.Further, starter culture promotes reduction in the ripening time and reduces the microbiological contaminant levels, increasing the product´s shelf life and safety (Tabanelli et al., 2012, Lorenzo et al., 2014).
Current research evaluates the use of Enterococcus faecium as starter culture in fermented sausage production, assesses the physicochemical and microbiological profile during ripening process and evaluates the sensorial acceptance of sausage after ripening.

Microorganism and material
Enterococcus faecium (ATCC 8459) was obtained from Tropical Culture Collection André Tosello Research and Technology Foundation in Campinas, São Paulo, Brazil.Spices and curing salts used were purchased from Fego ® Company, while pork meat (ham and bacon) was purchased in a local market in São José do Rio Preto, São Paulo, Brazil.

Fermented sausage production
A standard meat batter was used in fermented sausage manufacturing process, as shown in Table 1.Enterococcus faecium was reactivated in a BHI broth (50 mL) and incubated at 37ºC for 24 hours.The reactivated bacterium was then inoculated in a fresh BHI broth (500 mL) and incubated in same conditions above.E. faecium biomass was recuperated by centrifugation at 8000 rpm 5 min -1 at 4°C and the cell pellet was suspended in sterilized water (30 mL) and added to meat batter.Fermented sausage contained meat batter composed of ham, bacon, spices, curing salts and Enterococcus faecium as starter culture.Sausages were manually embedded in artificial collagen casings and incubated in heated ripening chamber for 28 days under controlled temperature and relative humidity, as shown in Table 2.

Physicochemical analysis
Physicochemical tests were performed after 1, 7, 14, 21 and 28 days of fermented sausage ripening.pH was measured using a digital pH-meter; Water activity (wa) was determined with Novasina-Axair/Switzerland-WA Sprint TH-500; Weight loss by fermented sausage was measured during every ripening period (1, 7, 14, 21 and 28 days).Proximal composition, comprising protein, lipids, moisture and ash, was determined in fermented sausage after 28 days of ripening, according to Cecchi (2001).

Enterococcus faecium survival in gastrointestinal conditions
Survival in gastrointestinal conditions after 28 days of ripening was estimated according to Liserre, Re, and Franco (2007), with modifications.Samples of fermented sausage were ground in a multiprocessor and 10 g were added in Erlenmeyer flasks containing 0.5% saline solution (90 mL -1 ) and four (10 mL -1 ) aliquots was pipetted into previously sterilized test tubes.Further, pH was adjusted to 2.0-2.5 with 0.5 N HCl and solutions of pepsin (3.0 g L -1 ) and lipase (0.9 mg L -1 ) were added in the test tubes and incubated at 37ºC under agitation.E. faecium resistance was determined by CFU containing BHI agar using the pour plate method.
Furthermore, pH was adjusted at 4.3-5.2 and solutions of pancreatin (1 g L -1 ) and bile salts (10 g L -1 ) were added.The tubes were incubated at 37ºC during 240 min, under agitation, whilst cell viability was determined as described previously.Finally, pancreatin (1 g L -1 ) and bile salts solutions (10 g L -1 ) were added and pH was adjusted in 6.7-7.5.Test tubes were then incubated during 360 min and E. faecium viability was evaluated as described above.For this analysis, was performed an independent fermentation.

Sensorial evaluation
Following (Brasil, 2005), current research was submitted to health risk evaluation by the Committee for Ethics in Research of the São Paulo State University (Unesp -Ibilce), São José do Rio Preto, São Paulo, Brazil, with protocol 13014913.7.0000.5466.Sensorial acceptation of sausage samples was assessed by a taste panel made up of one hundred 18-40 year-old untrained tasters.
Acceptance test evaluated appearance, color and aroma of two sausage samples (commercial sausage and sausage produced with E. faecium as starter culture).Sausage samples were presented in a monadic form, aleatory, coded by three-digit numbers in individual cabins.Acceptability index (AI) was evaluated according to the abovementioned attributes.Tasters evaluated whether they liked or disliked the product through a ninepoint hedonic scale (9 = 'I liked it extremely'; up to 1 'I disliked it extremely').

Statistical analysis
Physicochemical and microbiological parameters were submitted to statistical analysis, applying variance test (ANOVA) and Tukey's test at 5%.

Physicochemical analysis
Proximal composition of fermented sausage after 28 days of ripening complied with limits determined by Brazilian legislation (Brasil, 2000) for all evaluated parameters (Table 3).Meat in the manufacture of fermented sausages showed pH rates between 5.8 and 5.9 and thus the raw material was qualified for the elaboration of a safe product.According to Ordoñez et al. (2005) and Andrade (2006), pH rates for normal meat products must not exceed 6.0; rates outside this limit may indicate chemical and/or microbial deteriorations in the product and thus unfit for consumption.
Fermented sausage with E. faecium displayed pH rates between 5.96±0.005and 5.03 ±0.001 during 28 days of ripening.Significant differences (p < 0.05) were reported after seven days of ripening, or rather, pH rates decreased (Figure 1), due to hexoses fermentation and lactic acid formation from lactic acid bacteria.Lücke (2000) highlighted that decreases in pH rates promoted inhibition of undesirable microorganisms, maintaining the product´s microbiological safety.In addition, pH evaluation of meat products is extremely important for the formation of sensorial properties and for the maintenance of the final product´s microbiological safety (Terra, 1997).Initial mean water activity rate in raw meat was 0.99.Since raw meat naturally has higher water activity, it is susceptible to microbial spoilage and consequently yo a shorter shelf life.Since relative moisture is an important factor in the ripening process of fermented sausages, control started from 90% and reduced every two days until 75%, as Table 2 shows.Relative humidity was maintained at 75% to facilitate the drying and water activity reduction process.
Figure 2. Water activity profile in fermented sausages during the ripening process.Mauriello, Casaburi, and Villani (2004) reported that water activity decrease is related to pH decrease, or rather, when pH rates are close to the isoelectric protein point, the water retention capacity (WHC) decreases and dehydration occurs.
As shown in Figure 3, weight loss in the first seven ripening days reached 16.67%, followed by 23.9, 28.87 and 32.5% in 14, 21, 28 ripening days, respectively, with a statistically significant difference (p < 0.05) between the days evaluated.Sausages´ weight loss is a natural consequence during the ripening process and depends on such factors as pH, sausage diameter and incubation conditions in the ripening chamber.Terra (1998) mentioned that dehydration during fermented sausage ripening is essential for the product´s safety, quality and sensorial characteristics.
At the end of the ripening process, fermented sausage had 32.5% weight loss (Figure 3).Coelho, Santana, Terra, and Morandini (2000) reported that sausages may lose up to 40% of their weight during processing.Higher losses may interfere in texture, with the product´s deformation.

Microbiological analysis
Microbiological contaminants were monitored during the ripening process and results are shown in Table 4.Typical colonies of coagulase-positive Staphylococcus were found until the 14 th day of ripening, albeit negative by the coagulase test.The presence of coagulase-negative Staphylococcus may be due to meat batter transformation during the ripening process since the bacterium resists low water activity and high salt concentrations.In addition, starter culture may be inhibited by competition and other microorganisms sensitive to pH and water activity reduction.
Further, the presence of the same species of coagulase-negative Staphylococccus in meat products has been cited by Drosinos et al. (2005) and Cirolini et al. (2010) as desirable since the microorganisms cause color stabilization, rancidity prevention and enhancement of aromatic compounds.
As has been observed in current study, Campagnol, Fries, Terra, Santos, and Furtado (2007) verified that coagulase-positive Staphylococccus and Staphylococcus sp.decrease during the sausage´s ripening process with Lactobacillus plantarum as starter culture.
According to Brazilian legislation (Brasil, 2001), Salmonella sp. was absent in all sausage samples evaluated, during the ripening process.On the other hand, Hoffmann, Garcia-Cruz, Vinturim, and Carmello (1997) found Salmonella sp. in 13.3% of commercial fermented sausage samples evaluated.
Total coliforms in food products indicate hygienic conditions and their decrease during the ripening process has been reported in current analysis.According to Table 4, total and thermotolerant coliforms were found only on the first and seventh ripening days and E. coli was confirmed only in the initial period.
Similar results were found by Terra, Fries, and Terra (2004) who evaluated two fermented sausages samples, one using starter culture and another by spontaneous fermentation of raw material microorganisms.The authors reported coliform inhibition after 7 ripening days only in fermented sausages produced with starter culture.
On the other hand, Coloretti et al. ( 2014) registered increase in Enterobacteriacea from 2.6 on first day to 3.8 CFU g -1 after 30 ripening days in fermented sausage using Pediococcus pentosaceus and Staphylococcus xylosus as starter cultures.
As observed in current study, microorganisms of the Enterobacteriaceae family in raw material could be derived from contaminated animal tissues during slaughter, since several Enterobacteriaceae species have been naturally found in animal gastrointestinal tract (Fernández-López, Sendra, Sayas-Barberá, Navarro, & Pérez-Alvarez, 2008).Barbosa, Borges, and Teixeira (2014) and Bouton, Buchin, Pochet, and Beuvier (2009) highlighted the benefits of Enterococcus faecium use in fermented foods due to its ability to inhibit foodborne pathogens.

Enterococcus faecium viability during ripening process
The initial concentration of Enterococcus faecium in fermented sausage was 10.93±0.03log CFU g -1 and decreased to 10.22±0.07;9.68±0.02;8.61±0.19 and 8.47±0.21 in 7, 14, and 28 ripening days, respectively (Figure 4).Statistical analysis showed significant differences (p < 0.05) from the seventh until the last ripening day.After a 28-day ripening process, Enterococcus faecium remained viable in fermented sausages (8.47±0.21CFU g -1 ), confirmed by electronic scanning microscopy (Figure 5B) showing high cell concentration in the meat matrix after 28 days and fat globules typical of fermented sausages.Figure 5A shows raw material structure containing the same bacterial cells, fat globules and fibrous areas typical of meat muscle structure.

Enterococcus faecium survival during simulation of gastrointestinal condition: in vitro assay
The assay evaluates in vitro microbial resistance at conditions simulating the transit time in the human intestinal tract.As shown in Table 5, after 28 days of ripening, Enterococcus faecium in sausage matrix survived until 360 hours in simulation gastrointestinal conditions, but there was a reduction in E. faecium population during gastric (30-120 min), enteric I (240 min) and enteric II (360 min) phases, with no statistically significant difference (p > 0.05) between enteric phases I and II (Figure 6).
Same letters on the same column do not differ statistically (p < 0.05) by Tukey´s test.The above behavior was registered by Ji et al. ( 2013) using Leuconostoc citreum that showed viability of 2 log CFU mL -1 at enteric phase II, and by Buriti, Castro, and Saad (2010) who added Lactobacillus acidophilus in symbiotic cooled and frozen guava mousse.The authors reported best survival in presence of insulin as a prebiotic compound.
Results on E. faecium survival are very important for future uses of the strain as potentially probiotic bacteria, although specific and detailed tests should be undertaken to this end.

Sensorial acceptance
Table 5 shows results for sensory acceptance of sausages produced with E. facium and commercial sausages, with regard to appearance, aroma and color.According to Dutcosky (2007), a good acceptance range has to be higher or equal to 70%.
According to Simion et al. (2014), the sensorial profile of fermented products is a combination of physicochemical, biochemical and microbiological modifications.
Sausage formulated with E. faecium as starter culture presented an over 70% acceptance for all evaluated parameters, higher than that for commercial sausage, with statistical interference (p < 0.05) only for appearance (Table 5).Results indicate that sausages prepared with E. faecium as starter culture have been well received by tasters and thus have good commercialization potential.Simion et al. (2014) verified better results for aroma when Lactobacillus acidophilus CECT903 and Staphylococcus equorum SA25 were used as starter culture for fermented sausage production of a traditional Romanian dry sausage.

Conclusion
Enterococcus faecium was efficient as starter culture for the production of fermented sausages, with resistance to curing salt and sodium chloride in fermented sausage and viability during ripening process.Fermented sausages showed adequate microbial parameters from the seventh to the last ripening day.The bacterium also revealed to be in vitro resistant for human digestive tract simulation of and higher sensorial acceptance than for commercial sample in relation to aroma, color and appearance.

Figure 1 .
Figure 1.Evolution of pH rates in fermented sausage during ripening process.

Figure 3 .
Figure 3. Sausage weight loss profile in fermented sausage during ripening process.

Figure 5 .
Figure 5. SEM of meat batter (A) and fermented sausage produced with E. faecium as starter culture after ripening process (B).White arrow: same bacterial cells in raw material (A) and high microbial density adhered to fermented sausage matrix (B), grey arrow: meat structure of raw material (A) and fermented sausage matrix (B); black arrow: fat globules present in raw material (A) fermented sausage matrix (B).

Table 2 .
Ripening conditions for sausage incubation.

Table 3 .
Proximal composition of fermented sausage after ripening for 28 days.

Table 4 .
Microbiological contaminants in fermented sausages during ripening process.

Table 5 .
Sensorial acceptance of E. faecium sausages compared to commercial sausage.68.56 6.63 a 73.67 6.48 a 72.00 E. faecium sausage 6.50 b 71.22 6.68 a 74.22 6.70 a 74.44 a Figure 6.Results of in vitro Enterococcus faecium resistance in simulated gastrointestinal conditions.