Preparation, physicochemical and microbiological evaluations of italian type salami fermented with kefir

Industrialized meat product, known as salami, is composed of some types of meat, such as pork, beef or pork and beef together, added ingredients and bacon. This research aimed to elaborate Italian salami using the fermentation process with standard starter bacteria, such as control, water and milk kefir, and evaluate the physicochemical and microbiological characteristics of the final product. The use of kefir grains aimed to analyze the possibility for their use in the production of salami as starter cultures from their symbiotic association of yeasts, acid-lactic bacteria, acetic bacteria and also their probiotic and antimicrobial action. The microbiological and physicochemical composition indicates that kefir is a product with probiotic characteristics, because it has in its composition living microorganisms capable of improving the intestinal microbial balance producing beneficial effects on the health of the individual who consumes it. At the end of the analyses, it was observed that the salami based on standard starter bacteria culture presented greater diversity of fungi, and the fungus Exophiala spp was present in the 3 types of salamis. The samples showed absence of pathogenic bacteria such as Salmonella ssp, Shigella ssp and E.coli. There was little variation in the Research, Society and Development, v. 10, n. 16, e208101623581, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.23581 2 physical-chemical characteristics of the salamis evaluated. It was concluded that kefir can replace industrial fermentation culture without affecting the microbiological and physicochemical pattern of salami.


Introduction
The search of the population for new products for consumption motivates the food industries to commit themselves to the development of new alternatives with greater added value. The meat industry also seeks to be innovating, through new formulations of products with greater health benefits beyond the functional appeal, promoting an increasing demand and demand of the consumer market, thus there is a need for research that gives this support to these industries (Oliveira, et al, 2013;Tonollo, 2016).
Meat products or processed meats are the result of the need to preserve meat since ancient times. Meat, because it has high nutritional value, a high amount of free water and favorable pH is susceptible to microbial development favoring its deterioration. The conventional processes of meat conservation are salting, drying and fermentation, the latter dates back to the Babylonians reaching modern times through salamat (Terra, 2004).
In the fermentation process, microbial cultures are used for the development of the special flavor of the final product, for this we use initiating colonies called starter, these accelerate the maturation process and improve conservation by reducing the pH, developing an acidic flavor characteristic of fermented products, presenting two factors that makes the product differentiated from other sausages, the low moisture content and presence of acids (Leroy & de Vuyst, 2004). As an attractive alternative for the food industry, the total or partial replacement of starter-starter crops with others, for example kefir, can offer sensory, technological and nutritional advantages in addition to contributing to the safety of the elaborated product (Carasi et al, 2014;Muthukumarasamy & Holley, 2007).
The kefir is a probiotic mixture, has its origin from the Caucasian mountains of Russia, resulting from double fermentation: lactic and alcoholic. The beverage, after fermentation contains lactic, formic, succinic and propionic acids, CO2, ethyl alcohol, in addition to different aldehydes and traces of isoamyl alcohol, acetone and a variety of folates. The final product also consists of a suspension of symbiont microorganisms formed by acidophilic bacteria and yeasts, the association of all substances in the final product have several beneficial health effects presenting characteristics peculiar to kefir (Magalhães et al, 2011;Miguel et al, 2010;Moreira et al, 2008).
For the preparation of kefir, cow's milk is generally used, but the milk of buffalo, goat and sheep can be used, presenting as final product yellow grains composed of a heteropolysaccharide complex called kephyde. Water kefir is also found on the market, which is cultivated with brown sugar or fruit juices, obtaining a microbial composition product similar to the grains grown in milk, these have an ochre and brown coloration, being composed of dextran (Gulitz et al, 2011;Hsin-Hui et al, 2012;Otles & Cagindi, 2003;Weschenfelder, 2011;Witthunhn et al, 2004). Some research shows that kefir consumption improves digestion, as it is able to favor the multiplication of probiotic bacteria in the intestine and because it has biotherapeutic substances, due to these factors, stimulation of immune system cells occurs. (Foligne et al, 2010;Guzel-Seydim et al, 2012;Liu et al, 2002;Moreno de Leblanc et al, 2007;Vinderola et al, 2006;Wróblewska et al, 2009).
Thus, this research aimed to develop Italian type salamis using the fermentation process with standard starter bacteria, such as control, water and milk kefir, and the realization of evaluations, physicochemical and microbiological characteristics of the final product.

Drawing of salamis
The experiment was carried out at the Food Technology Laboratory of the Don Bosco Catholic University.
Three formulations of fermented meat were prepared, one control from standard starter culture (0.2%), one with fermentation with water kefir and one with milk kefir. The meat preparations contained 60% pork, 40% beef, and 5g of sodium chloride, 5g of colour fixator, 10g of special condiment for salami and water kefir (0.2%) and milk kefir (0.2%) were added.
The meat was ground in an industrial grinder and the condiments added after grinding. The inlay in synthetic wrap was performed by electric inlay and the salamis were packed in fermentation rooms for 45 days. After the fermentation period, physicochemical and microbiological analyses were performed.

Physical-chemical analysis
The centesimal composition was performed following the methodology described by AOAC (2002), being determined: the moisture content, by the drying method of the samples until constant weight, in an oven at 105 °C; the ashes in the samples, after complete carbonization in incineration in the muffle at 550 °C until the obtention of a coal-free residue, with grayish-white coloration; lipids, by the content of soluble substances in ethyl ether, by means of the Soxhlet lipid extractor apparatus; and the amount of protein, by the micro Kjeldahl method, multiplying the value by factor 6.25.
Water activity (aW) and color of ready-made products were analyzed.
Regarding color analysis, we have: -L: luminosity (the maximum value of L is 100, and represents a perfect diffuse reflection, while the minimum value is zero and constitutes black.); -a: red color intensity ("a" has no specific numerical limits. It ranges from red "+a" to green "-a"); -b: yellow color intensity ("b" has no specific numerical limits. Ranges from yellow "+b" to blue "-b"); -∆E: total color difference.

Microbiological analysis
Microbiological analyses were performed according to the recommendation and requirements of RDC no. 12, of January 2, 2001(Brazil, 2001. The methodology for performing microbiological analyses was based on Normative Instruction Nº 62, of August 26, 2003, from the Ministry of Agriculture, Livestock and Supply, which formalizes the official analytical methods for microbiological analyses for control of animal products and water (Brasil, 2003). The following microbiological analyses were performed: Salmonella ssp count, thermotolerant coliforms and Shiguella ssp.

Methodological report for research
The data presented followed the protocols of teaching, research and scientific methodology, as described by Estrela (2018).

Data analysis
The data obtained were submitted to analysis of variance and their means were compared by the Tukey test, a level of 5% significance. The results were arranged in tables.

Results and Discussion
Meat and its derivatives present a compensation system between protein, lipid and moisture levels (Rech, 2010). Table 1 shows the differences in the centesimal composition between salamis produced with standard starter bacteria and with water and milk kefir. The 3 formulations obtained water activity values lower than the maximum level of 0.90, recommended by Brazilian legislation for Italian type salami (Brazil, 2000). Water activity is a determining factor for the shelf life of products, as it makes the environment unfavorable to the growth and multiplication of decaying and pathogenic microorganisms (Macedo et al, 2008). The salami produced with milk kefir showed higher water activity, which is probably associated with higher luminosity and humidity found, but all samples are in the mean evaluated by the Tukey test. (Table 2). After 45 days, at the end of the maturation period, the color of the salami samples was monitored by means of a colorimeter. The analysis of L* values, referring to luminosity, it was observed that there was no significant difference (p>0.05), the color patterns showed higher *L in the salamis produced with kefir, both water and milk, which indicates higher luminosity of the product and may be associated with higher moisture content found in these products. Luminosity is considered a factor of attraction to the consumer, with color being the first attribute observed at the time of purchase (Gao, Li & Liu, 2014). The L* values presented in this study are below the values found by Campagnol et al, (2007) reported in their experiments at the end of the drying process, ranging from 37.22 to 38.70. Research, Society andDevelopment, v. 10, n. 16, e208101623581, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.23581 6 In this report, the values found a* and b* with the mean values of 7.15; 7.03 and 6.11 for a* and 2.51; 2.72 and 2.61 for b*, for the formulations of starter culture, kefir milk and water, respectively, did not differentiate between salamis, so all presented similar patterns of red and yellow. In Ruiz's work (2011) it was observed that there was a reduction in the value of a* for some Italian-type salami treatments. The attribution of the event may be related to the effect of lactic acid on the different chemical stages of myoglobin, because this acid can partially or totally denature the heme prosthetic group, because with the oxidation of the nitrosomyoglobin pigment the intensity of red color is observed due to the conversion of this into metamyoglobin (Wójciak et al., 2012). And for the yellow color index in the work of Nobile et al., (2009) higher results were reported for b* 3.46 and 4.91, in this experiment the salape were prepared with pork fat substitutes, however the authors report that there was no great evidence of the influence of this substitution on color parameters, possibly this occurred due to the typical heterogeneity in the color of these products. Table 3 shows the results of salmonella spp., thermotolerant coliforms and Shigella ssp for each formulation. In the results obtained, absence of Salmonella ssp was observed in 25g of the sample, the presence of thermotolerant coliforms and absence of Shigiella ssp were not detected. Among the most common bacteria in processed products, in addition to Salmonella ssp, is also Shigella ssp (Germano & Germano, 2003;Vieira et al., 2004), which although not mentioned in Brazilian legislation can generate problems of food infections.
These values found in microbiological analyses reveal that the processing of the 3 formulations of italian type salamis were performed with sanitary quality, a fact that is not always found, especially in small factories in Brazil, with disapproval in microbiological characteristics (Dalla Santa, 2008), so the 3 formulations met the microbiological standards regulated through Resolution nº12, of January 2, 2001, of the National Health Surveillance Agency (Brazil, 2001).
Three types of fungi were found in the samples, in which traditional salami samples showed a greater variety of fungi found and the fungus Exophiala spp was found in the 3 types of salami, as we can see in Table 4. The genus Exophiala is classified as a dimorphic fungus, composed of leveduriform colonies, presenting brown or black color and with dry aspect (Hoffmann, 2011).
Fungi of the genus Aspergillus are classified as filamentous and can be found in all seasons, in soil, vegetables or decomcomposition matter, thus occurring the dispersion of the coheads, which is the infecting form. The infection caused by the fungus is called aspergillosis, which is considered opportunistic, because patients with immunological dysfunction are more vulnerable than other individuals (Amorim et al., 2014;Kousha, Tadi & Soubani, 2011;Raja & Singh, 2006).
Lichtheimia species grow well in artificial media at temperatures between 30°C and 37°C, and can be found on a wide variety of substrates such as agricultural products, hay and straw, as well as food products, including processed and unprocessed ones such as flours and fermented soybeans (Schwartze & Jacobsen, 2014).

Conclusion
It was concluded that the use of kefir as initial fermentation bacteria for salami reached the expected result, being the suitability for use as fermenter, and also making available the probiotic characteristics for the fermented carthon produced product. The physical-chemical analysis showed similarity between the data obtained maintaining standardization between the samples.
The kefir grains of water and milk were able to ferment the bovine product, in the same way as the initial bacteria, usually used for its fermentation. The microbiota that grows in salamas is one of the main responsible for the formation of the characteristics of the final product. The results were satisfactory, resulting in the absence of Salmonella ssp, thermotolerant coliforms and Shigella ssp. The presence of fungal growth in some samples shows us a negative point, but they are opportunistic fungi, which generally do not cause problems for human health. Thus, for further research, it is suggested the study of sensory analysis of products to be observed the acceptance of final products.