Microorganism species most commonly used as starter cultures in fermented meat productsSOURCE: [15-17].a Used as probiotic cultures.b Used in commercial tests in industrial scale (Laboratorium Wiesby, Niebüll and Rudolf Müller and Co)c formerly known as
The growing concern of consumers regarding the food health and safety issues has led to the development of products that promote health and well-being beyond its nutritional effect . Functional foods are those which promote beneficial effects to human´s health beyond nutrition. Their effects are due to the addition of active ingredients, the removal or the replacement of undesirable compounds in its composition .
The marketing of food for health benefits began in 1960s. In 1970s the trend was to eliminate or reduce the harmful constituents like sugars and fats from food. In 1980s, the trend continued with the reduction or elimination of food additives, which led to the induction and addition of useful components like vitamins, minerals and probiotics in 1990s [1, 3].
Among the different types of functional food, probiotics represent a large share of the functional food market, being used mainly in dairy beverages, cereal products, infant feeding formulas, fruit juices and ice cream [4-7].
In meat industry, the demand for new products has greatly influenced its development, especially for sausage type products. However, lately, those meat products are considered unhealthy by a part of population because of their fat content and the use of additives and spices in their formulation. Therefore, the addition of probiotics to the fermented sausages could promote the health benefits associated with lactic acid bacteria and contribute to the increase in the consumption of such products [7, 8].
The use of probiotics seems more promising in raw fermented meat products like salami as they are made with raw meat and consumed without prior heating, which would kill the probiotic bacteria [9, 10]. However, the incorporation of probiotic bacteria to these products also represents a technological challenge because of the known sensitivity of probiotic to curing salts, spices and other ingredients used in the formulation of the fermented sausages . Furthermore, this addition requires the use of microorganisms that are resistant to the fermentation process and that remain in a minimal viable number of cells to survive the stomach pH and exert beneficial effects in the intestines .
Additionally, the processing of probiotic meat products implies taking into account the appropriateness of the probiotic culture to the target consumer, the intestinal functionality expected for the probiotic species, the rate of survival of probiotic during food processing and the need of maintenance in the probiotic product of the same sensory attributes that characterize the regular product [8, 10, 12].
This chapter presents the potential applications of probiotics in fermented meat products, focusing on the technological challenges, the functional effects of probiotics and on the researches that address the addition of probiotics in fermented meat products.
2. Fermented meat as a probiotic product
2.1. Fermented sausages
The quality of fermented sausages is closely related to the ripening process that gives color, flavor, aroma, and firmness to the product which are developed by a complex interaction of chemical and physical reactions associated with the fermentative action of the microbiological flora present in the sausage. In handmade production processes of fermented sausages, fermentation occurs spontaneously by the action of
Starter cultures are formed by mixing of different types of microorganisms, where each one has a specific function. Lactic bacteria are used in order to generate controlled and intense acidification which inhibits the development of undesirable microorganisms, and provides increased safety and stability to the product. On the other hand, coccus catalase positive type bacteria, as
Table 1 shows the microorganism species most commonly used as starter cultures to fermented meat products.
|Microorganism||Genus and Species|
|Lactic acid bacteria|
Pediococcus acidilactici, P. pentosaceus
Bifidobacterium sp. a
The selection of starter cultures for use in fermented meat products must be carried out according to the product formulation and the technological processing employed, since environmental factors can select a limited number of strains with the ability to compete and overcome on product. Typically, the species used as the starter culture are selected from strains naturally predominant in meat products and hence, well adapted to this environment. Therefore, these species present a tendency to have greater metabolic capacity which is reflected on the development of the proper sensory and physical-chemical characteristics on the product .
Given the adverse conditions of the meat matrix for a number of microorganisms, including those considered probiotics, several studies suggest the selection of probiotic properties in lactic bacteria from commercial starter culture traditionally used in fermented meat products and therefore, already adapted to grow in these conditions. These cultures will provide to the product the same sensory and technological characteristics than the traditional starter cultures, and exert beneficial effects to health [8, 15, 18]. Among the starter lactic acid bacteria,
2.2. Probiotic fermented sausages
Several meat products containing probiotics with claims for health beneﬁts have been commercialized. A salami containing three intestinal LAB (
Fermented sausages are suitable for the incorporation of probiotic bacteria since mild or no heat treatment is usually required by dry fermented meat products, thus providing the suitable conditions required for the survival of probiotics [3, 14, 26]. The sausage has to be designed in such a way as to keep the number and viability of probiotic strain in the optimum range. Thus, reduction in pH (e.g. < 5.0), extended ripening (e.g. >1 month), dry or excessive heating has to be avoided if the beneficial effects of probiotic are to be harvested [3, 7].
In meat sector, meat cultures are generally added to fermented meat products with the function of inhibiting pathogens and increasing shelf-life, rather than introducing functional or physiological qualities. Those cultures are called protective starter cultures and do not promote significant changes in physical and sensory characteristics of the product. On the other hand, probiotic cultures are, by definition, those that after ingestion in sufficient number employ health benefits in addition to their nutritional effects [6, 8, 15]. However, often, the probiotic cultures have also been used in meat products as protective cultures, since both of these cultures have the ability to survive in adverse environments and to produce organic acids and bacteriocins . Likewise, probiotics added to meat products are also known as functional starter cultures since they contribute to safety, can provide sensory and nutritional benefits and promote health .
The success of probiotics in other types of foods, especially dairy products, is based on scientific evidence of beneficial effects provided by some microorganisms. In meat products, the beneficial effects must be proven with the consumption of these products. From the good results obtained with dairy products it is not possible to conclude that a probiotic species will have the same effect on another type of product. This is due to the fact that the performance and properties of microorganisms are environment-dependent. Furthermore, there are few studies about the proper number of probiotic bacteria that should be ingested in meat products to achieve the desired effect [1, 15].
The estimated number of viable cells of probiotic bacteria to be ingested to obtain beneficial effects and temporary colonization of the intestine is around 109 to 1010 CFU/ g of product, in accordance with the counts of 106 to 108 viable cells found in 1 g of feces. Therefore, in a fermented meat product containing 108 CFU/ g, the minimum daily consumption might be 10-100 g of product [1, 29]. Rivera-Espinoza and Gallardo-Navarro  recommended the concentration of probiotic viable cells of at least 108 to 109 CFU/ g of the product to obtain the physiological effects associated with the use of probiotic food.
Despite the known health benefits provided by the use of probiotics such as the improvement of intestinal transit and digestion, improvement of symptoms of lactose intolerance, increase in immune response, reduction of diarrhea episodes, prevention or suppression of colon cancer and reduction of blood cholesterol [30, 31], much attention has paid to the use of probiotics in meat products in order to increase product safety and few studies evaluated the health benefits associated with the consumption of these products [7, 8, 15].
2.3. Most used probiotic cultures in meat products
Probiotics are mainly the strains from species of
In fermented meat products several studies have demonstrated the feasibility of using probiotic
Arihara et al.  studied the use of
Erkkilä et al.  conducted experiments using probiotic strains of
Andersen  demonstrated the ability of mix of a traditional starter culture, Bactoferm T-SPX (Chr Hansen), and the potential probiotic cultures of
Also Erkkilä et al.  used strains of
Macedo et al.  investigated the viability of the use of probiotic
Vuyst et al.  and Khan et al.  stated that
2.3.1. Criteria for the selection of probiotic cultures for meat products
The criteria for a microbial culture to be considered probiotic are the stomach acidity resistance, lysozyme and bile resistance and the ability to colonize the human intestinal tract using mechanisms of adhesion or binding to intestinal cells [7, 8, 23, 35]. Other authors have also included the ability to tolerate pancreatic enzymes as a required characteristic of probiotic cultures .
Additionally to the criteria described above, the probiotic bacteria need to have
However, among the criteria for the selection of probiotic cultures, the main condition to be evaluated is the ability of strains to promote beneficial effects in the host through interactions probiotic/ host and to prevent diseases . These effects on human health may occur in three different ways according to the specificity of the strain: the antagonist action against other microorganisms in the same environment (by nutrient competition, bacteriocin production or competitive exclusion), the barrier effect on the intestinal mucosa and the boosting of immune system [7, 36].
2.3.2. Technological characteristics of probiotic cultures for meat products
For addition in fermented meat products, the probiotic bacteria need to maintain their viability towards the adverse conditions generated during the fermented sausages manufacture: low pH (<5.0), high salt content (2-3%), high nitrite content (around 120 ppm) and low water activity (<0.85). The probiotic cultures should also be capable of growing fast during the fermentation, be easily cultivated on an industrial scale, resist to freezing and lyophilization processes, provide longer shelf life to the product as well as contribute to the sensory quality of the final product [7, 11].
Probiotic cultures can be added in fermented sausage as part of the starter culture or as an additional culture incorporated during the mass mixing (Figure 1).
Probiotic cultures may be added to the sausage batter as liquid inoculum, in high concentrations, or lyophilized. However, the addition of lyophilized culture can delay the fermentation time and reduce the culture viability in the final product. These effects can be reduced with the culture microencapsulation prior to lyophilization. This procedure is also indicated when probiotic strains are inhibited by ingredients of the sausage composition [6, 38].
Microencapsulation increases the viability of bacteria due to the protective effect of a polymeric membrane formed around the bacterial cells. The methods used for microencapsulation of lactic acid bacteria are extrusion and emulsification. Extrusion produces microcapsules with 2-3 mm in diameter which are 60 times greater than the microcapsule formed by emulsification. The materials most commonly used for the microencapsulation of probiotics include alginate, starch, k-carrageenan, guar gum, xanthan gum, gelatin and milk whey proteins. Muthukumarasamy and Holley  tested the microencapsulation of
Rivera-Espinoza and Gallardo-Navarro  encapsulated
22.214.171.124. Lactic acid production
One of the most important characteristics of
However, it is important to confirm that the lactic acid bacteria used as probiotic produce the L(+) isomer lactic acid and do not produce the D(-) isomer lactic acid, due to the higher inhibitory effect on undesirable microorganisms of the L(+) lactic acid. Moreover, the D(-) lactic acid form is not metabolized by the human body and may cause health problems in consumers [7, 14, 42].
126.96.36.199. Resistance to salt (NaCl) and nitrite (NO2)
According to Arihara and Itoh  and Sameshima et al. , the addition of 3% sodium chloride (NaCl) and 200 ppm sodium nitrite (NaNO2) to fermented sausage is mandatory in Japan in order to maintain the microbiological safety of the product. Thus, the use of cultures resistant to curing salts is the first condition for the production of sausage with probiotic properties .
Sameshima et al.  tested the resistance of 202
188.8.131.52. Bacteriocin production in meat products
Bacteriocins are peptides or proteins produced by microorganisms which destroy or inhibit the growth of gram positive bacteria, in particular
184.108.40.206 Resistance to low pH
The tolerance to acidity and bile salts are two fundamental properties that indicate the ability of a probiotic microorganism to survive through the gastrointestinal tract, resisting the acidic conditions of the stomach and the bile salts in the initial portion of the small intestine [22, 45].
The acidity is considered the most important deleterious factor that affects the viability and growth of lactic acid bacteria, since its growth is greatly inhibited at pH lower than 4.5. Such inhibition is related to a reduction in intracellular pH of the bacteria caused by non-dissociated lactic acid form, which due to its lipophilic nature, it diffuses through the cell membrane and causes collapse of the electrochemical gradient, promoting bacteriostatic or bactericidal effects [14, 36].
The survival of the probiotic to the gastric juice depends on its ability to tolerate low pH. At the time of hydrochloric acid excretion, the stomach pH is 0.9, however, during the digestive process the pH increases to around 3 due to the presence of food, remaining under this condition for a period of 2-4 hours [1, 22].
Due to the sensitivity of most bacteria to the low pH of the stomach, probiotic bacteria have to be ingested with food, because it acts as a buffer on the high acidity of the stomach, allowing the survival of the bacteria during gastric transit . Meat, as well as milk, has buffers characteristics in acid environment and can thereby protect the probiotic from the adverse environment of the stomach .
Erkkilä and Petäjä  reported the resistance of species of
Taking into account the pH conditions of stomach and the digestion time, probiotic bacteria ingested with food must be capable of resisting pH value 3 for a period of 2-4 hours to allow their survival during gastric transit. Macedo et al.  found that
Pennacchia et al.  tested the resistance of
220.127.116.11. Resistance to bile salts
Bile plays an important role in intestinal defense mechanism. The intensity of its inhibitory effect on microorganisms is determined by the concentration of salts in the bile composition . Bile salts act by destroying the lipid layer and the fatty acids of the cell membrane of microorganisms. However, some
According to Erkkilä and Petaja  and Pennacchia et al. , the average concentration of bile salts in the human intestinal tract is 0.3%, thus this is the critical concentration used for the selection of probiotic bacteria. Papamanoli et al.  consider as bile salts tolerance when a bacterial population reduces the number of viable cells from 106 - 107 CFU/ mL to 105 CFU/ mL in a 4 hour period.
Erkkilä and Petaja  observed a reduction of 1 log cycle in the initial number of viable cells of
From a total of 63 bacterial strains isolated from fermented sausages, canned fish, bakery dough and jellies, 9 strains of
Macedo et al.  found resistance of
Meat has also been reported to protect microbes against bile . During meat sausage processing,
18.104.22.168. Detoxification capacity of biogenic amines produced in meat products
The biogenic amines, organic bases with aliphatic, aromatic or heterocyclic structures, are produced by the microbial decarboxylation of amino acids present in meat products, either by naturally occurring microorganisms or from the starter culture. The biogenic amines such as histamine, tryptamine, tyramine, cadaverine, putrescine and spermidine can cause toxic effects, especially in consumers with amino oxidase deficiency. In fermented meat products, biogenic amines producing microorganisms have a favorable environment due to the high protein content and the intense proteolytic activity that occurs during the long ripening time of these products. However, some strains of
Ergönül and Kundakçi  found low biogenic amine contents in a Turkish fermented sausage manufactured by using three different probiotic starter culture combinations (
2.4. Beneficial effects associated with the consumption of probiotic meat products
As described earlier, most research involving probiotics in meat products focuses on the survival of probiotic species in the meat matrix and its influence on the technological and sensory characteristics of the final product. Few studies report the effects of consumption of these products on host health . This condition is mainly due to the fact that
One of the few studies reporting the effects of the consumption of probiotic meat product on the human health was carried out by Jahreis et al. . These authors evaluated the effect of daily consumption of 50g of probiotic salami containing
In laboratory animals probiotic administration has shown to decrease the blood cholesterol level and increase the feed-conversion rate 
Other important physiological properties to be considered for the potential probiotics are the adhesive capacity toward Caco-2 cells and the antagonism toward pathogenic organisms .
Klingberg et al.  evaluated the ability of probiotic cultures to colonize the human intestinal tract by
The majority of studies on probiotic meat products focuses on the inhibition of pathogens by probiotics, increasing the safety of meat products. Mahoney and Henriksson  tested the inhibition of colonization and virulence of
Autoaggregation of probiotic strains appears necessary for their adhesion to intestinal epithelial cells and coaggregation presents a barrier that prevents colonization by pathogenic microorganisms. Yuksekdag and Aslim  reported autoaggregation capacity of five
Growth inhibition of
Nedelcheva et al.  demonstrated the ability of
In addition to the studies related to the improvement of the safety of meat products with the use of probiotics, these bacteria have also been assessed for
The combined effect of the addition of probiotics and other active ingredients such as dietary fiber in meat products has also been studied. Sayas-Barberá et al.  reported that the addition of
The fermented sausages fit perfectly in the current consumption trend due to their ease of preparation (ready to eat), ease of conservation, versatility of use (individually or as an garnish in cooking plates), nutritional appeal and variety of forms of presentation . In this regard, probiotic fermented meat products might be the trend setters for development of innovative meat products.
Despite the selling of probiotic meat products occurs since 1998 in countries like Germany and Japan, further human-based studies are needed to establish documented proofs of the beneficial effect of these products, mainly with research on health promotion in humans . Only after these studies will be possible to confirm the intrinsic value of fermented meat products and contribute to the recognition of such products as health foods.