The hygienic quality of meat raw materials and ingredients is crucial to minimize the occurrence of microbial contaminants, and it thus constitutes a key point in controlling aminogenesis in fermented meat products (Maijala et al., 1995; Bover-Cid et al., 2000a,b; Naila et al., 2010). However, hygiene is a necessary, though not sufficient condition and additional technological measures focused on the control of aminogenic activity of endogenous microbiota are usually needed. Among the possible technological strategies, the use of starter cultures is one of the most important factors quantitatively affecting the accumulation of biogenic amines during sausage fermentation (Bover-Cid et al., 2000c; Suzzi and Gardini, 2003; Naila et al., 2010; EFSA Panel on Biological Hazards (BIOHAZ), 2011; Talon and Leroy, 2011). Indeed, the mechanism of starter cultures is based on preventing the outgrowth of the potential aminogenic endogenous bacteria together with their own inability to produce biogenic amines (Lonvaud-Funel, 2001; Suzzi and Gardini, 2003).

Strains of LAB and CNS specifically selected as starter cultures have to comply with some technological criteria, among which the adaptation to meat fermentation, the ability to compete with the natural (endogenous) microbiota of raw materials and the lack of amino acid decarboxylase capability are the most relevant for the control of biogenic amine production (Buckenhüskes, 1993; EFSA Panel on Biological Hazards (BIOHAZ), 2011; Talon and Leroy, 2011). Some LAB and CNS species usually used as meat starters (i.e., L. curvatus and S. carnosus) have been reported as strong biogenic amine producers, mainly of tyramine (Latorre-Moratalla et al., 2010a; Talon and Leroy, 2011). In contrast, species such as L. sakei, L. plantarum, and S. xylosus are usually described as weak or non-aminogenic microorganisms (Bover-Cid et al., 2001a; Aymerich et al., 2006; Latorre-Moratalla et al., 2010a; Linares et al., 2011). However, though some genera or species are more frequently reported than others to be able to produce specific biogenic amines, the ability to decarboxylate amino acids is a strain-dependent property (Bover-Cid and Holzapfel, 1999; Lonvaud-Funel, 2001; Linares et al., 2011). Therefore, it is necessary to carry out a case-by-case evaluation of the aminogenic activity of the strains to be selected as amine negative starter culture. For this purpose, several procedures have been reported (Marcobal et al., 2006; Landete et al., 2007; EFSA Panel on Biological Hazards (BIOHAZ), 2011). Amino acid decarboxylase potential might be tested by molecular techniques detecting specific genes coding for amino-acid decarboxylase. Nevertheless, the aminogenic potential of a given strain should be confirmed through the study of the phenotypic expression of this activity, both in vitro (as a screening procedure) and finally in real fermentation and ripening conditions (EFSA Panel on Biological Hazards (BIOHAZ), 2011).

Several studies have evaluated the use of commercial and experimental starter cultures in order to reduce aminogenesis during the fermentation of sausages. Although a number of studies have demonstrated the beneficial effect of starter cultures in reducing biogenic amine accumulation (Maijala et al., 1995; Hernández-Jover et al., 1997b; Bover-Cid et al., 1999b; González-Fernández et al., 2003; Gençcelep et al., 2007; Gücükoglu and Küplülü, 2010; Lu et al., 2010; Baka et al., 2011), other studies failed to demonstrate the efficiency of starter cultures to reduce the presence of biogenic amines in some fermented meat products (Rice and Koehler, 1976; Buncic et al., 1993; Bauer et al., 1994; Paulsen and Bauer, 1997; Roig-Sagués et al., 1997; Parente et al., 2001; Bozkurt and Erkmen, 2002).

Table 2 summarizes the relative reduction of biogenic amines by the use of negative amine producer starter cultures obtained from different experiments. In these studies, different percentages of biogenic amine reduction are observed depending on the bacterial species inoculated, varying from 9% to practically 100%. Moreover, some starters showed an ability to reduce the production of all amines and in other cases only reduce certain amines (Ayhan et al., 1999; Bover-Cid et al., 1999b; Gençcelep et al., 2007; Coloretti et al., 2008; Baka et al., 2011; Casquete et al., 2011).

In general, starters containing LAB species showed a higher effectiveness in biogenic amine reduction than starters including only CNS (Bover-Cid et al., 1999b, 2001b). LAB starter cultures could exert a more efficient replacement of endogenous microbiota with potential aminogenic ability, usually consisting of lactobacilli and enterococci. In particular within starters including LAB species, those containing L. sakei or L. plantarum are reported to significantly inhibit amine accumulation, though with different intensity depending on the strain and product (Hernández-Jover et al., 1997b; Coloretti et al., 2008; Latorre-Moratalla et al., 2010b; Baka et al., 2011; Tosukhowong et al., 2011). Within CNS, although their proteolytic activity could stimulate the aminogenesis by means of providing the amino acid precursors, Bover-Cid et al. (1999b) showed the potential of proteolytic staphylococci to inhibit biogenic amine production.

Several studies support a greater efficiency in biogenic amine reduction when L. sakei was used as the starter in comparison with other species (González-Fernández et al., 2003; Gençcelep et al., 2007; Latorre-Moratalla et al., 2010b; Baka et al., 2011). L. sakei are usually well adapted to the ecology of meat fermentation and are competitive between the temperatures of 15 and 25°C, which is the temperature range for sausages manufacture in European Countries (Hugas and Monfort, 1997; Bover-Cid et al., 2001b). In the study carried out by González-Fernández et al. (2003), among all the decarboxylase negative strains tested, L. sakei K29 showed the most efficiency in reducing amine production probably because this strain caused a rapid pH drop during sausage fermentation. Bover-Cid et al. (2001b) also described that the amino acid decarboxylase negative strain L. sakei CTC494 showed a strong ability to reduce biogenic amine formation in Spanish fermented sausage. However, when this same strain (L. sakei CTC494) was combined with S. carnosus LHT 2102, S. xylosus CTC3037 or S. xylosus CTC3050 an even more effective reduction of amine accumulation was achieved compared with the effect of each strain used alone (Bover-Cid et al., 1999b, 2000c). Similarly, Latorre-Moratalla et al. (2010b) described that the rate of reduction was improved when a mixed starter was used. Thus, after the addition of a single strain of S. equorum, the contents of cadaverine were reduced by 45% and the single strain of L. sakei inoculated in the same product was far more effective, reducing cadaverine by 75%. However, both strains (L. sakei and S. equorum) used together as a mixed starter reduced cadaverine by 89%. In fact, mixed starters may perform better than single starters being able to control the growth of different bacterial groups (Bover-Cid et al., 2000c; Latorre-Moratalla et al., 2010b; Naila et al., 2010).

Commercial starters usually used in industrial manufacture may not be fully adapted to the meat fermentation environment or more specifically to traditional fermenting conditions. Nowadays, for the fermentation of artisanal sausages the addition of the so-called autochthonous starter cultures consisting of selected strains originating from each specific fermented meat product is recommended (Benito et al., 2007; Talon et al., 2007; Casquete et al., 2011). Thus, an autochthonous starter helps to maintain the typical unique characteristics of artisanal products (Talon et al., 2007). This better adaptation and competitiveness of autochthonous starters compared with commercial ones could explain the greater reduction of biogenic amine contents in artisanal meat fermented products.

In this line, commercial mixed starters combining different LAB and staphylococci species (Hernández-Jover et al., 1997b; Ayhan et al., 1999; Gücükoglu and Küplülü, 2010) showed poorer amine reductions than those provided by mixed autochthonous starter cultures (Talon et al., 2008; Latorre-Moratalla et al., 2010b; Casquete et al., 2011). Talon et al. (2008) and Latorre-Moratalla et al. (2010b) evaluated the effect of autochthonous starter cultures without amino decarboxylase activity on biogenic amine reduction in different artisanal European fermented sausages. The work demonstrated the importance of case-by-case basis strain selection to obtain a good adaptation to the meat fermentation environment and in turn a good biogenic amine prevention.

It has been suggested that the use of bacterial strains with amine oxidase activity might enhance the reduction of biogenic amine accumulation by metabolizing the amines formed during the fermentation. Amine oxidase activity, metabolizing tyramine and/or histamine under in vitro conditions, has been described in microorganisms involved in sausage fermentation, such as specific strains of LAB (L. sakei or L. plantarum) and CNS (S. xylosus; Leuschner and Hammes, 1998; Martuscelli et al., 2000; Fadda et al., 2001; Gardini et al., 2002). However, under real conditions of sausage fermentation, amine-oxidizing microorganisms have shown a limited effect on tyramine and histamine levels, likely due to a low oxygen availability inside the sausage and/or an insufficient number of amine-oxidizing bacteria, e.g., below the minimum 10⁷ cfu/g, required for amine degradation (Leuschner and Hammes, 1998; Gardini et al., 2002).

The magnitude of reduction of biogenic amine accumulation achieved by a starter culture depends on the factors that determine the presence of endogenous microbiota as well as the competitiveness and implantation of the added starter culture.

It is clearly demonstrated that poor hygienic quality of raw materials and ingredients diminished the protective effect of the amine negative starter culture. Bover-Cid et al. (2001b) reported a significant reduction in the effectiveness of the amino acid decarboxylase negative strain L. sakei CTC494 when sausages were made from raw materials of relatively poor hygienic quality, since high bacterial loads (both of Gram-negative and Gram-positive) increase the number of potentially aminogenic microorganisms and also hamper the implantation and competitiveness of amine negative technological microbiota.

Different processing environment (e.g., pilot plant versus traditional processing plant) and the different type of formulation (e.g., chorizo or fuet) can also significantly influence the performance of the amine negative starter culture. The addition of a mixed starter culture including L. sakei (CTC6469 + CTC6626) and S. xylosus (CTC6013 + CTC6169) with no amino acid decarboxylase activity was successfully used in a fermentation study carried out in pilot plants by Garriga et al. (2005). The starter prevented the accumulation of tyramine, putrescine, and cadaverine by up to 90% of total amines. However, when the same strains (L. sakei CTC6626 + S. xylosus CTC6013) were inoculated into products produced in a traditional real processing plant, the starter culture showed a weaker effect, only slightly reducing the contents of tyramine and cadaverine, by 19 and 46%, respectively (Latorre-Moratalla et al., 2010b).

In the literature, several studies have evaluated the effect of factors that influence starter culture on biogenic amine reduction, such as the type and quantity of sugar added (González-Fernández et al., 2003) or the addition of some additives (Bozkurt and Erkmen, 2007; Coloretti et al., 2008). According to the results reported by González-Fernández et al. (2003), when the starter culture, especially L. sakei K29, was used with a sugar concentration of 0.5 or 1%, the presence of biogenic amines decreased considerably in comparison with the control and low sugar concentration sausage. Bozkurt and Erkmen (2007) described a higher decrease of tyramine, histamine and putrescine contents when a mixture of antimicrobials (nitrite and nitrate), antioxidants, and flavoring and coloring compounds were added with the starter culture mixture of Pediococcus acidilactici, L. plantarum, and S. carnosus.

The influence of processing temperatures on the reducing effect of starter cultures has been fully studied (Maijala et al., 1995; Komprda et al., 2001; Gücükoglu and Küplülü, 2010; Casquete et al., 2011). Gücükoglu and Küplülü (2010) reported that higher ripening temperatures (26°C versus 22°C) potentiated the reduction of putrescine by different starter cultures (L. sakei + S. xylosus B-FM, L. plantarum + S. carnosus TD66, and L. curvatus + S. carnosus + S. xylosus RM-10) in experimental Turkish fermented sausages. However, the fermentation temperatures did not have any significant effect on tyramine reduction. Similarly, Casquete et al. (2011) described an influence of temperatures on the development and activity of autochthonous starter cultures consisting of P. acidilactici MS200 + S. vitulus RS34 and P. acidilactici MS198 + S. vitulus RS34. In this case, higher fermentation temperature (12°C versus 7°C) also obtained the highest reduction of amines, probably due to a better adaptation of the starters to the meat fermentation environment.

In conclusion, the absence of decarboxylase activity should be a criterion for the selection of strains intended for use as starter cultures to obtain fermented sausages free of biogenic amines. Mixed starter cultures consisting of amine negative strains of LAB and CNS species, well adapted to the meat fermentation environment, seem the best alternative. Moreover, the use of autochthonous strains is presented as a promising control measure especially for the manufacture of traditional fermented sausages. High quality raw materials and optimal technological conditions are crucial factors to ensure a proper performance of starter cultures for the reduction of biogenic amine accumulation in fermented sausages.