**1. Introduction**

Biogenic amines (BAs) are organic bases with aliphatic, aromatic or heterocyclic structures that can be found in several foods, in which they are mainly produced by microbial decarboxylation of amino acids, with the exception of physiological polyamines. BAs may be of endogenous origin at low concentrations in non-fermented food such as fruits, vegetables, meat, milk and fish. High concentrations have been found in fermented foods as a result of a contaminating microflora exhibiting amino acid decarboxylase activity (Silla-Santos, 1996). However, BAs can also trigger human health problems leading to palpitations, hypertension, vomiting, headaches and flushing if food containing high concentrations are ingested. In fermented foods, some lactic acid bacteria (LAB) are able to convert available amino acid precursors into BAs via decarboxylase or deiminase activities during or following ripening processes. For this reason, amino acid catabolism by LAB can affect both the quality and safety of fermented foods (Verges et al., 1999). The amount and type of BAs formed depends on the nature of food and particularly on the kind of microorganisms present. Enterobacteriaceae and certain LAB are particularly active in the production of BA (Beutling, 1996). These amine-producing microorganisms either may form part of the food associated population or may be introduced by contamination before, during or after processing of the food product. Therefore, microorganisms naturally present in raw materials, introduced throughout the processing or added as starter culture can critically influence BA production during the manufacture of fermented products (Bover-Cid et al., 2000).

Nham is a Thai-style fermented pork sausage. Nham ripening generally takes 3-5 days and relies mainly on adventitious microorganisms, which are normally found in raw materials.

© 2013 Kongkiattikajorn, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

LAB produce organic acids from carbohydrates and cause the pH drop, which contribute to Nham formation. *Micrococcus* and *Staphylococcus* are capable of reducing nitrate to nitrite, which is important in producing the characteristic pigmentation. Also, as a source of lipolytic and proteolytic enzymes, they may contribute to flavor production. Therefore, the acidification and the proteolytic process occurring during Nham ripening make the environment particularly favorable for BAs production.

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 99

*Pediococcus*, *Micrococcus*, as well as to the species *S. carnosus* and *Brevibacterium linens*. They found that this enzymatic activity can be present at very different quantitative levels. Tyramine oxidase activity of several microbial strains was strictly dependent on pH (with an optimum at 7.0), temperature and NaCl, as well as glucose and hydralazine concentration. Moreover, this enzyme was characterised by a higher potential activity under aerobic conditions. Temperature has also an important effect on histamine degradation (Dapkevicius et al., 2000). The highest degradation rate of this amine was observed at 37 C, but at 22C and 15 C, degradation was still considerable. The AO responsible for this degradation has its optimum temperature at 37C and retains about 50% of its maximum activity at 20 C (Schomburg and Stephan, 1993). Many *S. xylosus* strains isolated from artisanal fermented sausages in southern Italy showed the ability to degrade BA in vitro (Martuscelli et al., 2000). Among the strains tested, *S. xylosus* S81 completely oxidised histamine, but it degraded, under the adopted conditions, also a part of tyramine. Even if the AO activity in vitro of microorganisms is not quantitatively reproducible in vivo (due to the more severe conditions and, in particular, to the low O2 tension, pH and salt concentration), reduction of histamine in dry sausages has been observed in the presence of AO-positive staphylococcal starter cultures (Leuschner and Hammes, 1998). In addition, important reduction of the concentration of tyramine and putrescine in the presence of AO positive *S. xylosus* starter cultures have been observed by Gardini et al. (2002). In other words, BA presence in foods is the consequence of a complex equilibrium between the composition of the food and the enzymatic activities of the microbial population. Together with the decarboxylating aptitude of the starter cultures, the presence and relative activity of AO should be considered as an important characteristic in the

selection of starter cultures used in the production of fermented foods.

subsequently during 28 days stored at different temperature was investigated.

**2. Materials and methods** 

**2.1. Microbiological analysis** 

Since Nham is normally consumed without cooking, proper acid production is important to determine the quality and safety of Nham for consumption. Depending on the initial number of contamination, the occurrence of pathogens such as *Salmonella* spp., *Staphylococcus aureus*, and *Listeria monocytogenes* was found specially in Nham with pH higher than 4.6. Due to inconsistency of product quality and ambiguous product safety, improved process of Nham ripening has been developed by using a starter culture technology. Starter cultures are applied to improve and stabilize the quality of the final product and to shorten the ripening period of Nham production. Meanwhile, only little information is available on the effect of starter culture on BA reduction in Nham. Therefore, the objective of this study was to investigate the effectiveness of AOs activity of LAB in inhibiting BA accumulation during Nham ripening. In addition, the change of chemical and microbial properties of Nham during ripening and

Nham sausages (25 g) were aseptically transferred into a stomacher bag, with 225 mL of peptone (0.85% of sodium chloride added) and then homogenized for two minutes. Further decimal dilutions were made and then 100 μL of each dilution was spread onto agar plates.

During meat ripening, microbial growth, acidification and proteolysis provide favourable conditions for BA production. The species of lactobacilli most commonly found in meat and meat products are *Lactobacillus sake* and *Lactobacillus cur*v*atus*, which together with *Lactobacillus ba*v*aricus* and *Lactobacillus plantarum* constitute the main microbial flora isolated from fermented sausages. Other bacteria that can be found in relatively high numbers include enterococci (*E. faecalis* and *E. faecium*), which also contribute to the ripening process. However, the presence of enterococci might also reflect a given level of contamination or a poor curing process. Salt-tolerant, nitrate-reducing coagulase-negative staphylococci are also detected in relatively high numbers in ripened meat products. *Staphylococcus xylosus* is the main species found in Spanish fermented sausages, although *S. carnosus* can also be used as a starter culture. BAs can be degraded through oxidative deamination catalyzed by amines oxidase (AO) with the production of aldehyde, ammonia and hydrogen peroxide. Monoamine oxidases (MAOs) and diamine oxidases (DAOs) had been described from some genus of the family Enterobacteriaceae (Yamashita et al., 1993). The potential role of microorganisms with AO activity had become a particular interest in the last few years to prevent or reduce BA accumulation in food products, especially fermented foods. Mah and Hwang (2009) investigated the effect of *Staphylococcus xylosus* to inhibit BA formation in a salted and fermented anchovy. Reduction of tyramine during ripening of fermented sausages was achieved when *Micrococcus varians* was applied as starter culture (Leuschner and Hammes, 1998). Inoculation of *L. plantarum* in sauerkraut effectively suppressed the production of tyramine, putrescine and cadaverine (Kalac et al., 2000).

BAs are physiologically inactivated by AO, which are enzymes found in bacteria, fungi, plant and animal cells able to catalyse the oxidative deamination of amines with production of aldehydes, hydrogen peroxide and ammonia (Cooper, 1997). The sequential action (in the presence of an electron acceptor, such as O2) of an AO and an aldehyde dehydrogenase leads to the production of an acid and ammonia, which can be used to support microbial growth (Parrot et al., 1987). MAO and DAO activity has been described in higher organisms as well as in bacteria (Murooka et al., 1976, 1979; Ishizuka et al., 1993). There are relevant differences between microbial AO in terms of substrate specificity and location, as stated by Cooper (1997). DAOs can oxidase several BA, such as putrescine and histamine, and their activity can be affected by substrate inhibition; aminoguanidine, antihistaminic drugs and foodborne inhibitors, such as ethanol, carnosine, thiamine, cadaverine and tyramine, reduce their activity (Lehane and Olley, 2000). The potential role of microorganisms involved in food ripenings with AO activity has been investigated with the aim to prevent or reduce the accumulation of BA in foods. Leuschner et al. (1998) tested in vitro the potential amine degradation by many bacteria isolated from foods and, in particular, in strains belonging to the genera *Lactobacillus*, *Pediococcus*, *Micrococcus*, as well as to the species *S. carnosus* and *Brevibacterium linens*. They found that this enzymatic activity can be present at very different quantitative levels. Tyramine oxidase activity of several microbial strains was strictly dependent on pH (with an optimum at 7.0), temperature and NaCl, as well as glucose and hydralazine concentration. Moreover, this enzyme was characterised by a higher potential activity under aerobic conditions. Temperature has also an important effect on histamine degradation (Dapkevicius et al., 2000). The highest degradation rate of this amine was observed at 37 C, but at 22C and 15 C, degradation was still considerable. The AO responsible for this degradation has its optimum temperature at 37C and retains about 50% of its maximum activity at 20 C (Schomburg and Stephan, 1993). Many *S. xylosus* strains isolated from artisanal fermented sausages in southern Italy showed the ability to degrade BA in vitro (Martuscelli et al., 2000). Among the strains tested, *S. xylosus* S81 completely oxidised histamine, but it degraded, under the adopted conditions, also a part of tyramine. Even if the AO activity in vitro of microorganisms is not quantitatively reproducible in vivo (due to the more severe conditions and, in particular, to the low O2 tension, pH and salt concentration), reduction of histamine in dry sausages has been observed in the presence of AO-positive staphylococcal starter cultures (Leuschner and Hammes, 1998). In addition, important reduction of the concentration of tyramine and putrescine in the presence of AO positive *S. xylosus* starter cultures have been observed by Gardini et al. (2002). In other words, BA presence in foods is the consequence of a complex equilibrium between the composition of the food and the enzymatic activities of the microbial population. Together with the decarboxylating aptitude of the starter cultures, the presence and relative activity of AO should be considered as an important characteristic in the selection of starter cultures used in the production of fermented foods.

Since Nham is normally consumed without cooking, proper acid production is important to determine the quality and safety of Nham for consumption. Depending on the initial number of contamination, the occurrence of pathogens such as *Salmonella* spp., *Staphylococcus aureus*, and *Listeria monocytogenes* was found specially in Nham with pH higher than 4.6. Due to inconsistency of product quality and ambiguous product safety, improved process of Nham ripening has been developed by using a starter culture technology. Starter cultures are applied to improve and stabilize the quality of the final product and to shorten the ripening period of Nham production. Meanwhile, only little information is available on the effect of starter culture on BA reduction in Nham. Therefore, the objective of this study was to investigate the effectiveness of AOs activity of LAB in inhibiting BA accumulation during Nham ripening. In addition, the change of chemical and microbial properties of Nham during ripening and subsequently during 28 days stored at different temperature was investigated.

### **2. Materials and methods**

98 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

environment particularly favorable for BAs production.

production of tyramine, putrescine and cadaverine (Kalac et al., 2000).

BAs are physiologically inactivated by AO, which are enzymes found in bacteria, fungi, plant and animal cells able to catalyse the oxidative deamination of amines with production of aldehydes, hydrogen peroxide and ammonia (Cooper, 1997). The sequential action (in the presence of an electron acceptor, such as O2) of an AO and an aldehyde dehydrogenase leads to the production of an acid and ammonia, which can be used to support microbial growth (Parrot et al., 1987). MAO and DAO activity has been described in higher organisms as well as in bacteria (Murooka et al., 1976, 1979; Ishizuka et al., 1993). There are relevant differences between microbial AO in terms of substrate specificity and location, as stated by Cooper (1997). DAOs can oxidase several BA, such as putrescine and histamine, and their activity can be affected by substrate inhibition; aminoguanidine, antihistaminic drugs and foodborne inhibitors, such as ethanol, carnosine, thiamine, cadaverine and tyramine, reduce their activity (Lehane and Olley, 2000). The potential role of microorganisms involved in food ripenings with AO activity has been investigated with the aim to prevent or reduce the accumulation of BA in foods. Leuschner et al. (1998) tested in vitro the potential amine degradation by many bacteria isolated from foods and, in particular, in strains belonging to the genera *Lactobacillus*,

LAB produce organic acids from carbohydrates and cause the pH drop, which contribute to Nham formation. *Micrococcus* and *Staphylococcus* are capable of reducing nitrate to nitrite, which is important in producing the characteristic pigmentation. Also, as a source of lipolytic and proteolytic enzymes, they may contribute to flavor production. Therefore, the acidification and the proteolytic process occurring during Nham ripening make the

During meat ripening, microbial growth, acidification and proteolysis provide favourable conditions for BA production. The species of lactobacilli most commonly found in meat and meat products are *Lactobacillus sake* and *Lactobacillus cur*v*atus*, which together with *Lactobacillus ba*v*aricus* and *Lactobacillus plantarum* constitute the main microbial flora isolated from fermented sausages. Other bacteria that can be found in relatively high numbers include enterococci (*E. faecalis* and *E. faecium*), which also contribute to the ripening process. However, the presence of enterococci might also reflect a given level of contamination or a poor curing process. Salt-tolerant, nitrate-reducing coagulase-negative staphylococci are also detected in relatively high numbers in ripened meat products. *Staphylococcus xylosus* is the main species found in Spanish fermented sausages, although *S. carnosus* can also be used as a starter culture. BAs can be degraded through oxidative deamination catalyzed by amines oxidase (AO) with the production of aldehyde, ammonia and hydrogen peroxide. Monoamine oxidases (MAOs) and diamine oxidases (DAOs) had been described from some genus of the family Enterobacteriaceae (Yamashita et al., 1993). The potential role of microorganisms with AO activity had become a particular interest in the last few years to prevent or reduce BA accumulation in food products, especially fermented foods. Mah and Hwang (2009) investigated the effect of *Staphylococcus xylosus* to inhibit BA formation in a salted and fermented anchovy. Reduction of tyramine during ripening of fermented sausages was achieved when *Micrococcus varians* was applied as starter culture (Leuschner and Hammes, 1998). Inoculation of *L. plantarum* in sauerkraut effectively suppressed the

### **2.1. Microbiological analysis**

Nham sausages (25 g) were aseptically transferred into a stomacher bag, with 225 mL of peptone (0.85% of sodium chloride added) and then homogenized for two minutes. Further decimal dilutions were made and then 100 μL of each dilution was spread onto agar plates.

Aerobic plate count agar was used to determine total aerobic. BA producing bacteria were counted using differential media supplemented with amino acids as precursor of BAs (Joosten and Northolt, 1989). The media contained of tryptone (0.5%), yeast extract (0.5%), sodium chloride (0.5%), glucose (0.1%), Tween 80 (0.05%), MgSO4•7 H2O (0.02%), CaCO3 (0.01%), MnSO4•4H2O (0.005%), FeSO4•7H2O (0.004%), bromocresol purple (0.006%), amino acid (2%) and agar (2%). The medium contained the precursor amino acids (0.5% tyrosine di-sodium salt and 0.25% L-histidine monohydrochoride, L-ornithine monohydrochoride, Llysine monohydrochoride, L- phenylalanine, and L-tryptophan), pyridoxal-5-phosphate as a codecarboxylase factor, growing factors and buffer compounds. All plates were then incubated for 48 h at 37 °C. Bacterial colonies which developed on each agar were then enumerated and expressed as log colony forming unit (CFU)/mL. Only bacterial colonies with purple halo in the differential media were counted as BAs producing bacteria.

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 101

were mixed thoroughly, packed into a plastic casing and sealed before incubation. Two separated batches of fermented sausage were prepared without starter culture and with different starter cultures (*L. plantarum* + *L. sake*) of approximately 107 cell/g. After incubation

The pH was measured directly from samples using a microcomputerized pH meter, inserting the electrode into the middle of the sausage. Moisture was determined by drying the sample at 100–105°C until a constant weight was achieved. The color of Nham was determined by Minolta Model DP-301 colorimeter. Color values (L, a, and b) were measured. A white standard tile was used to calibrate the colorimeter (L= 100.01, a= -0.01, b= -0.02) before measurements. Therefore L measures lightness (luminosity) and varies from white to black. The chromatically (a and b values) gives designations of color as follows; avalue measures redness when positive, gray when zero, and greenness when negative, bvalue measures yellowness when positive, gray when zero, and blueness when negative. The titratable acidity (TA) determined as total acid was estimated according to AOAC (2000) and expressed as g/100 g dry matter. TCA (trichloroacetic acid)-soluble peptide of the fermented sausages was measured by the method of Greene and Babbitt (1990). The oligopeptide content in the supernatant was determined according to by the method of Lowry et al (1951). Results were expressed as μmol/g (dry matter). Free -amino acid was measured using TNBS according to Benjakul and Morrissey (1997) Results were expressed

10 ML of 10% (w/v) trichloroacetic acid (TCA) were added to 3 g-samples, and homogenization of the mixture was effected via shaking for 1 h. The extract was then filtered through Whatman No. 1 filter paper. To remove any fat, the samples were kept at - 20 °C for 1 d, and then centrifuged at 7000 g for 15 min. The supernatants were collected and

Amines were determined by the high-performance liquid chromatography (HPLC) method described by Hernández-Jover et al. (1996). The method is based on the formation of ion pairs between amines extracted with 0.6 M perchloric acid from 5 to 10 g of sample, and octanesulphonic acid present in the mobile phase. Separation is preformed using a reversed phase column, then a postcolumn derivatization with *o*-phthalaldehyde (OPA) is followed by spectrofluorimetric detection. The method allows one to quantify, by an external standard procedure, 6 BAs, i.e., tyramine, histamine, tryptamine, phenylethylamine, putrescine, cadaverine. Samples for BA determination were stored at -15°C until

the fermented sausages were homogenized for analysis.

**2.6. Physical and chemical analyses** 

as μmol/g (dry matter).

**2.7. Extraction of amino acids and BAs** 

filtered through a 0.25 m membrane filter.

**2.8. Determination of BAs** 

required.
