**3. Results and discussion**

The effect of starter cultures of LAB on BAs and FAAs content was examined during the ripening process of Nham sausages. Microbial counts, pH and proteolysis-related parameters were also studied. The occurrence of amino acid-decarboxylase activity in 7 strains of LAB isolated from Nham sausages was investigated.


**Table 1.** Strains exhibiting the potential to degrade BAs in a buffer system within 24 h at 30°C

The presence of BAs in a decarboxylase synthetic broth was determined by high performance liquid chromatography with OPA derivatization. Among the 9 LAB strains tested, 5 lactobacilli (in particular, *L. cur*v*atus*) were amine producers and *L. plantarum* and *L.*

*sake*, were non-amine forming strains. The ability of AO exhibiting strains of LAB to degrade amine in vivo during sausage ripening was investigated.

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

Free amino acids (FAAs) in samples were determined using HPLC according to the method proposed by Rozan et al. (2000). A 20 μL aliquot of amino acid standard and digested sauce samples were transferred into vials and dried under vacuum. Then 20 μL of drying reagent containing methanol, water and triethylamine (ratio 2:2:1 v/v) was added. Then 20 μL of derivatizing reagent containing methanol, triethylamine, water and phenylisothiocyanate (PITC) (ratio 7:1:1:1 v/v) was added. The derivatized samples were then dissolved in 100 mL of buffer A that was used as mobile phase for HPLC. A Purospher® STAR RP-18e, 5 μm column was used with buffer A (0.1 M ammonium acetate, pH 6.5) and buffer B (0.1 M ammonium acetate containing acetonitrile and methanol, 44:46:10 v/v, pH 6.5) as mobile phase set for linier gradient at the flow rate of 1 mL/min. The injected sample volume was 20

Data was analysed by one-way ANOVA and differences among treatment means were

The effect of starter cultures of LAB on BAs and FAAs content was examined during the ripening process of Nham sausages. Microbial counts, pH and proteolysis-related parameters were also studied. The occurrence of amino acid-decarboxylase activity in 7

Starter culture Percent degradation (%)

Phenylet hylamine

*Lactobacillus curvatus* 1271 0 0 0 0 0 0 *Lactobacillus farciminis* 1452 0 0 0 0 0 5.7 *Lactobacillus kandleri* 2439 0 0 0 3.6 0 0 *Lactobacillus kefir* 2045 0 0 0 0 0 0 *Lactobacillus plantarum* 9825 0 0 12.6 9.3 0 19.4 *Leuconostoc mali* 7412 0 0 0 0 0 0 *Lactobacillus pentosus* 7054 0 0 5.2 4.7 0 0 *Lactobacillus reuteri* 7498 0 0 4.8 0 0 0 *Lactobacillus sake* 4127 0 0 17.3 8.2 0 14.5

**Table 1.** Strains exhibiting the potential to degrade BAs in a buffer system within 24 h at 30°C

The presence of BAs in a decarboxylase synthetic broth was determined by high performance liquid chromatography with OPA derivatization. Among the 9 LAB strains tested, 5 lactobacilli (in particular, *L. cur*v*atus*) were amine producers and *L. plantarum* and *L.*

Putrescine Cadaverine Histamine Tyramine

**2.9. Determination of amino acids** 

μL and monitored at 254 nm of wavelength.

determined by Duncan's new multiple-range test.

strains of LAB isolated from Nham sausages was investigated.

Tryptamine

**2.10. Statistical analysis** 

**3. Results and discussion** 

**Figure 1.** a\* Value during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

Fig. 1 showed a\* values represent red color of Nham during ripening time and temperature at 25°C, 30°C and 37°C, respectively. The results showed a value increased according to ripening and the a value of Nham control at 72 hours 37°C was higher than the other sample.

**Figure 2.** b\* Value during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

Fig. 2 shows b\* values represent yellow color of Nham during ripening time and temperature at 25°C, 30°C and 37°C, respectively. The results showed b value decreased according to storage and the b value of Nham with starters was lower than that of Nham control.

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 105

Fig. 3 is represent L\*values represent white color of Nham during ripening time and temperature at 25°C, 30°C and 37°C, respectively. The results showed L\* value increased

Fig. 4 shows that the initial pH of Nham samples ranged from 5.9 to 6.1. It then gradually decreased throughout the ripening process and there was significant difference at each time of sampling (*P* <0.05). The pH value reached 4.1 to 4.8 at the end of ripening (hour 72). However, there was significant difference (*P*<0.05) between the pH of Nham control and

**Figure 5.** Total acid content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham

Fig. 5 shows that the initial total acid content of Nham samples ranged from 0.5 to 0.55. It then gradually increased throughout the ripening process and there was significant difference at each time of sampling (*P* <0.05). The total acid content of Nham control and Nham with starters reached 0.95% to 1.57% and 1.04 %to 1.32% at the end of ripening (hour 72). However, there was significant difference (*P*<0.05) between the total acid content of Nham control and samples inoculated with starter culture after 48 hour of ripening. The results was shown that Nham control fermented at 37°C contained total acid content higher

Fig. 6 shows that TCA-soluble peptide content of Nham samples, the initial content was 9.02 mol/g dry matter. It then gradually increased throughout the ripening process. The TCAsoluble peptide content of Nham control and Nham with starters reached 23.6 to 87.2 mol/g dry matter and 24.1 %to 65.2 mol/g dry matter, respectively, at the end of ripening (hour 72). However, there was not significant difference (*P*<0.05) between the TCA-soluble peptide content of Nham control and samples inoculated with starter culture after 48 hour

according to storage during 72 hour of ripening.

samples inoculated with starter cultures after 48 hour of ripening.

with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

than the other Nham samples.

**Figure 3.** L\* Value during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

**Figure 4.** pH during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

Fig. 3 is represent L\*values represent white color of Nham during ripening time and temperature at 25°C, 30°C and 37°C, respectively. The results showed L\* value increased according to storage during 72 hour of ripening.

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

Fig. 2 shows b\* values represent yellow color of Nham during ripening time and temperature at 25°C, 30°C and 37°C, respectively. The results showed b value decreased according to

storage and the b value of Nham with starters was lower than that of Nham control.

**Figure 3.** L\* Value during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with

**Figure 4.** pH during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters

starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

(*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

Fig. 4 shows that the initial pH of Nham samples ranged from 5.9 to 6.1. It then gradually decreased throughout the ripening process and there was significant difference at each time of sampling (*P* <0.05). The pH value reached 4.1 to 4.8 at the end of ripening (hour 72). However, there was significant difference (*P*<0.05) between the pH of Nham control and samples inoculated with starter cultures after 48 hour of ripening.

**Figure 5.** Total acid content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

Fig. 5 shows that the initial total acid content of Nham samples ranged from 0.5 to 0.55. It then gradually increased throughout the ripening process and there was significant difference at each time of sampling (*P* <0.05). The total acid content of Nham control and Nham with starters reached 0.95% to 1.57% and 1.04 %to 1.32% at the end of ripening (hour 72). However, there was significant difference (*P*<0.05) between the total acid content of Nham control and samples inoculated with starter culture after 48 hour of ripening. The results was shown that Nham control fermented at 37°C contained total acid content higher than the other Nham samples.

Fig. 6 shows that TCA-soluble peptide content of Nham samples, the initial content was 9.02 mol/g dry matter. It then gradually increased throughout the ripening process. The TCAsoluble peptide content of Nham control and Nham with starters reached 23.6 to 87.2 mol/g dry matter and 24.1 %to 65.2 mol/g dry matter, respectively, at the end of ripening (hour 72). However, there was not significant difference (*P*<0.05) between the TCA-soluble peptide content of Nham control and samples inoculated with starter culture after 48 hour

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 107

respectively. It then gradually increased throughout the ripening process. The free -amino acid content of Nham control and Nham with starters reached 275.3 to 351.6 mmol/g dry matter and 262.4 to 302.2 mmol/g dry matter, respectively, at the end of ripening (hour 72). However, there was not significant difference (*P*<0.05) between the free -amino acid content of Nham control and samples inoculated with starter culture during ripening at 25°C. The results was shown that Nham control fermented at 37°C contained free -amino

acid content higher than the other Nham samples throughout the ripening process.

**Figure 8.** Total count of LAB during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and

starters (8.7 log CFU/g) were higher (*P* < 0.05) than in Nham control (7.7 log CFU/g).

The differences between Nham in counts of LAB during ripening are shown in Fig. 8. LAB in Nham with starters was increase until the 72 h of ripening. Counts of LAB in Nham with

Fig. 9 shows that cadaverine content of Nham samples, the initial content was 14.89 mg/kg dry matter. It then gradually increased throughout the ripening process. The cadaverine content of Nham control and Nham with starters reached 86.2 to 98.7 mg/kg dry matter and 42.4 to 51.6 mg/kg dry matter, respectively, at 72 hour of ripening. However, there was not significant difference (*P*<0.05) between the cadaverine content of Nham with starters during ripening at 25°C and 30°C. The results was shown that Nham control fermented at 37°C contained cadaverine content higher than the other Nham samples throughout the ripening

Fig. 10 shows that putrescine content of Nham samples, the initial content was 23.7 mg/kg dry matter. It then gradually increased throughout the ripening process and there was significant difference at each time of sampling (*P* <0.05). The putrescine content of Nham control and Nham with starters reached 115.4 to 242.6 mg/kg dry matter and 65.2 to 98.4 mg/kg dry matter, respectively, at 72 hour of ripening. However, there was not significant

Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

process.

**Figure 6.** TCA-soluble peptide content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

of ripening at each ripening temperature, and 72 hour of ripening at 25C and 30C. The results was shown that Nham control fermented at 37°C contained TCA-soluble peptide content higher than the other Nham samples after ripening for 72 hour.

**Figure 7.** Free -amino acid content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

Fig. 7 shows that free -amino acid content of Nham control samples and Nham with starters, the initial content were 216.2 mmol/g dry matter and 203.7 mmol/g dry matter, respectively. It then gradually increased throughout the ripening process. The free -amino acid content of Nham control and Nham with starters reached 275.3 to 351.6 mmol/g dry matter and 262.4 to 302.2 mmol/g dry matter, respectively, at the end of ripening (hour 72). However, there was not significant difference (*P*<0.05) between the free -amino acid content of Nham control and samples inoculated with starter culture during ripening at 25°C. The results was shown that Nham control fermented at 37°C contained free -amino acid content higher than the other Nham samples throughout the ripening process.

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

**Figure 6.** TCA-soluble peptide content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲)

of ripening at each ripening temperature, and 72 hour of ripening at 25C and 30C. The results was shown that Nham control fermented at 37°C contained TCA-soluble peptide

**Figure 7.** Free -amino acid content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲)

Fig. 7 shows that free -amino acid content of Nham control samples and Nham with starters, the initial content were 216.2 mmol/g dry matter and 203.7 mmol/g dry matter,

and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

content higher than the other Nham samples after ripening for 72 hour.

**Figure 8.** Total count of LAB during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

The differences between Nham in counts of LAB during ripening are shown in Fig. 8. LAB in Nham with starters was increase until the 72 h of ripening. Counts of LAB in Nham with starters (8.7 log CFU/g) were higher (*P* < 0.05) than in Nham control (7.7 log CFU/g).

Fig. 9 shows that cadaverine content of Nham samples, the initial content was 14.89 mg/kg dry matter. It then gradually increased throughout the ripening process. The cadaverine content of Nham control and Nham with starters reached 86.2 to 98.7 mg/kg dry matter and 42.4 to 51.6 mg/kg dry matter, respectively, at 72 hour of ripening. However, there was not significant difference (*P*<0.05) between the cadaverine content of Nham with starters during ripening at 25°C and 30°C. The results was shown that Nham control fermented at 37°C contained cadaverine content higher than the other Nham samples throughout the ripening process.

Fig. 10 shows that putrescine content of Nham samples, the initial content was 23.7 mg/kg dry matter. It then gradually increased throughout the ripening process and there was significant difference at each time of sampling (*P* <0.05). The putrescine content of Nham control and Nham with starters reached 115.4 to 242.6 mg/kg dry matter and 65.2 to 98.4 mg/kg dry matter, respectively, at 72 hour of ripening. However, there was not significant

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 109

**Figure 11.** Tyramine content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and

Fig. 11 shows that tyramine content of Nham samples, the initial content was 5.63 mg/kg dry matter. It then gradually increased throughout the ripening process and there was significant difference at each time of sampling (*P* <0.05). The tyramine content of Nham control and Nham with starters reached 17.6 to 46.4 mg/kg dry matter and 16.3 to 27.8 mg/kg dry matter, respectively, at 72 hour of ripening. However, there was not significant difference (*P*<0.05) between the tyramine content of Nham with starters during ripening at 30°C and 37°C and Nham control and Nham samples inoculated with starter culture during ripening at 25°C. The results was shown that Nham control fermented at 37°C contained tyramine content higher than the other Nham samples after 48 hour of the ripening process. The effect of temperature on BA content was evaluated (Fig. 6-9). The storage temperature of Nham with starters at 30°C and 37°C were shown higher BA oxidation comparing Nham control, a low content was observed at 25°C. This suggested that at ripening temperature of 30 °C and 37°C, a strong oxidation of the AO activity of the starters was evident, whereas at 25°C activity was low for amino acid decarboxylase for lysine (precursor of putrescine) and

Fig. 12 showed a\* values represent red color of Nham during stored at 15°C, 4°C and 25°C. The initial a\* values of Nham control ranged from 7.0 to 8.4. The results showed a\* value increased according to 4 week storage for storage temperature at 15°C and 25°C. However, there was significant decrease (*P*<0.05) between the a\* value of 4°C storage of the initial 1 week storage and after 4 week of storage. For Nham with starters, the initial a\* values ranged from 7.5 to 8.5. The a\* values of Nham with starter decreased according to 4 week storage for storage temperature at 4°C and 15°C. However, there was not significant difference (*P*<0.05) between the a\* value of 25°C storage of the initial 1 week storage and after 4 week of storage.

Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

tyrosine (precursor of tyramine) in Nham control.

**Figure 9.** Cadaverine content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

**Figure 10.** Putrescine content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

difference (*P*<0.05) between the putrescine content of Nham with starters during ripening at 25°C and 30°C. The results was shown that Nham control fermented at 37°C contained putrescine content higher than the other Nham samples throughout the ripening process.

**Figure 9.** Cadaverine content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and

**Figure 10.** Putrescine content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and

difference (*P*<0.05) between the putrescine content of Nham with starters during ripening at 25°C and 30°C. The results was shown that Nham control fermented at 37°C contained putrescine content higher than the other Nham samples throughout the ripening process.

Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

**Figure 11.** Tyramine content during ripening of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

Fig. 11 shows that tyramine content of Nham samples, the initial content was 5.63 mg/kg dry matter. It then gradually increased throughout the ripening process and there was significant difference at each time of sampling (*P* <0.05). The tyramine content of Nham control and Nham with starters reached 17.6 to 46.4 mg/kg dry matter and 16.3 to 27.8 mg/kg dry matter, respectively, at 72 hour of ripening. However, there was not significant difference (*P*<0.05) between the tyramine content of Nham with starters during ripening at 30°C and 37°C and Nham control and Nham samples inoculated with starter culture during ripening at 25°C. The results was shown that Nham control fermented at 37°C contained tyramine content higher than the other Nham samples after 48 hour of the ripening process.

The effect of temperature on BA content was evaluated (Fig. 6-9). The storage temperature of Nham with starters at 30°C and 37°C were shown higher BA oxidation comparing Nham control, a low content was observed at 25°C. This suggested that at ripening temperature of 30 °C and 37°C, a strong oxidation of the AO activity of the starters was evident, whereas at 25°C activity was low for amino acid decarboxylase for lysine (precursor of putrescine) and tyrosine (precursor of tyramine) in Nham control.

Fig. 12 showed a\* values represent red color of Nham during stored at 15°C, 4°C and 25°C. The initial a\* values of Nham control ranged from 7.0 to 8.4. The results showed a\* value increased according to 4 week storage for storage temperature at 15°C and 25°C. However, there was significant decrease (*P*<0.05) between the a\* value of 4°C storage of the initial 1 week storage and after 4 week of storage. For Nham with starters, the initial a\* values ranged from 7.5 to 8.5. The a\* values of Nham with starter decreased according to 4 week storage for storage temperature at 4°C and 15°C. However, there was not significant difference (*P*<0.05) between the a\* value of 25°C storage of the initial 1 week storage and after 4 week of storage.

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 111

values ranged from 5.5 to 6.2. The b\* values of Nham with starter increased according to 4 week storage for storage temperature at 4°C and 25°C. However, there was significant decrease (*P*<0.05) between the b\* value of 15C storage of the initial 1 week storage and after

**Figure 14.** L\* Value during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with

value of 4°C storage of the initial 1 week storage and after 4 week of storage.

25°C was lower than the other Nham samples throughout the storage process.

Fig. 14 showed L\* values represent white color of Nham during stored at 15°C, 4°C and 25°C. The initial a\* values of Nham control ranged from 55.1 to 56.4. The results showed L\* value decreased after 2 week storage and then increased after 3 week storage for each storage temperature. However, there was not significant difference (*P*<0.05) between the L\* value of 25°C storage of the initial 1 week storage and after 4 week of storage. For Nham with starters, the initial L\* values ranged from 55.0 to 55.6. The L\* values of Nham with starter decreased after 2 week storage then the L\* values increased after 3 week storage for each storage temperature and after 4 week storage at 15°C and 25°C, the L\* value was significant increased. However, there was no significant difference (*P*<0.05) between the L\*

Fig. 15 shows that the initial pH of Nham samples ranged from 4.3 to 4.5. It then gradually decreased throughout the storage. The pH value reached 4.1 to 4.5 at 4 week of storage. The pH values at each storage temperature of Nham with starter were higher than Nham control at each time of sampling. The results was shown that pH value of Nham control stored at

starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

4 week of storage.

**Figure 12.** a\* Value during storage of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

**Figure 13.** b\* Value during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

Fig. 13 showed b\* values represent yellow color of Nham during stored at 15°C, 4°C and 25°C. The initial a\* values of Nham control ranged from 5.0 to 6.1. The results showed a\* value increased according to 4 week storage for storage temperature at 4°C and 25°C. However, there was significant decrease (*P*<0.05) between the b\* value of 15°C storage of the initial 1 week storage and after 4 week of storage. For Nham with starters, the initial a\* values ranged from 5.5 to 6.2. The b\* values of Nham with starter increased according to 4 week storage for storage temperature at 4°C and 25°C. However, there was significant decrease (*P*<0.05) between the b\* value of 15C storage of the initial 1 week storage and after 4 week of storage.

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

starters (*L. plantarum* + *L. sake*) at 25°C (), 30°C (), 37°C ().

starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

**Figure 12.** a\* Value during storage of Nham control at 25°C (), 30°C (), 37°C (▲) and Nham with

**Figure 13.** b\* Value during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with

Fig. 13 showed b\* values represent yellow color of Nham during stored at 15°C, 4°C and 25°C. The initial a\* values of Nham control ranged from 5.0 to 6.1. The results showed a\* value increased according to 4 week storage for storage temperature at 4°C and 25°C. However, there was significant decrease (*P*<0.05) between the b\* value of 15°C storage of the initial 1 week storage and after 4 week of storage. For Nham with starters, the initial a\*

**Figure 14.** L\* Value during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

Fig. 14 showed L\* values represent white color of Nham during stored at 15°C, 4°C and 25°C. The initial a\* values of Nham control ranged from 55.1 to 56.4. The results showed L\* value decreased after 2 week storage and then increased after 3 week storage for each storage temperature. However, there was not significant difference (*P*<0.05) between the L\* value of 25°C storage of the initial 1 week storage and after 4 week of storage. For Nham with starters, the initial L\* values ranged from 55.0 to 55.6. The L\* values of Nham with starter decreased after 2 week storage then the L\* values increased after 3 week storage for each storage temperature and after 4 week storage at 15°C and 25°C, the L\* value was significant increased. However, there was no significant difference (*P*<0.05) between the L\* value of 4°C storage of the initial 1 week storage and after 4 week of storage.

Fig. 15 shows that the initial pH of Nham samples ranged from 4.3 to 4.5. It then gradually decreased throughout the storage. The pH value reached 4.1 to 4.5 at 4 week of storage. The pH values at each storage temperature of Nham with starter were higher than Nham control at each time of sampling. The results was shown that pH value of Nham control stored at 25°C was lower than the other Nham samples throughout the storage process.

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 113

content of Nham control stored at 4°C and 15°C and Nham with starters stored at 15°C and from the results, the total acid content of Nham with starters stored at 4°C was not significant difference (*P*<0.05) during storage process. The total acid content of Nham control stored at

**Figure 17.** TCA-soluble peptide during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and

Fig. 17 shows that the TCA-soluble peptide of Nham control and Nham with starters ranged from 45.2 to 98.4 and 46.3 to 79.6 mol/g dry matter, respectively. Nham control and Nham with starters stored at 25°C showed gradually increased throughout the storage process and there was significant difference at each time of sampling (*P* <0.05). The TCA-soluble peptide of Nham control and Nham with starters stored at 4°C and 15°C. However, there was not significant difference (*P*<0.05) between the TCA-soluble peptide of Nham control and samples inoculated with starters culture throughout the storage process at 4°C and 15°C. From the results, the TCA-soluble peptide of Nham control stored at 25°C was higher than

Fig. 18 shows that free -amino acid content of Nham control samples and Nham with starters, the initial ranged from 342.3 to 603.4 and 346.6 to 507.2 mmol/g dry matter, respectively. It then gradually increased throughout the storage process at 15°C and 25°C and there was significant difference at each time of sampling (*P* <0.05). The free -amino acid content of Nham control and Nham with starters reached 375.2 to 1867.6 mmol/g dry matter and 359.4 to 1252.4 mmol/g dry matter, respectively, at 4 week of storage. However, there was not significant difference (*P* <0.05) between the free -amino acid content of Nham control and samples inoculated with starters during storage at 4°C. The results was shown that Nham control stored at 25°C contained free -amino acid content higher than the other

Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

the other Nham samples throughout the storage process.

Nham samples after 3 week storage.

25°C was higher than the other Nham samples throughout the storage process.

**Figure 15.** pH during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

**Figure 16.** Total acid content during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

Fig. 16 shows that the initial total acid content of Nham samples ranged from 1.1 to 1.7. It then gradually increased throughout the ripening process and there was significant difference at each time of sampling (*P* <0.05). The total acid content of Nham control and Nham with starters reached 1.14% to 2.72% and 1.04 %to 2.32% at 4 week of storage, respectively. However, there was not significant difference (*P*<0.05) between the total acid content of Nham control stored at 4°C and 15°C and Nham with starters stored at 15°C and from the results, the total acid content of Nham with starters stored at 4°C was not significant difference (*P*<0.05) during storage process. The total acid content of Nham control stored at 25°C was higher than the other Nham samples throughout the storage process.

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

(*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

**Figure 15.** pH during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters

**Figure 16.** Total acid content during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham

Fig. 16 shows that the initial total acid content of Nham samples ranged from 1.1 to 1.7. It then gradually increased throughout the ripening process and there was significant difference at each time of sampling (*P* <0.05). The total acid content of Nham control and Nham with starters reached 1.14% to 2.72% and 1.04 %to 2.32% at 4 week of storage, respectively. However, there was not significant difference (*P*<0.05) between the total acid

**Figure 17.** TCA-soluble peptide during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

Fig. 17 shows that the TCA-soluble peptide of Nham control and Nham with starters ranged from 45.2 to 98.4 and 46.3 to 79.6 mol/g dry matter, respectively. Nham control and Nham with starters stored at 25°C showed gradually increased throughout the storage process and there was significant difference at each time of sampling (*P* <0.05). The TCA-soluble peptide of Nham control and Nham with starters stored at 4°C and 15°C. However, there was not significant difference (*P*<0.05) between the TCA-soluble peptide of Nham control and samples inoculated with starters culture throughout the storage process at 4°C and 15°C. From the results, the TCA-soluble peptide of Nham control stored at 25°C was higher than the other Nham samples throughout the storage process.

Fig. 18 shows that free -amino acid content of Nham control samples and Nham with starters, the initial ranged from 342.3 to 603.4 and 346.6 to 507.2 mmol/g dry matter, respectively. It then gradually increased throughout the storage process at 15°C and 25°C and there was significant difference at each time of sampling (*P* <0.05). The free -amino acid content of Nham control and Nham with starters reached 375.2 to 1867.6 mmol/g dry matter and 359.4 to 1252.4 mmol/g dry matter, respectively, at 4 week of storage. However, there was not significant difference (*P* <0.05) between the free -amino acid content of Nham control and samples inoculated with starters during storage at 4°C. The results was shown that Nham control stored at 25°C contained free -amino acid content higher than the other Nham samples after 3 week storage.

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 115

storage temperature were significant differences. In the present study in Nham are concerned, total LAB counts in Nham with starters on 3 week of storage were higher (*P* < 0.05) in comparison with the Nham control produced at the same storage temperature. An increase of LAB in Nham with starters until 3 week of storage and consecutive increase till 4 week of storage was significant. LAB of Nham with starters produced increase steadily during ripening and stored at different temperatures, however, at 4C storage, LAB counts

**Figure 20.** Cadaverine content during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and

Fig. 20 shows that cadaverine content of Nham samples, the initial ranged from 43.7 to 58.2 mg/kg dry matter. In Nham control stored at 15°C and 25°C, it then gradually increased throughout the storage process. The cadaverine content of Nham control and Nham with starters reached 58.4 to 91.2 mg/kg dry matter and 41.6 to 47.3 mg/kg dry matter, respectively, at 72 hour of storage. However, there was not significant difference (*P*<0.05) between the cadaverine content of Nham with starters during stored at 4°C. The results was shown that Nham control stored at 25°C contained cadaverine content higher than the other Nham samples throughout the storage process and there was significant decreased (*P*<0.05)

Fig. 21 shows that putrescine content of Nham control and Nham with starters, the initial ranged from 124.6 to 176.3 mg/kg dry matter and 126.2 to 98.3 mg/kg dry matter. Nham control stored at 4°C, 15°C and 25°C gradually increased throughout the storage process. The putrescine content of Nham control and Nham with starters reached 175.3 to 339.4 mg/kg dry matter and 122.6 to 129.3 mg/kg dry matter, respectively, at 4 week of storage. However, there were significant increase (*P*<0.05) between the putrescine content of Nham with starters at each storage temperature for 2 week and then the putrescine content

Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

in the cadaverine content of Nham with starters stored at 25°C for 4 week.

storage was not different significant.

**Figure 18.** Free -amino acid content during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

**Figure 19.** Total count of LAB during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

The differences between Nham in counts of LAB during ripening are shown in Fig. 19. LAB in Nham with starters was increase until the 4 week of storage. Counts of LAB in Nham with starters stored at 25C (9.4 log CFU/g) were higher (*P* < 0.05) than in Nham control stored at 25C (9.1 log CFU/g). LAB counts in Nham increased steadily during storage, the dependence of the LAB counts of Nham control and Nham with starters on ripening at each storage temperature were significant differences. In the present study in Nham are concerned, total LAB counts in Nham with starters on 3 week of storage were higher (*P* < 0.05) in comparison with the Nham control produced at the same storage temperature. An increase of LAB in Nham with starters until 3 week of storage and consecutive increase till 4 week of storage was significant. LAB of Nham with starters produced increase steadily during ripening and stored at different temperatures, however, at 4C storage, LAB counts storage was not different significant.

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

**Figure 18.** Free -amino acid content during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and

**Figure 19.** Total count of LAB during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham

The differences between Nham in counts of LAB during ripening are shown in Fig. 19. LAB in Nham with starters was increase until the 4 week of storage. Counts of LAB in Nham with starters stored at 25C (9.4 log CFU/g) were higher (*P* < 0.05) than in Nham control stored at 25C (9.1 log CFU/g). LAB counts in Nham increased steadily during storage, the dependence of the LAB counts of Nham control and Nham with starters on ripening at each

Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

**Figure 20.** Cadaverine content during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

Fig. 20 shows that cadaverine content of Nham samples, the initial ranged from 43.7 to 58.2 mg/kg dry matter. In Nham control stored at 15°C and 25°C, it then gradually increased throughout the storage process. The cadaverine content of Nham control and Nham with starters reached 58.4 to 91.2 mg/kg dry matter and 41.6 to 47.3 mg/kg dry matter, respectively, at 72 hour of storage. However, there was not significant difference (*P*<0.05) between the cadaverine content of Nham with starters during stored at 4°C. The results was shown that Nham control stored at 25°C contained cadaverine content higher than the other Nham samples throughout the storage process and there was significant decreased (*P*<0.05) in the cadaverine content of Nham with starters stored at 25°C for 4 week.

Fig. 21 shows that putrescine content of Nham control and Nham with starters, the initial ranged from 124.6 to 176.3 mg/kg dry matter and 126.2 to 98.3 mg/kg dry matter. Nham control stored at 4°C, 15°C and 25°C gradually increased throughout the storage process. The putrescine content of Nham control and Nham with starters reached 175.3 to 339.4 mg/kg dry matter and 122.6 to 129.3 mg/kg dry matter, respectively, at 4 week of storage. However, there were significant increase (*P*<0.05) between the putrescine content of Nham with starters at each storage temperature for 2 week and then the putrescine content

decreased and there was not significant difference (*P*<0.05) between the putrescine content of Nham with starters after 3 week storage. The results were shown that Nham control stored at 25°C contained putrescine higher than the other Nham samples throughout the storage process.

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 117

Fig. 22 shows that tyramine content of Nham control and Nham with starters, the initial ranged from 18.2 to 65.3 mg/kg dry matter and 19.2 to 22.4 mg/kg dry matter. Nham control stored at 15°C and 25°C gradually increased during storage process. The tyramine content of Nham control and Nham with starters reached 25.2 to 198.6 mg/kg dry matter and 21.4 to 27.6 mg/kg dry matter, respectively, at 4 week of storage. There was not significant difference (*P*<0.05) between the tyramine content of Nham control stored at 4°C and Nham with starters at each storage temperature during storage time. The results was shown that Nham control stored at 25°C contained tyramine higher than the other Nham samples

One of the most important factors influencing BA formation in Nham is starter culture (Maijalaet al., 1995). Increase of LAB starters culture in Nham resulted in overgrowth more than the microflora and LAB producing BAs in Nham control and caused decrease in BA contents in Nham during ripening and storage. A higher amount of BAs was formed in the Nham control than in starters culture-ones. However, strains of the starters showed lower decarboxylase activity (lower total free amino acid content in Nham) in comparison with the Nham control. Moreover, from the fact that BA production increased in Nham control after the ripening was finished and Nham was stored at the 15C and 25C, which coincided with the temporary increase of total LAB, the presence of spontaneous decarboxylating microflora can be inferred, and the refrigerated storage should be recommended. Simultaneously, higher concentration of BAs was found in Nham fermented at high 30C and 37C as compared to 25C at the end of ripening. However, as regards to the strongly hypothetical effects of some substances in the Nham spicing mixtures in connection with the BA formation, more research is needed. Decarboxylase activities present in microflora in Nham are influenced by pH, temperature (Gardini et al., 2001; Silla-Santos, 1996; Suzzi and Gardini, 2003). The decarboxylation of FAAs to BAs was found to be inhibited by low pH (Gardini et al., 2001). Though amino acid decarboxylase activities usually have acid pH optimum (Gale, 1946), the pH rise could favour the cell yield and growth (Maijala, 1994) of

Occurrence of toxic compounds such as BAs is favoured by a high concentration of substrates (i.e., free amino acids) together with environmental and technological factors (e.g. NaCl content, chemico-physical variables, hygienic procedure adopted during production) promoting microbial growth and the decarboxylase activity of microorganisms (Silla-Santos, 1996). In this study, a high correlation among total BAs and total FAAs content was observed. Temperature markedly influences the formation of BAs, and at 15C decarboxylases might be still active (Bover-Cid et al., 2001). During storage, the more temperature exceeds 14–15C the more decarboxylase activities might release BAs from FAAs. In this respect, processing procedures for Nham based on low salt addition, high ripening temperatures (over 20C), may favour proteolytic and decarboxylase activities. The high values of cadaverine, putrescine and tyramine detected in some Nham, may be ascribed to inadequate microflora and LAB producing BAs reduction occurring in some Nham control (Fig. 9- Fig. 11 and Fig. 20- Fig. 22).

throughout the storage process.

decarboxylase-positive microflora.

**3.1. BAs contents of Nham** 

**Figure 21.** Putrescine content during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

**Figure 22.** Tyramine content during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

Fig. 22 shows that tyramine content of Nham control and Nham with starters, the initial ranged from 18.2 to 65.3 mg/kg dry matter and 19.2 to 22.4 mg/kg dry matter. Nham control stored at 15°C and 25°C gradually increased during storage process. The tyramine content of Nham control and Nham with starters reached 25.2 to 198.6 mg/kg dry matter and 21.4 to 27.6 mg/kg dry matter, respectively, at 4 week of storage. There was not significant difference (*P*<0.05) between the tyramine content of Nham control stored at 4°C and Nham with starters at each storage temperature during storage time. The results was shown that Nham control stored at 25°C contained tyramine higher than the other Nham samples throughout the storage process.

One of the most important factors influencing BA formation in Nham is starter culture (Maijalaet al., 1995). Increase of LAB starters culture in Nham resulted in overgrowth more than the microflora and LAB producing BAs in Nham control and caused decrease in BA contents in Nham during ripening and storage. A higher amount of BAs was formed in the Nham control than in starters culture-ones. However, strains of the starters showed lower decarboxylase activity (lower total free amino acid content in Nham) in comparison with the Nham control. Moreover, from the fact that BA production increased in Nham control after the ripening was finished and Nham was stored at the 15C and 25C, which coincided with the temporary increase of total LAB, the presence of spontaneous decarboxylating microflora can be inferred, and the refrigerated storage should be recommended. Simultaneously, higher concentration of BAs was found in Nham fermented at high 30C and 37C as compared to 25C at the end of ripening. However, as regards to the strongly hypothetical effects of some substances in the Nham spicing mixtures in connection with the BA formation, more research is needed. Decarboxylase activities present in microflora in Nham are influenced by pH, temperature (Gardini et al., 2001; Silla-Santos, 1996; Suzzi and Gardini, 2003). The decarboxylation of FAAs to BAs was found to be inhibited by low pH (Gardini et al., 2001). Though amino acid decarboxylase activities usually have acid pH optimum (Gale, 1946), the pH rise could favour the cell yield and growth (Maijala, 1994) of decarboxylase-positive microflora.

### **3.1. BAs contents of Nham**

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

storage process.

decreased and there was not significant difference (*P*<0.05) between the putrescine content of Nham with starters after 3 week storage. The results were shown that Nham control stored at 25°C contained putrescine higher than the other Nham samples throughout the

**Figure 21.** Putrescine content during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham

**Figure 22.** Tyramine content during storage of Nham control at 4°C (), 15°C (), 25°C (▲) and Nham

with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

with starters (*L. plantarum* + *L. sake*) at 4°C (), 15°C (), 25°C ().

Occurrence of toxic compounds such as BAs is favoured by a high concentration of substrates (i.e., free amino acids) together with environmental and technological factors (e.g. NaCl content, chemico-physical variables, hygienic procedure adopted during production) promoting microbial growth and the decarboxylase activity of microorganisms (Silla-Santos, 1996). In this study, a high correlation among total BAs and total FAAs content was observed. Temperature markedly influences the formation of BAs, and at 15C decarboxylases might be still active (Bover-Cid et al., 2001). During storage, the more temperature exceeds 14–15C the more decarboxylase activities might release BAs from FAAs. In this respect, processing procedures for Nham based on low salt addition, high ripening temperatures (over 20C), may favour proteolytic and decarboxylase activities. The high values of cadaverine, putrescine and tyramine detected in some Nham, may be ascribed to inadequate microflora and LAB producing BAs reduction occurring in some Nham control (Fig. 9- Fig. 11 and Fig. 20- Fig. 22).

The toxicological level of BAs depends on the individual characteristics and the presence of other amines (Brink et al., 1990; Halasz et al., 1994). Toxic doses of tyramine in foods were reported in the range 100–800 mg/kg, but average amounts of tyramine detected in analysed samples (Fig. 9- Fig. 11 and Fig. 20- Fig. 22) were below this range, even if in case of a few samples, the 100 mg/kg value was exceeded. Putrescine has been regarded as not toxic by itself, but as a potentiator for the toxic effect of tyramine and histamine if present (Hui and Taylor, 1985). However, it was probable to demonstrate significant relationship between the concentration of a specific FAA and its corresponding BA in meat products (Eerola et al., 1998). Fig. 9- Fig. 11 and Fig. 20- Fig. 22 shows the BAs content of Nham evidence the effect of starters on the decrease of the BAs occurrence in Nham after ripening and storage. Histamine was always below the minimum detectable, in spite of the abundance of their precursors (histidine) released during the process; phenylethylamine was also not detected.

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 119

arginine, cysteine, serine, threonine and glycine, most FAAs displayed a rise during the extended storage. Arginine found in the most stored Nham was increase, due to changing of its content by proteolysis; and rise in arginine in stored Nham control was higher than stored Nham with starters. Arginine hydrolysis could be hydrolysed via the arginine deiminase pathway (ADI) leading to ammonia and ornithine. It seemed reasonable to postulate that ADI pathway enzymes (arginine deiminase and ornithine transcarbamylase) could be still active during storage times. Arginine catabolism, may be regarded as a source of the BA putrescine both via ADI ornithine generation (Montel and Champomier, 1987) and subsequent decarboxylation to putrescine, and via arginine decarboxylation to agmatine followed by deamidation to putrescine and removal of urea (Moreno-Arribas et al., 2003). The presence in Nham of environmental conditions suitable for decarboxylase activities together with large amounts of arginine may be consistent with the increase in putrescine.

The evolution during incubation/storage of the total free amino acid content, in both the Nham control and after inoculation with either of the two Lactobacillus strains selected, is shown in Table 2- Table 5, and encompassed 17 different amino acids. The control Nham showed the highest concentration of total amino acids at a 5% level of significance. The contents of total amino acids in Nham inoculated with *L. plantarum* and *L. sake*, increased throughout time, but at lower rates than the control. The contents of free amino acids and BAs in control and experimental Nham increase significantly throughout incubation/storage. However, specific lactic acid strains of the Lactobacillus genus can effectively prevent BAs from building-up excessively, putrescine (for quantitative reasons, owing to its level). This may lead to a favourable contribution to public health, especially in regions where Nham is frequently included in the diet. To have an overall evolution index of the proteases action in the Nham during processing the TCA-soluble peptide was evaluated (Fig. 6 and Fig. 17) (Toldr, 2005). More intense proteolytic activity occurred in the Nham control. The TCA-soluble peptide values of Nham control are quite high compared to those generally observed in other Nham with starters. This could be attributed to the microflora in Nham control slightly higher proteolytic activity during the process, in comparison with those Nham applied with starters. Proteolysis contributes to texture by

Table 2- Table 5 show the FAAs content of Nham during ripening and storage arginine and glutamic acid were the FFAs most representative; after ripening and storage a marked increase of alanine was observed. Table 6 shows the effect of the starters treatment on the evolution of the FAAs pattern of the Nham investigated during the ripening and storage: a significant increase in the concentration of all FAAs with respect to their initial occurred in Nham control and Nham with starters, resulting from the aminopeptidases activity of meat (Toldr, 2006) as well as microbial proteases (Dur et al., 2004; Molina and Toldrá, 1992; Rodrguez et al., 1998; Scannell et al., 2004). Moreover, starters in Nham seems to affect the production of some amino acids (Table 6). A lower concentration of lysine, threonine, glycine and proline was detected, after storage, in Nham processed. Arginine was the most abundant amino acid in all the final products, and its level was significantly higher in Nham control than in those subjected with starters to Nham. At the end of the ripening step,

breakdown of the muscle structure (Monin et al., 1997).

The concentration of tyramine was high in Nham control while low concentration, of their precursors (tyrosine) released during the process. Moreover, tryptamine resulted absent in all the investigated samples. The sum of vasoactive BAs, VBA; (tyramine, phenylethylamine, histamine and tryptamine) lower than 200 mg/Kg has been suggested by Eerola et al. (1998) as a quality index (VBA index) for ripened meat products. It is interesting to note that the computed VBA index of Nham with starters with differently processed resulted appreciable samples (3.70 2.46 mg/Kg). These results could be related to the specific characteristics of the product as well as to the process conditions adopted that could, in general, have limited the growth and activity of amino acid decarboxylase positive microorganisms (Suzzi and Gardini, 2003). Cadaverine, putrescine and tyramine were found in high amounts in Nham control. However, the occurrence of BAs in Nham control, and after the storage could be due to the microflora and LAB producing BAs that could have favoured their formation during ripening and storage. During ripening and storage of Nham control, putrescine and cadaverine show a marked increase with high amounts of their precursor, arginine and lysine, respectively, were detected. In fact, arginine may generate putrescine both via arginine deiminase pathway (ADI) leading to ornithine (Montel and Champomier, 1987) and their subsequent decarboxylation to putrescine, and via arginine decarboxylation to agmatine followed by deamination to putrescine and removal of urea (Moreno-Arribas et al., 2003). It seems reasonable to postulate that the large amounts of arginine could be the source of putrescine, which subsequently may be converted in spermine and spermidine by transamination reactions (Lehninger et al., 1999).

### **3.2. FAAs contents of Nham**

FAAs were reported in Table 2- Table 5 as net amounts (mmol/g dry matter) in order to investigate the differences in contents due to starters in Nham during ripening and storage. FAAs were compared to evaluate if the extended storage times gave a similar increase in all of them or different patterns were detectable. Most single FAAs increased during ageing with particular reference to the lipophylic ones; a rise in lypophilic valine, phenylalanine and tryptophan processed following a traditional prolonged way (Ruiz et al., 1999). In the present study, stored Nham showed a FAA pattern enriched with glutamic acid, alanine,

transamination reactions (Lehninger et al., 1999).

**3.2. FAAs contents of Nham** 

The toxicological level of BAs depends on the individual characteristics and the presence of other amines (Brink et al., 1990; Halasz et al., 1994). Toxic doses of tyramine in foods were reported in the range 100–800 mg/kg, but average amounts of tyramine detected in analysed samples (Fig. 9- Fig. 11 and Fig. 20- Fig. 22) were below this range, even if in case of a few samples, the 100 mg/kg value was exceeded. Putrescine has been regarded as not toxic by itself, but as a potentiator for the toxic effect of tyramine and histamine if present (Hui and Taylor, 1985). However, it was probable to demonstrate significant relationship between the concentration of a specific FAA and its corresponding BA in meat products (Eerola et al., 1998). Fig. 9- Fig. 11 and Fig. 20- Fig. 22 shows the BAs content of Nham evidence the effect of starters on the decrease of the BAs occurrence in Nham after ripening and storage. Histamine was always below the minimum detectable, in spite of the abundance of their precursors (histidine) released during the process; phenylethylamine was also not detected. The concentration of tyramine was high in Nham control while low concentration, of their precursors (tyrosine) released during the process. Moreover, tryptamine resulted absent in all the investigated samples. The sum of vasoactive BAs, VBA; (tyramine, phenylethylamine, histamine and tryptamine) lower than 200 mg/Kg has been suggested by Eerola et al. (1998) as a quality index (VBA index) for ripened meat products. It is interesting to note that the computed VBA index of Nham with starters with differently processed resulted appreciable samples (3.70 2.46 mg/Kg). These results could be related to the specific characteristics of the product as well as to the process conditions adopted that could, in general, have limited the growth and activity of amino acid decarboxylase positive microorganisms (Suzzi and Gardini, 2003). Cadaverine, putrescine and tyramine were found in high amounts in Nham control. However, the occurrence of BAs in Nham control, and after the storage could be due to the microflora and LAB producing BAs that could have favoured their formation during ripening and storage. During ripening and storage of Nham control, putrescine and cadaverine show a marked increase with high amounts of their precursor, arginine and lysine, respectively, were detected. In fact, arginine may generate putrescine both via arginine deiminase pathway (ADI) leading to ornithine (Montel and Champomier, 1987) and their subsequent decarboxylation to putrescine, and via arginine decarboxylation to agmatine followed by deamination to putrescine and removal of urea (Moreno-Arribas et al., 2003). It seems reasonable to postulate that the large amounts of arginine could be the source of putrescine, which subsequently may be converted in spermine and spermidine by

FAAs were reported in Table 2- Table 5 as net amounts (mmol/g dry matter) in order to investigate the differences in contents due to starters in Nham during ripening and storage. FAAs were compared to evaluate if the extended storage times gave a similar increase in all of them or different patterns were detectable. Most single FAAs increased during ageing with particular reference to the lipophylic ones; a rise in lypophilic valine, phenylalanine and tryptophan processed following a traditional prolonged way (Ruiz et al., 1999). In the present study, stored Nham showed a FAA pattern enriched with glutamic acid, alanine, arginine, cysteine, serine, threonine and glycine, most FAAs displayed a rise during the extended storage. Arginine found in the most stored Nham was increase, due to changing of its content by proteolysis; and rise in arginine in stored Nham control was higher than stored Nham with starters. Arginine hydrolysis could be hydrolysed via the arginine deiminase pathway (ADI) leading to ammonia and ornithine. It seemed reasonable to postulate that ADI pathway enzymes (arginine deiminase and ornithine transcarbamylase) could be still active during storage times. Arginine catabolism, may be regarded as a source of the BA putrescine both via ADI ornithine generation (Montel and Champomier, 1987) and subsequent decarboxylation to putrescine, and via arginine decarboxylation to agmatine followed by deamidation to putrescine and removal of urea (Moreno-Arribas et al., 2003). The presence in Nham of environmental conditions suitable for decarboxylase activities together with large amounts of arginine may be consistent with the increase in putrescine.

The evolution during incubation/storage of the total free amino acid content, in both the Nham control and after inoculation with either of the two Lactobacillus strains selected, is shown in Table 2- Table 5, and encompassed 17 different amino acids. The control Nham showed the highest concentration of total amino acids at a 5% level of significance. The contents of total amino acids in Nham inoculated with *L. plantarum* and *L. sake*, increased throughout time, but at lower rates than the control. The contents of free amino acids and BAs in control and experimental Nham increase significantly throughout incubation/storage. However, specific lactic acid strains of the Lactobacillus genus can effectively prevent BAs from building-up excessively, putrescine (for quantitative reasons, owing to its level). This may lead to a favourable contribution to public health, especially in regions where Nham is frequently included in the diet. To have an overall evolution index of the proteases action in the Nham during processing the TCA-soluble peptide was evaluated (Fig. 6 and Fig. 17) (Toldr, 2005). More intense proteolytic activity occurred in the Nham control. The TCA-soluble peptide values of Nham control are quite high compared to those generally observed in other Nham with starters. This could be attributed to the microflora in Nham control slightly higher proteolytic activity during the process, in comparison with those Nham applied with starters. Proteolysis contributes to texture by breakdown of the muscle structure (Monin et al., 1997).

Table 2- Table 5 show the FAAs content of Nham during ripening and storage arginine and glutamic acid were the FFAs most representative; after ripening and storage a marked increase of alanine was observed. Table 6 shows the effect of the starters treatment on the evolution of the FAAs pattern of the Nham investigated during the ripening and storage: a significant increase in the concentration of all FAAs with respect to their initial occurred in Nham control and Nham with starters, resulting from the aminopeptidases activity of meat (Toldr, 2006) as well as microbial proteases (Dur et al., 2004; Molina and Toldrá, 1992; Rodrguez et al., 1998; Scannell et al., 2004). Moreover, starters in Nham seems to affect the production of some amino acids (Table 6). A lower concentration of lysine, threonine, glycine and proline was detected, after storage, in Nham processed. Arginine was the most abundant amino acid in all the final products, and its level was significantly higher in Nham control than in those subjected with starters to Nham. At the end of the ripening step,

cysteine was also present in a relative higher concentration in Nham control, whereas significant larger amounts of proline, lysine, histidine, serine and threonine were reached in Nham control samples. The different profile of FAAs observed in Nham control and Nham with starters may be due to a different evolution of reactions and processes involving both production and consumption of amino acids that occur simultaneously during the various steps of the ripening process and storage and whose combined effects could give rise to an increase or, on the contrary, to a decrease of their concentration. The aminopeptidase activity is considered the main process implied in the FFA release in meat. Moreover, free amino acids concentration could be decreased either by chemical and enzymatic reactions where they act as substrates leading to the formation of secondary products (Ruiz et al., 1999; Ventanas et al., 1992) and/or by microbial amino acid decarboxylase activity with consequent BA production (Virgili et al., 2007).

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 121

Control (without starter culture).

temperature.

Results are expressed as means of three replicates in mmol/g dry matter. Means with different letters along rows are significantly different (*P*<0.05).

**Table 2.** Amino acid content of Nham without and with starter cultures during ripening at different

In Nham control, an effect due to higher concentration of decarboxylase than that of Nham with starters, thus, their reaction with the free amino acids causing an increase of their BA concentration in these samples.

The ripened taste could be related to lysine and glutamic acid, while isoleucine and aspartic acid are implied in acid taste and unpleasant aroma (Buscailhon et al., 1994; Flores et al., 1998). In this study, the increase in concentration of lysine and glutamic acid was observed. The changes in the contents of free amino acids observed in fermented sausages during ripening are given in Table 2. The total free amino acid contents of the Nham control and Nham with starters constituted 212.7–216.4 mmol/g and 197.2–203.4 mmol/g dry matter, respectively (before ripening) on 0 day. An increase in the content of amino acids of Nham control and Nham with starters was observed and ranged between 275.2–349.8 mmol/g and 259.8–300.3 mmol/g dry matter during the ripening on day 3, and a further increase up to the range of 377.6–1851.7 mmol/g and 348.1 nmol/g–1256.0 mmol/g dry matter of total free amino acids was observed during storage at 4C-25C of Nham control and Nham with starters (4 weeks). The highest total free amino acid concentration of 1867.2 mmol/g was observed with Nham control stored at 25 C for 4 week, whereas the lowest total free amino acid concentration of 359.6 mmol/g was observed with Nham with starters stored at 25 C for 4 week. The hydrolysis of meat proteins generates polypeptides that can be further degraded to smaller peptides and free amino acids. This degradation can be produced by endogenous and microbial enzymes (De Masi et al., 1990; Hughes et al., 2002; Molly et al., 1997). The increase in the total free amino acid concentration was detected in all batches (Hierro et al., 1999, Bruna et al., 2000, Bolumar et al., 2001 and Hughes et al., 2002).

The main differences in the content of total free amino acids among batches were detected during 72 hour of ripening and during 4 week of storage. The amino acids in which differences, which were primarily responsible for the increase in total free amino acids during ripening, were observed were Glu (glutamic acid), Ala (alanine) and Arg (arginine) in Nham control and Nham with starters. Mateo et al. (1996) reported an increase in the total free amino acid content during the ripening. The change occurred during ripening and storage process indicating that the highest enzymatic activity took place during these stages


Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 121

Control (without starter culture).

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

consequent BA production (Virgili et al., 2007).

concentration in these samples.

cysteine was also present in a relative higher concentration in Nham control, whereas significant larger amounts of proline, lysine, histidine, serine and threonine were reached in Nham control samples. The different profile of FAAs observed in Nham control and Nham with starters may be due to a different evolution of reactions and processes involving both production and consumption of amino acids that occur simultaneously during the various steps of the ripening process and storage and whose combined effects could give rise to an increase or, on the contrary, to a decrease of their concentration. The aminopeptidase activity is considered the main process implied in the FFA release in meat. Moreover, free amino acids concentration could be decreased either by chemical and enzymatic reactions where they act as substrates leading to the formation of secondary products (Ruiz et al., 1999; Ventanas et al., 1992) and/or by microbial amino acid decarboxylase activity with

In Nham control, an effect due to higher concentration of decarboxylase than that of Nham with starters, thus, their reaction with the free amino acids causing an increase of their BA

The ripened taste could be related to lysine and glutamic acid, while isoleucine and aspartic acid are implied in acid taste and unpleasant aroma (Buscailhon et al., 1994; Flores et al., 1998). In this study, the increase in concentration of lysine and glutamic acid was observed. The changes in the contents of free amino acids observed in fermented sausages during ripening are given in Table 2. The total free amino acid contents of the Nham control and Nham with starters constituted 212.7–216.4 mmol/g and 197.2–203.4 mmol/g dry matter, respectively (before ripening) on 0 day. An increase in the content of amino acids of Nham control and Nham with starters was observed and ranged between 275.2–349.8 mmol/g and 259.8–300.3 mmol/g dry matter during the ripening on day 3, and a further increase up to the range of 377.6–1851.7 mmol/g and 348.1 nmol/g–1256.0 mmol/g dry matter of total free amino acids was observed during storage at 4C-25C of Nham control and Nham with starters (4 weeks). The highest total free amino acid concentration of 1867.2 mmol/g was observed with Nham control stored at 25 C for 4 week, whereas the lowest total free amino acid concentration of 359.6 mmol/g was observed with Nham with starters stored at 25 C for 4 week. The hydrolysis of meat proteins generates polypeptides that can be further degraded to smaller peptides and free amino acids. This degradation can be produced by endogenous and microbial enzymes (De Masi et al., 1990; Hughes et al., 2002; Molly et al., 1997). The increase in the total free amino acid concentration was detected in all batches

(Hierro et al., 1999, Bruna et al., 2000, Bolumar et al., 2001 and Hughes et al., 2002).

The main differences in the content of total free amino acids among batches were detected during 72 hour of ripening and during 4 week of storage. The amino acids in which differences, which were primarily responsible for the increase in total free amino acids during ripening, were observed were Glu (glutamic acid), Ala (alanine) and Arg (arginine) in Nham control and Nham with starters. Mateo et al. (1996) reported an increase in the total free amino acid content during the ripening. The change occurred during ripening and storage process indicating that the highest enzymatic activity took place during these stages

Results are expressed as means of three replicates in mmol/g dry matter.

Means with different letters along rows are significantly different (*P*<0.05).

**Table 2.** Amino acid content of Nham without and with starter cultures during ripening at different temperature.

(Verplaetse et al., 1989). A major release of free amino acids at the beginning of the process have been studied in coincidence with the ripening stage (Diaz et al., 1997). This increase has been attributed to the higher temperatures applied during ripening compared to the low temperature. The most significant increases occurred in the content of Arg (arginine) in the sample. The decrease in the content of amino acids may indicate their metabolism by bacteria (Bover-Cid et al., 2000; Ordonez et al., 1999; Sekikawa et al., 2003).

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 123

His 10.4a 11.2a 16.2b 20.5c 11.3a 11.4a 13.8d 16.0b Leu 3.1a 3.3a 4.9b 6.0c 3.1a 3.2a 3.9a 4.6b Lys 7.1a 7.5a 11.3b 13.9c 7.5a 7.8a 9.2d 10.9e Ile 1.9a 2.1a 3.1ab 3.8b 1.9a 2.0a 2.5a 3.0ab Met 0.6a 0.6a 0.9a 1.2a 0.6a 0.6a 0.8a 0.9a Phe 1.4a 1.5a 2.3ab 2.9b 1.5a 1.5a 1.8a 2.2ab Pro 4.3a 4.5a 6.7b 8.3c 4.3a 4.6a 5.6a 6.7b Ser 12.8a 13.9a 20.7b 26.2c 13.0a 13.1a 15.8d 19.3b Thr 11.7a 12.5a 18.7b 23.1c 12.7a 12.8a 15.4d 18.4b Tyr 1.8a 1.9a 2.7b 3.4c 1.8a 1.9a 2.3ab 2.7b Val 3.7a 3.9a 5.9b 7.5c 3.8a 3.8a 4.6a 5.5b Total 348.8a 367.2a 542.4b 657.7c 359.1a 374.0a 461.2d 547.1e

**Table 4.** Amino acid content of Nham without and with starter cultures during stored at 15C.

Ala 35.6a 40.6b 103.8c 122.6d 31.5e 41.8b 68.2f 76.2g Arg 232.9a 280.6b 710.9c 832.7d 212.3e 291.3f 475.2g 531.2h Asp 4.7a 5.8ad 14.7b 17.1c 4.1a 5.6d 9.5e 11.0f Cys 28.7a 34.6b 86.9c 101.8d 24.0a 32.3b 55.1e 64.4f Glu 140.9a 174.2b 433.8c 512.6d 118.7e 160.1f 270.5g 313.2h Gly 16.9a 20.3b 50.6c 59.2d 14.2e 19.3b 32.9f 37.8g His 17.8a 21.0b 52.8c 62.4d 15.1a 20.4b 34.1e 39.5f Leu 4.8a 5.9a 14.6b 17.0c 4.1a 5.6a 9.4d 10.9e Lys 10.9a 13.5b 34.6c 39.8d 9.9a 13.3b 21.8e 25.0f Ile 3.0a 3.6a 8.9b 10.1c 2.6a 3.4a 5.9d 6.7d Met 0.9a 1.1ad 2.8bc 3.2c 0.8a 1.1ad 1.9d 2.1d Phe 2.3ac 2.8a 7.1b 8.2b 1.9c 2.6a 4.4d 5.1d Pro 6.5a 8.0b 20.5c 23.6d 5.9a 7.9b 13.5e 15.0f Ser 20.1a 24.2b 60.3c 68.2d 16.8e 22.8e 38.3f 44.7g Thr 17.7a 21.9b 56.0c 66.2d 16.4a 22.2b 36.8e 41.2f Tyr 2.8a 3.4a 8.4b 9.8c 2.4a 3.2a 5.5d 6.4d Val 5.7a 7.0b 17.8c 21.0d 5.0a 6.6b 10.9e 12.7f Total 541.0a 651.8b 1637.2c 1851.7d 472.0e 647.8b 1103.9f 1256.0g

Control *L. plantarum* + *L. sake*  Storage time (wk) Storage time (wk) 1 2 3 4 1 2 3 4

Control (without starter culture).

Control (without starter culture).

Results are expressed as means of three replicates in mmol/g dry matter. Means with different letters along rows are significantly different (*P*<0.05).

**Table 5.** Amino acid content of Nham without and with starter cultures during stored at 25C.

Amin o acid

Results are expressed as means of three replicates in mmol/g dry matter. Means with different letters along rows are significantly different (*P*<0.05).


Control (without starter culture).

Results are expressed as means of three replicates in mmol/g dry matter.

Means with different letters along rows are significantly different (*P*<0.05).

**Table 3.** Amino acid content of Nham without and with starter cultures during stored at 4C.



Control (without starter culture).

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

Amino acid

Control (without starter culture).

Amino acid

Results are expressed as means of three replicates in mmol/g dry matter. Means with different letters along rows are significantly different (*P*<0.05).

**Table 3.** Amino acid content of Nham without and with starter cultures during stored at 4C.

Control *L. plantarum* + *L. sake* Storage time (wk) Storage time (wk) 1 2 3 4 1 2 3 4

Ala 20.3a 21.7ad 31.2b 37.9c 23.0d 23.1d 27.3e 31.8b Arg 147.5a 159.4b 237.5c 288.0d 155.9b 162.4b 191.7e 231.4c Asp 3.2a 3.3a 5.0b 6.3c 3.1a 3.2a 3.9a 4.8b Cys 18.5a 19.5a 28.5b 36.1c 18.6a 19.0a 23.4d 27.8b Glu 91.6a 97.3b 146.3c 183.7d 91.1a 93.2a 114.0e 138.8f Gly 11.0a 11.5a 16.9b 21.1c 11.1a 11.5a 14.1d 16.8b

(Verplaetse et al., 1989). A major release of free amino acids at the beginning of the process have been studied in coincidence with the ripening stage (Diaz et al., 1997). This increase has been attributed to the higher temperatures applied during ripening compared to the low temperature. The most significant increases occurred in the content of Arg (arginine) in the sample. The decrease in the content of amino acids may indicate their metabolism by

Ala 21.4a 22.3a 23.0ab 24.8b 22.1a 20.7a 21.1a 20.2a Arg 151.9a 154.4a 154.9a 168.3b 149.8a 145.5a 153.8a 147.2a Asp 3.2a 3.4a 3.5a 3.7a 2.9a 2.9a 3.1a 3.0a Cys 18.2a 18.5a 18.6a 19.9b 17.8ac 17.0c 17.7ac 17.7ac Glu 96.0a 98.4a 99.6ab 103.6b 88.3c 86.5c 92.2d 91.4d Gly 11.2ac 11.4ac 11.6ac 12.4b 10.8c 10.4c 10.8c 10.6c His 10.5a 10.5a 10.6a 11.3a 10.8a 10.2a 10.7a 10.2a Leu 2.9a 3.0a 3.1a 3.3a 2.9a 2.8a 2.9a 2.9a Lys 7.7a 7.8a 7.9a 8.5a 7.4a 7.0a 7.5a 7.3a Ile 2.0a 2.0a 2.1a 2.2a 1.9a 1.8a 1.9a 1.9a Met 0.6a 0.6a 0.6a 0.7a 0.6a 0.6a 0.6a 0.6a Phe 1.5a 1.5a 1.6a 1.7a 1.4a 1.3a 1.4a 1.4a Pro 4.6a 4.6a 4.7a 4.8a 4.3a 4.2a 4.4a 4.3a Ser 12.7a 13.2a 13.6ab 14.4b 12.3ac 11.8c 12.4ac 12.1ac Thr 12.5ac 12.8ac 13.1ab 14.0b 12.2a 11.9c 12.3c 12.3c Tyr 1.8a 1.8a 1.8a 1.9a 1.7a 1.7a 1.8a 1.7a Val 3.7a 3.8a 3.9a 4.2a 3.6a 3.4a 3.5a 3.5a Total 352.9a 358.6a 363.1ab 377.6b 344.9ac 335.1c 357.3a 348.1ac

Control *L. plantarum* + *L. sake* Storage time (wk) Storage time (wk) 1 2 3 4 1 2 3 4

bacteria (Bover-Cid et al., 2000; Ordonez et al., 1999; Sekikawa et al., 2003).

Results are expressed as means of three replicates in mmol/g dry matter. Means with different letters along rows are significantly different (*P*<0.05).



Control (without starter culture).

Results are expressed as means of three replicates in mmol/g dry matter.

Means with different letters along rows are significantly different (*P*<0.05).

**Table 5.** Amino acid content of Nham without and with starter cultures during stored at 25C.

Two types of fermented sausage differing in starter culture were produced in parallel with two different starter cultures (no starter and *L. plantarum* + *L. sake*). The sausages were ripened 3 days and subsequently stored 7, 14, 21 and 28 days at the 4 °C, 15 °C and 25 °C. Concentration of three most abundant amines, cadaverine, putrescine and tyramine increased significantly (*P* < 0.05) in Nham during ripening and and also during storage. The dominant BAs in the control were cadaverine – and tyramine and putrescine, to a lesser extent; the cadaverine, putrescine and tyramine content were lower if inoculation had added with *L. plantarum* + *L. sake*; whereas they ranked above 300 mg/kg in the control by 3 d. At the end of ripening, cadaverine (98.7 mg/kg dry matter), putrescine (242.6 mg/kg dry matter) and tyramine (46.4 mg/kg dry matter) content in the A-samples-sausage was higher (*P* < 0.05) than in Nham with starters (51.6, 98.4 and 27.8 mg/kg dry matter, respectively). Starter culture influenced significantly in decrease of (*P* < 0.05) cadaverine, putrescine and tyramine content in the sausage. Due to the significant (*P* < 0.05) increase of total aerobic counts in the Nham control between the end of ripening and during storage, followed by the significant (*P* < 0.05) increase of the sum of total BAs between the 72 hour of ripening (387.7 mg/kg dry matter) and the 4th week of storage at 25°C (629.2 mg/kg dry matter).

Potential of Fermented Sausage-Associated Lactic Acid Bacteria to Degrade Biogenic Amines During Storage 125

ripening was further carried out with non-amine forming strain of *L. plantarum* + *L. sake* after ripening and stored at different temperature. The amount of amine in the product was significantly less than the control. The results obtained for BAs degradation by bacteria in a synthetic medium suggest that AO activity is strain dependent rather than being related to specific species. In all batchs, the total amino acid contents increased with time – and the predominant ones were arginine and glutamic acid. However, upon inoculation with nonamine forming strain, the total BAs contents remained considerably lower than those of the control. Hence, an efficient food-grade biological tool was made available that constrains

*School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Thailand* 

This study was supported by Office of the Higher Education Commission (OHEC), Thailand.

AOAC. (2000). *Official methods of analysis* (17th ed.). Gaithersburg, Maryland: Association of

Benjakul, S., & Morrissey, M. T. (1997). Protein hydrolysates from Pacific whiting solid

Beutling, D. (1996). Biogenic Amines in Nutrition (Biogene Amine in der Ernaehrung).

Bover-Cid, S., Hugas, M., Izquierdo-Pulido, M. & Vidal-Carou, M.C. (2000). Reduction of biogenic amine formation using a negative amino acid-decarboxylase starter culture for

Bover-Cid, S., Izquierdo-Pulido, M. & Vidal-Carou, M. C. (2000). Influence of hygienic quality of raw materials on biogenic amine production during ripening and storage of

Bover-Cid, S., Hugas, M., Izquierdo-Pulido, M. & Vidal-Carou, M. C. (2001). Amino aciddecarboxylase activity of bacteria isolated from fermented pork sausages. International

Bolumar, T., Nieto, P. & Flores, J. (2001). Acidity, proteolysis and lipolysis changes in rapid cured fermented sausage dried at different temperatures. *Food Science and Technology* 

Brink, B. ten., Damink, C., Joosten, H. M. L. J. & Huis in't Veld, J. H. J. (1990). Occurrence and formation of biologically active amines in foods. *International Journal of Food* 

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wastes. *Journal of Agricultural and Food Chemistry*, Vol.45, pp. 3423–3430.

buildup of BAs in fermented sausage during storage.

**Author details** 

Jirasak Kongkiattikajorn

**Acknowledgement** 

Official Analytical Chemists.

Springer-Verlag, Berlin, Germany, pp. 59–67.

*Journal of Food Microbiology*, Vol. 66, pp. 185–189.

*International*, Vol.7, pp. 269–276.

*Microbiology*, Vol.11, pp. 73–84.

**5. References** 

The main rate of BAs production was during the first two days, when a sharp pH decrease and the development of LAB occurred. Sausages fermented with starters had lower amounts of cadaverine, putrescine and tyramine than naturally fermented sausages (control) during storage at 15°C and 25°C. However, phenylethylamine, histamine and tryptamine were not detected.

Nham control showed proteolysis that was correlated with pH values higher than those with starters. However, no positive correlation was found between the proteolysis index and BAs production. Since proteolysis was stronger during the second half of the ripening process, the FAAs occurred later than the early amine production. No effect on pH development in the fermented sausage was observed when non-amine forming strain of *L. plantarum* + *L. sake* were present during 4 week of 4°C storage period. A study on the evolution of FAAs and BAs in Nham during 4 week at different temperatures of storage (4°C, 15°C and 25°C) was performed. FAAs and BAs were determined by RP-HPLC. Storage temperature of 15°C and 25°C promoted a significant increase of the contents of arginine, glutamic acid, cadaverine, putrescine and tyramine, expressed as g/kg of dry matter while storage temperature of 4°C decreased a significant of the contents of arginine, glutamic acid, cadaverine, putrescine and tyramine, expressed as g/kg of dry matter. These two amino acids and three BAs may serve as indicators of temperatures changes in stored fermented sausage.

### **4. Conclusions**

The aim of this study was to investigate the effect of non-amine forming LAB as starter culture during ripening and storage time and temperature on the evolution of FAAs of Nham during processing. The correlation between FAAs and BAs content was also investigated. Larger increases of FAAs occurred in Nham without starter in the ripening and storage step. Total FAAs content was highly correlated with total BAs amount. Sausage ripening was further carried out with non-amine forming strain of *L. plantarum* + *L. sake* after ripening and stored at different temperature. The amount of amine in the product was significantly less than the control. The results obtained for BAs degradation by bacteria in a synthetic medium suggest that AO activity is strain dependent rather than being related to specific species. In all batchs, the total amino acid contents increased with time – and the predominant ones were arginine and glutamic acid. However, upon inoculation with nonamine forming strain, the total BAs contents remained considerably lower than those of the control. Hence, an efficient food-grade biological tool was made available that constrains buildup of BAs in fermented sausage during storage.

### **Author details**

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

detected.

**4. Conclusions**

Two types of fermented sausage differing in starter culture were produced in parallel with two different starter cultures (no starter and *L. plantarum* + *L. sake*). The sausages were ripened 3 days and subsequently stored 7, 14, 21 and 28 days at the 4 °C, 15 °C and 25 °C. Concentration of three most abundant amines, cadaverine, putrescine and tyramine increased significantly (*P* < 0.05) in Nham during ripening and and also during storage. The dominant BAs in the control were cadaverine – and tyramine and putrescine, to a lesser extent; the cadaverine, putrescine and tyramine content were lower if inoculation had added with *L. plantarum* + *L. sake*; whereas they ranked above 300 mg/kg in the control by 3 d. At the end of ripening, cadaverine (98.7 mg/kg dry matter), putrescine (242.6 mg/kg dry matter) and tyramine (46.4 mg/kg dry matter) content in the A-samples-sausage was higher (*P* < 0.05) than in Nham with starters (51.6, 98.4 and 27.8 mg/kg dry matter, respectively). Starter culture influenced significantly in decrease of (*P* < 0.05) cadaverine, putrescine and tyramine content in the sausage. Due to the significant (*P* < 0.05) increase of total aerobic counts in the Nham control between the end of ripening and during storage, followed by the significant (*P* < 0.05) increase of the sum of total BAs between the 72 hour of ripening (387.7

mg/kg dry matter) and the 4th week of storage at 25°C (629.2 mg/kg dry matter).

indicators of temperatures changes in stored fermented sausage.

The main rate of BAs production was during the first two days, when a sharp pH decrease and the development of LAB occurred. Sausages fermented with starters had lower amounts of cadaverine, putrescine and tyramine than naturally fermented sausages (control) during storage at 15°C and 25°C. However, phenylethylamine, histamine and tryptamine were not

Nham control showed proteolysis that was correlated with pH values higher than those with starters. However, no positive correlation was found between the proteolysis index and BAs production. Since proteolysis was stronger during the second half of the ripening process, the FAAs occurred later than the early amine production. No effect on pH development in the fermented sausage was observed when non-amine forming strain of *L. plantarum* + *L. sake* were present during 4 week of 4°C storage period. A study on the evolution of FAAs and BAs in Nham during 4 week at different temperatures of storage (4°C, 15°C and 25°C) was performed. FAAs and BAs were determined by RP-HPLC. Storage temperature of 15°C and 25°C promoted a significant increase of the contents of arginine, glutamic acid, cadaverine, putrescine and tyramine, expressed as g/kg of dry matter while storage temperature of 4°C decreased a significant of the contents of arginine, glutamic acid, cadaverine, putrescine and tyramine, expressed as g/kg of dry matter. These two amino acids and three BAs may serve as

The aim of this study was to investigate the effect of non-amine forming LAB as starter culture during ripening and storage time and temperature on the evolution of FAAs of Nham during processing. The correlation between FAAs and BAs content was also investigated. Larger increases of FAAs occurred in Nham without starter in the ripening and storage step. Total FAAs content was highly correlated with total BAs amount. Sausage Jirasak Kongkiattikajorn *School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Thailand* 

### **Acknowledgement**

This study was supported by Office of the Higher Education Commission (OHEC), Thailand.

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**Chapter 5** 

© 2013 Kröckel, 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,

© 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,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

In food industry starter and protective cultures are currently used in a number of products to safeguard the microbial and sensory quality. Lactic acid bacteria (LAB) are the main players in the natural transformation of agricultural primary products into safe, delicious and shelf stable foods for human consumption. In meat products there are three basic fields of application for the targeted use of such cultures: raw fermented sausages, raw cured

Lactic acid bacteria (LAB) are widespread in nature and commonly occur on all kind of plant materials, on mucous membranes, in saliva and, in feces. Consequently and unavoidably they are part of the contamination flora of fresh meats after slaughter. Under certain conditions, e.g. in packaged refrigerated meats or raw sausage meats, they are able to compete efficiently with accompanying microorganisms for nutrients and may reach substantial viable counts. Their metabolic activities may ultimately result in either a desired preservative effect due to the repression of pathogenic and spoilage microorganisms, a desired tasty meat product, such as raw fermented sausage, or in meat spoilage through undesired transformations of raw and cooked meats. Heterofermentative LAB of the *Carnobacterium*, *Leuconostoc* and *Weissella* genera are usually more involved in meat spoilage than the homofermentative *Lactobacillus* and *Pediococcus* genera. Therefore, commercially available meat starter cultures for dry-fermented sausage production exclusively belong to the latter two. Homofermentative LAB produce almost exclusively lactic acid from fermentable carbohydrates present in meats, which is relatively mild and palatable, while heterofermentative species produce significant amounts of less desirable fermentation end products, such as CO2 gas, ethanol, acetic acid, butanoic acid and acetoin. However, under certain conditions *Lactobacillus* spp. may also produce significant amounts of acetic acid,

**The Role of Lactic Acid Bacteria in** 

**Safety and Flavour Development** 

**of Meat and Meat Products** 

Additional information is available at the end of the chapter

ropy slime and, discolouration (greening) of meats [1,2].

Lothar Kröckel

**1. Introduction** 

http://dx.doi.org/10.5772/51117


**Chapter 5** 
