**2.4. Preparation of starter culture**

Starter cultures used in this study were *L. plantarum* + *L. sake*, which were isolated from sausage. A loop from a slant tryptic soy agar culture of each culture was inoculated in 10 mL of tryptic soy broth and incubated at 37 °C for 24 h. Five milliliters of the culture was then transferred to 100 mL of tryptic soy broth and incubated at 37 °C for another 24 h. The culture was centrifuged at 10,000 g for 10 min at 4°C and then washed with broth. Broth was prepared by homogenizing 1 part with 9 part of distilled water, filtered, adjusted to pH 7.0 and then autoclaved at 121°C for 15 min. After centrifugation, the cell pellet was resuspended in sterile fish broth, adjusted to approximately 107 cell/g and used as starter culture in sauce ripening.

### **2.5. Nham preparation**

Minced pork (56%), pieced cooked pork skin (37%), garlic (3.2%), cooked rice (2%), sodium polyphosphate (0.15%), sodium chloride (1.5%) and sodium erythrobate (0.15%) chili (1%) 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 fermented sausages were homogenized for analysis.

### **2.6. Physical and chemical analyses**

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

**2.2. Bacterial strains and growth conditions** 

**2.3. Determination of amine degradation** 

frozen at -15°C until HPLC analysis.

**2.4. Preparation of starter culture** 

culture in sauce ripening.

**2.5. Nham preparation** 

MRS broth.

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.

Bacterial strains isolated from different fermented sausages were tested. LAB were grown in

An overnight culture was harvested, washed with 0.05 M phosphate buffer (pH 7) and the cell pellet resuspended in 0.05 M phosphate buffer supplemented with tyramine, histamine, tryptamine, phenylethylamine, putrescine, and cadaverine. The cell concentration was adjusted to 106, 107 and 108 CFU/mL. The cell suspensions (20 mL) were incubated in a 100 ml flask and shaken at 200 rpm. Samples were taken and added to an equal amount of 1 M HCl. The mixture was boiled for 10 min and centrifugated at 9000 g. The supernatant was

Starter cultures used in this study were *L. plantarum* + *L. sake*, which were isolated from sausage. A loop from a slant tryptic soy agar culture of each culture was inoculated in 10 mL of tryptic soy broth and incubated at 37 °C for 24 h. Five milliliters of the culture was then transferred to 100 mL of tryptic soy broth and incubated at 37 °C for another 24 h. The culture was centrifuged at 10,000 g for 10 min at 4°C and then washed with broth. Broth was prepared by homogenizing 1 part with 9 part of distilled water, filtered, adjusted to pH 7.0 and then autoclaved at 121°C for 15 min. After centrifugation, the cell pellet was resuspended in sterile fish broth, adjusted to approximately 107 cell/g and used as starter

Minced pork (56%), pieced cooked pork skin (37%), garlic (3.2%), cooked rice (2%), sodium polyphosphate (0.15%), sodium chloride (1.5%) and sodium erythrobate (0.15%) chili (1%) 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 as μmol/g (dry matter).

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

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 filtered through a 0.25 m membrane filter.

### **2.8. Determination of BAs**

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 required.
