Strains exhibiting the potential to degrade BAs in a buffer system within 24 h at 30°C
Biogenic amines (BAs) are organic bases with aliphatic, aromatic or heterocyclic structures that can be found in several foods, in which they are mainly produced by microbial decarboxylation of amino acids, with the exception of physiological polyamines. BAs may be of endogenous origin at low concentrations in non-fermented food such as fruits, vegetables, meat, milk and fish. High concentrations have been found in fermented foods as a result of a contaminating microflora exhibiting amino acid decarboxylase activity (Silla-Santos, 1996). However, BAs can also trigger human health problems leading to palpitations, hypertension, vomiting, headaches and flushing if food containing high concentrations are ingested. In fermented foods, some lactic acid bacteria (LAB) are able to convert available amino acid precursors into BAs via decarboxylase or deiminase activities during or following ripening processes. For this reason, amino acid catabolism by LAB can affect both the quality and safety of fermented foods (Verges et al., 1999). The amount and type of BAs formed depends on the nature of food and particularly on the kind of microorganisms present. Enterobacteriaceae and certain LAB are particularly active in the production of BA (Beutling, 1996). These amine-producing microorganisms either may form part of the food associated population or may be introduced by contamination before, during or after processing of the food product. Therefore, microorganisms naturally present in raw materials, introduced throughout the processing or added as starter culture can critically influence BA production during the manufacture of fermented products (Bover-Cid et al., 2000).
Nham is a Thai-style fermented pork sausage. Nham ripening generally takes 3-5 days and relies mainly on adventitious microorganisms, which are normally found in raw materials. LAB produce organic acids from carbohydrates and cause the pH drop, which contribute to Nham formation.
During meat ripening, microbial growth, acidification and proteolysis provide favourable conditions for BA production. The species of lactobacilli most commonly found in meat and meat products are
BAs are physiologically inactivated by AO, which are enzymes found in bacteria, fungi, plant and animal cells able to catalyse the oxidative deamination of amines with production of aldehydes, hydrogen peroxide and ammonia (Cooper, 1997). The sequential action (in the presence of an electron acceptor, such as O2) of an AO and an aldehyde dehydrogenase leads to the production of an acid and ammonia, which can be used to support microbial growth (Parrot et al., 1987). MAO and DAO activity has been described in higher organisms as well as in bacteria (Murooka et al., 1976, 1979; Ishizuka et al., 1993). There are relevant differences between microbial AO in terms of substrate specificity and location, as stated by Cooper (1997). DAOs can oxidase several BA, such as putrescine and histamine, and their activity can be affected by substrate inhibition; aminoguanidine, antihistaminic drugs and foodborne inhibitors, such as ethanol, carnosine, thiamine, cadaverine and tyramine, reduce their activity (Lehane and Olley, 2000). The potential role of microorganisms involved in food ripenings with AO activity has been investigated with the aim to prevent or reduce the accumulation of BA in foods. Leuschner et al. (1998) tested in vitro the potential amine degradation by many bacteria isolated from foods and, in particular, in strains belonging to the genera
Since Nham is normally consumed without cooking, proper acid production is important to determine the quality and safety of Nham for consumption. Depending on the initial number of contamination, the occurrence of pathogens such as
2. Materials and methods
2.1. Microbiological analysis
Nham sausages (25 g) were aseptically transferred into a stomacher bag, with 225 mL of peptone (0.85% of sodium chloride added) and then homogenized for two minutes. Further decimal dilutions were made and then 100 μL of each dilution was spread onto agar plates. Aerobic plate count agar was used to determine total aerobic. BA producing bacteria were counted using differential media supplemented with amino acids as precursor of BAs (Joosten and Northolt, 1989). The media contained of tryptone (0.5%), yeast extract (0.5%), sodium chloride (0.5%), glucose (0.1%), Tween 80 (0.05%), MgSO4•7 H2O (0.02%), CaCO3 (0.01%), MnSO4•4H2O (0.005%), FeSO4•7H2O (0.004%), bromocresol purple (0.006%), amino acid (2%) and agar (2%). The medium contained the precursor amino acids (0.5% tyrosine di-sodium salt and 0.25% L-histidine monohydrochoride, L-ornithine monohydrochoride, L-lysine 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.
2.2. Bacterial strains and growth conditions
Bacterial strains isolated from different fermented sausages were tested. LAB were grown in MRS broth.
2.3. Determination of amine degradation
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 frozen at -15°C until HPLC analysis.
2.4. Preparation of starter culture
Starter cultures used in this study were
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 (
2.6. Physical and chemical analyses
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; a-value measures redness when positive, gray when zero, and greenness when negative, b-value 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 α-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 μm 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
2.9. Determination of amino acids
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 μL and monitored at 254 nm of wavelength.
2.10. Statistical analysis
Data was analysed by one-way ANOVA and differences among treatment means were determined by Duncan’s new multiple-range test.
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.
|Starter culture||Percent degradation (%)|
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,
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.
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.
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.
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 (
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 (
Fig. 6 shows that TCA-soluble peptide content of Nham samples, the initial content was 9.02 μmol/g dry matter. It then gradually increased throughout the ripening process. The TCA-soluble peptide content of Nham control and Nham with starters reached 23.6 to 87.2 μmol/g dry matter and 24.1 %to 65.2 μmol/g dry matter, respectively, at the end of ripening (hour 72). However, there was not significant difference (
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.
Fig. 7 shows that free α-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 α-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 (
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 (
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 (
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 (
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 (
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 (
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 (
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 (
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.
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 (
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 μmol/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 (
Fig. 18 shows that free α-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 (
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 25ºC (9.4 log CFU/g) were higher (
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 (
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 (
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 (
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 15ºC and 25ºC, 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 30ºC and 37ºC as compared to 25ºC 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
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 15ºC decarboxylases might be still active (Bover-Cid et al., 2001). During storage, the more temperature exceeds 14–15ºC 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 20ºC), 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.
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, 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
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).
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 4ºC-25ºC 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 ºC 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 ºC 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
(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).
|Storage time (wk)||Storage time (wk)|
|Storage time (wk)||Storage time (wk)|
|Storage time (wk)||Storage time (wk)|
Two types of fermented sausage differing in starter culture were produced in parallel with two different starter cultures (no starter and
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
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
This study was supported by Office of the Higher Education Commission (OHEC), Thailand.