Effect of Salt Concentration and Incubation Temperature on Formation of Histamine, Phenethylamine, Tryptamine and Tyramine During Miso Fermentation

Transcription

1 423 Journal of Food Protection, Vol. 49, No. 6, Pages (June 1986) Copyright International Association of Milk, Food and Environmental Sanitarians Effect of Salt Concentration and Incubation Temperature on Formation of Histamine, Phenethylamine, Tryptamine and Tyramine During Miso Fermentation K.-D. HENRY CHIN and P. E. KOEHLER* Department of Food Science, University of Georgia, Athens, Georgia (Received for publication May 28, 1985) ABSTRACT Two factors, salt concentration and incubation temperature, were examined for their effect on the formation of histamine, phenethylamine, tryptamine and tyramine during miso (soybean paste) fermentation. Misos containing 5 and 10% NaCl were prepared and incubated at 25 and 35 C. The effect of each factor was determined from the chemical and microbiological changes in the misos during fermentation. Salt level was a significant factor in the formation of amines. Higher amine levels were found in low-salt (5% NaCl) formulations than in high-salt (10% NaCl) misos. Incubation temperature within the range of 25 to 35 C during fermentation had little effect on amine formation in misos. Histamine, tryptamine, tyramine and phenethylamine are biologically active (biogenic) amines which can have important psychoactive or vasoactive effects in humans (70). Tyramine, histamine and phenethylamine are pressor amines that cause changes in blood pressure (5,7,8). Tyramine and phenethylamine have also been implicated as causal agents in migraine headache (8,19,23). Biologically active amines have been found in many foods, especially fermented foods such as cheese, sauerkraut, wine and fermented meat (11,12,15-17,20,22,26,27). These biogenic amines are produced when specific amino acids undergo decarboxylation catalyzed by specific decarboxylase enzymes produced by microorganisms active during fermentation (13,18). Miso, an important and popular fermented soybean food in the Orient, is a semi-solid fermented soy paste produced through a fermentation similar to that of soy sauce (6). It is made from cereal grain, soybeans and salt through the action of molds, yeasts and bacteria (28). Because different types of miso are made by varying the ratio of raw materials, the composition of miso varies. The salt (NaCl) content of miso generally ranges from 5 to 13% (3). Yamamoto et al. (29) measured the tyramine content of several fermented food products and found that miso products contained 0.21 to (Jig of tyramine/g. Conditions that govern the formation of amines in fermented meat products have been investigated by Rice and Koehler (18). Significant factors included the availability of free amino acids, the presence of microorganisms that can decarboxylate amino acids, and favorable conditions for growth of microorganisms and production of decarboxylase enzymes. Because salt concentration and incubation temperature have significant effects on the activity and growth of microorganisms, it is possible that these two factors affect the formation of amines during miso fermentation. The objective of this study was to examine the effect of salt concentration and incubation temperature on the formation of amines during miso fermentations. MATERIALS AND METHODS Preparation of cultures Cultures of Aspergillus oryzae NRRL 1988 and Saccharomyces rouxii NRRL-Y-2547 were used in the fermentation of miso. A. oryzae was grown on YMPG agar plates (8) at 30 C for 4 d. Conidiospores of A. oryzae were then used to inoculate rice. YMPG broth (ph 7) containing 6% NaCl was used for culturing 5. rouxii (21). Cultures were grown under constant agitation on a gyratory waterbath shaker for 60 h at 30 C. Cells were collected by centrifugation (9000 x g, 10 min) and suspended in 0.1 M phosphate buffer (ph 7.0) containing 3% NaCl. The cellular suspensions were used as inocula for the miso formulations. Preparation of koji Enriched long-grain rice was mixed with water (2:1, wt/vol) and sterilized in an autoclave (121 C, 15 psi, 15 min). After cooling, conidiospores of A. oryzae were sprinkled over the cooked rice spread ca. 3 cm deep in shallow trays. The inoculated rice was incubated at 25 C for 2 to 3 d, with occasional stirring, until the grains were completely covered with creamy white mold mycelium but before the mold sporulated. Preparation of soybean paste Soybeans were soaked in water at room temperature for 18 h and then steamed for 60 min. The beans were spread out to

2 424 CHIN AND KOEHLER cool and then reduced to a paste by grinding in a Kolloid mill (Kolloid Mills Inc., Muncie, IN). Mixing of ingredients Rice koji was ground with a chopper (Enterprise Co., Philadelphia, PA) and then mixed with water, sodium chloride, S. rouxii inocula and soybean paste. Two different miso formulations were prepared with either 5 or 10% NaCl (Table 1). All misos contained 50% moisture. Conditions for fermentations After mixing the ingredients, each type (5 and 10% NaCl) of miso was divided into two equal parts and sealed in plastic bags. One part was incubated at 25 C and the other at 35 C. The fermentation period was 90 d. Sampling Samples were taken from each type of miso for chemical and microbiological analyses immediately after mixing (0 d) and after fermentation for 1, 2, 4, 8, 15, 30, 60 and 90 d. Amine analyses Histamine, tryptamine, phenethylamine and tyramine were determined in each miso sample. Sample (3 g) was mixed for 1 min with 3.5 ml of 0.1 N HC1 on a Genie Vortex mixer and then centrifuged (10,000 X g, 15 min, 4 C). The fat layer was removed and the aqueous phase collected. This procedure was repeated three times. The collected supernatant fluid was combined and made up to 12 ml with 0.1 N HC1. This amine extract was used in the preparation of dansyl derivatives. Dansyl derivatives of the amines were prepared, separated by thinlayer chromatography and quantitated by spectrofluorometry using the procedure developed by Chin and Koehler (2). Microbiological analyses The total bacterial, lactic acid bacteria, yeast, and mold populations in miso samples were determined with procedures described by Shieh and Beuchat (27). RESULTS AND DISCUSSION Typical bacteria and mold counts during a 90-d fermentation of misos with different salt concentrations and fermentation temperatures are shown in Figure 1. The curves for 25 and 35 C fermentation temperatures at both 5 and 10% NaCl levels were all nearly identical. The ph values of the misos during 90 d of fermentation are also shown in Figure 1. Acid-forming bacteria which are generally present in miso during fermentation produce organic acids (0.6 to 1.5%) such as lactic, succinic, acetic and phosphoric acid (9). Hence, the ph of Figure 1. Typical total bacterial and mold populations in misos during 90 d of fermentation: #, bacterial counts; A A, mold counts; O, ph. miso tends to decrease during fermentation. In addition to these organic acids formed by bacteria in miso, the accumulation of free fatty acids, amino acids and peptides containing carboxylic side chains as a result of hydrolysis of miso constituents could also contribute to changes in hydrogen ion concentrations (21). Incubation temperature (25 and 35 C) and salt concentrations (5 and 10%) had little or no effect on ph values. The ph of the miso samples ranged from 4.6 to 4.8 after 8 d of incubation. However, after 30 d, the ph of the misos increased slowly. This was probably due to decomposition of the organic acids previously formed. The final ph of all misos ranged from 5.1 to 5.3 after 90 d of fermentation. The initial mold population was ca. 3 x 10 6 CFU/g and consisted primarily of A. oryzae. During the fermentation, the mold population decreased rapidly. After 30 d of incubation, the mold count declined to less than 10 CFU/g. At 60 d of incubation, no molds were detected in 10"' dilutions of samples. The rapid decrease in mold population in the miso samples was probably due to the lethal effect of high salt concentration and anaerobic conditions (27). The initial population of yeasts was ca. 1 X 10 6 CFU/ g. The population of yeasts increased during the early fermentation period, then gradually declined (Fig. 2). Salt level had a significant influence on the yeast population. Higher yeast counts were noted in misos containing 5% TABLE 1. Formulations and fermentation temperatures for misos Ingredients NaCl (%) Rice koji (g) Soybean paste (g) Sodium chloride (g) Water (ml) Fermentation temperature ( C)

3 AMINES IN MISO Figure 2. Total yeast population in misos during 90 d of fermentation:, 5 % NaCl; A A, 10% NaCl. Figure 3. Lactic acid bacteria population in misos during 90 d of fermentation:, 5 % NaCl; A A, 10% NaCl. NaCl than in those containing 10% NaCl. Incubation temperature did not influence the yeast population. The change in the population of lactic acid bacteria during a 90-d miso fermentation is shown in Figure 3. The population curves at 25 and 35 C fermentation were nearly identical. The initial population of lactic acid bacteria was ca. 5 X 10 3 CFU/g. The population increased slowly during the early fermentation period but decreased gradually after 4 d of incubation. The counts declined to less than 10 CFU/g after 30 d of incubation. The low-salt (5% NaCl) formulae developed slightly higher lactic acid bacteria counts in the later stages of the fermentation than the high-salt (10% NaCl) formulae. Changes in the content of tyramine and histamine in misos during 90 d of fermentation are shown in Figures 4 and 5, respectively. These values were computed using the percent recovery determined for each amine in miso. In general, the amines reached their highest concentrations in 30 d and then decreased slightly. These amines are produced when specific amino acids undergo decarboxylation. This reaction is usually catalyzed by specific decarboxylase enzymes produced by microorganisms that are active during fermentation (13,18). The decrease in amine content during the later stages of fermentation was probably due to decomposition of the amines formed. In all cases, amine contents in 5% NaCl formulations were higher than those in 10% NaCl misos. Incubation temperature had almost no effect on amine contents in the final products. Tyramine was the major amine in miso samples (Fig. 4). All final miso products contained considerable amounts of tyramine. Tyramine was not detected until 48 h of incubation. Rapid tyramine production occurred between 2 and 8 d of incubation which corresponded to the time of rapid acid development, with the ph decreasing from ph 5.1 to 4.7. After reaching the maximum concentration, tyramine levels decreased slightly. Salt concentration affected tyramine levels significantly. The misos with 5% NaCl averaged 314 xg tyramine/g, whereas the misos with 10% NaCl averaged 176 xg/g. Several papers have been published concerning the effects of salt on histamine formation (14,24-26). Taylor and Woychik (25) found that levels of 3.5 to 5.5% NaCl could inhibit histamine production by Klebsiella pneumoniae, whereas lower levels were ineffective. Taylor and Speckhard (24) found similar results with Proteus morganii. Salt effects on tyramine formation have not been studied. Two commercial miso samples purchased locally contained 426 and 211 xg tyramine/g. Using gas-liquid chromatography in a survey of four brands of soy pastes, Yamamoto et al. (29) found tyramine concentrations ranging from 0.21 to xg/g. Histamine was present in detectable amount in 75% of the final miso products (Fig. 5). Considerable amounts of histamine were detected after only 4 d of incubation. Maximum levels were reached at 15 d of incubation, with levels then declining slowly. Much higher levels of histamine were found in the low-salt (5% NaCl) formulations than in high-salt (10% NaCl) misos. Incubation temperature had no effect on histamine levels in the misos. Tryptamine was detected in only two of the four final miso products and the levels of tryptamine were very low when compared to tyramine. The misos with 5% NaCl contained slightly higher tryptamine contents than those with 10% NaCl. Only one of the four final miso products contained detectable level of phenethylamine and the level was also very low compared to tyramine. In the low-salt (5% NaCl) formulae, a few xg of phenethylamine/g were found; however, in the high-salt (10% NaCl) misos, phenethylamine was never detected. From the results of this study, it is reasonable to conclude that salt (NaCl) level is a significant factor affect-

4 426 CHIN AND KOEHLER 100 Figure 4. Tyramine content of misos during 90 d of fermentation: (A) 5% NaCl, 25 C; (B) 5% NaCl, 35 C; (C) 10% NaCl, 25 C; (D) 10% NaCl, 35 C. 200 r B,o» loo- ^ 0 C 200 O 1Q o Figure 5. Histamine content of misos during 90 d of fermentation: (A) 5% NaCl, 25 C; (B) 5% NaCl, 35 C; (C) 10% NaCl, 25 C; (D) 10% NaCl, 35 C.

5 AMINES IN MISO 427 ing the formation of amines during miso fermentation. Tyramine and histamine production in misos paralleled bacterial growth, indicating that microbial decarboxylase enzymes probably play the major role in controlling amine formation in miso. Higher levels of amines are always formed in low-salt (5% NaCl) formulations than in high-salt (10% NaCl) misos. This probably relates to the inhibiting effect of higher salt concentration on the growth of many microorganisms, which in turn greatly decreases the likelihood of the production of decarboxylase enzymes responsible for the decarboxylation of amino acids to form amines. As these data have shown, incubation temperature has little effect on the formation of amines in misos during fermentation. Although temperature is also an important factor in the growth of microorganisms, the temperatures (25 and 35 C) used in this study were not extreme and most fermentation microorganisms can grow at either of these temperatures. The catalytic rates of decarboxylase enzymes are generally higher at elevated temperatures. However, the stability of decarboxylase enzymes is lower and the decomposition rates of amines are greater at higher temperature (4). As a result, temperature changes caused little change in amine content of misos during fermentation. In addition to salt concentration, other factors such as availability of various amino acids (type and ratios of raw materials) and use of starter cultures may also significantly affect amine formation in miso. REFERENCES 1. Beuchat, L. R Traditional fermented food products, pp In L. R. Beuchat (ed.) Food and beverage mycology. AVI Publishing Co., Westport, CT. 2. Chin, K.-D. H., and P. E. Koehler Identification and estimation of histamine, tryptamine, phenethylamine and tyramine in soy sauce by thin-layer chromatography of dansyl derivatives. J. Food Sci. 48: Ebine, H Miso. pp In M. Fujimaki and H. Mitsuda (eds.) 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