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Changes in microbial flora and enzyme activity during the aging of tou-pan-chiang, a Chinese fermented condiment
[j. Ferment. Technol., Vol. 66, No. 4, 473--478. 1988]
Note
Changes of Microbial Flora and Enzyme Activity during the Aging of Tou-Pan-Chiang, a Chinese Fermented Condiment CHENG-CHuN CHOU*, Gwo-RoNo HWANG, and FRANK-MING H o Graduate Institute of Food Science & Technology, National Taiwan University, Taipei, Taiwan, Republic of China
Investigation of the microbial flora, various enzyme activities, and acidity of mash during the aging oftou-pan-chiang, a Chinese traditional fermented condiment was done. During the 3-month aging period, the population of mold and aerobic microorganisms gradually decreased after 42 d of fermentation, while counts of lactic acid bacteria and yeasts increased. In general, the activities of proteases, amylases, cellulase and a-galactosidase rose, then declined. Changesof lipase activity showed no consistent trend. The acidity of mash increased from the initial value of 0.72 to 2.35%, while the pH declined from 6.2 to 5.4.
Many kinds of fermented foods such as
this study, we investigated the changes of
tou-yu or chiang-~u (soy sauce), in-yu (black microbial flora and activity of various bean sauce), tou-chiang (miso), tou-fu-ju enzymes during the aging of tou-pan-chiang (soybean cheese, sufu) as well as tou-pan- mash. chiang (fermented bean jam) are well known Processes f o r tou.pan.chiang p r o in Taiwan. duction Tou-pan-chiang was prepared in Although the manufacture processes for
a local plant by the traditional procedure.
tou-pan-chiang are similar to those for soy Soybeans were first soaked for 6 to 8 h , sauce, in-yu, and miso including koji-making dehulled, steamed, and cooled, then mixed and mash-aging (fermentation) periods, there are some differences among these fermented foods.x) The main raw materials of tou-pan-chiang are soybean plus wheat flour. They are mixed thoroughly with seed-koji in trays, then maintained at 25-30°C for about 72 h in the case of 3 d koji. After 72 h of incubation, the finished koji are mixed directly with salt water for fermentation. The form of the final product of tou-pan-chiang is semi-solid. Previously, we have studied the microbial flora and biochemical changes during the preparation of tou-pan-chiang koji.2) In * Corresponding author
with roasted wheat flour. The soybeanwheat flour mixtures were spreaded on trays, then mixed throughly with seed-koji. The ratios of soybeans to wheat flour and seed-koji to soybean-wheat flour mixtures were 10 : 2 (w/w) and 6 : 1000 (w/w), respectively. The seed-koji was bought from the Seedkoji Supply Center (Taipei, Taiwan). The koji-mold has been isolated and identified as Aspergillus oryzae. 2,3) For koji-making, the temperature and relative humidity of the incubator were kept at 25-30°C and more than 95%, respectively. During the 72 h koji-making period, the temperature of the soybean-
474
CHOU, I-{WANO,and Ho
wheat flour mixture on the trays rose to about 40°C around the 17thh as well as 25thh. The mixtures mere stirred at those times to remove the fermentation heat and to reduce the temperature to about 25-30°C. For aging, the finished koji was mixed with 20 °B6 salt water at a ratio o f l : 1.25 (w/v) and put in mash tanks. The aging period was about 100 d.
Enumeration
of
microorganism
Essentially, the media and culture methods described by Wang et al.8) as well as Shieh and Beuchat4) were followed to enumerate the total population of aerobic microorganisms, molds, yeasts, and lactic acid bacteria in the tou-pan-chiang mash.
Measurement
of
enzyme
activity
[J. Ferment. Technol.,
p-nitrophenyl-a-D-galactopyranose and 0.3 ml of 0 . 2 M phosphate buffer (pH6.4) were mixed and incubated at 37°C for 20 mitt. The reaction was stopped by adding 9.5 ml of 0.1 M Na,CO3 solution. The optical density was measured at 440 nm. s) A modification of Gaseoigne's method 9) was used to measme cellulase activity. A reaction mixture consisting of 0.5ml oI enzyme solution and 0.5 ml of 1% sodium earboxymethyl cellulose solution (dissolved in 0.01 M acetate buffer, p H 5 . 0 ) was incubated at 50°C for 10min. lipase activity was assessed by a modification of Bier's method. 10) Five ml ot enzyme solution, 10 ml of freshly prepared polyvinyl alcohol emulsion (pH6.0), and 5 ml of Mcllvaine buffel (pH 6.0) were mixed and shaken at 35°C for 60min. All the enzyme activity was expressed by dividing the enzyme activity in the aging m,~sh by that in the original mash sample.
Enzymes were extracted from 10 g of toupan-chian~ mash mith 100 ml of 0.5% NaC1 solution by shaking 3 h at 25°C then filtering through Whatman no. 1 filter paper. The Measurement of acidity and pH filtrate was then used as the enzyme solution. A modification of Anson's method~) was Five grams of mash was mixed with 100 ml used to measure protease activities. One of distilled water, boiled 2 min, cooled, and ml of enzyme solution and 1 ml of 2% then filtered. Acidity was measured by Hammarsten milk casein solution (dis- titrating the filtrate with 0.I N N a O H solved in glycine-HC1 buffer, p H 3.0, for solution and expressed as the percentage of acidic protease; phosphate buffer, p H 6.0, lactic acid, while p H was measured directly for neutral protease; and carbonate buffer, with a p H meter. p H 10.0, for alkaline protease) were mixed Changes of microbial populations and incubated at 37°C for 10 rain. Changes of microbial populations during A modification of Wohlgenuth's methode) tou-pan-chiang aging are presented in Fig. 1. was used to measure a-amylase activity. The total population of aerobic microA reaction mixture consisting of 0.5 ml of organisms increased slightly during the early enzyme solution, 10 ml of 3% soluble starch stage of fermentation. After 42 d of fersolution, and 5 ml of 0.2 M acetate buffer mentation, it gradually declined as fermen(pH 5.0) was incubated at 40°C for 5 rain. tation progressed. During the first 42 d aging period, the d-Amylase was assayed by mixing 2 m l of enzyme solution, 10ml of 3% soluble microbial populations in mash consisted starch solution, and 2 ml of 0.2 M acetate mainly ofkoji-molds since the total population buffer (pH 5.0). The leaction mixture of microorganisms was found almost was then incubated at 40°C for 30 rain and equivalent to the mold count. As aging tbe reaction was stopped by adding 2 ml further progressed, the mold count also of 2N N a O H solution. Maltose in the decreased as the total population of microreacted solution was measmed by the DNS organisms did. However, the mold count method 7) declined more drastically than the total To measure a-galactosidase activity, plate count did. 0.1 ml of enzyme solution, 0.1 ml of 0.01 M Anaerobic conditions and high salt content
Vol. 66, 1988]
Changes of Microbial Flora
475
9.0, 8.0 ¸ "~
7.0 6.0 5.0 4,0 ~ 3.0 2.01 1.0 t
7
1
28
42
56
7
84
98
Time (d) Fig. 1. Changesof microbial flora during mash-aging. Each value is an average of duplicate measurements. O, total aerobic microorganisms; O, molds; A, lactic acid bacteria; BI, yeasts. have been regarded as lethal, causing the decline of the mold count during the aging period of rniso and soy sauce.4,1x, 12) In this study, following the traditional tou-pan-chiang manufacture practise, mash was put in crocks loosely covered on top and the mash was stirred every day to make it uniform during the first-month aging period. This definitely would provide better aerobic conditions in the mash than the experimental conditions set by Shieh and Beuchat4~ for the aging of miso mash and thus allowed the survival of koji molds, as we observed. The existence of lactic acid bacteria and yeasts with an initial count of 1.6× 10 4 CFU/g and 3.0× 10 2 CFU/g, respectively, were noted in mash prepared in a local manufacturing plant which did not strictly follow aseptic procedures. On the other hand, preliminary experiments showed that the product prepared in the laboratory following strictly aseptic procedures without contamination by lactics and yeasts did not have the characteristic flavor of tou-panchiang. Therefore, the yeasts and lactic acid bacteria are being isolated and identified. T h e i r roles in the quality and manufacturing
process of tou-pan-chian# are also under investigation. During the aging period, the yeast count gradually increased as aging progressed and at the end of aging period it was 1.3× 10 7 CFU/g. This observation was, in general, in agreement with those reported by Yong and Wood is) as well as Hsu et aIA 4) However, it was in contrast to that reported by Shieh and Beuchat 4) who observed that the population of the inoculated Saccharomyces rouxii increased early during the miso fermenting period, then gradually declined as aging progressed. Yong and Wood ~s) and Hsu et alA*) studied the populations of inoculated lactic acid bacteria in soy-mash during fermentation and found that the number of lactic acid bacteria decreased as fermentation progressed. Shieh and Beuchat 4) reported no consistent trend with regard to populations of lactic acid bacteria in any of the sixteen miso formulae they prepared. However, we found that the number of viable salttolerant lactic acid bacteria slightly decreased from 1.6×10 4 C F U / g to 2 . 4 × 1 0 s C F U / g as measured after a 1 4 d aging period.
476
Ca~ou,I-Iw.~aqo,:andHo.
Thereafter, it gradually increased to 6.7× 106 CFU]g at the end of the aging period. Changes of acidity and pH Shieh and Beuchat 4) indicated that the final pH of miso they prepared with Rhizopus oligosporus or Aspergillus oryzae ranged from 4.71 to 5.15 after 9 0 d of aging. Shibasaki and Hesseltine ~5) also observed that the pH of fermented and aged miso ranged from 5.1 to 5.3. The initial pH and acidity of tou-pan-chiang mash was 6.2 and 0.72%, respectively. After 98 d of aging, the pH of the mash decreased to 5.4 while the acidity increased to 2.35% (Fig. 2). The decline of pH and increase of acidity may be attributed to the autolysis of microbial cells, accumulation of free fatty acids, amino acids, and peptides containing carboxylic side chains as a result of hydrolysis of mash constituents, as well as the microbial fermentation of carbohydrates 4) during aging (Fig. 1).
7.(] 6.0 5.0 4.0 t l l l
I
I
i
I
I
&
98
3.0 2.5
,~ 2.0 1.5 1.0 0.5
,,,, 0
7 1 21 2 8
, 42
Time
I
70
I
(d)
Fig. 2. Changes of pH and acidity during mashaging. Each value is an average of duplicate measurements.
[J. Ferment: Teehnol.,
Changes of enzyme activities Enzymes produced by koji-molds and other microorganisms, i.e., yeasts and lactic acid bacteria, are important for the production of the characteristic flavors, colors, and textures of oriental traditional fermented products of soybeans like soy sauce and miso.Xe-xs) Various investigators have reported changes of several enzyme activities during the aging of soy sauce. 9,1~,14) Changes of enzyme activities during the aging of tou-pan-chiang are presented in Fig. 3. The levels of a-amylase and fl-amylase increased in the initial stage and reached their maximum relative activities of 109% and 127%, respectively, at the 14th d of aging (Fig. 3-A). As aging further progressed, as in the results reported by Hsu et al., TM a- and fl-amylase activity decreased. Relative activities of 48.5% and 74.6% were found for a- and fl-amylase, respectively, at the end of the aging period. The levels of the three kinds of protease activity all increased at the initial stage of aging and all reached their maximum relative activities after 14 d of aging (Fig. 3-B). At this point the maximum relative activities were 134%, 118%, and 280%, respectively, for acidic, neutral, and alkaline protease. Of these three kinds of proteases, alkaline protease showed the highest activity. Thereafter, their activities decreased. The relative activity of a-galactosidase reached its maximum of 114% after 14d of aging. After this, its relative activity declined sharply and dropped to its minimum relative activity of 6.2% after 42 d of aging. Thereafter, it remained almost constant throughout the rest of the aging period (Fig. 3-C). A small fluctuation of relative cellulase activity in the mash was observed during the first 28 d of aging. Thereafter, it increased sharply to its maximum relative activity of 145% after 42 d of aging then decreased as aging further progressed (Fig. 3-C). At the end of aging, a relative activity of 55.0% in the mash was noted. There was no consistent trend in tou-pan-
Vol. 66, 1988]
Clmnges of Microbial Flora
477
c
200
150
100
50 i
~ 300
~_
250
..
200
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,
,
,
,
D
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50 i
,,,~,88
i
i
,2
~
7'0
8',
~8 0 Time ( d )
7 14 21 2 8
42
56
70
84
98
Fig. 3. Changes in enzyme activity during mash-aging. Each value is an average of duplicate measurements. The original enzyme activity was assigned a relative activity of 100%. A hundred percent corresponds to 2454 units of a-amylase; 1051 units of fl-amylase; 158 units of acidic protease; 411 units of neutral protease; 107 units of alkaline protease; 112 units of a-galactosidase; 727 units of cellulase; and 334 units of lipase. In Fig. 3A, @, a-amylase; II, fl-amylase; in Fig. 3B, @, acidic protease; II, neutral protease; A, alkaline protease; in Fig. 3C, @, cellulase; II, a-galactosidase; in Fig. 3D, lipase activity.
chiang m a s h w i t h r e g a r d to lipase a c t i v i t y d u r i n g the a g i n g period. L i p a s e relative a c t i v i t y increased to 151.6% on the 7 t h d o f a g i n g t h e n decreased to n o n e o n the 21th and 28thd. I t then increased steadly to its m a x i m u m a c t i v i t y o f 3 0 9 % on the 70th d t h e n decreased a g a i n (Fig. 3-D). T h e existence o f these enzymes in tou-panchiang koji h a d b e e n d e m o n s t r a t e d b y H w a n g et al.8) D u r i n g the a g i n g period, it is r e a s o n a b l e to expect t h a t the survival o f koji-molds a n d g r o w t h o f the c o n t a m i n a t i n g lactic a c i d b a c t e r i a as well as yeasts (Fig. 1) m a y further c o n t r i b u t e to the a c t i v i t y o f these enzymes. Besides, the acid, p H as well as the salt c o n t e n t m a y relate to the a c t i v i t y o f enzymes found in mash. H o w e v e r , the i n i t i a l increase o f proteases, amylases, a n d a - g a l a c t o s i d a s e could be a t t r i b u t e d to the release o f fresh enzymes from substrates o r u p o n lysis o f m o l d h y p h a e
in the b r i n e solution.4, aS)
Acknowledgment This research was supported by the National Science Council (ROC) grant NSC 75-0409-B002-37.
References 1) Su, Y.C.: Proceedings of the Oriental Fermented Foods, p. 15, Food Industry Research and Development Institute, Hsinchu, Taiwan, R.O.C. (1980). 2) I-Iwang, G.R., Chou, C.C., Ho, F . M . : Or. Chinese Agric. Chem. Society, 25, 47 (1987). 3) Wang, I.H., Chen, S.K., Hsu, W.H.: Rep. Food Industry Res. and Develop. Ins. No: 154, Hainchu, Taiwan, R.O.C. (1980). 4) Shleh, C.Y.S., Beuchat, L . R . : J. Food Sd., 47, 518 (1982). 5) Narahara, H., Koyama, Y., Yoshida, T., Pichangkura, S., Ueda, R., Taguchi, H.: Or. Ferment. Technol., 60, 311 (1982). 6) Tokyo University Press (ed.) : Experimental
478
7) 8) 9) 10)
11) 12)
Ca~ou, HWANO,and Ho
Agricultural Chemistry I, p. 279, Asakura Shoten, Tokyo (1962). Miller, G.L.: AnalyticalChem., 31,426 (1959). Kaeamura, S., Kasai, T., Tanusi, S.: Agric. Biol. Chem., 40, 641 (1976). Goel, S.K., Wood, B.J.B.: J. Food Technol., 13, 243 (1978). Bier, M.: Methods ofEnzymology, (Colwick, S. P., Kaplan, N. O.), Vol. 1, p. 627, Academic Press, N.Y. (1955). Susukida, W., Masttko, F., Matsuo, T.: jr. Ferment. Teehnol., 39, 1 (1961). Yong, F. M., Wood, B.J.B.: J. Food Teehnol., 12,
[J. Ferment. Teehnol.,
163 (1977). 13) Yong, F.M., Wood, B.J.B.: J. Food Technol., 11, 525 (1976). 14) Hsu, W. H., Lai, M. N., Lin, D. C., Liu, W. H., Jan, G.D.: FoodSd., 2, 7 (1975). 15) Shibasaki, K., Hesseltine, C.W. : Economic Botany, 16, 180 (1966). 16) Whitaker, J.R.: Food Technol., 32, 175 (1978). 17) Ota, A.: J. Brew. Soc. Japan, 73, 601 (1978). 18) Nakadai, N.: J. Jap. Soy Sauce Res. Inst., 11, 67 (1985). (Received January 27, 1988)
Note
Changes of Microbial Flora and Enzyme Activity during the Aging of Tou-Pan-Chiang, a Chinese Fermented Condiment CHENG-CHuN CHOU*, Gwo-RoNo HWANG, and FRANK-MING H o Graduate Institute of Food Science & Technology, National Taiwan University, Taipei, Taiwan, Republic of China
Investigation of the microbial flora, various enzyme activities, and acidity of mash during the aging oftou-pan-chiang, a Chinese traditional fermented condiment was done. During the 3-month aging period, the population of mold and aerobic microorganisms gradually decreased after 42 d of fermentation, while counts of lactic acid bacteria and yeasts increased. In general, the activities of proteases, amylases, cellulase and a-galactosidase rose, then declined. Changesof lipase activity showed no consistent trend. The acidity of mash increased from the initial value of 0.72 to 2.35%, while the pH declined from 6.2 to 5.4.
Many kinds of fermented foods such as
this study, we investigated the changes of
tou-yu or chiang-~u (soy sauce), in-yu (black microbial flora and activity of various bean sauce), tou-chiang (miso), tou-fu-ju enzymes during the aging of tou-pan-chiang (soybean cheese, sufu) as well as tou-pan- mash. chiang (fermented bean jam) are well known Processes f o r tou.pan.chiang p r o in Taiwan. duction Tou-pan-chiang was prepared in Although the manufacture processes for
a local plant by the traditional procedure.
tou-pan-chiang are similar to those for soy Soybeans were first soaked for 6 to 8 h , sauce, in-yu, and miso including koji-making dehulled, steamed, and cooled, then mixed and mash-aging (fermentation) periods, there are some differences among these fermented foods.x) The main raw materials of tou-pan-chiang are soybean plus wheat flour. They are mixed thoroughly with seed-koji in trays, then maintained at 25-30°C for about 72 h in the case of 3 d koji. After 72 h of incubation, the finished koji are mixed directly with salt water for fermentation. The form of the final product of tou-pan-chiang is semi-solid. Previously, we have studied the microbial flora and biochemical changes during the preparation of tou-pan-chiang koji.2) In * Corresponding author
with roasted wheat flour. The soybeanwheat flour mixtures were spreaded on trays, then mixed throughly with seed-koji. The ratios of soybeans to wheat flour and seed-koji to soybean-wheat flour mixtures were 10 : 2 (w/w) and 6 : 1000 (w/w), respectively. The seed-koji was bought from the Seedkoji Supply Center (Taipei, Taiwan). The koji-mold has been isolated and identified as Aspergillus oryzae. 2,3) For koji-making, the temperature and relative humidity of the incubator were kept at 25-30°C and more than 95%, respectively. During the 72 h koji-making period, the temperature of the soybean-
474
CHOU, I-{WANO,and Ho
wheat flour mixture on the trays rose to about 40°C around the 17thh as well as 25thh. The mixtures mere stirred at those times to remove the fermentation heat and to reduce the temperature to about 25-30°C. For aging, the finished koji was mixed with 20 °B6 salt water at a ratio o f l : 1.25 (w/v) and put in mash tanks. The aging period was about 100 d.
Enumeration
of
microorganism
Essentially, the media and culture methods described by Wang et al.8) as well as Shieh and Beuchat4) were followed to enumerate the total population of aerobic microorganisms, molds, yeasts, and lactic acid bacteria in the tou-pan-chiang mash.
Measurement
of
enzyme
activity
[J. Ferment. Technol.,
p-nitrophenyl-a-D-galactopyranose and 0.3 ml of 0 . 2 M phosphate buffer (pH6.4) were mixed and incubated at 37°C for 20 mitt. The reaction was stopped by adding 9.5 ml of 0.1 M Na,CO3 solution. The optical density was measured at 440 nm. s) A modification of Gaseoigne's method 9) was used to measme cellulase activity. A reaction mixture consisting of 0.5ml oI enzyme solution and 0.5 ml of 1% sodium earboxymethyl cellulose solution (dissolved in 0.01 M acetate buffer, p H 5 . 0 ) was incubated at 50°C for 10min. lipase activity was assessed by a modification of Bier's method. 10) Five ml ot enzyme solution, 10 ml of freshly prepared polyvinyl alcohol emulsion (pH6.0), and 5 ml of Mcllvaine buffel (pH 6.0) were mixed and shaken at 35°C for 60min. All the enzyme activity was expressed by dividing the enzyme activity in the aging m,~sh by that in the original mash sample.
Enzymes were extracted from 10 g of toupan-chian~ mash mith 100 ml of 0.5% NaC1 solution by shaking 3 h at 25°C then filtering through Whatman no. 1 filter paper. The Measurement of acidity and pH filtrate was then used as the enzyme solution. A modification of Anson's method~) was Five grams of mash was mixed with 100 ml used to measure protease activities. One of distilled water, boiled 2 min, cooled, and ml of enzyme solution and 1 ml of 2% then filtered. Acidity was measured by Hammarsten milk casein solution (dis- titrating the filtrate with 0.I N N a O H solved in glycine-HC1 buffer, p H 3.0, for solution and expressed as the percentage of acidic protease; phosphate buffer, p H 6.0, lactic acid, while p H was measured directly for neutral protease; and carbonate buffer, with a p H meter. p H 10.0, for alkaline protease) were mixed Changes of microbial populations and incubated at 37°C for 10 rain. Changes of microbial populations during A modification of Wohlgenuth's methode) tou-pan-chiang aging are presented in Fig. 1. was used to measure a-amylase activity. The total population of aerobic microA reaction mixture consisting of 0.5 ml of organisms increased slightly during the early enzyme solution, 10 ml of 3% soluble starch stage of fermentation. After 42 d of fersolution, and 5 ml of 0.2 M acetate buffer mentation, it gradually declined as fermen(pH 5.0) was incubated at 40°C for 5 rain. tation progressed. During the first 42 d aging period, the d-Amylase was assayed by mixing 2 m l of enzyme solution, 10ml of 3% soluble microbial populations in mash consisted starch solution, and 2 ml of 0.2 M acetate mainly ofkoji-molds since the total population buffer (pH 5.0). The leaction mixture of microorganisms was found almost was then incubated at 40°C for 30 rain and equivalent to the mold count. As aging tbe reaction was stopped by adding 2 ml further progressed, the mold count also of 2N N a O H solution. Maltose in the decreased as the total population of microreacted solution was measmed by the DNS organisms did. However, the mold count method 7) declined more drastically than the total To measure a-galactosidase activity, plate count did. 0.1 ml of enzyme solution, 0.1 ml of 0.01 M Anaerobic conditions and high salt content
Vol. 66, 1988]
Changes of Microbial Flora
475
9.0, 8.0 ¸ "~
7.0 6.0 5.0 4,0 ~ 3.0 2.01 1.0 t
7
1
28
42
56
7
84
98
Time (d) Fig. 1. Changesof microbial flora during mash-aging. Each value is an average of duplicate measurements. O, total aerobic microorganisms; O, molds; A, lactic acid bacteria; BI, yeasts. have been regarded as lethal, causing the decline of the mold count during the aging period of rniso and soy sauce.4,1x, 12) In this study, following the traditional tou-pan-chiang manufacture practise, mash was put in crocks loosely covered on top and the mash was stirred every day to make it uniform during the first-month aging period. This definitely would provide better aerobic conditions in the mash than the experimental conditions set by Shieh and Beuchat4~ for the aging of miso mash and thus allowed the survival of koji molds, as we observed. The existence of lactic acid bacteria and yeasts with an initial count of 1.6× 10 4 CFU/g and 3.0× 10 2 CFU/g, respectively, were noted in mash prepared in a local manufacturing plant which did not strictly follow aseptic procedures. On the other hand, preliminary experiments showed that the product prepared in the laboratory following strictly aseptic procedures without contamination by lactics and yeasts did not have the characteristic flavor of tou-panchiang. Therefore, the yeasts and lactic acid bacteria are being isolated and identified. T h e i r roles in the quality and manufacturing
process of tou-pan-chian# are also under investigation. During the aging period, the yeast count gradually increased as aging progressed and at the end of aging period it was 1.3× 10 7 CFU/g. This observation was, in general, in agreement with those reported by Yong and Wood is) as well as Hsu et aIA 4) However, it was in contrast to that reported by Shieh and Beuchat 4) who observed that the population of the inoculated Saccharomyces rouxii increased early during the miso fermenting period, then gradually declined as aging progressed. Yong and Wood ~s) and Hsu et alA*) studied the populations of inoculated lactic acid bacteria in soy-mash during fermentation and found that the number of lactic acid bacteria decreased as fermentation progressed. Shieh and Beuchat 4) reported no consistent trend with regard to populations of lactic acid bacteria in any of the sixteen miso formulae they prepared. However, we found that the number of viable salttolerant lactic acid bacteria slightly decreased from 1.6×10 4 C F U / g to 2 . 4 × 1 0 s C F U / g as measured after a 1 4 d aging period.
476
Ca~ou,I-Iw.~aqo,:andHo.
Thereafter, it gradually increased to 6.7× 106 CFU]g at the end of the aging period. Changes of acidity and pH Shieh and Beuchat 4) indicated that the final pH of miso they prepared with Rhizopus oligosporus or Aspergillus oryzae ranged from 4.71 to 5.15 after 9 0 d of aging. Shibasaki and Hesseltine ~5) also observed that the pH of fermented and aged miso ranged from 5.1 to 5.3. The initial pH and acidity of tou-pan-chiang mash was 6.2 and 0.72%, respectively. After 98 d of aging, the pH of the mash decreased to 5.4 while the acidity increased to 2.35% (Fig. 2). The decline of pH and increase of acidity may be attributed to the autolysis of microbial cells, accumulation of free fatty acids, amino acids, and peptides containing carboxylic side chains as a result of hydrolysis of mash constituents, as well as the microbial fermentation of carbohydrates 4) during aging (Fig. 1).
7.(] 6.0 5.0 4.0 t l l l
I
I
i
I
I
&
98
3.0 2.5
,~ 2.0 1.5 1.0 0.5
,,,, 0
7 1 21 2 8
, 42
Time
I
70
I
(d)
Fig. 2. Changes of pH and acidity during mashaging. Each value is an average of duplicate measurements.
[J. Ferment: Teehnol.,
Changes of enzyme activities Enzymes produced by koji-molds and other microorganisms, i.e., yeasts and lactic acid bacteria, are important for the production of the characteristic flavors, colors, and textures of oriental traditional fermented products of soybeans like soy sauce and miso.Xe-xs) Various investigators have reported changes of several enzyme activities during the aging of soy sauce. 9,1~,14) Changes of enzyme activities during the aging of tou-pan-chiang are presented in Fig. 3. The levels of a-amylase and fl-amylase increased in the initial stage and reached their maximum relative activities of 109% and 127%, respectively, at the 14th d of aging (Fig. 3-A). As aging further progressed, as in the results reported by Hsu et al., TM a- and fl-amylase activity decreased. Relative activities of 48.5% and 74.6% were found for a- and fl-amylase, respectively, at the end of the aging period. The levels of the three kinds of protease activity all increased at the initial stage of aging and all reached their maximum relative activities after 14 d of aging (Fig. 3-B). At this point the maximum relative activities were 134%, 118%, and 280%, respectively, for acidic, neutral, and alkaline protease. Of these three kinds of proteases, alkaline protease showed the highest activity. Thereafter, their activities decreased. The relative activity of a-galactosidase reached its maximum of 114% after 14d of aging. After this, its relative activity declined sharply and dropped to its minimum relative activity of 6.2% after 42 d of aging. Thereafter, it remained almost constant throughout the rest of the aging period (Fig. 3-C). A small fluctuation of relative cellulase activity in the mash was observed during the first 28 d of aging. Thereafter, it increased sharply to its maximum relative activity of 145% after 42 d of aging then decreased as aging further progressed (Fig. 3-C). At the end of aging, a relative activity of 55.0% in the mash was noted. There was no consistent trend in tou-pan-
Vol. 66, 1988]
Clmnges of Microbial Flora
477
c
200
150
100
50 i
~ 300
~_
250
..
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~8 0 Time ( d )
7 14 21 2 8
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84
98
Fig. 3. Changes in enzyme activity during mash-aging. Each value is an average of duplicate measurements. The original enzyme activity was assigned a relative activity of 100%. A hundred percent corresponds to 2454 units of a-amylase; 1051 units of fl-amylase; 158 units of acidic protease; 411 units of neutral protease; 107 units of alkaline protease; 112 units of a-galactosidase; 727 units of cellulase; and 334 units of lipase. In Fig. 3A, @, a-amylase; II, fl-amylase; in Fig. 3B, @, acidic protease; II, neutral protease; A, alkaline protease; in Fig. 3C, @, cellulase; II, a-galactosidase; in Fig. 3D, lipase activity.
chiang m a s h w i t h r e g a r d to lipase a c t i v i t y d u r i n g the a g i n g period. L i p a s e relative a c t i v i t y increased to 151.6% on the 7 t h d o f a g i n g t h e n decreased to n o n e o n the 21th and 28thd. I t then increased steadly to its m a x i m u m a c t i v i t y o f 3 0 9 % on the 70th d t h e n decreased a g a i n (Fig. 3-D). T h e existence o f these enzymes in tou-panchiang koji h a d b e e n d e m o n s t r a t e d b y H w a n g et al.8) D u r i n g the a g i n g period, it is r e a s o n a b l e to expect t h a t the survival o f koji-molds a n d g r o w t h o f the c o n t a m i n a t i n g lactic a c i d b a c t e r i a as well as yeasts (Fig. 1) m a y further c o n t r i b u t e to the a c t i v i t y o f these enzymes. Besides, the acid, p H as well as the salt c o n t e n t m a y relate to the a c t i v i t y o f enzymes found in mash. H o w e v e r , the i n i t i a l increase o f proteases, amylases, a n d a - g a l a c t o s i d a s e could be a t t r i b u t e d to the release o f fresh enzymes from substrates o r u p o n lysis o f m o l d h y p h a e
in the b r i n e solution.4, aS)
Acknowledgment This research was supported by the National Science Council (ROC) grant NSC 75-0409-B002-37.
References 1) Su, Y.C.: Proceedings of the Oriental Fermented Foods, p. 15, Food Industry Research and Development Institute, Hsinchu, Taiwan, R.O.C. (1980). 2) I-Iwang, G.R., Chou, C.C., Ho, F . M . : Or. Chinese Agric. Chem. Society, 25, 47 (1987). 3) Wang, I.H., Chen, S.K., Hsu, W.H.: Rep. Food Industry Res. and Develop. Ins. No: 154, Hainchu, Taiwan, R.O.C. (1980). 4) Shleh, C.Y.S., Beuchat, L . R . : J. Food Sd., 47, 518 (1982). 5) Narahara, H., Koyama, Y., Yoshida, T., Pichangkura, S., Ueda, R., Taguchi, H.: Or. Ferment. Technol., 60, 311 (1982). 6) Tokyo University Press (ed.) : Experimental
478
7) 8) 9) 10)
11) 12)
Ca~ou, HWANO,and Ho
Agricultural Chemistry I, p. 279, Asakura Shoten, Tokyo (1962). Miller, G.L.: AnalyticalChem., 31,426 (1959). Kaeamura, S., Kasai, T., Tanusi, S.: Agric. Biol. Chem., 40, 641 (1976). Goel, S.K., Wood, B.J.B.: J. Food Technol., 13, 243 (1978). Bier, M.: Methods ofEnzymology, (Colwick, S. P., Kaplan, N. O.), Vol. 1, p. 627, Academic Press, N.Y. (1955). Susukida, W., Masttko, F., Matsuo, T.: jr. Ferment. Teehnol., 39, 1 (1961). Yong, F. M., Wood, B.J.B.: J. Food Teehnol., 12,
[J. Ferment. Teehnol.,
163 (1977). 13) Yong, F.M., Wood, B.J.B.: J. Food Technol., 11, 525 (1976). 14) Hsu, W. H., Lai, M. N., Lin, D. C., Liu, W. H., Jan, G.D.: FoodSd., 2, 7 (1975). 15) Shibasaki, K., Hesseltine, C.W. : Economic Botany, 16, 180 (1966). 16) Whitaker, J.R.: Food Technol., 32, 175 (1978). 17) Ota, A.: J. Brew. Soc. Japan, 73, 601 (1978). 18) Nakadai, N.: J. Jap. Soy Sauce Res. Inst., 11, 67 (1985). (Received January 27, 1988)