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Role of the yeast maltose fermentation genes in CO2 production rate from sponge dough
Food Microbiology,
1990,7,43-47
Role of the yeast maltose fermentation production rate from sponge dough
genes in COP
Y. Oda* and K. Ouchi
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Machida-shi, Tokyo 194, Japan Received11 October 1989 The time course of CO2 production in sponge dough fermentation (Fermogram) was compared in yeast strains carrying various MAL (maltose fermentation) genes. Most MAL-constitutive strains showed curves with a significant increase after l-l -5 h, fermenting maltose liberated from starch by endogenous amylases. However, all MALinducible strains and maltose non-fermenting strains gave quite different curves which remained low, indicating theirpoor leavening ability in sponge dough. We were able to visualize the crucial role of MAL-constitutive genes in sponge dough leavening by the Fermogram.
Introduction Leavening ability in sponge dough without addition of sugar, is one of the most important properties in baker’s yeast, and is closely related to its maltose fermentability (Oda and Ouchi 1989a). In most maltose-fermenting strains of Saccharomyces cerevisiae, one or more of five unlinked genes (&G&Ll, MALZ, MAL3, MAL4 and MA&61 are contained, and expression of both a-glucosidase and maltose permqase are induce.d by maltose in the gr~th medium (Barnett 1976). Some strains can produce these two enzymes in the absence of maltose, and the gene responsible for this phenotype is defined as MAL-constitutive (MAL’) (Khan and Eaton 1971). The curve of COP production rate from sponge dough, which is called Zymotachygram (Ponte and Reed 1982) or Fermogram (Hino et al. 19881, have reflected the characteristics of baker’s yeast. In the present paper we show these curves obtained with the strains containing vari*Corresponding author 0740-0020/90/010043
+ 05 $02.0010
ous MAL genes, and visualize the crucial role of MAL’ genes in sponge dough fermentation.
Materials Yeasts
and Methods
Eight MATa and 8 MATu strains ofS. cerevisiap, as shown in Table 1, were crossed to construct 64 diploids in all combinations. These strains of 16 haploids and 64 diploids were used in the present experiments. The genetic notation for the MAL strains and the meaning of MALg and MAL’ have been reported elsewhere (Michels and Needleman 1983). Each functional MAL gene is comprised of two complementary genes; MALp gene encoding the positive regulatory protein and MALg gene encoding both a-glucosidase and maltose permease (Nhedleman et al. 1984). The strains containing MALg alone are defective in maltose fermentation. mal’ indicates that there are neither MALp nor MALg genes. MALI’ is the gene included in YOY34, a haploid strain derived from baker’s yeast (Oda and Ouchi 1989b1, and other genes are derived from laboratory stock strains (Khan and Eaton 1971). MALl’ and MALX are MAL-constitutive genes and were responsible for high levels of a-glucosidase activities in the molasses medium (Oda and Ouchi 1989c). Other MAL genes were distinguished as 0 1990 Academic Press Limited
44
Y. Oda and K. Ouchi Table
1. List
of Saccharomyces
Maltose fermentation
Strain YOY107 YOYlO8 X2180-1A X2180-1B YOY334 YOY329 YOY416 YOY328 YOY338 YOY339 YOY342 YOY344 YOY943 YOY374 YOY371 YOY370 MALI’
and
cerevisiae
MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa
+ + + + + + + + + + + + are
used.
Genotype
-
MAL.4’
strains
constitutive
alleles
of
mal” ma1 MALlgMAL3g MALlgMAL3g MALl’ MALl’ MALl MALl MAL2 MAL2 MAL3 MAL3 MAU’ MAL+P MAL6 MAL6 MALl
and
MAL4,
respectively.
MAL-inducible since they caused basal expression of enzyme activities similar to those of mol” and MALlgMAL3g in the molasses medium (Oda and Ouchi 1989c). FSC6001 was a commercial baker’s yeast. Culture Yeast cells were cultured in a molasses medium containing 3% sugar as cone molasses, 0.193% urea, and 0.046% KHsPO, (Oda and Ouchi 1989a). COP production rate The ingredients of sponge dough, containing 100 g of flour, O-5g of salt, 1.65 g of yeast cells as dry matter, and 65 ml of distilled water, were mixed for 2 min with a National Mixer (National Mfg Co., Lincoln, NE). A piece of dough based on 20 g flour was cut and placed in a bottle of the Fermograph (Atto Co., Ltd., Tokyo, Japan). The rate of COs gas production was recorded automatically at 30°C and depicted as the Fermogram (Hino et al. 1988).
Results and Discussion In the sponge dough, the main fermentable sugar for yeast is maltose liberated from the starch of the flour by amylases (Ponte and Reed 1982). When COs production rate from the dough is recorded
against time, baker’s yeast usually depicts curves with two distinct peaks, as shown in Fig. 1. The first increase represents the fermentation of sugars which pre-existed in the flour, and the following large increase corresponds to the fermentation of maltose. Therefore, the second increase is one of the characteristics in the fermentation of the dough with addition of little or no sugar.
f =o
10
‘:g .E E E
6
6
s 9 B B N s 6
4
% oz
0
2
0
100
200
300
Time (min)
Fig. 1. Time course of COs production rate
from sponge dough with a commercial baker’s yeast (FSC6001).
t MALlgMAL39 I h4AL1°
I I MAL2
haploid
Time (min)
I MALl
MATa I MAW
I MAL4 =
Fig. 2. Time course of CO2 production rate from sponge dough with haploids and their hydrids.
r mal?
3oolllin
1 MAL6
E
46
Y. Oda and K. Ouchi
The left-end column and the top-end row in Fig. 2 show the curves of COs production rate from sponge dough by MATa and MATa haploids, respectively. Those by the 64 diploids are placed in the points on which the column and the row of their parents cross. The curves in Fig. 2 were generally smaller in height than those by conventional baker’s yeasts, probably because tested strains were constructed from laboratory strains which had less leavening abilities (Oda and Ouchi 198913). In the dough containing 5% glucose based on flour, the amount of gas produced by the strains tested was about 80% of that by industrial strains of baker’s yeast (data not shown). MAL-inducible haploids showed curves which remained as low as those of maltose non-fermenting strains, and diploids of MAL-inducible x MAL-inducible, MAL-inducible x malo, and MALinducible x MALlgMAL3g also showed similar curves. Some strains had the first peak, but few possessed the second peak. On the other hand, the haploids carrying either MALI’ or MAL.4’ showed an obvious second increase. The diploids containing one or two MAL’ genes showed similar curves to MAL’ haploids. These observations were explained by the finding that the constitutivity of MALl”
and MAL4’ genes was dominant over the inducibility of other MAL genes (Oda and Ouchi 1989c). However, curves by MALl’ x mat’ and MAL4’ x mal” diploids were similar to those by mal” strains. Since a-glucosidase activities of MALI’ and MAL4’ strains were reduced when hybridized with mal” strains (Oda and Ouchi 1989c), an inhibitor against expression of a-glucosidase might be present in malo strains. The cell of MAL-inducible strains, when grown in the medium containing maltose as a sole carbon source, expressed much higher activity of aglucosidase by induction, and showed the distinguishable second increase in the curves (data not shown). However,
cane
or beet molasses, which contain no appreciable amount of maltose, are widely used in the production of baker’s yeast because of the convenience and economics (van Dam 1986). In conclusion, it is evident that MAL” gene is industrially essential for baker’s yeast and plays a crucial
role in the sponge
dough fermentation. Acknowledgements We thank K. Ohsawa-Ishii cal assistance.
References Bamett, J. A. (1976) The utilization of sugars by yeasts. Adv. Carbohydr.
for her techni-
Chem. Biochem.
32,
125-234. Hino, A., Takano, H., Kitabayashi, N., Nitta, F., Ohishi, T. and Tanaka, Y. (1988) Automatic measuring system for dough testing: design, construction and reproducibility. Nippon Syokuhin Kogyo Gakkaishi. 35,344-3X (in Japanese). Khan, N. A. and Eaton, N. R. (1971) Genetic control of maltase formation in yeast. I. Strains producing high and low basal levels of enzyme. Mol. Gen. Genet. 112,317-322. Michels, C. A. and Needleman, R. B. (1983) A genetic and physical analysis of the MALl and MAL3 standard strains of Saccharomyces cereuisiae. Mol. Gen. Genet. 191,225230. Needleman, R. B., Kaback, D. B., Dubin, R. A., Perkins, E. L., Rosenberg, N. G., Sutherland, K. A., Forrest, D. B. and Michels, C. A. (1984) MALG of Saccharomyces: a complex genetic locus containing three genes required for maltose fermentation. PFOC.Natl. Acad. Sci. U.S.A. 81, 2811-2815.
Significance
of yeast MAL genes
47
Oda Y. and Ouchi, K. (1989a) Principal-component analysis of the characteristics desirable in baker’s yeasts. Appl. Environ. Microbial. 55, 1495-1499. Oda Y. and Ouchi, K. (1989a) Genetic analysis of haploids from industrial strains of baker’s yeast. Appl. Environ. Microbial. 55, 1742-1747. Oda, Y. and Ouchi, K. (1989c) Maltase genes and a-glucosidase activities: their effects on the dough-leavening. Yeast 5, S125-Sl39. Ponte, J. G., Jr. and Reed, G. (1982) Bakery foods. In Prescott and Dunn’s industrial microbiology (4th ed.) (Ed. Reed, G.) pp. 246292. Westport, AVI Publishing Co., Inc. van Dam, H. W. (1986) The biotechnology of baker’s yeast: old or new business? In Chemistry andphysics ofbaking (Eds Blanshard, J. M. V., Frazier, P. J. and Galhard, T.) pp. 117-131. London, The Royal Society of Chemistry.
1990,7,43-47
Role of the yeast maltose fermentation production rate from sponge dough
genes in COP
Y. Oda* and K. Ouchi
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Machida-shi, Tokyo 194, Japan Received11 October 1989 The time course of CO2 production in sponge dough fermentation (Fermogram) was compared in yeast strains carrying various MAL (maltose fermentation) genes. Most MAL-constitutive strains showed curves with a significant increase after l-l -5 h, fermenting maltose liberated from starch by endogenous amylases. However, all MALinducible strains and maltose non-fermenting strains gave quite different curves which remained low, indicating theirpoor leavening ability in sponge dough. We were able to visualize the crucial role of MAL-constitutive genes in sponge dough leavening by the Fermogram.
Introduction Leavening ability in sponge dough without addition of sugar, is one of the most important properties in baker’s yeast, and is closely related to its maltose fermentability (Oda and Ouchi 1989a). In most maltose-fermenting strains of Saccharomyces cerevisiae, one or more of five unlinked genes (&G&Ll, MALZ, MAL3, MAL4 and MA&61 are contained, and expression of both a-glucosidase and maltose permqase are induce.d by maltose in the gr~th medium (Barnett 1976). Some strains can produce these two enzymes in the absence of maltose, and the gene responsible for this phenotype is defined as MAL-constitutive (MAL’) (Khan and Eaton 1971). The curve of COP production rate from sponge dough, which is called Zymotachygram (Ponte and Reed 1982) or Fermogram (Hino et al. 19881, have reflected the characteristics of baker’s yeast. In the present paper we show these curves obtained with the strains containing vari*Corresponding author 0740-0020/90/010043
+ 05 $02.0010
ous MAL genes, and visualize the crucial role of MAL’ genes in sponge dough fermentation.
Materials Yeasts
and Methods
Eight MATa and 8 MATu strains ofS. cerevisiap, as shown in Table 1, were crossed to construct 64 diploids in all combinations. These strains of 16 haploids and 64 diploids were used in the present experiments. The genetic notation for the MAL strains and the meaning of MALg and MAL’ have been reported elsewhere (Michels and Needleman 1983). Each functional MAL gene is comprised of two complementary genes; MALp gene encoding the positive regulatory protein and MALg gene encoding both a-glucosidase and maltose permease (Nhedleman et al. 1984). The strains containing MALg alone are defective in maltose fermentation. mal’ indicates that there are neither MALp nor MALg genes. MALI’ is the gene included in YOY34, a haploid strain derived from baker’s yeast (Oda and Ouchi 1989b1, and other genes are derived from laboratory stock strains (Khan and Eaton 1971). MALl’ and MALX are MAL-constitutive genes and were responsible for high levels of a-glucosidase activities in the molasses medium (Oda and Ouchi 1989c). Other MAL genes were distinguished as 0 1990 Academic Press Limited
44
Y. Oda and K. Ouchi Table
1. List
of Saccharomyces
Maltose fermentation
Strain YOY107 YOYlO8 X2180-1A X2180-1B YOY334 YOY329 YOY416 YOY328 YOY338 YOY339 YOY342 YOY344 YOY943 YOY374 YOY371 YOY370 MALI’
and
cerevisiae
MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa MATa
+ + + + + + + + + + + + are
used.
Genotype
-
MAL.4’
strains
constitutive
alleles
of
mal” ma1 MALlgMAL3g MALlgMAL3g MALl’ MALl’ MALl MALl MAL2 MAL2 MAL3 MAL3 MAU’ MAL+P MAL6 MAL6 MALl
and
MAL4,
respectively.
MAL-inducible since they caused basal expression of enzyme activities similar to those of mol” and MALlgMAL3g in the molasses medium (Oda and Ouchi 1989c). FSC6001 was a commercial baker’s yeast. Culture Yeast cells were cultured in a molasses medium containing 3% sugar as cone molasses, 0.193% urea, and 0.046% KHsPO, (Oda and Ouchi 1989a). COP production rate The ingredients of sponge dough, containing 100 g of flour, O-5g of salt, 1.65 g of yeast cells as dry matter, and 65 ml of distilled water, were mixed for 2 min with a National Mixer (National Mfg Co., Lincoln, NE). A piece of dough based on 20 g flour was cut and placed in a bottle of the Fermograph (Atto Co., Ltd., Tokyo, Japan). The rate of COs gas production was recorded automatically at 30°C and depicted as the Fermogram (Hino et al. 1988).
Results and Discussion In the sponge dough, the main fermentable sugar for yeast is maltose liberated from the starch of the flour by amylases (Ponte and Reed 1982). When COs production rate from the dough is recorded
against time, baker’s yeast usually depicts curves with two distinct peaks, as shown in Fig. 1. The first increase represents the fermentation of sugars which pre-existed in the flour, and the following large increase corresponds to the fermentation of maltose. Therefore, the second increase is one of the characteristics in the fermentation of the dough with addition of little or no sugar.
f =o
10
‘:g .E E E
6
6
s 9 B B N s 6
4
% oz
0
2
0
100
200
300
Time (min)
Fig. 1. Time course of COs production rate
from sponge dough with a commercial baker’s yeast (FSC6001).
t MALlgMAL39 I h4AL1°
I I MAL2
haploid
Time (min)
I MALl
MATa I MAW
I MAL4 =
Fig. 2. Time course of CO2 production rate from sponge dough with haploids and their hydrids.
r mal?
3oolllin
1 MAL6
E
46
Y. Oda and K. Ouchi
The left-end column and the top-end row in Fig. 2 show the curves of COs production rate from sponge dough by MATa and MATa haploids, respectively. Those by the 64 diploids are placed in the points on which the column and the row of their parents cross. The curves in Fig. 2 were generally smaller in height than those by conventional baker’s yeasts, probably because tested strains were constructed from laboratory strains which had less leavening abilities (Oda and Ouchi 198913). In the dough containing 5% glucose based on flour, the amount of gas produced by the strains tested was about 80% of that by industrial strains of baker’s yeast (data not shown). MAL-inducible haploids showed curves which remained as low as those of maltose non-fermenting strains, and diploids of MAL-inducible x MAL-inducible, MAL-inducible x malo, and MALinducible x MALlgMAL3g also showed similar curves. Some strains had the first peak, but few possessed the second peak. On the other hand, the haploids carrying either MALI’ or MAL.4’ showed an obvious second increase. The diploids containing one or two MAL’ genes showed similar curves to MAL’ haploids. These observations were explained by the finding that the constitutivity of MALl”
and MAL4’ genes was dominant over the inducibility of other MAL genes (Oda and Ouchi 1989c). However, curves by MALl’ x mat’ and MAL4’ x mal” diploids were similar to those by mal” strains. Since a-glucosidase activities of MALI’ and MAL4’ strains were reduced when hybridized with mal” strains (Oda and Ouchi 1989c), an inhibitor against expression of a-glucosidase might be present in malo strains. The cell of MAL-inducible strains, when grown in the medium containing maltose as a sole carbon source, expressed much higher activity of aglucosidase by induction, and showed the distinguishable second increase in the curves (data not shown). However,
cane
or beet molasses, which contain no appreciable amount of maltose, are widely used in the production of baker’s yeast because of the convenience and economics (van Dam 1986). In conclusion, it is evident that MAL” gene is industrially essential for baker’s yeast and plays a crucial
role in the sponge
dough fermentation. Acknowledgements We thank K. Ohsawa-Ishii cal assistance.
References Bamett, J. A. (1976) The utilization of sugars by yeasts. Adv. Carbohydr.
for her techni-
Chem. Biochem.
32,
125-234. Hino, A., Takano, H., Kitabayashi, N., Nitta, F., Ohishi, T. and Tanaka, Y. (1988) Automatic measuring system for dough testing: design, construction and reproducibility. Nippon Syokuhin Kogyo Gakkaishi. 35,344-3X (in Japanese). Khan, N. A. and Eaton, N. R. (1971) Genetic control of maltase formation in yeast. I. Strains producing high and low basal levels of enzyme. Mol. Gen. Genet. 112,317-322. Michels, C. A. and Needleman, R. B. (1983) A genetic and physical analysis of the MALl and MAL3 standard strains of Saccharomyces cereuisiae. Mol. Gen. Genet. 191,225230. Needleman, R. B., Kaback, D. B., Dubin, R. A., Perkins, E. L., Rosenberg, N. G., Sutherland, K. A., Forrest, D. B. and Michels, C. A. (1984) MALG of Saccharomyces: a complex genetic locus containing three genes required for maltose fermentation. PFOC.Natl. Acad. Sci. U.S.A. 81, 2811-2815.
Significance
of yeast MAL genes
47
Oda Y. and Ouchi, K. (1989a) Principal-component analysis of the characteristics desirable in baker’s yeasts. Appl. Environ. Microbial. 55, 1495-1499. Oda Y. and Ouchi, K. (1989a) Genetic analysis of haploids from industrial strains of baker’s yeast. Appl. Environ. Microbial. 55, 1742-1747. Oda, Y. and Ouchi, K. (1989c) Maltase genes and a-glucosidase activities: their effects on the dough-leavening. Yeast 5, S125-Sl39. Ponte, J. G., Jr. and Reed, G. (1982) Bakery foods. In Prescott and Dunn’s industrial microbiology (4th ed.) (Ed. Reed, G.) pp. 246292. Westport, AVI Publishing Co., Inc. van Dam, H. W. (1986) The biotechnology of baker’s yeast: old or new business? In Chemistry andphysics ofbaking (Eds Blanshard, J. M. V., Frazier, P. J. and Galhard, T.) pp. 117-131. London, The Royal Society of Chemistry.