Freeze resistant bakers' yeast

FIELD OF THE INVENTION
The present invention relates to a diploid hybrid strain characterized by at least having strong fermentative ability of "non-sugar bread dough" (hereinafter may be referred to as "non-sugar dough") and strong freeze-resistance and also relates to "frozen bread dough" (hereinafter may be referred to as "frozen dough") containing at least the hybrid strain and a dough composition. Furthermore, this invention relates to production of a variety of bread by using the frozen dough.
BACKGROUND OF THE INVENTION
Recently, frozen dough for bread has become very attractive to the baking industry because it has the following great advantages; i.e., (1) it may be useful in the supply of fresh-baked bread; and (2) it has vast merits for solving industrial productivity problems by shortening the baking process (savings of labor) and reducing the need for a night shift. Frozen dough is produced by kneading and fermenting materials for bread, keeping the material under freezing at around -20.degree. C. The frozen dough is subjected to baking after, if necessary, proofing. Common bakers' yeast is so likely damaged by fermentation prior to freezing that the use of yeast is limited to a case where the dough is enriched with a relatively large amount of materials such as sugar, fat, egg, milk products, etc. is not subjected to fermentation or subjected to fermentation for only a short time prior to freezing. When the dough containing common bakers' yeast which has been fermented for a short time before freezing is thawed and baked immediately after proofing, it cannot be sufficiently baked so that it causes some problems such that flavor and taste of bread may be deteriorated. A process wherein the frozen dough is thawed and subjected to proofing requires a long time for baking, so that the purpose of frozen dough method may be lost.
Thus, there has been a demand for a kind of bakers' yeast which has a great freeze-resistance, may be hardly damaged by storage under the frozen condition after fermentation, during preparation of frozen dough. Some reports have presented kinds of yeast having freeze-resistance, i.e., Saccharomyces rosei (Japanese Patent Kokoku No. 59-25584), Saccharomyces cerevisiae FTY (Japanese Patent Kokoku No. 59-48607), Saccharomyces cerevisiae IAM4274 (Japanese Patent Kokai No. 59-203442). Both Saccharomyces rosei and Saccharomyces cerevisiae FTY (FRI-413) do not have strong maltose-fermentative ability and therefore, they are not suitable for use in frozen dough with the sugar content in the range of 0 to 20% by weight based on flour, i.e., from non-sugar dough to dough with a moderate sugar level. Saccharomyces cerevisiae IAM4274 has maltose-fermentative ability but does not show sufficient freeze-resistance in the dough with the sugar content in the range of 0 to 20% by weight based on flour, i.e., from non-sugar dough, etc. to the dough with a moderate sugar level. Size of the yeast Saccharomyces rosei is smaller than that of common bakers' yeasts, so that it takes a long time for separation, washing and dehydration of yeasts. The other reports have presented bakers' yeast which is suitable for the dough for lean type bread and has maltose-fermentative ability and freeze-resistance, i.e. Saccharomyces cerevisiae KYF110 (Japanese Patent Kokai No. 62-208273), and a fusant strain Saccharomyces cerevisiae 3-2-6D (Japanese Patent Kokai No. 63-294778) which is obtained by the cell fusion technique and has strong maltose-fermentative ability and enhanced freeze-resistance. However, these are not satisfactory yet.
As heretofore mentioned, there has not been obtained bakers' yeast which has strong fermentative ability and great freeze-resistance of non-sugar dough up to the dough with a moderate sugar level (up to 20% by weight based on flour), and which is appropriate for the non-sugar dough without sugar such as French bread and bread crumb or the dough for a white bread. Therefore, it has been extremely difficult to produce frozen dough for such kinds of bread.
The common bakers' yeast commercially available for non-sugar dough has a drawback on storability because of the rapid decrease of its fermentative ability under storage in the form of products, compared with the deterioration rate of ordinary bakers' yeasts having high fermentative ability of dough of a higher sugar level and middle fermentative ability of non-sugar dough, which are widely used for the bread with the sugar content in the range of 5% to 30% by weight based on flour.
SUMMARY OF THE INVENTION
After intensive study to solve the above problems, the present inventors succeeded in a diploid hybrid strain which has both at least the same fermentative ability as the common bakers' yeasts for non-sugar dough, with respect to fermentation of dough having up to moderate sugar level (sugar content; 0 to 20% by weight based on flour), and has strong freeze-resistance. The "diploid hybrid strain" is obtained by conjugation between a "haploid yeast strain" (hereinafter may be referred to as "haploid strain") obtained from germination of spores from a diploid yeast strain belonging to Saccharomyces cerevisiae having at least strong fermentative ability on non-sugar dough and a haploid yeast strain obtained from germination of spores from a diploid yeast strain belonging to Saccharomyces cerevisiae having strong freeze-resistance but such a weak fermentative ability on non-sugar dough that it is hardly used for frozen dough of lean type bread. Furthermore, they found that the diploid hybrid strain was usable as bakers' yeast for non-sugar dough up to dough with a moderate sugar level, specifically for frozen dough. High quality bread can be obtained from frozen doughs which are made with the diploid hybrid strain and have non-sugar to a moderate sugar level, for example, non-sugar dough for such as French bread and bread crumb and dough for a white bread.
Additionally, the present diploid hybrid strain shows slower reduction in fermentative ability during storage than common bakers' yeast for non-sugar dough. Thus, the diploid hybrid strain provides great advantages in terms of storage.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows CO.sub.2 gas production as a function of the time of fermenting non-sugar dough for a yeast strain according to the invention (FTY-3) and for three comparative strains (KB-3, 237NG, FTY).
FIG. 2 shows the fermentation profile (CO.sub.2 v. time) for strain FTY-3 and four comparative strains (TFD-1, KYF110, 237NG, FD612) fermented on non-sugar dough after the yeast strains had been frozen for 7 days.
FIG. 3 shows the fermentation profile (CO.sub.2 v. time) for strain FTY-3 and three comparative strains (TFD-1, 3-2-6D, 237NG) on non-sugar dough after the yeast strains had been frozen for 7 days.

DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, a diploid hybrid strain characterized by at least having strong fermentative ability of non-sugar dough and strong freeze-resistance, which is provided by conjugation between a haploid yeast strain obtained from germination of spores of a diploid yeast strain belonging to Saccharomyces cerevisiae having at least strong fermentative ability on non-sugar bread dough and a haploid yeast strain obtained from germination of spores from a diploid yeast strain belonging to Saccharomyces cerevisiae which has strong freeze-resistance but weak fermentative ability on non-sugar bread dough. The present invention further provides frozen bread doughs containing at least the diploid hybrid strain and a dough composition.
In accompanying drawings, FIG. 1 shows that CO.sub.2 production every 10 minutes from non-sugar dough of Example 2 mentioned below. In the drawing, marks .quadrature., , .DELTA. and are for yeasts of the present diploid hybrid strain FTY-3 (FERM BP 2326), KB-3 (FERM BP 2742) or a mate for the FTY-3, 237 NG and FTY (FERM BP 2743) or a mate for the FTY-3, respectively. FIGS. 2 and 3 show the similar graphs to FIG. 1, refering to Example 10 mentioned below wherein each strain is allowed to ferment at 30.degree. C. for 2 hours in a non-sugar dough, in which the marks , , , * and .largecircle. in FIG. 2 are for FTY-3 (present diploid hybrid strain), KFI 110, FD 612 (heretofore known hybrid strains), TFD-1 (commercially available freeze-resistant yeast strain) and 237 NG (ordinary non-freeze-resistant yeast strain) strains, respectively, and the marks , , and * in FIG. 3 are for FTY-3, 3-2-6D (heretofore known hybrid strain), TFD-1 and 237 NG strains respectively.
The diploid hybrid strain according to the present invention is obtained by conjugation between two parent strains, i.e. a haploid strain from germination of spores from a diploid yeast strain belonging to Saccharomyces cerevisiae which has at least strong fermentative ability of non-sugar dough and a haploid strain from germination of spores from a diploid yeast strain belonging to Saccharomyces cerevisiae which has strong freeze-resistance. One such diploid hybrid strain is Saccharomyces cerevisiae FTY-3 (FERM BP-2326) deposited at the Agency of Industrial Science and Technology, the Fermentation Research Institute, 1-3, Higashi 1-chrome, Tsukuba-shi, Ibaraki-ken 305 Japan on Mar. 7, 1989 under the terms of the Budapest Treaty.
One of the parent strains, a diploid yeast strain belonging to Saccharomyces cerevisiae having at least strong fermentative ability of non-sugar dough, may be any bakers' yeast for non-sugar dough commercially available in the market or any yeast strains which exhibit equivalent fermentative ability to the yeast above but has weak freeze-resistance. One example thereof is Saccharomyces cerevisiae KB-3 (FERM BP 2742; product name, "45 Red Yeast"; manufactured by Toyo Jozo Company, Ltd.) deposited at the above depository on Jan. 24, 1990 under the terms of the Budapest Treaty.
Another parent diploid yeast strain belonging to Saccharomyces cerevisiae which has at least strong freeze-resistance, may be any yeast strains which have equivalent degree of freeze-resistance to that of the yeasts used in frozen rich bread dough and which exert weak fermentative ability of non-sugar dough but strong freeze-resistance. One example is Saccharomyces cerevisiae FTY (FIJI-413) (FERM BP 2743) deposited at the above depository on Feb. 24, 1982 under the terms of the Budapest Treaty.
Saccharomyces cerevisiae has the general properties mentioned below and the three diploid strains aforementioned above, namely, Saccharomyces cerevisiae FTY-3, Saccharomyces cerevisiae KB-3 and Saccharomyces cerevisiae FTY (FRI-413) have these characteristic properties.
______________________________________Fermentative ability of carbohydratesglucose +galactose +sucrose +maltose +lactose -Assimilation of carbon compoundsglucose +galactose +sucrose +maltose +lactose -raffinose +soluble starch "Certified" (Lot. No. +0178-15, manufactured by Difco, Co.Ltd.)Assimilation of nitrate (KNO.sub.3) -Vitamin auxotrophybiotin +folic acid -nicotinic acid -thiamin -riboflavin -Ca-pantothenate +inositol .+-.pyridoxine .+-.p-aminobenzoic acid -______________________________________
Separation of each haploid strain from the two parent strains is carried out by preculturing the parent strain each in a nutrient medium, inoculating the cells on a sporulation medium containing sodium acetate or potassium acetate, incubating the medium at 20.degree. to 25.degree. C. for 2 to 7 days until spores are formed, suspending the cells containing spores in a lyric enzyme solution and keeping the solution to incubate at 30.degree. C. for 30 minutes to 1 hour. After the enzyme-treatment, spores are isolated from ascus using a micro manipulator. The spores isolated are transferred to the nutrient medium and cultured at 30.degree. C. until germination is made and haploid strain is obtained. Each haploid strain is judged by testing whether or not conjugation occurs in the presence of a known mating type haploid strain.
Conjugation is conducted in such a manner as described in "Protein, Nucleic acid and Enzyme", Vol. 12, No. 12, pp. 1096-1099, Norio Gunke ed. (1967). Each haploid strain separated from the two parent strains is cultured in a nutrient medium at 30.degree. C. for 4 to 8 hours, separately, and then, the same amount of the each cultured medium is mixed and the mixture is incubated at 30.degree. C., to generate zygotes. The zygotes formed are isolated with a micro-manipulator and cultured. After several screenings of diploid hybrid strains having characteristics together of the parent strains, the present diploid hybrid strain Saccharomyces cerevisiae FTY-3 (FERM BP 2363) is obtained. The diploid hybrid strain was cultured with a fermenter and final product having water content of 64-73% was obtained after dehydration.
As mentioned above, the diploid hybrid strain according to the present invention is characterized by at east having strong fermentative ability of non-sugar dough and strong freeze-resistance. In particular, the diploid hybrid strain is distinguished from heretofore known diploid hybrid strains in having a non-freeze fermentative ability of not less than 140 ml and a residual fermentative ability rate after freezing of not less than 80%.
Herein, the term, "a non-freeze fermentative ability of not less than 140 ml", means that amount of carbon dioxide gas generated is not less than 140 ml when a strain is allowed to ferment at 30 .degree. C. for 2 hours in a non-sugar dough. The diploid hybrid strain FTY-3 according to the present invention produces not less than 140 ml of carbon dioxide gas, which is useful for dough fermentation, in a non-sugar dough, and is distinguished from other known hybrid strains, for example, Saccharomyces species FD 612 strain (FERM BP-742), Saccharomyces cerevisiae KHI 110 strain (FERM BP-972) and Saccharomyces cerevisiae 3-2-6D strain (FERM BP-1235), by this characteristic.
Also, the term, "a residual fermentative ability rate after freezing of not less than 80%", means that the residual fermentative ability rate after freezing which is calculated from measurements of amounts of produced carbon dioxide gas in the case (non-freezing) where a strain is allowed to ferment at 30.degree. C. for 60 minutes and subsequently, without freezing, at 38.degree. C. for 60 minutes, and in the case (freezing) where a strain is allowed to ferment at 30.degree. C. for 60 minutes, then frozen at -20.degree. C. for 7 hours, thawed at 30.degree. C. for 30 minutes, and allowed to ferment at 38.degree. C. for 60 minutes, both using a non-sugar dough, is not less than 80%. The FTY-3 strain according to the present invention shows a residual fermentative ability rate of not less than 80%, and is distinguished from these known strains by this characteristic. As obvious from FIGS. 2 and 3, the FTY-3 strain according to the present invention seems to be of an enzyme-existence type and the other strains seem to be of an enzyme-induction type. In the FTY-3 strain, accordingly, enzymes which act for the utilization in the fermentation of maltose produced from injured starch in flours by the action of flour-originated amylase (namely, maltase and maltose permease), exist originally in the cells, while, in the other strains, these enzymes are induced in the cells during the course of fermentation.
In order to prepare the frozen dough, dough fomula such as flour, sugar, salt, fat, skim milk, egg, yeast food, yeast, water, etc., may be appropriately used. Sugar content in the dough is 0-20% by weight based on flour. The diploid hybrid strain Saccharomyces cerevisiae FTY-3 as described above is produced in the compressed form of a 70% water content, and then usually added to the dough in an amount of 1 to 10% by weight based on flour.
The preparation of frozen dough, for instance, for a white bread, is conducted by a straight dough method wherein steps are as follows: mixing and kneading all of flour and other ingredients together with yeast, floor time, dividing, rounding, bench, intermediate proof, molding and freezing, sequentially. In a sponge dough method, steps are mixing and kneading a part of flour and other ingredients together with yeast, the first fermentation to prepare a sponge, and mixing and kneading the sponge with remaining ingredients, floor time, dividing, rounding, bench, molding and freezing, sequentially. In the case of straight dough method, the floor time is set at 0 to 240 minutes and frozen at -15.degree. to -40.degree. C., usually under the ambient pressure. The bread which is produced using the frozen dough of the present invention is quite superior in apparency, specific volume, grain and flavor to the bread produced from frozen dough made with the common bakers' yeast.
The frozen dough of the present invention may be used for doughs of steamed pork buns, yeast doughnut and the like in addition to a variety of non-sugar dough having up to moderate sugar level for a white bread, French bread, bread crumb, buns or raisin bread.
The following examples explain the present invention but they are not intended to limit the present invention thereto.
EXAMPLE 1
Conjugation
Production of the present diploid hybrid strain, Saccharomyces cerevisiae FTY-3, (hereinafter referred to as a FTY-3 strain).
Step 1. Presporulation
Each of Saccharomyces cerevisiae KB-3 (FERM BP 2742) having strong fermentative ability of non-sugar dough and Saccharomyces cerevisiae FTY (FRI-413) (FERM BP 2743) having strong freeze-resistance, was inoculated on a YPD agar medium plate and precultured at 30.degree. C. for 24 hours.
______________________________________Composition of YPD agar medium (pH 5.5)______________________________________yeast extract (Lot. 012701, manufactured by 5 gDifco, Co. Ltd.)peptone (Lot 018802, manufactured by Difco, 10 gCo. Ltd.)glucose (special grade, manufactured by Wako 40 gPure Chemical Industries, Ltd.)KH.sub.2 PO.sub.4 (Lot. CTJ3919, manufactured by Wako Pure 5 gChemical Industries, Ltd.)MgSO.sub.4.7H.sub.2 O (Lot. CTP2140, manufactured by Wako 2 gPure Chemical Industries, Ltd.)agar (Lot. 014001, manufactured by Difco, 20 gCo. Ltd.)distilled water 1000 ml______________________________________
Step 2. Sporulation
A loopful each of the two precultured strains was inoculated and cultured on Sherman's agar medium plate at 25.degree. C. for 6 days to induce sporulation.
______________________________________Composition of Sherman's agar medium (pH 7.2)______________________________________ potassium acetate (special grade, 1.0 g manufactured by Wako Pure Chemical Industries, Ltd.) yeast extract (Lot. 012701, manufactured 0.1 g by Difco, Co. Ltd.) glucose (special grade, manufactured 0.05 g by Wako Pure Chemical Industries, Ltd.) agar (Lot. 014001, manufactured by 2.0 g Difco, Co. Ltd.) distilled water 100 ml______________________________________
Step 3. Separation of spores
A loopful each of the two sporulated strains was suspended in a solution (2 ml) of a lytic enzyme (product name "Zymolyase-20T"; .beta.-1, 3-glucan lamunaripentaohydrase; 2-3 U/ml, manufactured by KIRIN BREWERY, Co., Ltd.), respectively, and incubated at 30.degree. C. for 30 minutes to 1 hour. After the enzyme treatment, spores were isolated from ascus with a micromanipulator.
Step 4. Germination and production of haploid strains
Each spore isolated was placed on the YPD agar medium plate and cultured at 30.degree. C. until the spore germinated to produce a haploid strain. Mating types of strains were judged by testing whether or not conjugation appears in the presence of a known mating type haploid strains.
Step 5. Conjugation
Each haploid strain thus obtained, i.e. the haploid strain obtained from germination of the diploid yeast strain having strong fermentative ability of non-sugar dough and the haploid strain obtained from germination of spores of the diploid yeast strain having strong freeze-resistance, was separately cultured on the YPD medium, i.e., a medium having no agar in the YPD agar medium at 30.degree. C. for 4 to 8 hours. Each cultured medium was mixed together and incubated at 30.degree. C. until zygotes were produced. The zygotes formed were isolated with a micromanipulator and cultured. A colony was formed on an agar medium plate containing maltose as a sugar source and the same agar medium was over laid to have it fermented. A colony which was able to produce the largest amount of CO.sub.2 therearound was selected, and was cultured in a liquid medium containing molasses as a sugar source. Cells obtained were frozen at -20.degree. C. for 7 days. Cells after being thawed were allowed to ferment in a liquid fermenting medium containing maltose as a sugar source and a strain which was able to produce the largest amount of CO.sub.2 was selected. The selected cell was mixed with non-sugar dough to prepare frozen dough. The dough was thawed and a strain which was able to produce a large amount of CO.sub.2 was selected.
Thus, the present diploid hybrid strain, Saccharomyces cerevisiae FTY-3 strain was obtained.
EXAMPLE 2
Fermentative Ability of Non-sugar Dough
Each strain was allowed to ferment in dough defined below at 30.degree. C. for 2 hours and the CO.sub.2 produced every 10 minutes for this period was measured as shown in the drawing attached.
______________________________________Composition of non-sugar dough______________________________________ flour (NISSHIN SEIFUN, Japan 20 g trademark "Camelia" (bread making protein flour) salt 0.3 g yeast 0.4 g water (Shizuoka prefecture, 13 ml Ohito-cho)______________________________________
TABLE 1______________________________________ CO.sub.2 production______________________________________ordinary bakers' yeast 78 mlyeast for non-sugar dough 152 mlFTY-3 strain 161 mlfreeze-resistant yeast 82 mlFTY (FRI 413) 32 ml______________________________________ Notes: *Ordinary baker's yeast: Saccharomyces cerevisiae 237NG (product name "45 Yeast", manufactured by Toyo Jozo Co., Ltd.). *Yeast for nonsugar dough; Sacharomyces cerevisiae KB3 (FERM BP 2742) (product name "45 Red Yeast", manufactured by Toyo Jozo Co., Ltd.). *Freeze-resistant yeast; a product marketed in Japan. *FTY (FRI 413); Saccharomyces cerevisiae FTY (FRI413) (FERM BP 2743).
The same was applied to hereinafter.
EXAMPLE 3
Fermentative Ability of Non-sugar Dough and Dough with a Moderate Sugar Level
Each strain was fermented in two varieties of dough with the following composition, i.e. dough with a low sugar level (sugar content, 5% based on flour) and dough with a moderate sugar level (sugar content, 20% based on flour) at 30.degree. C. for 2 hours. CO.sub.2 produced for this period was measured as shown in Table 2.
______________________________________Composition Dough with Dough with a low a moderate sugar level sugar level______________________________________flour (same above) 20 g 20 gsalt 0.3 g 0.3 gsugar 1 g 4 gyeast 0.4 g 0.4 gwater (same above) 12.4 ml 10 ml______________________________________
TABLE 2______________________________________ CO.sub.2 production CO.sub.2 production in dough with in dough with a low sugar a moderate level sugar level______________________________________ordinary bakers' 125 ml 220 mlyeastyeast for non-sugar 132 ml 195 mldoughFTY-3 strain 136 ml 215 mlfreeze-resistant 120 ml 200 mlyeast______________________________________
A strain FTY-3 showed weaker fermentative ability in dough with a high sugar level than in non-sugar dough and dough with a moderate sugar level.
EXAMPLE 4
Freeze-resistance (Non-sugar Dough)
The non-sugar dough shown in Example 2 was fermentated at 30.degree. C. for 60 minutes and frozen at -20.degree. C. for 7 days. After being thawed at 30.degree. C. for 30 minutes, the resulting dough was fermentated at 38.degree. C. for 60 minutes and CO.sub.2 produced was measured as shown in Table 3.
TABLE 3______________________________________ CO.sub.2 production CO.sub.2 production before freezing after thawing______________________________________ordinary bakers' 85 ml 35 mlyeastyeast for non-sugar 105 ml 40 mldoughFTY-3 strain 108 ml 88 mlfreeze-resistant 82 ml 65 mlyeast______________________________________
EXAMPLE 5
Freeze-resistance (Dough with a Low Sugar Level)
The dough with a low sugar level shown in Example 3 was fermentated at 30.degree. C. for 60 minutes and frozen at -20.degree. C. for 7 days. After being thawed at 30.degree. C. for 30 minutes, the resulting dough was fermented at 30.degree. C. for 120 minutes. CO.sub.2 produced in the latter fermentation was measured as shown in Table 4.
TABLE 4______________________________________ CO.sub.2 production CO.sub.2 production before freezing after thawing______________________________________ordinary bakers' 140 ml 42 mlyeastyeast for non- 150 ml 45 mlsugar doughFTY-3 strain 154 ml 118 mlfreeze-resistant 142 ml 85 mlyeast______________________________________
EXAMPLE 6
Shelf Life of Yeast
CO.sub.2 produced on the bakers' yeast for non-sugar dough was measured and that of a strain FTY-3, immediately after production thereof and after 4 days storage at 25.degree. C. The results are shown in Table 3.
TABLE 5______________________________________ CO.sub.2 production immediately CO.sub.2 production after production after storage______________________________________yeast for non- 150 ml 90 mlsugar doughFTY-3 strain 161 ml 142 ml______________________________________
EXAMPLE 7
Sporulation and Germination Ratio
Each loopful strain as in Example 2 was thinly inoculated on the YM agar medium plate (10 ml per sterilized petri dish of 85 mm diameter) using a flame-sterilized loop and precultured at 30.degree. C. for 24 hours. The resulting each strain was again thinly inoculated on the Sharman's agar medium plate (10 ml per sterilized petri dish of 85 mm diameter) using a flame-sterilized loop and incubated at 25.degree. C. for 7 days.
The strain each was subsequently stained according to the Moller's method in "Classification and Identification of Yeast", pp. 17-18, Hiroshi Iizuka and Shoji Goto ed., published by Tokyo University Press, on Mar. 21, 1969. The number of sporulating ascus every 100 cells was measured under a microscope.
Loopful cells each having sporulation ascus were suspended in a filtered solution (2 ml) of a lyric enzyme (3 mg/ml Zymolyase-20T in 0.05M Tris-HCl buffer, pH 7.5), in a sterilized tube (18 mm diameter). The tubes were incubated at 30.degree. C. for 1 hour and centrifugated (3000 rpm) for 10 minutes. The obtained cells were washed twice with sterilized water and suspended in 2 ml of sterilized water.
Spores were isolated from the enzyme-treated cells with a micromanipulator and germinated at 30.degree. C. on the YNB agar medium plate, a synthetic medium for yeasts. One hundred spores were separated from asci having 4 spores each inside.
______________________________________YM agar medium (pH 5.6)______________________________________ Yeast extract (Lot. 012701, manufactured by 0.3 g Difco, Co. Ltd. glucose (special grade, manufactued by Wako 1.0 g Pure Chemical Industries, Ltd.) maltose extract (Lot. 0186015, manufactured 0.3 g by Difco, Co. Ltd.) peptone (Lot. 018802, manufactured by 0.5 g Difco, Co. Ltd.) agar (Lot. 014001, manufactured by Difco, 2.0 g Co. Ltd.) distilled water 100 ml______________________________________
YNB agar medium (pH 5.6)
(Bacto-Yeast Nitrogen base in "Genetic experimental series, Vol. 3, Microbiological genetics research technique", pp. 186-188, Tatsuo Ishikawa ed., published by Kyoritsu Press, Japan, on Mar. 10, 1982).
______________________________________ YEAST NITROGEN BASE (Lot. 760960, manu- 0.67 g factured by Difco, Co. Ltd.) glucose (special grade, manufactured by Wako 2.0 g Pure Chemical Industries, Ltd.) agar (Lot. 014001, manufactured by Difco, 2.0 g Co. Ltd.) distilled water 100 ml______________________________________
The sporulation ratio and germination ratio of spores obtained above are shown in Table 6.
TABLE 6______________________________________ Sporulation GerminationStrain ratio (%) ratio (%)______________________________________ordinary bakers' yeast 2 4yeast for non-sugar 20 10doughFTY-3 strain 14 <1freeze-resistant yeast 12 5FTY (FRI 413) 2 8______________________________________
The FTY-3 strain of the present invention as described above showed a characteristic that its germination ratio of spores was so small in the YNB agar medium plate, a synthetic medium for yeasts, that the strain could be identified from other strains.
EXAMPLE 8
Serological Characteristic of FTY-3 Strain
Rabbit antisera generated against FTY-3 strain as antigen were immunologically examined in relation to each yeast strain as in Example 2.
The preparation of antigen and antibody and that of specific antibody absorpted with yeast strains (237 NG, KB-3, FTY-3 strain) were explained hereinafter. The agglutination was assayed as in the following.
Antigen: FTY-3 strain was suspended in physiological saline to a final concentration of about 10.sup.10 cells/ml and heated at 80.degree. C. for 30 minutes followed by centrifugation (1000 rpm) for 5 minutes to obtain 50 ml of the supernatant.
Antibody: Japanese white 5 rabbits aged 6 weeks were each injected 1 ml of the above antigen solution through ear vein using injectors. Additionally, 2 ml and 5 ml of the antigen solution were injected once and three times, respectively, with 4 days interval. On 8th day from the final immunization, blood was taken from the carotid artery of each rabbit. Whole blood was centrifuged (3000 rpm) for 15 minutes to yield about 20 ml of antiserum from each rabbit. Saturated ammonium sulfate solution (5 ml) was added to each antiserum (10 ml), and the mixture was kept at 5.degree. C. overnight and was centrifuged (5000 rpm) for 15 minutes. After each precipitated .gamma.-globulin was dissolved and dialyzed against physiological saline, it was adjusted to a final protein concentration of 20 mg/ml and designated as an antibody solution (A).
Specific antibody preparations absorbed with 237NG, KB-3 or FTY-3 strain: The ordinary bakers' yeast (300 mg each, Saccharomyces cerevisiae 237NG) was added to antibody solutions (A) (2 ml each), and reaction was allowed to proceed at 5.degree. C. for 15 minutes. Supernatant obtained by centrifugation of the reaction solution was subjected to agglutination with the ordinary bakers' yeast Saccharomyces cerevisiae 237NG This process was repeated until no agglutination was observed. Subsequently, the resulting antibody solution was diluted with physiological saline to a final protein solution of 10 mg/ml (B).
Yeast (Saccharomyces cerevisiae KB-3) for non-sugar dough was used in place of the ordinary bakers' yeast to carry out the above procedure similarly. The resulting solution was adjusted to a protein solution of 10 mg/ml (C).
Furthermore, the antibody solution (C) was subjected to an absorption treatment with FTY-3 strain, and then, the protein concentration of the resulting antibody solution was adjusted to 10 mg/ml (D).
Procedures for agglutination test: Each of the yeast cells was suspended in physiological saline to a concentration of about 10.sup.9 cells/ml. The suspension was divided to 40 .mu.l each per well of 96-well microtiter plates. Each antibody solution (A) or any one of antibody solutions (B) through (D) was added to wells at a volume of 40 .mu.l, separately, and shaken for 5 minutes. After these wells were kept to stand for 30 minutes, each well was examined in terms of the presence or absence of an agglutination mass. Tables 7, 8, 9 and 10 show the results of agglutination test using the antibody solution without absorption with yeast (A), the antibody solutions (B), (C) and (D), respectively. Normal serum shown in tables represents the serum from rabbits with no treatment.
TABLE 7______________________________________(A) Antibody solutionsSample 1 2 3 4 5 Normal serum______________________________________ordinary bakers' + + + + + -yeastyeast for non-sugar + + + + + -doughFTY-3 strain + + + + + -freeze-resistant + + + + + -yeastFTY (FRI 413) + + + + + -______________________________________
TABLE 8______________________________________(B) Antibody solutionsSample 1 2 3 4 5 Normal serum______________________________________ordinary bakers' - - - - - -yeastyeast for non-sugar - + + - - -doughFTY-3 strain - + + + - -freeze-resistant - - - - - -yeastFTY (FRI 413) - + + + - -______________________________________
TABLE 9______________________________________(C) Antibody solutionsSample 1 2 3 4 5 Normal serum______________________________________ordinary bakers' - - - - - -yeastyeast for non-sugar - - - - - -doughFTY-3 strain - + + + + -freeze-resistant - - - - - -yeastFTY (FRI 413) - + + + - -______________________________________
TABLE 10______________________________________(D) Antibody solutionsSample 1 2 3 4 5 Normal serum______________________________________ordinary bakers' - - - - - -yeastyeat for non-sugar - - - - - -doughFTY-3 strain - - - + + -freeze-resistant - - - - - -yeastFTY (FRI 413) - - - - - -______________________________________
______________________________________Example 9 Baking test1 French bread______________________________________ Composition flour (same above) 100 g yeast 4 g yeast foods (Toyo Jozo, trade 0.1 g name "Amila") malt extract (SANKYO FOODS, Japan, trade name "Sankyo Malt Ekisu B.sub.2 ") 0.3 g salt 2 g water (same above) 63 ml Procedure (Straight dough method) fermentation time 60 min. dividing 100 g bench time 20 min. freezing after molding at -20.degree. C. thawing 30.degree. C., 60 min. proofing 20.degree. C., 60 min. baking 230.degree. C., 15 min.______________________________________
TABLE 11______________________________________ Bread volume non- frozen for frozen for freezing 7 days 14 days______________________________________yeast for non- 350 ml 270 ml 250 mlsugar doughFTY-3 strain 360 ml 310 ml 300 mlfreeze-resistant 310 ml 280 ml 275 mlyeast______________________________________
______________________________________2 A white bread (sugar content: 5% based on flour)______________________________________ Composition flour (same above) 100 g yeast 4 g yeast foods (same above) 0.1 g fat 5 g sugar 5 g salt 2 g water (same above) 65 ml Procedure (straight dough method) fermentation time 40 min. dividing 50 g freezing after molding at -20.degree. C. thawing 30.degree. C., 90 min. proofing 38.degree. C., 40 min. baking 200.degree. C., 35 min.______________________________________
TABLE 12______________________________________ Bread volume non- frozen for frozen for freezing 7 days 14 days______________________________________yeast for non- 285 ml 180 ml 145 mlsugar doughFTY-3 strain 290 ml 265 ml 230 mlfreeze-resistant 280 ml 230 ml 195 mlyeast______________________________________
______________________________________3 Butter roll (sugar content: 10% based on flour)______________________________________ Composition flour (same above) 100 g yeast 4 g yeast foods (same above) 0.1 g fat 10 g sugar 10 g whole egg 10 g salt 1.5 g water (same above) 48 ml Procedure (straight dough method) fermentation time 45 min. dividing 50 g first bench time 10 min. second bench time 10 min. molding freezing after molding at -20.degree. C. thawing 30.degree. C., 30 min. proofing 38.degree. C., 80 % humidity, 40 min. baking 200.degree. C., 10 min.______________________________________
TABLE 13______________________________________ Bread volume non- frozen for frozen for freezing 7 days 14 days______________________________________yeast for non- 255 ml 185 ml 130 mlsugar doughFTY-3 strain 270 ml 255 ml 240 mlfreeze-resistant 250 ml 220 ml 170 mlyeast______________________________________
______________________________________4 Buns (sugar content: 15% based on flour)______________________________________ Composition flour (same above) 100 g, skim milk 3 g yeast 4 g whole egg 8 g yeast food (same above) 0.1 g fat 8 g sugar 15 g salt 1.3 g water (same above) 50 ml Procedure (straight dough method) fermentation time 40 min. dividing 50 g bench time 15 min. freezing after molding at -20.degree. C. thawing 30.degree. C., 30 min. proofing 38.degree. C., 50 min. baking 200.degree. C., 10 min.______________________________________
TABLE 14______________________________________ Bread volume non- frozen for frozen for freezing 7 days 14 days______________________________________yeast for non- 280 ml 250 ml 240 mlsugar doughFTY-3 strain 282 ml 278 ml 273 mlfreeze-resistant 270 ml 265 ml 260 mlyeast______________________________________
______________________________________5 Buns (sugar content: 20% based on flour)______________________________________ Composition flour (same above) 100 g, skim milk 3 g yeast 5 g whole egg 8 g yeast food (same above) 0.1 g fat 8 g sugar 20 g salt 1.3 g water (same above) 48 ml Procedure (straight dough method) fermentation time 60 min. dividing 50 g bench time 15 min. freezing after molding at -20.degree. C. thawing 30.degree. C. 30 min. proofing 38.degree. C. 50 min. baking 200.degree. C. 10 min.______________________________________
TABLE 15______________________________________ Bread volume non- frozen for frozen for freezing 7 days 14 days______________________________________yeast for non- 300 ml 265 ml 242 mlsugar doughFTY-3 strain 293 ml 292 ml 290 mlfreeze-resistant 276 ml 270 ml 265 mlyeast______________________________________
EXAMPLE 10
Comparative Tests on the FTY-3 Strain with Other Strains for the Freeze-resistance in a Non-sugar Dough
Non-freeze and after-freeze fermentative abilities of the present FTY-3 strain in a non-sugar dough were compared with those of other heretofore known hybrid strains, i.e., Saccharomyces species FD 612 (FERM BP-742) (hereinafter referred to as FD 612), Saccharomyces cerevisiae KFI 110 (FERM BP-972) (hereinafter referred to as KFI 110) and Saccharomyces cerevisiae 3-2-6D (FERM BP-1235) (hereinafter referred to as 3-2-6D), as well as an ordinary bakers' yeast strain (237 NG: non-freeze-resistant strain) and a commercially available freeze-resistant yeast strain used for frozen dough (TFD-1).
Each of the strains was cultured as follows:
______________________________________YPD liquid medium composition (pH 5.5)Yeast extract (manufactured by Difco Co., Ltd., 5 gLot 013402)Peptone (manufactured by Difco Co., Ltd., 10 gLot 020408)Glucose (manufactured by Wako Pure Chemical 40 gIndutries, Ltd., special grade)KH.sub.2 PO.sub.4 (manufactured by Wako Pure Chemical 5 gIndustries, Ltd., Lot CTJ 3919)MgSO.sub.4.7H.sub.2 O (manufactured by Wako Pure Chemical 2 gIndustries, Ltd., Lot CTP 2340)Distilled water 1000 mlMolasses medium (pH 5.6)Molasses (product from the Philippines, sugar 120 mlcontent 35%)KH.sub.2 PO.sub.4 (manufactured by Wako Pure Chemical 0.4 gIndustries, Ltd., Lot CTJ 3919)Urea (manufactured by JUNSEI KAGAKU 2.8 gKABUSHIKI KAISHA, special grade, Lot 3D1075)(NH.sub.4).sub.2 SO.sub.4 (manufactured by Wako Pure Chemical 1 gIndustries, Ltd., special grade,Lot CTJ 3814)MgSO.sub.4.7H.sub.2 O (manufactured by KANTO KAGAKU 0.3 gKABUSHIKI KAISHA, special grade,Lot 205A5993)Distilled water 880 ml______________________________________
To 5 ml of the above YPD liquid media (in a .phi. 16 mm.times.105 mm test tube) was inoculated a loopful each strain from a slant culture, and the medium was subjected to a shaken culture at 30.degree. C. for 24 hours.
One milliliter of the cultured medium was inoculated to 100 ml of the above molasses medium (in a 500 ml-volume Erienmeyer flask), and the medium was subjected to a shaken culture at 30.degree. C. for 48 hours.
The cultured medium was subjected to a centrifugation under 3000 rpm for 10 minutes to collect the cells. After rinsing under the same conditions, the cells (containing 67% water) were obtained by pressing dehydration using a filter paper.
______________________________________Non-sugar dough composition______________________________________Flour (manufactured by NISSHIN SEIFUN 20 gKABUSHIKI KAISHA, "Camelia",a bread making protein flour)Table salt 0.3 gYeast 0.4 gWater 13 ml______________________________________
The cells were added to the above non-sugar dough, and the dough was subjected to a mixing for 2 minutes. A divided 33.7 g (20 g as flour) portion of the dough was allowed to ferment at 30.degree. C. for 60 minutes. After degasification the dough was frozen at -20.degree. C. for 7 days. After being thawed at 30.degree. C. for 30 minutes, the dough was allowed to ferment at 38.degree. C. for 60 minutes, and the amount of gas generated was measured by a fermograph.
1 Non-freeze fermentative ability
Each strain was allowed to ferment at 30.degree. C. for 2 hours in the non-sugar dough (without freezing), the amount of carbon dioxide gas generated was measured. The results are comparatively shown in Tables 16 and 17 below.
TABLE 16______________________________________Strains Amount of gas generated______________________________________FTY-3 153 mlKFI 110 71 mlFD 612 48 ml237 NG 73 mlTFD-1 79 ml______________________________________
TABLE 17______________________________________Strains Amount of gas generated______________________________________FTY-3 156 ml3-2-6D 81 ml237 NG 75 mlTFD-1 78 ml______________________________________
As obvious from the tables, the present FTY-3 strain exhibited a strong fermentative ability in non-sugar dough composition as shown from the amount of generated gas of not less than 140 ml, which is useful for dough fermentation. While, the known strains, KFI 110 and FD 612, exhibited far less amount of the gas as compared with the FTY-3 strain, even lower than that of the commercially available freeze-resistant TFD-1 strain. The 3-2-6D strain exhibited less amount of the gas as compared with the FTY-3 strain, showing the similar fermentative ability to that of the TFD-1 strain.
2 Every 10 minutes gas generation in non-freeze fermentation
Each strain was allowed to ferment in the non-sugar dough at 30.degree. C. for 2 hours, and the amounts of gas generated every 10 minutes were measured with curves as shown in FIGS. 2 and 3.
The present FTY-3 strain exhibited a curve in which the amounts of gas generated gradually increased from the beginning of fermentation to after 50 minutes, and thereafter reached to a steady level, showing an enzyme-existence pattern. While, the curves from the KFI 110, FD 612 and 3-2-6D strains exhibited peaks of the gas amounts after about 30 minutes, then decreases and again increases after about 60 minutes, showing an enzyme-induction pattern which is similar to the 237 NG and TFD-1 strains.
3 Residual fermentative ability after freezing
In the case of non-freezing, each strain was allowed to ferment in the non-sugar dough at 30.degree. C. for 60 minutes, and subsequently (without freezing) allowed to ferment at 38.degree. C. for 60 minutes. In the case of freezing, each strain was allowed to ferment in the non-sugar dough at 30.degree. C. for 60 minutes, then the dough was frozen at -20.degree. C. for 7 days and thawed at 30.degree. C. for 30 minutes and then (after freezing) allowed to ferment at 38.degree. C. for 60 minutes. The amounts of carbon dioxide gas generated in both cases at the second fermentation were measured with the results shown in Tables 18 and 19.
TABLE 18______________________________________ Gas Gas generated generated Residual (non- (after fermentativeStrains (freeze) freezing) ability rate______________________________________FTY-3 101 ml 84 ml 83.2%KFI 110 73 ml 44 ml 60.3%FD 612 53 ml 41 ml 77.4%237 NG 80 ml 33 ml 41.3%TFD-1 81 ml 60 ml 74.1%______________________________________
TABLE 19______________________________________ Gas Gas generated generated Residual (non- (after fermentativeStrains freeze) freezing) ability rate______________________________________FTY-3 103 ml 86 ml 83.5%3-2-6D 82 ml 58 ml 70.7%237 NG 78 ml 31 ml 39.7%TFD-1 80 ml 57 ml 71.3%______________________________________
It is obvious from the tables that the present FTY-3 strain is an excellent freeze-resistant strain at least having a residual fermentative ability rate of not less than 80%. While, the known KFI 110 strain exhibited a far less amount of gas generated after freezing, as compared with that of the FTY-3 strain. Also, the known FD 612 strain exhibited far less amounts of gas generated both in non-freezing and after freezing, showing an insufficient fermentative ability to be used for producing non-sugar dough breads. Further, the known strains exhibited lower gas generation amounts in non-freezing and after freezing, when compared with those of the commercially available freeze-resistant TFD-1 strain. The known 3-2-6D strain exhibited a residual fermentative ability rate of as low as 70.7%, although it showed the similar degree of gas amounts generated in a non-freeze condition and after freezing as those of the commercially available TFD-1 strain.
As seen from the results of the above comparative tests, the FTY-3 strain according to the present invention is definitely superior to the other known strains, KFI 110, FD 612 and 3-2-6D, in each of its non-freeze fermentative ability, every 10 minutes gas generation in non-freeze fermentation, and residual fermentative ability after freezing. Accordingly, the present FTY-3 strain is distinguished from them in its characteristics of having a non-freeze fermentative ability of not less than 140 ml and a residual fermentative ability rate after freezing of not less than 80%, in a non-sugar dough.
According to the present invention, high-quality frozen bread dough having non-sugar for French bread and bread crumb and white bread having up to a moderate sugar level (sugar content: 0 to 20% based on flour) may be provided.
Furthermore, non-sugar dough containing the diploid hybrid strain according to the present invention has longer storage life than the same dough containing the conventional yeasts. Accordingly, the present yeast or dough containing the same is convenient when stored or transported.

Number	Name	Date
4318929	Clement et al.	Mar 1982
4547374	Nakatomi et al.	Oct 1985
4643901	Jacobson et al.	Feb 1987
4680182	Kawai	Jul 1987
4973560	Jacobson et al.	Nov 1990

Number	Date	Country
0196233	Oct 1986	EPX
59-25584	Jun 1984	JPX
59-48607	Nov 1984	JPX
59-203442	Nov 1984	JPX
60-15308	Apr 1985	JPX
62-208273	Sep 1987	JPX

Freeze resistant bakers' yeast

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Creuger et al., "Strain Development," In Biotechnology a Textbook of Industrial Microbiology, Oldenbourg Verlag, Muchen, 1989 pp. 9-58.
Spencer et al., "Genetic Manipulation of Non-Conventional Yeasts by Conventional and Non-Conventional Methods," J. Basic Microbiol., 28 (5) pp. 321-333 1988.