AU781331B2

AU781331B2 - Novel baker's yeast and doughs containing the same

Info

Publication number: AU781331B2
Application number: AU73209/00A
Authority: AU
Inventors: Norio Noda; Hayato Takata; Noritaka Tsujimoto; Yutaro Watanabe
Original assignee: Kaneka Corp
Current assignee: Kaneka Corp
Priority date: 1999-09-22
Filing date: 2000-09-22
Publication date: 2005-05-19
Anticipated expiration: 2020-09-22
Also published as: WO2001021763A1; CN100540651C; KR20020063163A; JP4565789B2; CN1382210A; AU7320900A

Description

1 KA023

DESCRIPTION

NOVEL BAKER'S YEAST AND DOUGH CONTAINING THE SAME TECHNICAL FIELD The present invention relates to novel baker's yeast and a method for breadmaking using the yeast. More particularly, the present invention relates to baker's yeast which is tolerant to osmotic pressure in fermentation of bread dough and a method for breadmaking with a straight method, a sponge dough method, and a freezing method using dough containing various breadmaking ingredients as well as the baker's yeast.

BACKGROUND ART Some bread is made of dough containing no or a small amount of sucrose added French bread, white bread, etc.), and other bread is made of dough containing a large amount of sucrose added sweet bread, etc.). Bread is thus made of various types of dough. Baker's yeasts having different levels of fermenting ability are used in breadmaking, depending on the amount of sugar. For bread dough containing a large amount of added sucrose, baker's yeast having high sugar tolerance has been selected.

Sugar tolerance conventionally means an ability to tolerate sucrose. The sucrose tolerance of baker's yeast has been long studied and there have been reports on the relationship with the activity of invertase. Invertase is an extracellular enzyme that degrades sucrose which is a disaccharide of the two monosaccharides, glucose and 2 KA023 fructose. After sucrose is extracellularly degraded by invertase into monosaccharides, the monosaccharides are taken into a yeast body and utilized as a nutrient. In the case of baker's yeast having a high level of invertase activity, degradation of sucrose into monosaccharides is accelerated, so that osmotic pressure around baker's yeast in dough is increased and suppresses fermentation by baker's yeast. Thus, it is suggested that there is a negative correlation between invertase activity and sucrose tolerance. Actually, baker's yeast which is currently used for sweet bread is selected from strains having a low level of invertase activity (Technical Report of The Japan Yeast Industry Association, 58, 77 (1988)).

Further, there were reports showing that sucrose tolerance was increased by actively breeding baker's yeast having a low level of invertase activity. Examples of such baker's yeast include: one that has a low level of invertase activity, is also freeze tolerant, and can be used in breadmaking using dough containing 25% sucrose (Japanese Laid-Open Publication No. 7-203952); one that has a low level of invertase activity, and has a high level of ability to ferment even in dough containing 30% sucrose (Japanese Laid-Open Publication No. 8-154666); and one that has a low level of invertase activity and a high level of maltase activity, and can be used in producing both white bread and sweet bread (Japanese Laid-Open Publication No. 9-149785).

Thus, it is indicated that the invertase activity is involved in an ability to ferment in dough containing a high concentration of sucrose. As described above, however, the baker's yeast having a low level of invertase activity is not tolerant to a high sucrose concentration per se, but decelerates degradation of sucrose into its monosaccharide 3 KA023 components glucose and fructose) suppressing an increase in osmotic pressure around the baker's yeast, resulting in sucrose tolerance.

There is a report indicating that in fermentation of sweet bread dough, sucrose tolerance is less affected by involvement of invertase activity than the osmotolerance of baker's yeast (Food Microbiol, 7, 241 (1990)). As an example showing involvement of osmotolerance in sucrose tolerance, there is a report indicating that the fermenting ability of baker's yeast in dough having a relatively high sucrose concentration is strongly correlated with the amount of glycerol in the baker's yeast body (Appl. Environ.

Microbiol, 63, 145 (1997)) and another report indicating that glycerol was externally added and taken into baker's yeast bodies, so that the fermenting ability of the baker's yeast is improved (Food Microbiol, 15, 51 (1998)). Further, as an example showing that sucrose tolerance is improved, there is a report indicating that the osmotolerance of baker' s yeast could be improved by adding inorganic salt, such as NaCl, KC1, etc. into a culture of baker's yeast, thereby enhancing the fermenting ability of the baker's yeast in sweet bread dough (US Patent No. 4,420,563 (1983)).

Thus, the examples which have been reported on sucrose tolerance indicate that invertase activity is involved in sucrose tolerance, and that the involvement of invertase is limited. In other words, sucrose tolerance is a combined property of invertase activity and osmotolerance. To improve sucrose tolerance, most reports have intended to breed strains having a low level of invertase activity. In these cases, sucrose tolerance along with a practical fermenting ability level has been exhibited only when the 4 KA023 sucrose content was 30% or less.

There are two representative methods for breadmaking. One is a straight dough method in which the fermenting abilityof baker's yeast is immediatelyreflected.

In this method, all ingredients for bread dough are mixed at one time and allowed to ferment, followed by baking. The other is a sponge dough method in which bread dough is made in two steps. In this method, pre-fermented dough sponge dough) is made and fermented at first, another dough is made and mixed with the sponge dough, and the resultant mixture (herein referred to as a final dough) is made and allowed to ferment. The sponge dough method is often used in breadmaking since the method has the advantages of an increase in bread volume due to the flexibility and gas retaining ability of the bread, an improvement in the machine tolerance of bread dough, etc.

As an example of preparation of baker's yeast suitable for these two breadmaking methods, there is a report indicating yeast having sucrose tolerance in the straight dough method and the sponge dough method (Japanese Laid-Open Publication No. 10-191964). In this example, sucrose tolerance was exhibited in dough containing 30% sucrose in the straight dough method and in final dough containing sucrose in the sponge dough method.

Further, baker's yeast having freeze tolerance as well as sucrose tolerance has been developed. There are the following examples of the baker's yeast having freeze tolerance and sucrose tolerance: IAM4274 (Japanese Laid-Open Publication No. 59-203442) having an excellent final proof after freezing and specific volume in sweet bread dough 5 KA023 (sucrose FTY-2 (Japanese Laid-Open Publication No. 7-203952) having freeze tolerance (it is described that the amount of gas produced in dough containing 30% sucrose was slightly less than when commercially available baker's yeast was used); baker's yeast (US Patent No. 4,547,374) having a satisfactory result in a freeze preservation test of dough having 25% sucrose; baker's yeast (Japanese Laid-Open Publication No. 7-203952) having a low level of invertase activity and freeze tolerance, and able to be used in breadmaking using dough containing 25% sucrose; baker's yeast (Japanese Laid-Open Publication No. 8-154666) having fermenting ability after thawing at a residual rate of at least 90% in dough containing 30% sucrose. Further, based on the idea that the intracellular trehalose content is involved in freeze tolerance, the NTH or ATH gene encoding an enzyme degrading the trehalose was destroyed by a recombinant technique to suppress degradation of trehalose in order to improve freeze tolerance (Japanese Laid-Open Publication No. 10-117771 and Japanese Laid-Open Publication No. 11-169180).

In most of the above-described examples, freeze tolerance was obtained in sweet bread containing 25% sucrose with respect to flour. The greatest amount of sucrose added was (Problem to be Solved by the Invention) The breadmaking industry often uses not only sucrose but also isomerized liquid sugar, which is converted from a part of glucose to fructose by isomerase treatment. In sucrose-added dough, as sucrose is degraded to monosaccharides by the invertase of baker's yeast, osmotic pressure is gradually increased. In isomerized sugar-added 6 KAO 23 dough containing glucose and fructose, and sucrose-mixed and isomerized sugar-added dough, baker's yeast ferments a dough having a high level of osmotic pressure from the beginning.

Further, salt and other sub-ingredients for breadmaking other than sugars are added to bread dough so asto improve flavor and taste, thereby producing distinctive bread. Representative examples of the bread sub-ingredients mixed to bread dough other than sugars include fats, dairy products such as milk, skim milk powder, etc., and eggs. Some of these bread sub-ingredients added to bread dough affect osmotic pressure similarly to salt.

Therefore, the coexistence of such a bread sub-ingredient and sugar or salt increases osmotic pressure, whereby the fermenting ability of baker's yeast is suppressed.

As described above, in the conventional straight dough method and freezing method, examples of baker's yeast exhibited sucrose tolerance when the sucrose content of dough was 30% or less. In the conventional sponge dough method, there were only reports that sucrose tolerance was exhibited in final dough containing 25% sucrose. In dough containing at least 30% of sucrose, or dough containing 30% or less of sugar but further containing isomerized sugar, and high osmotic pressure dough containing salt or bread sub-ingredients so that osmotic pressure is increased, fermentation is suppressed, thereby making it more difficult to obtain a sufficient volume of bread. There was no known baker's yeast which is suitable for fermentation in high osmotic pressure dough. The fermentation of known baker's yeast was insufficient in any of the straight dough method, the sponge dough method and the freezing method for 7 KA023 breadmaking.

The objective of the present invention is to prepare novel osmotolerant baker's yeast capable of strong fermentation in high osmotic pressure dough which is conventionally difficult, and novel baker's yeast which can be used in the straight dough method, the sponge dough method and the freezing method for breadmaking in various high osmotic pressure dough.

DISCLOSURE OF THE INVENTION (Means for Solving the Problem) Strains were isolated fromnature andwere repeatedly subjected to screening for osmotolerance, sponge dough tolerance and freeze tolerance. The present invention has succeeded in preparing practical strains having excellent levels of these functions.

In one aspect, the present invention relates to baker's yeast having osmotolerance in fermentation of bread dough.

In one embodiment, the present invention relates to baker's yeast having the above-described properties in the straight dough method. In another embodiment, the present invention relates to baker's yeast having the above-described properties in the final fermentation of the sponge dough method. In a still another embodiment, the present invention relates to baker's yeast having the above-described properties in final dough, both in the straight dough method and the sponge dough method. The present invention also relates to baker's yeast having freeze tolerance.

8 KA023 In one embodiment, as to the baker's yeast of the present invention, the amount of carbon dioxide gas generated at 38 0 C for 2 hours is at least 140 ml for every 50 g of isomerized sugar containing dough, corresponding to 35% of sugar.

In another embodiment, the present invention relates to bread dough containing the baker's yeast of the present invention. In still another embodiment, the present invention relates to a method for breadmaking using the baker's yeast of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

First, the terms as used herein will be described below. The terms as used herein have the same meanings as those usually used in the art, except for the terms particularly specified below.

In the present specification, the percentage of sucrose, salt, and other bread sub-ingredients refers to percentage by weight with respect to flour. For example, sucrose means that 30 g of sucrose is used with respect to 100 g of flour. The term "bread sub-ingredient" as used herein refers to ingredients which may be used in breadmaking, other than flour, salt and water. Examples of the bread sub-ingredients include, but are not limited to, sucrose, isomerized sugar, dairy products, eggs, fats.

9 KA023 The term "high osmotic pressure dough" as used herein refers to dough having the number of moles corresponding to at least 30% of sucrose with respect to flour where the number of moles is calculated by summing the number of moles of sucrose sucrose, isomerized sugar, etc.), salt, and ingredients involved in osmotic pressure which are contained in dairy products or the like, in breadmaking ingredient. The term "osmotolerant baker's yeast" as used herein refers to baker's yeast having strong fermenting ability in high osmotic pressure dough containing breadmaking ingredients sucrose, glucose, fructose, sucrose-mixed isomerized sugar, isomerized sugar, salt, dairy products, etc.). Further, the term "strong fermenting ability" refers to, for example, that the amount of carbon dioxide gas generated at 38 0 C for 2 hours is at least 140 ml in every 50 g of isomerized sugar containing dough, corresponding to 35% of sugar.

The term "isomerized sugar" as used herein refers to sugar containing glucose and fructose which sugar is obtained by converting a part of glucose with isomerase.

A mixture of glucose and fructose can also be included in isomerized sugar. The term "isomerized sugar containing dough" as used herein refers to dough containing glucose and fructose. An example of such dough is, but not limited to, one that contains 50% glucose and 50% fructose. Further, in the bread-making industry, "sucrose-mixed isomerized sugar" containing sucrose is often used as isomerized sugar.

The term "sucrose-mixed isomerized sugar containing dough" as used herein refers to dough containing sucrose, glucose and fructose. For example, the sucrose-mixed isomerized sugar containing dough is, but not limited to, dough containing 50% sucrose, 25% glucose and 25% fructose.

10 KA023 The baker's yeast of the present invention having osmotolerance in bread dough fermentation is tolerant to high osmotic pressure in bread dough made of various breadmaking ingredients. For example, such tolerance includes, but is not limited to, glucose tolerance, fructose tolerance, isomerized sugar tolerance, salt tolerance, dairy product tolerance, etc.

The baker's yeast of the present invention having excellent osmotolerance exhibits sucrose tolerance even in sucrose-containing dough containing at least 30% sucrose.

Conventionally bred baker's yeast strains having sucrose tolerance due to a reduction in invertase activity acquire sucrose tolerance by suppressing an increase in osmotic pressure around the baker's yeast due to degradation of sucrose. Thus, it is clear that such conventionally bred baker's yeast is different from the osmotolerant baker's yeast of the present invention.

In one embodiment, the osmotolerant baker's yeast of the present invention is characterized in that the amount of carbon dioxide gas generated at 38 0 C for 2 hours is at least 140 ml, more preferably at least 150 ml, and even more preferably at least 160 ml, in every 50 g of isomerized sugar containing dough, corresponding to 35% of sugar. More preferably, in addition to the amount of carbon dioxide gas generated in isomerized sugar containing dough of 35% sugar concentration, the baker's yeast of the present invention is characterized in that the amount of carbon dioxide gas generated at 38 0 C for 2 hours is at least 180 ml, preferably at least 190 ml, and more preferably at least 200 ml, in dough containing 40 g of sucrose with respect to 100 g of 11 KA023 flour.

In one aspect, the baker's yeast of the present invention may have sponge dough tolerance. The term "sponge dough tolerance" refers to osmotolerance in the final dough fermentation subsequent to fermentation of sponge dough.

Usually, in the sponge dough method, dough having a low sugar concentration (about is prepared at first and is allowed to ferment (referred to as first fermentation or sponge dough fermentation). It is believed that this sponge dough fermentation allows the baker's yeast to be active. In final fermentation after the sponge dough fermentation, high osmotic pressure is created due to added sugar ingredients and other breadmaking ingredients. The active baker's yeast is suddenly allowed to ferment in high osmotic pressure dough. Therefore, baker's yeast having osmotolerance after activation due to sponge dough fermentation first fermentation) is suitable for the sponge dough method. Such baker's yeast is referred to as sponge dough tolerant baker's yeast.

The baker's yeast of the present invention exhibits strong fermenting ability and remains osmotolerant in final fermentation after sponge dough fermentation in spite of the presence of a high concentration of added sucrose, isomerized sugar, and breadmaking ingredients, such as salt, etc. which increase the osmotic pressure of dough.

Specifically, the sponge dough tolerant baker's yeast of the present invention which is tolerant to osmotic pressure in final fermentation after sponge dough fermentation is characterized in that the amount of carbon dioxide gas generated at 38 0 C for 2 hours is at least 140 ml, more 12 KA023 preferably at least 150 ml, in every 50 g of final dough whose composition is shown in Table 1 after sponge dough fermentation at 30 0 C for 150 minutes.

As described above, the baker's yeast of the present invention exhibits osmotolerance in any of the straight dough method and the sponge dough method.

Table 1 Ingredients Sponge dough Final dough Flour 70 g 30 g Yeast 3 g Glucose 3 g 15 g Fructose 15 g Water 39 ml 15 ml Steps 1) Mixing sponge dough 2) Sponge doughfermentationat 30°Cfor2.5 hours 3) Mixing final dough 4) The amount of gas generated in every 50 g of dough was measured with a Fermograph (made by ATTO Co.) at 38 0 C for 2 hours. The resultant value is regarded as the amount of gas generated in final dough.

In another aspect, the baker's yeast of the present invention is also characterized by freeze tolerance in high osmotic pressure dough. Conventionally, there was only a report that freeze tolerance was obtained in dough having 30% or less of sucrose. There was no example reported that has freeze tolerance when containing over 30% of sucrose, or in high osmotic pressure dough containing isomerized sugar, salt, or a dairy product, etc.

The fermenting ability before freezing are important 13 KA023 for the fermenting ability after freezing as well as the strength of freeze tolerance. The baker's yeast of the present invention has strong freeze tolerance, and strong fermenting ability in high osmotic pressure dough before freezing, so that the strong fermenting ability is maintained after freezing. The fermenting ability after freezing of the baker's yeast of the present invention is represented by the amount of carbon dioxide gas generated in every g of dough which is mixed and is allowed to ferment at for 60 minutes, followed by freeze preservation for a given time, and after thawing at 25 0 C for 60 minutes, is allowed to ferment at 38 0 C for 2 hours. The osmotolerant and freeze-tolerant baker's yeast of the present invention is characterized in that the amount of carbon dioxide gas generated in 35%-sucrose dough after freezing for 4 weeks is at least 300 ml, preferably at least 320 ml, and that the amount of carbon dioxide gas generated in dough containing of sucrose-mixed isomerized sugar and 1% salt after freezing for 4 weeks is at least 270 ml, preferably at least 280 ml.

The present invention relates to baker' s yeast having osmotolerance in the straight dough method. Further, the present invention also relates to sponge dough tolerant baker's yeast having osmotolerance in final dough fermentation in the sponge dough method. Preferably, the present invention relates to baker's yeast having osmotolerance both in the straight dough method and a sponge dough method. Further, preferably, the present invention relates to baker's yeast having osmotolerance in the straight dough method and the sponge dough method and having freeze tolerance.

14 KA023 The present invention relates to bread dough containing flour and breadmaking ingredients sugars, salt, eggs, fats, dairy products, emulsifiers, etc.), and the baker's yeast of the present invention. The baker' syeast of the present invention can be used in various bread dough ranging from low sugar concentration to high sugar concentration. Particularly, the baker's yeast of the present invention is suitable for dough having high osmotic pressure due to the presence of sugars and various breadmaking ingredients. The baker's yeast of the present invention is suitable for any of the straight dough method and the sponge dough method, and can be sufficiently used after freeze preservation.

The baker's yeast of the present invention is not particularly limited as long as it has osmotolerance in the straight dough method and/or the sponge dough method for breadmaking, and has freeze tolerance. The baker's yeast of the present invention can be obtained from nature by screening and obtained by a breeding technique for baker's yeast, such as crossing, mutation, cell fusion, etc.

Preferably, the baker's yeast of the present invention can be prepared by cross breeding of a plurality of strains including a strain having freeze tolerance. Crossed strains are screened to obtain osmotolerant and sponge dough tolerant strains, and are further screened to obtain freeze tolerant strains. From these strains, baker's yeast can be prepared by a culture method shown in the Examples.

A preferable baker's yeast of the present invention is Saccharomyces cerevisiae. A representative example of a crossed strain selected by the above-described method is the KKK47 strain, which is a strain of Saccharomyces 15 KA023 cerevisiae. The KKK47 strain is deposited at the National Institute of Bioscience and Human-Technology, the Agency of Industrial Science and Technology, the Ministry of International Trade and Industry (1-1-3 Higashi, Tsukuba, Ibaraki, Japan) under the depository number FERM BP-7267 on August 31, 1999 (receipt of request for deposit dated August 7, 2000).

Examples Hereinafter, examples of the present invention will be described. These examples are only intended for illustrative purposes. The present invention is not limited to the examples. Ingredients used in the examples are the following: flour is Camellia manufactured by Nisshin Flour Milling; yeast food is Kaneplus C (manufactured by Kaneka Corporation); shortening is Snow Light (manufactured by Kaneka Corporation); and margarine is Nova 11 (manufactured by Kaneka Corporation). Other breadmaking ingredients and bread sub-ingredients are available from general stores.

As control strains, three baker's yeast strains commercially available from Kaneka Corporation were used.

Commercially available baker's yeast A (regular yeast manufactured by Kaneka) Commercially available baker's yeast B (freeze tolerant yeast manufactured by Kaneka) Commercially available baker's yeast C (freeze tolerant yeast manufactured by Kaneka) Example 1: Crossed breeding 16 KA023 Among Saccharomyces cerevisiae stock strains processed by the Applicant three strains, including two strains having freeze tolerance, were used as original strains. All of these original strains were diploid. The original strains were allowed to form spores in sporulation medium, followed by cross breeding.

1) Spores derived from the two freeze tolerant strains were crossed with spores derived from a common yeast strain, thereby preparing a number of first-generation crossed strains.

2) The first-generation crossed strains were allowed to form spores. The spores derived from the different original freeze tolerant strains were crossed with each other, thereby preparing second-generation crossed strains.

3) The second-generation crossed strains were allowed to form spores. Crossing was conducted in various combinations, thereby preparing third-generation crossed strains.

A number of crossed strains of each generation were screenedforosmotolerance, sponge dough tolerance and freeze tolerance, thereby obtaining parent strains for next-generation crossed strains. An attempt was made to improve these capabilities for each generation. The KKK47 strain of the present invention having the intended capabilities was eventually acquired in the third-generation crossed strains.

Example 2: A method for producing baker's yeast Medium having a composition shown in Table 2 was 17 KA023 prepared as volumes of 5 ml/large test tubes and 50ml/500ml Sakaguchi flasks, which were in turn autoclaved. The resultant medium was used.

A platinum loop of the cross-bred strain was inoculated into the large test tube, followed by shaking culture at 30 0 C for one day. The resultant culture was transferred to the 500 ml Sakaguchi flask, followed by shaking culture at 30 0 C for one day. The resultant yeast was subjected to 5L jar culture.

Table 2 Flask seed yeast culture Medium composition Sugar (molasses) Urea 0.3% Ammonium sulfate 0.08% Potassium 0.04% dihydrogen-phosphate Zinc sulfate 5 ppm 2L of medium having a composition shown in Table 3 was poured into a 5L jar, and was autoclaved. The yeast collected from five 500 ml Sakaguchi flasks was inoculated and cultured under the conditions shown in Table 4.

Table 3 jar seed yeast culture Medium composition Sugar (molasses) 90 g Urea 6.75 g Ammonium sulfate 1.8 g Potassium 0.9 g dihydrogen-phosphate Zinc sulfate 11.25 mg Water 2250 ml 18 KA023 Table 4 Airlation volume 2.0 nl/min Agitation 650 rpm Temperature 33 0

C

pH 4.7 control (using 14% ammonium water) 5L jar main culture g of seed yeast bodies cultured in the 5L jar was added as wet yeast bodies to starting liquid having a medium composition shown in Table 5, and cultured under the conditions shown in Table 6.

Table 5L jar main culture Medium composition Sugar (molasses) 230 g Urea 4.9 g phosphate 1.4 ml Zinc sulfate 20 mg Copper sulfate 3.15 mg Vitamin B1 10.5 mg Water 2000 L Table 6 Airlation volume 2.5 nl/min Agitation 650 rpm Temperature 33 0

C

pH 4.7 control (using 14% ammonia water) Culture was conducted for 13 hours. Sugars were added portion by portion during 12-hour culture.

The cultured yeast was centrifuged immediately after 19 KA023 the culture, and were suctioned and dehydrated with a suction funnel, thereby preparing wet yeast bodies. The wet yeast body was used in the examples below. When used in an experiment, the moisture content of the wet yeast was measured, and the amount used was calculated based on moisture.

The amount of gas generated was compared between the thus-obtained baker's yeast of the present invention and the commercially available strains using the straight dough method (Examples 3 to The amount of carbon dioxide gas generated was measured as follows: dough ingredients described in each example were mixed with a Hobart desk-top mixer for 3 minutes, the amount of gas generated in every 50 g of the dough was measured at 38 0 C for 2 hours with Fermograph (manufactured by ATTO).

Example 3: Straight dough method Osmotolerance (1) The amounts of carbon dioxide gas generated in glucose containing dough and fructose containing dough having a composition shown in Table 7 were measured and compared between the KKK47 strain, and commercially available baker's yeasts A, B and C. The results are shown in Table 8.

Table 7 Dough composition Glucose Fructose dough dough Flour 100 g 100 g Baker's yeast 4 g 4 g Glucose 35 g Fructose 35 g Water 50 ml 50 ml 20 KA023 Table 8 Amount of gas generated Glucose Fructose dough dough KKK47 231 ml 163 ml Commercially 151 ml 86 ml available baker's yeast A Commercially 160 ml 90 ml available baker's yeast B Commercially 186 ml 115 ml available baker's yeast C Fermenting ability in high osmotic pressure dough containing 35% sugars (glucose and fructose) with respect to flour was compared between the KKK47 strain of the present invention, and the commercially available baker's yeasts to find that the KKK47 strain generated a larger amount of gas than the commercial yeasts. Thus, the KKK47 strain is considered to have superior osmotolerance.

Example 4: Straight dough method Osmotolerance (2) The amounts of carbon dioxide gas generated in sucrose-mixed isomerized sugar containing dough and isomerized sugar containing dough (sugar concentration: having compositions shown in Table 9 were measured and comparedbetween the KKK47 strain, and commercially available baker's yeasts A, BandC. The resultsareshowninTable 21 KA023 Table 9 Dough composition Sucrose-mixed Isomerizedsugar isomerized sugar containing dough containing dough Flour 100 g 100 g Baker's 4 g 4 g yeast Sucrose 17.5 g Glucose 8.75 g 17.5 g Fructose 8.75 g 17.5 g Water 50 ml 50 ml Table Amount of gas generated Sucrose-mixed Isomerized isomerized sugar sugar containing containing dough dough KKK47 239 ml 187 ml Commercially 163 ml 116 ml available baker's yeast A Commercially 172 ml 116 ml available baker's yeast B Commercially 211 ml 135 ml available baker's yeast C The KKK47 strain generated a larger amount of gas compared to the commercially available yeasts. In this case, the sugar concentration was 35%. The sucrose-mixed isomerized sugar containing dough and the isomerized sugar containing dough had higher osmotic pressure than sucrose containing dough having the same sugar concentration. Thus, the KKK47 strain is considered to have superior osmotolerance in sucrose-mixed isomerized sugar containing dough and 22 isomerized sugar containing dough, as found in the results for glucose containing dough and fructose containing dough.

Example 5: Straight dough method Osmotolerance (3) The amounts of carbon dioxide gas generated in dough and 30%-sucrose 3 %-salt dough shown in Table 11 were measured and compared between the KKK47 strain, and commercially available baker's yeasts A, B and

C.

Table 11 Dough composition dough 3%-salt dough Flour 100 g 100 g Baker's yeast 4 g 4 g Sucrose 30 g 30 g Salt 3 g Water 52 ml 52 ml Table 12 Amount of gas generated 30%-sucrose dough 3%-salt dough KKK47 366 ml 182 ml Commercially 322 ml 95 ml available baker's yeast A Commercially 341 ml 98 ml available baker's yeast B Commercially 313 ml 128 ml available baker's yeast C__ In addition to 30% sucrose, salt was added to increase 0 0 0000 0 00 0 0 0 00 0 0 0 0 0 00 0 0 0 00 0 0 23 KA023 osmotic pressure, and salt tolerance was examined. Among the three commercially available baker's yeast products, their ranks of fermenting ability were different between the 30%-sucrose dough and the dough further containing salt.

This indicates that sucrose tolerance to up to 30% sucrose and salt tolerance (osmotolerance) are different. TheKKK47 strain of the present invention has superior tolerance to osmotic pressure due to salt.

Example 6: Straight dough method Osmotolerance (4) The amounts of carbon dioxide gas generated in dough and 30%-sucrose dairy product dough shown in Table 13 were measured and compared between the KKK47 strain, and commercially available baker's yeasts A, B and

C.

The results are shown in Table 14.

Table 13 Dough composition dough dairy product dough Flour 100 g 100 g Baker's yeast 4 g 4 g Sucrose 30 g 30 g Skim milk powder 4 g Milk 50 ml Water 52 ml 5 ml 24 KA023 Table 14 Amount of gas generated dough dairy product dough KKK47 355 ml 240 ml Commercially 322 ml 180 ml available baker's yeast A Commercially 340 ml 185 ml available baker's yeast B Commercially 320 ml 216 ml available baker's yeast C Fermenting ability was compared between the 30%-sucrose dough and the 30%-sucrose dough mixed with a dairy product in which dairy product tolerance (osmotolerance) was examined. The ranks of the sucrose tolerance and the fermenting ability of the three commercially available baker's yeasts were similar to those in Example 5. The KKK47 strain of the present invention is considered to be superior to the commercially available yeasts in dough which is mixed with a dairyproduct to increase osmotic pressure.

Example 7: Straight fermentation Sucrose tolerance The amounts of carbon dioxide gas generated in dough and 40%-sucrose dough shown in Table were measured and compared between the KKK47 strain, and commercially available baker's yeasts A, B and C.

The results are shown in Table 16.

25 KA023 Table Dough composition Amount of sucrose added Flour 100 g 100 g Baker's yeast 4 g 4 g Sucrose 30 g 40 g Water 52 ml 47 ml Table 16 Amount of gas generated Amount of sucrose added KKK47 362 ml 218 ml Commercially 303 ml 119 ml available baker's yeast A Commercially 346 ml 135 ml available baker's yeast B Commercially 315 ml 174 ml available baker's yeast C Similar to Examples 5 and 6, commercially available baker's yeast B had the highest level of fermenting ability in the 30%-sucrose dough of the three commercially available baker's yeasts. The 40%-sucrose dough resulted in different ranking of fermenting ability. In this case, commercially available baker's yeast C had the highest fermenting ability. The sucrose tolerance in the 30%-sucrose dough does not necessarily mean sucrose tolerance in dough having a sucrose concentration of at least 30%. The osmotolerant KKK47 strain has excellent fermenting ability in dough. It is therefore considered that the fermenting ability of the KKK47 strain was not much suppressed when the amount of sugar added was increased from 30% to 26 KA023 and that the KKK47 strain has excellent sucrose tolerance.

Example 8: Sponge dough method Next, the amount of carbon dioxide gas generated was compared among final dough having different compositions in the sponge dough method.

Sponge dough ingredients showninTable 17weremixed for 3 minutes with a Hobart desk-top mixer, followed by sponge dough fermentation at 30 0 C for 150 minutes. The fermented sponge dough was mixed with final dough ingredients for 3 minutes with the Hobart desk-top mixer. The amount of carbon dioxide gas generated in every 50 g of the dough was measured at38 0 C for 2 hours using a Fermograph.

The results are shown in Table 18.

Table 17 Dough composition Sponge Final dough composition dough (3) composition Flour 70 g 30 g 30 g 30 g Baker's 3 g yeast Sucrose 25 g 12.5 g Glucose 3 g 6.25 g 15 g Fructose 6.25 g 15 g Water 39 ml 15 ml 15 ml 15 ml Final dough composition: 25%-sucrose dough sucrose-mixed isomerized sugar containing dough having a sugar concentration of isomerized sugar containing dough having a sugar concentration of 27 KA023 Table 18 Amount of gas generated Final dough (3) KKK47 301 ml 264 ml 179 ml Commercially 248 ml 198 ml 109 ml available baker's yeast A Commercially 305 ml 235 ml 130 ml available baker's yeast B Commercially 258 ml 219 ml 82 ml available baker's yeast C The ranking of osmotic pressure, from lowest to highest, is 25%-sucrose dough, (2)25%-sugar sucrose-mixed isomerized sugar containing dough and (3) isomerized sugar containing dough. Although commercially available baker's yeast C had the greatest amount of gas generated in the high osmotic pressure dough of the straight dough method, commercially available baker's yeast B had the greatest amount of gas generated in the high osmotic pressure final dough in the sponge dough method.

Different levels of osmotolerance were exhibited between the methods.

For any of the baker's yeasts, it was observed that as the osmotic pressure of dough was increased, the amount of gas generated was decreased. The KKK47 strain of the present invention generated the greatest amount of gas even in the high osmotic pressure dough, and the fermenting ability of the KKK47 strain was not much suppressed with an increase in osmotic pressure. Thus, the KKK47 strain is considered to be osmotolerant even after activation due to sponge dough 28 KA023 fermentation.

Example 9: Freeze tolerance Freeze tolerance was examined.

dough and sucrose-mixed isomerized sugar containing dough shown in Table 19 were prepared. The amounts of carbon dioxide gas generated in the dough were measured and compared between the KKK47 strain, and commercially available baker's yeasts A, B and C before freezing and after freezing for 4 weeks.

The results are shown in Table 29 KA023 Table 19 Dough composition sucrose Sucrose-mixed isomerized sugar dough composition sugar) Flour 100 g 100 g Baker's yeast 5 g 4 g Sucrose 35 g 15 g Glucose 7.5 g Fructose 7.5 g Salt 0.5 g 1 g Water 50 ml 52 ml Steps Mixing Hobart desk-top mixer 3 minutes Dividing A loaf of dough 50 g Pre-fermentation 30 0 C 60 minutes Measurement Measure the amount of gas before freezing generated at 38 0 C for 2 hours Freezing -30 0 C, 1 hour 0 C, a given time Thawing 25 0 C 1 hour Measuring Measure the amount of gas generated at 38 0 C for 2 hours 30 KA023 Table Amount of gas generated before and after freezing dough Before freezing After freezing for 4 weeks KKK47 362 ml 351 ml Commercially 294 ml 254 ml available baker's yeast B Commercially 310 ml 272 ml available baker's yeast C isomerized sugar dough Before freezing After freezing for 4 weeks KKK47 302 ml 299 ml Commercially 246 ml 228 ml available baker's yeast B Commercially 287 ml 260 ml available baker's yeast C The KKK47 strain had excellent osmotolerance.

Therefore, the KKK47 strain had strong fermenting ability before freezing, and excellent freeze tolerance in sucrose-rich dough and high osmotic pressure dough, and therefore retained fermenting ability to a major extent after freeze preservation.

Example 10: Baking test with straight dough method The KKK47 strain and commercially available baker's yeast C were subjected to a baking test with the straight dough method using a dough composition shown in Table 21, in which specific volume was measured. Specific volume was 31 KA023 measured with a rapeseed replacement method.

The results are shown in Table 22.

Table 21 Dough composition Flour 100% Baker's yeast 3 Isomerized liquid sugar 28 Salt Shortening 8 Yeast food 0.1 Skim milk powder 2 Hen egg Water 48 Steps Mixing L3M3H1 4 L2M2H2 Dough temperature 28 0

C

Floor time 90 minutes (28 0

C)

Dividing 400 g Bench time 40 minutes (28 0

C)

Moulding Final proof 38 0 C 45 minutes Baking 200 0 C 30 minutes Table 22 Specific volume of bread KKK47 5.2 Commercially 4.8 available baker's yeast C The KKK47 strain resulted in bread having a good specific volume in a baking test with the straight dough method using dough mixed with sucrose-mixed isomerized sugar and bread sub-ingredients such as salt, shortening, skim milk powder, etc.

32 KA023 Example 11: Baking test Sponge dough (1) The KKK47 strain and commercially available baker's yeasts B and C were subjected to a baking test with the sponge dough method using a dough composition shown in Table 23, in which specific volume was measured.

The results are shown in Table 24.

33 KO2 KA023 Table 23 Dough composition_______ dough Final dough Flour 70% Baker's yeast 3 Isomerized 3 liquid sugar Salt Shortening 8 Yeast food 0.1 Skim milk 2 Hen egg Water 140 8 Sponge dough L3M3 mixing Dough 24 0

C

temperature Sponge dough 28 0 C 2 hours fermentation Mixing final L2M2H2 I L3M3H1 dough Dough 28 0

C

temperature Floor time 50 minutes Dividing 400 g Bench time 15 minutes Final proof 38 0 C 45 minutes Baking 7 200 0 C 30 minutes 34 KA023 Table 24 Specific volume of bread KKK47 5.3 Commercially available baker's yeast B Commercially 4.8 available baker's yeast C The final dough contained sucrose-mixed isomerized sugar which causes higher osmotic pressure than the same sugar concentration of sucrose. In this baking test using dough mixed with bread sub-ingredients, such as salt, skim milk powder, etc., the KKK47 strain resulted in bread having a larger specific volume even in the sponge dough method.

Thus, it was confirmed that the KKK47 strain is suitable for breadmaking using high osmotic pressure dough.

Example 12: Sponge dough method (2) The KKK47 strain and commercially available baker's yeasts B and C were subjected to a baking test with the sponge dough method using a dough composition shown in Table in which specific volume was measured.

The results are shown in Table 26.

35 KG2 KA023 Table Dough composition Sponge dough Final dough Flour 70% Baker's yeast Sucrose 3 Salt Margarine Yeast food 0.1 Milk Emulsifier 0.25 Hen egg Water 28 8 Steps Sponge dough L3M3 mixing Dough 24 0

C

temperature Sponge dough 28 0 C 2 hours fermentation Mixing final L3M5 4. M5 4 M5H1 dough Dough 27 0

C

temperature Floor time 28 0 C 60 minutes Dividing 400 g Bench time 28 0 C 20 minutes Moulding Final proof 38 0 C 50 minutes Baking 200 0 C 30 minutes 36 KA023 Table 26 Specific volume of bread KKK47 5.1 Commercially 4.4 available baker's yeast B Commercially 4.2 available baker's yeast C The KKK47 strain exhibited excellent capability for breadmaking by the sponge dough method in which the final dough composition had a sucrose concentration of as high as 35%. Thus, it was confirmed that the KKK47 strain is tolerant to high sucrose concentrations in the sponge dough method as well as the straight dough method.

Example 13: Baking test for frozen dough The KKK47 strain and commercially available baker's yeast C were subjected to a baking test for frozen dough using a dough composition shown in Table 27, in which the specific volume was measured.

The results are shown in Table 28.

37 KA023 Table 27 Dough composition Flour (strong flour) 100% Baker's yeast 6 Sucrose Salt Margarine Dough modifier 2 Milk Hen egg Water 33 Steps Mixing L3M5 4 M5 4 Dough temperature 22 0

C

Floor time 28 0 C 30 minutes Dividing 60 g Bench time 15 minutes Moulding Roll type Freezing Rapid freezing at 0 C for 1 hour freeze preservation at -20 0

C

Thawing 25 0 C 60 minutes Final proof 38 0 C 60 minutes Baking 200 0 C 12 minutes Table 28 Specific volume of bread Freeze time 1 week 2 weeks 4 weeks KKK47 6.2 6.2 6.1 Commercially 5.8 5.7 available baker's yeast C This example shows freeze tolerance in the sucrose-rich dough of 35% sucrose. The KKK47 strain retained strong fermenting ability even in such sucrose-rich dough, 31/03 '05 THUI 17:46 FAX 61 3 9288 1567 FREEHILLS PATENT TRADE @008 38 KA023 and excellent freeze tolerance, so that a reduction in the volume of bread due to freeze preservation was not substantially observed.

INDUSTRIAL APPLICABILITY The baker's yeast of the present invention has excellent osmotolerance, and can be effectively used in high osmotic pressure dough for any of straight dough method bread, sponge dough method bread, and freezing method bread.

Further, the baker's yeast of the present invention has excellent fermenting ability irrespective of high osmotic pressure due to isomerized sugar, salt, and other bread sub-ingredients in addition to sucrose-rich dough.

15 Therefore, with the baker's yeast of the present invention, bread having a good volume can be produced and the range S* of combinations of various bread sub-ingredients can be broadened, thereby making it possible to produce a greater variety of bread products than ever before.

Various aspects of the present invention are herein described by use of the particular embodiments. Changes and *o modifications will be apparent from the disclosure. It will be understood that the disclosure falls within the scope 25 and spirit of this invention as indicated in the claims appended hereto.

As used herein, the term "comprise" and variations S* of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

COMS ID No: SBMI-01185019 Received by IP Australia: Time 16:50 Date 2005-03-31