JPH0116154B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a large freeze-resistant baker's yeast belonging to the genus Torulaspora, which has diploid chromosomes. Yeasts belonging to the genus Torulaspora, such as Torulaspora delbrucki (Torulaspora
delbrueckii: Former name Satsukaromyces rosei
Saccharomyces rosei has disappeared taxonomically and has been given a new name. “JA Barnett et al., Yeasts:
Characteristics and Identification, 508 pages,
Cambridge University Press, Cambridge,
1983” and “NJWKreger-van Rij, ed.
The Yeasts, 3rd revised edition, 453 pages, Elsevier
Science Publishers, Amsterdam, 1984) is excellent for producing sweet breads with high sugar concentration (Tokuko Showa).
No. 54-13491), and freeze-resistant yeast (JP-A-Sho
56-144036). Freezing resistance means that bread dough containing fermentation yeast cells must be mixed in advance and fermented for a while, or it can be kept at -20°C as is.
It has the property of being able to be stored frozen, thawed and re-fermented as needed to bake bread. The ability to freeze and preserve bread eliminates the process of having to start making bread early in the morning, and also has the advantage of being able to bake bread in anticipation of demand. However, Torulaspora delbrutskii generally has small bacterial cells, so it has the disadvantage that operations such as bacterial collection, washing, and dehydration after culturing take time. The present inventors irradiated yeast belonging to the genus Torulaspora with ultraviolet light (UV) and obtained a large cell line with a culture of chromosomes. As a result, the above work time was significantly shortened without changing the properties of baker's yeast. After discovering what could be done, the present invention was completed. It has been known that in filamentous fungi, heterokaryons, which have two nuclei within the cell, undergo nuclear fusion and become diploid when irradiated with ultraviolet light (C. Ishitani,
Nature 178 , 706, 1956). This time, we investigated the yeast Torulaspora delbrucki, which normally grows in a haploid manner (NJWKregervan mentioned above).
Rij, The Yeasts, p. 434) was irradiated with ultraviolet light, and unexpectedly it was possible to create and establish a diploid strain. The diploid yeast strain obtained according to the present invention stably maintained its ploidy and remained frozen tolerant. In addition, the bread dough swelling strength was slightly improved as the polyploidy increased. The general production method of the present invention is to obtain a diploid yeast strain by irradiating yeast cells belonging to the genus Torulaspora with ultraviolet rays, and then selecting those that have grown into large cells from among the remaining colonies that have grown. Select wild-type strains that have the same growth rate as the parent strain and do not have auxotrophic artificially mutated genetic traits on their chromosomes from among the strains that have grown into large cells, and then This is achieved by selecting and acquiring plants that appear to be undamaged. Examples of polyploid strain construction using Torulaspora delbrucki and the properties of the resulting bacterial cells will be described below. The cell volume of the parent strain Torulaspora delbrutskii Y-134-5 using the present invention is approximately 18 ^μm3 ,
The bacterial strain created as described below is approximately 52 μm ³ , which is approximately 3 times larger. Example (1) Acquisition of large cell line Torulaspora delbrutskii Y-134-5 (February Institute of Microbiological Sciences No. 905) The bacterial cells were irradiated with ultraviolet light according to the conventional method (edited by Tatsuo Ishikawa, Experimental Methods of Microbial Genetics) , 194 pages,
Kyoritsu Shuppan, Tokyo, 1982). That is, YPD medium (yeast extract 1%, polypeptone 1%, glucose 2
%), take a bacterial culture in the early stationary phase grown at 30°C, and add to sterilized 67mM KH ₂ PO ₄ to a bacterial concentration of 10 ⁵ cells/
diluted to ml. While stirring 10ml with a magnetic stirrer, UV light (2000 ergs/sec/
cm ² ) was irradiated. At time intervals, the bacterial suspension was taken out, diluted appropriately with 67mM KH ₂ PO ₄ and a small amount was spread on a YDP agar medium. After culturing at 30°C for 2 days, colonies on an agar plate with a survival rate of 2 to 10% were collected and examined under a microscope. Among the colonies that had grown into large cells, those in which no auxotrophic mutation was observed were purified by repeating single colony isolation twice in a conventional manner. (2) Properties of the large cell lines obtained Among the large cell lines obtained as in (1), one strain that had excellent resistance to bread dough expansion tests and freezing was inoculated with Torulaspora delbrutskii SANK52084 (microengineering). It was named Kenbokuyori No. 7898). The cell volume is approximately 3 times larger than that of the parent strain Y-134-5 (Table 1), and the intracellular
The DNA content was also approximately doubled (Table 2). Larger strains have good sporulation, and agar plate medium for sporulation (1% potassium acetate, 0.1% yeast extract,
0.05% glucose, 2% agar) for 2 days at 30℃
When cultured in , many cells came to have 1 to 2 spores, and in rare cases 3 to 4 spores.
Collect the bacterial cells containing these asci and apply them twice in hypertonic solution A.
(PH7.5 with 0.6M KCl, 20mM Tris HCl), and then suspended in 5ml of hypertonic solution A. At this time, the number of bacteria was adjusted to approximately 4×10 ⁸ cells/ml. 2-
Add 15μ of mercaptoethanol per ml of bacterial suspension.
and incubated at 30°C for 30 minutes. The treated bacterial cells were collected by centrifugation and washed twice with hypertonic solution A to completely remove 2-mercaptoethanol. Suspend the bacterial cells in 5 ml of hypertonic solution,
Add 3 mg of Zymolyase 60000 (manufactured by Kirin Beer Co., Ltd.) and
After incubation for hours, more than 99% became protoplasts. The protoplast formation rate was determined by placing one drop of the suspension on two places on a slide glass, and placing it on one side at 10%.
N-lauroylsarcosine sodium solution
Estimated by adding 1μ and comparing both with a microscope. The generated protoplast is 500×G, 10
Collected by centrifugation for 1 minute. After washing with hypertonic solution A twice under the same centrifugation conditions, the protoplasts were
Place in 6.7mM Tris-HCl buffer (PH7.8),
It burst. The obtained spore mass was washed twice with the same buffer and separated into single spores by ultrasonication. When these spores were cultured, the cells had become almost the same size as the parent strain Y-134-5, and it was confirmed that they had returned to haploid state. However, as long as normal culture is continued,
The large cell line SANK52084 is a stable strain that maintains its cell size and can be cultured on a large scale.
The doubling time of SANK52084 is
It took 84 minutes under aerobic conditions at 30°C in YPD medium, which was comparable to the parent strain Y-134-5, and the final bacterial yield was also comparable (Table 3). Carbon compound materialization is
SANK52084 and Y-134-5 are exactly the same (Table 4). From cell size and intracellular DAN content,
It was concluded that the obtained large cell line SANK52084 was diploid. Other mycological properties are consistent with those of haploid yeasts (NJWKregervan Rij, ed., cited above).
(See The Yeasts, p. 435), differ in that pseudozygotic tubes are not formed during sporulation, and the cells convert directly into sporangia. In general, when cultured, the parent strain may aggregate under poor conditions, making collection and dehydration impossible. On the other hand, the enlarged cell line SANK52084 has almost no aggregation. Reference Example (1) Preparation of cake yeast from large cell line SANK52084 Baker's yeast is commercially available in the form of a cake.
Large cell lines that reduce the time required for cake preparation
A comparative test was conducted on SANK52084 and the parent strain Y-134-5. SANK52084 and Y-134-5 were cultured under the same conditions. That is, 35 kg of seeds and live yeast were placed in a 10 ton culture tank, and while adding and feeding blackstrap molasses, urea, and dibasic sodium phosphate, the temperature was 30°C, pH 5.0 to 5.5,
Aeration and agitation culture was carried out for 16 hours at an aeration rate of 1 v.vm. The yeast cells were collected by centrifugation, washed four times, and concentrated to obtain yeast cream 1000 (containing 520 kg of fresh yeast). It was treated with a salt solution and made into cake yeast using a rotary vacuum dehydrator (dehydrator, manufactured by Alfa-Laval).
As shown in Table 5, the processing area of the rotary vacuum dehydrator is 8m ²
The amount of cream yeast that can be processed per hour is Y
-134-5 is 1000, while SANK52084
is 2470, and the amount of cake yeast obtained per unit area/time is approximately 2.5 times. In other words, the working time required for dehydration was significantly shortened to about 1/2.5. (2) Freezing resistance of large cell line SANK52084 Using the cake yeast obtained as above, bread dough was prepared according to the formulation and process shown in Table 6, and was taken out on the 8th and 19th day after freezing at -20°C. and unzip it,
I was baking bread. When using SANK52084, the baked bread volume is 2125ml even after 19 days of freezing.
2064 ml was obtained by the parent strain Y-134-5 (Table 6).

[Table] The long axis and short axis of the cell are 2a for the long axis and 2b for the short axis.
50 cells were measured from photographs and the average value and standard deviation were calculated. The volume was calculated using the formula V=4/3πab ² assuming that the cell was a perfect ellipsoid.

[Table] Using calf thymus DNA as a standard, measurements were performed by colorimetry using diphenylamine. Y-134-5
DNA content is approximately 23fg/cell. (fg= ^10-15 g).

[Table] Table 4 Test strain for carbon compound assimilation: Y-134-5 and SANK52084 Assimilation: glucose, inulin, lactic acid, D-mannite, raffinose, D-sorbitol,
No assimilation of L-sorbose, sucrose, trehalose: D-arabinose, L-arabinose, cellobiose, citric acid, erythritol, galactose, galactose, lactose, maltose, melezitose, melibiose, α-methyl-D-glucoside, rhamnose, adonite , D-ribose, salicin,
Starch, succinic acid, D-xylose

[Composition]

Flour 100 Sugar 4 Salt 2 Dough conditioner 1.2 Shortening 4 Yeast 6 Water 63 [Process] Mixing time: 3 minutes on low speed, 3 minutes on medium speed, 2 minutes on low speed, 5 minutes on medium speed, 3 minutes on high speed Top temperature: 28 ℃ Pre-fermentation: 30℃, 30 minutes → degassing, molding Freezing: -30℃, 60 minutes quick freezing → -20℃ storage Thawing: 26℃, 90 minutes Incubation: 38℃, humidity 90%, 90 minutes Baking: 220℃ °C, 25 minutes

[Table] *1 Testing is carried out in a metal box with a fixed volume. The pad that protrudes from the top of the box
Measure the height of the dough and use it as an indicator of fermentation capacity.
*2 Specific volume is the value obtained by dividing the volume of baked bread by the weight.

Claims (1)

[Scope of Claims] 1. The cells of a yeast strain belonging to the genus Torulaspora are irradiated with ultraviolet light, and then the cell suspension irradiated with ultraviolet light is cultured, and the chromosomes of the culture become diploid. A method for producing yeast, which is characterized by obtaining a large cell strain belonging to the genus Torulaspora by isolating yeast that does not have an artificially mutated genetic trait of auxotrophy and has growth comparable to that of the parent strain. 2 Yeast belonging to the genus Torulaspora is Torulaspora delbrutskii Y-134-5 (Feikoken Bacteria Serial No. 905).
No. 1). 3. The production method according to claim 1 or 2, wherein the large-celled yeast belonging to the genus Torulaspora is Torulaspora delbrutskii SANK52084 (Feikoken Bibori No. 7898).