JPH01191679A - Protoplast-fused yeast, its preparation and production of seasoning - Google Patents

Abstract

NEW MATERIAL:Yeast produced by the protoplast fusion of a yeast belonging to genus Zygosaccharomyces and having excellent ethanol productivity with a yeast belonging to genus Candida and capable of producing 4-ethyl.guaiacol (abbreviated as 4-EG). USE:Capable of producing a seasoning having excellent flavor and taste in a short time. It has excellent productivity of ethanol and 4-EG. PREPARATION:A yeast belonging to genus Zygosaccharomyces and having excellent ethanol productivity (e.g., Zygosaccharomyces rouxii IFO 1877) and a yeast belonging to genus Candida and having 4-EG productivity (e.g., Candida versatilis FRZ451) are subjected to protoplast fusion and the objective yeast is separated from the obtained fused protoplast.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a protoplast-fused yeast obtained by protoplast fusion, a method for producing the same, and a method for producing a seasoning using the protoplast-fused yeast.

[Conventional technology]

As a conventional yeast breeding method, spore-based hybridization has been known, and crosses were carried out between spore-forming Satucharomyces cerevisiae and its closely related strains.

In recent years, prodoblast fusion has attracted attention as a yeast breeding method that does not rely on hybridization, and until now, for example, fusion methods between closely related species belonging to the genus Satucharomyces (Special Publication No. 57-3499
5, etc.), and a protoplast fusion method between different layers, for example, a yeast belonging to the genus Torulopsis and a yeast belonging to the genus Pichia (Japanese Patent Application Laid-Open No. 59-21386, etc.).

[Problems to be solved by the invention] However, in general, the probability of fusion between yeast protoplasts is low, and the resulting fused yeast are often unstable, and it is even more difficult to efficiently obtain a novel fused yeast with new properties. was extremely difficult.

As a result of various studies regarding the fusion method between different layers of yeast protoplasts, the present inventor discovered that yeast belonging to the genus Thigosaccharomyces and the genus Candeida, which has excellent ethanol production ability, and 4-ethyl guaiacol (hereinafter referred to as 4) - By fusing yeast with protoplast production ability (abbreviated as EC), a novel fused yeast with excellent ethanol and 4-EC production ability can be obtained, and the cells of the fused yeast are adsorbed and immobilized on a ceramic carrier. The present invention was completed based on the knowledge that seasonings with good flavor can be obtained in a short period of time by fermentation using a fermentation device that has been developed.

[Means for Solving the Problems] That is, the present invention is created by protoplast fusion of a yeast that belongs to the genus Chigosaccharomyces and has an excellent ability to produce ethanol, and a yeast that belongs to the genus Candeida and has an ability to produce 4-ethyl guaiacol. The fused protoplast obtained by fusion of a yeast that belongs to the genus Chigosaccharomyces and has an excellent ability to produce ethanol and a yeast that belongs to the genus Candeida and has an ability to produce 4-ethyl guaiacol. This is a method for producing protoplast fusion yeast, which is characterized by separating and collecting the desired protoplast fusion yeast from inside, and furthermore, aluminum oxide (AL+Oi) and silicon oxide (Sing
) and alumina fibers having 30 to 3
00 Alumina fiber walls bonded to form micron pores are coated with yeast belonging to the genus Chigosaccharomyces, which has excellent ethanol production ability, and yeast belonging to the genus Candeida.
- After adsorbing and immobilizing the protoplast-fused yeast cells obtained by fusing yeast capable of producing ethyl guaiacol with protoplasts, the substrate is fermented by passing the substrate through the immobilized cells under anaerobic conditions. This is a method for producing a seasoning characterized by the following.

The present invention will be explained in detail below.

First, the protoplast fusion yeast of the present invention and its production method will be explained.

In the creation of the fusion yeast of the present invention, examples of yeast having excellent ethanol production ability belonging to the genus Chigosaccharomyces, which is one of the parent yeasts, include Tygosaccharomyces ruxii IF01877, Tygosaccharomyces ruxii ATC.
C10383, Chigosaccharomyces ruxii ATCC
13356 etc. are mentioned.

Further, examples of yeast having 4-ethylguaiacol-producing ability belonging to the genus Candeida, which is the other parent yeast, include Candeida versatilis FRZ451, Candeida versatilis ATCC 20190, Canandida versatilis ATCC 20191, and the like.

In cell fusion, selection and isolation of the fusion strain created are as follows:
This can be carried out based on the differences in nutritional requirements, drug resistance, and temperature sensitivity between the parent strains and the fused yeast strain used, or can be carried out in combination. auxotrophic,
To take advantage of the differences between the two, for example, the protoplasts of the strain containing the fused protoplasts described above are cultured on a selective medium with different growth characteristics for the parental strains and the fused strain, and the desired fused strain is identified, isolated, and collected. What is done is done.

Examples of the parent strain of yeast belonging to the genus Thigosaccharomyces include its histidine auxotrophic mutants, and examples of the yeast belonging to the genus Candeida include its arginine auxotrophic mutants.

The yeast cells used for protoplast fusion were 7M
Medium [glucose 5 (W/V) χ, malt extract 0.
5 (W/V)χ, yeast extract 0.5 (w/V)
! , Polypebut 70.5 (W/V)L pH 5,6
) etc., and is preferably cultured at 25 to 30°C until the logarithmic growth phase.

Each of the obtained yeast cells (parent strain) was incubated at 30-60mM E.
Pretreatment is carried out with a solution containing 50-100 μM 2-mercaptoethanol containing DTA at a temperature of 25-30°C.

Next, the bacterial cells are treated with a yeast cell wall lytic enzyme, and as the lytic enzyme, for example, Cymolyase 20T (manufactured by Kirin Brewery Co., Ltd.) or the like is suitably used.

The enzyme treatment is carried out by using a yeast cell wall lytic enzyme in a phosphate buffer (around pH 7.5) containing sorbitol, EDTA, etc. as an osmotic pressure stabilizer, using the bacterial cells that have been pretreated for protoplast formation. It is preferable to process at 11°C for about 2 hours. The enzyme-treated bacterial cells (10 each
Mix 7-108 cells, 10-50 u
Protoplast fusion can be carried out by suspending in an aqueous solution of 30-35 (W/V) x polyethylene glycol containing M CaC1z or 0.5-1.0 M sorbitol and treating it for about 40 minutes at 20-30°C. desirable.

Next, the regeneration of protoplasts is carried out on fused protoplasts, e.g. 2-4 (W/V
) χ Sodium alginate was added, and this was added dropwise to the solution containing CaCI □ to immobilize the protoplasts of both yeasts, and this immobilized gel was added to the regeneration medium [glucose 2 (匈/V) χ, malt extract 0.25 (匈/V)]. V) χ, East exhaust 7! ,
0.25 (W/V) χ, polypebuty 0.25 (W
/V)2:, 0.8M sorbitol, pH 5, '6), and is preferably statically cultured at 25-30°C for 13-5 days to regenerate the cell wall.

Next, the fixed gel was washed with 0.5 to 1.0 M sorbitol and then placed in 0.1 M citrate buffer (pH 6.0).
It is desirable to dissolve the gel and obtain yeast fused cells by reacting at around 0°C for about 5 to 20 minutes.

These fused cells are grown in minimal medium [B
act-Yeast Nitrogen Ba5e W
lo Am1-no acid (manufactured by Deifico) 0
．． 67% glucose plus 0.5%], the first step is to isolate colonies that grow in the minimal medium, and the second step is to use methods such as water valves (water valve). The desired fused yeast can be obtained by isolating a colony that exhibits a dye staining property different from that of the parent strain using the dye plate culture method described by Benshin, Methodology in Yeast Research, Gakkai Publishing Center Publishing, 1982).

Protoplast-fused yeast can be isolated by the above two-step selection method.

The mycological properties of Candeida H-1, a strain of the protoplast fusion yeast of the present invention obtained by the above procedure, are described below.

Growth status of the medium (1) YM liquid medium After culturing at 30°C for 15 days, the cells are spherical to elliptical, (2,0-3.5) x (3,0-4.5)μ, with membrane production. Not formed.

(2) YM agar medium Slant culture cultured at 30°C for 12 weeks is grayish brown, slightly glossy, and the surface is smooth.

2. Physiological properties Pseudo-mycel production; - Ascospore formation; - 3. Sugar assimilation; glucose; + sucrose; + lactose; + maltose; The mycological properties of Candeida H-1 will be explained below in comparison with the parent strain.

(Margins below this page) fl) Mycological properties Table 1 [Medium used: YM medium with 5 (V/V)% raw soy sauce added] (2) Sugar fermentability Table 2 Versatillis FR245
1) and one strain of the fusion yeast of the present invention, Candeida
H-1 was submitted to the Institute of Microbiological Technology, Agency of Industrial Science and Technology (FERM P-9794).
Also, FEI-KEN BIYORI No. 9795 (FEI?M P-97
No. 95).

The present invention also includes a method for producing a seasoning using the protoplast-fused yeast cells described above.

The method for producing the seasoning will be explained below.

First, the raw materials for seasoning production used in the present invention are those normally used for soy sauce production, that is, those obtained by adding starchy raw materials to protein raw materials. Examples of protein raw materials include defatted soybeans, whole soybeans, and wheat gluten. , corn gluten,
Preferred examples of the starchy raw material include soybean purified protein, soluble isolated protein, seafood, meat, yeast extract, etc., and wheat, barley, corn, etc. as the starchy raw material.

These raw materials are subjected to conventional raw material processing, such as softening of the raw material structure, denaturation of proteins, gelatinization of starch, and sterilization.

Next, the enzyme-based hydrolysis of the raw material for seasoning production may be performed using either an enzyme-based method or a method in which the raw material is hydrolyzed using koji, but from the viewpoint of the hydrolysis operation, the former is particularly preferred. .

Next, the above-mentioned raw materials are enzymatically or chemically hydrolyzed, and if the pH is not around 3-7, the pH is adjusted to around 3-7 by lactic acid fermentation or by adding acid.

In addition, lactic acid fermentation is carried out using the hydrolyzate, for example, Pedeiococcus soae IAM 1673 (ATCC 13621
), Pedeiococcus soae IAM 1681 (AT
CC13622), Pedeiococcus soae IAM
1685 (ATCC 13623), Pedeiococcus
Halophilus IAM 1693, etc., or a culture solution thereof is added and kept at 25 to 35°C under anaerobic conditions with occasional or continuous mechanical stirring to carry out lactic acid fermentation.

At this time, the lactic acid bacteria cells may be prepared using a conventional method or immobilized lactic acid bacteria cells immobilized on a fibrous ceramic or spherical ceramic packing may be used, and the hydrolyzate may be brought into contact with the lactic acid bacteria cells to perform lactic acid fermentation.

Alternatively, instead of the lactic acid fermentation described above, an organic acid such as lactic acid or acetic acid or an inorganic acid such as hydrochloric acid or sulfuric acid is added to the hydrolyzed fermentation raw material, and the pH of the hydrolyzed product is adjusted to about 3 to 7. Good too.

If the hydrolyzed product is in a liquid state containing little or no decomposition residue (solid content), use it as is; if not, use the above-mentioned lactic acid fermentation or add acid, and press or filter using conventional methods. Solid-liquid separation is performed by operations such as centrifugation to obtain a liquid substrate.

Next, a liquid state obtained by hydrolyzing the above fermentation raw material,
The aforementioned protoplast-fused yeast cells are suspended by a conventional method, and the suspension is absorbed onto the alumina fiber wall to adsorb and immobilize the cells.The immobilized cells are then heated at an appropriate temperature, e.g.
Yeast fermentation is carried out while contacting under anaerobic conditions at around °C.

The alumina fiber wall is made of aluminum oxide (AlzO2
) and silicon oxide (SiO□) are bonded to each other using colloidal silica or a sulfate-based binder to form pores of 30 to 300 microns to form a wall shape, and the angle is 700 to 1400°. It is made by firing with C, and its main component is aluminum oxide (^
1203) and silicon oxide ((Sin2)) because they are easy to mold into fibers and have good affinity with yeast.

Further, the pore diameter was set in the range of 30 to 300 microns because yeast cells of approximately 5 microns can easily pass through and be immobilized. Since the alumina fibers described above as the immobilization carrier are bonded to each other so as to form pores of 30 to 300 microns to form a wall-shaped alumina fiber wall, it is possible to obtain the effect that yeast is easily adsorbed.

Next, a method for obtaining immobilized microbial cells by absorbing the above suspension of microbial cells onto the alumina fiber wall and adsorbing and identifying the microbial cells will be described.

First, a suspension obtained by adding, for example, a solution of glucose, yeast extract, etc. to the above-mentioned bacterial cells is sent by an appropriate means such as a peristal pump, and the bacterial cells are adsorbed and immobilized by being absorbed into the alumina fiber wall. to obtain the above-mentioned immobilized bacterial cells.

Next, the immobilized bacterial cells obtained by the above operation are placed in a fermentation container, and the hydrolyzed fermentation raw material is poured into the container, and fermentation is carried out under anaerobic conditions while contacting with the immobilized bacterial cells. Obtain the fermentation liquid.

In addition, as the above-mentioned fermentation container, for example, a film reaction tank,
Any type of tank may be used, such as a stirred tank, a cylindrical tank, a tank partitioned with ceramic adsorption plates, and a small spherical ceramic filled tank.

Moreover, the above-mentioned anaerobic conditions particularly mean a state in which no aeration is performed or a state in which the space of the fermentation vessel is replaced with carbon dioxide gas, nitrogen gas, or the like.

The fermentation time under the anaerobic conditions is preferably 5 hours or more, preferably about 20 to 120 hours. The above fermentation types include batch type,
It can be carried out by appropriately selecting a continuous method or the like.

In addition, if the number of bacteria is insufficient at the time of adsorption of the bacteria, the bacteria can be pre-cultured for an appropriate period of time under conditions suitable for the growth of the bacteria. After that, the fermentation raw material may be brought into contact with a hydrolyzed product for fermentation.

The seasoning obtained by the above operation can be used as it is, but if necessary, it can be subjected to conventional treatments such as filtration, pasteurization, shoplifting, etc. to make a seasoning product with excellent flavor.

〔Example〕

Hereinafter, the present invention will be specifically illustrated by examples.

Example 1 Stygosaccharomyces ruxii IF01877 (histidine demanding strain) and Candeida versatilis FRZ
451 (Feikoken Bacterial Serial No. 9794) (arginine auxotrophic strain) was cultured in YM medium [glucose 5.0 (revised V) χ,
Malt extract 0.5 (W/V)χ, yeast extract 0
．． 5 (W/V) L Polyhept: / 0.5 (W/V
) χ, pH 5, 6), shaking culture was performed at 30°C for 220 hours.

The obtained yeast cells were collected, washed with water, and then treated with 5mMED.
After pretreatment with 100mM mercaptoethanol solution containing TA for 15 minutes, centrifugation and washing with 0.8M sorbitol, the yeast cells were treated with 10mM ED of 0.8M sorbitol.
Suspended in phosphate buffer (pH 7.5) containing TA (2X
Zymolyase 20T (manufactured by Kirin Brewery Co., Ltd.) was added at a rate of 0.5 μ/rd, and the mixture was reacted at 30° C. for 2 hours.

Then, the protoplasts of both yeasts obtained by washing with 0.8M sorbitol were mixed with approximately 5 XIO7/mf (1:1
Mix at a mixing ratio of 50mM Ca
A reaction was carried out at 20° C. for 30 minutes in an aqueous solution 3- of 30 (W/V) χ polyethylene glycol 6000 containing C1z. Next, after collecting protoplast-fused yeast cells, 0
．． After washing with 8M sorbitol, 2rnl of 2% sodium alginate solution was added to this, and this was mixed with 50mM CaC.
1 g solution to immobilize the protoplast fusion suspension of both yeasts, this immobilized gel was cultured for regeneration [gluco-
, 2. 2 (W/V)χ, maltoeki, 2. 0.
25 (W/v) Z, East exhaust 7! ,0.25(
W/V)X, polypeptone 0.25(W/V)X, 0.
8(W/V)') rubitol, p) I5.6), and cultured at 30°C for 4 days.

The gel washed with 0.8 M sorbitol was then incubated in 0.1 citrate buffer (pH 6.0) at 30°C for 1 hour.
After standing for 0 minutes to dissolve the gel, the regenerated yeast cells were collected by centrifugation.

After diluting the obtained regenerated yeast cells, it was added to minimal medium (
Bact-Yeast Nitrogen Ba5e
O, 67 (W/V) χ, glucose 0.5 (W/V)
χ] colonies were isolated as fused yeast.

The obtained fused yeast was placed on a 2% agar medium (glucose 2(4/4)).
V) χ, polypeptone 0.1 (1~/V) χ, yeast exhaust, 2.0.1 (W/V) χ, 7/L/I-exhaust, 2.0.1 (W/v) χ , ammonium sulfate 0.15 (W/V)
χ, calcium phosphate 0.15 (W/V)%, magnesium sulfate O,l (W/V)%, water blue 3
0 μg/-, Tsuksin, Rhodamine B, Safranin T, and Safranin O were added to 50 μg/+ rl in a medium (pH 5, 6)), and colonies showing a different color tone from the parent strains were isolated and protoplast fused. The strain, Candeida H-1 (Feikoken Bibori No. 9795) was obtained.

Example 2 9 kg of water was added to a mixture of 6 kg of defatted soybeans and 1.3 kg of wheat.
, 61 and put this in a 6042 airtight container to weigh 1 kg.
/c+fl・g water tofu for 30 minutes, then loosen well.
After further heating and post-treatment with 1 kg/ci·g of water vapor for 45 minutes, it was cooled.

On the other hand, Aspergillus oryzae ATC was found on 3 kg of barley skin.
C20386 was inoculated and koji was made at 30 to 35°C for 42 hours to obtain a solid oil, and the solid oil was extracted with 5 times the amount of cold water. The obtained enzyme solution was prefiltered with a filter press, and further 5A
A sterile enzyme solution was obtained by filtration using a model-451 sterile filter (manufactured by Nippon Rosuiki Kogyo A Kiku).

This sterile enzyme solution at 9.6°C was added to the entire amount of the above-mentioned cooled raw material, and the mixture was enzymatically decomposed at 40°C for 64 hours while being shaken.

After adding 2 kg of salt to the obtained hydrolyzate (salt concentration: 9 (W/V)χ), it was squeezed to obtain 20. I.J.
2 was collected and adjusted to pH 6.0 with caustic soda, soy sauce lactic acid bacteria Pedeiococcus herophilus IA) 11693 was added to the lactic acid bacteria medium (dark raw soy sauce 10 (V)).
/V)χ,g/l/ , : ] -su1 (W/V)χ,
Salt 8 (W/V) χ, Sodium acetate 3.5 (W/V)
V) χ, yeast extract 0.3 production C) χ, pH 7,0) 3
Lactic acid bacteria culture solution cultured at 0°C for 14 days (number of viable cells: 1.
Add 100 mQ of I
Lactic acid fermentation was carried out at 0°C for 1120 hours.

Next, this lactic acid fermentation liquid was heated at 80°C for 20 minutes to stop lactic acid fermentation, and the produced lactic acid bacteria cells were filtered through diatomaceous earth in a conventional manner to obtain an enzymatically decomposed filtrate (liquid component value: TN 2).
．． 05 (W/V) Z, RS 8.83 (W/V) L N
aC19,00(W/V)χ, pH5,06, TA2
．． 15) Obtained 19.4ffi.

On the other hand, the protoplast fusion strain obtained in Example 1, Candeida H-1, and FAIKEN Bacteria No. 9795 were cultured in a yeast culture liquid medium (dark soy sauce 5-10 (V/V)!, glucose 5
~7 (W/V)′1, salt 0~8 (W/V)χ, 1 potassium phosphate 0.1 (W/V)! , magnesium sulfate 0.05 (W/V) %, yeast extract 71.0.1-0
．． 5 (W/V)χ, calcium chloride O.

OHW/V)X, Polypebut 70~0.5 (W/V)
After shaking culture at 30'C for 48-72 hours at χ, p, H5゜0-5.5), each yeast cell obtained by centrifugation at 8000XG for 10 minutes was incubated once in sterile water. After washing and centrifuging again to separate the yeast cells, the yeast cells were added to the above-mentioned yeast culture liquid medium and mixed well to obtain a yeast suspension. Next, this yeast suspension was absorbed onto an immobilization carrier.

As the immobilization carrier material, aluminum oxide (A1203
) and silicon oxide (Si(h)) are bonded together to form pores of 30 to 300 microns.

Further, the shape of the immobilization carrier includes columnar, cylindrical, plate-shaped, and granule-shaped ones prepared for filling.

Next, for the reaction, the lactic acid fermentation finished liquid is circulated through the immobilized carrier at a rate of 10 to 100 ml/hr using a pellicle pump to promote the reaction. Fermentation is carried out at a temperature of 20 to 30''C. The desired seasoning liquid 1.5% is obtained in 48 to 72 hours. The seasoning liquid is subjected to pasteurization treatment in a conventional manner to obtain a product.

〔Effect of the invention〕

According to the present invention, yeast belonging to the genus Chigosaccharomyces and having excellent ethanol production ability and 4-EC belonging to the genus Candeida
By fusing capable yeast with protoplasts, a new fused yeast with excellent ethanol and 4-EC producing ability can be efficiently obtained, and furthermore, by using a fermentation apparatus in which the cells of the protoplast-fused yeast are immobilized. By fermenting, seasonings with good flavor can be obtained in a short period of time.

Claims (1)

[Scope of Claims] 1. A protoplast-fused yeast created by protoplast fusion of a yeast that belongs to the genus Chigosaccharomyces and has an excellent ability to produce ethanol, and a yeast that belongs to the genus Candeida and has an ability to produce 4-ethyl guaiacol. 2. Protoplast fusion of yeast that belongs to the genus Thigosaccharomyces and has an excellent ability to produce ethanol and yeast that belongs to the genus Candeida and has an ability to produce 4-ethyl guaiacol, and from the resulting fused protoplasts, obtain the desired protoplast-fused yeast. A method for producing protoplast fusion yeast, characterized by separation and collection. 3. Aluminum oxide (Al_2O_3) and silicon oxide (
The alumina fiber wall, which is made by bonding alumina fibers mainly composed of SiO_2) to each other to form pores of 30 to 300 microns, is coated with yeast belonging to the genus Chigosaccharomyces, which has excellent ethanol production ability, and yeast of the genus Candeida. After adsorbing and immobilizing the cells of the protoplast-fused yeast, which is produced by fusing yeast that has the ability to produce 4-ethyl guaiacol with protoplasts, the substrate is passed through the immobilized cells under anaerobic conditions. A seasoning manufacturing method characterized by fermenting.