JP2006042827A - Bioreactor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluidized bed-type bioreactor; and to provide a bioreactor device capable of carrying out a continuous fermentation or the like by a comparatively simple structure. <P>SOLUTION: The bioreactor has a water flow-generating means for generating the water flow from the lower part of a pipe body to the upper part, and a taking-out port for taking out a treated liquid treated with a biocatalyst, installed in the upper part of the pipe body, and treats a liquid to be treated by flowing the biocatalyst by the water flow-generating means. A cylindrical partitioning wall is formed at the upper inner part of the pipe body so as to form a distance from the inner periphery surface of the pipe body, and the lower end part of the partitioning wall is extended to the lower part of the taking-out port. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention is a novel yeast belonging to the genus Tigosaccharomyces that has a cell mass formation ability and can be fermented with high efficiency even in a medium with a high salt concentration, for example, and a method for producing a fermented seasoning using the yeast strain, and The present invention relates to a fluidized bed type bioreactor.

Yeast has been used for various productions such as the production of alcohol such as Japanese sake, the production of fermented seasonings such as soy sauce, and the fermentation of bread.
For example, soy sauce brewing is made by mixing cooked whole soybeans or processed defatted soybeans with roasted wheat, inoculating seed koji and making koji, and then adding salt to create flavors, fermenting and aging Later, soy sauce is produced by pressing this moromi (honjozo).
However, since it takes about 6 months to ferment and ripen moromi, the fermentation and ripening period occupies most of the production process. Therefore, if the fermentation / ripening period can be shortened, the production cost can be greatly reduced in soy sauce production.

Therefore, studies have been made to efficiently perform the fermentation process using yeast by a bioreactor.
In general, in a fermentation process using a bioreactor, a method in which a biocatalyst is immobilized is used.

As a method for immobilizing a biocatalyst, a binding method for supporting on an insoluble carrier, a cross-linking method for binding biocatalysts to each other, a comprehensive method for wrapping inside a polymer gel, a composite method of these, and the like are known.
However, in these methods, the biocatalyst is immobilized such as purchase of an immobilization carrier, sterilization of the immobilization carrier, immobilization process of the biocatalyst, maintenance of the immobilization carrier, and disposal / treatment of the immobilization carrier. The above cost is required.
Also, in the production of fermented foods and the continuous production of alcohol that require immobilization of living cells (proliferating cells), it is due to cell growth or the disintegration of an immobilization carrier such as a polymer gel due to chemical components in the raw material liquid. In general, the carrier binding method (physical adsorption) to porous inorganic materials is used because of problems such as clogging. However, when porous inorganic materials such as porous ceramics are used as immobilized carriers Has a small amount of cells to be immobilized and has problems such as an increase in fermentation rate and resistance to microbial contamination.

  On the other hand, in the method using aggregating yeast that allows easy separation and recovery of bacterial cells, the yeast cell immobilization step and the separation step between the yeast cells and the fermentation broth can be omitted. The system is simplified, and low-cost and efficient fermentation production is possible.

The aggregating yeast strains reported so far are yeasts belonging to Saccharomyces cerevisiae, and these are bred mainly by cell fusion technology and gene recombination technology.
However, these strains improved from Saccharomyces cerevisiae have low osmotic pressure resistance (including salt tolerance), so although raw materials with low salt and sugar concentration can be used as fermentation raw materials, molasses (eg, potassium, magnesium) When using as a raw material high-concentration salts such as sodium and other salts such as sodium), saccharide hydrolysates, and liquids containing saccharides, it is necessary to dilute the raw materials. This leads to an increase in the amount of waste liquid treated after completion of fermentation / distillation, which contributes to an increase in industrial production costs.

  In addition, in industrial ethanol production by fermentation methods, inexpensive fermentation raw materials such as molasses are used, and batch fermentation methods and continuous fermentation methods such as the yeast circulation method and the immobilized yeast method have been developed. However, in the conventional technique, since yeast cells are maintained at a high density, the apparatus and process are complicated, and the maintenance cost is high.

This invention makes it a subject to provide the manufacturing method of the fermented seasoning using the novel yeast which does not use an immobilization support | carrier, and has high osmotic pressure tolerance in view of the above points.
Furthermore, this invention makes it a subject to provide the bioreactor apparatus which can process a continuous fermentation etc. with a comparatively simple structure.

Yeast belonging to the genus Tigosaccharomyces used for soy sauce brewing has high osmotic pressure resistance, for example, it grows and ferments in the presence of 2.5M sodium chloride and / or 50% (W / W) glucose medium. It can be performed.
And when the present inventors screened yeast from soy sauce moromi containing yeast of the genus Tigosaccharomyces, a novel soy sauce main fermenting yeast strain (Tigo Saccharomyces ruxii R-2 strain having a predetermined cell mass formation ability ( Zygosaccharomyces rouxii R-2 strain) was named: Seikoku Kenkyu No. 18251 [FERM P-18251]).
The cell mass formed by this strain was hard and did not collapse even by shearing force by aeration stirring, and the cell mass formation ability (including aggregation ability and adhesion ability) was superior to commercially available yeast. However, the cell mass was relatively small, and sedimentation was not always sufficient when assumed to be used in a bioreactor.
Therefore, as a result of further earnest research, the present inventors obtained an excellent yeast that has high pressure resistance and can form a larger cell mass by cell fusion of this strain and soy sauce main fermentation yeast. Completed the invention.

That is, the present invention provides a novel yeast (Nippon Kogyo Bacteria No. 18252 [FERM P-18252]) belonging to Zygosaccharomyces having a cell mass formation ability (including aggregation ability and adhesion ability). .
Furthermore, the present invention provides a method for producing a fermented seasoning such as soy sauce using the novel yeast.

Further, the present invention is provided with water flow generating means for generating a water flow from the lower side to the upper side of the tube body 2, and the outlet 6 from which the processing liquid processed by the biocatalyst is taken out above the tube body 2. And a bioreactor in which the biocatalyst flows by the water flow generating means to process the liquid to be processed, and a cylindrical partition wall 7 is provided in the inner periphery of the pipe body 2 above the pipe body 2. Provided is a bioreactor that is provided at a distance from the surface and in which the lower end of the partition wall 7 extends below the outlet 6.
Further, the present invention provides the bioreactor in which a passage port 8 through which a treatment liquid can pass is formed at a position that is a peripheral wall of the partition wall 7 and is not opposed to the outlet 6.
Furthermore, the present invention is provided with water flow generating means for generating a water flow from the lower side to the upper side of the tube body 2, and the outlet 6 from which the processing liquid processed by the biocatalyst is taken out above the tube body 2. And a bioreactor in which the biocatalyst flows by the water flow generating means to process the liquid to be processed, and a small hole plate 5 that divides the inside of the pipe body 2 is provided in the vertical direction. A multi-stage bioreactor is provided.

Since the novel yeast according to the present invention has a high self-aggregation property and becomes a large lump, efficient fermentation using a bioreactor or the like is possible without immobilization on a carrier.
In particular, it is suitable for continuous production of fermented seasonings such as soy sauce.
In addition, since the novel yeast of the present invention can be fermented with high efficiency even in a medium with a high salt concentration, it can be fermented using various liquids to be treated. Furthermore, the bioreactor of the present invention can separate the treatment liquid and the biocatalyst with a relatively simple structure.

Hereinafter, embodiments of the present invention will be described.
As described above, the novel yeast of the present invention is a yeast strain having a cell mass formation ability (Zygosaccharomyces rouxii R-2 strain) from soy sauce moromi containing yeast of the genus Tigosaccharomyces: Biotechnological No. 18251 [FERM P-18251] (hereinafter sometimes referred to as “R-2 strain”), and this strain and a commercially available strain of Zygosaccharomyces rouxii (Zygosaccharomyces rouxii) (Hereinafter may be abbreviated as “commercial yeast strain”) by cell fusion.

In cell fusion, the parent strain is usually subjected to a mutation treatment and labeled with genetic properties, but it is preferable not to perform the labeling treatment from the viewpoint that the excellent properties of yeast are not impaired. As the cell fusion method, a known method can be adopted, but the high voltage pulse cell fusion method is preferable from the viewpoint of high fusion efficiency. After culturing the obtained fused strain a plurality of times, a strain suitable for the novel yeast of the present invention, particularly the soy sauce main fermentation yeast, can be obtained by selecting a strain capable of forming a larger cell mass.
This new yeast strain was named Zigogosaccharomyces rouxii ZR-2f (hereinafter sometimes abbreviated as “ZR-2f”), and this was researched by the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology. Was deposited as Life Engineering Research Bacteria 18252 [FERMP-18252].

The bacteriological properties of this Tigosaccharomyces luxi ZR-2f strain (FERM P-18252) and its parent strains (commercial yeast strain and R-2 strain) were examined under experimental agricultural chemistry, 3rd edition (Faculty of Agriculture, Faculty of Agriculture, University of Tokyo) By the author, published by Asakura Shoten Co., Ltd.), it is as follows.
[ZR-2f strain]
(A) Cell morphology when cultured for 2 days at 30 ° C. using a YPD medium containing 15% NaCl:
1) Vegetative cell size: 4-8 μm.
2) Vegetative cell morphology: egg shape.
3) Form of growth: budding.
(B) Colony morphology when cultured on a YPD agar medium containing 15% NaCl at 30 ° C. for 4 days:
1) Form: Circle.
2) Uplift: Head shape.
3) Perimeter: Smooth.
4) Size: 1-3 mm.
5) Color tone: white and opaque.
6) Surface: Smooth and semi-glossy.
(C) Carbon source assimilation: glucose, galactose, sucrose, maltose, cellobiose, soluble starch, sorbitol, methanol, fructose, mannose, mannitol and dextrin are assimilated to ferment glucose and maltose. α-methyl glycoside, trehalose, lactose, melibiose, raffinose, inulin, sodium citrate, ethanol, xylose are not assimilated.
(D) Salt tolerance: Can grow on YPD medium containing 25% NaCl.
[Commercial yeast strain]
(A) Cell morphology when cultured for 2 days at 30 ° C. using a YPD medium containing 15% NaCl:
1) Vegetative cell size: 4-8 μm.
2) Vegetative cell morphology: egg shape.
3) Form of growth: budding.
(B) Colony morphology when cultured on a YPD agar medium containing 15% NaCl at 30 ° C. for 4 days:
1) Form: Circle.
2) Uplift: Head shape.
3) Perimeter: Smooth.
4) Size: 1-3 mm.
5) Color tone: white and opaque.
6) Surface: Smooth and glossy.
(C) Carbon source assimilation: glucose, galactose, sucrose, maltose, cellobiose, soluble starch, sorbitol, methanol, fructose, mannose, mannitol and dextrin are assimilated to ferment glucose and maltose. α-methylglucoside, trehalose, lactose, melibiose, raffinose, inulin, sodium citrate, ethanol and xylose are not assimilated.
(D) Salt tolerance: Although it can grow on a YPD medium containing 25% NaCl, it takes time.
[R-2 stock]
(A) Cell morphology when cultured for 2 days at 30 ° C. using a YPD medium containing 15% NaCl:
1) Vegetative cell size: 4-8 μm.
2) Vegetative cell morphology: egg shape.
3) Form of growth: budding.
(B) Colony morphology when cultured on a YPD agar medium containing 15% NaCl at 30 ° C. for 4 days:
1) Form: Circle.
2) Uplift: Head shape.
3) Perimeter: Smooth.
4) Size: 1-3 mm.
5) Color tone: white and opaque.
6) Surface: Smooth and semi-glossy.
(C) Carbon source assimilation: glucose, galactose, sucrose, maltose, cellobiose, soluble starch, sorbitol, methanol, fructose, mannose, mannitol and dextrin are assimilated to ferment glucose and maltose. α-methylglucoside, trehalose, lactose, melibiose, raffinose, inulin, sodium citrate, ethanol and xylose are not assimilated.
(D) Salt tolerance: Can grow on YPD medium containing 25% NaCl.

Since the novel yeast of the present invention itself becomes a large lump, since the yeast cell immobilization process and the separation of the yeast cell and the treatment liquid (fermented liquid) can be easily performed, in particular, soy sauce, mirin, etc. It can be suitably used in a fluidized bed bioreactor in the production of fermented seasonings, the production of alcohols such as sake, shochu and wine, and the production of bacterial cells during sugar nucleotide production. Furthermore, since fermentation can be performed even in a relatively high salt concentration environment, various treatment liquids can be subjected to fermentation treatment.
The novel yeast of the present invention can also be used for fermentation of bread, miso and the like.

  The novel yeast of the present invention is used by culturing the yeast, and it can be used for any system of a batch fermentation system in which the whole liquid is replaced after the fermentation process, or a continuous fermentation system in which the liquid to be treated is continuously supplied to perform the fermentation process. In particular, it can be suitably used for a continuous fermentation system.

As the bioreactor used in the continuous fermentation system, a conventionally known bioreactor may be used, but in the present invention, the following fluidized bed bioreactor is preferably used.
Specifically, as shown in FIGS. 1 and 2, the bioreactor 1 includes, for example, a lower part 2a (introduction part 2a), an intermediate part 2b (biological treatment part 2b), and an upper part 2c (solid-liquid separation part 2c). ) A plurality of cylindrical members 2, a supply port 3 provided in a lower portion 2 a of the tube 2, and a small hole plate 5 for partition provided in an intermediate portion 2 b of the tube 2. And an outlet 6 provided in the upper part 2c of the tube body 2 and a cylindrical partition wall 7 provided in the upper part 2c of the tube body 2 and in the center of the upper part 2c. .

The tubular body 2 is made of a known material having impact resistance such as, for example, impact resistant glass, metal such as stainless steel, and is formed in an appropriate size according to the processing amount of the liquid to be processed.
As shown in FIG. 1, the tubular body 2 may have a configuration in which a plurality of cylindrical members of the lower portion 2 a, the intermediate portion 2 b, and the upper portion 2 c are connected, or may be configured by an integral cylindrical member. .
The supply port 3 is formed in the bottom surface of the lower part 2a of the tube body 2, for example. A supply path (not shown) for supplying air and liquid to be processed is attached to the supply port 3 so that air and liquid to be processed are supplied from the supply port 3 to the inside of the tube body 2. (Arrow in FIG. 1).
The supply port 3 may be formed in the peripheral wall of the lower portion 2a, or two or more supply ports 3 may be provided to supply air and the liquid to be processed separately.

The small hole plate 5 has a hole having a size through which the liquid to be treated filled in the tube body 2 and all or a part of the biocatalyst (for example, the cell mass according to the yeast of the present invention) can pass. The member is not particularly limited as long as it is a member having, for example, a mesh plate (for example, a metal net-like plate) having a size of 1.5 to 3 mm, more preferably 2 to 2.5 mm.
By providing the small hole plate 5, a concentration gradient of the biocatalyst and / or a concentration gradient of the product in the tube body 2 can be formed. Further, by providing the small hole plate 5, fluid convection is generated in each region partitioned by the small hole plate 5 (indicated by arrows in FIG. 1). One small hole plate 5 may be used, but it is preferable that the small hole plate 5 is provided in multiple stages in the vertical direction in order to form a good concentration gradient.
Specifically, as shown in FIG. 1, two mesh plates 5 having a mesh size of 2 mm are provided at predetermined intervals.

The outlet 6 is provided in the middle portion of the peripheral wall of the upper portion 2c of the tube body 2, and the processing liquid after processing is taken out from the outlet 6.
A cap 15 is fitted to the outlet 6, and a discharge pipe 16 is provided at the center. In this embodiment, the pipe 16 substantially serves as the outlet 6 and the processing liquid and air are discharged from the pipe 16 (arrows in FIG. 1).
In this way, the air supplied from below is discharged upward (by the air flow), so that a water flow is generated in the pipe body 2 from below to above (to the water flow generating means). Equivalent to).
The water flow generating means is not limited to the configuration by supplying and discharging air, and for example, a configuration in which a rotating blade is provided in the tube body 2 and rotated to generate a water flow may be used.

Next, the partition wall 7 is formed of, for example, a cylindrical body formed integrally with the tube body 2, and an upper end portion of the partition wall 7 is connected to the tube body 2. On the other hand, the lower end portion of the partition wall 7 is a free end, and extends downward from the lower end portion 6a of the outlet 6 as shown in FIG.
The partition wall 7 has the same opening as the intermediate part 2b or an opening larger than the intermediate part 2b in order to receive the water flow rising from the intermediate part 2b. As shown in FIG. It arrange | positions so that the opening part 14 of the part 2b may be surrounded (when it sees from a plane, it arrange | positions so that the intermediate part 2b may be enclosed by the partition 7). With this configuration, most of the fluid (treatment liquid and biocatalyst) rising from the intermediate portion 2 b is first concentrated inside the partition wall 7.
Further, the partition wall 7 is disposed with an interval (gap 11) at which the outer peripheral surface thereof flows with respect to the inner peripheral surface of the tube body 2.

Further, in the partition wall 7, a passage port 8 through which at least the treatment liquid can pass is formed at a position that does not face the outlet 6 (which means that the partition wall 7 is not a position facing the straight direction). Examples of the passage port 8 include a notch portion 8 where a part of the passage port 8 is located below or at substantially the same height as the lower end of the outlet 6 as shown in the figure. A collection of a plurality of small holes may be configured as the passage port 8.
As shown in the drawing, the non-opposing position where the passage port 8 is formed is preferably a position opposite to the outlet 6 (rotated 180 degrees horizontally), but is perpendicular to the outlet (shown as B in FIG. 2). It may be. Moreover, although it is sufficient if the passage port 8 is formed at one place in the opposite position, it may be formed at a plurality of places.

Reference numeral 10 denotes an operation port that communicates with the inside of the tube body 2 and is provided inside the partition wall 7. Reference numeral 11 denotes a cap that closes the operation port. Reference numeral 12 denotes a passage through which hot water or the like is passed in order to maintain the temperature in the tube body 2.
Further, a watertight packing is provided between the respective parts such as the lower part 2a, the intermediate part 2b and the upper part 2c of the tubular body 2.

The bioreactor 1 is filled with a liquid to be treated such as (waste) molasses and a biocatalyst such as ZR-2f strain inside, and by supplying air, the biocatalyst flows to ferment the liquid to be treated. Then, biological treatment such as decomposition and generation is performed, and the obtained treatment liquid is discharged from the outlet 6.
At this time, as shown in FIGS. 1 to 3 (indicated by arrows of the fluid flow), the bioreactor 1 of the present invention allows the biocatalyst and the treated treatment liquid to be placed above the tubular body 2 by the water flow generating means. It rises to the part 2c.
The rising processing liquid and biocatalyst flow inside the partition wall 7, and the processing liquid and a part of the biocatalyst flow out to the outside of the partition wall 7 through the passage port 8, and between the partition wall 7 and the tubular body 2 (gap 11). ) Through the outlet 6 side. Since the gap 11 is not easily affected by the upward water flow by the water flow generating means due to the presence of the partition wall 7, the biocatalyst descends due to its own weight while passing through the gap 11, and almost only the treatment liquid is present. Move to the outlet 6. Therefore, the treatment liquid and the biocatalyst can be easily separated.

As the liquid to be treated, known ones such as (waste) molasses and saccharide hydrolysates can be used.
In addition, the biocatalyst is not limited to the novel yeast (ZR-2f strain) of the present invention, but is known flocculent yeast, cells and enzymes immobilized on a carrier, cells and enzymes included in a gel, and the like. A conventionally well-known thing can also be used.

Hereinafter, selection of a fusion strain excellent in cell fusion and cell mass formation ability, batch fermentation and continuous fermentation test of the selected fusion strain will be specifically described.
However, the technical scope of the present invention is not limited by these examples. In the description of the bioreactor and the like, those having the same names as those in the above embodiment are the same as those in the above embodiment unless otherwise specified.
Preparation of Protoplasts Protoplasts were basically prepared according to the method of Noda et al. (Agr. Biol. Chem., 54, 2023 (1990)).
That is, a cell mass-forming yeast Tigosaccharomyces ruxii R-2 that quickly settles from the fermentation liquid discharged from a conventional bioreactor (using a porous ceramic carrier on which a commercially available yeast strain is supported) The yeast strain Tigosaccharomyces luxi (Bioc, product name: soy sauce main fermenting yeast) was shaken and cultured at 30 ° C. in a known YPD medium containing 10% NaCl at 30 ° C. until the early stationary phase (1 × 10 8 cells / ml). The obtained cells were washed with sterilized 10% saline. Subsequently, it was suspended in 50 mM sodium phosphate buffer (pH 7.5) containing 1.2 M sorbitol, 5 mM EDTA, and 0.5% mercaptoethanol. To this cell suspension, a zymolyce solution (manufactured by Seikagaku Corporation) was added to a final concentration of 1 mg / ml and treated at 30 ° C. for 90 minutes to produce a protoplast. The protoplasts were collected by centrifugation (1,200 × g), washed 3 times with 1.2 M sorbitol solution and suspended at a concentration of 5 × 10 7 protoplasts / ml.

Cell fusion cell fusion was performed by the high voltage pulse cell fusion method (electric cell fusion method).
That is, a cell fusion device (manufactured by Shimadzu Corporation, product name: SSH-10) was used, and fusion conditions were as follows: electrode spacing 1 mm, AC frequency 1 MHz, AC initial applied voltage 40 V, pulse width 500 μS, pulse voltage 450 V ( The electric field intensity was 4.5 kV / cm), and the AC secondary applied voltage was 40 V. The protoplast suspension after the application treatment was incubated at 30 ° C. for 30 minutes, and this was stored in an SD plate (minimum medium: 0.67% yeast nitrogen base, no amino acid, 2% glucose, 1.2 M sorbitol, The protoplasts were regenerated by applying 0.1 ml to 2% agar) and culturing at 25 ° C. for 3 to 7 days. Next, the regenerated cells are placed in 10 ml of YPD liquid medium (pH 5.0) containing 15% NaCl, and 5 ml of the medium is added to the surface of the SD plate, and then the grown colonies (cells) are dispersed. 0.5 ml of the cells were inoculated and subjected to shaking culture at 30 ° C. until a stationary phase was reached (2 to 3 days). This culture solution is allowed to stand for 5 minutes, then the floating cells are removed, and 10 ml of a new YPD medium containing 15% NaCl is added thereto, followed by further shaking culture at 30 ° C. until reaching the stationary phase. It was. This operation was repeated 5 to 9 times to concentrate strains having improved cell mass formation ability. Thereafter, the settled cells were washed three times with sterilized 15% saline, and then applied to a YPD plate containing 15% NaCl (applying 0.1 ml of a 1000-fold diluted suspension), Growing colonies were obtained. Furthermore, these colonies were inoculated into 10 ml of YPD liquid medium (pH 5.0) containing 15% NaCl (inoculate one colony), and cultured with shaking at 30 ° C. for 3 days. The colony having (ZR-2f strain) was selected.

Evaluation of the cell mass sedimentation rate The cell mass sedimentation rate was evaluated by the rate of decrease in turbidity (OD660 nm) by a spectrophotometer (product name: SPECTORONIC 20 manufactured by MILTONROY).
The test strain is inoculated into 10 ml of YPD medium containing 15% NaCl (inoculated with 0.5 ml as a bulk of aggregated cells), and cultured with shaking at 30 ° C. for 48 hours. This is vortex mixer (manufactured by iuchi). , Product name: AUTOMATIC LABO.MIXER), and after stirring for 10 seconds, the change in absorbance at 660 nm was measured over time. The result is shown in FIG.
In addition, the test strain used three types, the commercially available yeast strain, R-2 strain, and the colony (ZR-2f strain) obtained by the said cell fusion.

In the commercial yeast strain, no change was observed in the absorbance at 660 nm, and a slight change was observed in the R-2 strain, whereas the absorbance at 660 nm rapidly decreased in the ZR-2f strain. From this, it was found that the cell mass of ZR-2f strain settles quickly.
Moreover, visually, the ZR-2f strain formed a bacterial mass having a diameter of about 0.5 to 3 mm.
In order to clarify the cell mass formation effect of this ZR-2f strain, R-2 strain, ZR-2f strain, and commercially available yeast strain were taken in a test tube having a diameter of 18 mm, and the appearance of those cell masses was shown in Reference Photo 1. Shown in
As is clear from Reference Photo 1, the ZR-2f strain formed a much larger cell mass than the R-2 strain.
In the R-2 strain, although it was relatively small, the formation of cell mass was clearly observed in the photograph of the bottom of the test tube.
On the other hand, in the commercially available yeast strain, the formation of the cell mass was not recognized at all in the photograph of the side surface portion of the test tube and the photograph of the bottom surface portion of the test tube.
As a result, it was found that the ZR-2f strain has a very characteristic cell mass formation ability, and the R-2 strain has at least a cell mass formation ability that can be clearly distinguished from a commercially available yeast strain.

Pulse field electrophoresis Next, 0.5 ml of each test strain is shaken and cultured in 10 ml of YPD medium containing 15% NaCl at 30 ° C. for 48 hours, and then collected and washed with a centrifuge Used. Sample preparation was carried out in an agarose gel and the gel block was adjusted to prevent fragmentation of chromosomal DNA. As the gel, 1% agarose (Pulsed Field Centrified Agarose manufactured by Bio-Rad) gel was used, and 0.5 × TBE buffer (pH 8.0) was used for electrophoresis. For electrophoresis, CHEF-DR III System manufactured by Bio-Rad was used, electrophoresis voltage 6 V / cm, pulse interval (start: INT) 60 to (end: FIN) 120 seconds, pulse angle 120 degrees, electrophoresis time 22 hours, bath The internal temperature was 14 ° C.
The band after electrophoresis is shown in Reference Photo 2. As is clear from Reference Photo 2, the R-2 strain, the ZR-2f strain, and the commercially available yeast strain had almost the same chromosome separation pattern. However, in the R-2 and ZR-2f strains, chromosomal DNA was observed at a position of about 800 kb, which was not observed in the commercially available yeast strain.
From this, although it was confirmed that the ZR-2f strain and the R-2 strain are the same strains, they are clearly different from the commercially available yeast strains.

Example 1
(Batch fermentation test with jar fermenter)
As a pre-culture, 20 ml of the ZR-2f strain obtained by the cell fusion was cultured with shaking in 500 ml of YPD medium containing 15% NaCl at 30 ° C. for 48 hours.
Thereafter, the medium for fermentation test (yeast) was left as it was without being supported on an immobilization carrier, and about 50 ml of the ZR-2f strain obtained by removing the cells that did not settle together with the culture solution by standing for 10 minutes. To a jar fermenter (product name: MINI FERMENTOR M-100, manufactured by Tokyo Rika Kikai Co., Ltd.) containing 1 liter of extract 0.5%, polypeptone 1%, glucose 10%, NaCl 15%, pH 5.0) The mixture was filled and fermented at 30 ° C., and the alcohol concentration was measured over time by a gas chromatograph (manufactured by Shimadzu Corporation, product name: GC-9A). The result is shown in FIG.
Although the first fermentation had a slightly longer delay time, the fermentation proceeded smoothly, and 3.7% (W / V) of alcohol was produced on the fifth day of fermentation. At this time, although a strain that became non-aggregating appeared, aggregates settled after the end of fermentation, and repeated batch fermentation was possible. In addition, the delay time was shortened in the second and third batch fermentations, and 3.8% (W / V) alcohol was produced on the third fermentation day.

Example 2
(Ethanol continuous fermentation test using bioreactor)
Example 2-1.
As shown in FIG. 6, the entire tubular body 20 is a cylindrical bioreactor 100 (specifications: the lower part 20a has a capacity of about 74 ml, the intermediate part 20b has an inner diameter (diameter) of 45 mm and a capacity of about 286 ml, and the upper part 20c has a capacity. The following test was performed using about 60 ml (total volume 420 ml) (manufactured by Tokyo Rika Kikai Co., Ltd., special order product). The bioreactor 100 is not provided with a partition wall and a small hole plate.
The bioreactor 100 was filled with 1 liter of a fermentation test medium (yeast extract 0.5%, polypeptone 1%, glucose 10%, NaCl 15%, pH 5.0), and the same as in Example 1 above. About 50 ml of the pre-cultured ZR-2f strain was filled as it was without being supported on an immobilization carrier, and subjected to circulation fermentation at 30 ° C. for 2 days with an aeration rate of 0.1 vvm. Thereafter, the same medium was supplied to the bioreactor apparatus at a flow rate of 10 ml / hr to 25 ml / hr for continuous fermentation.
The fermentation broth produced over time was sampled, and the alcohol concentration was analyzed by a gas chromatograph (manufactured by Shimadzu Corporation, product name: GA-9A) to measure alcohol productivity. As a result, the agglomerate increased with the growth of the bacterial cells, but this increased bacterial mass adhered to the outlet 6 of the bioreactor on the 18th day, and the outlet 6 was clogged, resulting in stable continuous fermentation. Was impossible.

Example 2-2.
Next, a continuous fermentation test was similarly performed using the bioreactor 1 described in the above embodiment. That is, the bioreactor 1 having the shape shown in FIG. 1 (specifications: the lower part 2a has a capacity of about 74 ml, the intermediate part 2b has an inner diameter (diameter) of about 45 mm and a capacity of about 286 ml, the upper part 2c has a capacity of about 270 ml and a total volume of 630 ml. 7 has an inner diameter (diameter) of 70 mm, a height difference H of the lower end portion of each wall 7 and the lower end portion 6a of the outlet 6 is about 20 mm, and the gap width is about 10 mm. Was not provided.
And when circulating fermentation and continuous fermentation were performed similarly to the said Example 2-1, using this, separation with a fermented liquor, a non-aggregated microbial cell, and a microbial cell lump is performed rapidly, and the outlet 6 No obstruction occurred.
In addition, although the non-aggregating strain is discharged together with the fermentation broth, the bacterial mass is retained in the reactor and the growth of the aggregate is observed, so the lack of stability of the fused strain is a problem did not become.

  The result of Example 2-2 is shown in FIG. Alcohol productivity gradually improves with the growth of the cell mass after the start of fermentation, and after 20 days, about 1.4 g / liter · hr or more (1.4 g or more alcohol per hour by 1 liter reactor reaction tank) Means that will be produced). This shows that stable continuous fermentation is possible without using an immobilization support. The ethanol concentration at this time was 3.8% (W / V), and the ethanol conversion rate was 74%.

Example 2-3.
Furthermore, the continuous fermentation test was conducted by increasing the cell density.
That is, as the cells, only about 30 ml of the bulk capacity obtained in Example 2-1 was taken out, and cyclic fermentation and continuous fermentation were performed in the same manner as Example 2-2 except that this was used. . The result is shown in FIG. As a result, it was possible to improve the ethanol concentration to 4.1% and the conversion rate to 80%.

Example 2-4.
Further, only about 60 ml of the bulk capacity obtained in Example 2-1 was taken out, and between the lower part 2a and the intermediate part 2b and between the intermediate part 2b and the upper part 2c under the same conditions as in Example 2-3. The influence of the stainless steel mesh was examined using the bioreactor 1 (the one shown in FIG. 1) in which a stainless steel mesh (small hole plate 5) having an opening of 2 mm was interposed in each flange. The result is shown in FIG. By interposing a stainless steel mesh, the product ethanol concentration and alcohol productivity that did not intervene with the stainless steel mesh hardly increased during the fermentation period of 19 days, whereas those with the stainless steel mesh intervened. It turns out that it improves rapidly. This was thought to be due to the increase in cell growth and decrease in the amount of discharged cells by interposing a stainless steel mesh (the amount of aggregated cells after fermentation was The bulk capacity was about 70 ml without intervening mesh, and the intermediary volume was about 120 ml). In addition, the production ethanol concentration and the alcohol production rate were less varied when stainless steel mesh was interposed, and stable fermentation was possible.

Example 3
(Continuous fermentation test of ultra-low-mouthed deep-fried soy sauce by bioreactor)
Example 3-1.
In this embodiment, except that stainless steel mesh (small hole plate 5) having a mesh size of 2 mm is interposed between the flanges between the lower part 2a and the intermediate part 2b and between the intermediate part 2b and the upper part 2c, respectively. The same bioreactor 1 (in FIG. 1) used in Example 2-2 was used.
This bioreactor was charged with 1 liter of raw soy sauce decolorization solution (total nitrogen 1.41%, salt content 17%, straight sugar content 4.5%, alcohol 1.65%, pH 5.1), and Similarly, about 50 ml of the pre-cultured ZR-2f strain was filled as it was without being supported on an immobilization carrier, and circulating fermentation was performed at 30 ° C. for 2 days with an aeration rate of 0.1 vvm. Thereafter, the same-colored soy sauce decolorization solution was supplied to the bioreactor at a flow rate of 30 to 60 ml / hr and continuously fermented so that the ethanol concentration increased by 1%.
The fermentation broth produced over time was sampled, and the alcohol concentration was analyzed by a gas chromatograph (manufactured by Shimadzu Corporation, product name: GC-9A) to measure alcohol productivity. The result is shown in FIG.
Alcohol productivity improved rapidly after the start of fermentation, maintained about 1.1 g / l · hr after 14 days, and stable continuous fermentation was possible without using an immobilized carrier.

Example 3-2.
Next, a test similar to Example 3-1 was performed except that a bioreactor in which the small hole plate 5 was removed from the bioreactor 1 (that is, the same bioreactor as that of Example 2-2) was used. The result is shown in FIG.

Example 3-3.
Example 3-3 was performed by a conventional method. That is, about 400 ml (bulk volume) of an immobilization carrier in which a commercially available yeast strain was supported on a porous ceramic carrier (product name: ceramic carrier) as a biocatalyst in the same bioreactor 1 as in Example 3-1. ) After that, the same test as in Example 3-1 was performed. The results are also shown in FIG.
In the conventional method using this immobilized carrier, the alcohol productivity was about ½ times that of the ZR-2f strain. In addition, the obtained fermented broth showed no sensory difference from a known fermented broth (product name: ultra-thin fried).

Example 4
(Continuous fermentation test of ultra-fresh raw soy sauce by bioreactor)
Using the bioreactor (specification: the lower part has a capacity of 3000 ml, the middle part has a capacity of 3000 ml, the upper part has a capacity of 3000 ml and has a total volume of 9 liters) (product name: MBRB-303, manufactured by Tokyo Rika Kikai Co., Ltd.) Went. In this reactor, stainless steel meshes (small hole plates) having a mesh size of 2 mm were interposed between the flanges between the lower part and the intermediate part and between the intermediate part and the upper part, respectively.
In this bioreactor, about 2000 ml of the ZR-2f strain that had been pre-cultured in the same manner as in Example 1 and conditioned by standing at 30 ° C. for 2 days with fresh soy sauce decolorizing solution (sometimes stirred) was immobilized. The sample was filled as it was without being supported on a carrier, and cultured for 2 days at 30 ° C. with aeration of 9 liters of fresh soy sauce decolorizing solution. Thereafter, the same-colored soy sauce decolorizing solution was supplied to the bioreactor at a flow rate of 9 liters to 27 liters / day, and continuous fermentation was performed for 30 days.
The alcohol concentration was measured over time by an oxidation method, and alcohol productivity was measured. The result is shown in FIG.

  Alcohol productivity improved steadily after the start of fermentation, showing a maximum of 1.4 g / l · hr. Stable continuous fermentation was possible, and a fermented liquid of 3 times the volume of the bioreactor could be obtained in one day. Moreover, functionally, the obtained fermentation broth did not show a difference from that obtained by the conventional method.

Example 5
(Continuous fermentation test of ultra-fresh mouth raising by bioreactor)
A bioreactor 110 having a cylindrical shape as a whole shown in FIG. 12 (specifications: inner diameter (diameter) of about 300 mm, height of about 1500 mm, total volume of 100 liters) (Tokyo Rika Kikai Co., Ltd.) The following test was conducted using a specially manufactured product.
The reactor 120 is provided with a plurality of (six) upper surface opening type mesh rods 30 having openings larger than the small hole plate 5 (a cylinder with a bottom surface having a diameter of 280 mm and a height of 210 mm. 6 The third one from the bottom is 280mm in height). Further, a stainless mesh (small hole plate 5) having a mesh size of 1.5 to 2 mm is provided on the outer side of the bottom surface of the mesh cage 30 shown in the mesh mesh 30. Further, the supply port 3 of the reactor 110 is divided into a liquid supply port 3a to be processed and an air supply port 3b. Further, the outlet 6 is provided above the tube body 120, and similarly, an air exhaust port 31 is separately provided above the tube body 120. In addition, the partition is not provided.
The bioreactor 110 was filled with 100 liters of fresh soy sauce decolorizing solution, and about 70 liters of the ZR-2f strain pre-cultured in the same manner as in Example 4 was filled as it was without being supported on the immobilization carrier, and aerated. The amount was 8.0 liters / min and cultured at 30 ° C. for 2 days. Thereafter, the same-colored soy sauce decolorizing solution was supplied to the bioreactor 110 at a flow rate of 50 liters to 200 liters / day, and continuous fermentation was performed for about 225 days.
As a comparative example, about 70 liters (bulk capacity) of a conventional method (in the same bioreactor 110, a commercially available yeast strain as a biocatalyst supported on a porous ceramic carrier (product name: ceramic carrier). In the same manner, continuous fermentation was performed for about 56 days. In the conventional method, fermentation could not be performed after 56 days.
The alcohol concentration was measured over time by an oxidation method, and alcohol productivity was measured. The result is shown in FIG.

  Alcohol productivity improved steadily after the start of fermentation, and it was possible to obtain a fermented liquor twice the bioreactor capacity in one day. The obtained fermented liquor did not show a difference in functionality from that obtained by the conventional method. In addition, after 3 weeks from the start, the operation was interrupted for about 2 weeks and then restarted. However, the recovery was quicker than the conventional method, and the alcohol productivity improved steadily after the return and stable operation was possible. there were. After that, the operation was interrupted intermittently, but it returned in the same way. Moreover, since it has aggregability, the amount of leaking bacteria was reduced, so the filterability was improved and the workability was improved.

The longitudinal cross-sectional view which shows one Embodiment of the bioreactor of this invention. AA sectional view taken on the line AA of FIG. The reference perspective view which shows the solid-liquid separation part (upper part) of a bioreactor. The graph which shows the sedimentation rate of a test strain. The graph which shows the degree of fermentation in the batch fermentation test of Example 1. The longitudinal cross-sectional view which shows the conventional bioreactor. The graph which shows the fermentation degree in the continuous fermentation test of Example 2-2. The graph which shows the fermentation degree in the continuous fermentation test of Example 2-3. The graph which shows the fermentation degree in the continuous fermentation test of Example 2-4. The graph which shows the fermentation degree in the continuous fermentation test of Example 3. FIG. The graph which shows the fermentation degree in the continuous fermentation test of Example 4. FIG. FIG. 6 is a longitudinal cross-sectional reference diagram showing the bioreactor of the present invention used in Example 5. The graph which shows the fermentation degree in the continuous fermentation test of Example 5. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Bioreactor 2 Tubing body 2a Lower part 2b Middle part 2c Upper part 5 Small hole board 6 Outlet 7 Bulkhead 8 Passing port 11 Crevice

Claims (3)

Water flow generating means for generating a water flow from the lower side to the upper side of the pipe body is provided, and an outlet for taking out the processing liquid treated with the biocatalyst is provided above the pipe body, A bioreactor in which a biocatalyst flows by means to process a liquid to be processed,
A cylindrical partition is provided in the upper interior of the tube with a space from the inner peripheral surface of the tube, and the lower end of the partition extends below the outlet. A characteristic bioreactor.   The bioreactor according to claim 1, wherein a passage port through which a treatment liquid can pass is formed in a peripheral wall of the partition wall at a position not facing the outlet. Water flow generating means for generating a water flow from the lower side to the upper side of the pipe body is provided, and an outlet for taking out the processing liquid treated with the biocatalyst is provided above the pipe body, A bioreactor in which a biocatalyst flows by means to process a liquid to be processed,
A bioreactor characterized in that inside the tube body, small hole plates for partitioning the inside are provided in a multi-stage shape in the vertical direction.

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