JP2013150599A - Fermentation method for coculturing antimicrobial substance-producing microorganism and fermentation microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fermentation method capable of stably producing useful substances over a long period without causing microorganismal effluence with no concern of miscellaneous germs contamination.SOLUTION: The objective fermentation method is capable of stably producing useful substances over a long period by using agglomerates obtained by cocultivation of yeast and bacteriocin-producing lactobacillus without concern miscellaneous germs contamination nor microorganismal effluence.

Description

  The present invention relates to a method for performing fermentation using an aggregate obtained by co-culturing lactic acid bacteria producing bacteriocin as an antibacterial substance-producing bacterium and yeast having resistance to the bacteriocin.

Conventionally, methods for producing useful substances by fermentation of microorganisms such as organic acids such as ethanol and lactic acid, and perfume ingredients such as phenethyl alcohol have been used. As such a fermentation method, a batch method in which raw materials and microorganisms are prepared once, a repeated batch culture method (semi-continuous fermentation method) incorporating a microbial cell collection system, or a raw material for the first prepared microorganisms A continuous fermentation method is known in which fermentation is continuously performed by sequentially adding.
Among them, the repeated batch culture method (semi-continuous fermentation method) and the continuous fermentation method are expected to be industrially used as a method for producing a large amount of useful substances by continuing the fermentation continuously. When fermentation is performed for a long period of time, contamination with various bacteria is likely to occur, and there is a problem that a useful substance cannot be stably obtained in a desired yield. Further, when these fermentation methods are carried out and contamination with germs occurs, there is a risk that the cost for disposal becomes very high because a large amount of waste is generated.
Furthermore, in these fermentation methods, when recovering useful substances, microorganisms also flow out together (wash out), and there is a problem that fermentation cannot continue stably, and there is no concern about contamination with bacteria. Therefore, it has been desired to provide a fermentation method that can be carried out for a long period of time without microbial runoff.

The present inventors have found that biofilms in which these microorganisms are immobilized on a carrier can be obtained by co-culturing yeast and lactic acid bacteria (for example, see Patent Document 1), and this biofilm is useful. It has been confirmed that it can be used in a repeated batch culture method for the purpose of production of other substances (such as ethanol) (see Non-Patent Document 1).
In the fermentation method using this biofilm, since a useful substance can be recovered in a state where the outflow of microorganisms is avoided, fermentation can be performed stably for a long period of time. However, just using such a biofilm could not sufficiently solve the problem of contamination with bacteria.

In addition, simply co-culturing lactic acid bacteria and yeast does not form a biofilm. Instead, a sugar solution is fed into a bioreactor filled with lactic acid bacteria, yeast and ceramic beads (carrier), and a fermentation solution containing ethanol and lactic acid. In this system, since lactic acid bacteria and yeast do not form a biofilm, a fermented liquid is produced. With recovery, lactic acid bacteria and yeast were likely to flow out.
Moreover, in order to suppress the contamination of various bacteria during fermentation, it is necessary to use a heat-sterilized raw material solution or to carry out fermentation under conditions such that the salt concentration is 2 to 16% by weight. However, it could not be said that contamination with bacteria would not occur unless strict management was performed.

Therefore, the present inventors have solved the problem of contamination by various bacteria, and the present invention aims to perform a fermentation method such as a repeated batch culture method (semi-continuous fermentation method) or a continuous fermentation method stably in the long term. Focused on the utilization of lactic acid bacteria that produce bacteriocin, an antibacterial substance.
Conventionally, such lactic acid bacteria have been cultured for the purpose of obtaining bacteriocin, and the bacteriocin produced by this lactic acid bacterium is an antibacterial agent for livestock, a methane production inhibitor or feed composition for ruminants, or a storage property. Have been used for the production of foods and the like with high quality (see, for example, Patent Documents 4 to 7). However, use of lactic acid bacteria that produce bacteriocin to obtain useful substances other than bacteriocin has not been studied.

Japanese Patent No. 4667791 JP-A-8-9936 JP-A-6-189722 JP 2006-169197 A International Publication WO2005 / 045053 Pamphlet Japanese Patent Application Laid-Open No. 2010-200730 JP 2006-166853 A

Abstracts of Annual Meeting of the Biotechnology Society of Japan, page 82, 3P-1098

  An object of the present invention is to provide a fermentation method capable of producing a useful substance stably for a long period of time without causing concern about contamination with microorganisms and without causing the outflow of microorganisms.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have obtained agglutination obtained by co-culturing lactic acid bacteria that produce bacteriocin as antibacterial substance-producing bacteria and yeast that is resistant to the bacteriocin. By using the product, it was found that a fermentation method capable of producing a useful substance stably for a long period of time without causing microbial outflow without concern for contamination by microorganisms was obtained, and the present invention was completed.
In the fermentation method of the present invention, since lactic acid bacteria and yeast form aggregates that maintain a stable flora, there is no possibility that microorganisms will flow out. In addition, since bacteriocin produced by lactic acid bacteria itself suppresses contamination of bacteria, it is necessary to maintain the pH of the fermentation broth or to sterilize the raw material and use it for fermentation. In addition, it is possible to simply continue the repeated batch culture method (semi-continuous fermentation method) and the continuous fermentation method as they are.

That is, the present invention relates to the following fermentation methods (1) to (11), a method for producing ethanol, and the like.
(1) A fermentation method using an aggregate obtained by co-culturing lactic acid bacteria that produce bacteriocin and yeast.
(2) The fermentation method according to (1) above, wherein the lactic acid bacterium producing bacteriocin is a lactic acid bacterium that exhibits coaggregation with yeast and forms an aggregate composed of the lactic acid bacterium and yeast when co-cultured with yeast.
(3) The fermentation method according to (1) or (2) above, wherein the aggregate is a biofilm or an aggregate.
(4) The fermentation method according to any one of (1) to (3), wherein the lactic acid bacterium producing bacteriocin is a lactic acid bacterium belonging to Lactobacillus plantarum.
(5) The fermentation method according to any one of (1) to (4) above, wherein the lactic acid bacterium producing bacteriocin is Lactobacillus plantarum HM23 strain (reception number NITE AP-1168).
(6) The fermentation method according to any one of (1) to (5) above, wherein the yeast is a yeast belonging to Saccharomyces cerevisiae.
(7) The fermentation method according to any one of (3) to (6), wherein a carrier is used in forming the biofilm.
(8) The fermentation method according to (7), wherein the carrier is a carrier containing glass or cellulose.
(9) The fermentation method according to any one of the above (1) to (8), which is performed by a repeated batch culture method or a continuous fermentation method.
(10) A method for producing ethanol by the fermentation method according to any one of (1) to (9) above.
(11) Lactic acid bacteria deposited as receipt number NITE AP-1168 (HM23).

  In the present invention, since fermentation can proceed in the state where bacteriocin, an antibacterial substance, is produced, it is possible to reduce ethanol contamination in a long term and in a large amount while suppressing contamination by repeated batch culture methods or continuous fermentation methods. Useful substances such as alcohols, organic acids such as lactic acid, and perfume ingredients such as furanones can be produced. In addition, since fermentation is carried out in a state where bacteriocin is produced and contamination with bacteria can be sufficiently suppressed, sterilization treatment of materials for fermentation can be reduced, and burdens such as cost and time can be reduced for a long time. It can also be reduced to

It is the figure which showed the outline | summary of the apparatus for continuous ethanol fermentation using a bifilm reactor (Example 1). It is the figure which showed the biofilm formation ability of the Lactobacillus plantarum HM23 strain | stump | stock (reception number NITE AP-1168) (it may be hereafter described as HM23 strain | stump | stock) (Test Example 3).

The “fermentation method” of the present invention may be any method as long as it is carried out under the condition that “use an aggregate obtained by co-culturing a lactic acid bacterium producing bacteriocin and yeast”, and includes other methods. May be.
The “aggregate” of the present invention refers to an “aggregate” or “biofilm” formed by lactic acid bacteria and yeast.
An “aggregate” is an aggregate formed by solids adhering (attaching) to each other by some kind of interaction, and here, it is formed by adhesion between different types of cells, yeast and lactic acid bacteria. A “biofilm” is a cell aggregate formed at an interface such as a solid-liquid interface or a gas-liquid interface, and an “aggregate” formed by adhesion between cells of yeast and lactic acid bacteria is a carrier such as a solid. It is formed on and attached to the surface or inside of the.
Specific “fermentation method” of the present invention includes “repetitive batch culture method” or “continuous fermentation method”.
In a normal fermentation method, inoculate and inoculate a culture medium sterilized in a culture tank, collect the fermentation liquid in the fermentation tank outside the culture tank after culturing, and then wash and ferment the fermentation tank. However, the batch culture method in which the inoculum is inoculated again and cultured is mainly performed.
In contrast, in the “repeated batch culture method”, first, 1) culture is started in the same manner as in the normal batch culture method, and after completion of the culture, the fermentation broth is discharged outside the culture tank and collected. Next, 2) without rinsing and sterilizing the culture tank from which the fermentation broth has been drawn, a new medium is filled, and the culture is started again without inoculating a new inoculum. 3) The operations 1) and 2) are repeated.
In the “repetitive batch culture method” performed by the procedure of 1) to 3), in order to omit the work of inoculating the inoculum, the fermentation broth containing the bacteria is partially left in the fermenter as an inoculum for the next culture. Method is used. However, in the fermentation method according to the “repetitive batch culture method” of the present invention, even if the entire fermentation broth is discharged outside the culture vessel and recovered, “lactic acid bacteria producing bacteriocin” and “yeast” remain on the aggregates. Since it is maintained and retained in the fermenter, it is not necessary to inoculate a new inoculum. Moreover, in the “repeated batch culture method” of the present invention, it is not necessary to leave the fermentation solution in the tank, so that the fermentation can be performed efficiently.
Furthermore, in the “continuous fermentation method”, a sterilized medium is continuously supplied to “bacteriocin-producing lactic acid bacteria and yeast” maintained on the aggregate, and in parallel with this, “lactic acid bacteria producing bacteriocin”. It is possible to continuously recover the fermented liquid obtained by fermenting the medium with “Yeast and Yeast”, and the fermentation can be performed more efficiently than the “repetitive batch culture method”.

Of the “fermentation method” of the present invention, an outline of the “continuous fermentation method” is shown as arrows (1) to (7) in FIG. 1 as an example. In this schematic diagram, “biofilm” is used as the aggregate. In (1), the raw material is stored in the raw material storage tank, and in (2) to (4), the raw material is used from the raw material storage tank through a tube pump and used in the present invention. The raw material fermented liquid fermented by the biofilm reactor used in the present invention in (5) to (6) flows out of the biofilm reactor and is stored in the fermented liquid storage liquid. And in (7), it consists of the inflow path | route of a raw material and the outflow path | route of a fermented liquid of collect | recovering the fermented liquid stored in the fermented liquid storage liquid.
When performing the “continuous fermentation method” of the present invention in the form shown in FIG. 1 and recovering the target substance contained in the fermentation broth, the concentration of the target substance contained in the fermentation liquid recovered from the biofilm reactor It is preferable to inject the raw material so as not to decrease. The higher the raw material injection rate, the greater the amount of the target product obtained. Therefore, it is particularly preferable to adjust the raw material injection rate within a range in which the maximum concentration of the target material can be maintained. The same applies when “aggregates” are used as the aggregates.

In FIG. 1, the raw material and the fermentation broth are shown in a form that flows through a tube or the like, but the “continuous fermentation method” of the present invention is not necessarily required, for example, in a state in which the raw material is added. Any form that can continuously supply the raw material to the biofilm and recover the obtained fermented liquid, such as changing the raw material storage tank or the fermented liquid storage for recovery as required It may be.
As a device for performing such a “continuous fermentation method” of the present invention, any conventionally known device can be used. Moreover, you may use the apparatus etc. which were produced uniquely and the biofilm does not necessarily need to be produced using the reactor.
In addition, when “aggregate” is used as the aggregate, any conventionally known apparatus can be used as well, but it is preferable to use an apparatus that does not break the aggregate when performing the “continuous fermentation method”. Even equipment that does not have a function such as stirring, or equipment that has a function such as stirring, use equipment that has been adjusted so as to minimize the collapse caused by the mechanical force of the “aggregates” due to stirring. It is preferable.

Among the “aggregates” of the present invention, a “biofilm” is a cell aggregate formed at an interface such as a solid-liquid interface or a gas-liquid interface, and is formed by adhesion between lactic acid bacteria and yeast cells. “Agglomerates” refers to those in the form of adhering to the surface or inside of a solid carrier.
This “biofilm” can be formed by co-culturing under conditions that allow both lactic acid bacteria and yeast to grow stably in a situation that includes a carrier. As the “carrier” used herein, any “carrier” that can form the “biofilm” of the present invention can be used. A “carrier” containing a porous substance that has as large a surface area as possible to which microorganisms can adhere and does not cause clogging is preferable. Examples of such substances include glass, cellulose, latex, zeolite, ceramic, sintered glass, and the like, and “carriers” containing these include glass beads, cellulose beads, latex beads, zeolites, ceramic beads, Sintered glass, rice bran, bagasse, wood chips and the like can also be used as the “carrier” of the present invention. Also, a microorganism-immobilized carrier for water treatment such as pumice, activated carbon, urethane foam or urethane sponge can be used as the “carrier” of the present invention. A plurality of these “carriers” may be used in combination to form a biofilm, such as glass beads and cellulose beads.
The shape of the “carrier” may be any shape, and examples thereof include a bead shape, a fiber shape, a thin plate shape, and a film shape. The size of the “carrier” can be adjusted according to the scale for carrying out the “continuous fermentation method” of the present invention. For example, glass beads having a diameter of 1 mm to 100 mm can be mentioned. In addition, when using Inawara or the like as a “carrier”, it may be used as it is. However, it may be cut to an appropriate length, for example, about 5 mm, and may be sterilized. May be used.

The “biofilm” may be formed by any conventionally known method. For example, a material such as a stainless steel container, a plastic container, a glass container, or the like is added to the container. A biofilm can be formed on a carrier by adding a culture solution containing “lactic acid bacteria belonging to Lactobacillus plantarum” and a culture solution containing “yeast” that can form aggregates and co-culturing for a certain period of time. .
The co-culture for a certain period of time here may be a time when a biofilm can be formed in a temperature range in which “bacteriocin-producing lactic acid bacteria” and “yeast” capable of forming aggregates can be grown. For example, co-culture is performed at -35 degrees for 16 to 48 hours.
Further, a carrier added to a container such as a metal such as stainless steel, plastic or glass may be appropriately added according to the type of carrier used. For example, when using glass beads, cellulose beads or the like as a carrier. In order to make the best use of the capacity of the container, it is preferable that the container is filled to the full extent.

Among the “aggregates” of the present invention, the “aggregates” are aggregates formed by solids adhering (adhering) to each other by some kind of interaction, and here, between different types of cells such as yeast and lactic acid bacteria. The one formed by bonding.
“Aggregates” can be formed by co-culturing lactic acid bacteria and yeast capable of forming aggregates under conditions where both lactic acid bacteria and yeast can stably grow.
This co-culture may be performed for a period of time during which aggregates can be formed in a temperature range in which both “lactic acid bacteria belonging to Lactobacillus plantarum” and “yeast” can grow. For example, co-culture for 48 hours may be mentioned.
For co-cultivation, it is only necessary to mix the bacterial solution of lactic acid bacteria and yeast, or to culture the bacterial solution containing yeast and lactic acid bacteria as they are, and co-culture while gently stirring these bacterial solutions as necessary. Then, the opportunity of contact between cells can be increased. The stirring may be performed under conditions suitable for forming “aggregates” depending on the apparatus to be used.

As the “lactic acid bacterium producing bacteriocin” of the present invention, any lactic acid bacterium can be used as long as it is a “lactic acid bacterium producing bacteriocin” capable of forming an aggregate.
Furthermore, the “lactic acid bacterium producing bacteriocin” of the present invention is preferably a “lactic acid bacterium producing bacteriocin” that exhibits coaggregation with yeast. Here, “coaggregation with yeast” refers to the property that when coexisting with yeast, cells of “lactic acid bacteria producing bacteriocin” and yeast cells adhere to each other to form an aggregate. Say. Specifically, a cell suspension containing “bacteriocin-producing lactic acid bacteria” and yeast cultured overnight (12 to 16 hours) is mixed in a medium and allowed to stand, and then visually within 30 minutes. When the formation of aggregates is observed, such a “lactic acid bacterium producing bacteriocin” can be said to be a “lactic acid bacterium producing bacteriocin” that exhibits coaggregation with yeast.

One such “lactic acid bacterium producing bacteriocin” of the present invention is a lactic acid bacterium belonging to Lactobacillus plantarum. More specifically, it is a strain isolated by the present inventors from sushi, and in the independent microorganisms product evaluation technology base mechanism patent microorganism deposit center (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan) Examples include Lactobacillus plantarum HM23 strain (reception date: November 29, 2011) which has received a receipt number as NITE AP-1168.
This “lactic acid bacteria producing bacteriocin” that can be used in the fermentation method of the present invention is represented by 366 partial nucleotide sequences of 16S rDNA shown in SEQ ID NO: 1 (derived from HM23 strain) and SEQ ID NO: 2 in SEQ ID NO: 2. It is preferable that it has 150 partial base sequences (derived from HM23 strain) of 16S rDNA.
Furthermore, the “lactic acid bacterium producing bacteriocin” of the present invention is a “lactic acid bacterium producing bacteriocin” capable of forming an aggregate consisting of a lactic acid bacterium producing bacteriocin and yeast. In the 16S rDNA base sequence, It may include a partial base sequence shown in SEQ ID NO: 1 and a partial base sequence having high homology (identity) with the partial base sequence shown in SEQ ID NO: 2.
Here, “high homology (identity)” usually means 97% or more homology, preferably 99% or more homology, and most preferably 100% homology. Say.
Such a “lactic acid bacterium that produces bacteriocin” indicating the base sequence of 16S rDNA may be a strain closely related to the Lactobacillus plantarum HM23 strain, or may be a strain other than the related strain. .

In addition, the “yeast” of the present invention is not limited to a yeast having an aggregating property, as long as it is a “yeast” capable of forming an aggregate by co-culture with lactic acid bacteria. The yeast can also be used. Examples of such yeast include Saccharomyces cerevisiae BY4741 strain or Saccharomyces cerevisiae NBRC0282 strain.
Any strain may be combined as long as it is a “lactic acid producing bacteriocin” and “yeast” capable of forming an aggregate. In addition, for example, when ethanol is used as the target product recovered from the fermentation broth by the “fermentation method” of the present invention, it is excellent in ethanol production ability, and it is suitable for “yeast” and high-concentration ethanol. It is preferable to use in combination “resistant bacteriocin-producing lactic acid bacteria”.

In the “fermentation method” of the present invention, any material can be used as long as the desired product can be recovered from the fermentation broth.
When the target product to be recovered from the fermentation broth is, for example, ethanol, examples thereof include raw materials containing sugar, such as a saccharified solution of starch and cellulose, and a medium or fruit juice mainly composed of a solution containing biomass-derived sugar, Wastes such as vegetable scraps, seaweeds and the like obtained by saccharification using acids and enzymes, and the like can also be used as raw materials. These raw materials may be unsterilized or sterilized.
The raw material used in the “fermentation method” of the present invention is preferably in contact with the aggregate in an aqueous solution state. Such an aqueous solution-like raw material is preferably used after adjusting to pH 5 or lower in order to improve the storage stability after preparation, whether it is sterilized or unsterilized.
In addition, the raw materials are preferably prepared and used at appropriate intervals, for example, once every day or every other day, and it is not preferable to make a large amount.
When the “continuous fermentation method” is performed using “aggregates” as aggregates, it is preferable to stir to the extent that “aggregates” do not settle in order to avoid a decrease in fermentation efficiency due to sedimentation of “aggregates”. . As a stirring method, any method such as stirring using stirring blades, stirring using the gas lift effect by generated carbon dioxide may be used, but in the apparatus used in “continuous fermentation method”, “aggregates” are allowed to settle. In addition, it is preferable to perform stirring within a range of conditions that do not cause destruction.

  EXAMPLES Examples and test examples are shown below, and the present invention is further described in detail. However, the present invention is not limited to these descriptions.

Isolation and Identification of Lactic Acid Bacteria Producing Bacteriocin Numerous lactic acid bacteria were isolated and screened using a new source of strawberry. As a result, the strain HM23 was obtained as a lactic acid bacterium having the ability to form aggregates in co-culture with yeast and producing bacteriocin (antibacterial substance).
As a result of the identification test, HM23 strain was Gram-positive lactobacilli, catalase-negative, homolactic fermentation, and peptidoglycan type was DAP type.
Moreover, as a result of analyzing the base sequence of 16S rDNA, it had the partial base sequence shown in SEQ ID NO: 1 in the sequence listing and the partial base sequence shown in SEQ ID NO: 2 in the sequence listing. From these results, the HM23 strain isolated by the present invention was identified as a strain belonging to Lactobacillus plantarum.
The present inventors deposited this Lactobacillus plantarum HM23 strain with the National Institute for Product Evaluation Technology Patent Microorganisms Deposit Center (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba, Japan). -Received the receipt number as 1168 (Reception date: November 29, 2011).

[Test Example 1]
Antibacterial activity of Lactobacillus plantarum HM23 strain Sample 1) Medium (1) MRS liquid medium (manufactured by DIFCO)
Proteose peptone no. 3, 10.0 g: cattle extract, 10.0 g: yeast extract, 5.0 g: dextrose, 20.0 g: polysorbate 80, 1.0 g: ammonium citrate, 2.0 g: sodium acetate, 5.0 g: magnesium sulfate , 0.1 g: Manganese sulfate, 0.05 g: Dipotassium phosphate, 2.0 g: (in 1 L)
(2) Tomato juice agar (made by DIFCO)
Agar plate (3) Lactobacillus agar AOAC medium prepared by dissolving 20 g of tomato juice, 10 g of peptone, 10 g of milk peptone, and 11 g of agar in a pH 6.5 MES buffer (manufactured by Dojin Chemical) at 0.06 molar concentration and sterilizing by autoclave. (Made by DIFCO)
Milk peptone 15 g / l, yeast extract 5 g / l, dextrose 10 g / l, tomato juice 5 g, 1 potassium phosphate 2 g, polysorbate 80 1 g, agar 10 g
2) Microorganisms (1) Lactic acid bacteria Lactobacillus plantarum HM23 strain and Pediococcus pentosaseus JCM5883 strain (obtained from Microorganism Materials Development Office, RIKEN Bioresource Center) as test bacteria Using.
(2) Yeast Saccharomyces cerevisiae BY4741 strain (obtained from National BioResource Project (NBRP)) was used.

2. Evaluation of antibacterial activity 1) A culture solution (5 μl) obtained by pre-culturing Lactobacillus plantarum HM23 strain in MRS liquid medium at 30 ° C. overnight was inoculated into tomato juice agar medium and cultured at 30 ° C. for 20 hours. Thus, a culture plate A was prepared.
2) After sterilization of Lactobacillus agar AOAC medium to about 50 ° C after autoclaving, inoculate 1/100 volume of fermentation broth precultured Pediococcus pentosaceus JCM5883 strain at 30 ° C overnight in MRS liquid medium The culture plate A prepared in 1) above was immediately overlaid.
3) The culture plate A prepared in 2) above was cultured at 30 ° C. for 20 hours, and formation of a blocking circle was observed.
In the preparation of the culture plate A, an agar plate containing 200 u / ml protease (Umamizyme manufactured by Amano Enzyme) was prepared, and it was examined whether or not a blocking circle was formed in the presence of the protease, so that the strain HM23 It was also examined whether or not the bacteriocin produced by the product was degraded by proteases.

3. Results As shown in Table 1, the Lactobacillus plantarum HM23 strain formed a blocking circle against Pediococcus pentosaceus JCM5885 strain and showed antibacterial activity, but it was antibacterial against Saccharomyces cerevisiae BY4741 strain. It did not show sex.
In addition, a similar antibacterial substance was produced against Pediococcus pentosaceus JCM5885 during co-culture with Saccharomyces cerevisiae BY4741 strain.
In addition, since the inhibition circle | round | yen by HM23 strain | stump | stock was not formed in the presence of protease, it was clearly shown that the antibacterial substance produced by HM23 strain | stump | stock is a bacteriocin decomposed | disassembled by protease.

[Test Example 2]
Evaluation of coaggregation with yeast of lactic acid bacteria producing bacteriocin Sample 1) Medium (1) MRS liquid medium (manufactured by DIFCO)
(2) YPD medium It was prepared to be 1% yeast extract, 2% polypeptone, 2% glucose (pH 5.8), and sterilized in an autoclave at 120 ° C. for 20 minutes as necessary.
2) Microorganism (1) Lactic acid bacteria Lactobacillus plantarum HM23 strain was used.
In addition, for comparison, Lactobacillus plantarum NCIMB8826 strain (NCIMB: Avagene AB21 9YA, obtained from Scotland), Lactobacillus casei subsissis Ramnosus NBRC3831 strain (obtained from National Institute of Biotechnology, National Institute of Technology and Evaluation) Was used.
Each of these bacteria was statically cultured overnight in 10 ml of MRS liquid medium, and the cells were collected by centrifugation. Then, after washing twice with YPD medium, each was suspended in 10 ml of YPD medium to prepare a lactic acid bacteria suspension.
(2) Yeast Saccharomyces cerevisiae BY4741 strain (obtained from National BioResource Project (NBRP)) was used. This was statically cultured overnight in 10 ml of YPD medium, and the cells were collected by centrifugation. Then, after washing twice with YPD medium, it was suspended in 10 ml of YPD medium to prepare a yeast suspension.
3) Evaluation of coaggregation property After mixing 1.0 ml of the lactic acid bacteria suspension prepared in 2) above and 1.0 ml of the yeast suspension in a mini petri dish (40 mmφ), the mixture is allowed to stand at room temperature. After 30 minutes, the presence or absence of aggregate formation was visually observed, and the degree of aggregate formation was evaluated.
Measured using 1.0 ml of YPD medium mixed with yeast suspension instead of lactic acid bacteria suspension and one mixed with 1.0 ml of YPD medium instead of yeast suspension as controls did.
As a result, as shown in Table 2, the HM23 strain showed strong aggregability with the yeast (BY4741 strain), and formed an aggregate that could be visually observed within 30 minutes after mixing the suspension. In contrast, no clumps were observed in the NCIMB8826 strain and the NBRC3831 strain.

<Creation of biofilm reactor>
1. Sample 1) Medium (1) YPD medium A sample prepared in the same manner as in Test Example 2 was used.
(2) YPD medium (glucose 10%)
It was prepared to be 1% yeast extract, 2% polypeptone, and 10% glucose (pH 5.8), and sterilized in an autoclave at 120 ° C. for 20 minutes as necessary.
2) Microorganism (1) Yeast Saccharomyces cerevisiae BY4741 strain (obtained from National BioResource Project (NBRP)) was used.
(2) Lactic acid bacteria Lactobacillus plantarum HM23 strain was used.
3) Carrier (1) Cellulose beads (4 mmφ): Viscopearl A (AH4050L) (manufactured by Rengo Co., Ltd.)

2. Preparation of biofilm reactor (A) A biofilm reactor (A) was prepared according to the following procedures 1) to 3) using the above sample, Lactobacillus plantarum HM23 strain as lactic acid bacteria, and Saccharomyces cerevisiae BY4741 strain as yeast.
1) Cellulose beads were filled in a 50 mL column reactor prepared using a plastic syringe, 20 mL of unsterilized YPD medium was added, and sterilized at 120 ° C. for 20 minutes in an autoclave.
2) A culture solution of Lactobacillus plantarum HM23 strain and a culture solution of yeast Saccharomyces cerevisiae BY4741 strain, which were cultivated by sterilization in a sterilized YPD medium for 24 hours, in a column reactor sterilized in 1) above, 2 mL each was inoculated.
3) The biofilm was formed on each support | carrier by culturing said 2) at 30 degreeC for 24 hours, and the biofilm reactor (A) was obtained.

[Test Example 3]
Confirmation of biofilm-forming ability of Lactobacillus plantarum HM23 strain Using Lactobacillus plantarum HM23 strain as lactic acid bacteria and Saccharomyces cerevisiae BY4741 strain as yeast, Lactobacillus plantarum HM23 strain according to the following procedures 1) to 4) The biofilm forming ability of the HM23 strain by co-culture with yeast was examined. As a comparison, biofilm-forming ability was tested using only Lactobacillus plantarum HM23 strain or yeast alone.
1) Yeast was precultured at 27 ° C. for 24 hours using a YPD medium prepared in the same manner as in Test Example 2.
The Lactobacillus plantarum HM23 strain was pre-cultured at 27 ° C. for 24 hours using MRS medium (manufactured by DIFCO).
2) Each of the precultured bacterial solutions obtained in 1) above was inoculated into a YPD medium and dispensed into a 96-well titer plate at 100 μL per well. When inoculating only one strain, the precultured bacterial solution was inoculated in a one-hundredth amount of YPD medium, and in the case of inoculating two strains, a one-200th amount of YPD medium was inoculated per strain.
3) After inoculation with the precultured bacterial solution, static culture was performed at 30 ° C. for 24 hours.
4) After the above 3), the biofilm was stained by a crystal violet (0.1%) staining method. The dye was eluted from the stained biofilm with 200 μl of 70% (v / v) ethanol solution, and the absorbance at 590 nm of the eluted dye was measured and evaluated as the amount of biofilm formation.

  As a result, as shown in FIG. 2, it was confirmed that the Lactobacillus plantarum HM23 strain (FIG. 2, HM complex) exhibits high biofilm-forming ability by co-culture with yeast. In FIG. 2, Lactobacillus plantarum HM23 strain alone is shown as HM alone, and yeast alone is shown as yeast alone.

Ethanol production by fermentation (repeated batch culture) (1)
In the biofilm reactor (A) prepared in Example 2, ethanol was produced by the repeated batch culture method according to the following procedures 1) to 3).
1) The cultured fermentation broth is removed from the biofilm reactor (A) prepared in Example 2, and replaced with fresh YPD medium (glucose 10%) or YPD medium (glucose 20%) prepared in the same manner as in Example 2. And it culture | cultivated at 30 degreeC for 24 hours.
The YPD medium (glucose 20%) is prepared to be 1% yeast extract, 2% polypeptone, 20% glucose (pH 5.8), and sterilized in an autoclave at 120 ° C. for 20 minutes as necessary. Was prepared.
2) The fermentation broth after the culture in 1) above was collected, replaced with fresh YPD medium (glucose 10% or 20%), and cultured at 30 ° C. for 24 hours.
3) The above 2) was repeated and a repeated batch culture method was performed so that the total number of cultures was 10.

Measurement of ethanol production In the above, when all the cultures were completed, the ethanol concentration in the fermentation broth extracted from each biofilm reactor (A) every 24 hours was measured using an alcohol concentration meter “Alcomate AL-3” (stock) Analyzed by the company Woodson Riken Keiki Co., Ltd.).

  Tables 3 and 4 show the ethanol production (w / v%) when the biofilm reactor (A) of the present invention was used. As a result, when the biofilm reactor (A) of the present invention is used, it can be confirmed that ethanol can be stably produced in spite of the repeated batch culture of 10 times in any YPD medium. It was.

[Test Example 4]
Measurement of Viable Lactic Acid Bacteria and Yeast Yeast in Fermentation Method (Repeated Batch Culture Method) The change in the number of viable bacteria in the fermentation method (repeated batch culture method) of Example 3 was analyzed.
YPD medium (prepared in the same manner as in Test Example 2) supplemented with chloramphenicol 100 μg / ml and sodium propionate 10 μg / ml was used to measure the number of viable bacteria in yeast. MRS liquid medium (manufactured by DIFCO) supplemented with 10 μg / ml sodium azide and 10 μg / ml cycloheximide was used. The viable cell count was shown as the viable cell count per cellulose bead.
As a result, as shown in Table 5, when YPD medium (glucose 10%) is used as a raw material, yeast maintains 10 ⁷ or more per bead and 10 ⁶ or more lactic acid bacteria from the 2nd to 10th days of fermentation. The stability of the flora was confirmed. In addition, when YPD medium (glucose 20%) was used as the raw material, the yeast on the second day was less, but it was stable at 10 ⁶ or more after the fifth day, and 10 ^{6 for} both yeast and lactic acid bacteria on the 10th day. The above values were shown.
Therefore, from this result, it was clarified that the biofilm produced by combining HM23 strain and yeast has stable bacteria and is suitable for the fermentation method (repetitive batch culture method) of the present invention.

Ethanol production by fermentation (repeated batch culture) (2)
Biofilm reactor (A) was prepared using BY4741 strain and HM23 strain in the same manner as in Example 2, and YPD medium (glucose 10%), YPD medium (glucose 15%) prepared in the same manner as in Example 2 or Using YPD medium (glucose 20%) prepared in the same manner as in Example 3, a long-term repeated batch culture method over 20 days was performed.
The YPD medium (glucose 15%) is prepared to be 1% yeast extract, 2% polypeptone, 15% glucose (pH 5.8), and sterilized in an autoclave at 120 ° C. for 20 minutes as necessary. Was prepared.

As a result, as shown in Table 6, even when any YPD medium was used, it was confirmed that fermentation was performed extremely stably after the fourth day and ethanol could be produced.
TSB plate medium (casein peptone 17 g / l, soy peptone 3 g / l, sodium chloride 5 g / l, dipotassium phosphate 2.5 g / l, glucose 2.5 g / l, agar 15 g / l, pH 7.3) When the bacterial flora in the fermented liquid after 20 days was examined, no bacteria other than yeast and lactic acid bacteria were found.
Therefore, from these results, it was confirmed that the biofilm reactor using lactic acid bacteria producing bacteriocin such as HM23 strain is excellent in stability in fermentation and bacteria elimination ability and high in usefulness.

Ethanol production by fermentation method (continuous culture method) Preparation of Biofilm Reactor (B) Using a 670 mL glass container as a column reactor container, Lactobacillus plantarum HM23 as a lactic acid bacterium, and Saccharomyces cerevisiae NBRC0282 as a yeast (Biotechnology Center, National Institute of Biotechnology, Biotechnology Center) The biofilm reactor (B) was prepared according to the following procedures 1) to 3).
1) Cellulose beads (4 mmφ) were filled in a glass container having a total volume of 670 mL, and sterilized in an autoclave at 120 ° C. for 20 minutes.
2) Saccharomyces cerevisiae NBRC0282 strain or Lactobacillus plantarum HM23 strain was inoculated into 500 mL of sterilized YPD medium (prepared in the same manner as in Test Example 2), and each was statically cultured for 24 hours.
3) After inoculating 5 mL each of the cultured medium obtained in 2) above, the mixture was injected into the column reactor prepared in 1) above.
4) After filling the column reactor of 3) above with 350 mL of sterilized YPD medium, a biofilm was formed on the cellulose beads by culturing at 30 ° C. for 24 hours to obtain a biofilm reactor (B).

2. Ethanol production by fermentation method (continuous fermentation method) Using the biofilm reactor (B) prepared in step 1, ethanol was produced according to the following procedure.
That is, YPD medium (glucose 10%) prepared and sterilized in the same manner as in Example 2 was continuously injected into the biofilm reactor (B) at a rate of 18 mL / h using a tube pump. The fermentation broth was withdrawn from the biofilm reactor (B).
In addition, as a comparative example 1, a yeast single biofilm reactor prepared by inoculating only the culture solution of Saccharomyces cerevisiae NBRC0282 strain by the same procedure as in the above 1, using the same method as above, by a continuous fermentation method Ethanol production was also attempted.

3. Measurement of ethanol production amount 2. The ethanol concentration in the fermentation broth extracted from each biofilm reactor every 24 hours was analyzed with an alcohol concentration meter “Alcomate AL-3” (manufactured by Woodson Riken Keiki Co., Ltd.).
As a result, as shown in Table 7, when the biofilm reactor (B) was used, the ethanol concentration reached about 3.6% (w / v) on the 5th day of culture, and 12 days from the 5th day. It was confirmed that about 4% (w / v) ethanol was continuously and stably produced until the day.
Moreover, when the pH of the extracted fermentation broth was measured, it was maintained in the range of pH 4 or lower in all of the period from the 2nd day to the 12th day of continuous fermentation without artificially adjusting the pH. I understood that. Furthermore, when the bacterial flora in the fermentation broth after 12 days was analyzed, no contamination was observed in any of the carriers.
On the other hand, in the case of using the yeast-only biofilm reactor, the ethanol concentration reached 3.5% (w / v) on the fifth day from the start of continuous fermentation, but the ethanol concentration was higher than that in the case of using the biofilm reactor (B). Concentration was low, and contamination of various germs occurred after the 8th day, and continuous fermentation had to be stopped.
Therefore, from these results, it was confirmed that the biofilm reactor using lactic acid bacteria producing bacteriocin such as HM23 strain is excellent in stability in fermentation and bacteria elimination ability and high in usefulness. Moreover, it was shown that ethanol can be stably produced for a long period of time by a continuous fermentation method by combining lactic acid bacteria that produce bacteriocin and yeast.
An outline of an apparatus for continuous ethanol fermentation using the bifilm reactor of the present invention is shown in FIG.

In FIG. Is a raw material storage tank, b. Is a tube pump, c. A biofilm reactor for use in the present invention, and d. Indicates a fermentation broth storage solution. Moreover, the arrows of (1) to (7) indicate the following raw material inflow path and fermentation broth outflow path.
(1) The raw material is a. Stored in raw material storage tank.
(2) to (4) a. From raw material storage tank b. Via tube pump c. It flows into the biofilm reactor used in the present invention.
(5) to (6) c. The raw material fermented liquid fermented by the biofilm reactor used in the present invention flows out of the biofilm reactor, d. Stored in fermentation broth storage.
(7) d. Fermentation liquid is collected from the fermentation liquid storage.

Production of ethanol (beer) by continuous fermentation using wort as raw material Stable ethanol by continuous fermentation using wort (sugar concentration 11.5% (w / v)) as a feed medium as raw material We examined whether it is possible to produce (beer). The raw wort was prepared by dissolving a commercially available malt extract (a malt extract of a handmade beer kit purchased from Advanced Brewing). The wort was prepared, heated at 100 ° C. for 3 minutes, cooled at room temperature, transferred to a medium storage tank without aseptic operation, and used for continuous fermentation. The feed medium was prepared once a day.

Example 2, 2. Was continuously injected at a rate of 10 mL / h into a biofilm reactor using the Lactobacillus plantarum HM23 strain and Saccharomyces cerevisiae NBRC0282 strain prepared in the same manner as above, and the fermentation solution from the biofilm reactor at the same rate. Pulled out. The supply side medium reservoir was kept at room temperature. The continuous fermentation method was performed for 7 days, and the ethanol concentration in the drawing solution was measured in the same manner as in Example 2.
The results are shown in Table 8. Even when wort was used as a raw material, it was confirmed that ethanol could be produced continuously and stably. Moreover, the pH of the culture solution after culturing obtained by the continuous fermentation method was maintained between pH 3 and 5 throughout the entire operation period.

Production of Ethanol by Biofilm Reactor (C) Using Inawara as Carrier A biofilm reactor was prepared in the same manner as in Example 2 using Saccharomyces cerevisiae BY4741 strain and Lactobacillus plantarum HM23 strain as microorganisms.
Specifically, in the same manner as in Examples 2 and 2.1), a 50 mL column reactor made by using a plastic syringe was cut into a length of about 5 mm instead of cellulose beads. Sterilized), 20 mL of unsterilized YPD medium was added, and sterilized in an autoclave at 120 ° C. for 20 minutes, and then a biofilm reactor (C) was obtained by the procedures of Examples 2, 2.2) and 3). .

By using this biofilm reactor (C) and, as a control, a biofilm reactor (A) prepared using cellulose beads as a carrier in the same manner as in Example 2, and using the same fermentation method (repetitive batch culture method) as in Example 3, Ethanol production was performed.
As a result, as shown in Table 9, it was confirmed that ethanol can be produced stably even when Inawara is used as a carrier, similarly to the case where cellulose beads are used as a carrier.
In addition, when the bacterial flora in the fermentation broth 10 days after the start of the culture was analyzed, no contamination of germs was observed in any of the carriers.
From these results, it was confirmed that the biofilm reactor using lactic acid bacteria that produce bacteriocin is excellent in stability in fermentation and the ability to eliminate germs, and is highly useful even when Inawara is used as a carrier.
Therefore, from this result, by utilizing the present invention, ethanol is produced by forming a biofilm using Inawara, which is an agricultural waste that can be easily obtained in a rural area, as a carrier. It was thought that the establishment of biomass-based bioethanol fermentation system would be facilitated.

Ethanol production by biofilm reactor (D) using Inawara as a carrier Sample 1) Microorganism (1) Yeast Saccharomyces cerevisiae BY4741 strain was repeatedly cultured twice at 30 ° C. for 24 hours in a YPD medium (manufactured by DIFCO) and pre-cultured to obtain a seed culture solution.
(2) Lactic acid bacteria Lactobacillus plantarum HM23 strain was subjected to static culture twice at 30 ° C. for 24 hours in MRS medium (manufactured by DIFCO) and pre-cultured to obtain a seed culture solution.

2. Preparation of Biofilm Reactor (D) 2 g of Inawara (unsterilized) cut to a length of about 5 mm was filled in a 50 mL Falcon tube and fixed with a wire mesh.
To this, 20 ml of a sterilized YPD medium inoculated with 1/200 of yeast and lactic acid bacteria seed culture solution was added and co-cultured at 30 ° C. for 24 hours to form a biofilm on the surface of the unsterilized inowara. A biofilm reactor (D) using (unsterilized) as a carrier was obtained.

3. Ethanol production by repeated batch fermentation 1) 2 above. The medium in the biofilm reactor (D) prepared in step 1 was replaced with 20 ml of YPD medium (glucose 10%) prepared in the same manner as in Example 2 and cultured at 30 ° C. for 24 hours.
2) The fermentation broth after the culture in 1) above was collected, replaced with fresh YPD medium (glucose 10% or 20%), and cultured at 30 ° C. for 24 hours.
3) The above 2) was repeated and a repeated batch culture method was performed so that the total number of cultures was 10.

4). Measurement of ethanol production The concentration of produced ethanol was analyzed with an alcohol concentration meter “Alcomate AL-3” (manufactured by Woodson Riken Keiki Co., Ltd.). The fermentation results from the 2nd day to the 10th day after the start of the fermentation test are shown in Table 10. Stable fermentation results were obtained from the 3rd day after the start, and during this time the pH was maintained at 3 or more and 4 or less. Therefore, from this result, it was confirmed that ethanol can be produced stably even when fermentation is performed without using sterilization (non-sterilized) as a carrier.

5. Examination of elimination of various bacteria 3. Take 10 grams of wet straw in the reactor biofilm reactor (D) before the start of the fermentation test for repeated batch fermentation (when not inoculated with yeast or lactic acid bacteria) and on the 10th day of the fermentation test, and add 90 ml of physiological saline. In addition, after sufficiently homogenizing, dilute appropriately, PCA medium (for general live bacteria; peptone 5.0 g, yeast extract 2.5 g, glucose 1.0 g, agar 15.0 g, distilled water 1 L, pH 7.0), Lactic acid bacteria selection medium (MRS + sodium azide 10 mg / L, cycloheximide 10 mg / L + agar 15.0 g, pH 6.5) or yeast selection medium (YPD + sodium propionate 2 g / L + chloramphenicol 100 mg / L + agar 15.0 g, It was smeared at pH 6.5) and the viable cell count was measured.

As a result, as shown in Table 11, 2.5 × 10 ⁵ miscellaneous bacteria were attached to 1 g (wet weight) of non-sterilized rice straw before the start of the fermentation test. It was thought to be a fungus.
On the other hand, after the end of the fermentation test, the number of general viable bacteria per 1 g (wet weight) of Inowara increased to 4.6 × 10 ⁷ , but lactic acid bacteria was 5.1 × 10 ⁷ and yeast was 1.7 × 10 ^7. Since it was measured, it was considered that all the bacteria measured as general viable bacteria were lactic acid bacteria or yeast. This could also be confirmed from the shape of the colonies that appeared on the plate medium.
Therefore, from these results, even in the case where fermentation was carried out without using sterilization (unsterilized) as a carrier, in the co-culture system of yeast and lactic acid bacteria, various bacteria such as Bacillus subtilis originally attached to the larvae. It was confirmed that high yield ethanol fermentation was possible.

<Creation of aggregate>
1. Sample 1) Microorganism (1) Yeast Saccharomyces cerevisiae BY4741 strain was repeatedly cultured twice at 30 ° C. for 24 hours in a YPD medium (manufactured by DIFCO) and pre-cultured to obtain a seed culture solution.
(2) Lactic acid bacteria Lactobacillus plantarum HM23 strain was subjected to static culture twice at 30 ° C. for 24 hours in MRS medium (manufactured by DIFCO) and pre-cultured to obtain a seed culture solution.

2. Creation of aggregates Breakage of aggregates due to agitation by removing all stirring blades of 1L jar fermenter (BMJ-01, manufactured by Able Co., Ltd.) and using only a stirring bar at the bottom to minimize agitation A reactor was used as a reactor.
This was sterilized by adding 500 ml of YPD medium, and then inoculated with 1/200 volume of yeast and lactic acid bacteria seed culture solution at 30 ° C. and 150 rpm for 24 hours while gently stirring to such an extent that these bacteria would not settle. Co-cultured. Microscopic observation after co-culture revealed the formation of coaggregates of yeast and lactic acid bacteria.

3. Ethanol production by continuous fermentation 2. The ethanol was produced by the following procedure using the agglomerate reactor prepared in (1).
That is, a YPD medium with a glucose concentration of 10% is continuously supplied to the reactor at a rate of 20 ml / h using a tube pump, and the uppermost culture solution is supplied by a tube pump via a tube installed on the culture solution surface. I pulled it out.
Thus, continuous culture was performed at 30 ° C. while keeping the culture medium volume constant while leaving most of the aggregates inside the reactor.

4). Measurement of ethanol production 3. The concentration of ethanol produced in ethanol production by continuous fermentation was analyzed with an alcohol concentration meter “Alcomate AL-3” (Woodson Riken Keiki Co., Ltd.).
The ethanol concentration of 3.9% (w / v) was reached on the second day after the start of continuous culture, and stable fermentation results were obtained until the tenth day thereafter. During this period, the average concentration of ethanol for 9 days was 4.2% (w / v). Further, the pH of the culture solution was maintained in the range of 3.5 to 4.0 from the 2nd day to the 10th day when the continuous culture was started. As a result of microscopic observation, it was also confirmed that significant coaggregates were retained in the reactor from the 2nd day to the 10th day after the start of continuous culture.
Therefore, from these results, it was confirmed that ethanol fermentation by continuous fermentation was possible with aggregates of yeast and lactic acid bacteria.

5. Ethanol production by fermentation method (repeated batch culture method) The ethanol was produced by the repeated batch culture method according to the following procedures 1) to 3) using the aggregate reactor prepared in (1).
1) 2. After co-culturing from the aggregate reactor prepared in step 1, stirring was stopped and the mixture was allowed to stand for 30 minutes to precipitate the aggregate. Thereafter, the supernatant fermentation solution was taken out, replaced with a fresh YPD medium (glucose 10%) prepared in the same manner as in Example 2, and cultured at 30 ° C. for 24 hours.
2) The fermentation broth after culturing in 1) above is collected, replaced with fresh YPD medium (glucose 10%), and gently stirred so that these bacteria do not settle at 30 ° C. and 150 rpm. Incubate for hours.
3) The above 2) was repeated and a repeated batch culture method was performed so that the total number of cultures was 10.

6). Measurement of ethanol production amount 5. When all the cultures were completed, the alcohol concentration meter “Alcomate AL-3” (manufactured by Woodson Riken Keiki Co., Ltd.) was used to determine the ethanol concentration in the fermentation broth drawn from the aggregate reactor every 24 hours. Analyzed in
Table 12 shows the ethanol production (w / v%) when the aggregate reactor of the present invention was used. As a result, it was confirmed that when the aggregate reactor of the present invention was used, ethanol could be stably produced despite the repeated batch culture of 10 times.

The fermentation method of the present invention makes it possible to perform a repeated batch culture method or a continuous fermentation method while suppressing contamination with various bacteria, and in the long term alcohols such as ethanol, organic acids such as lactic acid, perfume ingredients such as furanones, etc. It becomes possible to produce useful substances.
In the fermentation method of the present invention, since a lactic acid bacterium that produces bacteriocin is used, the treatment for suppressing contamination with bacteria is reduced, and the fermentation can proceed in a simpler process than the conventional method. In addition, the heat energy required for sterilization can be greatly reduced. Such a fermentation method of the present invention can be utilized as an extremely important elemental technology for producing biomass energy which is renewable energy.

(1) NITE AP-1168

[Reference to deposited biological materials]
(1) Lactobacillus plantarum HM23 strain (i) Name and address of the depositary institution that deposited the biological material National Institute of Technology and Evaluation Technical Microbiology Deposit Center 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture (zip code 292-0818) )
Date of deposit of biological material at Loi depository (Receipt date)
Deposit number (receipt number) given by the depositary institution on November 29, 2011 for deposit
NITE AP-1168

Claims (11)

A fermentation method using an aggregate obtained by co-culturing lactic acid bacteria that produce bacteriocin and yeast. The fermentation method according to claim 1, wherein the lactic acid bacterium producing bacteriocin is a lactic acid bacterium that exhibits coaggregation with yeast and forms an aggregate composed of the lactic acid bacterium and yeast when co-cultured with the yeast. The fermentation method according to claim 1 or 2, wherein the aggregate is a biofilm or an aggregate. The fermentation method according to any one of claims 1 to 3, wherein the lactic acid bacterium producing bacteriocin is a lactic acid bacterium belonging to Lactobacillus plantarum. The fermentation method according to any one of claims 1 to 4, wherein the lactic acid bacterium producing bacteriocin is Lactobacillus plantarum HM23 strain (reception number NITE AP-1168). The fermentation method according to any one of claims 1 to 5, wherein the yeast belongs to Saccharomyces cerevisiae. The fermentation method according to any one of claims 3 to 6, wherein a carrier is used in forming the biofilm. The fermentation method according to claim 7, wherein the carrier is a carrier containing glass or cellulose. The fermentation method according to any one of claims 1 to 8, which is carried out by a repeated batch culture method or a continuous fermentation method. The method to produce ethanol by the fermentation method in any one of Claims 1-9. Lactic acid bacteria deposited as receipt number NITE AP-1168 (HM23).