JP2010193846A - Lactic acid fermentation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To realize a lactic acid fermentation method which facilitates the form control of a mold. <P>SOLUTION: The lactic acid fermentation method uses a carrier made of a spongelike porous material by making it bear mold having a lactic acid generation ability for lactic acid fermentation. Consequently, the method limits the growth of hyphae of the mold within the carrier, permits the mold to take the form of a uniform pellet of a fixed size without depending on the inoculation quantity of spores and the flow conditions of culture and can prevent adhesion to a culture vessel. Since made of a porous material having communicating pores, the carrier facilitates the growth of the hyphae of the mold. Since the carrier can bear a large quantity of the hyphae, it helps increase the manufacturing efficiency of the lactic acid. Having the communicating pores and compressibility, the carrier permits air in the interior to be easily released. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a lactic acid fermentation method using filamentous fungi.

Lactic acid is used in various fields such as foods such as brewing, pickles and sour seasonings for soft drinks, medical use, industrial use such as paints, solvents and biodegradable plastics, and its demand is increasing. Conventionally, as a method for producing lactic acid, a method of fermenting and producing lactic acid bacteria using edible biomass as a main raw material has been performed. Here, the production of lactic acid by lactic acid bacteria has the advantage that the fermentation conditions are easy to control and the yield of lactic acid is high, but it requires many nutrients for culturing lactic acid bacteria, and the medium composition is complicated and expensive. Because lactic acid bacteria are acidic and easily killed by the lactic acid they produce, it is necessary to perform fermentation while neutralizing the lactic acid produced by adding alkali such as calcium carbonate. There were problems such as high costs.
In addition, when a large amount of auxiliary material remains in the medium, it is difficult to obtain high purity lactic acid. In order to solve these problems, various methods for producing lactic acid using filamentous fungi have been developed. In the method for producing lactic acid using filamentous fungi, the medium components may be a carbon source and an inorganic salt, and the production cost can be reduced. Moreover, L-lactic acid with high optical purity can be produced. Furthermore, non-edible biomass can be used as a raw material. As such a technique, for example, Patent Document 1 discloses that a bacterial cell aggregate (pellet) generated by inoculating filamentous fungal spores in a liquid medium and culturing them while stirring by aeration in the liquid medium. A method for producing lactic acid is disclosed, which is characterized in that fermentation is carried out by floating.
Japanese Patent Laid-Open No. 6-253871

However, the form of filamentous fungi varies greatly depending on the culture conditions such as the amount of spore inoculation, medium composition, pH change, and strength of stirring, and the mycelium floats on the liquid surface of the medium or adheres to the inner wall of the fermentation apparatus. Or the fermentation efficiency tends to deteriorate. For this reason, the above-described technique has a problem that it is difficult to perform the culture operation because it is necessary to control the culture conditions highly in order to form the filamentous fungus into a pellet.

Then, an object of this invention is to implement | achieve the lactic-acid-fermentation method with easy form control of filamentous fungi.

In order to achieve the above object, according to the first aspect of the present invention, in the lactic acid fermentation method, in the lactic acid fermentation method, the carrier made of a sponge-like porous material having continuous pores and having compressibility has a filamentous form having lactic acid-producing ability. A technical means is used in which bacteria are carried and the carrier is suspended in a liquid medium to cause lactic acid fermentation.

According to the invention described in claim 1, since the filamentous fungus having the ability to produce lactic acid is supported on a carrier made of a sponge-like porous material and used for lactic acid fermentation, the growth of mycelium of the filamentous fungus is limited within the carrier, The filamentous fungus can be made into a uniform pellet form of a certain size without depending on the spore inoculation amount or the culture flow conditions, and adhesion to a culture vessel or the like can be prevented. Since the carrier is made of a porous material having continuous pores, the mycelium of filamentous fungi is easy to grow and can be supported in a large amount, so that the production efficiency of lactic acid can be increased. In addition, since it has communicating pores and has compressibility, the air inside can be easily removed, and the carrier can be suspended in the liquid medium without floating on the surface of the liquid medium. . Furthermore, since only the liquid medium can be easily separated, it is possible to easily perform repeated batch culture and continuous culture using the bacterial cells repeatedly.

According to a second aspect of the present invention, in the lactic acid fermentation method according to the first aspect, a technical means that the carrier has a porosity of 90% or more is used.

In the invention described in claim 2, since the carrier has a very high porosity of 90% or more, and thus can support a large number of filamentous fungi, the production efficiency of lactic acid can be increased.

According to a third aspect of the present invention, in the lactic acid fermentation method according to the first or second aspect, a technical means is used in which the carrier is formed in a lump shape having an outer dimension of 1 to 7 mm.

In lactic acid fermentation using filamentous fungi, it is known that the lactic acid yield is high and the fermentation rate is high when the bacterial appearance is in the form of pellets of 1 to 7 mm. Since the carrier formed in a lump shape with a size of 1 to 7 mm can increase the production efficiency of lactic acid, it can be suitably used. The lump includes various forms, and forms such as a cube, a sphere, and an indeterminate form can be adopted.

In the invention according to claim 4, in the lactic acid fermentation method according to any one of claims 1 to 3, technical means that the filamentous fungus is a microbial cell belonging to the genus Rhizopus. Is used.

Like invention of Claim 4, the microbial cell which belongs to Rhizopus (Rhizopus) genus can grow only with an inorganic salt and a carbon source, and the purity of L-lactic acid produced | generated is high. Moreover, since lactic acid can be obtained not only from hexose but also from pentose, non-edible biomass (woody / herbaceous biomass) can be used as a raw material. In particular, Rhizopus oryzae can be suitably used because it can produce a large amount of L-lactic acid.

The lactic acid fermentation method in the present invention will be described with reference to the drawings. FIG. 1 is an explanatory view showing the structure of the carrier.

The carrier used in the lactic acid fermentation method of the present invention is, for example, a carrier 10 made of a porous material having continuous pores as shown in FIG. Since the carrier 10 is made of a porous material 12 having a high porosity having continuous pores 11, the mycelium of filamentous fungi is easy to grow and can be supported in large quantities, so that the production efficiency of lactic acid can be increased. . Here, when the porosity is 90% or more, it is preferable that the mycelium of the filamentous fungus is easily proliferated.

Since the carrier 10 has pores 11 communicated with each other and has compressibility, the air inside can be easily removed, and the carrier 10 does not float on the surface of the liquid medium, and the carrier 10 is suspended in the liquid medium. Can be made.

The carrier 10 as described above is formed in a cubic shape by a sponge-like porous material such as polyurethane foam.

Lactic acid fermentation is performed according to the following procedure. Here, Rhizopus oryzae (Japanese name Kumonosukabi) belonging to the genus Rhizopus is used as the filamentous fungus. Bacteria belonging to the genus Rhizopus can grow only with inorganic salts and a carbon source, and the purity of the produced L-lactic acid is high. Moreover, since lactic acid can be obtained not only from hexose but also from pentose, non-edible biomass (woody / herbaceous biomass) can be used as a raw material. Rhizopus oryzae can be suitably used because it can produce a large amount of L-lactic acid.

First, Rhizopus oryzae spores are cultured in a potato dextrose agar (PDA) medium or the like, and mixed with sterile water to prepare a spore suspension having a predetermined initial concentration. Tween surfactants such as Tween 80 can be added to the sterile water. Thereby, spores can be effectively dispersed in sterile water, and a homogeneous suspension can be obtained.

Next, a liquid medium used for lactic acid fermentation is prepared. The liquid medium is prepared by adding a small amount of inorganic salt such as ammonium nitrate or ammonium sulfate to a carbon source such as glucose or xylose. The liquid medium may be of any type as long as it is a normal liquid medium that can grow filamentous fungi.

Subsequently, a liquid medium and a carrier are placed in a fermentation vessel, sterilized by an autoclave, and then a spore suspension is aseptically inoculated. The carrier can be easily degassed by pressing in the liquid medium. Here, since the carrier is heated by sterilization (121 ° C.), durability at the temperature is required.

Since lactic acid fermentation by Rhizopus oryzae is aerobic fermentation, lactic acid fermentation is carried out while growing Rhizopus oryzae according to fermentation conditions such as predetermined temperature and pH while stirring the liquid medium and the carrier with a stirring blade or air lift. I do. Here, since the carrier is deaerated, it is well suspended in the liquid medium.

Here, since Rhizopus oryzae is supported on a carrier and growth is limited within the carrier, it can be made into a uniform pellet form of a certain size without depending on culture conditions such as spore inoculation amount and flow. Adhesion to a container or the like can be prevented. Here, the carrier is preferably formed in a lump shape having an outer dimension of 1 to 7 mm. This is because, in lactic acid fermentation using filamentous fungi, the yield of lactic acid is high and the fermentation rate is high when the bacterial appearance is in the form of pellets of 1 to 7 mm. At this time, for example, 10 ⁵ to 10 ⁶ filamentous fungi are carried per ml of the carrier. The lump includes various forms, and forms such as a cube, a sphere, and an indeterminate form can be adopted.

Since Rhizopus oryzae mostly grows only on the carrier, the liquid medium and Rhizopus oryzae can be easily separated by separating the liquid medium and the carrier. Thereby, since the microbial cells remain in the carrier, repeated batch culture and continuous culture using the microbial cells repeatedly can be easily performed.

In the above-described process, Rhizopus oryzae was supported on a carrier together with fermentation, but it can also be supported on a carrier before fermentation. Moreover, as a carrying | support method, it can also be made to adsorb | suck physically besides the above-mentioned method, and another method can also be employ | adopted.

(Example)
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the support | carrier, microbial cell, fermentation method, etc. which concern on this invention are not limited to an Example.

As the carrier particles, a polyurethane foam having continuous pores and having a porosity of 95 to 96% molded into a 3.5 mm square cubic shape was used.

As a strain, Rhizopus oryzae NBRC4707 was obtained from the National Institute of Technology and Evaluation Biotechnology Headquarters.

The liquid medium used was a mixture of each stock solution prepared with the composition shown in Table 1 at a ratio shown in Table 2. The initial pH was 5.26.

A method for preparing spores is shown below. Spores of the obtained strain were suspended in a condensate, applied to two PDA agar media, and cultured in an incubator at 30 ° C. for 1 week. Appropriate amount of 0.03% Tween 80 aqueous solution sterilized in the spore grown on the obtained agar medium is collected, collected with a spreader or a spatula, applied on 20 PDA agar mediums, and kept at 30 ° C. for 1 week in an incubator. In culture. The obtained spores were 0.03% Tween 80 aqueous solution + 40%
A spore suspension was made with a solution of aqueous glycerol (1: 1). The spore suspension thus obtained was appropriately diluted with a 0.01% Tween 80 aqueous solution, and the number of bacteria in a fixed volume was repeatedly counted under a microscope with a toma hemocytometer to obtain the concentration of the spore suspension. This spore suspension was stored at -80 ° C. The initial inoculation amount of spore was 2 × 10 ⁶ / mL.

Example 1
After the spore suspension is thawed, 2 × 10 ⁶ / mL of the spore suspension in a uniform state is inoculated into a sterilized liquid medium in a 300 ml Erlenmeyer flask at 37 ° C. and a stirring speed of 150 rpm. Shaking culture was performed. The carrier was used at a ratio of 300 to 50 mL of liquid medium. A sponge carrier was placed in a liquid medium other than the carbon source, pressed through a punching metal, and the air in the carrier was deaerated. The amount of L-lactic acid produced by fermentation was analyzed by atomic absorption method by collecting 500 μL of the fermentation broth every day. Conical flasks with and without baffles were prepared, and the influence of the presence or absence of baffles as well as the influence of the carrier was examined.

FIG. 2 shows the change over time in the amount of L-lactic acid produced. When a conical flask with a carrier and a baffle was used, the yield of L-lactic acid was high and the maximum lactic acid production rate was also fast. It is thought that the yield increased due to the fact that the microbial cells entered the carrier and became uniform, and the baffle improved the agitation / aeration effect compared to normal. When the carrier was used, the cells grew only inside the carrier, but when the carrier was not used, the cells adhered to the inner wall of the Erlenmeyer flask or floated in the liquid medium as a large lump. .

(Example 2)
Example 1 was scaled up and cultured in a fermenter. After sterilization, the apparatus was set, and A (carbon source) shown in Table 1 was poured from the inoculation port at about 37 ° C. Then, it inoculated from the inoculation opening so that it might become uniform spore suspension 2 * 10 < ⁶ > / mL, and culture | cultivation was started on 37 degreeC and the conditions of stirring speed 250rpm. The total amount was 1.5 L. The carrier was used at a ratio of 300 to 50 mL of liquid medium.

  FIG. 3 shows the form of the cells after culturing. FIG. 3 (A) shows the form of bacterial cells when no carrier is used, and FIG. 3 (B) shows the form of bacterial cells when a carrier is used. As shown in FIG. 3 (A), in the case where no carrier was used, the bacterial cells floated in a lump in the liquid medium, or adhered to the inner wall, stirring blade, stirring rod in the form of pulp. The device has stopped working. On the other hand, as shown in FIG. 3B, when the carrier was used, the cells were uniformly contained in the carrier, and the cells did not adhere to the inner wall or the like.

(Example 3)
The time course of the amount of cells in the carrier was examined. The culture conditions were 37 ° C. and a stirring speed of 150 rpm for 9 days. FIG. 4 shows the change over time in the amount of cells supported on the carrier. The amount of cells rapidly increased until 3rd day, and then increased gradually and became constant at about 0.5 mg / carrier. The liquid medium was almost transparent, and it was confirmed that the cells grew only in the carrier.

Example 4
Repeated batch culture was performed under the same culture conditions as in Example 1. When the production amount of L-lactic acid reaches 12 to 13 g / L, remove the liquid medium in the clean bench, add the sterilized liquid medium, wash the carrier once, and add a new sterilized liquid medium. Returned to the incubator. This operation was repeated 10 times. The amount of L-lactic acid produced by fermentation was analyzed by atomic absorption method by collecting 500 μL of the fermentation broth every day. Note that the concentration of xylose was 60 g / L only in the fourth culture.

  FIG. 5 shows changes with time in the amount of L-lactic acid produced by repeated batch culture. The change over time in the amount of L-lactic acid produced did not depend on the number of repetitions, showed almost constant behavior, and the lactic acid yield was about 0.4 g / g except for the fourth time, indicating a good result. Thereby, it was confirmed that repeated batch cultures can be performed under good conditions using the carrier of the present invention.

(Example 5)
The effect of the amount of initial spores on L-lactic acid production was examined. The amount of initial spores is 2 × 10 ⁴ / mL, 2 × 10 ⁵ / mL, 2 × 10 ⁶ / mL, 2 × 10 ⁷ / mL, and Example 1 using a conical flask with a carrier and a baffle. The culture was performed under the same culture conditions. The amount of L-lactic acid produced by fermentation was analyzed by atomic absorption method by collecting 500 μL of the fermentation broth every day.

FIG. 6 shows the change with time in the amount of L-lactic acid produced by different amounts of initial spores. The yield of L-lactic acid and the maximum lactic acid production rate showed the highest values when the amount of initial spores was 2 × 10 ⁵ / mL. As the amount of initial spores decreased, the time at which L-lactic acid began to be produced was also delayed. Moreover, visually, when the amount of initial spores was 2 × 10 ⁷ / mL, a large amount of cells adhered to the upper inner wall of the Erlenmeyer flask. As the number of inoculated spores increases, the production rate and yield of L-lactic acid do not increase, so there is an upper limit on the amount of initial spores per carrier particle. It was thought that I could not fit in.

(Example 6)
The time course of L-lactic acid production with different carbon sources was examined. In addition to Xylose, which is a carbon source hexose used in Examples 1 to 5, culturing was performed for up to 10 days under the same conditions as in Example 3 using Glucose, which is pentose. FIG. 7 shows changes with time in the amount of L-lactic acid produced. The straight line in the figure is an approximate line for obtaining the maximum lactic acid production rate. Table 3 shows the maximum lactic acid production rate and the yield of L-lactic acid.

The maximum lactic acid production rate was about twice as fast when using Glucose than when using Xylose. The yield of lactic acid is about 0.5 g / g, and both Glucose and Xylose can be suitably used for lactic acid fermentation. Thereby, it was confirmed that lactic acid can be obtained not only from hexose but also from pentose.

In each example, Rhizopus oryzae NBRC4707 was used as a strain, but the present invention is not limited to this. For example, Rhizopus oryzae NBRC5384 can also be suitably used.

[Effect of Best Embodiment]
(1) According to the lactic acid fermentation method of the present invention, filamentous fungi having a lactic acid-producing ability are supported on a carrier made of a sponge-like porous material and used for lactic acid fermentation. Thus, the filamentous fungus can be made into a uniform pellet form of a certain size without depending on the spore inoculation amount or the flow conditions of the culture, and can be prevented from adhering to the culture vessel. Since the carrier is made of a porous material having continuous pores, the mycelium of filamentous fungi is easy to grow and can be supported in a large amount, so that the production efficiency of lactic acid can be increased. In addition, since it has communicating pores and has compressibility, the internal air can be easily removed, and the carrier can be suspended in the liquid medium without floating on the surface of the liquid medium. . Furthermore, since only the liquid medium can be easily separated, repeated batch culture or continuous culture using the cells can be easily performed.

(2) Since the carrier has a very high porosity of 90% or more, it can carry a large number of filamentous fungi, so that the production efficiency of lactic acid can be increased.

(3) In lactic acid fermentation using filamentous fungi, it is known that the yield of lactic acid is high and the fermentation rate is high when the appearance of the bacteria is in the form of pellets of 1 to 7 mm, as in the invention of claim 3 In addition, since the carrier formed in a lump shape with an outer dimension of 1 to 7 mm can increase the production efficiency of lactic acid, it can be preferably used. The lump includes various forms, and forms such as a cube, a sphere, and an indeterminate form can be adopted.

(4) Bacteria belonging to the genus Rhizopus can grow only with inorganic salts and carbon sources, and the purity of the produced L-lactic acid is high. Moreover, since lactic acid can be obtained not only from hexose but also from pentose, non-edible biomass (woody / herbaceous biomass) can be used as a raw material. In particular, Rhizopus oryzae can be suitably used because it can produce a large amount of L-lactic acid.

It is explanatory drawing which shows the structure of a support | carrier. FIG. 3 is an explanatory diagram showing a change with time in the amount of L-lactic acid produced in Example 1. It is explanatory drawing the form of the microbial cell after the culture | cultivation in Example 2. FIG. FIG. 3 (A) shows the form of bacterial cells when no carrier is used, and FIG. 3 (B) shows the form of bacterial cells when a carrier is used. FIG. 6 is an explanatory diagram showing a change with time of the amount of bacterial cells carried on a carrier in Example 3. It is explanatory drawing which shows the time-dependent change of the L-lactic acid production amount in the repeated batch culture in Example 4. It is explanatory drawing which shows the time-dependent change of the L-lactic-acid production amount by the different amount of initial spores in Example 5. It is explanatory drawing which shows the time-dependent change of the L-lactic acid production amount in Example 6.

Claims (4)

A carrier made of a sponge-like porous material having pores connected to each other and having a compressibility supports a filamentous fungus capable of producing lactic acid, and the carrier is suspended in a liquid medium so that lactic acid fermentation is performed. Lactic acid fermentation method. The lactic acid fermentation method according to claim 1, wherein the carrier has a porosity of 90% or more. The lactic acid fermentation method according to claim 1 or 2, wherein the carrier is formed in a lump shape having an outer dimension of 1 to 7 mm. The lactic acid fermentation method according to any one of claims 1 to 3, wherein the filamentous fungus is a cell belonging to the genus Rhizopus.

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