JP2003310296A - Method for producing riboflavin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing riboflavin in a high yield and a high production rate by improving dispersibility of a vegetable oil or an animal oil to a culture medium in the method for culturing a riboflavin producing microorganism in the culture medium comprising the vegetable oil or the animal oil as a carbon source, producing, accumulating the riboflavin and then collecting. <P>SOLUTION: The method allows the coexistence of a support comprising a clay mineral having an oil absorbing property or a product of a chemical treatment on the mineral or a calcium compound in the culture medium. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing riboflavin, more specifically, a clay mineral having an oil adsorbing property, a chemically treated product thereof or a carrier composed of a calcium compound is allowed to coexist in a riboflavin production medium. Thus, it relates to a method for producing riboflavin from a vegetable or animal oil carbon source in high yield and high production rate.

[0002]

2. Description of the Related Art Riboflavin-producing bacteria are cultured in a medium,
A method for producing riboflavin, which comprises producing and accumulating riboflavin and collecting it, has been known for a long time. As a riboflavin-producing bacterium, Eremothecium a
shbyii, Ashbya gossypii, Candida flareri, Mycocand
ida riboflavina, Clostridium acetobutylicum, etc.,
Riboflavin-producing bacterium of the genus Bacillus (JP-A-49-6689)
No. 4), a mutant strain of Streptomyces testasseus (Japanese Unexamined Patent Publication No. 50-116690), an Achromobacter riboflavin-producing bacterium (Japanese Unexamined Patent Publication No. 52-54094), and a Prebibacterium riboflavin-producing bacterium ( It is also known to use riboflavin-producing bacteria of the genus Saccharomyces (JP-A-60-241895), Candida famata (ATCC20849) (JP-A-5-509221), and the like. There is.

Further, as a carbon source in the medium, sugars such as glucose and sucrose, starch and its hydrolysates, acetic acid, citric acid, ethanol, hydrocarbons for certain microorganisms, It is known to use benzoic acid and the like.

[0004]

However, when using fats and oils as a carbon source, it is necessary to disperse the fats and oils in an aqueous medium, and therefore, stirring the medium or
Alternatively, it is necessary to add a dispersant or an emulsifier to the system. However, under stirring conditions in which oil is emulsified or suspended in water, the cells themselves are killed, and it is observed that the yield of riboflavin decreases. In addition, the method of adding a dispersant or an emulsifier to the system cannot avoid the adverse effects of these drugs on the bacterial cells, and also has the problem that the added drug is mixed with the riboflavin produced.

The present inventors have proposed a method for producing riboflavin, which comprises culturing a riboflavin-producing bacterium in a medium containing a vegetable oil or an animal oil as a carbon source, producing and accumulating riboflavin, and collecting the riboflavin. When a carrier comprising a clay mineral or a chemically treated product thereof or a calcium compound is coexistent in the medium, vegetable oil or animal oil can be stably dispersed in the medium without an excessive stirring operation that damages the cells. It was found that riboflavin can be produced in high yield and high production rate.

The present inventors further provide a method for producing riboflavin, which comprises culturing a riboflavin-producing bacterium in a medium containing vegetable oil or animal oil as a carbon source, producing and accumulating riboflavin, and collecting the riboflavin. It has been found that the waste vegetable oil or waste animal oil to be disposed of can be effectively used by using the waste clay containing the vegetable oil or animal oil as the source.

That is, the object of the present invention is to improve the dispersibility of a vegetable oil or an animal oil in a medium in a method for producing a riboflavin by culturing a riboflavin-producing bacterium in a medium containing a vegetable oil or an animal oil as a carbon source. The present invention provides a method capable of producing riboflavin with high yield and high production rate. Another object of the present invention is to provide a method for producing riboflavin, which can be carried out at low cost without requiring a troublesome operation for concentration and recovery of riboflavin. Still another object of the present invention is to provide a method capable of effectively utilizing waste vegetable oil or waste animal oil to be disposed of and recovering riboflavin therefrom.

[0008]

According to the present invention, riboflavin is produced by culturing a riboflavin-producing bacterium in a medium containing vegetable oil or animal oil as a carbon source, producing and accumulating riboflavin, and collecting the riboflavin. In the method
There is provided a method for producing riboflavin, characterized in that a carrier comprising a clay mineral having oil adsorbability, a chemically treated product thereof, or a calcium compound is allowed to coexist in the medium. In the method for producing riboflavin of the present invention, it is preferable that the carrier is present in the medium in an amount of 0.1 to 10% by weight. In a preferred embodiment of the present invention, the carrier is preferably a chain clay mineral, and particularly, the carrier is a clay mineral having a fiber diameter of 70 to 400 angstrom and a fiber length of 0.2 to 400 μm. Preferably there is. In another preferred aspect of the present invention, at least a part of the carrier is preferably a smectite group clay mineral and an acid-treated product thereof. A calcium compound can also be used as the carrier, and the calcium compound is preferably calcium carbonate. In the present invention, the manner of coexistence of the carrier in the medium is not particularly limited, but in some cases it is preferable to allow the carrier to coexist in the medium in the state of encapsulating the vegetable oil or the animal oil, and as a carrier encapsulating the vegetable oil or the animal oil. Waste clay can also be used. Further, according to the present invention, a riboflavin-producing bacterium is cultivated in a medium containing a vegetable oil or an animal oil as a carbon source, riboflavin is produced and accumulated, and in the method for producing riboflavin comprising collecting the riboflavin, the carbon source is Provided is a method for producing riboflavin, which is waste clay containing vegetable oil or animal oil. According to the present invention, further, in the method for producing riboflavin, which comprises culturing a riboflavin-producing bacterium in a medium containing vegetable oil or animal oil as a carbon source, producing and accumulating riboflavin, and collecting the riboflavin, the carbon source is Provided is a method for producing riboflavin, which is an oil component extracted from waste clay containing vegetable oil or animal oil.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION [Action] In the present invention, a riboflavin-producing bacterium is cultured in a medium containing a vegetable oil or an animal oil as a carbon source to produce and accumulate riboflavin, which is collected. It is characterized in that a carrier composed of a clay mineral having adsorptivity or a chemically treated product thereof or a calcium compound is allowed to coexist. For example, sterilizing a medium containing pH-adjusted vegetable oil or animal oil, and then inoculating and culturing a riboflavin-producing bacterium that has been pre-cultured, promotes consumption of vegetable oil or animal oil by allowing the above carrier to coexist in this medium. Therefore, the yield and production rate of riboflavin can be improved.

In the present invention, basically, when a riboflavin-producing bacterium is cultured in a medium containing a vegetable oil or an animal oil, a clay mineral having an oil adsorbing property or a chemically treated product thereof or a calcium compound is allowed to coexist in the medium. And the production yield of riboflavin is improved. For example, when the production amount of riboflavin in a medium not containing clay mineral is 100%, 1% by weight of sepiolite is added to the medium. , Even if the other conditions are the same,
The amount of riboflavin produced is improved to 159% (about 1.6 times). (See Example 1 for details of the experiment)

In riboflavin fermentation, vegetable or animal oils that are organic substances are decomposed by riboflavin-producing bacteria,
Although it accumulates riboflavin which is a metabolite, according to the study of the present inventors, in the process leading to this decomposition, first, a process in which vegetable oil or animal oil is taken up into the microbial cells,
It was found that there is a process in which the vegetable oil or animal oil taken into the cells is decomposed.

FIG. 1 of the accompanying drawings is a graph plotting the relationship between culture time and the amount of residual oil and the amount of riboflavin produced when soybean vegetable oil was present in a 50 cc medium, and FIG. It is a plot of the relationship with weight gain. According to FIG. 1, the amount of oil in the medium monotonously decreases with the culture time, but there is a constant time lag between the decrease in the amount of oil in the medium and the amount of riboflavin produced. The production of riboflavin has started 48 hours after the start.

On the other hand, FIG. 3 of the accompanying drawings shows a micrograph (A) (× 600 times) of the cells after 48 hours, and the oil content of the cells was stained with Nile red, and the stained cells were Is a photomicrograph (B) (× 600 times) measured with a fluorescence microscope. In FIG. 3 (B), the portion indicated by the arrow on a white background is the dyed oil. Referring to these photographs, the vegetable oil in the medium is first taken up by the cells and then decomposed to accumulate riboflavin which is a metabolite.

The oil-adsorptive clay mineral or a chemically treated product thereof used in the present invention promotes the uptake of vegetable oil or animal oil in the medium into the microbial cells, and further increases the production yield of riboflavin. FIG. 4 of the accompanying drawings shows the relationship between the culture time and the increase in the cell weight, when the agitation speed of the medium was 600 rpm, when sepiolite was added to the medium at 1% (●) and when it was not added (▲) ( A), a relationship between culture time and residual oil concentration (B), and a relationship between culture time and riboflavin concentration (C) are plotted. According to this result, the absorption of oil was
It can be seen that the production of riboflavin is enhanced, although at the same level.

The oil-adsorptive clay mineral or a chemically treated product thereof or a carrier composed of a calcium compound, which is used in the present invention, is stably present in the aqueous phase which is the medium like the riboflavin-producing bacterium, and its oil-adsorptive property. The oil component is taken in and dispersed in water into fine particles, facilitating the uptake of the oil component by the bacterial cells, and further promoting this. This is considered to be the effect of addition of the clay mineral or its chemically treated product as a carrier.

In order to coexist the clay mineral or its chemically treated product and the vegetable oil or animal oil in the medium, various means and measures can be adopted. For example, the clay mineral or a chemically treated product thereof and the vegetable oil or animal oil can be added to the medium independently, or can be added to the medium in the form of a composition. In one embodiment of the present invention, a carrier made of clay mineral or the like is allowed to coexist in a medium in the state of containing vegetable oil or animal oil. One of the most readily available such carriers encapsulating vegetable or animal oils is waste clay. Acid clay or activated clay which is chemically treated is widely used for decolorization and purification of fats and oils, but waste clay is generated during the treatment, and the treatment is a problem. That is, this waste white clay contains 20 to 60% by weight of oils.
It is contained in a certain amount, and since this waste clay is a sticky paste, it is extremely difficult to handle. Moreover, this waste white clay reaches a large amount of 50,000 tons per year, but according to the present invention, by using this waste white clay as a vegetable oil or animal oil storage carrier for fermentation production of riboflavin, It becomes possible to effectively use the vegetable oil or animal oil mentioned above as a resource, and to reuse the white clay from which the vegetable oil or animal oil has been removed. By effectively recycling the resource, it is possible to effectively prevent pollution of the natural environment. It will be possible.

In the riboflavin fermentation method of the present invention, it has been found that the produced riboflavin exhibits a very convenient action of being adsorbed to a clay mineral or the like as a carrier. That is, in the usual fermentation method, since the produced riboflavin is contained in the medium in a dilute state, considerable energy cost is required for its concentration. On the other hand, in the fermentation method of the present invention, since the riboflavin to be produced exists by being adsorbed on the clay mineral which is the carrier, the carrier on which the riboflavin is adsorbed is separated from the medium, and the riboflavin is extracted from the separated carrier. In this case, the operation required for concentration can be simplified and the cost can be saved. Further, in the present invention, an oil component extracted from waste white clay encapsulating vegetable oil or animal oil as a carbon source can be preferably used, and it is also excellent in effective use of the waste white clay.

When calcium carbonate, which is a typical example of a calcium compound, is used as the above-mentioned carrier, riboflavin is prevented by preventing calcium carbonate from lowering the pH due to fatty acids produced by the decomposition of fats and oils and preventing the bacteria from being damaged. It is considered that the production yield of the is increased (see Example 13). Further, as can be seen from Comparative Example 8, when magnesium hydroxide, which is a magnesium compound, was used as the same alkaline earth metal as calcium, there was no problem in the growth of bacteria, but there was no effect in the production of riboflavin. It is considered that this is because the fatty acids produced by the decomposition of fats and oils are not used for the production of riboflavin, but are used as an energy source for the growth of bacteria, and when the carbon source is depleted thereafter, the formation of spores is promoted.

[Clay Mineral or Chemically Treated Product Thereof] The clay mineral or chemically treated product used as a carrier in the present invention has an oil adsorbing property and generally has a fibrous, scaly or layered appearance. And is finely dispersed in any of the water-oil phases.

As a preferred example of the oil-adsorptive clay mineral, a chain clay mineral can be mentioned. The chain clay mineral used in the present invention is a fibrous magnesium silicate clay mineral represented by sepiolite, attapulgite, palygorskite and the like, and these have a three-dimensional chain structure and have a two-dimensional structure such as talc. Unlike a three-dimensional crystal structure, the voids formed in the chain structure have a BET specific surface area of 1
It is a porous clay mineral having a large specific surface area such that it falls within the range of 00 to 350 m ² / g and having an adsorbing action.

Unlike ordinary layered clay minerals represented by montmorillonite, which is also a porous clay mineral, sepiolite and the like are also characterized in that they do not swell in an aqueous system.

Due to the characteristics of chain-like clay minerals such as sepiolite, that is, the fact that they are fibrous and that they are porous, compared to ordinary clay minerals and other inorganic carriers, The entanglement with the body is improved, and the fixed cells are firmly and stably fixed, and the fixed cells are made porous so that the supply of the culture solution and oxygen to the cells is improved and the filterability is also improved. Can be done.

In the present invention, the chain clay mineral can be used alone or in combination with a double-layered fibrous mineral such as halloysite and asbestos, or a volcanic fibrous mineral including Kanuma soil, Akadama soil and the like. Can be used in. If necessary, zeolite or an adsorbent clay mineral represented by acid clay and rocks such as cristobalite, quartz and feldspar may be used in combination with this fibrous clay mineral.

[0024] In the present invention, sepiolite which are preferably used are chemical represented by the formula _{(1) (OH 2) 4} (OH) 4 Mg 8 Si 12 O 30 · 6~8H 2 O ‥‥ (1) It has a structure, and a two-dimensional crystal structure such as talc forms a chain crystal structure in which bricks are alternately stacked. In addition, it is fibrous due to the pores formed in the chain-like gap, but unlike other fibrous minerals, it also has a large specific surface area and adsorptivity.

In the present invention, the specific surface area is in the range of 100 to 350 m ² / g and the oil absorption is 100 to 30.
Sepiolite in the range 0 ml / 100 g is preferably used. If the specific surface area is smaller than this range, the oil adsorbability is low, while if it is larger than this range, no further effect can be obtained.

The fibrous sepiolite preferably used in the present invention generally has a fiber diameter of 70 to 400 angstroms, a fiber length of 0.2 to 400 μm, and an aspect ratio of 5 to 500. preferable.

Table 1 below shows an example of the general chemical composition of sepiolite (dry product at 110 ° C. for 2 hours).

[Table 1]

In the present invention, smectite clay minerals such as acid clay (montmorillonite), bentonite, saponite, hectorite, stevensite, and acid-treated products thereof can be used as carriers. Among these clay minerals, montmorillonite clay minerals and acid-treated products thereof are suitable for the purpose of the present invention, and these are widely used for decolorizing and refining oils and fats.

A montmorillonite clay mineral such as acid clay has a basic unit of a three-layer structure in which two SiO ₄ tetrahedral layers are sandwiched with an AlO ₆ octahedral layer sandwiched therebetween. It is an aluminosilicate in which a number of layered structures are further laminated in the C-axis direction to form a layered crystal structure. This layered crystal structure is common to montmorillonite clay minerals.

Among the montmorillonites, the acid clay that is widely produced in Japan is, due to weathering, the AlO ₆ octahedral layer Al in the three-layer structure which is the basic unit of montmorillonite.
Some of the atoms are replaced with alkaline earth metals such as magnesium and calcium, and hydrogen ions are bonded so as to supplement their valences. Therefore, when the acidic clay is suspended in a saline solution and the pH thereof is measured, the hydrogen ions are replaced with sodium (Na) ions, and the acidic clay exhibits acidity. on the other hand,
Bentonite has mostly exchangeable cations of sodium (Na)
Therefore, the pH shows neutral to slightly alkaline, and the water swelling property is large, whereas in the acid clay, sodium ions are replaced by alkaline earth metals, the alkali metal component is small, and the water swelling property is also low. In addition, since the silicic acid content is high, it is extremely advantageous in terms of adsorptivity.
Thus, as the montmorillonite, any acid clay produced in Japan is widely used, and a montmorillonite group clay mineral called so-called subbentonite (Ca-type bentonite) is also used.

Table 2 below shows an example of the general chemical composition of acid clay (100 ° C. dried product).

[Table 2]

When acid clay is used, the rocks such as cristobalite, quartz and feldspar contained in the clay should be easily separated by a separation method (classification means such as elutriation or elutriation) utilizing the difference in specific gravity. You can Further, among these, cristobalite of crystalline silicic acid can be easily removed by this method because it can easily react with alkali to be converted into alkali silicate. These methods can improve the purity of the layered crystal structure.

On the other hand, the acid-treated clay of acid clay is generally known as activated clay which is a refining agent for fats and oils. This acid-treated product is easily prepared by treating acidic clay with a mineral acid solution such as sulfuric acid or hydrochloric acid to elute a part of the basic component contained therein and washing. By this acid treatment, part of the layered crystal structure originally possessed by the acid clay was destroyed, but the content of silicic acid (SiO ₂ ) increased,
As a result, the specific surface area is increased and the physical properties such as adsorption ability are improved. The acid-treated substance of acid clay, the activated clay which is generally commercially available, and the intermediate product for production thereof are refiners having excellent properties.

The chemical composition of this acid-treated product varies depending on the type of the starting acid clay and the acid-treatment conditions, but generally has the composition shown in Table 3 below.

[Table 3]

The above-mentioned acid clay and activated clay are preferably used in the present invention in the form of so-called waste clay which occludes vegetable oil or animal oil. This will be described in detail later.

[Calcium compound] Specific examples of the calcium compound usable as the carrier include natural calcium carbonate (vaterite, calcite, aragonite),
Synthetic calcium carbonate, calcium silicate, calcium hydroxide, calcium phosphate (apatite etc.) is used,
Calcium carbonate is particularly preferable.

[Other carriers] As the carrier, natural silica, synthetic silica, silas balloon, diatomaceous earth, perlite, magnesium carbonate, magnesium silicate, magnesium phyllosilicate, hydrotalcite, synthetic magnesium hydroxide and the like are used in combination with the above-mentioned carriers. Can be used.

[Vegetable Oil and Animal Oil] The vegetable oil used as a carbon source in the present invention is widely present in the natural plant kingdom and mainly contains an ester of fatty acid and glycerin, for example, safflower oil, soybean oil, Rapeseed oil, palm oil,
Palm kernel oil, cottonseed oil, coconut oil, rice bran oil, sesame oil, castor oil, linseed oil, olive oil, tung oil, camellia oil, peanut oil, kapok oil, cacao oil, tree wax, sunflower oil, corn oil, etc. The vegetable oil used in the present invention is preferably one mainly composed of an ester of fatty acid at least partially unsaturated and glycerin. Further, as the animal fats and oils, sardine oil, fish oil such as herring oil, squid oil and saury oil, liver oil, whale oil, beef tallow, beef tallow, lard, chicken oil, horse oil, sheep oil and the like are used.

[Waste White Clay] The waste white clay used in the method of the present invention is a white matter for decolorization or refining used for decolorization or refining of oils and fats, which is a by-product separated in this step, and contains oil. Disposal of this was not allowed from the viewpoint of environmental pollution, and there was a strong desire for its effective use.

That is, the oil or fat to be decolorized or refined is treated with a montmorillonite group clay mineral such as acid clay or an activated clay obtained by subjecting these clay minerals to an acid treatment and / or an alkali treatment in the form of a powder or a decolorizing agent. By adding as a refining agent and stirring the both uniformly, the coloring components and impurity components contained in the fat and oil are adsorbed in the clay particles.
20 to 6 in white clay separated after decolorization or purification treatment
Oil content of about 0% by weight is retained.

The decolorizing treatment of fats and oils is a condition known per se, for example, 0.1 to 5% of white clay on a weight basis per fat and oil is added as a decolorizing or refining agent, and 90 to 150 ° C
The decolorization or purification treatment can be completed by stirring both compositions at the temperature of 5 to 30 minutes.

The mixture that has been decolorized or purified is treated with any filter such as a filter press, a belt filter, an Oliver filter, an American filter, or the like.
By supplying to a reduced pressure or pressure type filtration machine such as a centrifugal filtration machine, refined oil and fat and used so-called waste clay which is a decolorizing or refining agent are obtained. Depending on the type of raw material oil to be refined, the waste white clay generally has an oil content of 20 to 60, which is retained in the particles.
It contains about wt%. In addition, the waste clay is considered to retain the catalytic ability. In the present invention, this waste clay can be used as a carbon source for riboflavin fermentation.

[Riboflavin-producing bacterium] In the present invention,
As the riboflavin-producing bacterium, any bacterium known per se known to produce riboflavin can be used. Eremothecium as a riboflavin-producing bacterium
ashbyii, Ashbya gossypii, Candida flareri, Mycoca
ndida riboflavina, Clostridium acetobutylicum, etc., Bacillus riboflavin-producing bacteria, Streptomyces testaceus mutants, Achromobacter riboflavin-producing bacteria, Previbacterium riboflavin-producing bacteria, Saccharomyces riboflavin-producing bacteria, Candida
Famata (ATCC20849) and the like can be mentioned. Among these riboflavin-producing bacteria, those capable of incorporating vegetable oil or animal oil as a carbon source are preferable, and suitable examples thereof include Ashbya gossypii ATCC 10895.
Is used.

[Culture Medium] The fermentation method of the present invention uses vegetable oil or animal oil as the carbon source in the medium, but it is of course possible to use a carbon source other than vegetable oil or animal oil in combination, and such combination is possible. Glucose as a carbon source,
Galactose, maltose, cellobiose, arabinose, sugars such as sucrose, sugar alcohols such as glycerin, acetic acid, gluconic acid, succinic acid, formic acid, citric acid, fumaric acid, glutamic acid, organic acids such as lactic acid and salts thereof, methanol, ethanol And the like.

On the other hand, examples of the nitrogen source in the medium include organic or inorganic ammonium compounds such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium phosphate, and ammonium acetate, urea and its derivatives, and other natural nitrogen sources. used.

As the inorganic substances, salts of metals such as sodium, potassium, magnesium, calcium, zinc, cobalt, nickel, copper and manganese, chloro, sulfuric acid,
Salts such as phosphoric acid and nitric acid are used alone or in combination.

When the bacteria used show auxotrophy, the required substance is added to the medium. In addition, natural nutrients, cell hydrolysates of various microorganisms, yeast extract, sake lees extract, meat extract, peptone, soybean meal decomposition products and the like can be added to the medium, if necessary.

In the medium, various substances such as purines such as guanine, adenine, hypoxanthine, thymine,
In some cases, the amount of riboflavin produced can be increased by adding pyrimidines such as uracin and cytosine, and their sugars or phosphorylated sugar derivatives.

In the present invention, the riboflavin culture is
It can be carried out under conditions known per se. Culture conditions such as temperature are known per se, but it is generally preferably 20 to 35 ° C, particularly preferably 25 to 30 ° C.

There is also an optimum range for the range of the water phase and the oil phase in the medium. The water phase and the oil phase are, by weight ratio, generally 200: 1 to 3: 1, especially 50: 1 to 10: 1.
It should be in the range of. When the amount of the oil phase is larger than the above range or when the amount is smaller than the above range, the production rate of riboflavin tends to decrease.

In the present invention, the quantitative range of the carrier in the medium has an optimum range. The amount of carrier is preferably present in an amount of 0.1 to 10% by weight, especially 0.5 to 5% by weight, relative to the medium. When the amount of the carrier is small, the productivity of riboflavin decreases, and when the amount of the carrier is too large, the growth of riboflavin tends to be inhibited, and the productivity of riboflavin tends to decrease.

In one preferred embodiment of the present invention, when waste clay containing oil is used as a raw material and carrier, the aqueous phase and the waste clay containing oil are 100: 1 in weight ratio.
It should be in the range of 5: 1 to 5: 1, especially 20: 1 to 5: 1. When the amount of the waste clay phase is larger than the above range or smaller than the above range, the production rate of riboflavin tends to decrease.

In the emulsification method of the present invention, the order of addition of the above three is not particularly limited, but it depends on the mixing ratio of both phases of oil and water, its application, and the type of oil depending on use conditions. It depends, but preferably, after adding the carrier to water and stirring,
A method of adding oil and stirring and emulsifying is selected, but more preferably, a more stable emulsion composition can be obtained by adding a carrier to oil and stirring and then adding water and stirring and emulsifying. The stirring speed is preferably 400 to 700 rpm.

The emulsification operation in the present invention may be carried out by a generally employed physical method such as stirring, and in the laboratory, a mechanical force having a shearing force comparable to that of an ordinary household mixer is used for several tens of seconds to 10 seconds. A treatment time of a few minutes is sufficient, and industrially, a large amount can be treated by a homogenizer, a colloid mill, a jet flow mixer, a voterator, etc., but in the present invention, such a physical method is not necessarily used. The method is not limited, and if necessary, a chemical method such as an inversion emulsification method, a gel emulsification method, an HLB-temperature emulsification method or the like may be adopted.

In culturing, the emulsified composition formed as described above by adding the emulsifier consisting of the clay mineral of the present invention to the culture medium containing useful bacteria, fats and oils is filled in a conventional aeration and stirring tank. Using a reactor such as a bed-type reactor or an air-lift type bubble column, the fermentation can be performed in a batch, semi-batch, repetitive batch or continuous manner depending on the characteristics of the fermentation product.

[0056]

EXAMPLES The present invention will be described in the following examples, but the present invention is not limited to these examples. (Measurement method) (1) Measurement method of oil consumption 5 ml of hexane was added to 5 ml of the culture solution, mixed for 2 minutes, and centrifuged at 3,000 rpm for 15 minutes. The hexane layer was taken and dried at 105 ° C for 3 hours to obtain the amount of residual oil, which was subtracted from the amount of oil used first to obtain the amount of consumption. (2) Measurement of riboflavin concentration Dilute the culture solution so that the riboflavin concentration is 30 mg / L or less, and add 0.8 ml of the diluted culture solution and 0.2 ml of 1N-NaOH and mix. Take 0.4 ml of the culture solution mixed with NaOH, and
Add 1 ml of 0.1 M phosphate buffer (pH 6.0), and add 10 ml at 11,000 rpm.
Centrifugation was performed, the supernatant was taken, and the absorbance at a wavelength of 444 nm was measured. The concentration of riboflavin was calculated in the unit of mg / L by the absorbance at a wavelength of 444 nm × dilution rate × 127.2971. (3) Absorbance It was measured using U-2001 type manufactured by Hitachi, Ltd. (4) Fluorescence microscope photograph It was observed using IX-70 manufactured by Olympus Optical Co., Ltd.

(Example 1) <Flask culture> (Pre-culture) 30g CSL (Corn Steep Liquor), 9g yeast exhaust
(Yeast extract), 15g soybean oil is dissolved and distilled water is added.
To 1 liter, then add 5N-KOH to adjust the pH to 6.
The medium was adjusted to 8. 100 ml of the medium prepared above
Ashbya gossyp after sterilization in a 500 ml flask
ii ATCC 10895 was inoculated and pre-cultured at 28 ° C and 220 rpm for 24 hours
Then, the preculture liquid was obtained. (Main culture) 50g soybean oil and sepiolite (Mizusawa Chemical Co., Ltd.)
Product name: Adeplus ML-50D) mixed with 11.1 g
Then, with this mixture, 30g gelatin, 60g CSL, 1.5g K
H_TwoPO_Four, 1.5g glycine, 2mg Co²⁺, 5 mg
 Mn²⁺, 10 mg Zn²⁺, And 1 mg Mg²⁺ 1 liter of distilled water
Dissolve in a tor and adjust the pH to 6.8 with 5N-KOH to adjust the medium.
Made The sepiolite concentration in this medium is 1.1% by weight.
Is. (Note that sepiolite is also used in Examples described later.
The above product manufactured by Mizusawa Chemical Co., Ltd. was used. ) 500 ml
Add 50 ml of the above medium to the flask and sterilize it, and then
Inoculate 1 ml of the nutrient solution and culture at 28 ° C and 220 rpm for 7 days. Obtained
Riboflavin concentration, yield (%) and improvement rate (%)
It shows in Table 4. The improvement rate is Improvement rate (%) = (A−B) × 100 / B Where A: riboflavin concentration with the addition of sepiolite B: Riboflavin concentration without addition of sepiolite I asked more. In addition, the yield is raw oil (in this case, soybean oil)
The amount (%) of riboflavin produced by 1 g is shown.

(Examples 2 to 4) For main culture in which the pH was adjusted to 6.8 in the same manner as in Example 1 except that the amount of sepiolite used was changed to 22.3 g, 33.4 g or 55.7 g. The medium was prepared. The sepiolite concentrations in the obtained medium are 2 wt%, 3 wt% and 5 wt%, respectively. 50 ml of the above medium was placed in a 500 ml flask for sterilization, and then 1 ml of the preculture liquid prepared in Example 1 was inoculated and cultured at 28 ° C. and 220 rpm for 7 days. The concentration, yield (%) and improvement rate (%) of the obtained riboflavin are shown in Table 4.

Comparative Example 1 A medium for main culture, the pH of which was adjusted to 6.8, was prepared in the same manner as in Example 1 except that sepiolite was not used. 50 ml of the above medium was placed in a 500 ml flask for sterilization, and then 1 ml of the preculture liquid prepared in Example 1 was inoculated and cultured at 28 ° C. and 220 rpm for 7 days. The concentration, yield (%) and improvement rate (%) of the obtained riboflavin are shown in Table 4.

[0060]

[Table 4]

(Examples 5 to 7) <Jar fermenter culture> (pre-culture) 2 g yeast extract (yeast extract), 20 g peptone, 0.6 g myo-inosito (myo-inosito)
l), dissolve 10 g glucose in 1 liter and add 5N-KOH to pH
Was adjusted to 6.8 to obtain medium A. Also, 30g CSL (Corn S
teep Liquor), 9g yeast extract, 15g
Dissolve soybean oil, add distilled water to make 1 liter, 5N-K
The medium B was obtained by adjusting the pH to 6.8 with OH. 50 ml of medium A is put into a 500 ml Erlenmeyer flask and sterilized.
Ashbyagossypii ATCC 10895 was inoculated, 28 ° C, 220 rpm for 2
Preculture was performed for 4 hours to obtain a culture solution A. Next, 100m
After 1 liter of the medium B was placed in a 500 ml Erlenmeyer flask and sterilized, 1 ml of the culture medium A obtained above was inoculated and secondary preculture was carried out for 24 hours to obtain a secondary culture medium. (Main Culture) A medium for main culture (sepiolite concentration: 1% by weight) was prepared in exactly the same manner as in Example 1. This medium
After 2 liters were placed in a 5 liter jar fermenter for sterilization, 100 ml of the secondary culture solution obtained above was inoculated and cultured for 5 days. The stirring speed was changed to 400 to 700 rpm, and oxygen (1 vvm) was supplied. Table 5 shows the concentration and the improvement rate (%) of the obtained riboflavin. In addition, in Table 5,
The results obtained twice at each stirring speed are shown below.

Comparative Examples 2 to 4 In exactly the same manner as in Comparative Example 1, a medium for main culture containing no sepiolite was prepared. After 2 liters of this medium was placed in a 5 liter jar fermenter for sterilization, 100 ml of the secondary culture solution prepared in Example 5 was inoculated and cultured for 5 days. The stirring speed was changed to 400 to 700 rpm, and oxygen was supplied. Table 5 shows the concentration and the improvement rate (%) of the obtained riboflavin.

[0063]

[Table 5]

Example 8 Strain Ashbya gossypii ATCC 1
Using 0895, a solid medium (pH 6) containing 10 g of yeast extract, 10 g of glucose, 3 g of glycine and 20 g / L of agar was added at 30 ° C.
The cells were grown for 2 days (solid culture). Next, using a medium containing 30 g / L of CSL, 15 g / L of yeast extract, and 6 g / L of soybean oil in distilled water, and sterilizing this, inoculate the above solid culture one platinum loop amount, 28 ° C Preculture was performed at 220 rpm for 24 hours to obtain a preculture liquid. Next, 30g gelatin, 60g CSL, 1.5g KH ₂ PO
₄ , 1.5g glycine, 2mg Co ²⁺ , 5mg Mn ²⁺ , 10mg Zn
A medium for main culture was prepared by dissolving ²⁺ , 1 mg Mg ²⁺ , and a carbon source in 1 L of distilled water and adjusting the pH to 6.8 with 5N-KOH. As the carbon source, 125 g of waste white clay containing 40 wt% of rapeseed oil was used. To the medium for main culture prepared above, inoculate 2 ml of the preculture liquid, and use a rotary shaker to prepare 200
Culture was performed at 28 ° C. for 7 days at rpm. After the completion of the culture, the waste clay was separated, and a wet weight of 1.5 g (a dry weight of 0.636 g after drying at 105 ° C. for 2 hours) was taken to extract riboflavin. As for the extraction method, 0.4N-NaOH was added to the waste clay and the mixture was vigorously stirred, and then the riboflavin concentration in the supernatant was measured. As a result, 80% or more of riboflavin was recovered by two extractions and 90% or more of three or more extractions. Table 6 shows the concentration and yield (%) of the obtained riboflavin.

Example 9 Strain Ashbya gossypii ATCC 1
Using 0895, solid culture and preculture were performed in the same manner as in Example 8 to obtain a preculture liquid. Further, main culturing was carried out in the same manner as in Example 8 except that 125 g of waste white clay containing 40 wt% of palm oil was used as a carbon source, and riboflavin was extracted in the same manner. Table 6 shows the concentration and yield (%) of the obtained riboflavin.

(Examples 10 to 11) Strain Ashbya gossypi
Using iATCC 10895, solid culture and preculture were performed in the same manner as in Example 8 to obtain a preculture liquid. Further, main culture was carried out in exactly the same manner as in Example 8 except that 50 g of rapeseed oil or palm oil extracted from waste clay was used as a carbon source.
After the culture, the oil concentration and the riboflavin concentration were measured, and the yield (%) was calculated from the results. The concentration of oil is
Add the same amount of hexane to 2 ml of culture solution and mix well for 2 minutes, then centrifuge at 3,000 rpm and remove the supernatant.
It was calculated by evaporating hexane, then drying, and weighing the oil. The riboflavin concentration was calculated by the method described above. Table 6 shows the concentration and yield (%) of riboflavin.

[0067]

[Table 6]

(Comparative Examples 5-6) Strain Ashbya gossypii AT
Using CC 10895, solid culture and preculture were performed in the same manner as in Example 8 to obtain a preculture liquid. Further, main culture was carried out in exactly the same manner as in Example 8 except that 50 g of virgin soybean oil (reagent manufactured by Wako Pure Chemical Industries) or rapeseed oil (reagent manufactured by Nacalai Tesque) was used as a carbon source. After culturing, Examples 10 and 11
The oil concentration and the riboflavin concentration were measured in the same manner as in, and the yield (%) was calculated from the results. Table 7 shows the concentration and yield (%) of riboflavin.

[0069]

[Table 7]

Example 12 Strain Ashbya gossypii ATCC
Using 10895, solid culture and preculture were performed in the same manner as in Example 8 to obtain a preculture liquid. Further, main culture was carried out in the same manner as in Example 8 except that 188 g of waste clay containing 40 wt% of palm oil was used as a carbon source and the main culture time was set to 10 days, and riboflavin was extracted in the same manner. It was Table 8 shows the concentration and yield (%) of the obtained riboflavin.

Comparative Example 7 Strain Ashbya gossypii ATCC 1
Using 0895, solid culture and preculture were performed in the same manner as in Example 8 to obtain a preculture liquid. Further, the main culture was carried out in the same manner as in Example 12 except that 75 g of virgin palm oil (Spectrum Chemical Mfg. Corp.) was used as the carbon source. After the culture, after the culture, the oil concentration and the riboflavin concentration were measured in the same manner as in Examples 10 and 11, and the yield (%) was calculated from the results. The riboflavin concentration and yield (%) are shown in Table 8.

[0072]

[Table 8]

(Example 13) Charcoal having 50 g of rapeseed oil adsorbed
Calcium acid (PC manufactured by Shiraishi Calcium Industry) 125g
Was prepared. 30g gelatine with this calcium carbonate
60g CSL, 1.5g KH_TwoPO_Four, 1.5g glycin
e), 2mg Co²⁺, 5mg Mn²⁺, 10 mg Zn²⁺, And 1 mg
Mg²⁺ Is dissolved in 1 liter of distilled water and the pH is adjusted to 6 with 5N-KOH.
The medium was adjusted to 8 to prepare a medium for main culture. 500 ml of frass
After sterilizing by adding 50 ml of the above-mentioned medium to the co.
Inoculate 1 ml of the prepared pre-culture liquid and culture at 28 ° C, 220 rpm for 7 days.
went. The concentration and yield (%) of the obtained riboflavin
It shows in Table 9.

Comparative Example 8 Main culture was carried out in the same manner as in Example 13 except that 125 g of magnesium hydroxide having 50 g of rapeseed oil adsorbed therein was used in place of the above calcium carbonate. The concentration and yield (%) of the obtained riboflavin are shown in Table 9.

(Comparative Example 9) 125 g Silica (Mizukasil P707 manufactured by Mizusawa Chemical Co., Ltd.) adsorbed with rapeseed oil 125 g
Was cultured in the same manner as in Example 13 except that the above calcium carbonate was used instead of the above calcium carbonate. The concentration and yield (%) of the obtained riboflavin are shown in Table 9.

[0076]

[Table 9]

Example 14 Strain Ashbya gossypii ATCC
Using 10895, solid culture and preculture were performed in the same manner as in Example 8 to obtain a preculture liquid. In addition, waste clay containing 40 wt% of beef tallow as a carbon source (beef tallow: litter = 1: 1 mixture) 1
Main culture was carried out in the same manner as in Example 8 except that 25 g was used, and riboflavin was extracted in the same manner. The concentration and yield (%) of the obtained riboflavin are shown in Table 10.
Shown in.

Comparative Example 10 Strain Ashbya gossypii ATCC
Using 10895, solid culture and preculture were performed in the same manner as in Example 8 to obtain a preculture liquid. Further, the main culture was carried out in exactly the same manner as in Example 14 except that 50 g of virgin beef tallow was used as the carbon source. The concentration and yield (%) of the obtained riboflavin are shown in Table 10.

Comparative Example 11 Strain Ashbya gossypii ATCC
Using 10895, solid culture and preculture were performed in the same manner as in Example 8 to obtain a preculture liquid. Further, main culture was carried out in exactly the same manner as in Comparative Example 10 except that 50 g of virgin lard was used as the carbon source. The concentration and yield (%) of the obtained riboflavin are shown in Table 10.

[0080]

[Table 10]

[0081]

According to the present invention, a method for producing riboflavin, which comprises culturing a riboflavin-producing bacterium in a medium containing a vegetable or animal oil as a carbon source to produce and accumulate ribflavin, and collecting the ribflavin, When a clay mineral having oil adsorption property or a chemically treated product thereof or a carrier composed of a calcium compound coexists in the medium, the vegetable oil is stably dispersed in the medium without an excessive stirring operation that damages the cells. Riboflavin can be produced in high yield and high production rate.

Further, according to the present invention, in the method for producing riboflavin by culturing a riboflavin-producing bacterium in a medium containing a vegetable oil or an animal oil as a carbon source, the dispersibility of the vegetable oil or the animal oil in the medium is improved. , A method capable of producing riboflavin at high yield and high production rate is provided. According to the present invention, there is further provided a method for producing riboflavin, which can be performed at low cost without requiring a troublesome operation for concentration and recovery of riboflavin. Furthermore, according to the present invention, waste vegetable oil or waste animal oil to be disposed of can be effectively utilized, and riboflavin can be effectively recovered therefrom. The recovered riboflavin is used as a pharmaceutical or animal feed additive, a food colorant, and a nutritional supplement. Further, those supported on clay minerals are useful as animal feed as they are, and can be expected to have nutritional supplementation and intestinal regulating action. In particular, when waste clay is used, the waste clay can be effectively used and at the same time the environmental load can be reduced.
Furthermore, it is also possible to produce riboflavin by adding the carrier used in the present invention to a waste edible oil produced from households, fast food stores, bakery companies, etc., which has recently been a problem of disposal, and treating it. .

[Brief description of drawings]

FIG. 1 shows the presence of soybean vegetable oil in a 50 cc medium,
It is the graph which plotted the relationship between the culture time, the residual oil amount, and the produced riboflavin amount. Riboflavin production starts 48 hours after the start.

FIG. 2 is a graph plotting the relationship between culture time and increase in cell weight.

FIG. 3 is a micrograph (A) of a bacterial cell after 48 hours (×)
600 times) and the oil content of the cells was stained with Nile red,
Fluorescent micrograph (B) of this stained cell (× 600
Times).

FIG. 4 shows the relationship between the culture time and the increase in cell weight (A), when the agitation speed of the medium was 600 rpm, when 1% sepiolite was added to the medium (●) and when it was not added (▲). It is the graph which plotted the relationship (B) between culture time and residual oil concentration, and the relationship (C) between culture time and riboflavin concentration.

Continued front page    (72) Inventor Park Ryusoo             836 Otani, Shizuoka City, Shizuoka Prefecture Faculty of Agriculture, Shizuoka University             Department of biochemistry (72) Inventor Zheng Silver ▲ Hi ▼             836 Otani, Shizuoka City, Shizuoka Prefecture Faculty of Agriculture, Shizuoka University             Department of biochemistry (72) Inventor Minghua             836 Otani, Shizuoka City, Shizuoka Prefecture Faculty of Agriculture, Shizuoka University             Department of biochemistry F-term (reference) 4B064 AH02 CA07 CC03 CD01 CD05                       CD21 CE20 DA01 DA20

Claims (10)

[Claims] 1. A method for producing riboflavin, which comprises culturing a riboflavin-producing bacterium in a medium containing a vegetable oil or an animal oil as a carbon source, producing and accumulating riboflavin, and collecting the riboflavin, which is a clay mineral having an oil adsorption property. Alternatively, a method for producing riboflavin, characterized in that a chemically treated product thereof or a carrier composed of a calcium compound is allowed to coexist in the medium. 2. The method according to claim 1, wherein the carrier is present in the medium in an amount of 0.1 to 10% by weight. 3. The production method according to claim 1, wherein the carrier is a chain clay mineral. 4. The carrier according to claim 1, wherein the carrier is a clay mineral having a fiber diameter of 70 to 400 Å and a fiber length of 0.2 to 400 μm.
The manufacturing method according to any one of 1. 5. The method according to claim 1, wherein at least a part of the carrier is a smectite group clay mineral and an acid-treated product thereof. 6. The method according to claim 1, wherein the carrier is allowed to coexist in the medium in a state of containing vegetable oil or animal oil.
6. The manufacturing method according to any one of 5 to 5. 7. The method according to claim 6, wherein the carrier containing the vegetable oil or the animal oil is waste clay. 8. The manufacturing method according to claim 1, wherein the calcium compound is calcium carbonate. 9. A method for producing riboflavin, which comprises culturing a riboflavin-producing bacterium in a medium containing vegetable oil or animal oil as a carbon source, producing and accumulating riboflavin, and collecting the riboflavin, wherein the carbon source is vegetable oil or animal oil. A method for producing riboflavin, characterized in that it is waste clay containing 10. A method for producing riboflavin, which comprises culturing a riboflavin-producing bacterium in a medium containing vegetable oil or animal oil as a carbon source, producing and accumulating riboflavin, and collecting the riboflavin, wherein the carbon source is vegetable oil or animal oil. A method for producing riboflavin, characterized in that it is an oil component extracted from a waste white clay encapsulating.