JP6860284B2 - AGP-degrading enzyme-producing bacteria - Google Patents

Description

The present invention relates to arabinogalactan or a microorganism capable of assimilating arabinogalactan protein. Further, the present invention relates to a method for decomposing arabinogalactan or arabinogalactan protein using such a microorganism or an arabinogalactan or arabinogalactan proteolytic enzyme produced by the microorganism, arabinogalactan or arabinogalactan protein degradation. The present invention relates to a method for producing a food or drink in which an agent, arabinogalactan or arabinogalactan protein is decomposed. Furthermore, the present invention relates to a method for screening arabinogalactan or a microorganism capable of assimilating arabinogalactan protein.

Polysaccharides such as galactomannan, arabinogalactan (hereinafter, also referred to as AG), arabinogalactan protein (hereinafter, also referred to as AGP), and cellulose are known as cell wall constituents of plants. For example, in green coffee beans, the content of insoluble polysaccharides is extremely high, and the insoluble polysaccharides and proteins form a complicated structure, so that it is difficult to solubilize green coffee beans.

Solubilizing insoluble polysaccharides is important from the viewpoints of elucidation of the structure and constituents of plant cell walls, application to food materials and functional foods, and effective utilization of unused agricultural products. Specifically, it is conceivable to improve the conversion efficiency of the raw material into a product, and to improve the preferable taste and flavor due to the solubilization of the raw material. For coffee beans, efficient extraction of functional materials from raw bean raw materials, unprecedented changes in the flavor and texture of roasted beans, and effective use of extraction slag, which is an industrial waste, can be considered. ..

AG and AGP are basic sugars found in plants, and although structural models have been shown, techniques for reducing the molecular weight by enzymes and the like are still insufficient. For example, it is speculated that the cell wall of coffee beans has a "repeated multi-layered structure" in which a galactomannan layer, an AGP layer, and a cellulose layer are combined and stuck together, and it is very hard and insoluble, making it a general cellulase. It shows resistance to it.

As a method for enzymatically decomposing the cell wall of coffee beans, a method of repeatedly and stepwise performing a dilute alkali treatment and a cellulase treatment has been reported (Patent Document 1).

Japanese Patent No. 5172095

However, in the AGP of the solubilized fraction after the stepwise enzyme treatment in which the dilute alkali treatment-cellulase treatment is repeated, the terminal arabinose is almost removed, but the molecular weight is hardly reduced. The molecular weight of AGP in this solubilized fraction remains above 1.5 million Da. AGP is present throughout the tissue because the cross section and deep crushed portion formed by cutting or crushing the residue after enzymatic decomposition are also dyed in vermilion with the Yarib reagent. Therefore, the conventional method is not preferable in food processing because the enzyme treatment is very time-consuming and a kind of chemical treatment is performed.

The technique for safely reducing the molecular weight of the cell wall of a plant containing AG or AGP by enzymatic decomposition or the like in terms of food processing by the above-mentioned conventional technique is still insufficient.

An object of the present invention is to provide a microorganism capable of efficiently decomposing, reducing the molecular weight of AG or AGP, particularly high molecular weight, and assimilating it, or a method for screening such a microorganism. Is. Another object of the present invention is to provide a method for decomposing AG or AGP using such a microorganism, and a method for producing a food or drink in which AG or AGP is decomposed.

As a result of diligent studies to solve the above problems, the present inventors conducted screening using a medium containing partially purified AGP as the sole carbon source, and assimilated AG or AGP, which has not been reported so far. We have found a microorganism that has the ability and produces AG or AGP degrading enzyme in the culture medium, and have completed the present invention.

That is, the present invention is a microorganism capable of assimilating AG or AGP, and is obtained by culturing at least 90% or more of the carbon source in a medium derived from arabinogalactan or arabinogalactan protein. It relates to a microorganism belonging to the genus Citinofaga, the genus Ensifer, or the genus Burkholderia.

The present invention also comprises the step of allowing at least one selected from the group consisting of the above-mentioned microorganisms, bacterial cell cultures, and bacterial cell extracts to act on the AG or AGP-containing material, and the AG in the contained material. Or, it relates to the decomposition method of AGP.

Further, the present invention comprises the step of allowing at least one selected from the group consisting of the above-mentioned microorganism, cell culture, and cell extract to act on the AG or AGP-containing material, and the AG or AGP is decomposed. Regarding the manufacturing method of food and drink.

Furthermore, the present invention is a method for screening a microorganism having the ability to assimilate AG or AGP.
A culture step of culturing candidate microorganisms in a medium in which at least 90% or more of the carbon source is derived from AG or AGP, and
The present invention relates to a method comprising an evaluation step of comparing the content of the AG or AGP in the sample before and after the culture.

Furthermore, the present invention relates to an arabinogalactan or arabinogalactan proteolytic agent, which comprises at least one selected from the group consisting of the above-mentioned microorganisms, cell cultures, and cell extracts.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a microorganism belonging to the genus Sinorhizobium, Ensifer, or Burkholderia, which has an assimilation ability for AG or AGP. Further, by utilizing these microorganisms, it is possible to provide a method for decomposing AG or AGP or a method for reducing the molecular weight, and it is possible to produce foods and drinks in which AG or AGP is decomposed or reduced in molecular weight. Furthermore, by utilizing these microorganisms, AG or AGP is decomposed, so that the constituent polysaccharides of the raw material to which AG or AGP is stuck can be solubilized to a higher degree.

It is a figure which shows the HPLC analysis result about the prepared AGP solution. It is a figure which shows the HPLC analysis result in 2 cases of the strain which a reduction of 80% or more with respect to the peak of AGP of a blank was observed as a result of screening with the culture medium containing AGP as the sole carbon source. Chinophaga sp. It is a figure which shows the result of HPLC analysis of the resolution of AGP derived from green coffee beans in KSM45 strain. Chinophaga sp. It is a graph which shows the relationship between the enzyme concentration and the AGP reduction rate about the culture supernatant obtained from the KSM45 strain. Ensifer sp. It is a figure which shows the result of HPLC analysis of the resolution of AGP derived from green coffee beans in KSM81 strain. Ensifer sp. It is a graph which shows the relationship between the enzyme concentration and the AGP reduction rate about the culture supernatant obtained from the KSM81 strain. It is a figure which shows the result of HPLC analysis of the resolution of AGP derived from green coffee beans in Burkholderia stabilis KSM97 strain. It is a graph which shows the relationship between the enzyme concentration and the AGP reduction rate about the culture supernatant obtained from Burkholderia stabilis KSM97 strain. Chinophaga sp. It is a photograph which shows the result of the enzymatic reaction of the alkali-treated coffee bean section in the KSM45 strain. Chinophaga sp. KSM45 strain, Sinorhizobium sp. It is a photograph which shows the result of the enzymatic reaction of the alkali-treated coffee bean section in KSM81 strain, or Burkholderia stabilis KSM97 strain. Using various cellulases, Chinophaga sp. It is a photograph which shows the result of having performed the enzymatic reaction of the coffee bean section in the KSM45 strain. Chinophaga sp. KSM45 strain, Sinorhizobium sp. It is a figure which shows the result of HPLC analysis of the resolution of gum arabic-derived AGP in KSM81 strain, or Burkholderia stabilis KSM97 strain.

[Microorganisms]
The microorganism of the present invention has the ability to assimilate AG or AGP, and is obtained by culturing at least 90% or more of the carbon source in a medium derived from AG or AGP. It belongs to the genus, or the genus Burkholderia.

As used herein, "having the ability to assimilate AG or AGP" means having the ability to grow using AG or AGP as a carbon source. Specifically, for example, when a microorganism can grow in a medium containing AG or AGP as the sole carbon source, it can be said that the microorganism has the ability to assimilate AG or AGP. The period during which the microorganism can grow and grow in a medium containing AG or AGP as the sole carbon source can be at least 24 hours, preferably 2 days or more, more preferably 3 days or more, still more preferably 5 days. It can be the above.

In the present invention, the "medium in which at least 90% or more of the carbon source is derived from AG or AGP" is not limited as long as the medium contains a high concentration of AG or AGP as the carbon source of the medium, but the carbon of the medium is not limited. A medium in which 90% of the source is derived from AG or AGP can be used, preferably 95%, more preferably 98%, and a medium in which AG or AGP is substantially the only carbon source is used. Is particularly preferred. For example, by using a medium containing AGP as a carbon source at a high concentration, a microorganism capable of proliferating can be obtained by assimilating AGP and using it as a nutrient source, instead of simply a microorganism capable of metabolizing and decomposing AGP. Be done.

The Kichinofaga (Chitinophaga) microorganism used in the present invention, as long as the effect of the present invention, but are not limited to, for example, Chitinophaga arvensicola, Chitinophaga ginsengisegetis, Chitinophaga niastensis, Chitinophaga terrae, Chitinophaga eiseniae, Chitinophaga japonensis, Chitinophaga sancti, Chitinophaga rupis, Chitinophaga filiformis, Chitinophaga ginsengisoli, Chitinophaga niabensis, Chitinophaga oryziterae, Chininophia These Variant of Concernophila microorganisms may be naturally occurring strains, variants of natural strains, or genetically modified species as long as the effects of the present invention are exhibited. Catabolism of AG or AGP, from the viewpoint of efficient AG or AGP degradation effects, Chitinophaga arvensicola, Chitinophaga ginsengisegetis, Chitinophaga niastensis, Chitinophaga terrae, Chitinophaga eiseniae, is Chitinophaga japonensis or closely related strains thereof Preferably, Chitinophaga arvensicola or The closely related strain is more preferable.

Further, as the microorganism of the genus Chitinofaga used in the present invention, the effect of the present invention is exhibited in the molecular phylogenetic analysis based on the 16S rDNA base sequence based on the "Rapid identification method of microorganism by gene analysis" of the Japanese Pharmacy. In the range, a strain having a sequence highly homologous to the sequence shown in SEQ ID NO: 1 in the sequence listing is preferable, 90% or more homology is more preferable, and 95% or more homology is further preferable. , 98% or more homology is particularly preferable. A strain having the sequence shown in SEQ ID NO: 1 is particularly preferable as the base sequence of 16S rDNA. In the molecular phylogenetic analysis based on the 16S rDNA base sequence, after determining the 16S rDNA base sequence of the strain, the obtained DNA base sequence is searched for homology using an international base sequence database such as GenBank, DDBJ, or EMBL. It can be done by.

As a specific example of the microorganism belonging to the genus Chitinofaga used in the present invention, Chinophaga sp. A strain having a sequence highly homologous to the entire base sequence of the KSM45 strain (accession number: NITE P-01889) is preferable, 95% or more homology is more preferable, and 98% or more homology is required. Is more preferable, and it is particularly preferable that the homology is 99% or more.

The microorganism of the genus Sinorhizobium used in the present invention is not limited as long as it exerts the effects of the present invention, but is, for example, Examples thereof include Sinorhizobium, Sinorhizobium, Sinorhizobium, Sinorhizobium, Sinorhizobium, Sinorhizobium, or strains closely related biologically and genetically. These microorganisms of the genus Ensifer may be naturally occurring strains, variants of natural strains, or genetically modified species as long as the effects of the present invention are exhibited. From the viewpoint of the assimilation ability of AG or AGP and the efficient decomposition effect of AG or AGP, Ensifer agent, Ensifer mexinanus, Ensifer fredii, Ensifer siherhi, Ensifer medicae, Ensiferhizobium, , Sinorhizobium or a closely related strain thereof is preferable, and Ensifer adhaerens or a closely related strain thereof is more preferable.

Further, as the Ensifer genus microorganism used in the present invention, the effect of the present invention is exhibited in the molecular phylogenetic analysis based on the 16S rDNA base sequence based on the "Rapid identification method of microorganism by gene analysis" of the Japanese Pharmacy. In the range, a strain having a sequence highly homologous to the sequence shown in SEQ ID NO: 2 is preferable, 90% or more homology is more preferable, 95% or more homology is further preferable, and 98%. The above homology is particularly preferable. A strain having the sequence shown in SEQ ID NO: 2 is particularly preferable as the base sequence of 16S rDNA.

Specific examples of the microorganism belonging to the genus Ensifer used in the present invention include Ensifer sp. A strain having a sequence highly homologous to the entire base sequence of the KSM81 strain (accession number: NITE P-01890) is preferable, 95% or more homology is more preferable, and 98% or more homology is required. Is more preferable, and it is particularly preferable that the homology is 99% or more.

The Burkholderia (Burkholderia) microorganism used in the present invention, as long as the effect of the present invention, but are not limited to, for example, Burkholderia stabilis, Burkholderia ambifaria, Burkholderia pirrocinia, Burkholderia metallica, Burkholderia diffusa, Burkholderia contaminans, Burkholderia arboris, Burkholderia seminalis, Burkholderia cepacia, Burkholderia latens, Burkholderia anthina, Burkholderia vietnamiensis, Burkholderia dolosa, Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia oklahomensis, Burkholderia ubonensis, Burkholderia gladioli, Burkholderia glumae, Burkholderia thailandensis, Burkholderia plantarii, Burkholderia pseudomallei, Burkholderia mallei, Burkholderia soridicola, Burkholderia caryophylli, Burkolderia soli, Burkholderia sediminicola, Burkholderia unamae, Burkholdia unanamee, Burkholdia These Burkholderia microorganisms may be naturally occurring strains, variants of natural strains, or genetically modified species as long as the effects of the present invention are exhibited. Catabolism of AG or AGP, from the viewpoint of efficient AG or AGP degradation effects, Burkholderia stabilis, Burkholderia ambifaria, Burkholderia pirrocinia, Burkholderia metallica, Burkholderia diffusa, Burkholderia contaminans, Burkholderia arboris, Burkholderia seminalis, Burkholderia cepacia, Burkholderia latens , Burkholderia anthina, Burkholderia vietnamiensis or its closely related strains are preferred, and Burkholderia stabilis or its closely related strains are even more preferred.

Further, as the Burkholderia genus microorganism used in the present invention, 16S conforming to the "Rapid identification method of microorganism by gene analysis" of the Japanese Pharmacopoeia.
In the molecular phylogenetic analysis based on the rDNA base sequence, a strain having a sequence highly homologous to the sequence shown in SEQ ID NO: 3 is preferable, and 90% or more homology is more preferable, as long as the effect of the present invention is exhibited. , 95% or more homology is more preferable, and 98% or more homology is particularly preferable. A strain having the sequence shown in SEQ ID NO: 3 is particularly preferable as the base sequence of 16S rDNA.

As a specific example of the Burkholderia genus microorganism used in the present invention, it is homologous to the entire base sequence of the Burkholderia stabilis KSM97 strain (accession number: NITE P-01891) within the range in which the effect of the present invention is exhibited. Strains having a high sequence are preferable, 95% or more homology is more preferable, 98% or more homology is further preferable, and 99% or more homology is particularly preferable.

The method for degrading AG or AGP of the present invention is a microorganism itself belonging to the genus Sinorhizobium, Ensifer, or Burkholderia, its cell culture, and / or its cell extract. Includes the step of acting on AG or AGP-containing material.

As used herein, "decomposition" of AG or AGP means that the content of the polymeric AG or AGP in the AG or AGP content is reduced after use as compared to before use of the method of the present invention. Say that. When a microorganism is used, "degradation" of AG or AGP means that AG or AGP is enzymatically decomposed or reduced in molecular weight by an enzyme produced by the microorganism, and AG or AGP is decomposed and metabolized by the microorganism. Including being done or being assimilated. Here, the "polymeric AG or AGP" refers to an AG or AGP having a weight average molecular weight of at least 1.5 million Da or more. The weight average molecular weight is a value measured by a gel permeation chromatogram (GPC) device. Further, here, "reducing the molecular weight of high-molecular-weight AG or AGP" means that AG or AGP having a molecular weight of 1.5 million Da or more changes so as to reduce the molecular weight. For example, it means that AG or AGP having a molecular weight of 1.5 million Da or more is decomposed to about 10 to 100,000 Da. Compared with before use of the decomposition method of the present invention, after use, the AG or AGP content in the AG or AGP content is preferably reduced by 50% or more, preferably 70% or more by weight. Is more preferable, and it is further preferable that the amount is reduced by 80% or more. The AG or AGP content in the AG or AGP content can be analyzed using a known measurement method, and is not limited, but can be obtained from the peak area value of the analysis result when the HPLC method is used. ..

As used herein, the term "AG or AGP-containing substance" refers to a plant-derived substance such as a plant or a part of its constituents, which contains AG or AGP as a constituent polysaccharide. Generally, AG or AGP is known as a major constituent polysaccharide of a plant, but in particular, AGP is very difficult to be decomposed by an enzyme because the polysaccharide and the protein form a complicated constituent. In particular, high molecular weight AG or AGP can hardly be decomposed by an enzyme. Further, the more the AG or AGP-containing substance contains an insoluble polysaccharide component, and the more the high molecular weight polysaccharide component is contained, the more difficult it is to decompose by an enzyme. For example, coffee beans have an extremely high content of insoluble and high molecular weight polysaccharides, and these polysaccharides are adhered to proteins and AGPs to form a complex structure, so that they are decomposed by enzymes. It is said to be very difficult.

[Food and drink in which AG or AGP is decomposed]
According to the present invention, it is possible to produce foods and drinks in which AG or AGP is decomposed. Decomposing AG or AGP in food and drink raw materials has the advantages of disrupting the structure of the constituent polysaccharides, facilitating enzymatic decomposition, facilitating processing, and increasing the solubilization of the constituent polysaccharides of the raw materials. Since it is expected, it will be possible to manufacture processed products that have never existed before. For example, by decomposing plant seeds such as coffee beans and AG or AGP contained in the leaves, stems, roots, flowers, etc. of plants, the cell wall is formed by enzymatic decomposition of cell wall-constituting polysaccharides using cellulase or the like. Since it is easily disintegrated and decomposed, it is possible to provide a confectionery material with reduced hardness. In addition, the constituent polysaccharides are decomposed into oligosaccharides and monosaccharides, which makes it possible to improve the extraction efficiency and the conversion efficiency into products.

Further, according to the present invention, AG or AGP is decomposed to enable at least partial solubilization and low molecular weight of food and drink raw materials. Therefore, a product having a taste and aroma different from that of conventional foods and drinks can be produced. Is possible. Further, according to the present invention, since the plant can be partially or completely solubilized, there is an advantage that the components contained in the raw material plant can be easily extracted. It is also possible to use a solubilized product of a raw material plant to make a functional food or drink, or to use it as a raw material for cosmetics, clothing, and pharmaceuticals.

The solubilized plant product can be provided in various forms such as liquid, gel, solid and powder. The solubilized plant product can also be formed into granules, granules, tablets, capsules, pastes and the like by appropriately using known additives. In addition, solubilized plant products can be used by adding to various foods such as processed meat foods such as ham and sausage, processed marine foods such as kamaboko and chikuwa, bread, confectionery, butter, and milk powder, and water. It can also be used by adding it to beverages such as fruit juice, milk, soft drinks, and tea beverages.

When the microorganisms belonging to the genus Sinorhizobium, Ensifer, or Burkholderia themselves are allowed to act on the AG or AGP-containing material, the method is not limited, but for example, these microorganisms and AG. Alternatively, the AGP-containing material may be cultured in the same container. Since the microorganism of the present invention can proliferate by assimilating AG or AGP, there is an advantage that only the inoculum (starter) needs to act.

When the microorganism of the present invention and the AG or AGP-containing material are cultured in the same container, the weight ratio thereof is appropriately changed depending on the type of the AG or AGP-containing material and the like. For example, when the AG or AGP-containing substance is green coffee beans, about 100 g to 10000 g of a section or pulverized product of green coffee beans can be added to the medium for culturing with respect to 1 g of the dry weight of the microorganism of the present invention. it can.

As a type of medium, a known nutrient medium can be used, but a medium having a small amount of carbon source components other than AG or AGP is preferable, and a medium containing no carbon source component other than AG or AGP is preferable. More preferred. Specifically, a liquid medium containing 0.1% by weight of AGP, 0.01% by weight of polypeptone, and 0.1% by weight of NH ₄ NO _{3 can be used.}

When a cell culture of a microorganism belonging to the genus Chitinophaga, Enzyme, or Burkholderia is allowed to act on an AG or AGP-containing material, the method is not limited, but for example, a microorganism. The culture supernatant of the above is directly or diluted to react with AG or AGP-containing material, or the enzyme in the culture supernatant is extracted, and in some cases, the enzyme is purified and then the enzyme is directly or diluted. Reaction with AG or AGP-containing material can be mentioned.

The culture supernatant of the microorganism can be obtained, for example, by culturing the microorganism in a known liquid for 1 day and then centrifuging. The period for culturing to obtain this culture supernatant is appropriately changed depending on the type of microorganism, enzyme production ability, and the like, but can be at least 1 day, preferably at least 3 days, and more preferably at least 5 days.

Although not limited, for example, about 2 mg of microorganisms are inoculated with a platinum loop into a liquid medium containing 0.1% by weight of AGP, 0.01% by weight of polypeptone, and 0.1% by weight of NH ₄ NO _{3 and cultured for 1 day.} After that, the culture supernatant of the microorganism can be obtained by performing centrifugation.

When a bacterial cell extract of a microorganism belonging to the genus Sinorhizobium, Ensifer, or Burkholderia is allowed to act on an AG or AGP-containing substance, the method is not limited, but for example, a microorganism. The cells of Sinorhizobium can be crushed with a homogenizer or the like, and the contents of the cells can be dissolved in an appropriate solvent such as water, an organic solvent or a hydrous organic solvent before use. Examples of these extraction solvents include water, alcohol, or a mixture thereof, preferably water, methanol, ethanol, 1,3-butylene glycol, propylene glycol, glycerin, or a mixture thereof, and water, ethanol, or a mixture thereof. Ethanol is more preferred.

These cell cultures or cell extracts can be appropriately diluted or concentrated depending on the mode of use. In addition, these cell cultures or cell extracts can be purified by further subjecting them to purification treatment before and after dilution or concentration. In the purification treatment, known separation / purification methods can be appropriately combined and used. For example, salting out such as ammonium sulfate precipitation method, gel filtration method by sephadex or the like, diethylaminoethyl group or carboxymethyl group and the like can be used. Ion exchange chromatography method using a carrier, hydrophobic chromatography method using a carrier having a hydrophobic group such as butyl group, octyl group, phenyl group, dye gel chromatography method, electrophoresis method, dialysis, limitation It is possible to use an external filtration method, an affinity chromatography method, a high-speed liquid chromatography method, or the like.

When the chromatograph method is used, for example, either column chromatography using a normal phase or reverse phase carrier or an ion exchange resin, high performance liquid chromatography, thin layer chromatography, centrifugal liquid chromatography, or the like, or A method of using them in combination can be mentioned. When the chromatographic method is used, the purification conditions such as the carrier and the elution solvent can be appropriately selected according to various chromatographic methods.

When the microorganism of the present invention itself, its cell culture or its cell extract is allowed to act on an AG or AGP-containing substance, the number of microorganisms is not limited as long as the effect of the present invention is exhibited, but for example, for example. about 10 ⁵ cells / g to about ^{10 14} / g, preferably possible to use the number of bacteria which corresponds to about 10 ⁸ / g to about ^{10 12} / g.

When the microorganism of the present invention itself, its cell culture, and / or its cell extract is allowed to act on an AG or AGP-containing substance, the action time is the amount of the microorganism cells and the amount of the enzyme in the culture supernatant. It can be appropriately changed depending on the reaction temperature, pH of the reaction solution, etc., but for example, it can be at least 1 hour, preferably at least 10 hours or more, more preferably at least 24 hours or more, and at least 2 hours. It is more preferably days or more, more preferably at least 3 days or more, particularly preferably at least 4 days or more, and most preferably at least 5 days or more.

When the microorganism of the present invention itself, its cell culture, and / or its cell extract is allowed to act on an AG or AGP-containing substance, the operating temperature depends on the temperature suitable for the growth of the microorganism and the optimum temperature of the enzyme. Although it can be changed as appropriate, it can be operated at, for example, 5 ° C to 50 ° C, preferably 10 ° C to 45 ° C, more preferably 20 ° C to 40 ° C, and 25 ° C to 38 ° C. More preferred.

When the microorganism of the present invention itself, its cell culture, and / or its cell extract is allowed to act on an AG or AGP-containing substance, the pH in the medium or solution is a pH suitable for the growth of the microorganism or an enzyme. Although it can be appropriately changed depending on the optimum temperature, it can be usually 6 to 8, preferably 6.8 to 7.5. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

The weight ratio when the microorganism of the present invention itself, the cell culture thereof, and / or the cell extract thereof is allowed to act on the AG or AGP-containing material depends on the type of the microorganism and the type and state of the AG or AGP-containing material. Although it is changed as appropriate, when the microorganism itself is allowed to act, the dry weight can be about 100 to 10,000 g of AG or AGP-containing substance with respect to about 0.1 to 1.0 g of the microorganism, and the bacterial cells of the microorganism. When the culture or bacterial cell extract is allowed to act, the dry weight can be about 100 to 10,000 g of AG or AGP-containing material with respect to about 0.1 to 1.0 g of microorganisms.

In the present invention, as the AG or AGP-containing material, it is preferable to use a cut product or a pulverized product in order to increase the contact area with microorganisms and enzymes. A known method such as a mixer can be used for cutting or crushing, but when the cut material is a large scale such as 1 kg to 10 kg, a large vertical cutter mixer or the like can be used, and several tens of g to In the case of a small scale of several hundred g, a household mixer or the like can be used. For example, when applying crushing conditions on a large scale, the crushing time is tens of seconds to several minutes, and the rotation speed of the mixer is 10 to 3000 rpm, although it is appropriately changed depending on the hardness of the raw material, the water content, etc. It is possible to do.

In the present invention, it is possible to include a step of subjecting the AG or AGP-containing material to a cellulase treatment for the purpose of further enhancing the decomposition effect of AG or AGP. By performing the cellulase treatment, polysaccharides such as cellulose, mannan and galactomannan, which are constituent sugars of the plant, are decomposed, the contact area between the plant and the enzyme produced by the microorganism is increased, and the AG or AGP assimilation of the present invention is carried out. It is expected that it will be easier to demonstrate its ability. The step of applying the cellulase treatment can be carried out simultaneously or after the step of allowing the microorganism of the present invention itself, the cell culture thereof, and / or the cell extract thereof to act on the AG or AGP-containing material. .. When AG or AGP is decomposed by the enzyme produced by the microorganism of the present invention and the cell wall constituents are loosened, cellulase easily permeates. Therefore, the step of performing the cellulase treatment includes the microorganism itself of the present invention, its cell culture, and the cell culture. / Or the cell extract thereof is preferably carried out at the same time as or after the step of acting on the AG or AGP-containing material.

The cellulase that can be used in the cellulase treatment step can be used by separating and purifying the cellulase in the natural world, or a commercially available product can be purchased and used. The cellulase is appropriately changed depending on the type of AG or AGP-containing substance used, and is not limited, but for example, Trichoderma viride-derived cellulase, Trichoderma reesei-derived cellulase, Trichoderma sp. Cellulase derived from Aspergillus niger, cellulase derived from Aspergillus oryzae r, cellulase derived from Aspergillus aculeatus, Cellulase derived from Aspergillus sp. Examples thereof include cellulase derived from Hirondellea gigas and cellulase derived from Hirondellea gigas. Examples of commercially available cellulase products include GODO-TCF (manufactured by Godo Sake Seisha), Sumiteam C (manufactured by Shin Nihon Kagaku Kogyo Co., Ltd.), Meiserase (manufactured by Meiji Seika Co., Ltd.), Multifect (manufactured by Danisco Japan), and Cellulase A " Amano ”3 (manufactured by Amano Enzyme), cellulase XL-531 (manufactured by Nagase ChemteX) and the like can be mentioned.

In the cellulase treatment, the reaction temperature can be, for example, 5 ° C. to 50 ° C., preferably 10 ° C. to 45 ° C., more preferably 20 ° C. to 40 ° C., and 25 ° C. to 38 ° C. ℃ is more preferable. In the cellulase treatment, the reaction time can be at least 1 hour, preferably at least 10 hours or more, more preferably at least 24 hours or more, further preferably at least 2 days or more, and at least. It is more preferably 3 days or more, particularly preferably at least 4 days or more, and most preferably at least 5 days or more.

In the present invention, it is possible to include a heating step for the AG or AGP-containing material for the purpose of further enhancing the decomposition effect of AG or AGP. As a result, it is expected that the enzymes originally contained in the AG or AGP-containing material will be reduced in activity or inactivated, and the enzymes produced by the microorganisms will be more likely to act. In addition, it is expected that the above-mentioned cellulase will be more likely to act by reducing or inactivating the enzymes originally contained in the AG or AGP-containing material.

The heating temperature and heating time in the heating step are not limited as long as the temperature and time are such that the enzymes originally contained in the AG or AGP-containing material cause a decrease in activity or deactivation, but for example, at 60 ° C. for 15 minutes or more. It is preferable that the temperature is 70 ° C. for 15 minutes or longer.

A known heating method and heating device can be adopted in the heating step, and the heating step is not limited, and examples thereof include a high-pressure steam sterilizer (autoclave), a constant temperature bath, and a heat block. When a high-pressure steam sterilizer is used, it can be heated at 121 ° C., 2 atm, and 20 minutes.

When the AG or AGP-containing material contains chlorogenic acids, it is possible to further include a hot water extraction step for the AG or AGP-containing material for the purpose of extracting the chlorogenic acids as a functional component without being oxidized in advance. .. The AG or AGP-containing material containing chlorogenic acids is not limited, and examples thereof include green coffee beans. As a result, it is expected that the decomposition of the AG or AGP-containing material can proceed without oxidizing the chlorogenic acids contained in the AG or AGP-containing material.

In the present specification, in the hot water extraction step for AG or AGP-containing material containing chlorogenic acids, known methods can be used as long as chlorogenic acids can be reduced. Although not limited, hot water extraction can be performed by adding hot water to the AG or AGP-containing material, mixing the AG or AGP-containing material with water, and then heating the mixture. Is.

Although not limited, the temperature of hot water in the hot water extraction step can be, for example, 25 ° C to 100 ° C, preferably 45 ° C to 99 ° C, and even more preferably 80 ° C to 98 ° C. In the hot water extraction step for AG or AGP-containing material containing chlorogenic acids, the extraction time can be at least 3 minutes, preferably at least 5 minutes or more, and more preferably at least 10 minutes or more. It is more preferably at least 15 minutes or more, further preferably at least 20 minutes or more, particularly preferably at least 25 minutes or more, and most preferably at least 30 minutes or more.

After the hot water extraction step, the extracted supernatant contains chlorogenic acid, polyphenols, sucrose, trigonelline, caffeine, etc., and chlorogenic acid is obtained by separating the extract supernatant and the residue. Can be removed together with the extract supernatant. A known method can be used for such a separation step. Although not limited, for example, centrifugation and the like can be mentioned.

By allowing the microorganism of the present invention itself, its cell culture, and / or its cell extract to act on the AG or AGP-containing material, the polysaccharides, oligosaccharides, which constitute them from the AG or AGP-containing material, Alternatively, it is possible to obtain monosaccharides. Although not limited, the microorganism itself of the present invention, its cell culture, and / or its cell extract are allowed to act on an AG or AGP-containing substance to carry out an enzymatic reaction to obtain the enzymatic reaction product. By performing a separation step such as centrifugation on the enzyme reaction product, polysaccharides, oligosaccharides, monosaccharides and the like can be obtained as water-soluble solids. It is also possible to obtain polysaccharides, oligosaccharides, monosaccharides and the like by adding an organic solvent that can be mixed with alcohol such as ethanol or water such as acetone to the enzyme reaction product and precipitating it.

The more the AG or AGP-containing material contains an insoluble polysaccharide component and the more the high-molecular-weight polysaccharide component is contained, the more difficult it is to decompose by a conventionally used enzyme. Therefore, the AG or AGP-containing material is classified according to the present invention. If polysaccharides, oligosaccharides, or monosaccharides can be decomposed to obtain polysaccharides, oligosaccharides, or monosaccharides, it is possible to develop new products using AG or AGP-containing substances, to recycle previously discarded AG or AGP-containing substances, and the like.

It is also possible to produce instant products using the obtained polysaccharides and oligosaccharides. The instant product is not limited, and examples thereof include instant foods and instant coffee.

For example, instant coffee is produced by leaching the extraction solution into a cell (column) filled with roasted and crushed coffee beans, concentrating the obtained coffee extract, and then freeze-drying or spray-drying (see Sivetz as a reference). , Coffee Processing Technology, Volume 1, p262-p263, AVI, 1963, etc.). There are various manufacturing methods for instant coffee, but the common one is the "extraction process that mainly recovers aroma from coffee beans" performed under normal pressure conditions at 100 ° C, and the temperature of 150 to 180 ° C, for example. The point is that there is a "step of extracting hydrolyzed coffee bean cell wall components" performed under pressure conditions. The reason why there is an extraction process under pressure conditions of a temperature of 150 to 180 ° C. is to secure the coffee bean cell wall component necessary for forming a solid of instant coffee after drying and for preventing the dissipation of aroma components. To do. There is always a concentration step in the production of instant coffee, and in order to form hard instant coffee, a certain solid concentration is required at the liquid stage before drying. However, if the extract obtained by atmospheric pressure extraction is excessively concentrated, the aroma component of coffee disappears. On the other hand, if an attempt is made to excessively extract the extract (coffee bean cell wall component) from the pressure extraction, the taste is deteriorated by various substances produced by hydrolyzing the coffee beans. That is, the extract obtained under high temperature and high pressure conditions adversely affects the flavor and aroma of instant coffee. Therefore, by using green coffee bean components such as polysaccharides produced by an enzymatic reaction instead of high temperature and high pressure conditions for the production of instant coffee, the taste and aroma of coffee are not adversely affected, and a large-scale plant is not required. Manufacture under conditions becomes possible.

Examples of the method for producing instant coffee include mixing an extract of roasted bean coffee with a polysaccharide or oligosaccharide obtained by enzymatically decomposing an AG or AGP-containing substance. This makes it possible to produce instant coffee while retaining the original flavor and aroma of green coffee beans. By drying the mixture of the obtained polysaccharide or oligosaccharide and the roasted bean coffee extract, solid instant coffee can be obtained. Therefore, in another embodiment of the present invention, a step of enzymatically reacting at least one selected from the group consisting of the above-mentioned microorganism, bacterial cell culture, and bacterial cell extract with green coffee beans, said coffee. It is possible to provide a method for producing instant coffee, which comprises a step of mixing an enzymatically decomposed product of raw beans and a coffee extract, and a step of drying the mixture. The same applies to the preparation of microorganisms, cell cultures, or cell extracts that can be used.

In the step of mixing the enzymatic decomposition product of green coffee beans and the coffee extract, the coffee extract can be obtained by extracting roasted coffee beans with hot water, and the concentrate thereof can also be used. Is. When the enzymatic decomposition product of green coffee beans and the coffee extract are mixed, the mixing ratio is not limited. For example, when a roasted bean coffee extract (concentrate) having a Brix of 25.3 is used, this roasting is performed. The enzymatic decomposition product of green coffee beans can be mixed so as to have a ratio of 1 to 20% (w / w) with respect to the solid content dissolved in the roasted soybean coffee extract (concentrate). It is preferably ~ 15% (w / w), preferably 2-15% (w / w), and even more preferably 5-10% (w / w).

In the step of drying the mixture, a known method can be used as the drying method, and examples thereof include, but are not limited to, natural drying, spray drying, heat drying, vacuum drying, vacuum drying, and freeze drying. These can be used alone or in combination.

In addition, polysaccharides and oligosaccharides obtained from AG or AGP-containing substances are useful as prebiotic materials, and are applied to food materials, supplement materials, cosmetic materials, or in the pharmaceutical field such as sugar chain engineering. Can also be used. The obtained polysaccharides and oligosaccharides can be blended in, for example, processed foods and cosmetics, and can be used for stabilizing the shape and emulsifying oil and water. These polysaccharides and oligosaccharides are useful as excipients and stabilizers. When green coffee beans are used as the AG or AGP-containing material, for example, polyphenols and the like that are susceptible to oxidation are extracted before the enzymatic reaction, and the microorganism itself of the present invention itself and its cells are subjected to the extraction residue. Prebiotics containing polyphenols, etc., whose deterioration due to oxygen is prevented by mixing the polyphenols, etc. extracted earlier after performing an enzymatic reaction by allowing the culture and / or the bacterial cell extract thereof to act. It is possible to manufacture the material. In addition, polysaccharides and oligosaccharides obtained from green coffee beans are various health-related polysaccharides derived from green coffee beans and oligosaccharides derived from green coffee beans due to their expected indigestibility and improvement of intestinal bacteria. Can be used for food and drink.

[Screening method]
The present invention also provides a method for screening a microorganism having an AG or AGP assimilation ability. The screening method of the present invention includes a culturing step of culturing a candidate microorganism in a medium in which at least 90% or more of the carbon source is derived from AG or AGP.

The medium used in the culturing step is not limited as long as it contains a high concentration of AG or AGP as the carbon source of the medium, but a medium in which 90% of the carbon source of the medium is derived from AG or AGP should be used. It can be preferably 95%, more preferably 98%, and it is particularly preferable to use a medium in which AG or AGP is substantially the only carbon source. For example, by using a medium containing AGP as the sole carbon source, it is possible to select microorganisms capable of growing by assimilating AGP and using it as a nutrient source, not simply microorganisms capable of metabolizing and decomposing AGP. Is possible.

AG or AGP can be extracted from a plant and used, or a commercially available product can be purchased and used. It is also possible to utilize a plant tissue containing AG or AGP. When AG or AGP is extracted from a plant and used, it is preferable to use AGP derived from green coffee beans for the purpose of selecting microorganisms having higher AG or AGP assimilation ability. Since AGP derived from green coffee beans is insoluble and has a high molecular weight, it is presumed that it is more difficult for microorganisms to assimilate it.

In the present invention, the content of AG or AGP contained in the medium containing AG or AGP as the main carbon source is not limited, but is 0 from the viewpoint of selecting microorganisms having higher AG or AGP assimilation ability. It can be .001 to 10% by weight, preferably 0.01 to 1% by weight, more preferably 0.05 to 0.5% by weight.

In the present invention, the medium containing AG or AGP as the main carbon source contains an ammonium salt of an inorganic acid or an organic acid such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, sodium nitrate, and ammonium phosphate as the nitrogen source. Alternatively, in addition to other nitrogen-containing compounds, urea, peptone, polypeptone, meat extract, corn steep liquor, yeast extract, dried yeast, soybean flour, cazamino acid, amino acids and the like are used. Of these nitrogen sources, peptone and polypeptone are preferable, and polypeptone is more preferable, from the viewpoint of ease of handling. Examples of inorganic salts include primary potassium phosphate, dipotassium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, and copper sulfate. , Calcium carbonate and the like are used.

In the present invention, the pH of the medium containing AG or AGP as the main carbon source can be usually 6 to 8, preferably 6.8 to 7.5. The pH of the medium can be adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

The culture temperature can be appropriately changed depending on the temperature suitable for the growth of microorganisms and the optimum temperature for the enzyme, but can be allowed to act at, for example, 5 ° C. to 50 ° C., preferably 10 ° C. to 45 ° C., 20 ° C. More preferably, it is ℃ -40 ℃.

The culturing time can be appropriately changed depending on the type of microorganism and the like, but can be, for example, at least 1 hour, preferably at least 10 hours or more, more preferably at least 24 hours or more, and at least 2 days or more. It is more preferable that it is at least 3 days or more, and it is particularly preferable that it is at least 5 days or more.

The culture can also be aerated or agitated, if desired.

The screening method of the present invention includes an evaluation step of comparing the content of AG or AGP in the sample before and after culturing the candidate microorganism.

In the present invention, the AG or AGP content in the sample in the evaluation step can be analyzed by a known method and is not limited, for example, by high performance liquid chromatography (HPLC) method, chemical phenol sulfate. It can be analyzed by a method, an immunoblotting method, or the like.

In the evaluation step, when the content of AG or AGP in the sample after culturing the cells of the candidate microorganism as compared with that before culturing shows a decrease of at least 50% by weight, the microorganism is selected. It can be determined that it has the ability to assimilate AG or AGP. In the present invention, in the evaluation step, it is possible to add that the candidate microorganism can grow and grow for at least 5 days as a criterion for determination.

The screening method of the present invention can further include an enzymatic reaction step of acting on AG or AGP to decompose at least one selected from the group consisting of bacterial cells of candidate microorganisms and bacterial cell cultures thereof.

In the present invention, in the enzymatic reaction step, it is possible to evaluate the AG or AGP resolution of the candidate microorganism by the enzymatic reaction using the bacterial cells and / or the culture supernatant of the candidate microorganism as a substrate. For example, AGP can be added to a phosphate buffer solution (0.1 M, pH 7) so as to have 0.1% AGP, and an enzymatic reaction can be carried out with the bacterial cells and / or the culture supernatant of the candidate microorganism.

The reaction time can be appropriately changed depending on the temperature suitable for the growth of microorganisms and the optimum temperature for the enzyme, but can be at least 1 hour, preferably at least 3 hours, and more preferably at least 24 hours. ..

The reaction temperature can be appropriately changed depending on the temperature suitable for the growth of microorganisms and the optimum temperature for the enzyme, but can be allowed to act at, for example, 5 ° C. to 50 ° C., preferably 10 ° C. to 45 ° C., 20 ° C. More preferably, it is ℃ -40 ℃.

The evaluation method in the enzymatic reaction can be carried out by comparing the content of AG or AGP before and after the reaction in the same manner as the evaluation method in the above-mentioned culture step.

[AG or AGP decomposing agent]
INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an AG or AGP degrading agent containing at least one selected from the group consisting of the above-mentioned microorganism, cell culture, and cell extract. The same applies to the preparation of microorganisms, cell cultures, or cell extracts that can be used. By using a known method, such AG or AGP decomposing agent can be used as a food waste treatment agent, a plant waste treatment agent, a compost decomposition accelerator, a deodorant, a food or drink, a health food, a food additive, or the like. It can be provided as a cosmetic, a pharmaceutical product, a quasi-drug, or the like.

In the present specification, AG or AGP decomposing agents are used for food waste treatment agents, plant waste treatment agents, compost decomposition accelerators, deodorants, pharmaceuticals, non-pharmaceutical products, cosmetics, food additives, health foods, etc. In the case, it can be processed by a known method, and can be used as, for example, tablets, powders, powders, granules, fine granules, capsules, beverages, dairy products, gums, kitchen wastes and the like. When used as a health food, it can be used as a food for specified health use, a dietary supplement food, a functional food, a food with a functional claim, a food for the sick, and the like.

When an AG or AGP decomposing agent is used as a food waste treatment agent, the food waste includes, but is not limited to, plant-derived food waste, and in particular, plant-derived food waste rich in AG or AGP. preferable. Plant-derived food waste and vegetable waste include, for example, coffee grounds, beer grounds, shochu grounds, whiskey grounds, sake grounds, okara, green tea grounds, tea grounds, wheat tea grounds, corn starch by-products, potato ground meal, and cansho. Examples include starch dregs, paper sludge, bark, ogakuzu, fir, rice bran, wheat straw and the like. Coffee waste is preferable as kitchen waste and vegetable waste from the viewpoint that the content of the insoluble polysaccharide is extremely high and the insoluble polysaccharide and the protein form a complicated structure.

In recent years, consumption of coffee has been increasing at home and abroad, mainly in developed countries, and especially in Japan, it is also provided in large quantities at convenience stores and fast food stores. At convenience stores and fast food stores, a large amount of kitchen waste after coffee is extracted is discarded at each store. It is estimated that more than 100,000 tons of plant waste related to coffee beans, including coffee bean processing factories, is produced annually. These wastes can be decomposed and processed into other products, or the waste can be converted into biomass. It would be industrially useful if sugars such as polysaccharides and oligosaccharides could be extracted and used.

When the AG or AGP degrading agent is applied to the AG or AGP-containing material, the method is not limited as long as the effect of the present invention is obtained, but it is selected from the group consisting of the above-mentioned microorganisms, cell cultures, and cell extracts. It is possible to mix with at least one of the above. Alternatively, at least one selected from the group consisting of microorganisms, bacterial cell cultures, and bacterial cell extracts is processed into a liquid preparation by a known method, or is appropriately dried into tablets, powders, or powders. It can also be mixed with AG or AGP-containing materials by processing. In addition, when the above-mentioned microorganism itself is applied to an AG or AGP-containing substance, due to the property that the above-mentioned microorganism can assimilate the AG or AGP-containing substance, the above-mentioned microorganism can be continuously administered only by a single administration at the start of treatment. It is possible to use it. It is also possible to administer and use the new microorganisms on a regular basis, such as once every few months or years.

When an AG or AGP degrading agent is applied to an AG or AGP-containing material, a group consisting of microorganisms, a cell culture, and a cell extract while stirring by a known method in order to uniformly exert the effect of the present invention. It is also possible to mix at least one selected from the AG or AGP-containing material. In this case, the temperature conditions, pH conditions, mixing time conditions, etc. are the same as described above.

When the above AG or AGP degrading agent is allowed to act on AG or AGP-containing substances, it is useful to use it together with cellulase. The types and conditions of cellulase that can be used are the same as above. The AG or AGP degrading agent and cellulase can be processed as one preparation, or can be processed as separate preparations and used in combination at the time of use. When the AG or AGP degrading agent and cellulase are used in combination as separate preparations, either one can be allowed to act on the AG or AGP-containing material first, and at the same time, it can be allowed to act on the AG or AGP-containing material at the same time. Is.

According to another embodiment of the present invention, an AG or AGP-containing product containing an AG or AGP degrading agent, which comprises at least one selected from the group consisting of the above-mentioned microorganism, cell culture, and cell extract. It is possible to provide a kit for disassembling. Such a kit can further include cellulase, a container for an enzyme reaction, a product description, and the like.

With respect to the embodiments described above, the present invention discloses the following microorganisms and methods.

A microorganism capable of assimilating AG or AGP, obtained by culturing at least 90% or more of the carbon source in a medium derived from AG or AGP, and is obtained by culturing in a medium derived from AG or AGP. A microorganism belonging to the genus Burkholderia.

The microorganism in which the AG or AGP is polymer.

The microorganism in which the AG or AGP has a molecular weight of at least 1.5 million Da.

The microorganism according to any one of the above, wherein the AG or AGP is derived from green coffee beans.

The microorganism according to any one of the above, wherein the microorganism belonging to the genus Chinophaga is a strain closely related to Chinophaga arvensicola.

The microorganism belonging to the genus Chinophaga is a strain having 90% or more homology with the base sequence set forth in SEQ ID NO: 1 in the sequence listing in a molecular phylogenetic analysis based on the 16S rDNA base sequence. The microorganism according to any of the above.

The microorganism according to any one of the above, wherein the microorganism belonging to the genus Sinorhizobium is a strain closely related to Sinorhizobium.

The microorganism belonging to the genus Ensifer is a strain having 90% or more homology with the base sequence shown in SEQ ID NO: 2 in the sequence listing in a molecular phylogenetic analysis based on the 16S rDNA base sequence. The microorganism according to any of the above.

The microorganism according to any one of the above, wherein the Burkholderia genus microorganism is Burkholderia stabilis or a strain closely related thereto.

The Burkholderia genus microorganism is characterized in that it is a strain having 90% or more homology with the base sequence shown in SEQ ID NO: 3 in the sequence listing in the molecular phylogenetic analysis based on the 16S rDNA base sequence. The microorganism according to any of the above.

The sequences of the 16S-rDNA genes in the microorganisms belonging to the genus Chitinophaga, Ensifer, or Burkholderia are listed in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively, in the sequence listing. The microorganism according to any of the above, which comprises the described base sequence.

Contains AG or AGP at least one selected from the group consisting of microorganisms belonging to the genus Sinorhizobium, Ensifer, or Burkholderia, bacterial cell cultures, and bacterial cell extracts. A method for decomposing AG or AGP in a substance, which comprises a step of causing the substance to act on the substance.

Contains AG or AGP at least one selected from the group consisting of microorganisms belonging to the genus Sinorhizobium, Ensifer, or Burkholderia, bacterial cell cultures, and bacterial cell extracts. A method for producing a food or drink in which AG or AGP is decomposed, which comprises a step of acting on an object.

At least one selected from the group consisting of microorganisms belonging to the genus Sinorhizobium, Ensifer, or Burkholderia, bacterial cell cultures, and bacterial cell extracts, AG or AGP. A method for decomposing AG or AGP and solubilizing the constituent polysaccharides of the plant tissue, which comprises a step of acting on the contained plant tissue.

The above method, wherein the AG or AGP is polymeric.

The method according to any of the above, wherein the AG or AGP has a molecular weight of at least 1.5 million Da.

The method according to any one of the above, wherein the AG or AGP-containing material is green coffee beans.

The method according to any of the above, further comprising a heating step of heating the AG or AGP-containing material.

The method according to any one of the above, wherein the heating step is carried out by a high pressure steam sterilizer under the conditions of 121 ° C., 2 atm and 20 minutes.

The method according to any one of the above, further comprising a step of subjecting the AG or AGP-containing material to a cellulase treatment.

The cellulase is Trichoderma viride-derived cellulase, Trichoderma reesei-derived cellulase, Trichoderma sp. Cellulase derived from Aspergillus niger, cellulase derived from Aspergillus oryzae, cellulase derived from Aspergillus aculeatus, Cellulase derived from Aspergillus sp. The method according to any of the above, which is a cellulase derived from a cellulase derived from Hirondellea gigas or a cellulase derived from Hirondellea gigas.

The step of performing the cellulase treatment is simultaneous with or at the same time as the step of allowing at least one selected from the group consisting of the microorganism, the bacterial cell culture, and the bacterial cell extract according to any one of the above to act on the AG or AGP-containing material. The method according to any of the above, characterized in that it is performed later.

The method according to any one of the above, wherein the heating step is performed before or at the same time as the step of performing the cellulase treatment.

A screening method for microorganisms having the ability to assimilate AG or AGP.
A culture step of culturing candidate microorganisms in a medium in which at least 90% or more of the carbon source is derived from AG or AGP, and
A method comprising an evaluation step of comparing the content of the AG or AGP in a sample before and after the culture.

The method according to any one of the above, wherein the AG or AGP contained in the medium is polymer.

The method according to any of the above, wherein the AG or AGP contained in the medium has a molecular weight of at least 1.5 million Da.

The method according to any one of the above, wherein the AG or AGP contained in the medium is derived from green coffee beans.

The method according to any of the above, wherein the only carbon source in the medium is derived from AG or AGP.

The method according to any one of the above, wherein the culturing time in the culturing step is at least 10 hours or more.

The method according to any one of the above, wherein the evaluation step is based on the fact that the content of AG or AGP shows a reduction amount of at least 50% by weight.

The method according to any one of the above, wherein the evaluation step is further based on the fact that the candidate microorganism can proliferate and grow for at least 5 days in the culture step.

The method according to any one of the above, further comprising an enzymatic reaction step of reacting and degrading at least one selected from the group consisting of bacterial cells of a candidate microorganism and a bacterial cell culture thereof on AG or AGP.

The method according to any one of the above, wherein the action time in the enzyme reaction step is at least 1 hour or more.

Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following Examples.

1. 1. Preparation of AGP from Green Coffee Beans 506.84 g of green coffee beans (UCC Ueshima Coffee Co., Ltd., Indonesia AP-1) was degreased with 1 L of hexane overnight to obtain 468.39 g of green coffee beans. It was. 2.5 L of 0.1 M NaOH was added thereto, and the mixture was autoclaved (121 ° C., 20 minutes). Next, the residue was washed with tap water and distilled water, and the residue was collected by suction filtration (the supernatant was disposed of) and divided in half (A1, A2). To each of these, 2.5 L of acetic acid buffer (0.1 M (after addition), pH 5.0) and 1% by weight of cellulase were added, and the mixture was reacted overnight (37 ° C., 24 hours). After completion of the reaction, the supernatant was collected by decantation, the cellulase was inactivated by autoclave (121 ° C., 20 minutes), the protein was precipitated, and the supernatant was obtained by centrifugation (10000 rpm, 7 minutes) (A1: Approximately 2800 mL, A2: Approximately 2000 mL). This supernatant was adjusted to pH 4.5 to prevent spoilage, and concentrated with an ultrafiltration membrane (manufactured by SARTORIUS, VIVAFLOW 200) to about 500 mL each. This was centrifuged (10000 rpm, 10 minutes), and the supernatant was placed in a centrifuge tube and dialyzed overnight (water was exchanged twice) to obtain an internal dialysis solution. This was used as the prepared AGP below (A1: about 580 mL, A2: about 660 mL).

2. Measurement of total sugar amount (TS: Total Sugar) The total sugar amount in the obtained prepared AGP solution was measured by the phenol-sulfuric acid method. 12.5, 25, 50, 100, and 200 μg / mL standard sugars by appropriately diluting a stock solution with a concentration of 10.0 mg / mL, which is obtained by dissolving 10.0 mg of dried special grade glucose in distilled water to make 1 mL. The solution was prepared. 100 μL of standard sugar solution and 100 μL of 5% phenol were taken in a test tube, 500 μL of concentrated sulfuric acid was added so as to directly hit the liquid surface, and the mixture was mixed well. Since it generates heat strongly, after allowing it to cool at room temperature, 200 μL was taken on a microplate, and the absorbance at 492 nm was measured with a microplate reader (Micro Plate Reader EZS-ABS manufactured by Asahi Techno Glass Co., Ltd.) to prepare a calibration curve. Next, the prepared AGP prepared above was similarly colored, the total sugar amount was determined from the calibration curve, and the results are shown in Table 1.

As shown in Table 1, the total sugar content of A1 is 139 μg / mL, the total sugar content of A2 is 120 μg / mL, and the yield (assuming the weight of defatted pulverized coffee green beans is 100%) is 3.41%. there were.

3. 3. High Performance Liquid Chromatography (HPLC) Analysis The obtained prepared AGP solution was analyzed by HPLC, and the results are shown in FIG.

<HPLC measurement conditions (same below)> Detector: SPD-10A UV-VIS DETECTOR (manufactured by SHIMADZU) Column: TSK-gel G-3000 SWXL (ID 7.8 mm x 300 mm) Column temperature: 30 ° C. Phase: 0.1M phosphate buffer (pH 7) + 0.1M Na ₂ SO ₄ Flow rate: 1 mL / min Detection: UV280 nm Injection amount: 20 μL

As shown in FIG. 1, peaks (5 minutes) containing AGP were detected in A1 and A2.

4. Screening for AGP-degrading enzyme-producing bacteria (1) Screening using synthetic medium Soil samples were collected in Osaka Prefecture. 206 kinds of soil samples were put into 3 mL of distilled water for 3 to 4 kinds each for one microspattera, and the suspension was used as a soil suspension. A liquid synthetic medium (Table 2) was prepared, and 3 mL each was dispensed into a test tube and sterilized in an autoclave (121 ° C., 20 minutes). After allowing to cool at room temperature, about 100 μL of each of the prepared soil suspensions was added, the lid was closed, and the cells were cultured in a shaking incubator (30 ° C., 200 osc / min) for 14 days. During the culturing, although the color of the medium changed to yellow or yellowish green, about 100 μL of the culture broth was placed in a newly prepared liquid synthetic medium (Table 2) and cultured.

A plate synthetic medium (Table 2) was prepared, sterilized in an autoclave (121 ° C., 20 minutes), placed in a petri dish in an amount of about 10 mL, cooled in a clean bench, and solidified. About 20 μL of the above culture solution was sprinkled on this, and the bacteria were isolated by streaking with a platinum loop. Next, each of the isolated bacteria was inoculated into 3 mL of the prepared liquid nutrient medium (Table 3). Incubate in a shaking incubator (30 ° C., 200 osc / min) for 4 to 6 days, take 1.5 mL from the culture solution, centrifuge (13200 rpm, 5 minutes), pass through a filter having a pore size of 0.20 μm, and perform HPLC analysis. , AGP degradation reduction was evaluated.

In addition, about 2 weeks after the culture was stopped, all the culture solutions were subjected to HPLC analysis, and a decrease in AGP was observed. Therefore, about 10 mL of medium cooled to about 40 ° C after sterilization was poured into each petri dish in which 100 μL was dropped, and then slowly mixed. The medium was allowed to solidify at room temperature and cultured at 30 ° C. A plate synthetic medium (Table 2) was prepared, sterilized in an autoclave (121 ° C., 20 minutes), placed in a petri dish in an amount of about 10 mL, cooled in a clean bench, and solidified. The separated bacteria were taken with a sterilized toothpick, suspended in 1 drop of distilled water passed through a filter having a pore size of 0.20 μm, and this was streaked with a platinum loop to separate the bacteria. Strains in which the medium around the colony turned yellow or yellow-green were inoculated into 3 mL of newly prepared liquid nutrient medium (Table 3). Incubate in a shaking incubator (30 ° C., 200 osc / min) for 4 to 6 days, take 1.5 mL from the culture solution, centrifuge (13200 rpm, 5 minutes), pass through a filter having a pore size of 0.20 μm, and perform HPLC analysis. It was.

As a result of HPLC analysis of the culture medium, the strains isolated from the culture medium whose color changed to yellow or yellowish green during the culture and the strains isolated from the culture medium about 2 weeks after the culture was stopped showed a blank AGP peak. 33 strains were selected from which a decrease of 80% or more was observed. These strains were subjected to an enzymatic reaction using AGP as a substrate, and further strains were selected. Among the strains in which a decrease in AGP was observed, the results of HPLC analysis of the culture solutions of two strains are shown in FIG. 2 as an example.

(2) Screening using nutrient medium The same soil suspension as in 4 (1) above was used. A plate nutrient medium (Table 3) was prepared and sterilized in an autoclave (121 ° C., 20 minutes). About 10 mL of the medium cooled to about 40 ° C. after sterilization was poured into a petri dish in which 100 μL of each of the prepared soil suspensions was dropped, and the medium was allowed to stand at room temperature to solidify the medium and culture was started at 30 ° C. Strains in which the medium around the grown colony turned yellow or yellowish green were selected and isolated. A plate nutrient medium (Table 3) was prepared, sterilized in an autoclave (121 ° C., 20 minutes), placed in a petri dish in an amount of about 10 mL, cooled in a clean bench, and solidified. The separated bacteria were taken with a sterilized toothpick, suspended in 1 drop of distilled water passed through a filter having a pore size of 0.20 μm, and this was streaked with a platinum loop to separate the bacteria. Strains in which the medium around the colony turned yellow or yellow-green were inoculated into 3 mL of a newly prepared liquid nutrient medium (Table 3). Incubate in a shaking incubator (30 ° C., 200 osc / min) for about 5 days, take 1.5 mL from the culture solution, centrifuge (13200 rpm, 5 minutes), pass through a filter having a pore size of 0.20 μm, and perform HPLC analysis. The reduction in AGP degradation was evaluated.

As a result of HPLC analysis of the culture medium of the strains isolated from those whose medium color changed to yellow or yellowish green during culturing, 7 strains showed a decrease of 80% or more with respect to the peak of AGP in the blank. Stock selection. These strains were subjected to an enzymatic reaction using AGP as a substrate to further select them.

5. Screening based on AGP degradation reaction using bacterial cells and / or culture supernatant (1) Screening based on AGP degradation reaction using supernatant and bacterial cells A total of 40 strains selected in 4 (1) and (2) above were used as a liquid nutrient medium (1). After culturing in Table 3) for 5 days, centrifugation (13200 rpm, 10 minutes) was performed to obtain a supernatant and bacterial cells (precipitation). As for the supernatant, 0.25 mL was placed in an Eppen tube, the prepared AGP solution was mixed with 1 mL of phosphate buffer (0.1 M, pH 7) added so as to contain 0.1% of AGP, and the solution was allowed to stand in an incubator for reaction. (37 ° C., 24 hours). As a blank, 0.25 mL of distilled water was reacted in the same manner. After removing the supernatant as much as possible, 0.25 mL of phosphate buffer (0.1 M, pH 7) was added to the cells, and 0.1% AGP-added phosphate buffer (0.1 M, pH 7) was added in the same manner as the enzymatic reaction of the supernatant. ) Was mixed with 1 mL and reacted with a shaker (37 ° C., 24 hours). As a blank, only 0.25 mL of phosphate buffer and 1 mL of 0.1% AGP-added phosphate buffer were mixed and reacted. After the reaction, the mixture was centrifuged (13200 rpm, 5 minutes), and the supernatant was passed through a filter having a pore size of 0.20 μm for HPLC analysis.

As a result of performing an enzymatic reaction and HPLC analysis on the 40 strains selected in 4 (1) and (2) above separately for the supernatant and the cells, AGP in which only the supernatant or both the supernatant and the cells are blank 17 strains were selected from the strains in which a decrease of 50% or more was observed with respect to the peak of.

(2) Screening based on AGP degradation reaction with diluted supernatant The strains selected in 5 (1) above were cultured again in a liquid nutrient medium (Table 3) for 5 days, then centrifuged (13200 rpm, 10 minutes) and the supernatant was obtained. After that, the supernatant was diluted 2-fold and 4-fold with phosphate buffer (0.1 M, pH 7). Put 0.25 mL each of the supernatant stock solution, the 2-fold diluted supernatant and the 4-fold diluted supernatant into an Eppen tube, mix with 1 mL of 0.1% AGP-added phosphate buffer (0.1 M, pH 7), and in the incubator. It was allowed to stand and reacted (37 ° C., 18 hours). As a blank, 0.25 mL of distilled water was reacted in the same manner. After the reaction, the enzyme was inactivated by heating with a heat block (100 ° C., 20 minutes). This was centrifuged (13200 rpm, 5 minutes), and the supernatant was passed through a filter having a pore size of 0.20 μm for HPLC analysis.

As a result of HPLC analysis, three strains producing an enzyme having a particularly strong AGP degrading activity were selected and used as KSM45, KSM81, and KSM97. The HPLC analysis results and the AGP reduction rate of these enzyme reaction samples are shown in FIGS. 3 to 8, respectively.

As shown in FIGS. 3 and 4, in KSM45, the first peak (5 minutes) containing AGP in the enzyme reaction solution almost disappeared at all dilution ratios, and the second peak (11 minutes) became larger. ing. Therefore, it was determined that AGP was degraded by the enzyme produced by the strain.

As shown in FIGS. 5 and 6, in KSM81, as the enzyme concentration increased, the first peak (5 minutes) containing AGP in the enzyme reaction solution became smaller. On the contrary, the second peak (11 minutes) became larger as the enzyme concentration increased. Therefore, it was determined that AGP was degraded by the enzyme produced by the strain.

As shown in FIGS. 7 and 8, in KSM97, as the enzyme concentration increased, the first peak (5 minutes) containing AGP in the enzyme reaction solution became smaller. The second peak (11 minutes) was not significantly different from the blank, but it was judged that AGP was degraded by the enzyme produced by the strain.

6. Identification of selected strains (base sequence analysis of 16S rDNA)
Technosuruga Lab Co., Ltd. was requested to identify KSM45, KSM81, and KSM97, which produce enzymes with particularly strong AGP degrading activity, which were selected as described above. As a result of the total base sequence analysis of 16S rDNA, since the base sequence of 16S rDNA of KSM45 has the sequence shown in SEQ ID NO: 1, Chinophaga sp. It was found to be a strain (also referred to herein as Chinophaga sp. KSM45 strain). This strain was issued to the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture) on June 25, 2014 with the accession number NITE P-01889 (identification). Indication: Deposited as Chiba phaga sp. KSM45).

Since the base sequence of 16S rDNA of KSM81 has the sequence shown in SEQ ID NO: 2, Ensifer sp. It was found to be a strain (also referred to herein as the Ensifer sp. KSM81 strain). This strain was issued to the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture) on June 25, 2014 with the accession number NITE P-01890 (identification). Display: Deposited as Ensifer sp. KSM81).

Since the nucleotide sequence of 16S rDNA of KSM97 has the sequence shown in SEQ ID NO: 3, it was found to be Burkholderia stabilis (also referred to as Burkholderia stabilis KSM97 strain in the present specification). This strain was issued to NITE P-01891 (identification) on June 25, 2014 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture). Display: Deposited as Burkholderia stabilis KSM97).

Further analysis was performed on these three strains, such as examination of AGP degradation products and enzymatic reaction with coffee bean slices.

7. Enzyme reaction of coffee bean sections About 20 green coffee beans were soaked in distilled water, autoclaved (121 ° C., 20 minutes), and then thinly sliced one by one using a simple microtome and a shaving sword. At this point, several sections were collected as untreated alkali sections. Next, the sections were placed in 50 mL of 0.1 M NaOH and treated with alkali (121 ° C., 20 minutes). After autoclaving, it was washed with distilled water three times for 5 minutes each. This was used as an alkali-treated section.

After culturing the strain in a liquid nutrient medium (Table 3) for 5 days, the strain was centrifuged (13200 rpm, 10 minutes) to obtain a culture supernatant (the OD value of the culture at 660 nm was 0.160 for KSM45 and 0 for KSM81. .160, KSM97 is 0.216). For each acquired strain, 200 μL of culture supernatant, 200 μL of saline (blank), and 200 μL of saline containing 1% GODO-TCF (Trichoderma reesei-derived cellulase, manufactured by Godo Shusei Co., Ltd.) for the alkali-treated section and the alkali-treated section. 200 μL of 1% GODO-TCF-added culture supernatant was placed in a microplate together with the prepared coffee bean sections, and an enzymatic reaction was carried out at 37 ° C. for 7 days. The results of suspending with a toothpick and observing the state of collapse are shown in FIGS. 9 and 10.

As shown in FIG. 9, regarding the reaction between the enzyme of KSM45 and the alkali-untreated section, the section to which only the blank and the culture supernatant were added did not disintegrate. Sections supplemented with 1% GODO-TCF-added physiological saline (hereinafter referred to as [1% TCF only]) and 1% GODO-TCF-added culture supernatant (hereinafter referred to as [1% TCF + KSM45 (KSM81, KSM97)]]. ) Was added to both of the sections, but on the 6th day, the sections of [1% TCF + KSM45] were almost completely disintegrated, shattered, and partially solubilized.

Similarly, for the reaction of the enzyme of KSM81 with the alkali-untreated section, the section to which only the blank and the culture supernatant were added did not disintegrate. Both the [1% TCF only] and [1% TCF + KSM81] sections were disintegrated, but on day 8, the [TCF + KSM81] section was almost completely disintegrated, shattered, and partially solubilized.

Similarly, regarding the reaction between the enzyme of KSM97 and the alkali-untreated section, the section to which only the blank and the culture supernatant were added did not disintegrate. Both the [1% TCF only] and [1% TCF + KSM97] sections were only slightly disintegrated.

As shown in FIG. 10, in the reaction between the enzyme of KSM45 and the alkali-treated section, the section to which only the blank and the culture supernatant were added did not disintegrate. However, both the sections to which [1% TCF only] and [1% TCF + KSM45] were added disintegrated into pieces, but when observed under a microscope, the sections to which [1% TCF + KSM45] disintegrated more finely. From the above, it was inferred that the section was almost completely disintegrated and solubilized by the decomposition of AGP by the enzyme of KSM45, the loosening of the cell wall constituents, and the ease with which cellulase worked in every corner.

Similarly, in the reaction between the enzyme of KSM81 and the alkali-treated section, the section to which only the blank and the culture supernatant were added did not disintegrate. However, both the sections to which [1% TCF only] and [1% TCF + KSM81] were added disintegrated into pieces, but when observed under a microscope, the sections to which [1% TCF + KSM81] disintegrated more finely. From the above, although the effect was smaller than that of KSM45, an effective synergistic effect of cellulase and enzyme was observed.

Similarly, in the reaction between the enzyme of KSM97 and the alkali-treated section, the section to which only the blank and the culture supernatant were added did not disintegrate. However, both the sections to which [1% TCF only] and [1% TCF + KSM97] were added disintegrated into pieces, but when observed under a microscope, the sections to which [1% TCF + KSM97] were added were slightly disintegrated. From the above, although the effect was smaller than that of the above two strains, an effective synergistic effect of cellulase and the enzyme was observed.

8. Enzyme reaction using various cellulase Soak green coffee beans in distilled water, autoclave (121 ° C, 20 minutes), and then put the sections prepared by a simple microtome into the holes of a 96-hole microplate reader one by one. Then, 125 μL of the culture supernatant of KSM45 and 125 μL of a 1% solution of the cellulase enzyme preparation were added and mixed, and the effect of section solubilization was tested. For the effect of cellulase alone, 125 μL of a 1% solution of cellulase enzyme preparation and 125 μL of water were mixed and reacted in the same manner. The blank was similarly tested by adding 250 μL of section and water. The reaction was carried out at a reaction temperature of 37 ° C., and the reaction time was 3 days. As an evaluation method, a 96-well microplate was directly photographed in a dark field and observed with a microscope at a magnification of 50 times.

The results are shown in FIG. 11. When various cellulase were evaluated, the cellulase derived from Trichoderma viride, the cellulase of the genus Trichoderma, the cellulase derived from Aspergillus niger, etc. showed remarkable cell disruption as compared with the case of the cellulase solution alone. A solubilizing effect was observed. That is, it was shown that the combination of AGP degrading enzyme and some cellulase preparations is effective in efficiently solubilizing coffee bean sections.

9. Enzymatic reaction of gum arabic solution 0.4% gum arabic prepared by dissolving 0.25 mL each of the culture supernatants of the selected strains (KSM45, KSM81, KSM97) in an Eppen tube and dissolving 0.4 g of gum arabic in 100 mL of distilled water. It was mixed with 1 mL of gum solution and allowed to stand in an incubator for reaction (37 ° C., 18 hours). As a blank, 0.25 mL of distilled water was reacted in the same manner. After the reaction, the enzyme was inactivated by heating with a heat block (100 ° C., 20 minutes). This was centrifuged (13200 rpm, 10 minutes), and the supernatant was passed through a filter having a pore size of 0.20 μm for HPLC analysis. The result is shown in FIG.

Gum arabic is said to contain about 20-25% AGP. As shown in FIG. 12, the 5-minute peak on the polymer side was not observed in the samples other than the blank, and the 8-minute peak was smaller in the sample than in the blank. Since AGP was contained in either of these peaks, it was considered that AGP in gum arabic was decomposed. In addition, since the peak of the sample was larger at the peak of 12 minutes than that of the blank, it was considered that the decomposition products increased in the same manner as the enzymatic decomposition of coffee-derived AGP. From the above, it was considered that the enzyme produced by the acquired strain also decomposes gum arabic-derived AGP.

Claims (10)

It is a microorganism capable of assimilating arabinogalactan protein, and is used in Chinogalactan sp. KSM45 strain (accession number NITE P-01889), Ensifer sp. A microorganism that is a KSM81 strain (accession number NITE P-01890) or a Burkholderia stabilis KSM97 strain (accession number NITE P-01891). The microorganism according to claim 1 , wherein the arabinogalactan protein has a molecular weight of at least 1.5 million Da. The microorganism according to claim 1 or 2 , wherein the arabinogalactan protein is derived from green coffee beans. At least one selected from the group consisting of the microorganism according to any one of claims 1 to 3 , the cell culture of the microorganism , and the cell extract of the microorganism is added to the arabinogalactan protein-containing substance. A method for degrading arabinogalactan protein in the content, which comprises a step of acting. At least one selected from the group consisting of the microorganism according to any one of claims 1 to 3 , the cell culture of the microorganism , and the cell extract of the microorganism is added to the arabinogalactan protein-containing substance. A method for producing foods and drinks in which arabinogalactan protein is decomposed, including a step of acting. A screening method for microorganisms capable of assimilating arabinogalactan protein.
A culture step of culturing candidate microorganisms in a medium in which at least 90% or more of the carbon source is derived from arabinogalactan protein, and
A method comprising an evaluation step of comparing the content of the arabinogalactan protein in a sample before and after the culture. The method according to claim 6 , wherein the culturing step is a culturing time of at least 10 hours or more. The method according to claim 6 or 7 , wherein the evaluation step is based on the fact that the content of the arabinogalactan protein in the sample shows a decrease of at least 50% by weight. Further, any one of claims 6 to 8 , further comprising an enzymatic reaction step of reacting and degrading at least one selected from the group consisting of bacterial cells of candidate microorganisms and bacterial cell cultures on arabinogalactan protein. The method described. An arabinogalactan protein-degrading agent containing at least one selected from the group consisting of the microorganism according to any one of claims 1 to 3 , a cell culture of the microorganism, and a cell extract of the microorganism. ..

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