JP5680313B2 - Proteolytic treatment method and composition for proteolytic treatment - Google Patents

Description

  The present invention relates to a proteolytic treatment method and a composition for proteolytic treatment. More specifically, the present invention relates to a proteolytic treatment method and a proteolytic treatment composition suitable for use in, for example, food waste containing a large amount of protein.

  As a conventional technique related to a protein processing method, for example, a method described in Patent Document 1 is known. Specifically, it describes that organic waste containing proteins is decomposed in the presence of a thermophilic proteolytic bacterium belonging to the genus Bacillus such as Bacillus stearothermophilus.

  Thus, by using thermophilic proteolytic bacteria, it can be decomposed at a temperature of 50 ° C. or higher, so that vegetable oils and animal fats and oils contained in organic waste can be dissolved and proteolytic The contact area between the bacteria and the protein can be increased, and the protein can be easily decomposed.

JP 2003-274936 A

  However, the conventional proteolytic treatment method using a thermophilic proteolytic bacterium has a problem that the proteolytic proteolytic bacterium itself has a low protein resolution, and the proteolytic treatment cannot be performed efficiently.

  Moreover, in the conventional proteolytic treatment method, although wastes containing proteins can be decomposed, energy cannot be recovered therefrom.

  An object of this invention is to provide the proteolytic processing method which can improve the proteolytic rate by proteolytic bacteria.

  Another object of the present invention is to provide a proteolytic treatment method capable of improving the rate of proteolysis by thermophilic proteolytic bacteria at a temperature of 50 ° C. or higher at which vegetable oils and animal fats are dissolved. .

  Furthermore, an object of the present invention is to provide a method capable of recovering energy while improving the rate of proteolysis by proteolytic bacteria.

  Another object of the present invention is to provide a composition for proteolytic treatment capable of recovering energy while efficiently decomposing protein.

As a result of intensive studies by the inventors of the present invention in order to solve such problems, the thermophilic proteolytic bacterium (CT-1 strain) isolated by the inventors of the present application and methano, which is a thermophilic hydrogen-assimilating methane-producing bacterium, are obtained. When the protein is decomposed in a co-culture environment with Thermobacter thermautotrophicus, compared to the case where the protein is decomposed in a single culture of a thermophilic proteolytic bacterium (CT-1 strain), It came to know that the degradation rate of protein improves significantly. Furthermore, the present inventors have found that biogas containing methane gas is generated by thermophilic hydrogen-assimilating methane-producing bacteria and can be recovered as energy. Based on these findings, the present invention has been completed.

That is, the proteolytic treatment method according to claim 1 is directed to the production of a thermophilic hydrogen-assimilating methane and a microorganism belonging to the genus Coprothermobacter which is entrusted with a thermophilic proteolytic bacterium, accession number FERM P-21909. In a co-culture environment of Methanothermobacter thermautotrophicus, which is a fungus, the temperature of the co-culture environment is set to 50 ° C. or higher to decompose the protein or protein-containing material.

Further, as described in claim 3 , in the proteolytic treatment method according to claim 1 , it is preferable to recover biogas containing methane gas generated during the proteolytic treatment.

Next, the composition for proteolytic treatment according to claim 2 comprises a microorganism belonging to the genus Coprothermobacter (Coprothermobacter) and thermophilic hydrogen assimilation, which are entrusted with the accession number FERM P-21909, which is a thermophilic proteolytic bacterium. It contains methanothermobacter thermautotrophicus, which is a methanogenic bacterium .

  According to the proteolytic treatment method of the present invention, the proteolytic rate can be greatly improved as compared to the case where the proteolytic treatment is performed by a proteolytic bacterium alone. Therefore, it is possible to reduce the volume by decomposing organic waste containing much protein, for example, food waste, more efficiently than conventional methods.

Moreover , according to the proteolytic treatment method of the present invention, proteolysis is performed as compared with the case where the proteolytic treatment is performed alone at a temperature of 50 ° C. or higher at which vegetable oils and animal fats are dissolved. The processing speed can be greatly improved. Therefore, vegetable oils and animal fats and oils contained in organic waste can be dissolved to increase the contact area between proteolytic bacteria and proteins, and protein degradation can be performed more efficiently.

Furthermore, according to the energy recovery method of the present invention, a microorganism belonging to the genus Coprothermobacter and the thermophilic hydrogen-assimilating methanogen that are entrusted with the thermophilic proteolytic bacteria under the accession number FERM P-21909. Methanothermobacter thermautotrophicus (Methanothermobacter thermautotrophicus) is a thermophilic hydrogen-assimilating methanogen because it decomposes proteins in the co-culture environment of Methanothermobacter thermautotrophicus Biogas containing methane gas produced by the above can be recovered as energy. Therefore, methane gas can be recovered as energy while improving the protein degradation rate, and it is possible to effectively use the protein or protein-containing material as a resource.

Moreover, according to the composition for proteolytic treatment of the present invention, microorganisms belonging to the genus Coprothermobacter (Coprothermobacter) and thermophilic hydrogen-assimilating methane, which are entrusted with the thermophilic proteolytic bacterium, accession number FERM P-21909. Since it contains methanoothermobacter thermautotrophicus, which is a producer, it is possible to easily form an environment in which methane gas can be recovered as energy while promoting protein degradation. Become.

It is a figure which shows the time-dependent change of the number of bacteria in a culture test. It is a figure which shows the time-dependent change of the ammonium ion concentration of the casein culture medium in a culture test. It is a figure which shows the time-dependent change of the TOC density | concentration of the casein culture medium in a culture test. It is a figure which shows a time-dependent change of the lower fatty acid (VFA) density | concentration of the casein culture medium in a culture test. It is a figure which shows the production | generation process of the methane gas from protein.

  Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings.

  In the proteolytic treatment method of the present invention, a protein or a protein-containing material is decomposed in a co-culture environment of proteolytic bacteria and methanogenic bacteria.

  Decomposing proteins or protein-containing materials in a co-culture environment of proteolytic bacteria and methanogens consumes hydrogen, lower fatty acids, and carbon dioxide generated by the proteolytic bacteria as they are decomposed by the proteolytic bacteria. As a result, the growth of proteolytic bacteria is promoted, and the proteolytic treatment rate is improved.

  In the proteolytic treatment method of the present invention, a protein or a protein-containing material is a treatment target. Examples of the protein-containing material include, but are not limited to, organic waste such as food waste containing a lot of protein.

  Here, the proteolytic bacteria and the methanogen are preferably thermophilic. In this case, since the co-culture environment can be set to 50 ° C. or higher, vegetable oils and animal fats and oils contained in protein-containing materials such as organic waste are dissolved to contact the proteolytic bacteria with the protein. The area can be increased, and the protein can be decomposed more efficiently.

As the thermophilic proteolytic bacterium, a proteolytic bacterium belonging to the genus Coprothermobacter, which is entrusted with the accession number FERM P-21909 is preferable.

As the thermophilic methanogen used in the present invention, Methanothermobacter thermautotrophicus which is a thermophilic hydrogen-utilizing methanogen is preferable.

  Examples of the co-culture environment for proteolytic bacteria and methanogenic bacteria include a culture solution containing nutrient sources, trace elements, vitamins, and the like necessary for these bacteria. In this case, the protein or protein-containing material can be suspended and treated in the culture solution, and the protein or protein-containing material can easily be brought into contact with the proteolytic bacteria. However, the co-culture environment is not limited to the culture solution, and may be a solid medium such as an agar medium or sludge. In addition, the proteolytic treatment can be performed by spraying a co-culture solution of proteolytic bacteria and methanogenic bacteria on the protein or the protein-containing material and impregnating it. In addition, a co-culture environment may be formed by adding methanogenic bacteria to an environment containing proteolytic bacteria or treatment equipment. Also in this case, the growth of proteolytic bacteria in the environment and processing equipment can be promoted, and the proteolytic processing rate can be improved. Further, a co-culture environment may be formed by adding a proteolytic bacterium to an environment containing methanogen or a processing facility. In this case, it is possible to efficiently decompose proteins by imparting proteolytic processing ability to an environment or processing facility containing methanogens.

Here, when thermophilic proteolytic bacteria and thermophilic hydrogen-assimilating methanogens are used, the temperature of the co-culture environment can be set to 50 ° C. or higher. Since it may be inactivated, the temperature of the co-culture environment is preferably 50 ° C to less than 100 ° C, more preferably 50 ° C to 80 ° C, and 50 ° C to 70 ° C. Is more preferred.

In the proteolytic treatment method of the present invention, since a thermophilic hydrogen-assimilating methanogen is used, biogas containing methane gas is consumed by consuming hydrogen produced by protein degradation by the proteolytic bacterium. Generated. Therefore, there is an advantage that methane gas can be recovered from the protein as energy.

  Next, the composition for proteolytic treatment of the present invention will be described.

  The composition for proteolytic treatment of the present invention contains the above-mentioned proteolytic bacterium and methanogenic bacterium, and its form is not particularly limited. For example, cryopreserved products that have been cryopreserved by adding proteolytic bacteria and methanogens to nutrient solutions, trace elements, vitamins, etc. necessary for growth, and nutrient sources and traces necessary for growth A solution in which proteolytic bacteria and methanogens are added to a culture solution containing elements and vitamins, etc., and proteolytic bacteria and methanogens are freeze-dried together with nutrients, trace elements and vitamins necessary for growth It can be in various forms such as freeze-dried products.

  By introducing the composition for proteolytic treatment of the present invention into, for example, a culture solution or the like, a co-culture environment of proteolytic bacteria and methanogenic bacteria can be easily formed. Therefore, it is possible to easily form a proteolytic processing environment that has high proteolytic processing ability and that can recover biogas containing methane gas as energy.

  The above-described embodiment is an example of a preferred embodiment of the present invention, but is not limited thereto, and various modifications can be made without departing from the gist of the present invention.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1
We examined the proteolytic processing capacity of proteolytic bacteria and methanogens in a co-culture environment.

(1) Proteolytic bacteria In this example, proteolytic bacteria that were present in the methane fermentation broth were isolated and used.

  The methane fermentation broth was collected from a thermophilic methane fermentation tank that had been operated at a volume of 2 L and a temperature of 55 ° C. In addition, the methane content rate of the biogas generated from this methane fermenter was 61%, the lower fatty acid concentration of the methane fermentation liquid was 3.2 mM, and the chemical oxygen demand removal rate was 76%.

  Proteolytic bacteria were isolated using enrichment culture and solid culture.

For enrichment culture, the collected methane fermentation solution is inoculated into a liquid medium placed in a vial, and the head space above the liquid surface is filled with N ₂ and CO ₂ (N ₂ : CO ₂ = 80: 20 (volume ratio)). Then, the lid was covered and the temperature was 55 ° C.

The liquid medium used for enrichment culture is a basic medium having the following composition with 3 g / L of casein (manufactured by Sigma) and 1.0 g / L of yeast extract (manufactured by Wako Pure Chemical Industries, Ltd.). Using. In the following description, this liquid medium is referred to as casein medium. As a reducing agent, 1.0 g / L of cysteine-HCl · H ₂ O and 1.0 g / L of Na ₂ S · 9H ₂ O were added to the casein medium. Moreover, the medium 131 trace element solution made from DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen) was used for the trace element solution, and the medium 141 vitamin solution made from DSMZ was used for the vitamin solution.
[Composition of basic medium (composition in 1 L of distilled water)]
・ KH ₂ PO ₄ : 0.8 g
・ K ₂ HPO ₄ : 1.6 g
・ NH ₄ Cl: 1.0 g
・ NaHCO ₃ : 2.0 g
MgCl ₂ · 6H ₂ O: 0.1 g
・ CaCl ₂ · 2H ₂ O: 0.2 g
・ NaCl: 0.8g
・ Resazurin: 0.1mg
Trace element solution: 10 mL
・ Vitamin solution: 10mL

  After performing enrichment culture, after confirming that the ammonium ion produced by the degradation of casein by proteolytic bacteria is present in the casein medium, inoculate a part of the casein medium on the agar medium. Solid culture was performed.

  Solid culture was performed using AnaeroPack (manufactured by Mitsubishi Gas Chemical Co., Ltd.) in an anaerobic environment at 55 ° C. Ten days later, a colony was picked up, and this was smeared again on the same medium to generate colonies twice.

  The agar medium used for the solid culture was the above basic medium supplemented with 3.0 g / L of gelatin (manufactured by Sigma) and 10.0 g / L of gellan gum.

  The finally obtained colony was put into casein medium and cultured under the same conditions as enrichment culture. As a result, the production of ammonium ions was confirmed by the indophenol method. That is, it was confirmed that casein was decomposed by the isolated microorganism to produce ammonium ions. From this result, it was confirmed that the isolated microorganism was a proteolytic bacterium having a protein resolution. Moreover, since it has protein resolution at 55 degreeC, it has also confirmed that it was a thermophilic proteolytic microbe.

Next, the base sequence of 16S rRNA gene was analyzed for the isolated proteolytic bacteria. Specifically, PCR amplification of 16S rRNA gene was performed using primers 27f and 1492r (see Reference 1). The internal 16S rRNA primer Bac806R was used for full-length base sequence analysis (see Reference 2). The obtained base sequence was compared with the base sequence in the base sequence database of GenBank / EMBL / DDBJ by the BLAST program.
・ Reference 1: Lane, DJ: 16S / 23S rRNA sequencing. P.115-175. In Stackebrandt, E. and Goodfellow, M. (eds.), Nucleic acid techniques in bacterial systematics. John Wiley & Sons, New York (1991).
Reference 2: Takai, K. and Horikoshi, K .: Rapid detection and quatification of members of archaeal community by quantitative PCR using fluorogenic probes. Appl. Environ. Microbiol., 66, 5066-5072 (2000).

The base sequence of the 16S rRNA gene of the isolated proteolytic bacterium is shown in SEQ ID NO: 1 in the sequence listing. The isolated proteolytic bacterium is a microorganism belonging to the genus Coprothermobacter, and has been reported to be a proteolytic bacterium using a protein as a substrate. Coprothermobacter proteolyticus: Accession number; CP001145, Reference 3 and Reference 4) and 99.4% homology with the 16S rRNA gene. In the following description, the isolated proteolytic bacterium is referred to as CT-1 strain.
・ Reference 3: Ollivier, BM, Mah RA, Ferguson TJ, Boone DR, Garcia JL, Robinson R (1985). Emendation of the genus Thermobacteroides: Thermobacteroides proteolyticus sp. Nov., A proteolytic acetogen from a methanogenic enrichment. Int. J. Syst. Bacteriol. 35: 425-248.
Reference 4: Kersters, I., Maestrojuan GM, Torck U, Vancanneyt M, Kersters K and Verstraete W (1994). Isolation of Coprothermobacter proteolyticus from an anaerobic digest and further characterization of the species. Syst. Appl. Microbiol. 17 : 289-295.

  As a result of observing the CT-1 strain with an optical microscope, it was found to be a Gram-negative bacterium in which a linear rod shape, sometimes slightly curved, was observed. In this respect as well, it was similar to Copro Thermobacter proteolyticus (Reference 4).

CT-1 strain, and it has been assigned accession number FER M P -21909 in the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center in February 2010 9 dated.

(2) Methanogen In this example, Methanothermobacter thermautotrophicus (JCM1004), which is a thermophilic hydrogen-assimilating methane-producing bacterium, was used as a methane-producing bacterium. In the following explanation, the Methanothermobacter saum autotrophica is referred to as MT strain.

(3) Co-culture test 60 mL of a casein medium containing 1.0 g / L of cysteine-HCl · H ₂ O and 1.0 g / L of Na ₂ S · 9H ₂ O as a reducing agent in a serum vial for 120 mL The culture test was carried out under the following three conditions. The initial pH of the medium was 7.8, and the temperature was 55 ° C. The head space above the liquid surface was filled with N ₂ and CO ₂ (N ₂ : CO ₂ = 80: 20 (volume ratio)) and capped. The culture period was 7 days.
・ Condition 1: No bacterial cells added ・ Condition 2: Only CT-1 strain added ・ Condition 3: CT-1 strain and MT strain added

The initial addition density of the CT-1 strain in conditions 2 and 3 was 7.1 × 10 ⁵ cells / mL. The initial addition density of the MT strain in Condition 3 was 2.4 × 10 ⁵ cells / mL.

  The measurement items were (a) change in the number of bacteria over time, (b) product gas composition, (c) change in ammonium ion concentration over time, (d) decomposition rate of insoluble fraction of casein, (e) change in TOC concentration over time, (F) A culture test was performed as a change with time in the lower fatty acid concentration.

(A) Change in the number of bacteria over time On the second day, the fourth day, and the seventh day of the culture, the casein medium was collected from the vial, and the total number of bacteria was counted with a microscope. The number of methanogenic bacteria (MT strain) was determined from the ratio of autofluorescence from coenzyme F420 and DAPI (4 ′, 6-diamino-2-phenylindole) using a fluorescence microscope. The number of proteolytic bacteria (CT-1 strain) was determined by subtracting the number of methanogenic bacteria from the total number of bacteria. The time course of the number of bacteria is shown in FIG. ♦ is the number of bacteria in CT-1 strain under condition 2, ■ is the number of bacteria in CT-1 strain under condition 3, and ▲ is the number of bacteria in MT strain under condition 3. From this result, it was clarified that by co-culturing the CT-1 strain and the MT strain, the amount of proliferation of the CT-1 strain can be increased by about 4 times compared to the case where only the CT-1 strain is cultured alone. .

(B) Product gas composition The composition of the product gas was analyzed on the 7th day of culture by gas chromatography (manufactured by GL Science, GC390B). The results are shown in Table 1. In condition 2 where only the CT-1 strain was cultured alone, carbon dioxide was detected together with hydrogen, but methane gas was not detected. On the other hand, in condition 3 where the CT-1 strain and the MT strain were co-cultured, hydrogen was not detected, but methane gas and carbon dioxide were detected. From this result, it is clear that in the condition 3 in which the CT-1 strain and the MT strain were co-cultured, hydrogen produced by decomposition of casein by the CT-1 strain was consumed by the MT strain and methane gas was generated. became.

(C) Time-dependent change of ammonium ion concentration On the second day, the fourth day, and the seventh day of culture, the casein medium was collected from the vial and the ammonium ion concentration was measured. The ammonium ion concentration was measured by ion exchange chromatography (ICS-1500, manufactured by Dionex) equipped with a CS16 cation column after filtration through a membrane having a pore size of 0.2 μm. The results are shown in FIG. It was clarified that the ammonium ion concentration was overwhelmingly higher in the condition 3 in which the CT-1 strain and the MT strain were co-cultured than in the condition 2 in which only the CT-1 strain was cultured alone.

(D) Degradation rate of insoluble fraction of casein About the casein medium on the 7th day of culture in conditions 2 and 3, the precipitate obtained by shaking vigorously and standing for 1 hour is defined as undigested casein. The amount of the precipitate in condition 1 was compared. Specifically, the suspension was carefully removed, the precipitate was filtered through a glass fiber filter (0.45 μm), dried at 105 ° C. for 90 minutes, and the dry weight was measured. And the insoluble fraction decomposition rate of casein was calculated from the dry weight obtained in each condition. The results are shown in Table 2. Compared with condition 2 in which only the CT-1 strain was cultured alone, condition 3 in which the CT-1 strain and MT strain were co-cultured was found to have an overwhelmingly higher degradation rate of casein insoluble fraction. It was.

(E) Change in TOC concentration over time On the second day, the fourth day, and the seventh day of culture, the casein medium was collected from the vial and the TOC concentration was measured. The TOC concentration was measured with a TOC analyzer (manufactured by Toray, TNC-6000). The results are shown in FIG. It was clarified that the TOC concentration was overwhelmingly higher in the condition 3 in which the CT-1 strain and the MT strain were co-cultured than in the condition 2 in which only the CT-1 strain was cultured alone.

(F) Change with time of lower fatty acid concentration On the second day, the fourth day, and the seventh day of the culture, the casein medium was collected from the vial, and the lower fatty acid concentration was measured. The lower fatty acid concentration was measured by high pressure liquid chromatography (GL Science, GL-7400). The results are shown in FIG. It was clarified that the lower fatty acid concentration was overwhelmingly higher in the condition 3 in which the CT-1 strain and the MT strain were co-cultured than in the condition 2 in which only the CT-1 strain was cultured alone.

(G) Summary From the results of the culture test, compared to condition 2 in which only the CT-1 strain was cultured alone, condition 3 in which the CT-1 strain and the MT strain were co-cultured was more proliferating in the CT-1 strain. It became clear that can be promoted. Moreover, it became clear that the ammonium ion concentration, the TOC concentration, and the lower fatty acid concentration were increased, and the decomposition rate of the insoluble fraction of casein was also improved. Therefore, by co-culturing the CT-1 strain and the MT strain, it is possible to promote the growth of the CT-1 strain and promote the degradation of the protein. As a result of the accelerated degradation of the protein, the degradation production of the protein It turned out that the density | concentration of a thing ammonium ion, TOC, and a lower fatty acid improves.

  As is clear from the analysis results of the product gas composition, the co-culture of the CT-1 strain and the MT strain consumes hydrogen, which is a protein degradation product, and converts it into methane gas. It was thought that protein degradation was promoted by the consumption of the product.

  Here, the protein degradation process when co-cultivating proteolytic bacteria and methanogenic bacteria can be expressed as shown in FIG. In this example, CT-1 strain was used as a proteolytic bacterium, MT strain was used as a methanogenic bacterium, hydrogen as a protein degradation product of CT-1 strain was consumed by MT strain, It succeeded in promoting proliferation and proteolysis by the CT-1 strain. From this result, the same effect can be obtained by using not only hydrogen-utilizing methanogens that consume hydrogen, such as the MT strain, but also acetic acid-assimilating methanogens that consume lower fatty acids. It was thought to be. In this example, thermophilic proteolytic bacteria and thermophilic methanogenic bacteria were used. However, the proteolytic process shown in FIG. 5 is limited to the case where the thermophilic proteolytic bacteria and thermophilic methanogenic bacteria are co-cultured. This is a reaction process that can occur even when co-cultured with general proteolytic bacteria and methanogens. It was considered that the same effect as the case where the MT strain was used was exhibited when the methanogen was used in general.

Claims (3)

Microbes of the genus Coprothermobacter (Coprothermobacter) entrusted with accession number FERM P-21909 which is a thermophilic proteolytic bacterium and Methanothermobacter thermautotrophicus (Methanothermobacter thermautotrophicus) which is a thermophilic hydrogen-assimilating methanogen In the co-culture environment , a protein or protein-containing material is decomposed at a temperature of the co-culture environment of 50 ° C. or higher . Microbes of the genus Coprothermobacter (Coprothermobacter) entrusted with accession number FERM P-21909 which is a thermophilic proteolytic bacterium and Methanothermobacter thermautotrophicus (Methanothermobacter thermautotrophicus) which is a thermophilic hydrogen-assimilating methanogen A composition for proteolytic treatment, comprising: A biogas recovery method comprising recovering biogas containing methane gas generated during proteolytic treatment by the method according to claim 1 .

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