JP2008199938A - Method for treating organic wastes using bacillus microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism which can continuously degrade organic wastes containing much proteins and fats over long periods, and to provide a method for treating organic wastes, comprising continuously degrading the organic wastes such as meats and meat processed product residues with the microorganism over long periods. <P>SOLUTION: This microorganism has been obtained by separating microorganisms from many samples collected from soils, composts, food factory drainage treatment facilities and the like in many places in Japan, performing protein degradation activity tests using pork proteins and milk proteins as substrates, and fat degradation activity tests using animal fats (lards) as substrates, for the separated microorganisms, selecting 50 strains having high degradation abilities for these three substrates, performing tests for degrading meats and meat processed product residues for the 50 strains in flasks, selecting 19 strains excellent in degradation abilities, identifying three strains excellent in salt resistance and acid resistance among the 19 strains, and then confirming that the three strains can continuously degrade organic wastes comprising meats and meat processed product residues over 60 days or more. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a Bacillus microorganism that has protein and fat resolution, salt resistance, and acid resistance, and is suitable for decomposition of meat and processed meat products, and organic waste using such a Bacillus microorganism. In particular, it relates to a method of continuous decomposition treatment.

  As for the decomposition treatment of organic waste using microorganisms, various studies have already been made on devices and microbial preparations for the purpose of improving treatment capacity, and many reports have been made. However, many of these technologies are for organic waste (so-called garbage) discharged from homes, restaurants, supermarkets, and the like. These organic wastes are often composed of various foods, so there are a wide variety of components that serve as nutrients for microorganisms, and there are various types of microorganisms derived from foods. In many cases, degradation proceeds naturally while the adapted microorganisms develop.

  On the other hand, with regard to organic waste discharged from food factories, etc., the constituents are single or limited (for example, limited to processed meat products such as meat, ham and sausage in a processed meat product factory) ) In that case, the types of microorganisms derived from food and the types of microorganisms that can grow in the environment are few, and if treated for long periods in such an environment, the environment becomes difficult for microorganisms to grow, and the processed product is sufficiently decomposed. Often fail to proceed In fact, there are very few studies on organic waste that is continuously discharged at specific food factories. For example, the majority of organic waste discharged from meat processing factories and related offices is meat and processed meat residues that are high in protein, fat, and salt. For such organic wastes, the accumulation of fat during treatment causes viscosity in the treated product, making it difficult to contain air inside, resulting in a lack of oxygen, and the treated product tends to clump and prevent decomposition. In the past, continuous decomposition treatment using microorganisms has been difficult due to the fact that the pH of the processed product tends to be acidic (prone to rancidity) and the salt contained in the processed meat product residue accumulates. It was.

  Conventionally, as a method for treating these organic wastes using microorganisms, Bacillus stearothermophilus No. 38 (FERM P-17939), which efficiently decomposes oils in organic wastes, is used as an organic waste containing oils. Organic waste treatment method (for example, refer to Patent Document 1), food waste (grains such as rice, noodles and bread, meat such as cattle, pigs, chickens and fish) Fats and oils, vegetables in general, fruits and fruit skins, seaweeds, crab and shrimp shells, fish and other small bones, egg shells, etc., sawdust and other cellulose and lignin, environmental hormones (salts, basic compounds) ), Is resistant to acid (the ability to decompose salt under acidic conditions), and is a mixture of four Bacillus microorganisms and one Arthrobacter microorganism that are resistant to heavy metals. Organic waste using In addition to the treatment method (for example, see Patent Document 2), an organic waste treatment apparatus operating method (for example, see Patent Document 3) using seven kinds of microorganisms including Bacillus, Bacillus subtilis) and thermophilic bacteria (Bacillus pallidus) are treated with organic waste (for example, see Patent Document 4), garbage processing methods using novel Bacillus strains (for example, see Patent Document 5), thermostable enzymes A method for treating food-related waste using a high-temperature bacterium (genus Bacillus) that produces a group (amylase, protease, lipase) is known (for example, see Patent Document 6).

JP 2002-17341 A JP 2002-51767 A JP 2003-274939 A WO2004 / 067197 JP 200521010 A JP 2003-305444 A

  An object of the present invention is to provide microorganisms capable of continuously decomposing organic waste containing a large amount of protein and fat such as meat and processed meat residues, etc. over a long period of time, and processed meat and processed meat residues using microorganisms. An object of the present invention is to provide a method for treating organic waste that continuously decomposes organic waste over a long period of time.

  The present inventors have conducted intensive research to solve the above-mentioned problems, and separated nearly 1,000 microorganisms from many samples collected from soil, compost, wastewater treatment facilities of food factories, etc. in various parts of Japan, and the separated microorganisms. Proteolytic activity test using pork protein and milk protein as substrate, and lipolytic activity test using animal fat (lard) as substrate, and each of the three substrates of pork protein, milk protein and animal fat (lard) as On the other hand, 50 strains with high degradability were selected, and about 50 strains were subjected to meat and processed meat residue decomposition tests using a flask system, and 19 strains with excellent resolution were selected. Among them, salt resistance and acid resistance were selected. Identify three excellent strains, and these three strains can continuously decompose organic waste consisting of meat and processed meat products over 60 days or longer. Verify, which resulted in the completion of the present invention.

  That is, the present invention (1) has protein and fat resolution, can grow under the conditions of a salt concentration of 6%, pH 7.3, and a temperature of 32 ° C., and a salt concentration of 0.5%, pH 4.0 to 8.0. , Salt and acid resistance that can grow under conditions of temperature 32 ° C, and meat and processed meat residue (analytical values: protein 16%, fat 23%, salinity 1.5%) cultured for 7 days Decrease rate (%) [(weight before culturing−weight on the seventh day of culture) / weight before culturing × 100] is 5.0% or more, (2 (1) Bacillus microorganisms as described in (1) above, and (3) Bacillus licheniformis or Bacillus subtilis characterized by being able to grow under conditions of a salt concentration of 0.5% and pH 7.3, 50 ° C. A bee according to (1) or (2) above, characterized in that Scan a microorganism belonging to the genus and, (4) Bacillus licheniformis No. 3 strains (FERM AP-21208), Bacillus subtilis No. 132 strain (FERM AP-21210) or Bacillus licheniformis No. It is 146 strains (FERM AP-21209), It is related with the Bacillus genus microorganisms of the said (3) description characterized by the above-mentioned.

  In addition, the present invention uses (5) one or more strains of the Bacillus genus described in any one of (1) to (4) above, and continuously decomposes organic waste containing a large amount of protein and fat over a long period of time. And (6) Bacillus licheniformis No. 6 as a microorganism belonging to the genus Bacillus. 3 strains (FERM AP-21208), Bacillus subtilis No. 132 strain (FERM AP-21210) and Bacillus licheniformis no. One or two or more strains selected from 146 strains (FERM AP-21209) are used, and the organic waste treatment method according to (5) above, and (7) an organic material rich in protein and fat (8) The organic waste processing method according to (5) or (6) above, wherein the waste is meat and processed meat products, and (8) continuous decomposition over 60 days or longer The method for treating organic waste according to any one of (5) to (7) above.

  When the microorganism of the genus Bacillus of the present invention is used, organic waste containing a large amount of protein and fat such as meat and processed meat residue can be continuously decomposed over a long period of time.

  The microorganism belonging to the genus Bacillus of the present invention has a protein and fat resolution, can grow under conditions of a salt concentration of 6%, pH 7.3, and a temperature of 32 ° C., and a salt concentration of 0.5%, pH 4.0 to 8. 0, salt and acid resistance that can grow under conditions of 32 ° C., and meat and processed meat residue (analytical values: protein 16%, fat 23%, salinity 1.5%) for 7 days Especially if it is a microorganism belonging to the genus Bacillus having a reduction rate (%) after culture treatment [(weight before culture−weight on culture day 7) / weight before culture × 100] of 5.0% or more. The determinations of “resolution”, “growable” and “5.0% or more” are “(1) Degradation activity test” and “(3) Growth characteristic test” in the description of Example 1 described later, respectively. And "(2) Decomposition of meat and processed meat residue using flask system" Definitive if it can be determined similarly to "was isolated as these Bacillus microorganisms No. 3 strains, no. 132 strains, no. 146 strain, No. 502, No. Examples of 5 strains of 515 strains can be given.

  These five strains can grow under the conditions of a salt concentration of 0.5% and a pH of 7.3 and 50 ° C. Among these five strains, Bacillus licheniformis No. 3 which is vigorously grown at pH 4.0. 3 strains (FERM AP-21208), Bacillus subtilis No. 132 strain (FERM AP-21210), Bacillus licheniformis no. The 146 strain (FERM AP-21209) can be preferably exemplified.

  As a method for treating organic waste according to the present invention, organic waste containing a large amount of protein and fat (main component) is used for a long time using one or more strains of the Bacillus genus of the present invention. The organic waste is not particularly limited as long as it is a continuous decomposition method. Examples of the organic waste include livestock excreta, carcass, skin, organs, blood, meat residues, and meat processing from the livestock and meat industries. Suitable examples include animal residues and food waste that contain a large amount of protein and fat, such as product residues, and in particular, in the molding process discharged from meat factories and related establishments that contain a large amount of protein, fat, and salt. Meat residues and processed meat residues discharged after processing can be particularly preferably exemplified during processing. In addition, the long-term continuous decomposition method is 10 days or more, preferably 30 days or more, particularly preferably 60 days or more, and decomposition treatment while adding additional organic waste in one treatment tank. Say.

The Bacillus microorganism of the present invention used in the method for treating organic waste of the present invention may be a single strain, but preferably two or more strains are used simultaneously. For example, Bacillus licheniformis no. 3 strains, Bacillus subtilis No. No. 132 strain and Bacillus licheniformis no. If 3 strains of 146 strains are used at the same time, it can be continuously decomposed over a long period of 60 days or longer. Further, the Bacillus microorganism of the present invention may be added at the beginning of the decomposition treatment, but can be additionally input during continuous decomposition as required. When the organic waste is, for example, meat consisting of 16% protein, 23% fat, and 1.5% salinity, the initial number of bacteria of the genus Bacillus is 1.0 × per 1 g of waste. 10 ⁵ CFU or more, for example, 1.0 × 10 ⁵ to 10 ⁷ CFU is preferable. Although these Bacillus microorganisms can be administered as they are, they may be mixed with a carrier such as wood chips and administered.

  In the continuous decomposition treatment of organic waste, a carbon source, a nitrogen source, etc. can be blended as necessary. In the continuous decomposition treatment, the stirring plate installed in the treatment tank is rotated continuously or intermittently to feed air into the entire treated product and simultaneously exhaust through the exhaust port. The decomposition treatment temperature and pH do not usually need to be controlled, but can be controlled to 20 to 50 ° C. and pH 4 to 8 as necessary.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Screening of strains]
Each sample collected from soil, compost, wastewater treatment facilities of food factories, etc. in various places in Japan was diluted 10 times, streaked on a standard agar medium (manufactured by Nissui Pharmaceutical), and cultured at 32 ° C. for 48 hours. After the culture, 421 strains of bacteria were isolated from the colonies that appeared. Using these isolates, screening was conducted in stages by the following (1) degradation activity test, (2) meat and meat processed product residue degradation test using a flask system, and (3) growth characteristic test.

(1) Degradation activity test 1) Proteolytic activity A well having a diameter of 5 mm is aseptically opened on an agar medium using pork protein and milk protein (casein) as substrates, and 1.0 × 10 ⁶ CFU / in sterile saline. 30 μl of each bacterial solution prepared in ml was added to the well. After culturing at 32 ° C. for 48 hours, the proteolytic activity was evaluated using as an index the size of the clear zone formed by decomposing pork protein and milk protein. “−” Indicates that no clear zone was generated, “+” indicates a clear zone, and “+” indicates a diameter of less than 10 mm, “++” indicates a diameter of 10 mm or more and less than 25 mm, and “++++” indicates a diameter of 25 mm or more. And shown in Tables 1-3. A method for preparing an agar medium using pork protein and milk protein (casein) as substrates is shown below.

(Method for preparing agar medium using pork protein as substrate)
After immersing the pork loin red meat in an equal amount of sterile physiological saline overnight, the supernatant obtained by centrifugation at 6000 rpm for 20 minutes was heat sterilized at 80 ° C. for 30 minutes to obtain a pork protein suspension. Add 20.0 g of agar to 1 L of 0.1 M phosphate buffer solution (pH 6.8), dissolve by heating, sterilize at 120 ° C. for 15 minutes, add 10% pork protein suspension and stir well, then sterilize It was dispensed into a petri dish and solidified.

(Method for preparing agar medium using milk protein as substrate)
Add 2.5 g of yeast extract, 5.0 g of tryptone, 1.0 g of glucose, 15.0 g of agar, 10.0 g of sodium caseinate, 20.0 ml of 1M calcium chloride solution to 1 L of 0.4% sodium citrate solution and dissolve by heating. After sterilizing at 120 ° C. for 15 minutes, the solution was dispensed into a sterilized petri dish and solidified.

2) Lipolytic activity 30 μl of each bacterial solution prepared to 1.0 × 10 ⁶ CFU / ml was added and smeared on an agar medium using animal fat (Lard) as a substrate. After culturing at 32 ° C. for 48 hours, lipolytic activity was evaluated by lowering the pH due to lipolysis (indicator blue). "-" Indicates that no blue discoloration was observed, and "-" indicates that blue discoloration was observed, depending on the degree of blue discoloration, slightly "+" for blue discoloration, "++" for blue discoloration, and "++" for strong blue discoloration. The results are shown in Tables 1 to 3. A method for preparing an agar medium using animal fat as a substrate is shown below.

(Method for preparing agar medium using animal fat as substrate)
Add 100 ml of Lard's filtrate, which has been heated and melted, to a mortar kept at 45 ° C., add 75 mg of Victoria Blue adjusted to weak alkalinity, grind it, keep it at 90 ° C., and stir and filter until pigment particles disappear. And a pigment fat liquid. Add 10.0 g of agar, 5.0 g of peptone, and 5.0 g of meat extract to 1 L of distilled water, dissolve by heating, sterilize at 120 ° C. for 15 minutes, add 5% pigment fat solution and stir well. The solution was dispensed and solidified.

  From the results of the degradation activity test, 50 strains having high degradation ability for each of the three substrates were selected and subjected to the following (2) meat and processed meat residue degradation test using a flask system.

(2) Decomposition test of meat and processed meat product residue using flask system To a 500 ml Erlenmeyer flask, 60 g of wood waste as a carrier, 30 ml of water, 30 g of meat and processed meat product residue as decomposition targets were added and mixed, and then flasked with aluminum foil The sample was capped and sterilized at 120 ° C. for 30 minutes and used as a test system. The meat and processed meat residue used as the decomposition target were mixed so that the weight ratio was 10% meat, 30% ham, 30% sausage, 30% bacon, and then chopped to 5 mm. (16% protein, 23% fat, 1.5% salt, 58% moisture). 50 strains selected in the degradation activity test were prepared at 1.0 × 10 ⁸ CFU / ml, and ⁸ ml was aseptically added to the flask. In addition, 8 ml of sterilized water was added to the control. Each flask was cultured at 32 ° C. for 7 days, and changes in weight and appearance were observed. The whole flask was agitated twice daily during the culture to ensure that the contents were fully oxygenated. The rate of decrease was calculated from the weight of the contents before culturing and on the seventh day of culturing using the following formula. In addition, since the wetness or liquefaction of the contents is observed due to the decomposition action of the microorganisms, “−” indicates that the contents are not changed in appearance, and “+” indicates that the contents are not wet or liquefied. “,” Where strong wetting or liquefaction was observed is shown in Table 4 as “++”.
Decrease rate (%): (weight before culture−weight on the seventh day of culture) / weight before culture × 100

  From the results of the meat and processed meat residue decomposition test using the flask system, 19 strains with a reduction rate of 5.0% or more and wet or liquefaction of “+” or more were selected. (3) It used for the growth characteristic test.

(3) Growth characteristic test The growth characteristic test in the salt concentration of each selected strain, pH, and culture | cultivation temperature was done. Tryptosoya broth (manufactured by Nissui Pharmaceutical Co., Ltd., salt concentration 0.5%, pH 7.3) is used as the basic medium, and the salt concentration is 0.5, 2.0, 4.0, 6.0, 8.0%. 10 ml each of the medium prepared in (1) and the media prepared at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 were prepared. 100 μl of each bacterial solution prepared to 1.0 × 10 ³ CFU / ml was added to each medium, and the degree of growth after culturing at 32 ° C. for 48 hours was evaluated. Further, the growth degree after culturing for 48 hours while changing the temperature to 20, 30, 40, 50, and 60 ° C. using a basic medium was evaluated. Evaluation of growth is shown in Table 5 with an absorbance meter (wavelength of 660 nm) with an absorbance value of 0.0 indicating no growth “−”, 0.0 to 0.3 as growth “+”, and 0.3 or more as strong growth “++”. Show.

  From the results of the growth characteristics test, it was determined that No. 1 was a strain that strongly developed “++” at a salt concentration of 0.5% to 6.0%, pH 4.0 to 8.0, and a temperature of 20 ° C. to 50 ° C. 3 strains, no. 132, No. 146 strains were selected. Based on the results of the screening test, the selected No. 3 strains, no. 132, No. Three of the 146 strains have high protein and lipolytic activity, efficiently decompose meat and processed meat products, and have salt resistance (growth at 6.0% salt) and acid resistance (growth at pH 4.0). Have.

[Identification of strain]
Selected No. 3 strains, no. 132, No. About 146 strain | stump | stock, as a result of identifying the genus from the morphological and physiological property according to the conventional method, all strains were genus Bacillus (Bacillus). As a result of discriminating species from biochemical properties using API 50CHB (manufactured by BioMerieux) which is a Bacillus genus identification kit, 3 shares and No. 146 strain is Bacillus licheniformis, No. 146. The 132 strain was identified as Bacillus subtilis. The results are shown in Table 6. These three strains are registered with the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (Central 6th, 1st East, Tsukuba City, Ibaraki, Japan), respectively. 3 strains (FERM AP-21208), Bacillus subtilis No. 132 strain (FERM AP-21210), Bacillus licheniformis no. It has been deposited as strain 146 (FERM AP-21209).

[Degradation test of meat and processed meat residue using actual machine]
The test was performed using two commercially available household garbage processing machines. This machine is a so-called bio-type, which is decomposed by microorganisms derived from food (wood waste) or foods to be introduced, and has a processing capacity of about 800 g (manufacturer recommended) per day. One test was conducted as a control group and the other as a strain addition group. In order to eliminate the influence of mixed microorganisms, the carrier used in the strain addition group was previously sterilized and dried at 120 ° C. for 50 minutes, and then No. 1 prepared to 10 ⁶ CFU / g with respect to the carrier. 3 strains, no. 132, No. 30 g of 146 strains were added. In the control group, the carrier specified by the manufacturer was used as it was.

  The decomposition target was the meat and processed meat residue described in Example 1 (2) described above, and 700 to 800 g was introduced into the processing tank of each machine every two days, and the treatment was performed for 60 days. . During processing, the weight of the contents was measured about every two days. Moreover, 20g of contents were extract | collected for every week, and the water | moisture content and the number of heat-resistant bacteria were measured.

  The water content was measured according to a conventional method using 10 g of the contents by a normal pressure heat drying method. The number of heat-resistant bacteria was determined by diluting 10 g of the contents with sterile physiological saline, taking 10 ml, heat-treating at 75 ° C. for 30 minutes, and measuring the number of general viable bacteria in the solution. The number of general viable bacteria was measured according to a standard method.

  FIG. 1 shows the input amount and the weight change of the contents during the treatment period. The total input amount of the degradation target was 21.6 kg. Throughout the period, the content weight was kept lower in the strain addition group than in the control group.

The weight reduction rate by dry weight (dry weight reduction rate) was calculated from the weight and moisture content of the contents by the following formula. The value obtained by this formula represents only the reduction due to the decomposition of the organic matter in order not to take into account the reduction due to the evaporation of the water contained in the decomposition target.
Drying loss ratio (%): (Dry weight of input-dry weight of input after treatment) / Dry weight of input x 100

  The change in the weight reduction rate due to the dry weight during the treatment period is shown in FIG. It was found that the strain addition group maintained a high reduction rate throughout the treatment period compared to the control group.

The behavior of the number of heat-resistant bacteria during the treatment period is shown in FIG. The number of heat-resistant bacteria that were present at 10 ⁶ CFU / g after addition in the strain addition group was stable throughout the treatment period.

  From these results, the obtained No. 3 strains, no. 132, No. By adding 146 strains to the garbage processing machine, it was possible to improve the decomposition efficiency of the input meat and processed meat residue and to perform stable processing.

The Bacillus genus no. 3 strains, no. 132, No. It is a figure which shows the weight change of the content in the processing period using the mixed bacteria of 146 strains. The Bacillus genus no. 3 strains, no. 132, No. It is a figure which shows the change of the weight loss rate by the dry weight in the processing period using the mixed bacteria of 146 strains. The Bacillus genus no. 3 strains, no. 132, No. It is a figure which shows the behavior of the number of heat-resistant bacteria in the process period using the mixed bacteria of 146 strains.

Claims (8)

It has protein and fat resolution, can grow under conditions of salt concentration 6%, pH 7.3, temperature 32 ° C and salt concentration 0.5%, pH 4.0-8.0, temperature 32 ° C Decrease rate (%) when cultivated for 7 days with growth-resistant salt and acid resistance and meat and processed meat residues (analytical values: protein 16%, fat 23%, salinity 1.5%) [(Weight before culturing−Weight on the seventh day of culturing) / Weight before culturing × 100] is 5.0% or more. The microorganism of the genus Bacillus according to claim 1, which can grow under conditions of a salt concentration of 0.5% and a pH of 7.3 and 50 ° C. The microorganism belonging to the genus Bacillus according to claim 1 or 2, which is Bacillus licheniformis or Bacillus subtilis. Bacillus licheniformis no. 3 strains (FERM AP-21208), Bacillus subtilis No. 132 strain (FERM AP-21210) or Bacillus licheniformis No. The microorganism of the genus Bacillus according to claim 3, which is 146 strain (FERM AP-21209). An organic waste containing a large amount of protein and fat is continuously decomposed over a long period of time using one or two or more strains of the Bacillus genus microorganism according to any one of claims 1 to 4. Processing method. As Bacillus microorganisms, Bacillus licheniformis no. 3 strains (FERM AP-21208), Bacillus subtilis No. 132 strain (FERM AP-21210) and Bacillus licheniformis no. The method for treating organic waste according to claim 5, wherein one or more strains selected from 146 strains (FERM AP-21209) are used. The organic waste processing method according to claim 5 or 6, wherein the organic waste containing a large amount of protein and fat is a residue of processed meat and processed meat. The method for treating organic waste according to any one of claims 5 to 7, wherein the organic waste is continuously decomposed over a long period of 60 days or longer.