JP2000102378A - High-density bacillus sp and highly concentrated bacillus sp metabolite-including material, and production and utilization thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the subject material useful for therapy of infectious diseases of animals (e.g. human beings, livestock or the like) and plants or the like by increasing the concentration of metabolite or products through the repetition of a multiplication cycle consisting of germination, growth, disassembly and sporulation of spores of microorganisms belonging to bacillus genus. SOLUTION: This method for producing a material including high-density Bacillus sp. and highly concentrated Bacillus sp. metabolite is to increase the concentration of metabolite or products secreted from Bacillus sp. due to the activity and/or sporulation of cells through the repetition of a multiplication cycle consisting of germination, growth, disassembly and sporulation of spores of Bacillus sp. and is as follows: (1) multiplication of Bacillus sp. in drainage system such as effluent treatment facility, a culture vessel or the like; (2) collecting the precipitate when the spore concentration of Bacillus sp. in precipitate such as sludge become ca. 108/g and subjecting the precipitate to solid-liquid separation, followed by drying it; (3) imparting nutrient, oxygen and heat to it, followed by fermentation; (4) repeating the above processes to increase the spore density of Bacillus sp. as well as the concentration of its metabolite and products.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention is based on a novel concept to increase the density of Bacillus sp and to increase the concentration of metabolites and products thereof, and further to reduce the density of Bacillus sp and its metabolites and formation Along with a new method to increase the concentration of substances, this increased concentration of metabolites and produced substances is used to control pathogenic microorganisms and pests against animals and plants such as humans, livestock, poultry, seafood, and agricultural products. And treatment of infectious diseases,
The present invention relates to a method for improving antiviral immunity, expressing an antistress effect, and a novel method for decomposing pesticides contained in soil to purify the soil.

[0002]

2. Description of the Related Art It has been known that Bacillus sp and particularly its metabolites (products) are effective in preventing epidemics of animals and plants, treating infectious diseases, and improving immune functions. In this case, the higher the Bacillus sp density and its metabolite concentration, the more effectively it can be used. However, conventionally, a large amount of wood was required due to low Bacillus sp density and metabolite concentration, and there was a great limitation in its use (the conventional product was one hundredth or less of the product of the present invention). In the case of metabolites, because of treatment in a liquid, they always flowed out, making it impossible to increase the concentration. In the present invention, this was successfully achieved by dry propagation.

[0003] In agriculture and aquaculture, at present, the pursuit of ad hoc productivity is pursued, and the spraying of antibiotics and pesticides, the administration of excess nutrients, and the cultivation of drug pickles are being carried out. Such a tendency is progressing worldwide, and causes a problem like O157. In addition, to remove dirt and pathogens from the matrix of the field, replacement and sterilization of the culture area, insecticide treatment, adsorption removal of ammonia and ammonium salts by spraying zeolite, incineration disinfection, excessive administration of antibiotics, administration of hormones, Physical filtration by filtration media,
Although such methods have been implemented, they all require a great deal of cost and labor, but because they always balance the target treatment, it is difficult to obtain decisive effects.

[0004]

Therefore, the present inventor has studied and studied the characteristics and functions of Bacillus sp for the first problem, and has investigated the densification of Bacillus sp spores and the high concentration of metabolites and produced substances. We have developed a method that simultaneously increases the concentration of metabolites and produced substances as well as a method that simultaneously increases the density of spores that have been increased in density.

[0005] In addition, a method has been developed to solve the second problem by feeding the enriched Bacillus sp metabolites and products, or the mixture itself, as feed or fertilizer to animals and plants. did.

[0006]

The Bacillus sp (Bacillus bacterium) survives all over the earth, but its distribution density is extremely low, and its density is usually less than 10 ³ / g. In addition, even in a limited place where the survival condition and the growth condition are accidentally set, it is only about 10 ⁴ cells / g. On the other hand, the metabolite concentration of Bacillus sp is limited to those secreted from the dilute Bacillus sp phase, and is extremely low, but in the natural world, it works to control and lyse pathogenic bacteria and maintain healthy nature. Working for. Therefore, the present inventors artificially extremely increased the density of Bacillus sp, thereby effectively utilizing the metabolites and the produced substances having a high concentration, and making humans, livestock, poultry, fish and shellfish, and It can be used for improving plant immunity, preventing epidemics, treating infectious diseases, promoting growth, preventing spoilage of residual feed, and decomposing pesticide residues in soil.

Therefore, the present inventors have proposed that Bacillus
The Bacillus sp was collected and grown in a liquid medium under the growth conditions of Bacillus sp. The density of the Bacillus sp in the medium was successfully increased to 10 ⁸ cells / ml or more. For this, sporulation of Bacillus sp is an essential condition.
The growth and growth conditions of Bacillus sp are not only nutrients but also appropriate temperature and oxygen, moisture and carbon sources, but when nutrients are consumed and the medium becomes oligotrophic, Bacillus sp.
sp sporulates. Then, when given nutrients, they germinate and gradually become denser. As the density of Bacillus sp increases, the bacterium in the medium also lyses or suppresses anaerobic bacteria such as spoilage bacteria, pathogenic bacteria, Nocardia-type bacteria, etc., so that Bacillus sp becomes dominant and Bacillus sp becomes dominant. The occupation rate will exceed 98%. In addition, about 1% of yeasts were mixed in this bacterial flora. The densification of Bacillus sp
It is possible only by sporulating p-bacilli, and sporulation proceeds under oligotrophic conditions, aerobic atmosphere, and activation temperatures. In addition, sludge from a wastewater treatment plant or a human waste treatment plant originally contains Bacillus sp of about 10 ³ / g.
Therefore, this sludge may be collected and multiplied to about 10 ⁸ / g, or a condition required for the growth of Bacillus sp (addition of silica or magnesium) in a wastewater treatment facility or the like.
Bacillus sp High density sludge may be produced.

[0008] In the case of sludge, the sporulated Bacillus s
Separately dehydrate the sludge of p, and dehydrate the Bacillussp dehydrated sludge (10
⁸ / g). The water content of the dewatered sludge is 75-80%
It is about. Further, the dewatered sludge is dried (natural drying, etc.) to have a water content of 55 to 65% and a Bacillus sp density of 10 ⁹ / g.
And fermented by stacking them in a fermenter heat insulation box.
Decompose the cell membrane of the filamentous body of llus sp (the spore husk) and the carcass of the protozoa to obtain a high-density Bacillus sp and a highly concentrated metabolite-containing material. The fermentation is performed by feeding the dried sludge with nutrient materials, starch, saccharides, proteins, lipids, moisture, oxygen, and heat, which also function as a void material. As a nutrient source material, for example, soybean meal, corn flakes, oil cake, and other dried vegetable chips are used. When the fermentation decomposition is completed, both the spore density and the metabolite concentration increase. By repeating this fermentation operation, the spore density becomes 10 ¹¹ / g or more, and the metabolite concentration also exceeds 2% in the sludge.

Thus, germination → proliferation → of Bacillus sp
Bacillus s
Both p density and metabolite concentration increase. However, at present, the density of Bacillus sp is limited to 10 ¹¹ to 10 ¹² cells / g, but the concentration of metabolites and produced substances further increases. In addition, as Bacillus sp becomes denser, pathogenic bacteria, anaerobic bacteria, spoilage Nocardia-type bacteria, etc. are lysed or suppressed, and Ba
Dominance of cillus sp progresses, and the dominance rate exceeds 98%.

The metabolite-containing material referred to in the present invention is Baci
This refers to a mixture of fermentation intermediates and residues, spores of Bacillus and cell walls of dead bacteria, including metabolites and products obtained as a result of the growth cycle of llus sp. The metabolites are those secreted by the activity and sporulation of the bacterial cells, particularly sporulation, and include cyclic peptides and polypeptides having an antibiotic action. The product includes degradation products of nutrients and reaction products of the degradation products, such as ammonia, hydrogen sulfide, amino acids, and polypeptides produced by the reaction of amino acids with substances having a carbosyl group. Then, metabolites and product substances such as polypeptides and cyclic peptides contained in the metabolite-containing material are eluted in a solvent such as dilute ethanol or water, and only these can be recovered and used as drugs.

As described above, in the propagation method of the present invention, it is not always necessary to use sludge collected from nature or sludge from a treatment plant as a starting material. The same steps (cycles) are repeated to grow to a sufficient spore density.

Further, when the oligotrophic growth cycle is repeated after the above-described growth cycle (vegetative growth cycle), the concentrations of metabolites and produced substances can be further increased. That is, Bacill by the vegetative growth cycle described above
When it is determined that the us sp density has reached or reached the limit, the germination under oligotrophic conditions is repeated, and the digestion and fermentation treatment is repeated. In this case, the oligotrophic treatment is different from the oligotrophic treatment for promoting spore formation described above, in which the supply of nutrients from the outside is continued while the temperature, oxygen, moisture and carbon source are supplied. Then, the spores will germinate, but some will be killed and the other will be digested using the cell wall of the killed bacteria as the only nutrient. And
Bacillus sp spore density gradually decreases, decreases to 10 ² to 10 ⁴ / g close to natural state. By doing so, the cell walls and the like in the metabolite-containing material decrease, and the concentrations of metabolites and product substances as active ingredients increase relatively.

The bacterium belonging to the genus Bacillus (Bacillus s.
p) In the presence of silica and magnesium, sporulation is greatly promoted. That is, silica is an important substance constituting a cell solution, a cell membrane, and a spore membrane of useful bacteria. If this is insufficient, useful bacteria cannot be proliferated. Magnesium is also the generation time of this useful bacterium (growth cycle)
Has an effect of extremely shortening. This function of silica and magnesium was first found by the present inventors. The higher the temperature, the faster the growth rate. The preferred upper limit of the temperature depends on the type of Bacillus sp.
It is about 2 ° C or 45 ° C. Fermentation by Bacillus sp combined with fermentation heat by yeast.
Since the temperature is raised to about 0 ° C. (it may reach 75 ° C. depending on auxiliary raw materials and fermentation conditions), Bacillus sp grows very quickly. In addition, Bacillussp activities require more oxygen. Oxygen is supplied by stirring the fermentation bed, but frequent stirring lowers the temperature.
Therefore, it is necessary to stir once or twice a day for several minutes. If these conditions are met, it has the ability to greedily consume nutrients and become dominant, killing other bacteria such as staphylococci and streptococci. Also,
When they become oligotrophic, they sporulate and germinate again when supplied with nutrients.

(Organic matter treatment by Bacillus bacterium) Some of the treated bacteria degrade hydrocarbons such as starch and carbohydrates, while others degrade proteins and lipids. Although there is also a difference, if stable treatment is always carried out when decomposing given nutrients, more effective bacteria will become dominant. For example, the sludge water in which Bacillus is dominating to a high concentration and performing very good treatment in a treatment plant where the BOD of the inflowing sewage always exceeds 12,000 ppm was subjected to dilution cultivation to examine the Bacillus concentration. , Always 5 × 10 ⁸ / ml ~
The density was 5 × 10 ¹⁰ cells / ml.

In order to make (magnesium) Bacillus dominant, the generation time related to the growth rate of Bacillus must first be shortened, but the purpose can be achieved by supplying magnesium ions. It is not always necessary to supply magnesium ions with ions, but magnesium compounds and contents are mixed into the fermentation bed,
The purpose can also be achieved by slowly melting magnesium. However, the generation time varies depending on the species of the genus Bacillus. Then, seeds with a short generation time are given priority as a result.

(Silica) Next, in order to increase the concentration of the grown Bacillus, it is necessary to respore-form the Bacillus after the generation time and preserve it. Normally, Bacillus grows under the supply of nutrients, water, oxygen, and heat to form filamentous bodies, reaches oligotrophic conditions, forms endospores, dissociates and spores, and sustains life, but is metastable. This tendency is particularly remarkable in the presence of type silicate minerals, silicate ions, and silanols, and shows extremely high sporulation.

[0017]

EXAMPLES (Example 1: Production of a metabolite-containing material) 1000 kg of dehydrated sludge is spread on a concrete slab and air-dried to a water content of about 60%. As a nutrient for each fermentation, glucose 10 kg skim milk 10 kg mince 10 kg of meat 10 kg of corn flakes 3 kg of salt were added, and water was further added so that the water content became 65%. Oxygen supply and hydration during fermentation were performed by turning over once every three days. The added nutrients are not limited to the above. Further, by adding about 10 ppm or more of dissolved silica, the increase in the cell density and the metabolite concentration was accelerated. Primary fermentation treatment days 42 days Secondary fermentation treatment days 28 days

The Bacillus sp density was determined by a dilution culture method using a flat medium, and the concentration of Bacillus sp metabolites and produced substances was measured by weighing out and weighing 10.0 g of a representative sample.
After adding 00 ml and eluting in an ultrasonic bath (50 Kz) for 3 minutes, the solution was suction-filtered with a Nuclepore filter or a GF-F filter (0.7 μm), weighed as a constant weight in a 105 ° C. constant temperature bath, and eluted. Metabolites due to weight difference before and after
The amount of product was determined. The results are as follows. Dehydrated and dried sludge Bacillus sp Density 12 × 10 ⁸ / g (Comparative material) Metabolite% 0.12% Color Black Primary fermentation treatment Bacillus sp Density 16 × 10 ⁹ / g Metabolite% 1.34% Color Gray Secondary Fermentation treatment Bacillus sp Density 8 × 10 ¹¹ / g Metabolite% 2.44% Color White (The metabolite dissolves and becomes black when hydrated) Tertiary fermentation treatment Bacillus sp Density 5 × 10 ⁶ / g Metabolite% 8.03% digestion and fermentation treatment Bacillus sp Density 7 × 10 ³ cells / g Metabolite% 11.36% Repeated treatment increases the metabolite concentration. Thus, compared to the comparative material (dewatered and dried sludge), Bacillus sp.
Both the density and the metabolite concentration increase as the treatment progresses. Bacillus sp density is expected to be limited (10 ¹¹ -1
0 about ¹² / g) but, for the metabolite concentration, it is possible to further increase. Also, depending on the purpose, reduce its density or remove aseptic fermentation residues and cell wall fragments with sterilization, such as polypeptides and cyclic peptides, metabolites and metabolites and products to prevent epidemics of humans and animals and plants, treatment of infectious diseases,
It achieves the objectives of improving immunity and anti-stress effect. Therefore, high-density Bacillus sp and high-concentration Baci
The use of llus sp metabolites directly to achieve their purpose is widened, and they are also extremely effective as a basic process and material for industrially extracting metabolites. The production of the metabolite-containing material is not limited to this example.

(Changes in Concentrations of Metabolites and Produced Substances such as Polypeptides and Cyclic Peptides) At the beginning of the drug production process of the present invention,
Metabolites and product substances such as polypeptides and cyclic peptides in the middle stage, terminal stage, and final stage are contained in each fermentation residue. Such as polypeptides and cyclic peptides,
In order to investigate changes in metabolites and product content, sampling was performed at each time point of the end of the primary process, the end of the secondary process, the end of the tertiary process, and the end of the digestive fermentation process, and 10 g each
Weighed and weighed, and diluted ethanol (1: 3) 50 ml
And ultrasonically dissolve (50 kHz × 10 minutes). Then, it is filtered and washed with GF-F (0.7 μm) to remove the fermentation residue, thereby obtaining a lysate, Bacillus sp spores passing through GF-F, and a cell wall. Two 0.1 ml portions of this mixed solution are collected on a slide glass and dried at room temperature in a clean bench to precipitate eluted substances. This deposited substance was observed with a microscope. At one other point, one drop of sterile water was dropped to dissolve the precipitate, and observed under a microscope. As a result, the residual at the primary residual spore density in step higher 10 ⁷ / g cell wall pieces often deposited material remaining spore density was less secondary step reduction 10 ⁵ / g cell wall pieces decreases precipitated substance increasing new product appeared tertiary step Spore density decreases 10 ³ / g Cell wall fragment disappears Precipitate increase New product growth Residual spore density decreases in digestion and fermentation process 10 ² / g Cell wall fragment disappears Precipitate further increases New product further grows

Next, the whole eluate is transferred to an evaporating dish, evaporated to dryness at a low temperature, kept in a desiccator for at least 24 hours and dried, and the collected precipitates (metabolites such as polypeptides and cyclic peptides) are removed. ) Was weighed to determine the content of metabolites and product substances such as polypeptides and cyclic peptides in each process sample. As a result, the primary process sample was 6.90%, the secondary process sample was 8.30%, the tertiary process sample was 10.36%, and the recycled sample was 11.41%. This result is a behavior consistent with the previous microscopic observation results. Thus, by repeating the cycle of proliferation → sterilization → digestion → proliferation, or proliferation → sterilization → digestion → germination → sterilization → digestion, the polypeptide was obtained. Concentration of metabolites and products such as liposomes and cyclic peptides and decrease the spore density of Bacillus sp.
Protein). (Crystals have disappeared and become monosaccharides (glucose).)

(Example 2) Culture of Prawns Prawns and other shrimp farms in Japan and abroad
It is a well-known fact that shrimp infectious diseases often cause the loss of reared seedlings. Therefore, the sediment of a shrimp culture pond (area 100,000 m ² ) that was devastatingly hit by the outbreak of infectious diseases was collected, and Bacillus sp.
Alternatively, a culture test was carried out in a sterile culture tank to confirm that the pathogenic microorganisms could not be eliminated by the metabolites, and the effectiveness was confirmed. Such a phenomenon can be said to be the result of Bacillus sp directly attacking microorganisms or synergistically eliminating pathogenic microorganisms by releasing metabolites and products such as cyclic peptides and polypeptides. Therefore, Bacill is directly applied to the cultured seedlings.
In order to supply us sp, its metabolites, and products, it was given in feed.

The fish meal, seaweed powder, clay powder, to feed consisting mainly of starch, 10 ⁶ to 10 ⁷ cells / g of Bacillus sp metabolites containing material to produce aquaculture feed for mixed about 5~10ppm ponds Sprayed. The prawns responded well to this feed, and they became well adapted. Through this experiment, it was also found that, in the case of epidemics of animals and fish and shellfish, an effect of 1 to 100 mg / kg, particularly 5 to 20 mg / kg / day of body weight can sufficiently exert its effects. As a result, the shrimp shrimp seedlings were completely eliminated, the shrimp growth was improved, the life was shortened by about 2 months, and the yield was greatly improved (about 8%). The pathogenic microorganism in the sediment was 3 × 10 ² cells / g, and the density of Bacillus sp was 15 × 1.
0 ⁸ / g and the sediment has been improved.

Example 3 Improving Immunity of Shrimp to PRDV Virus Bacillus sp metabolite-containing materials or metabolites / producers tend to improve the immune function of organisms or suppress pathogenic viruses. Therefore, the following experiment was performed, and good results were obtained. That is, 30 liters of bottom sediment of a culture pond in which prawns were completely destroyed due to a viral disease were taken, prawn seedlings were put into the ponds, and the prawns were cured for 2 days to attach acute viremia (PRDV) virus to the gills.
The virus-infected shrimp seedlings were then split in two, one fed normal diet. On the other hand, Bacillus sp.
A diet containing ¹¹ g / g and its metabolite in 1% was prepared and fed with Bacillus sp and its metabolite at 10 mg / day per 1 kg of cultured shrimp. As a result, as shown in FIG.
In the case of cillus sp and the metabolite-containing diet, the depletion rate was 25% for 5 days, 30% for 10 days, and 31% for 20 days, indicating that the antiviral immunity was significantly improved. Therefore, it was revealed that the yield of cultured shrimp can be significantly improved by administering such a bait containing the Bacillus sp metabolite-containing material as appropriate.

(Example 4 Enhancement of Immunity by Bacillus sp Metabolite and Product: Chicken, Pig) Infectious diarrhea of a collective chicken that has already occurred in a large-scale poultry
A metabolite was given as drinking water and cured in three days.
Japanese Patent Application No. 7-276461. afterwards,
At the poultry farm, chicken water was not given at all and only the above drinking water was given without giving any drug. Also, in pig farms, Bacillus sp10 ¹¹ /
When g and metabolites were given in a mixed amount of 0.001% of the body weight, the same effect was observed. These phenomena are considered to be the result of the marked improvement in immunity of pigs, especially by giving trace amounts of metabolites and produced substances including polypeptides and cyclic peptides. Recently, it has been experimentally confirmed that by providing a small amount of a produced substance to organisms other than mammals, particularly to fish and shellfish, particularly shrimp, sharks, and bachinella, it is possible to prevent viral diseases. It is thought that immunity is also improved.

Example 5 Prevention and Treatment of Chicken Diseases by Metabolites and Products such as Polypeptides and Cyclic Peptides Prevention and Treatment of Chicken Diseases by Drugs of the Present Invention Containing Metabolites and Products such as Polypeptides and Cyclic Peptides Was tested. The drug of the present invention was added to 1200 chickens in the poultry house in the experimental plot.
Drinking water containing 01% was continued to be administered. In addition, ordinary tap water was given as drinking water to 1,000 chickens in the poultry house in the control plot. On the 39th day (June 10) from the experiment, diarrhea occurred in the control group, and after 3 days, it spread to the entire control group. Immediately, the administration was switched to a diet containing a commercially available antibiotic, but diarrhea did not converge, and switching to a diet containing a different type of antibiotic had no effect.

At this time, since no diarrhea occurred in the experimental plot and the chickens were in good health, drinking water containing 0.01% of the drug of the present invention was also prepared in the control plot, and the water supply pipe was provided. Started water supply. Two days after the start of watering, most chickens had diarrhea, and on the third day the whole house was cured. Thereafter, when this water supply was continued, no collective diarrhea occurred. In addition, when the diarrhea stool was cultured and examined, Salmonella was detected. It was not detected in the experimental plot. From these results, it is clear that metabolites and products such as polypeptides and cyclic peptides containing no Bacillus sp spores or cell walls (peptidoglycan or protein) can prevent, treat, and improve immunity of chickens' infectious diseases. It became.

Example 6 Protection of Pigs Average Weight 12K
g piglets × 10 experimental pigs and 10 control pigs were prepared, and in each experimental plot, a drug (digestion / fermentation → centrifugation) consisting of a metabolite or product such as the polypeptide or cyclic peptide of the present invention was prepared. ) Was administered, and a control group was administered a diet containing no drug, and bred for 60 days. The level of drug contamination in the experimental plot was 0.5 per kg of body weight in the experimental plot.
mg, and 1.0 mg in the experimental two sections. No. Level of metabolites, etc. Type of incidence Remarks Experiment 1 group 0.5 mg 0 Experiment 2 group 1.0 mg 0 Control group 0 mg 6 Antibiotic administration As a result, no disease occurred in the experimental group.
In the control group, diarrhea caused by intestinal vibrios occurred. This indicates that a drug comprising a metabolite or a product such as the polypeptide or cyclic peptide of the present invention is effective for improving immunity of piglets and preventing infectious diseases.

(Example 7: Immunostimulation / NK activation)
A terminal cancer patient (85-year-old woman) who complained of pain was allowed to drink a supernatant liquid obtained by dissolving 2 g of a metabolite-containing material in 500 cc of distilled water every day. I no longer feel pain.

On the other hand, for a healthy male at the age of 57, the measured value of natural killer cell activity (NK activity) which is said to attack cancer cells (measured by Mitsubishi Chemical) is usually 10%.
-20, the amount of the metabolite-containing material of the present invention was 1
g was measured after drinking for 3 days and found to be 60. Normally, such high values (especially in the case of Mitsubishi Chemical measurements) are unthinkable.

(Example 8 treatment of rheumatism) When a 60-year-old woman who has been suffering from rheumatoid arthritis for many years has consumed 500 mg / day of the metabolite-containing material of the present invention for one month continuously, the pain completely disappeared. did.

Example 7 Treatment of athlete's foot, insect bites, atopic dermatitis 2 g of a metabolite-containing material was added to 500 cc of distilled water.
Was applied to the athlete's foot each evening, and after 7 days, the redness and itch disappeared completely. Since then, there has been no recurrence. An old man with a constitution in which a mosquito bite was swollen showed no swelling and no itching when the supernatant was applied.

The present inventors have determined that the body weight of 0.001 against severe diarrhea with fever, stomatitis, gingivitis, epidemic cold, bronchitis, allergic dermatitis, dermatitis due to floor rubbing, and the like.
They share the experience of quickly improving or eliminating these symptoms by drinking or applying Bacillus sp and metabolites equivalent to 0.0001%. In addition, the human body cells destroyed or damaged by dermatitis or the like can be promptly restored as healthy cells by performing the above-mentioned treatment, because the metabolites and produced substances of the present invention enhance the human body activity, This is considered to be the result of improving immunity.

Example 8 Cosmetics 100 mg of the powdered drug of the present invention is dissolved in 800 ml of distilled water to prepare a base solution. Into this, 100 ml of ethyl alcohol and 20 g of glycerin are dissolved, and 0.6 g of monomer silica (a substance produced by dissolving slag in acid) is further added and mixed. Furthermore,
Add fragrance if necessary. When this cosmetic was used daily for one month, the skin became smooth and glossy, and fine lines were eliminated.

Example 9 Confirmation of antibacterial activity Escherichia coli was extracted from an agar plate medium (starch 1.5%, nutrient broth 0.8%, salt 0.5%) from the sediment of a shrimp farm. Bacteria and bacteria extracted from cabbage field soil are inoculated, and each sample is prepared for (A) Bacillus sp + metabolite, (B) water containing Bacillus sp metabolite, and (C) untreated (comparative level) Then, the cells were cultured in a sterile culture box at 32 ° C. for 72 hours. As a result, E. coli is A,
Disappeared in each level of B, were detected 10 ⁸ / g at C levels. Shrimp farm bottom sediment is Staphylococcus aureus at A level,
E. coli and streptococci disappeared, and only Bacillus sp grew. At the B level, Staphylococcus aureus, Escherichia coli and Streptococcus were suppressed, and Bacillus sp germinated. At the C level, each of the above pathogenic bacteria grew. Cabbage field soil
At the A and B levels, rhizobia and actinomycetes are suppressed, and Bacillus s
p grew. At the C level, rhizobia and actinomycetes grew, and Bacillus sp remained at only 10 ³ / g. From the above results, it is clear that Bacillus sp and its metabolites have an antibacterial effect.

Example 10 Decomposition of Pesticide Residue and Recovery of Ground Strength Recently, at a golf course, a herbicide was erroneously sprayed, and a green turf was devastated and became irrecoverable. Therefore, a solution of the same concentration of the herbicide sprayed on the green was obtained, and the herbicide in this solution was replaced with Baci.
After confirming and testing the possibility of decomposition by llus sp and confirming the possibility, the following remedies were taken. Water containing trace amounts of Bacillus sp spores and their metabolites was periodically sprayed on the green to decompose the herbicide remaining in the green. Frequency of spraying Spray every 3 days Spray volume 100 liters / 1 are Spraying period Until the green recovery 10 / 27- By this method, 14 green / 18 holes 2 sub greens 2 putting greens remarkable recovery after 2 months 3 months Later, 2 greens / 18 holes were remarkably recovered. 2 greens / 18 holes were recovering. Next, the density of Bacillus sp in soil on the green was investigated. A soil sample was collected from a position 15 mm deep from the green surface, diluted and cultured, and the density thereof was examined. As a result, 10 ⁸ / g / 2 holes, 10 ⁷ / g / 12 holes, 10 ⁶ / g / g 2 holes, 10 ⁴ pieces / g / 2 holes, 10 ⁷ pieces / g / Sub2, Pat2. Ba
cillus sp Only 2 holes with a density of 10 ⁴ / g seemed to be delayed in recovery, but by continuing spraying,
It was presumed to recover. Also, Bacill in surface soil
A remarkable correlation was found between the us sp density and the recovery status of the green grass, and it became clear that the herbicide decomposition had progressed.
A petri dish test also demonstrated herbicide degradation.

Example 11 Natural Cultivation of Tea Leaves Bacill
It was investigated whether or not pesticide-free or reduced-pesticide cultivation is possible by providing us sp or its metabolite to tea plants, tea leaves, and fields, and whether or not there is an effect on yield. Experiment A Conventional conventional farming (spraying of chemical pesticides) Experiment B Fertilization of Bacillus sp dominated compost Experiment CB Each level of CB level + diluted secretion solution spraying was carried out in the following manner. In addition, Bacillus sp releases secretions to specific pathogens, pathogens and general pests upon sporulation, but the concentration increases as Bacillus sp becomes a high-density spore population. Therefore, in this example, dilution water containing the metabolites of Bacillus sp in the range of 0.01 to 0.03 ppm was prepared and used as spray water, or compost containing Bacillus sp 10 ¹⁰ cells / g or more was produced. And sprayed on the field.

(Preparation of Dilute Metabolites for Spraying on Soil and Foliage) Bacillus sp. Seed material having a density of 10 ¹¹ spores / g was kept in a sterilization box at 121 ° C. for 60 minutes, and Bacillus sp.
Bacteria other than us sp are sterilized, dissolved in 100-fold normal water, gently stirred for 10 minutes to elute metabolites, allowed to stand to sediment the seed material, and the supernatant water is used as a stock solution to give leaves. A 100-fold dilution water was used for surface application and a 200-fold dilution water for soil application. (Achievement of Bacillus sp compost and dilution water spraying) Date Compost spraying Dilute metabolite spraying (L / 10a) Remarks Volume Kg / 10a Soil spraying Tea leaf spraying 10/20 ~ E 1000 1000 1000 1/16 ~ E 3000 2000 * 1000 * On warm days 1 / E * 1000 Spray 2 / C * 1000 3 / R to E 1000 x 3 times 4 / R to E 1000 1000 x 2 times 5 / E 1000 Spray after tea picking Total 4000 4000 10000 Above The following effects were obtained as a result of spraying in the following manner. Yield (kg / a) Increase / decrease ratio Number of sprout / 30 g Remarks Experiment A 701 1.0 165 General compost + pesticide Experiment B 960 1.37 129 10 ¹⁰ / G Experiment C 1080 1.54 110 Stock solution 20L + 100L

As described above, the yield increased as in the case of other agricultural products, but the characteristic feature was that the thickness of the tea leaves was thick and the yield was increased. Both B and C levels were pesticide-free cultivation, but no disease or pests were found. Bacillus s
p Compost not only contains Bacillus sp and its metabolic secretions, but also consumes the necessary minerals of the silicate-Mg-Ca-Fe party as well as the N-P-K, which is essential for plant growth. It contains an appropriate amount in a form that is easy to be used, and has a fertilizer effect and suppresses the propagation of pathogenic microorganisms. Furthermore, the dilute metabolite directly sprayed has an effect of directly exerting toxicity on pathogenic bacteria and pathogens to suppress or eliminate them, resulting in increased harvest. Bacillus s
The shape of p is larger than Bacillus pumilus, etc.
Bacillus, which mainly degrades starch, sugars, cellulose, etc.
It is desirable that Thuringiensis has a high secretion amount, and that among these strains, there are many strains having a high degradation rate such that starch is degraded in one day.

[0039]

As described in detail above, the present invention relates to Bacillus
By repeating the growth cycle of sp spore germination, proliferation, demolition, and sporulation, it increases the concentration of Bacillus sp metabolites and product secreted with sporulation as well as Bacillus sp density. Therefore, high concentrations of Bacillus s
p Metabolites and products can be easily obtained. As a result, the amount of Bacillus sp metabolites and product substances added to livestock, poultry, and fish and shellfish feeds is less than one-hundredth of conventional levels, and the taste is not impaired. effective. In addition, it has an excellent effect on improving human immune activity and treating skin disorders, and has characteristics that it can be used in many aspects such as growth of plants and removal of residual pesticides.

Furthermore, by repeating the growth cycle under oligotrophic conditions, the density of Bacillus sp can be reduced below the natural distribution density. As a result, Bacillus sp spores and cell walls (peptidoglycan and protein) are not contained at all, and It is possible to obtain a metabolite or a product such as a polypeptide or a cyclic peptide which is more effective.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing a change in consumption rate of virus-infected juvenile shrimp.

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[Procedure amendment]

[Submission date] November 16, 1998 (1998.11.
16)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction contents]

The Bacillus sp density was determined by a dilution culture method using a flat medium, and the Bacillus sp density was determined.
Measurement of metabolites and product concentrations is based on a representative sample 10.0
g of pure water, 500 ml of pure water was added thereto, and an ultrasonic bath (5
0 Kz) for 3 minutes, then suction-filtered with a Nuclepore filter or GF-F filter (0.7 μm), weighed as a constant weight in a thermostat at 105 ° C. The amount of product was determined. The results are as follows. Dehydrated and dried sludge Bacillus sp density 12 × 10 ⁸ / g (comparative material) Metabolite% 0.12% Color Black Primary fermentation treatment Bacillus sp density 16 × 10 ⁹ / g Metabolite% 1.34% Color Gray Secondary Fermentation treatment Bacillus sp density 8 × 10 ¹¹ / g metabolite% 2.44% Color White (metabolite dissolves and becomes black when hydrated) Tertiary fermentation treatment Bacillus sp density 5 × 10 ⁶ / g metabolite% 8.03% digestive fermentation treatment Bacillus sp density 7 × 10 ³ cells / g Metabolite% 11.36% Repeated treatment increases the metabolite concentration. Thus, compared to the comparative material (dewatered and dried sludge), Bacill
Both the us sp density and the metabolite concentration increase with the progress of the treatment. Although the Bacillus sp density is expected to have a limit (approximately 10 ¹¹ to 10 ¹² cells / g), the metabolite concentration can be further increased. In addition, depending on the purpose, reduce its density, remove fermentation residues and cell wall fragments with sterilization, and prevent epidemics of humans and animals and plants with metabolites and products such as polypeptides and cyclic peptides, treat infectious diseases, It achieves the objectives of improving immunity and anti-stress effect. Therefore, high density Ba
Cillus sp and high concentration Bacillus sp
The range of achieving the purpose by directly using the metabolites is widened, and it is also extremely effective as a basic process and a raw material for industrially extracting metabolites. The production of the metabolite-containing material is not limited to this example.

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(Changes in Concentrations of Metabolites and Produced Substances such as Polypeptides and Cyclic Peptides) At the beginning of the drug production process of the present invention,
Metabolites and product substances such as polypeptides and cyclic peptides in the middle stage, terminal stage, and final stage are contained in each fermentation residue. Such as polypeptides and cyclic peptides,
In order to investigate changes in metabolites and product content, sampling was performed at each time point of the end of the primary process, the end of the secondary process, the end of the tertiary process, and the end of the digestive fermentation process, and 10 g each
Weighed and weighed, and diluted ethanol (1: 3) 50 ml
And ultrasonically dissolve (50 kHz × 10 minutes). Then, it was filtered and washed with GF-F (0.7 μm) to remove fermentation residues, and the lysate and Bacill passing through GF-F were used.
Obtain us sp spores and cell wall. Two points each of 0.1 ml of this mixed solution are collected on a slide glass, and dried at room temperature in a clean bench to precipitate an eluted substance. This deposited substance was observed with a microscope. At one other point, one drop of sterile water was dropped to dissolve the precipitate, and observed under a microscope. as a result,

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Example 6 Protection of Pigs Average Weight 12K
g piglets × 10 experimental pigs and 10 control pigs were prepared, and in each experimental plot, a drug (digestion / fermentation → centrifugation) consisting of a metabolite or product such as the polypeptide or cyclic peptide of the present invention was prepared. ) Was administered, and a control group was administered a diet containing no drug, and bred for 60 days. The level of drug incorporation in the experimental plot was 0.1 kg / kg body weight in the experimental plot.
5 mg, and 1.0 mg in the second experiment. No. Level of metabolites, etc. Type of incidence Remarks Experiment 1 section 0.5 mg 0 Experiment 2 section 1.0 mg 0 Control section 0 mg 6 Antibiotic administration As a result, no disease occurred in the experimental section,
In the control group, diarrhea caused by intestinal vibrios occurred. This indicates that a drug comprising a metabolite or a product such as the polypeptide or cyclic peptide of the present invention is effective for improving immunity of piglets and preventing infectious diseases.

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Example 10 Decomposition of Pesticide Residue and Recovery of Ground Strength Recently, at a golf course, a herbicide was erroneously sprayed, and a green turf was devastated and became irrecoverable. Therefore, a solution of the same concentration of the herbicide sprayed on the green was obtained, and this herbicide was applied to Ba.
A confirmation test was performed to determine whether or not it could be degraded by C. sp., and the possibility was confirmed. Therefore, rescue treatment was performed by the following method. Water containing trace amounts of Bacillus sp spores and their metabolites was periodically sprayed on the green to decompose the herbicide remaining in the green. Frequency of spraying Spray every 3 days Spray volume 100 liters / 1 are Spraying period Until green recovery October 27- By this method, 14 green / 18 holes 2 sub greens 2 putting greens remarkable recovery after 2 months 3 months Later, 2 greens / 18 holes were remarkably recovered. 2 greens / 18 holes were recovering. Then Bacillus sp in soil on green
Was investigated for density. A soil sample was taken from a position 15 mm deep from the green surface, diluted and cultured, and the density thereof was examined. As a result, 10 ⁸ pieces / g / 2 holes, 10 ⁷ pieces / g
/ 12 holes, 10 ⁶ / g / 2 holes, 10 ⁴ / g
/ 2 hole was 10 ⁷ / g / Sub2, pat2. Bacillus sp density of 10 ⁴ / g 2
The recovery was likely to be delayed only in the hole, but it was assumed that the recovery would be continued by continuing spraying. Also,
A remarkable correlation was observed between the Bacillus sp density in the surface soil and the recovery status of the green turf, and it became clear that the herbicide decomposition had progressed. A petri dish test also demonstrated herbicide degradation.

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(Preparation of Dilute Metabolites for Soil and Foliar Spraying) Bacillus sp spores were kept at a density of 10 ¹¹ spores / g in a sterilization box at 121 ° C. for 60 minutes, except for Bacillus sp. Sterilize various bacteria
Dissolve in 00-fold normal water, gently stir for 10 minutes to elute metabolites, allow to stand to sediment the bacterial seed material, and use the supernatant water as a stock solution, 100-fold diluted water for foliar spraying, For soil application, 200 times dilution water was used. (Results of Bacillus sp compost and dilution water spraying) Date Compost spreading Dilute metabolite spraying (L / 10a) Remarks Volume Kg / 10a Soil spraying Tea leaf spraying 10/20 to E 1000 1000 1000 1/16 to E 3000 2000 * 1000 (* sprayed on warm days) 1 / E * 1000 2 / C * 1000 3 / R to E 1000 × 3 times 4 / R to E 1000 1000 × 2 times 5 / E 1000 (sprayed after tea removal) Total 4000 4000 10000 The following effects were obtained as a result of spraying as described above. Yield Increase / decrease ratio Number of sprout Remarks (kg / a) (/ 30g) Experiment A 701 1.0 165 General compost + pesticide Experiment B 960 1 .37 129 10 ¹⁰ / g Experiment C 1080 1.54 110 Stock solution 20L + 100L

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 35/74 ABE A61K 35/74 ABE 4D004 ABG ABG 4D027 ADA ADA B09C 1/10 ZAB C02F 3/00 C02F 3 / 00 C12P 1/04 Z C12P 1/04 B09B 3/00 ZABE // (C12N 1/00 C12R 1:07) F term (reference) 2B104 EF09 2B150 AC02 4B064 AH19 CA02 CC04 CC05 CD23 DA01 DA11 DA16 4B065 AA15X BA22 BC41 BC50 BD02 CA43 CA44 CA50 CA56 4C087 AA01 AA02 AA03 AA10 BC64 CA08 CA10 MA01 MA16 MA63 NA14 ZA89 ZA90 ZB02 ZB09 ZB13 ZB15 ZB32 ZB33 ZC61 ZC65 4D004 AA41 AB10 BA10 CA18 CC02 CC07 CC08 4D027 CA03

Claims (14)

[Claims] The present invention increases the concentration of metabolites and metabolites of Bacillus sp, which are secreted with the activity and sporulation of bacterial cells, by repeating the growth cycle of germination, growth, demolition and sporulation of spores of Bacillus sp. High density Bacill
Method for producing material containing metabolites of us sp and high concentration Bacillus sp 2. Bacill is used in water systems such as wastewater treatment facilities and culture tanks.
When the concentration of Bacillus sp spores in the sediment such as sludge reaches about 10 ⁸ / g, the sediment is collected, and the solid-liquid separated dehydrated sediment is further dried. The high-density Bacillus sp and the high-concentration Bacillus sp metabolism according to claim 1, wherein the fermentation step of fermenting by applying nutrients, oxygen, and heat is repeated to increase the concentration of metabolites and produced substances as well as the density of Bacillus sp spores. Method of manufacturing material containing material 3. Spore germination, proliferation, disassembly of Bacillus sp.
High concentration Baci obtained by repeating the growth cycle called sporulation
For the llus sp and the high-concentration Bacillus sp metabolite-containing material, the growth cycle was further repeated under oligotrophic conditions to reduce the Bacillus sp density to the natural distribution density of 10 ⁴
A method for producing a material containing a high-concentration Bacillus sp metabolite, which is intended to reduce the Bacillus sp cell wall and to increase the concentration of a Bacillus sp metabolite and a produced substance while reducing the number of Bacillus sp cell walls to not more than the number per cell / g. 4. The germination, proliferation, demolition, and sporulation of spores of Bacillus sp are repeated to increase the concentration of metabolites and product of Bacillus sp secreted with the activity of cells and sporulation. High-density Bacill
Us sp and high concentration Bacillus sp metabolite containing material. 5. Spore germination, growth, disintegration of Bacillus sp.
A low-density Bacillus sp. Characterized by repeating a growth cycle called sporulation and further repeating a growth cycle under oligotrophic conditions to achieve low-density sporulation of Bacillus sp and high concentration of metabolites and produced substances. And high concentration Bacillus sp metabolite-containing material. 6. The Bacillus s according to claim 4 or claim 5.
p A liquid drug in which a metabolite or product such as a polypeptide or a cyclic peptide is eluted from a metabolite-containing material using a solvent, and the solution is removed as it is or by further centrifugation or filtration to remove spores and fermentation residues. 7. Bacillus s according to claim 4 or claim 5.
From the metabolite-containing material, a solvent is used to elute metabolites and product substances such as polypeptides and cyclic peptides using a solvent, and the solution is used as it is, or after spores and fermentation residues are removed by centrifugation or filtration, the solvent is removed. Evaporated powdered drug. 8. The livestock, poultry, fish and shellfish and the like, wherein the metabolite-containing material according to claim 4 or 5 or the drug according to claim 6 or 7 is mixed directly or in feed or drinking water. For preventing epidemics and treating infectious diseases, improving antiviral immunity, and expressing an antistress effect by giving the composition to animals and plants. 9. An immunostimulant comprising the metabolite-containing material according to claim 4 or 5 or the drug according to claim 6 or 7 as a main component. 10. An NK activity activator comprising the metabolite-containing material according to claim 4 or 5 or the drug according to claim 6 or 7 as a main component. 11. A therapeutic agent for rheumatism containing the metabolite-containing material according to claim 4 or 5 or the drug according to claim 6 or 7 as a main component. 12. A therapeutic agent for athlete's foot, insect bites, and atopic dermatitis, comprising the agent according to claim 6 or 7 as a main component. 13. A cosmetic product comprising, as a main component, a solution obtained by dissolving the liquid drug according to claim 6 or the powder drug according to claim 7 in distilled water, to which ethyl alcohol and glycerin are added and dissolved. 14. A soil containing at least 10 ⁶ / g of Bacillus sp, a Bacillus sp of at least 10 ⁶ / ml and 0.1% of Bacillus sp.
A soil purification method characterized in that Bacillus sp decomposes pesticide residues as nutrients using water containing Bacillus sp metabolites and product substances of not less than 01 ppm and eliminates its toxicity.