KR20030002398A - Novel Bacillus amyloliquefaciens B4 strain producing fermented swine feed utilizing food waste - Google Patents

Abstract

PURPOSE: Provided is a novel Bacillus amyloliquefaciens B4 strain producing swine feeds by wet fermentation of food wastes. The novel strain grows rapidly, and is excellent in enzyme activity and antagonism. CONSTITUTION: The novel Bacillus amyloliquefaciens B4 strain(KCTC 18083P) has enzyme activities of amylase, protease, cellulase and lipase, and has antagonistic effects on E.coli, Salmonella, Staphylococcus and Candida, without oral toxicity. A microbial preparation contains the novel Bacillus cereus NK 204strain(KCTC 18083P) adsorbed to a growth medium selected from one of diatomite, zeolite and rice bran and a mixture thereof, and to a carrier.

Description

Novel Bacillus amyloliquefaciens B4 strain producing fermented swine feed utilizing food waste}

The present invention relates to a microbial preparation for the wet fermentation of food waste, which is a waste organic resource in household wastes, and more particularly, to a bacterium amylolycifaciens ratio as a novel microorganism with fast cell growth and excellent enzymatic activity and antagonism. It relates to 4 (KCTC 18083P) fungicide. Using the novel strain of the present invention can be prepared by using a pig feed by wet fermentation of food waste.

The present inventors completed the present invention by successfully incubating the novel strain of the present invention by using skim steel and wet fermentation of food waste using the same.

The daily discharge of food wastes ranges from 12,000 to 20,000 tons, and only a few of them are recycled by feed or compost, but most of them rely on landfilling or incineration, resulting in serious environmental problems. Recycling of residues to livestock feed is an important means to simultaneously reduce production costs and environmental pollution in the livestock industry along with saving foreign currency by import substitution of compound feeds, which rely on imports for 96% of raw materials.

In Korea, there are many difficulties in practical use in the recycling of leftovers of food waste as feed, due to the lack of excellent technology that has been proven effective and effective by systematic technology development and field application. Therefore, although food wastes have been made a lot of efforts to solve the environmental problems as well as to enhance the import substitution effect by the technology that ensures the value and stability as feed (see Patent No. 46453, etc.). In Japan, the recycling of food waste as waste organic resources is developing a weight loss technology centered on the food service industry. The final goal is to compost the waste organic resources by ripening. Meanwhile, in the United States and Europe, the recycling of waste organic resources is used for energy production by anaerobic fermentation and compost production by aerobic fermentation, and the use of feedstock is legally allowed only within a very limited range. to be.

Accordingly, it is an object of the present invention to provide a novel strain suitable for pig feeding of food waste. Another object of the present invention to provide a microbial preparation and pig feed using the strain.

The object of the present invention was achieved by selecting a novel microbial strain and using a wet fermentation by the microorganisms in order to use waste organic resources such as food waste as pig feed. Wet fermentation of food waste converts high-molecular substances such as carbohydrates, crude protein, crude fat and crude fiber into low molecules to improve digestive absorption rate of livestock, and secondary metabolites and microbial cells synthesized in the growth process of microorganisms. It can be used as an animal booster and nutrition source to maximize the value of feed. Therefore, the value of livestock feed is largely influenced by the type and characteristics of microorganisms involved in fermentation, so only microorganisms suitable for feed purposes should be selected.

Hereinafter, the specific configuration and operation of the present invention.

The present invention consists of a new strain Bacillus amylolyticeciens ratio 4 (KCTC 18083P) and a microbial agent comprising the same.

The present invention is to isolate the microbial strain with excellent antagonistic ability and rapid growth rate for the feed of food waste according to the manual manual (Bergy's Manual of Determinative Bacteriology) and the identification of medical bacteria (Manual for the Identification of Medical Bacteria) Strain identification was carried out by examining the morphological, physiological and biochemical characteristics of the present invention strains. The bacterial fatty acid analysis was performed by extracting fatty acids from 40-50 mg of wet cells in logarithmic growth phase and using the MIS (Microbial Identification System, Hewlett-Packard 5890A). ) Was analyzed using gas liquid chromatography.

As a result, as shown in Table 1, the novel strain of the present invention forms a circular colony in a solid medium, and the colony color was yellow. The microbial strain of the present invention is a Gram-positive bacterium, and the pH range suitable for growth was 6.0-10.0, and the growth temperature range was 20-37 ° C. Catalase produced, no oxidase, and did not produce organic acid when using glucose. Gelatin liquefaction, casein and starch hydrolysis and nitrate reduction were negative, and methylred reaction, indole production and urease production were negative. Carbohydrate could not produce acid from maltose and glucose under aerobic conditions.

From the above results, the microorganism strain of the present invention was estimated to be Bacillus amylolyticipeciens, but as a result of analyzing the cell fatty acid composition for more accurate strain identification, the main component was identified as C 15: 0 anteiso, and the morphological, cultural and physiological characteristics described above. Based on the physiological characteristics, this strain exhibits a cell fatty acid that is most similar to Bacillus amyloliquefaciens, so the microbial strain isolated from the present invention is named Bacillus amyloliquefaciens B4. It was deposited on March 16, 2001 by the Korea Institute of Biotechnology and Gene Gene Bank, and was given accession number KCTC 18083P.

Characteristics of the novel microbial strain (KCTC 18083P) of the present invention factor Characteristics Morphological characteristics Gram staining Motile sporulation + Bacterium ++ Culture Characteristics Aerobic Culture Anaerobic Culture Growth Temperature 5 ℃ 30 ℃ 40 ℃ 5% Sodium Chloride +-+++ Physiological Properties Catalase Production Oxidase Production Acid Production from Glucose Carbohydrate (O / F ¹ /-) Acid Production from Carbohydrate Nitrate Reduced Gelatin Liquefaction Casein Hydrolyzed Starch Hydrolyzed Arginine Dihydrolazelysine Dicarboxylase V-P Test Fatty Acid Main Ingredient +++ O ++++++++ 15: 0 Anteiso (34%) 15: 0 Iso (29%), 17: 0 Iso (14%)

¹oxidation-fermentation test

The culture of the present invention microbial strain Bacillus amylokifaciens ratio 4 (KCTC 18083P) was used for the liquid and solid medium of LB, NB, growth was vigorous, and colony forming ability was excellent. As a result of the experiment, as shown in [Table 2], it showed high antagonistic activity against Escherichia coli and Salmonella.

Antagonistic investigation result of the present invention microbial strain Antagonistic strain Escherichia coli Salmonella Staphylococcus Candida Bacillus Antagonism ++ +++ + + -

On the other hand, as a result of culturing the microbial strain of the present invention using the liquid and solid medium of LB, NB, the growth was vigorous at medium temperature (28 ℃) and high temperature (50 ℃), colony (colony) forming ability is excellent, the experimental results, As shown in Table 3, the enzyme activity of amylase, protease, cellulase and lipase was high.

Enzyme Activity of the Microbial Strains of the Invention enzyme Amylase Protease Cellulase Lipase activation ++ +++ ++ +

Using the novel microbial strain Bacillus amylolyticipeciens ratio 4 (KCTC18083P) of the present invention will be described in detail the microbial preparation and pig feed preparation method of the present invention through the following examples, the present invention these examples It is not limited to. It goes without saying that the scope of the present invention also extends to any step change and numerical limitation.

Example 1 Preparation of Microbial Preparation for Pig Feeding by Wet Fermentation of Food Waste

The new strain Bacillus amylolyticipeciens ratio 4 (KCTC 18083P) of the present invention is excellent in growth, antagonistic ability to E. coli, and appeared to be harmless even in oral toxicity test results of any one of diatomaceous earth, zeolite, degreasing steel Adsorbed onto a carrier or a composite carrier to prepare a microbial agent.

The preparation method of the microbial preparation of the present invention is inoculated with a seed culture medium in a primary liquid medium and incubated at 30 ° C. for 24 hours. Microbial preparations were prepared. In solid culture, 5% glucose, 1% yeast extract, 0.14% potassium monophosphate, and 0.06% potassium diphosphate were added to supply carbon, nitrogen, and phosphorus, which are essential for the growth of microorganisms, and to act as cofactors for enzymes. . In this Example, the cultured degreased steel as a medium was cultured for 48 hours at 40 ℃ after the total number of bacteria was investigated was 2.0 × 10 ¹⁰ CFU / g and confirmed as the strain of the present invention.

Example 2 Acute Oral Toxicity of the Microorganisms of the Present Invention

The acute oral toxicity test was performed on the microbial preparation prepared by the method of Example 1 to confirm the presence of toxicity. Microbial stock solution (10 ⁹ cfu / ml) was used as a test substance, and the test substance was refrigerated until just before use. Experimental animals were 5-6 week old female ICR mice in the absence of specific pathogens (abbreviated as 'SPF') maintained by the Korea Research Institute of Bioscience and Biotechnology. The experimental animals were maintained in an animal room of temperature 22 ± 1 ° C., humidity 55 ± 5%, and illumination 12L / 12D. Mice were acclimated one week prior to use in the experiment. Feed for experimental animals [for CheilJedang Co., Ltd., mice and rats] and negative water were supplied after sterilization and freely ingested. The administration method was first divided into seven mice per group, and the feed by-product stock solution (10 ⁹ cfu / ㎖) was orally administered at 0.2 ml / 20g body weight. Samples were administered orally once and observed for changes in mouse weight and death for 10 days. On the day of dosing, the change of general symptoms and the presence or absence of dead animals were observed at least once in the morning and afternoon every day from 1 day, 4 hours, 8 hours, 12 hours after the next day to 10 days. On the 10th day of administration, the animals were killed and dissected, and the internal organs were visually examined. In addition, by measuring the change in body weight every two days from the day of administration was observed the weight change of the animal by the by-product.

The acute toxicity of the microbial agent of the present invention is shown in [Table 4]. Acute oral toxicity of the sample resulted in no mortality for 10 days after oral administration of the sample stock solution. There was no gross lesion on necropsy at the time of autopsy, and normal weight gain was observed without any weight loss for 10 days from the day after the administration [Table 5]. In the sample product of the present invention, no toxicity was observed upon oral administration.

Acute Oral Toxicity of Microorganisms of the Invention sample gender Marisu Number of lethal animals (day) Remarks 0 (hours) One 2 3 4 5 6 7 8 9 10 One 4 8 12 Saline solution Female 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Microbial agents Female 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Extending Effect of Samples of the Present Invention sample gender Marisu Average weight (g) Remarks 0 2 4 6 8 10 Saline solution Female 7 29.9 ± 1.2 30.1 ± 1.8 30.2 ± 2.0 30.8 ± 2.4 31.0 ± 2.6 31.6 ± 3.3 Microbial agents Female 7 30.4 ± 1.6 30.6 ± 1.4 30.7 ± 1.4 30.8 ± 1.7 31.1 ± 1.2 31.7 ± 1.2

[Example 3] Improvement of palatability of the wet fermented feed of the present invention (improvement of diet and increase of growth rate)

In this example, palatability-improving effect experiment was conducted using the microbial preparation of the present invention prepared in Example 1. The experiment was carried out to investigate the changes of enzyme activity and organic matter at each fermentation process.

[Table 6] shows the activity of amylase, protease, cellulase and lipase enzymes using a solid plate containing each substrate for each sample. This is the result measured. In the two farms (Icheon and Hwaseong, Gyeonggi-do), where the microorganisms were used for the production of hog feed, the samples were sampled at each fermentation step and the enzyme activity was measured in most samples. appear. However, after culturing each sample in a liquid medium for 1 day, the enzyme activity was measured, and the lipase and cellulase activity was also found to be the strains used in the microbial preparations of the present invention.

Enzyme Activity by Fermentation Process sample Amylase Protease Cellulase Lipase Y (food waste) 7 mm 4 mm - - Y (storage) 10 mm 6 mm - - Y (fermentation tank) 10 mm - - - Y (aging mat) - - - - W (food waste) 8 mm 8 mm - - W (storage) 10 mm 8 mm - - W (fermentation tank) - 8 mm - 6 mm W (aging mat) 6 mm 10 mm - -

[View] Y; Icheon Farm Sample, W; Hwaseong Farm Sample

Table 7 below shows the results of examining changes in organic matter before and after fermentation of food waste using the microbial agent of the present invention. As a result, in both Icheon and Hwaseong samples, the water content after fermentation decreased by 77% and 73%, respectively, and the salt content decreased by 0.41% and 0.47%, respectively. In general, fermentation using microorganisms has been considered to be problematic due to the high amount of salt due to the nature of food waste in Korea, but the amount of salt did not matter. When the amount of salt was controlled by adjusting the amount of additives in the field, the total amount of salt before fermentation was considerably low as shown in [Table 7]. Crude protein, crude fat and crude fiber contents were increased in both samples after fermentation. These results indicate that the polymer material was decomposed into small molecules by the enzymatic activity of the added microbial agent. In addition, after fermentation, the calorie increased from about 851 calories to about 1,350 calories, indicating that the dietary properties of food waste feed were greatly improved.

Organic Matter Change Before and After Fermentation of Food Waste Using Microbial Preparation of the Present Invention sample moisture(%) Crude protein Crude fat Crude fiber View minutes calcium sign Salinity Calorie Y / Ex 80.45 4.24 3.63 0.61 2.68 0.41 0.22 0.66 After Y / 77.06 5.18 2.31 1.31 2.36 0.38 0.20 0.41 W / Ex 82.55 4.62 3.75 0.73 2.87 0.55 851.40 W / after 72.99 6.23 4.06 0.98 2.80 0.47 1349.60

[View] Y; Icheon Farm Sample, W; Hwaseong Farm Sample

Example 4 Efficacy Effect of the Complex Microbial Agent

In this example, after examining the microbial changes and antagonistic activity in each step of the fermentation process, the efficacy and effects of the composite microbial preparation of the present invention were examined. [Table 8] is the result of measuring the number of viable cells using NA and LB solid medium to change the microorganisms in each step (fermentation) and the fermentation process added to food waste feed. The number of viable cells measured from the added raw material ranged from 1.4 × 10 ⁶ / g to 2.0 × 10 ⁷ / g, but the viable cell count of the microbial agents used for food waste was 5.8 × 10 ⁷ / g. In addition, the results of measuring the viable cell counts from the samples of the fermentation process of Icheon and Hwaseong farms showed that the viable cell count of microorganisms decreased as the high number of viable cells were shown in the initial food and then moved to the fermenter and the aging tank. These results indicate that the temperature and reaction time of the fermenter were 55 ° C. and about 2-3 hours, respectively, and the mesophilic microorganisms were killed except for the high temperature microorganisms. From these results, most of the microbial strains of the microorganisms used in the present invention have a high growth rate at high temperature, so that they are grown even during food waste fermentation and produce enough enzymes to have the effects and efficacy as feed microorganisms. . [Table 9] shows the antagonistic activity by measuring the size of the transparent ring around the colony (colony) by using a test plate containing a variety of microorganisms by taking a sample of food waste fermentation step by step. The samples from both Icheon and Hwaseong farms showed no antagonistic activity in the initial diet, but since the storage, fermentation and aging tanks have shown high antagonistic activity in Salmonella sp. And E. coli. The samples of Hwaseong farm showed high antagonistic activity even in Bacillus sp. However, the microorganisms of the genus Staphylococcus sp. And Candida sp. Showed no activity in most samples. From these results, it can be seen that the fermentation of food waste by the microbial agent of the present invention prevents the decay of food by other microorganisms and removes odors and prevents the appearance of flies and pupa. In particular, it is possible to prevent diarrhea caused by pathogenic microorganisms of pigs, and has the advantage of improving the preservation of food waste fermented feed. Therefore, the feed method using the present invention microbial preparation for food waste can be said to be of great value in terms of recycling as well as the dual effect of improving the palatability of the feed and increase the rate of increase of feed by increasing the palatability of the feed.

Microbial change by fermentation stage badge Y (food waste) Y (storage) Y (fermentation tank) Y (aging mat) NA 2.4 × 10 ⁸ 4.0 × 10 ⁷ 2.0 × 10 ⁴ 8.0 × 10 ⁴ LB 1.5 × 10 ⁸ 5.6 × 10 ⁷ 3.0 × 10 ⁴ 8.5 × 10 ⁴ Badge W (food waste) W (storage) W (fermentation tank) W (aging mat) NA 5.1 × 10 ⁷ 4.6 × 10 ⁸ 2.0 × 10 ⁴ 7.0 × 10 ⁴ LB 3.6 × 10 ⁷ 3.8 × 10 ⁸ 1.1 × 10 ⁵ 7.0 × 10 ⁵

[View] Y; Icheon Farm Feed, W; Hwaseong Farm Feed

Microbial Antagonism by Fermentation Stage sample C SL R209 209 BE BS Y (food waste) - - - - - - Y (storage) - ++ - - ++ - Y (fermentation tank) - ++ - - ++ + Y (storage) - ++ - - ++ + W (food waste) - - - - - - W (storage) - ++ + + ++ + W (fermentation tank) + ++ - - ++ ++ W (aging mat) + + - - ++ ++

[View] 1. Y; Icheon Farm Feed, W; Martian Farm Feed, C; Candida, SL; Salmonella,

2. R209, 209; Staphylococcus, BE; Escherichia coli, BS; Bacillus

3.-; None, +; Weak, ++; good

As will be apparent from the above description, the present invention has the effect of providing a novel microbial strain having excellent antagonistic ability and no acute oral toxicity, and added to the final reservoir by using 0.02% of the present invention by adsorbing a suitable carrier. Wet fermentation of food wastes among domestic wastes has an excellent effect of providing acute oral toxicity and excellent pig feed. In addition, the wet feed prepared by the method of the present invention has high enzymatic activity and greatly improved diet, resulting in carcass grades A and B of about 80% when fed with swine feed, flesh, year, sirloin pH, sirloin moisture. In the quantitative, protein content and meat content, there is no significant difference from pigs reared in the general formulated feed, which is a very useful invention for the feed manufacturing industry and the livestock industry.

Claims (4)

Bacillus amyloliquefaciens B4 strain KCTC 18083P with amylase, protease, cellulase, lipase enzyme activity and antagonistic activity against Escherichia coli, Salmonella, Staphylococcus, and Candida, and without oral toxicity. A microbial agent obtained by adsorbing the microorganism Bacillus amylolyticeciens B4 strain according to claim 1 to a growth substrate and a storage carrier. The microorganism preparation according to claim 2, wherein the growth substrate and the preservation carrier are selected from any one of diatomaceous earth, zeolite, degreasing steel or mixtures thereof. Pig feed that is obtained by wet fermentation by combining one or more strains of the above-described bacterial strains that exhibit the decomposition activity of waste organic resources (food waste) by using the ground skimmed steel as a growth substrate and a carrier for microbial strains.