TW201915165A - Bacillus subtilis strain for preparing fermented feather meal and use thereof for producing feather powder and proteolytic enzyme and keratinolytic enzyme strains - Google Patents

Abstract

There are two purposes of the present invention, the first purpose is to assist in improving the shortcomings of a hydrolyzed feather powder, which is to add bacillus subtilis into a hydrolyzed feather powder, and after one day of reaction, the pepsin digestibility of the feather powder is increased. The second purpose is that the strain also has a proteolytic enzyme and a keratinolytic enzyme, which can be used as an additive for animal feed to increase nutritional value.The bacillus subtilis 531-7 of the present invention is screened out from compost, then subjected to comparisons of feather decomposition and enzyme activity to confirm its commercial value. The novel Bacillus subtilis strain of the present invention is identified and confirmed by 16S rDNA gene sequence analysis and gyrB gene sequence analysis.

Description

Bacillus subtilis strain for preparing fermented feather meal and use thereof

The present invention relates to a novel strain of Bacillus subtilis isolated from compost for producing feather meal and keratinase and protease enzyme activity. The invention also relates to a preparation method for reprocessing hydrolyzed feather powder.

The main protein in feathers is keratin, which is composed of insoluble structural protein-keratin. Keratin contains a high amount of disulfide and hydrophobic and hydrogen bonds, and is not easily broken down by general proteolytic enzymes. The animal body lacks such proteolytic enzymes, so for poultry and livestock, keratin cannot be used directly.

The method for hydrolyzing feather powder in the prior art is mainly realized by using steam to hydrolyze feathers. Specifically, as shown in FIG. 13, after the wet feathers are dewatered (step 301), the feathers are input into a refining furnace, and after the steam is injected, the feathers are uniformly heated and stirred. When the feather and water are in the cooking machine, add 10 bar steam and control the temperature at 187 ° C; at this time, the hydration in the cooking machine turns into water vapor, and the volume of water vapor increases to generate pressure, helping Hydrolysis of feathers; Then, the steamer was maintained at 3 bar, 145 ° C for 50 minutes, and the feathers were hydrolyzed to preserve the available protein. After the high-pressure hydrolysis, the feathers become a paste (step 302), and after being dried in a vacuum, the dry feather powder with a moisture content of less than 12% is extracted (step 303). After the dry feather powder is obtained, put it in a collection tank to cool it down to reduce the temperature. After the dry feather powder has cooled down, send it to the vibrating screen and sifter out coarse blocks or waste. Then, send it to the crusher, bagging and The weighed amount is the hydrolyzed feather meal (step 304).

However, according to the previous method of hydrolyzing feather powder, the control of vapor pressure and time will affect the quality of hydrolyzed feather powder. Excessive hydrolysis will degrade a large amount of amino acids and cause nutrient loss; but insufficient hydrolysis will incompletely dissolve the disulfide bonds, resulting in poor protein quality.

The prior art points out that although the water bath heating method can increase the nutritional content to a certain extent, it will lose the content of essential amino acids [such as lysine, methionine, tryptonphan, etc.] And lead to an increase in the content of non-essential amino acids such as lysinoalanine and lanthionine. Even if the feather is hydrolyzed by a water bath heating method, if it is hydrolyzed excessively, there will be problems such as destruction of amino acids, degradation of protein quality, or low digestibility of the obtained feather meal.

Furthermore, the quality of hydrolyzed feather meal produced from different sources or batches may be different. Existing feed company Fushou Industry provides 8 different sources or different batches of hydrolyzed feather meal products. After analysis, the pepsin digestibility of hydrolyzed feather meal is about 23.5% to 92.8%, and the crude protein content is about 79.7% to 93.3%. The feed industry attaches great importance to pepsin digestibility, and poor digestion often causes illness in poultry or livestock. The general government requires pepsin digestibility in hydrolyzed feather meal products to be higher than 70% or 75%. The crude protein content of domestic or imported hydrolyzed feather meal is 79.7% to 93.3%, but the digestibility is between 23.5% and 92.8%, which shows that the digestibility of hydrolyzed feather meal cannot be judged by the crude protein content. Regarding the amino acid content of the hydrolyzed feather meal, the feed industry believes that it can be adjusted by subsequent additions. Therefore, the amino acid content of the hydrolyzed feather meal is less important to poultry or livestock than pepsin digestibility.

In recent years, due to the high price of feed fishmeal, feather meal converted from discarded feathers is one of the industry-locked alternatives. However, the quality of hydrolyzed feather meal is currently unstable, resulting in unstable digestibility of feeds formulated with it. Big trouble. In addition, animals such as feathers are not easily digested by steam hydrolysis, so they seek biodegradation to improve the difficulty of digestion. However, it takes time to biodegrade, and the waste feathers in the slaughterhouse need to be treated immediately to avoid odor.

In view of this, the prior art needs to be improved. To provide rapid preparation of stable and high-quality feather meal quality, while avoiding the occurrence of feather accumulation, remains to be developed.

In order to overcome the shortcomings of the prior art, the purpose of the present invention is twofold. The first purpose is to help improve the disadvantages of hydrolyzed feather meal. The hydrolyzed feather meal is added to Bacillus subtilis to increase the pepsin digestibility of the feather meal. The second purpose is to use this strain to produce proteolytic enzymes and keratinolytic enzymes, which can be used as animal feed additives to increase nutritional value. The Bacillus subtilis of the present invention does have commercial value.

In order to achieve the above-mentioned object of the present invention, the present invention provides a Bacillus subtilis 531-7 strain, which is deposited in the Hsinchu Food Industry Development Research Institute of Taiwan Consortium, the deposit number is BCRC 910793, and the deposit date is 2017. August 09; also deposited in China Type Culture Collection Center, the deposit number is CCTCC M 2017495, and the deposit date is September 6, 2017.

The present invention also provides a microbial preparation, which comprises a culture of the aforementioned Bacillus subtilis 531-7 strain as an active ingredient.

The present invention further provides the use of the Bacillus subtilis 531-7 strain as the feed or feed additive; preferably, the feed is animal feed or fish feed.

The invention further provides the use of the Bacillus subtilis 531-7 strain as the fertilizer or fertilizer additive. The Bacillus subtilis 531-7 strain of the present invention can be added to organic compost to promote composting efficiency.

The invention further provides a method for preparing bacterial fermented feather powder, which comprises the following steps: (1) preparing feathers (such as white broiler feathers, loess chicken feathers, and black-bone chicken feathers), cutting the feathers and placing them in a culture medium; The medium includes, but is not limited to, a basic salt medium or groundwater (groundwater pH value 7.72, conductivity 657 μS / cm, salinity 325 ppm, total dissolved solids (TDS) 456 mg / L), and a weight-volume ratio of 1% (w / v) to 6.5% (w / v) feather medium; (2) adding the Bacillus subtilis 531-7 strain to the feather medium as described above, so that the concentration of the Bacillus subtilis 531-7 strain is 10 per ml ⁵ to 10 ⁷ colony units (CFU / mL) for 3 to 8 days at a temperature between 20 ° C and 40 ° C; (3) sterilize the cultured feather medium containing Bacillus subtilis 531-7 strain Dry, and then crush with a homogenizer and grind to less than 0.15mm with a ball mill to obtain bacterial yeast feather powder. In addition, when the medium in the step (1) of the present invention is groundwater, a similar effect can be achieved.

Preferably, in the step (1), the weight-volume ratio is 5% (w / v) to 6.5% (w / v) feather medium. Preferably, the feathers of the feather medium include, but are not limited to, chicken feathers; more preferably, the chicken feathers include, but are not limited to, white broiler chicken feathers.

Preferably, in the step (2), the subtilis 531-7 strain is added to the feather medium and cultured for 1 to 8 days, more preferably 6 days.

Preferably, in the step (2), the culture temperature is 37 ° C.

The invention provides a bacterial fermented feather powder, which is prepared by the above method. The pepsin digestibility is 86.3%, the crude protein is 94.7%, and the total amino acid content is 99.1%. The hydrolyzed amino acid content value (amino acid divided by crude protein, aa mg / CP mg,%) is: methyl sulfur Amino acid (Met) 0.3%, Lysine (Lys) 1.6%, Histidine (His) 0.5%, Tryptophan (Trp) 0.5%, Thramine (Thr) 4.6%, Valine (Val) 6.3%, Cystine (Cys) 6.7%, Isoleucine (Ile) 4.4%, Leucine (Leu) 8.1%, Tyrosine (Tyr) 3.3%, Phenylalanine (Phe) 5.4%, Spermine Acid (Arg) 6.4%, alanine (Ala) 4.5%, aspartic acid (Asp) 6.6%, glutamic acid (Glu) 10.4%, glycine (Gly) 7.6%, proline (Pro) 11.6 %, Serine (Ser) 10.5%.

The present invention further provides the use of the bacterial fermented feather powder as the feed or feed additive.

The invention further provides the use of the bacterial fermented feather powder as the fertilizer or fertilizer additive.

The present invention further provides a method for preparing fermented feather powder, which comprises the following steps: (1) Completely hydrolyzed feather powder is provided, and the hydrolyzed feather powder is placed in a culture medium, including but not limited to a basic salt medium or groundwater, to obtain The weight-volume ratio is from 1% (w / v) to 20% (w / v) hydrolyzed feather meal medium; (2) the aforementioned Bacillus subtilis 531-7 strain is added to the hydrolyzed feather meal medium to make Bacillus subtilis 531 -7 strain per ml concentration of ¹⁰⁵ to ¹⁰⁷ colony-forming units (CFU / mL) were cultured 1-3 days (37 ° C); (3 ) cultured for 1 day to 3 days, containing the B. subtilis 531-7 After the strain's hydrolyzed feather meal culture medium is sterilized and dried, it is broken (not ground) by a homogenizer to obtain a processed fermented feather meal. In addition, when the medium in the step (1) of the present invention is groundwater, a similar effect can be achieved.

Preferably, the hydrolyzed feather powder includes, but is not limited to, a commercially available hydrolyzed feather powder.

The "commercially-available hydrolyzed feather meal" in the present invention refers to feather meal that has not been cultured by the Bacillus subtilis 531-7 strain of the present invention, and the feathers are hydrolyzed to hydrolyzed feather meal under high pressure.

Preferably, in the step (2), the culture time is 72 hours. After 72 hours of low-hydrolyzed feather meal incubation, its digestibility improved from 23.5% to 83.4%. Such as culture for 24 hours, the digestibility improves to 62.8%. After 72 hours of cultivation of highly hydrolyzed feather meal, its digestibility improved from 78.9% to 84.9%.

The invention also provides a processed fermented feather powder, which is prepared by the above method.

The invention further provides a method for preparing an enzyme, which comprises the following steps: (1) preparing a raw material, placing the raw material in a culture medium, and obtaining a raw material culture medium having a weight-volume ratio of 1% (w / v) to 10% (w / v); ; (2) the request entry as Bacillus subtilis strain 1531-7 of the material added to the medium so that the concentration of Bacillus subtilis strain 531-7 becomes ¹⁰⁵ per ml to 10 ⁷ colony-forming units (CFU / mL) cultured. (3) The raw material medium containing the Bacillus subtilis 531-7 strain is dried to obtain an enzyme.

Preferably, in step (1), the raw material is feather; in steps (1) to (3), the raw material medium is feather medium; in step (3), the enzyme comprises keratinase.

Preferably, in step (1), the raw material is soybean; in steps (1) to (3), the raw material culture medium is soybean culture medium; in step (3), the enzyme comprises caseinase.

The advantages of the invention are:

1. The Bacillus subtilis 531-7 strain of the present invention is a strain having protease, which mainly includes two functional activities, such as keratinase and casein. The protease is produced by the strain as fertilizer or animal feed. In addition, Feather meal quality is stable (Table 4 below), such as consistent pepsin digestibility (Table 4 below). Based on the above characteristics, the fungus can be developed for use in feather waste disposal, animal feed addition, fertilizer, and the like.

2. In the present invention, the method for preparing bacterial fermented feather meal can be used to collect enzymes. Bacteria were added to the basic salt medium (containing 2% feathers) and cultured for 6 days (37ºC). After filtration, the filtrate was taken to obtain the crude enzyme. Keratinase was obtained without sterilization. Protease was collected by the following methods: Bacteria were added to the basic salt medium (containing 1% soybean flour), cultured for 2 days (37ºC), and the filtrate was taken to obtain the crude enzyme without sterilization to obtain protease. The protease obtained by the present invention (mainly including keratinase and casein) can be used as an animal feed additive to increase nutritional value, and can also be used as fertilizer.

3. The method for preparing processed fermented feather powder of the present invention, by adding low-hydrolyzed feather powder to the aforementioned strain, the feather powder quality (such as pepsin digestibility, crude protein content) is improved after the action, and the commercially-available hydrolyzed feather is improved. The shortcomings of powder make processed fermented feather powder as an animal feed additive more nutritious and easy to digest, and can also be used as fertilizer.

In the following, with reference to the drawings and the preferred embodiments of the present invention, the technical measures adopted by the present invention to achieve the purpose are further explained.

Preparation Example 1 Source and isolation of bacterial strain

The novel strain in this case, code-named Bacillus subtilis 531-7, was isolated from compost, and then compared with the efficiency of feather decomposition, and deposited in the Center for Bioresources Conservation and Research, Taiwan Food Industry Development Institute, Hsinchu, Taiwan. , The serial number is BCRC 910793, the deposit date is August 09, 2017; it is also deposited at the China Type Culture Collection Center, the deposit number is CCTCC M 2017495, and the deposit date is September 6, 2017. The following is a three-stage screening method for strains:

Phase 1: Comparison of Casein Breakdown Power

Take 1 gram (g) of soil and add 9 milliliters (mL) of sterile water, treat it at 80 ° C for 20 minutes with water bath or directly (room temperature) diluted to 0.01 times and 0.001 times, and then apply it on the base salt with 1% casein Medium (basal salt medium) [contains 0.7 g / L potassium dihydrogen phosphate (KH ₂ PO ₄ ), 1.4 g / L dipotassium hydrogen phosphate (K ₂ HPO ₄ ), 0.5 g / L sodium chloride (NaCl), 0.1 g / L magnesium sulfate (MgSO ₄ ), 10 g / L casein, pH 7.2, 15 g / L agar gel (agar)], select a single colony with a transparent ring in LA medium [LB medium (Lysogeny broth plus ampicillin) after activation for 20 hours, pick a single colony with a sterilized toothpick, spot it on the casein medium, and incubate at 37 ° C for 24 hours, observe whether a transparent ring appears around the colony, take a picture and Record the size of the colonies and transparent rings. Choose the larger transparent ring and colonies to enter the second stage.

Phase II: Visual comparison of the resolution of a single feather

Preparation of phosphate buffered saline (PBS): 10 mL of PBS was placed in a spiral test tube, and a whole chicken feather was added to weigh about 0.05 grams, and then sterilized by autoclaving. After the test strain was activated on LA and cultured in 5 mL of LB for 20 hours, the amount of bacteria was adjusted to OD ₆₀₀ = 0.3 (10 ⁸ cfu / mL) and 100 microliters (μl) was pipetted into a test tube at 37 ° C. After 6 days of down culture, the integrity of the feathers was observed, and those who did not add the test strain were used as blanks. Take the third or fourth class of decomposing strains to compare the bacterial decomposition rate of 4 grams of feathers in the next stage. Decomposition force is divided into four levels, zero level: no obvious decomposition. Level 1: Partially decomposed, with only a few feather debris suspended. Level 2: Feathers are clearly decomposed, and the amount of debris is large. Level 3: Feathers are shattered, with only shafts remaining. Level 4, fully decomposed feathers, no residual appearance (see Figure 1).

Phase III: Comparison of bacterial decomposition rate (%) of 4 grams of feathers

Prepare 200 mL of basal salt medium (0.7 g / L KH ₂ PO ₄ , 1.4 g / LK ₂ HPO ₄ , 0.5 g / L NaCl, 0.1 g / L MgSO ₄ , pH 7.2), and add 4 grams of white broiler feathers to 500 The 2 mL feather flask becomes 2% feather medium. After high temperature sterilization, add the test strain that has been activated in LB for 24 hours. The bacterial solution is OD ₆₀₀ = 0.3 (10 ⁸ CFU / ml). The volume percentage is 2% (v / v). After incubating the Erlenmeyer flask at 37 ° C and 150 rpm for 6 days, the filter paper [ADVANTEC No. 1, pore size> 10 micrometers (μm)] was filtered (the collected filtrate was a crude enzyme), and the trapped feather residue was put into After drying in a 65 ° C oven, weigh and calculate the feather decomposition rate (%), strains with a decomposition rate greater than 80%, and then make feather meal and two crude enzyme activity tests (keratinase and protease). Strains and enzymes with decomposed feathers of commercial value are screened out.

Preparation Example 2 Test of Basic Physiological Characteristics of Strains

1. Basic characteristics of the strain:

Appearance of Bacillus subtilis 531-7 strain: as shown in Figure 2 is a germ-positive bacterium, the single cell size is about 0.4 μm to 0.6 μm × 1.0 μm to 1.6 μm, the end of the bacteria is semicircular and single or short chain. As shown in FIG. 3, spores are formed at the apical part of the cell, and the spores are oval or round, and the size is about 0.6 μm to 0.7 μm × 1.0 μm to 1.6 μm. As shown in FIG. 4, the electron micrograph of the Bacillus subtilis 531-7 strain showed prospores. As shown in FIG. 5, when the Bacillus subtilis 531-7 strain of the present invention is grown in LA medium, the initial (24 hours) fungus-shaped ridges are round and white, with raised colonies formed on the surface, irregular colonies or smooth edges. As shown in Figure 6, the colony surface was dry and wrinkled after 3 to 4 days.

2. pH measurement

The 3 days and 6 days of culture were taken from the filtered feather decomposition filtrate to measure the pH. The results showed that the pH of the feather decomposition solution was 7.6 to 7.7 after 3 days, and the pH was 8.5 to 8.7 after 6 days.

3. Growth curve determination-turbidity (OD) value and number of colonies (CFU)

Take a single intact colony with an inoculating loop in a 15 mL centrifuge tube containing 6 mL of LB broth, and shake culture at 37 ° C (150 rpm) for 24 hours. Adjust the bacterial liquid OD ₆₀₀ <0.06. Take the adjusted 1 mL of bacterial solution (OD ₆₀₀ <0.06) and place it in 50 mL of LB culture solution (OD ₆₀₀ <0.01). Shake the culture at 37 ° C (speed 150 rpm), and then set it at 0, 1, 2, respectively. 4, 6, 8, 24, 26, 28, 30, and 32 hours (adjust the back-end time point according to the growth curve). Count the number of bacteria and measure the OD value. Take 2 mL of each time point to measure its OD value (correction solution is LB culture solution). And take another 0.1 mL to count the number of bacteria (37 ° C, 24 hours) (as shown in Figure 7). The bacterial solution is in the logarithmic growth phase after 2 hours to 6 hours of culture, and enters the lag phase after 6 hours of culture.

Preparation Example 3 Strain Identification Method

1. Identification of BIOLOG strains and determination of carbon source and energy

(1) The BIOLOG identification system is used to determine the strain of the strain of the present invention, and the obtained threshold value is compared with that of the BIOLOG company's resource bank. The strain has 71 carbon sources and 23 OD ₅₉₅ values for chemical sensitivity tests and strains from the resource bank. The similarity value (above 0.50) gave the close identification ID of the tested strain. Based on the analysis of the software of the MicroStation ^TM system provided by BIOLOG, it was found that its phenotype was the highest similar to that of Bacillus subtilis strain, with a similarity of 0.64, and possibly an organism of 0.77. Therefore, the strain was named as Bacillus subtilis 531-7 strain ( Bacillus subtilis 531-7).

(2) Available carbon sources and chemical sensitivity test for Bacillus subtilis 531-7 strain: 23 kinds of available carbon sources. Α-D-Glucose, Dextrin, D-Mannose, D-Maltose, D-Trehalose, D-Celloblose, Gentioblose, Sucrose, L-Malic Acid, D-Gluconic Acid, L-Lactic Acid, Citric Acid, Alanine (L -Alanine), L-Aspartic Acid, L-Glutamic Acid, D-Sorbitol, D-Mannitol, Glycerol, Muscle Myo-inositol, L-Galactonic Acid Lactone, D-Sallcin, β-methyl-D-Glucoside, and D-Saccharic acid) 23 species in total.

The results of the chemical sensitivity test were salinity resistance 1, 4, 8% NaCl, pH 5 and pH 6, resistance to 1% Sodium Lactate, Aztreonam, Lithium Chloride, butyric acid Sodium Butyrate and Potassium tellurite are chemically sensitive. The carbon source energy use and chemical sensitivity test results can be used for the incremental cultivation of Bacillus subtilis 531-7 strain.

5. Identification of API-50CHB strains and determination of carbon source and energy

(1) The API-50CHB kit (bioMerieux, Inc.) was used to determine the strain and carbohydrate energy of the strain of the present invention. Each test well of API 50 CH strip contains 49 different carbohydrates, which can be used to determine the use of carbon sources by bacteria. After inoculation with bacteria, the growth of bacteria in it will cause the pH of the medium to change. The color changes to determine the type of bacteria. According to the method described in the booklet provided by bioMerieux, Inc., Hazelwood, MO, the results showed that 531-7 strain was identified as Bacillus licheniformis (99.9%) by API 50CHB. However, API 50CHB is mainly used to analyze the availability of different carbohydrates. The accuracy of bacteria identification is not as accurate as DNA sequence identification.

(2) Energy source for carbon source: The 531-7 strain screened by the present invention can be analyzed by API 50CHB. It can be seen that the bacteria can use 34 of 49 carbon sources, including glycerol, L-arabinose, D-ribose, and D- Xylose, L-xylose, D-ribitol, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, galactitol, inositol, D-mannitol, D -Sorbitol, methyl α-D-glucopyranoside, N-acetamylglucosamine, mandelin, arbutin, chestnut / iron citrate, salicin, D-cellobiose, D-maltose, D -Melibiose, D-sucrose, D-trehalose, inulin, D-raffinose, starch, liver sugar, gentiobiose, D-melosylose, D-tagatose, potassium gluconate.

2. 16S ribosomal DNA (rDNA) identification of bacterial strains

Strain DNA was extracted and polymerase chain reaction (PCR) was performed with a bacterial 16S rDNA universal primer pair: forward primer (F: SEQ ID NO.1) and reverse primer (R: SEQ ID NO.2). The length of the product was 1481 base pair (bp). The amplified DNA products are sequenced by nucleotides, and the query and comparison of the online gene bank (GenBank) is performed by the BLAST program to identify the genus. The isolated strain was amplified by 16S rDNA gene with bacterial universal primers. As a result, it was found that the isolate could be amplified to 16S rDNA gene fragment, sequenced as Bacillus subtilis (1504bp, SEQ ID NO. 3, similarity 99%). .

3. Bacterial strain gyrB gene identification method

Strain DNA was extracted and bacterial gyrB gene primer pairs: forward primer (F: SEQ ID NO. 4) and reverse primer (F: SEQ ID NO. 5) were used to identify species of Bacillus species. The amplified DNA product was subjected to nucleotide sequencing and alignment, and the fragment sequence of this strain was confirmed to be Bacillus subtilis, with a size of about 1.3 kb. The sequencing result was Bacillus subtilis (1259bp, SEQ ID NO. 6, similarity is 99%).

The strains were compared with 16S rDNA (Figure 8) and gyrB gene (Figure 9): the strains included the Bacillus subtilis 531-7 strain and 8 different spores deposited at the American Type Culture Collection (ATCC) Bacillus, the phylogenetic tree was analyzed by Neighbor-joining (NJ) method; the 數 character on the branch represents the bootstrap value.

Example 1 Enzyme activity test

(1) Analysis of keratinase activity in proteases obtained from feather medium

Take the Bacillus subtilis 531-7 strain selected in Preparation Example 3, culture it in LB and adjust the bacterial solution to OD ₆₀₀ = 0.3 (10 ⁸ CFU / mL). Take 4 mL of the bacterial solution to 2% feather medium (4 grams of feathers). A total of 200 mL, so that the concentration of Bacillus subtilis 531-7 strain in the feather medium became 2 × 10 ⁶ CFU / mL, and then cultured at 37 ° C and 150 rpm for 3 days, the culture solution was placed on ice, and Brinell Funnel filtration (ADVANTEC No. 1 filter paper), collect the liquid on ice to be the first crude enzyme solution (about 180 mL), and immediately take 1 mL of fresh first crude enzyme solution to analyze keratinase activity (U / mL) and protein The content is converted into specific activity (U / mg). Among them, Bacillus subtilis 531-7 strain has a variety of proteases cultured through feather medium. Here, the main protease-keratinase is used as the activity analysis. The remaining first crude enzyme solution was sterilized and lyophilized to become the first freeze-dried enzyme. The sterilization method is to sterilize the filtrate: after centrifugation at 4 ° C, 8500 × g, take the supernatant (approximately 1 mL to 2 mL) and extract it on ice with a 0.45 μm filter membrane (mixed cellulose ester, ADVANTEC ^® ) Air filtration (full ice). Table 1. Specific activity of the first crude enzyme of Bacillus subtilis 531-7 and commercially available keratinase * Commercially available products are taken from Taiwan Yongxin Pharmaceutical Industry Co., Ltd. for the production of Fusuo enzyme 600 powder (versazyme ^® 600, 1 kg, produced by American BioResource International (BRI) OEM, Taiwan Province Agriculture and Animal Feed Addition No. AN00561; use Target: poultry, livestock; use: improve feed efficiency, promote animal growth and increase poultry egg production rate).

It can be known from the above Table 1 that the first crude enzyme solution of the Bacillus subtilis 531-7 strain of the present invention increases the specific activity of keratinase due to freeze-drying to a concentrated state, and the first crude enzyme solution of the Bacillus subtilis 531-7 strain is fresh or After freeze-drying, the specific activity of keratinase is better than the commercial products, about 3 to 5 times higher.

(2) Analysis of caseinase activity in protease obtained from soybean culture medium:

Take the Bacillus subtilis 531-7 strain screened in Preparation Example 3, culture it in LB and adjust the bacterial solution to OD ₆₀₀ = 0.3 (10 ⁸ CFU / mL). Take 4 mL of the bacterial solution to 1% soy bean culture solution (2 grams of soybeans). 200 ml of basic salt medium was added to the powder), so that the concentration of Bacillus subtilis 531-7 strain in the soybean medium was 2 × 10 ⁶ CFU / mL, cultured at 37 ° C, 150 rpm for 2 days, and then filtered with a filter ( ADVANTEC No. 1 filter paper) The liquid collected by filtration is the second crude enzyme solution (about 180 mL), and the second crude enzyme solution is used to analyze caseinase activity (U / mL) and protein content, which is converted into specific activity (U / mg) Among them, there are many kinds of proteases that are cultivated by soybean culture solution of Bacillus subtilis 531-7 strains. Here, the main protease-casein protease is used as the activity analysis. After the remaining second crude enzyme solution is sterilized, it is freeze-dried to become a freeze-dried second crude enzyme. Table 2. Specific activity of the second crude enzyme of Bacillus subtilis 531-7 and commercially available caseinase * Commercially available products are taken from Taiwan Yongxin Pharmaceutical Industry Co., Ltd. for the production of Fusuo enzyme 600 powder (versazyme ^® 600, 1 kg, produced by American BioResource International (BRI) OEM, Taiwan Province Agriculture and Animal Feed Addition No. AN00561; use Target: poultry, livestock; use: improve feed efficiency, promote animal growth and increase poultry egg production rate).

From Table 1 above, it can be known that the first crude enzyme solution of the Bacillus subtilis 531-7 strain of the present invention does not have a specific activity of keratinase due to freeze drying, and the first crude enzyme solution of the Bacillus subtilis 531-7 strain is either fresh or After freeze-drying, the specific activity of keratinase is better than the commercial products. From Table 2 above, it can be known that the second crude enzyme solution of the Bacillus subtilis 531-7 strain of the present invention will not affect the specific casein activity due to freeze-drying, and whether the second crude enzyme solution of the Bacillus subtilis 531-7 strain is fresh or After freeze-drying, the specific activity of casein is better than the commercial products, which is about 2 times higher.

(3) Concentration of crude enzymes and analysis of protein molecular weight .

The experiment was divided into 4 groups: (a) 11594 crude enzyme: BCRC 11594 ( Bacillus licheniformis ) was obtained in the same manner as in Example 1 (1) to obtain 11594 crude enzyme . (b) 531-7 crude enzyme: taken from the first crude enzyme solution of Example 1 (1). (c) 531-7 Precipitation enzyme: Put the first undiluted fresh crude enzyme solution [from Example 1 (1), in which the bacterial solution is cultured with 2% or 10% feather medium] 50 mL in an ice bath Medium, slowly add solid ammonium sulfate 40% to 80% saturation (approximately 10 mL increase), leave to stand for 20 minutes after dissolution, centrifuge 9000 × g, remove the supernatant after 20 minutes, and dissolve the precipitate in 10 mL 0.1 M PBS (pH 7.2) to obtain 531-7 precipitated enzyme. The above three groups were subjected to polyacrylamide colloid electrophoresis (SDS-PAGE), and stained with coomassie blue dye.

Please refer to Figure 10, lines 1 and 8 for protein standards (Protein Ladder, Thermo, # 26616LCS). Lines 2 and 4 are the crude enzyme solution of 531-7 crude enzyme (2% feather medium) diluted 0.2 times, and the band is not obvious. Lines 3 and 5 are 531-7 precipitated enzyme (2% feather medium) diluted 5 times, about 4 distinct bands; one of them is between 55-70 kDa, and one is The bands are between 55-40 kDa and there are 2 bands between 35-25 kDa. Line 6: 531-7 precipitated enzyme (10% feather medium) is diluted 5 times, and there are about 7 bands; 1 band is located at 130 kDa, and 1 band is between 55-70 kDa. One band is located at 35 kDa, two bands are between 35-25 kDa, and two bands are between 15-25 kDa. The seventh line is 11594 crude enzyme diluted 0.2 times, about 5 bands; 1 band is between 55-40 kDa, 1 band is at 40 kDa and 1 band is between 35 -Between 25 kDa, one band is located at 15 kDa and one band is located at 10 kDa. Most literatures indicate that the molecular weight of keratinase is between 30 and 40 kDa. Therefore, the molecular weight of keratinase of Bacillus subtilis 531-7 is between 25 and 35 kDa, which is alkaline keratinase (pH 8.9). SDS PAGE analysis showed a large amount of low molecular weight polypeptide (less than 13 kDa) in feather decomposition.

Example 2 Preparation of Bacterial Fermented Feather Powder

(1) Please refer to FIG. 11, take 25 grams of chicken feathers which have been washed, sterilized and dried, and cut into pieces, and add 500 mL of basic salt medium (become 5% to 6.5% (w / v) feather medium) (step 101), The bacterial solution containing the Bacillus subtilis 531-7 strain of Preparation Example 3 was adjusted to OD600 = 0.3 (10 ⁸ CFU / mL), and 5 mL of the bacterial fluid was added to 500 mL of 5% feather medium to make the Bacillus subtilis 531-7 strain After the concentration of the feather medium became 10 ⁶ CFU / mL, and then cultured at 37ºC and 150 rpm for 1 to 6 days (the decomposition started on 1 day, the decomposition power was about 60% after 3 days, and the decomposition power was greater than 86% after 6 days. ) (Step 102), sterilize the culture medium for 15 minutes (121ºC, 15psi per square inch), and then dry it at 80ºC for 48 hours. Collect the dried material and grind it to an appropriate size to obtain bacterial yeast feather powder (Step 103). ). Its quality analysis is shown in Table 3: Table 3. Analysis of digestibility, crude protein and amino acids of 531-7 bacterial fermented feather meal Table 4. Comparison of the quality of commercially available hydrolyzed feather meal with bacterial fermented feather meal produced by the strains of the invention * The average value of 9 batches of commercially available hydrolyzed feather powder. The statistical method is mean ± standard deviation (mean ± SD).

The quality of bacterial fermented feather powder (Table 3) is better than the commercially available hydrolyzed feather powder (Table 4), and as can be seen from Table 4 above, the standard deviation of the commercially available hydrolyzed feather powder is large, indicating that the quality of each batch is quite inferior. Stable; Compared with the bacterial fermented feather powder of the present invention, the standard deviation is small, indicating that the quality is stable. The most important thing is to improve the digestibility of gastric protein, from 65.6% to 86.3%. And the crude protein content also increased, while the total amino acid content was slightly higher than the commercially available hydrolyzed feather meal.

(2) In the step of Example 2 (1), the decomposing solution after the bacterial solution was cultured and fermented in 2% or 10% feather medium for 6 days was allowed to stand in two layers, and the upper layer liquid and the lower layer liquid were filtered. Among them, the decomposition liquid, the upper liquid and the lower liquid contain a large amount of amino acids (Table 5), which can be used as additives for organic fertilizers. Table 5.Amino acid analysis of the decomposition solution obtained from 2% or 10% feather medium

Example 3 Preparation of processed fermented feather powder

Please refer to FIG. 12, take 10 g of commercially available low-hydrolyzed feather meal, add 200 mL of basic salt medium (5% hydrolyzed feather meal medium) (step 201), and add a strain containing Bacillus subtilis 531-7 strain of Preparation Example 3 The bacterial solution was adjusted to OD ₆₀₀ = 0.3 (10 ⁸ CFU / mL), and 10 mL of the bacterial solution was taken into 200 mL of 5% hydrolyzed feather meal medium, so that the concentration of Bacillus subtilis 531-7 strain in the hydrolyzed feather meal medium became 5 ×. 10 ⁶ CFU / mL, and then culture the yeast at 37 ° C and 150 rpm for 1 day (step 202), sterilize the culture solution for 15 minutes, and then dry it at 80 ° C for at least 48 hours. Collect the dried material and grind it. The leaven feather meal is processed (step 203). The commercially available low-grade hydrolyzed feather powder (codes L5, L6, and M3) (taken from Taiwan Fushou Industrial Co., Ltd.) was compared with the processed peptone feather powder for pepsin digestibility. The pepsin decomposition test is as follows: Take 0.3 g of feather powder and add it to 50 mL of freshly prepared and pre-heated 0.075N hydrochloric acid (HCl) of 0.2% pepsin (active 1: 10000) at 45 ° C. After mixing at 45 ° C, The reaction was shaken at 150 rpm for 16 hours, filtered through Whatmen # 541 filter paper, the remaining insoluble matter after decomposition was dried and weighed at 80 ° C, and the remaining insoluble matter was analyzed for protein content and pepsin digestibility.

As shown in Table 6 below, after comparing the digestibility of pepsin, the digestibility of the commercially available hydrolyzed feather powder L5, L6, and M3 was 23.5, 29.2, and 68.3%, respectively. The digestibility of the processed fermented feather powder obtained after processing was improved. To 62.8, 67.4 and 79.2% , the digestibility increased by approximately 2.3, 2.7 and 1.2 times. Table 6. Comparison of digestibility of commercially available hydrolyzed feather meal and processed fermented feather meal

As shown in Table 7 below, the crude protein content of processed fermented feather meal is about 3.5% to 13.8% higher than that of hydrolyzed feather meal before processing. Table 7.Effects of commercially available hydrolyzed feather meal and processed fermented feather meal on crude protein

It will be apparent to those skilled in the art that various modifications and variations can be made in accordance with the present invention without departing from the scope and spirit of the invention. Although the invention has been described with specific preferred specific facts, it must be understood that the invention should not be unduly limited to such specific specific facts. In fact, in implementing the described mode of the present invention, different modifications obvious to those skilled in the art are also included in the scope of the following patent applications.

S101‧‧‧Step 101

S102‧‧‧Step 102

S103‧‧‧Step 103

S201‧‧‧Step 201

S202‧‧‧Step 202

S203‧‧‧Step 203

S301‧‧‧Step 301

S302‧‧‧Step 302

S303‧‧‧Step 303

S304‧‧‧Step 304

Figure 1 is a photo of the degree of feather decomposition. FIG. 2 is a Gram-stained microscopic examination of the Bacillus subtilis 531-7 strain of the present invention. Figure 3 is a microscopy image of endospore staining of the Bacillus subtilis 531-7 strain of the present invention. FIG. 4 is an electron microscope photograph of the Bacillus subtilis 531-7 strain of the present invention. FIG. 5 is a photograph of the colony morphology of the Bacillus subtilis 531-7 strain cultured for 24 hours. FIG. 6 is a photograph of the colony morphology of a 72-hour culture of the Bacillus subtilis 531-7 strain of the present invention. FIG. 7 is a line chart showing the relationship between the OD ₆₀₀ value of the bacterial solution and the colony forming unit (CFU) at various time points after cultivating the Bacillus subtilis 531-7 strain of the present invention. FIG. 8 is a tree evolution diagram of the parental evolution of 16S rDNA of the Bacillus subtilis 531-7 strain and 8 public strains of the invention. FIG. 9 is a tree evolution diagram of the parental evolution of the gyrB gene of the Bacillus subtilis 531-7 strain and eight public strains of the present invention. FIG. 10 is a protein electrophoresis staining diagram of Bacillus subtilis 531-7 strain of the present invention. FIG. 11 is a flowchart of preparing fermented feather powder; S101 to S103 represent steps 101 to 103. FIG. 12 is a preparation flowchart of processing feather powder; S201 to S203 represent steps 201 to 203. FIG. 13 is a flowchart of the preparation of the prior art hydrolyzed feather powder; S301 to S304 represent steps 301 to 304.

TW Republic of China, Bioresources Conservation and Research Center of Food Industry Development Institute, 2017/08/09, BCRC 910793.

CN China Mainland, China Type Culture Collection, 2017/09/06, CCTCC M 2017495.

＜ 110 ＞ Agricultural Drugs Toxicology Laboratory of the Agricultural Commission of the Executive Yuan ＜ 120 ＞ Bacillus subtilis strains for preparing fermented feather meal and its uses ＜ 130 ＞ P117537 ＜ 160 ＞ 6 ＜ 170 ＞ PatentIn version 3.5 ＜ 210 ＞ 1 ＜ 211 ＞ 17 ＜ 212> DNA <213> Artificial sequence <220><223> 16S rDNA forward primer <400> 1 gtttgatcct ggctcag 17 <210> 2 <211> 16 <212> DNA <213> Artificial sequence <220><223> 16S rDNA reverse primers <400> 2 taccttgtta cgactt 16 <210> 3 <211> 1504 <212> DNA <213> Bacillus subtilis <400> 3 gtttgatcct ggctcaggac gaacgctggcgggggggtgtccta atacatgcag gcctggcctggcctgcctgcctgcc 120 gcctgtaaga ctgggataac tccgggaaac cggggctaat accggatggt tgtttgaacc 180 gcatggttca aacataaaag gtggcttcgg ctaccactta cagatggacc cgcggcgcat 240 tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg cgtagccgac ctgagagggt 300 gatcggccac actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa 360 tcttccgcaa tggacgaaag tctg acggag caacgccgcg tgagtgatgt aggttttcgg 420 atcgtaaagc tctgttgtta gggaagaaca agtaccgttc gaatagggcg gtaccttgac 480 ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540 gcaagcgttg tccggaatta ttgggcgtaa agggctcgca ggcggtttct taagtctgat 600 gtgaaagccc ccggctcaac cggggagggt cattggaaac tggggaactt gagtgcagaa 660 gagagagtgg aattccacgt gtagcggtga aatgcgtaga gatgtggagg aacaccagtg 720 gcgaaggcga ctctctggtc tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca 780 ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt agggggtttc 840 cgccccttag tgctgcagct aacgcattaa gcactccgcc tggggagtac ggtcgcaaga 900 ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 960 aagcaacgcg aagaacctta ccaggtcttg acatcctctg acaatcctag agataggacg 1020 tccccttcgg gggcagagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat 1080 gttgggttaa gtcccgcaac gagcgcaacc cttgatctta gttgccagca ttcagttggg 1140 cactctaagg tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca 1200 tgccccttat gacctgggct acacacgtgc tacaatgga c agaacaaagg gcagcgaaac 1260 cgcgaggtta agccaatccc acaaatctgt tctcagttcg gatcgcagtc tgcaactcga 1320 ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1380 gggccttgta cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgagg 1440 taacctttta ggagccagcc gccgaaggtg ggacagatga ttggggtgaa gtcgtaacaa 1500 ggta 1504 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <220><223> gyrB forward primer <220><221> misc_feature <222> (27) .. (27) <223> n is a, c, g, or t <220><221> misc_feature < 222 ＞ (30) .. (30) ＜ 223 ＞ n is a, c, g, or t ＜ 220 ＞ ＜ 221 ＞ misc_feature ＜ 222 ＞ (33) .. (33) ＜ 223 ＞ n is a, c, g, or t ＜ 400 ＞ 4 gaagtcatca tgaccgttct gcaygcnggn ggnaarttyg a 41 ＜ 210 ＞ 5 ＜ 211 ＞ 44 ＜ 212 ＞ DNA ＜ 213 ＞ Artificial sequence ＜ 220 ＞ ＜ 223 ＞ gyrB reverse primer ＜ 220 ＞ ＜ 221 ＞ misc_feature ＜ 222 ＞ (27) .. (27) ＜ 223 ＞ n is a, c, g, or t ＜ 220 ＞ ＜ 221 ＞ misc_feature ＜ 222 ＞ (33) .. (33) ＜ 223 ＞ n is a, c, g , or t ＜ 220 ＜ 221 ＞ misc_feature ＜ 222 ＞ (39) .. (39) ＜ 223 ＞ n is a, c, g, or t ＜ 400 ＞ 5 agcagggtac ggatgtgcga gccrtcnacr tcngcrtcng tcat 44 ＜ 210 ＞ 6 ＜ 211 ＞ 1259 ＜ 212 ＞ DNA <213> Bacillus species (Bacillus subtilis) <400> 6 gaagtcatca tgaccgttct gcacgccgga ggcaaatttg acggaagcgg ctataaagta 60 tccggaggat tacacggtgt aggtgcgtcg gtcgtaaacg cactatcaac agagcttgat 120 gtgacggttc accgtgacgg taaaattcac cgccaaacct ataaacgcgg agttccggtt 180 acagaccttg aaatcattgg cgaaacggat catacaggaa cgacgacaca ttttgtcccg 240 gaccctgaaa ttttctcgga aacaaccgag tatgattacg atctgcttgc taaccgcgtg 300 cgtgaattag cctttttaac aaagggcgta aacatcacga ttgaagataa acgtgaagga 360 caagagcgca aaaatgaata ccattacgaa ggcggaatta aaagttatgt agagtattta 420 aaccgctcta aagaggttgt ccatgaagaa ccgatttaca ttgaaggcga aaaggacggc 480 attacggttg aagtggcttt gcaatacaat gacagctaca caagcaacat ttactcgttt 540 acaaacaaca ttaacacgta cgaaggcggt acccatgaag ctggcttcaa aacgggcctg 600 actcgtgtta ttaacgatta cgccagaaaa aaagggctta ttaaagaaaa tgatcc aaac 660 ctaagcggag atgacgtaag ggaagggctg acagcgatta tttcaatcaa acaccctgat 720 ccgcagtttg agggccaaac gaaaacaaag ctgggcaact cagaagcacg gacgatcacc 780 gatacgttat tttctacggc gatggaaaca tttatgctgg aaaatccaga tgcagccaaa 840 aaaattgtcg ataaaggctt aatggcggca agagcaagaa tggctgcgaa aaaagcccgt 900 gaactaacac gtcgtaagag tgctttggaa atttcaaacc tgcccggtaa gttagcggac 960 tgctcttcaa aagatccgag catctccgag ttatatatcg tagagggtga ctctgccgga 1020 ggatctgcta aacaaggacg cgacagacat ttccaagcca ttttgccgct tagaggtaaa 1080 atcctaaacg ttgaaaaggc cagactggat aaaatccttt ctaacaacga ggttcgctct 1140 atgatcacag cgctcggcac cggtattggg gaagacttca accttgagaa agcccgttac 1200 cacaaagttg tcattatgac ggatgctgg tgggggat

Claims (15)

A strain of Bacillus subtilis 531-7 is deposited with the Hsinchu Food Industry Development Research Institute of Taiwan Consortium and the deposit number is BCRC 910793. A microbial preparation comprising a culture of the Bacillus subtilis 531-7 strain according to claim 1 as an active ingredient. A use of the Bacillus subtilis 531-7 strain according to claim 1 as a feed or a feed additive. A use of the Bacillus subtilis 531-7 strain according to claim 1 as a fertilizer or a fertilizer additive. A method for preparing bacterial fermented feather powder, comprising the following steps: (1) prepare feathers, cut the feathers and place them in the culture medium to obtain a weight-volume ratio of 1% (w / v) to 20% (w / v) feather medium; (2) the request entry of the B. subtilis strain 1531-7 feather added to the medium so that the concentration of Bacillus subtilis strain 531-7 becomes ¹⁰⁵ per ml to 10 ⁷ colony-forming units (CFU / mL) cultured (3) dry and grind the feather medium containing Bacillus subtilis 531-7 strain to obtain bacterial fermented feather meal. The method according to claim 5, wherein in step (2), the subtilis 531-7 strain is added to the feather medium and cultured for 1 to 8 days. A bacterial fermented feather meal prepared by the method described in claim 5 or 6. The use of the bacterial fermented feather meal as claimed in claim 7 as a feed, a fertilizer or an additive thereof. A method for preparing fermented feather powder, which comprises the following steps: (1) Completely hydrolyzed feather powder, placing the hydrolyzed feather powder in a culture medium, and obtaining a weight-volume ratio of 1% (w / v) to 20% (w / v) ) hydrolyzed feather meal medium; (2) the request entry of the B. subtilis strain 1531-7 hydrolyzed feather meal is added to the medium so that the concentration of Bacillus subtilis strain 531-7 becomes ¹⁰⁵ per ml to 10 ⁷ colony-forming units (CFU / mL) for culture; (3) dry-grind the hydrolyzed feather meal medium containing Bacillus subtilis 531-7 strain to obtain processed fermented feather meal. The method according to claim 9, wherein in step (2), the Bacillus subtilis 531-7 strain is added to a hydrolyzed feather meal culture medium for 24 to 72 hours. A processed fermented feather powder prepared by the method according to claim 9 or 10. The use of the processed fermented feather powder as claimed in claim 11 as a feed, a fertilizer or an additive thereof. A method for preparing an enzyme, comprising the following steps: (1) preparing a raw material, placing the raw material in a culture medium, and obtaining a raw material culture medium having a weight-volume ratio of 1% (w / v) to 10% (w / v); (2) the requested item as Bacillus subtilis strain 1531-7 of the material added to the medium so that the concentration of Bacillus subtilis strain 531-7 becomes ¹⁰⁵ per ml to 10 ⁷ colony-forming units (CFU / mL) were cultured; (3) containing The raw material culture medium of Bacillus subtilis 531-7 is dried to obtain an enzyme. The method according to claim 13, wherein in step (1), the raw material is feather; wherein in steps (1) to (3), the raw material medium is feather medium; and in step (3), the enzyme contains horns Protease. The method according to claim 13, wherein in step (1), the raw material is soybean; wherein in steps (1) to (3), the raw material culture medium is soybean culture medium; and in step (3), the enzyme comprises casein Protease.