KR20080009935A - Feed additive for broiler feeding mud flat bacteria origin protease as effective ingredient - Google Patents

Abstract

A feed additive for broiler chicks containing a protease agent derived from a lugworm as an active ingredient is provided to improve the rate of weight gain, feed requirement rate and digestibility and to increase total protein availability in blood when fed to broiler chicks. A feed additive for broiler chicks contains a protease agent separated from a lugworm. The active ingredient of the protease is 1.0x10^8 CFU/g. For an example, a lugworm is finely cut, stirred with 20mM phosphate buffer solution(pH 7.5) for 16hr and centrifuged at 10,000rpm for 30min. The supernatant is heated at 40deg.C for 10min and centrifuged at 10,000rpm for 30min to give a crude enzyme solution. The crude enzyme solution is treated with ammonium sulfate and a Sephacryl S-200 column(2.5x110cm) equilibrated with 10 mM KH2PO4 to give a protease agent derived from the lugworm.

Description

Feed additive for broiler containing lug-derived proteolytic enzymes {Feed additive for broiler feeding mud flat bacteria origin protease as effective ingredient}

The present invention relates to feed additives for broilers, and relates to feed additives for broilers comprising the worm-derived protease.

At present, industrial enzymes depend on imports for most industrial enzymes in Korea, as the scope of application is increasing in the fields of food, medicine, pharmacy, chemistry and environment. In addition, as the question of the enzymatic ability of imported enzymes is amplified, localization of industrial enzymes is emerging as an urgent task.

Soybean meal has been widely used as a source of vegetable protein for pig feed because of its high protein content and excellent amino acid composition. However, when soybean meal is used as a natural feed, it is not efficiently broken down in the intestines of livestock, especially short livestock such as pigs and chickens. Therefore, when feeding soybean meal to livestock, it is necessary to find a way to ensure that the livestock can digest it sufficiently.

Therefore, the purpose of adding enzyme to livestock feed is to increase the utilization of feed nutrients, improve growth rate and increase feed efficiency by effectively decomposing anti-effect factors in feed.

Lumber-derived protease is our unique enzyme and has a competitive edge. Korean Patent Registration No. 10-0326941 discloses a protease isolated from a worm, and the protease is capable of inhibiting enzyme degradation, stability, long-term storage and soybean trypsin inhibitor (SBTI) in soybean meal. In this case, it is possible to solve the environmental problems caused by excess protein design, micronization and excretion of surplus nitrogen compounds in feedstock, and improve feed density by improving biomass. In addition, the degradation of trypsin inhibitors is expected to promote diarrhea prevention and growth, and to obtain high competitiveness in the livestock sector such as reducing feces and environment-friendly breeding.

The characteristics of lugworm-derived protease are that they maintain activity in the presence of surfactants or heavy metals, extreme pH conditions, extreme oxidative or reducing environments, and enzyme activity at low temperatures.

Accordingly, an object of the present invention is to provide a feed additive for broilers to improve the increase rate, feed demand and digestion rate by using a domestic native enzyme as an active ingredient, and increase the availability of total protein in the blood.

The present invention provides a feed additive for broilers containing a proteolytic enzyme isolated from the worms as an active ingredient.

The effective dose of the protease is 1.0 × 10 ⁸ CFU / g.

The protease of the present invention improves the weight gain, feed demand and digestibility in broiler feeding, and increases the availability of total protein in the blood.

Hereinafter, the present invention will be described in detail through examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Example 1 Protease Isolation

The worms were finely chopped and 2 volumes of 20 mM sodium phosphate buffer (pH 7.5) were added, followed by stirring for 16 hours. The supernatant was taken by centrifugation at 10,000 rpm for 30 minutes, and then the supernatant was heated at 40 ° C for 10 minutes. This was again centrifuged at 10,000 rpm for 30 minutes to take a supernatant to prepare a coarse enzyme solution of the earthworm.

Ammonium sulfate was added to the final concentration of the crude enzyme solution to 30%, stirred for 30 minutes, and centrifuged at 10,000 rpm for 30 minutes to obtain a supernatant. After adding ammonium sulfate so that the final concentration was 80% again, the mixture was stirred for 30 minutes, centrifuged at 10,000 rpm for 30 minutes, and the precipitate was recovered and dissolved in distilled water. This was again centrifuged at 10,000 rpm for 30 minutes to obtain a supernatant, thereby obtaining a 30-80% ammonium sulfate fraction.

The 30-80% ammonium sulfate fraction obtained above was equilibrated with a Sephacryl S-200 column (2.5 × 110 cm, Amersham-Pharmacia Co.) with 100 mM sodium phosphate buffer (pH 7.5), then added to the column and the buffer Developed at a flow rate of 20 mL / hr to obtain the active fraction. Again the active fractions obtained above were equilibrated with DEAE-Sepharose column (2.5 × 10 cm, Amersham-Pharmacia) with sodium phosphate buffer and then added. After washing with the buffer until the absorbance of 280 nm became nearly zero, the active fractions were eluted with 100 mM sodium phosphate buffer containing 500 mM sodium chloride. Again equilibrate the benzamidine sepharose column (1.0 × 10 cm, Amersham-Pharmacia) with 100 mM sodium phosphate buffer and add the active fraction to the column until the absorbance at 280 nm is nearly zero. After washing, the active fraction was eluted with 100 mM glycine-hydrochloric acid (pH 3.0) buffer to separate protein fermentase derived from worms.

Experimental Example 1

The effective dose of protease is 1.0 × 10 ⁸ CFU / g. When the protease was added in the feed, 0.1% was added to the feed content.

Materials and methods

(1) Test animal and test design

This test revealed 480 chicks of two-day-old Arbor Acre broiler (male) chicks. The body weight was 44.22 ± 1.90 g at the start of the test and the specification test was carried out for five weeks. The trial design included 1) PC (positive control: high nutrient density diet), 2) PCP (PC diet + 0.1% protease), 3) NC (negative control: low nutrient density diet), and 4) NCP (NC diet + 0.1% 4 treatments with protease) followed by 6 repetitions per treatment and 20 repetitions per repetition.

(2) Test feed and specification management

Test feed was formulated based on corn-soybean meal (Table 1). The chicks were reared in plains, and the feed and water were allowed to be vegetarian.

TABLE 1

Feed Ingredient Name (g) High Nutrition Diet Low Nutrition Diet At the start At the end At the start At the end corn 50.25 50.68 55.50 55.35 Soybean meal (46% CP) 22.10 18.10 19.80 16.50 Wheat 15.00 18.00 15.00 18.00 Corn gluten 5.00 5.00 2.75 2.37 Chaejongbak 2.00 3.00 2.00 3.00 Uji 1.80 1.60 1.00 1.00 Calcium phosphate 1.55 1.29 1.57 1.32 Limestone 0.99 1.02 0.99 1.01 L-Lysine-HCL 0.30 0.33 0.33 0.35 Salt 0.20 0.20 0.20 0.20 Sodium Bicarbonate 0.20 0.20 0.20 0.20 DL-Methionine 0.19 0.15 0.24 0.21 Choline liquid 0.15 0.12 0.12 0.12 Mineral premix 0.12 0.12 0.12 0.12 Vitamin Premix 0.07 0.06 0.07 0.06 Salinomycin - 0.10 - 0.10 Linacox 0.05 - 0.05 - Avilamycin 0.03 0.03 0.03 0.03 Sum 100.00 100.00 100.00 100.00

(3) Survey items and methods

(3-1). Weight gain , Feed intake and feed demand

Weight gain was measured at the beginning of the test, after 3 weeks, and at the end of the test, respectively. Feed intake was calculated by subtracting the remaining amount from feed intake when measuring body weight. Feed demand was calculated by dividing feed intake by weight gain.

Table 2 shows the effect of dietary protease supplementation on the productivity in broilers. The proteolytic enzyme added group improved the weight gain and feed requirement compared to the NC group.

TABLE 2

Item PC PCP NC NCP  0-3 weeks    Weight gain (g) 676.53 662.36 631.37 666.09    Feed Intake (g) 952.33 934.17 951.84 929.83    Feed rate 1.41 1.41 1.51 1.40  4-5 weeks    Weight gain (g) 875.00 887.35 771.53 855.72    Feed Intake (g) 1626.42 1604.25 1583.08 1632.92    Feed rate 1.86 ^b 1.81 ^b 2.05 ^a 1.91 ^b  0-5 weeks    Weight gain (g) 1551.53 1549.71 1402.90 1521.81    Feed Intake (g) 2578.75 2538.42 2534.92 2562.75    Feed rate 1.66 1.64 1.81 1.68

(3-2). Nutrient digestibility

In order to measure the nutrient digestibility, a total of 40 waters, 10 for each test area, were published in the metabolism cage 7 days before the end of the specification test, and a preliminary period was set for 3 days to adapt to the changed environment. After drying for 72 hours in a drier, it was ground and used for analysis. General composition and chemical analysis of feed were analyzed by the Association of Official Analytical Chemists (AOAC).

Table 3 shows the effects of dietary protease supplementation on nutrient digestibility in broilers. The proteolytic enzyme added improved dry matter and nitrogen digestibility.

TABLE 3

Item(%) PC PCP NC NCP  Building (DM) 71.69 72.68 70.32 71.83  Nitrogen (N) 67.35 69.81 65.94 67.08

(3-3). In the blood  Total protein Blood urea nitrogen  Research

At the end of the specification test, blood samples were randomly selected for each treatment, and blood was collected from the alveolar veins and centrifuged at 2,000 × g for 30 minutes at 4 ° C using a total biochemical analyzer (HITACHI 747, Japan). Protein and blood urea nitrogen (BUN) were investigated.

The effects of dietary protease supplementation on total protein and blood urea nitrogen content in broilers are shown in Table 4. The total protein content tended to decrease with the addition of protease, which is thought to improve protein availability in broilers.

TABLE 4

Item PC PCP NC NCP Total Protein (g / dL) 3.68 2.93 3.05 2.91 Blood Urea Nitrogen (mg / dL) 1.74 1.86 1.88 1.67

The feed additive containing the protease isolated from the wormworm of the present invention as an active ingredient improves the weight gain, feed demand and digestion rate when feeding broiler meat, increases the total protein availability in the blood, and is useful as a feed additive, You will be able to get high competitiveness in

Claims (2)

A feed additive for broilers containing a protease isolated from wormworms as an active ingredient. The feed additive for broilers according to claim 1, wherein the active ingredient of the protease is 1.0 × 10 ⁸ CFU / g.