METHOD OF MANUFACTURING FERMENTED PORCINE BLOOD AND ANTIBACTERIAL COMPOSITION AND POULTRY FEED COMPOSITION USING SAME

Abstract

Disclosed are a method of manufacturing fermented porcine blood having antibacterial activity and poultry egg-laying enhancement and weight gain effects, and an antibacterial composition and a poultry feed composition using the fermented porcine blood.

Claims

1. A method of manufacturing fermented porcine blood, the method comprising: (a) preparing porcine blood; and (b) subjecting the porcine blood to inoculation with a fermentation strain selected from among Lactobacillus plantarum, Lactobacillus agilis, Lactobacillus alimentarius, Lactobacillus fermentum and a strain mixture of Lactobacillus fermentum and Saccharomyces cerevisiae, and to fermentation culture.

2. The method of claim 1, wherein the porcine blood is added with a blood anticoagulant, and hydrolyzing the porcine blood with a proteolytic enzyme is performed after step (a) and before step (b), whereby the inoculation with the fermentation strain is carried out for the porcine blood hydrolyzed with the proteolytic enzyme.

3. The method of claim 2, wherein the hydrolyzed porcine blood is added with 5 to 7 wt % of a carbon source, and the culture is carried out at 30 C. to 45 C.

4. A fermented porcine blood, obtained by the method of claim 1.

5. An antibacterial composition, comprising the fermented porcine blood of claim 4 as an active ingredient.

6. The composition of claim 5, which has antibacterial activity against E. coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella typhimurium.

7. The composition of claim 5, wherein the composition is a quasi-drug composition.

8. The composition of claim 5, wherein the composition is a pharmaceutical composition.

9. The composition of claim 5, wherein the composition is a cosmetic composition.

10. The composition of claim 5, wherein the composition is a food composition.

11. The composition of claim 5, wherein the composition is a feed composition.

12. A poultry feed composition for egg-laying enhancement, comprising the fermented porcine blood of claim 4 as an active ingredient.

13. A poultry feed composition for weight gain, comprising the fermented porcine blood of claim 4 as an active ingredient.

14. A poultry egg-laying enhancement method, the method comprising feeding the poultry feed composition of claim 12 to poultry.

15. A poultry weight gain method, the method comprising feeding the poultry feed composition of claim 13 to poultry.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIGS. 1 and 2 show the growth characteristics of fermentation strains in the hydrolyzed blood sample and the non-hydrolyzed blood sample, respectively;

[0074] FIG. 3 shows the growth characteristics of the strain mixture depending on the amount of a carbon source that is added; and

[0075] FIG. 4 shows the growth characteristics of the strain mixture depending on the fermentation temperature.

DETAILED DESCRIPTION

[0076] Hereinafter, a detailed description will be given of the present invention through the following examples. However, these examples are not to be construed as limiting the scope of the present invention.

EXAMPLES

[0077] Preparation of Fermented Porcine Blood and Experiment on Effect of Fermented Porcine Blood as Feed Additive for Poultry

[0078] 1. Preparation of Fermented Porcine Blood

[0079] Porcine blood samples were obtained from a slaughterhouse on Jeju Island, and these blood samples were added with 10% (v/v) of a 4% sodium citrate solution, serving as a blood anticoagulant, and stored frozen until the experiment.

[0080] The frozen blood sample was thawed at room temperature and added with 1% (w/v) of a proteolytic enzyme Provia (Novozyme, Co.), and thus hydrolyzed for 3 hr at 55 C., which is the optimal activity condition of the enzyme.

[0081] The blood sample not treated with the proteolytic enzyme or the blood sample treated with the proteolytic enzyme was added with sugar as a carbon source, mixed, inoculated with 6% (v/v) of each of five fermentation strains, namely (i) a Lactobacillus plantarum KCTC 21004 culture broth (2.310.sup.8 cells/ml), (ii) a Lactobacillus agilis KCTC 3606 culture broth (3.610.sup.8 cells/ml), (iii) a Lactobacillus alimentarius KCTC 3593 culture broth (2.610.sup.8 cells/ml), (iv) a Lactobacillus fermentum KACC 15736 culture broth (1.510.sup.8 cells/ml), and (v) a mixture of a Lactobacillus fermentum KACC 15736 culture broth (1.510.sup.8 cells/ml) and a Saccharomyces cerevisiae KCTC 7083 culture broth (4.510.sup.7 cells/ml) at 1:1, and cultured with stirring at 50 to 70 rpm, thereby manufacturing fermented porcine blood. The Lactobacillus culture broth was prepared in an MRS broth medium and the yeast culture broth was prepared in a YM broth medium.

[0082] 2. Bacterial Count

[0083] For bacterial count, the prepared culture broth sample was homogenized into a stock solution, and the stock solution was then diluted tenfold with a sterile saline solution (0.85% NaCl solution) to afford a working solution. 1 ml of the working solution was inoculated in a petri film medium and cultured in an incubator at 35 C. for 48 hr, and the number of colonies was determined.

[0084] 3. Strain Growth and Fermentation Characteristics

[0085] 3.1 Growth Characteristics Depending on Presence or Absence of Proteolytic Enzyme

[0086] The results of culture of the hydrolyzed blood sample and the non-hydrolyzed blood sample at 30 C. for 7 days using 6% (w/v) of sugar as the carbon source are shown in FIGS. 1 and 2, respectively.

[0087] As shown in FIG. 1, the hydrolyzed blood sample exhibited rapid growth of all fermentation strains and no blood coagulation.

[0088] As shown in FIG. 2, the non-hydrolyzed blood sample exhibited slow growth of all of five fermentation microorganisms, but the Lactobacillus fermentum strain and the strain mixture of Lactobacillus fermentum and Saccharomyces cerevisiae showed relatively fast growth. Furthermore, the culture broth began to coagulate from the 2.sup.nd day after culturing and intensively coagulated on the 3.sup.rd day.

[0089] In consideration of these growth characteristics, investigation of subsequent growth characteristics was performed using the strain mixture.

[0090] 3.2 Growth Characteristics Depending on Amount of Added Carbon Source

[0091] As the blood sample, the hydrolyzed blood sample was used, and as the carbon source, sugar was used in amounts of 1%, 3%, 5%, 7% and 10%, and the culturing temperature was set to 30 C. Fermentation was carried out using the strain mixture. The results of culturing for 5 days are shown in FIG. 3.

[0092] With reference to FIG. 3, in the treatment group added with 1% sugar, the growth increased until the 1.sup.st day after culturing but slowed significantly from the 2.sup.nd day. In the treatment group added with 3% sugar, rapid growth appeared until the 3.sup.rd day, after which growth also slowed significantly. The groups added with 5% or more of sugar manifested continuous growth after culturing and rapid growth up to the 4.sup.th day. Therefore, for economic fermentation, the concentration of sugar that was added was determined to be in the range of 5 to 7%.

[0093] 3.3 Growth Characteristics Depending on Fermentation Temperature

[0094] As the blood sample, the hydrolyzed blood sample was used, the carbon source was 6% sugar, and the fermentation temperature was set to 15 C., 20 C., 30 C. and 40 C. Fermentation was carried out using the strain mixture. The results of culturing for 8 days are shown in FIG. 4.

[0095] With reference to FIG. 4, the treatment group at 15 C. showed gentle growth during the culturing period and the lowest growth characteristics at the end of culturing. The treatment group at 20 C. manifested gentle growth and then the growth slowed and appeared to be stable on the 4.sup.th day. In the treatment group at 30 C., relatively fast growth was observed, and on the 4.sup.th day, the stable stage appeared, and the bacterial count reached a level of 10.sup.8 cells/ml. The treatment group at 40 C. showed rapid growth until the 2.sup.nd day, reaching a level of 10.sup.8 cells/ml, and from the 3.sup.rd day it reached the stable stage. Therefore, growth characteristics tended to increase with an elevation in the temperature. The appropriate fermentation temperature was determined to be 30 C. or higher taking into consideration economic benefits and fermentation rate.

[0096] 3.4 Fermentation Characteristics Under Optimal Fermentation Conditions

[0097] The characteristics of the fermented porcine blood obtained under the optimal fermentation conditions confirmed above, particularly use of the hydrolyzed blood sample, use of the strain mixture, use of 6% carbon source sugar, a fermentation temperature of 30 C. and fermentation for a total of 6 days, are summarized in Table 1 below.

[0098] As is apparent from Table 1 below, on the 3.sup.rd day after the start of fermentation (October 27), the fermentation was judged to reach an end point based on the number of bacteria, pH, fermentation odor and color. From the 4.sup.th day to the 6.sup.th day, the number of bacteria increased slightly, and the pH was kept constant at 4.0, which appeared to indicate a stable stage, rather than a growth stage. In particular, long-term storage of about 12 months at room temperature was determined to be possible because of the constant pH of 4.0.

TABLE-US-00001 TABLE 1 October October October October October October October Culture date 24 25 26 27 28 29 30 Time (d) 0 1 2 3 4 5 6 pH 7.5 6.9 4.7 4.2 4 4 4 Viscosity 85 80 77 79 70 78 72 Color Red Red Blackish Dark Dark Dark Dark brown blackish blackish blackish blackish brown brown brown brown Odor Blood Blood Fermentation Fermentation Fermentation Fermentation Fermentation odor odor odor odor odor odor odor Number of 3.5 10.sup.5 2.1 10.sup.6 7.3 10.sup.7 2.9 10.sup.8 3.8 10.sup.8 3.0 10.sup.8 3.6 10.sup.8 micro- organisms (cpu/ml)

[0099] Also, the results of general analysis are shown in Table 2 below. As is apparent from Table 2, heavy metals such as lead, mercury and the like were not detected, and no antibiotics were detected. These results were found to satisfy the Standards and Specifications of Feed and the like under the Ministry of Agriculture, Food and Rural Affairs.

TABLE-US-00002 TABLE 2 Analysis items Unit Fermentation stock solution Water % 80.11 Crude protein % 14.97 Crude fat % 0.43 Crush ash % 0.75 Calories cal/g 1157 Calcium (Ca) mg/kg 602.21 Chromium (Cr) mg/kg Not detected Copper (Cu) mg/kg Not detected Iron (Fe) mg/kg 176.51 Potassium (K) mg/kg 1055.87 Magnesium (Mg) mg/kg 123.54 Manganese (Mn) mg/kg Not detected Sodium (Na) mg/kg 488.83 Phosphorus (P) mg/kg 403.56 Zinc (Zn) mg/kg 5.46 Salt % 0.32 Cadmium (Cd) mg/kg 0.07 Lead (Pb) mg/kg Not detected Mercury (Hg) mg/kg Not detected Arsenic (As) mg/kg 1.68 Sulfur (S) % 0.12 Acetic acid % 0.45 Butyric acid % 0.13 L-lactic acid % 1.76 Propionic acid % Not detected Beta-lactam Negative Macrolide Negative Sulfonamide Negative Aminoglycoside Negative Tetracycline Negative

[0100] 4. Antibacterial Experiment

[0101] 4.1 Antibacterial Experiment Method

[0102] An antibacterial experiment was performed in accordance with ASTM E149, and is as follows.

[0103] 1 ml of the sample was mixed with a phosphate buffer (pH 7.0) and added with the test bacterial culture broth so as to attain a concentration of 2.510.sup.5 cfu/ml. The total volume of the phosphate buffer to which the sample and the test bacteria were added was adjusted to 50 ml so that the concentration of the sample that was added was 2% (v/v), and 1 ml of the diluted solution was spread on a film medium for bacterial culture and then cultured for 24 hr. Finally, the number of test bacteria in the cultured film medium was measured.

[0104] 4.2 Antibacterial Experiment Results

[0105] The antibacterial experiment results are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Antibacterial experiment results 40Bx 50Bx Classification Blank sample sample E. coli Initial 2.1 10.sup.5 2.1 10.sup.5 2.1 10.sup.5 bacterial count After 24 hr 1.3 10.sup.5 <30 <30 Bacterial 99.9 99.9 reduction rate S. aureus Initial 2.1 10.sup.5 2.1 10.sup.5 2.1 10.sup.5 bacterial count After 24 hr 1.3 10.sup.5 2.8 10.sup.2 3.5 10.sup.4 Bacterial 99.8 73.1 reduction rate K. pneumoniae Initial 2.1 10.sup.5 2.1 10.sup.5 2.1 10.sup.5 bacterial count After 24 hr 1.7 10.sup.5 <30 <30 Bacterial 99.9 99.9 reduction rate P. aeruginosa Initial 2.1 10.sup.5 2.1 10.sup.5 2.1 10.sup.5 bacterial count After 24 hr 1.3 10.sup.5 <30 <30 Bacterial 99.9 99.9 reduction rate S. typhimurium Initial 2.1 10.sup.5 2.1 10.sup.5 2.1 10.sup.5 bacterial count After 24 hr 1.7 10.sup.5 <30 <30 Bacterial 99.9 99.9 reduction rate * Sample concentration: 2% (v/v) Bacterial reduction rate unit: % Bacterial count unit: cells/ml Buffer solution: 50 ml of Phosphate buffer (pH 7.2) Control sample: non-fermented blood (sample vacuum-concentrated to 40 Brix)

[0106] The results of Table 3 show that the fermented blood sample exhibit very high antibacterial activity against all bacteria tested. For reference, the non-fermented blood sample (obtained through treatment with sodium citrate and then hydrolysis with proteolytic enzyme) showed no antibacterial activity at all, but all of the above bacteria proliferated, and moreover, severe decay was visible with the naked eye (data not shown).

[0107] 5. Poultry Farm Site Evaluation

[0108] 5.1 Experiment Method

[0109] The weight evaluation of broilers (variety: Hanhyup 3) was performed from Nov. 1, 2018 to Mar. 2, 2019.

[0110] The evaluation of egg-laying rate of laying hens (variety: Hy-Line Brown, type: brown species, brown eggs) was carried out from Feb. 1, 2019 to Mar. 2, 2019.

[0111] As feed, feed for laying hens and broilers from Dongyang Feed (Gyeonggi, Korea) (control group) and a mixture thereof with 1% (w/w) of the lyophilized powder of fermented porcine blood obtained under optimal fermentation conditions (treatment group) were used.

[0112] The feed was freely provided during the experiment, and drinking water was freely provided using an automatic water dispenser.

[0113] 5.2 Experiment Results

[0114] The results of weight evaluation of broilers are shown in Table 4 below, and the results of evaluation of egg-laying rate and the like of laying hens are shown in Table 5 below.

[0115] As is apparent from Table 4, the weight gain rate during the experiment was 6.9% in the control group but was apparently increased to 13.3% in the treatment group.

[0116] As is apparent from Table 5, the number of eggs and the egg-laying rate during the experiment were significantly increased.

TABLE-US-00004 TABLE 4 Weight at Weight at start date of end date of Weight Weight Classification experiment (g) experiment (g) gain (g) gain rate Control group 40,150 42,900 2,750 6.8% (20 broilers) Treatment group 40,300 45,630 5,330 13.2% (20 broilers)

TABLE-US-00005 TABLE 5 Total Daily Egg feed feed Egg- Breeding Eggs weight intake intake per laying Feed Feed Classification Mortality (hens) (number) (40, g) (40, g) hen (g) rate (%) efficiency requirement Control 1 39 37.sup.a 2,278* 4,613 115.33.sup.a 94.43.sup.+ 0.494 2.029 group Treatment 0 40 39.sup.a,b 2,416** 4,919 122.97.sup.a,b 96.83.sup.++ 0.491 2.038 group .sup.a, b, abT-Test, P < 0.05 *, **T-Test, P < 0.05 .sup.+, ++T-Test, P < 0.05

[0117] Although preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications are possible without departing from the scope and spirit of the invention as disclosed in the accompanying claims, and such modifications should not be understood separately from the technical ideas or essential characteristics of the present invention.