JP2023055600A - Feed additive for promoting growth, and preventing and/or treating respiratory diseases, and method for preparing the same - Google Patents

Abstract

To provide a feed additive that can be added to an animal feed, and exerts beneficial effects of promoting growth, and preventing respiratory diseases.SOLUTION: A method for adding a feed additive includes: (a) process of preparing a fermentation substrate including coffee powder and an auxiliary material; and (b) process of fermenting a fermentation substrate with Aspergillus oryzae to obtain a feed additive. In the fermentation substrate, the content of coffee powder is 45 wt.% to 80 wt.% relative to the overall weight, a carbon-nitrogen ratio of the fermentation substrate is 35 to 65, an auxiliary material includes corn grains, rice husks, flour of soybeans with husks, crushed rice or a combination thereof. The feed additive includes high specific activity digestive enzyme, and has selective bacteriostatic action and a resistive effect to respiratory pathogens.SELECTED DRAWING: Figure 1

Description

The present invention relates to a feed additive and its preparation method, and more particularly to a feed additive for promoting growth and preventing and/or treating respiratory diseases and its preparation method.

Coffee beans contain different types of chemicals such as caffeine and polyphenols. Among these, caffeine promotes metabolism and blood circulation, has a stimulating effect on the central nervous system, and polyphenols have antioxidant and anti-inflammatory beneficial effects that contribute to good health. In addition, the coffee beverage after roasting the coffee beans has a unique flavor and aroma. Therefore, in recent years, coffee has become one of the mainstream beverages that are widely loved by people.

However, coffee beans typically require processes such as cooking and brewing to produce coffee beverages. Coffee grounds left over after coffee beverages have been manufactured do not contain high levels of functional nutrients and are rarely utilized further. Currently, about 90,000 tons of ground coffee are produced domestically each year. Serious environmental protection and waste disposal problems arise if these coffee grounds cannot be properly disposed of and safely emplaced.

On the other hand, currently, fish meal and soybean meal are commonly used as the main nutritional sources for animal feed in the animal feed industry. Fishmeal is generally considered a growth-enhancing source for animals due to its high protein content. However, fishmeal is expensive, increasing costs and making it difficult to use widely in animal feed. Although soybean meal is inexpensive, its nutritional value is incomparable to that of fishmeal and cannot be used as animal feed to effectively promote animal growth.

Taiwan Patent Publication No. 201722285

From the above, in order to solve the environmental protection and waste disposal problems caused by coffee grounds, develop technical means for applying coffee grounds to animal feed, and at the same time reduce the cost of animal feed. It has become necessary to achieve the goal of improving and promoting animal growth.

In order to overcome the shortcomings of the prior art, the object of the present invention is a method for preparing a feed additive that contains digestive enzymes with higher specific activity, can be applied to animal feed, and can exert the effect of promoting animal growth. , and to provide feed additives thereof.

Another object of the present invention is to provide a method for preparing a feed additive with selective bacteriostatic and resistant effects against respiratory pathogens and the feed additive.
Yet another object of the present invention is to provide a method of preparing a feed additive that can solve the problem of coffee grounds disposal and re-establishment and reduce the cost of preparing animal feed. be.

To achieve the above objectives, the present invention provides a method of preparing a feed additive comprising the steps of: Step (a): A fermentation substrate containing coffee grounds and auxiliary materials, wherein the content of coffee grounds is 45% by weight or more and 80% by weight or less based on the total weight of the fermentation substrate, and the fermentation substrate is carbon- A step of providing a fermentation substrate having a nitrogen ratio of 35 to 65 and a supplementary material comprising corn grains, rice husks, soybean flour with husks, broken rice, or a combination thereof, and step (b): the fermentation substrate comprising Aspergillus oryzae ( Aspergillus oryzae) to obtain a feed additive.

A feed additive prepared by this method by making the fermentation substrate contain coffee grounds in a specific content range and a carbon-nitrogen ratio in a specific range, and employing Aspergillus oryzae to ferment the fermentation substrate. has the advantage of containing digestive enzymes with relatively high specific activity, selective bacteriostasis and resistance to respiratory pathogens. Therefore, this feed additive can be applied to animal feed to promote animal growth, reduce its cost and reduce the risk of animal infection with respiratory diseases. Moreover, the coffee grounds can be reused, thereby avoiding environmental protection and waste disposal problems.

In the present invention, the carbon-nitrogen ratio (C/N ratio) indicates the ratio of the total carbon content to the total nitrogen content in the organic material. For example, when the carbon-nitrogen ratio is 20, it means that the content of carbon element is 20 times the content of nitrogen element in the organic material.

According to the invention, the fermentation substrate may be sterilized prior to fermentation. In one embodiment, the fermentation substrate can be sterilized at a temperature between 120° C. and 130° C. and under a pressure between 1 bar and 1.5 bar for 30 minutes to 90 minutes. However, it is not limited to this.

Preferably, the carbon-nitrogen ratio of the fermentation substrate is 35-60. More preferably, the carbon-nitrogen ratio of the fermentation substrate is 37-60.
Preferably, the content of the auxiliary material is ≧20% and ≦55% by weight, based on the total weight of the fermentation substrate.

Preferably, the carbon-to-nitrogen ratio of the fermentation substrate is between 46 and 53, and the auxiliary material comprises rice husks. The content of rice husk is 7% or more and 10% or less by weight, based on the total weight of the fermentation substrate. The specific activity of protease and amylase in the feed additive can be further increased by including rice husks in a specific content range in the fermentation substrate.

In some embodiments, the supplemental material is corn grits, and the corn grits content is 25% or more and 40% or less by weight, based on the total weight of the fermentation substrate. In another embodiment, the auxiliary material is corn grits and rice husks, and based on the total weight of the fermentation substrate, the corn grits content is 25 wt% or more and 38 wt% or less, and the rice husk content is is 7% by weight or more and 15% by weight or less. In yet another embodiment, the auxiliary material is corn grits, rice hulls and hulled soy flour, and the corn grits content is 14% or more and 20% or less by weight, based on the total weight of the fermentation substrate. , the content of rice husk is 8% or more and 12% or less by weight, and the content of soybean flour with husk is 1% or more and 5% or less by weight.

Preferably, the content of Aspergillus oryzae is 0.02 wt% or more and 0.2 wt% or less, based on the total weight of the fermentation substrate. Specifically, Aspergillus oryzae is 6.5×10 ⁷ spores/g or more.

Preferably, the fermentation substrate has a moisture content of 50% by weight or more and 75% by weight or less. More preferably, the fermentation substrate has a water content of 60% by weight or more and 75% by weight or less. Even more preferably, the moisture content of the fermentation substrate is 65% by weight.

In some embodiments, in step (a), the fermentation substrate can be subjected to a cell wall-breaking treatment (wall-breaking treatment). It includes a step of leaving for 2 to 5 minutes at a temperature of 150° C. or higher and a rotation speed of 230 rpm or higher and 250 rpm or lower. By pre-wall-disrupting the fermentation substrate, intracellular substances are released and contribute to the fermentation process by Aspergillus oryzae, increasing the content of proteases in the fermentation.

Preferably, in step (b), the temperature for fermenting the fermentation substrate with Aspergillus oryzae is 25°C or higher and 40°C or lower, and the fermentation time is 2 days or longer and 12 days or shorter. More preferably, the temperature for fermenting the fermentation substrate with Aspergillus oryzae is 25° C. or higher and 30° C. or lower, and the fermentation time is 4 days or longer and 6 days or shorter.

Preferably, in step (b), the fermentation substrate may be fermented with Aspergillus oryzae followed by a drying step to obtain the feed additive. The drying step can be performed at a temperature of 45° C. or higher and 60° C. or lower for 8 hours or longer. More preferably, the drying step can be performed at a temperature of 50° C. for 12 to 14 hours.

Furthermore, the present invention provides a feed additive prepared by the aforementioned method. The feed additive has the effect of containing digestive enzymes with higher specific activity, selective bacteriostasis and resistance to respiratory pathogens, is applicable to animal feed, promotes animal growth, It is effective in reducing the risk of animal respiratory disease infection.

According to the invention, digestive enzymes refer to proteins capable of degrading certain substances. Preferably, digestive enzymes include proteases, amylases, cellulases and xylanases.

Preferably, the feed additive has a protease specific activity of 355.80 Unit/g or more. More preferably, the feed additive has a protease specific activity of 463.86 units/g or more. Even more preferably, the feed additive has a protease specific activity of 854.74 units/g or more.

The present invention also provides a feed additive for promoting the growth of animals prepared by the method described above. The effect of promoting animal growth has been demonstrated to be suitable for both normal growing animals and growth retarded animals.

Suitably the animal comprises poultry or livestock.

Preferably, the effect of promoting animal growth is suitable for normal growing animals.

Furthermore, the present invention provides a feed additive for preventing and/or treating respiratory diseases, prepared by the method described above. Suitably, the respiratory disease refers to a disease caused by infection with Mycoplasma hyopneumoniae or Mycoplasma pneumoniae.

Further, the present invention provides a feed additive for inhibiting the growth of pathogens prepared by the method described above and comprising Streptococcus suis, Escherichia coli, Salmonella spp., Riemerella anatipestifer, Gallibacterium anatis, Staphylococcus spp., Mycoplasma hyopneumoniae or Mycoplasma pneumoniae. offer things.

Further, the present invention provides a feed additive for inhibiting infection with pathogens, including Mycoplasma hyopneumoniae or Mycoplasma pneumoniae, prepared by the method described above.

Further, the present invention provides a feed additive for suppressing inflammation prepared by the method described above. Preferably, the inflammation is pneumonia, more preferably pneumonia induced by Mycoplasma hyopneumoniae or Mycoplasma pneumoniae.

In the present specification, the range represented by "from the lower limit to the upper limit" means from the lower limit to the upper limit, unless otherwise specified. For example, "30 to 90 minutes" indicates "30 minutes or more and 90 minutes or less".

Other objects, advantages and novel features of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings.

1 is a flow chart of a method of preparing a feed additive of the present invention; Figure 2 shows results of specific protease activity at different fermentation times during the fermentation process of Examples 10-14. Fig. 2 shows amylase specific activity results at different fermentation times during the fermentation process of Examples 10-14. The bacteriostatic results of Example 1 and Comparative Example 1 for Escherichia coli are shown. The results of bacteriostasis of Example 1 and Comparative Example 1 for Streptococcus suis are shown. Shown are the bacteriostatic results of Example 1 and Comparative Example 1 for Salmonella choleraesuis. 1 shows the bacteriostatic results of Example 1 and Comparative Example 1 for Lactobacillus casei. Figure 2 shows the results of inhibiting the growth of Mycoplasma hyopneumoniae under treatment with different concentrations of ME. The results of inhibiting the growth of Mycoplasma pneumonia under treatment with different concentrations of ME are shown. The results of inhibiting Mycoplasma hyopneumoniae infection under treatment with an ME concentration of 800 µg/ml are shown. Shown are the cell viability results of the PK-15 cell line under treatment with different ME concentrations. Fig. 3 shows the results of inhibition of Mycoplasma pneumoniae infection under treatment with an ME concentration of 1600 µg/ml. Figure 2 shows cell viability results of A549 cell line under treatment with different ME concentrations. TNF-α release results when MH-S cells were treated with different concentrations of ME. Figure 2 shows IL-6 release results when MH-S cells were treated with different concentrations of ME. Figure 2 shows cell viability results of MH-S cells under treatment with different ME concentrations. The results of total cell counts analyzed by flow cytometry are shown. Fig. 2 shows the results of neutrophil cell counts analyzed by flow cytometry. The results of monocyte cell counts analyzed by flow cytometry are shown.

Several examples are provided below to illustrate the practice of the invention. A person skilled in the art can easily achieve the effects of the present invention according to the contents of this specification. Various modifications and variations may be made in implementing or applying the present invention without departing from the spirit and scope of the present invention.

<Example 1: feed additive>

600 g of coffee powder, 300 g of corn coarse grains and 100 g of rice husks were weighed and mixed, and then water was added to adjust the water content to obtain a mixture with a water content of 65%. After that, 3 kg of the mixture was placed under conditions of a temperature of 121° C. and a pressure of 1.2 bar and sterilized for 90 minutes. After cooling the sterilized mixture to room temperature, the fermentation substrate was obtained.

Next, 1.5 g to 3 g of Aspergillus oryzae powder of 6.5×10 ⁷ spores/g or more was added to the fermentation substrate and fermented at 30° C. for 4 days to obtain a crude product. After drying the crude product at 50° C. for 12 to 14 hours to a moisture content of less than 7.5%, the crude product is ground until it passes through a sieve with a pore size of about 830 micrometers (μm), The feed additive of Example 1 was obtained.

The proportion of each component of Example 1 (based on the total weight of the fermentation substrate) and the carbon-to-nitrogen ratio (C/N ratio) of the fermentation substrate are listed in Table 1 below. In Table 1, the C/N ratio of the fermentation substrate was obtained by adding the C/N ratio of each component multiplied by the ratio of each component. Here, the C/N ratio of coffee grounds is about 20, the C/N ratio of corn coarse grains is about 97.3, the C/N ratio of soybean flour with husks is about 4.7, and the C/N ratio of rice husks is about 4.7. The N ratio was about 90. For example, the fermentation substrate of Example 1 contains 60% by weight coffee grounds (C/N ratio: 20), 30% by weight corn grits (C/N ratio: 97.3), and 10% by weight rice husks (C/N ratio: 97.3). /N ratio: 90), thus the C/N ratio of the fermentation substrate of Example 1 was about 50.2.

<Examples 2 to 9: feed additives>

The preparation methods of Examples 2 to 9 were the same as in Example 1, but the main differences were the component ratios and C/N ratios of the fermentation substrates employed. The proportion of each component in Examples 2-9 (based on the total weight of the fermentation substrate) and the C/N ratio of the fermentation substrate are also listed in Table 1 below.

<Examples 10 to 14: feed additives>

The preparation method of Examples 10-14 is the same as that of Example 1, but the main differences are the ratio of the components of the fermentation substrate and the C/N ratio used, and the fermentation time of Examples 10-14 is 7. days. The proportion of each component in Examples 10-14 (based on the total weight of the fermentation substrate) and the C/N ratio of the fermentation substrate are also listed in Table 1 below.

<Comparative Example 1: Unfermented coffee powder>

Comparative Example 1 used the same coffee powder as in Example 1, that is, unfermented coffee powder.

<Test Example 1: Evaluation of Specific Activity of Digestive Enzymes>

In Test Example 1, for Examples 1-9 and Comparative Example 1, the specific activities of protease, amylase, cellulase and xylanase were used to evaluate the specific activity of different groups of digestive enzymes.

(1) Evaluation of protease specific activity Evaluation of protease specific activity was performed by mixing protease and hemoglobin for 30 minutes at a pH of 4.7 and a temperature of 50°C to generate proteolytic hemoglobin. , the amount of proteolyzed hemoglobin produced was determined by analyzing the amount of tyrosine released after hemoglobin was proteolyzed. Here, the protease activity unit (U) was expressed as the hemoglobin unit (HUT) of tyrosine. One HUT indicates that 1.10 micrograms per milliliter (μg/ml) of tyrosine is released from hemoglobin per minute in a 0.006 normality (N) hydrochloric acid solution.

Specifically, tyrosine standard solutions with concentrations of 25 μg/ml, 50 μg/ml, 75 μg/ml and 100 μg/ml were prepared in advance. Then, after analyzing the absorbance at 275 nm of these standard solutions with an enzyme immunoassay device (purchased from BioTek Instruments, Inc.; model: MQX200), the absorbance at 275 nm of a tyrosine solution with a concentration of 1.10 μg/ml was obtained. After that, 4 g of hemoglobin was added to 100 ml of distilled water, and the pH value was adjusted to 1.7 using 0.3N hydrochloric acid solution. After 10 minutes, 0.5 molar (M) sodium acetate solution was used to adjust the pH value to 4.7, and distilled water was added to a total volume of 200 ml to obtain a hemoglobin solution. On the other hand, 5 g of the feed additives of Examples 1 to 9 (Examples 1 to 9) and unfermented coffee powder (Comparative Example 1) were added to 45 ml of distilled water, respectively, and the temperature was 30 ° C. and the rotation speed was 130 rpm. After spinning at high speed for an hour, it was centrifuged at 12000 rpm for 5 minutes. Supernatants were collected as samples for Examples 1-9 and Comparative Example 1.

The samples of Examples 1-9 and Comparative Example 1 were then diluted 5-fold with distilled water. 100 microliters (μl) of the diluted samples of Examples 1-9 and Comparative Example 1 were then added to 500 μl of hemoglobin solution (which had already been placed in a 40° C. water bath for 5 minutes) and placed in a 40° C. water bath. After reacting for 30 minutes in the medium, 500 μl of trichloroacetic acid was added to stop the reaction. After 30 minutes at room temperature, the reaction mixture was centrifuged at 12000 rpm for 5 minutes, the supernatant of each group was collected and analyzed with an enzyme immunoassay analyzer to obtain the absorbance at 275 nm for each group. Here, since the color of each sample is different, a blank group prepared by adding each sample to the hemoglobin solution after adding trichloroacetic acid is prepared for each group, thereby providing a background when analyzing the absorbance. to be able to represent values.

Protease specific activity was determined as described below. After subtracting the background value of the corresponding blank group from the absorbance of Examples 1 to 9 and Comparative Example 1, the absorbance of the tyrosine solution at a concentration of 1.10 μg/ml was divided by the total volume of the reaction solution and the reaction time. 1 to 9 and Comparative Example 1 protease activities per unit volume were obtained. The unit is HUT/ml. Further, after multiplying the result of protease activity by the dilution rate of each group and dividing by the concentration of each sample, the specific activity of proteases of Examples 1 to 9 and Comparative Example 1 was obtained. The unit is HUT/g, and the samples of Examples 1 to 9 and Comparative Example 1 show the protease activity per unit weight of protein. The specific activity results of the proteases of Examples 1 to 9 and Comparative Example 1 are shown in Table 2 below, and HUT/g is expressed in units/g.

(2) Evaluation of amylase specific activity In the evaluation of amylase specific activity, the samples of Examples 1 to 9 and Comparative Example 1 were reacted with starch, and then the absorbance at 600 nm from the color indicated by adding an iodine solution. was analyzed. The decrease in absorbance of the samples compared to the control group indicated the percentage of amylase in the sample, which allowed to obtain the specific activity of amylase for each group.

Specifically, 0.04 g of starch was weighed and dissolved in 100 ml of phosphate buffer to obtain a starch solution. Further, 0.277 g of potassium iodide was weighed and mixed with 4.6 ml of 0.1N potassium iodate solution, 0.93 ml of hydrochloric acid solution and 100 ml of ultrapure water to obtain a colored liquid. In addition, 5 g of the feed additives of Examples 1 to 9 and the unfermented coffee powder of Comparative Example 1 were each added to 45 ml of distilled water, stirred at high speed for 2 hours at a temperature of 25 ° C. and a rotation speed of 130 rpm, and then stirred at 12000 rpm. Centrifuged for 5 minutes. Supernatants were collected as samples for Examples 1-9 and Comparative Example 1.

The samples of Examples 1-9 and Comparative Example 1 were then diluted 5-fold with distilled water. After that, 10 μl of the diluted samples of Examples 1-9 and Comparative Example 1 were added to 500 μl of starch solution (already preheated in a 37° C. water bath for 2 minutes), respectively, and reacted in a 37° C. water bath for 8 minutes. let me Next, after adding 500 µl of a coloring solution to the reaction solution, 4000 µl of ultrapure water was added, reacted for 5 minutes, and centrifuged at 8000 rpm for 5 minutes. The supernatant of each group was collected and centrifuged again at 8000 rpm for 5 minutes. The supernatant was then collected and analyzed with an enzyme immunoassay to obtain the absorbance at 660 nm for each group. Here, the blank group was a group to which no amylase-containing sample was added.

The amylase specific activity was determined as follows. After subtracting the absorbance of Examples 1-9 and Comparative Example 1 from the absorbance of the blank group, the result is divided by the absorbance of the blank group, multiplied by 800, and amylase per unit volume of Examples 1-9 and Comparative Example 1 activity was obtained. Units are units/dl. Further, the amylase activity results were multiplied by the dilution rate of each group and divided by 100 to obtain the amylase specific activities of Examples 1 to 9 and Comparative Example 1. The unit is unit/g. The amylase specific activity results of Examples 1-9 and Comparative Example 1 are shown in Table 2 below.

3) Evaluation of Cellulase Specific Activity One unit of cellulase activity was defined as 1 μmole of glucose released from cellulose under the conditions of pH 5.0 and temperature 37°C. This experiment was performed according to the procedure described in "Enzymatic Assay of Cellulase" by Sigma-Aldrich Corporation and gave amylase specific activity results for Examples 1-9 and Comparative Example 1. Table 2 shows the results.

(4) Evaluation of Specific Activity of Xylanase One unit of xylanase activity was defined as 1 μmol of xylanose released from xylan under conditions of pH 4.5 and temperature 30°C. This experiment was performed according to the procedure set forth in Sigma-Aldrich Corporation's "Enzymatic Assay of XYLANASE (EC 3.2.1.8)" (enzymatic assay of xylanases) and determined the specific activity of the xylanases of Examples 1-9 and Comparative Example 1. got the result. Table 2 shows the results.

As can be seen from the results in Table 2 above, in the test evaluating the specific activities of protease, amylase, cellulase and xylanase, no specific activity of each digestive enzyme of Comparative Example 1 was detected. In other words, it was revealed that the unfermented coffee powder of Comparative Example 1 did not have a measurable level of specific activity of these digestive enzymes. In the results of Examples 1 to 9, specific activities of proteases, amylases, cellulases and xylanases of these groups were all detected. These groups had high protease specific activities (355.80 Units/g to 973.77 Units/g). Thus, the feed additives of Examples 1-9 actually had higher specific activities of digestive enzymes compared to the unfermented coffee grounds of Comparative Example 1.

Further, referring to the component proportions and fermentation substrate C/N ratios listed in Table 1 above, Examples 1 and 5 have fermentation substrate C/N ratios similar to 50.2 and 50.9, respectively. However, the fermentation substrate of Example 1 contained an additional 10% by weight of rice husks, which further increased the protease and amylase specific activities of Example 1 compared to Example 5. . Thus, adding a specific content range of rice husks to the fermentation substrate could actually further increase the specific activities of proteases and amylases.

<Test Example 2: Effect of fermentation time on specific activity of digestive enzyme>
In Test Example 2, Examples 10 to 14 were used. Specifically, in the preparation steps of Examples 10 to 14, 1.5 g to 3 g of Aspergillus oryzae at 6.5×10 ⁷ spores/g or more was added to 3 kg of sterilized fermentation substrate, and fermented at 30° C. for 7 days. Fermentation substrate samples were collected at different fermentation times of 3 days, 4 days, 5 days, 6 days and 7 days. After drying the sample at 50°C for 12-14 hours to a moisture content of less than 7.5%, the proteases and amylases of the feed additives of Examples 10-14 were tested according to the same method as in Test Example 1 for different fermentation times. of specific activity was obtained. The results are shown in Tables 3 and 4, respectively.

According to the results in Table 3, the protease specific activities of the feed additives of Examples 10-14 were all higher than 340 units/g by fermentation over 3 days, After fermentation, the protease specific activities were as high as 697.6 units/g and 825.88 units/g, respectively. In addition, the protease specific activities of Examples 10 to 14 tended to increase as the fermentation time lengthened.

To clearly show the relationship between protease specific activity and fermentation time, the results of Table 3 are also shown in FIG. 2A. According to FIG. 2A, the protease specific activity actually increased with increasing fermentation time, with a clear increase after 4-6 days of fermentation time.

According to the results in Table 4, the amylase specific activities of the feed additives of Examples 10-14 were all higher than 89 units/g over 3 days of fermentation, and in Examples 10 and 11, 3 After fermenting for days, they exhibited high amylase specific activities of 167.89 units/g and 165.02 units/g, respectively. The amylase specific activities of Examples 10 to 14 also tended to increase as the fermentation time lengthened.
To clearly show the relationship between amylase specific activity and fermentation time, the results of Table 4 are also shown in FIG. 2B. As shown in Figure 2B, the amylase specific activity actually increased with increasing fermentation time, with a clear increase after 4-6 days of fermentation time.

<Test Example 3: Evaluation of growth promoting effect on animals>
In Test Example 3, the feed additive of Example 1 was used. Specifically, 120 weaned piglets (60 male piglets, 60 female piglets, average weight 7.15 kg), with 40 piglets in each group (20 male piglets, 20 female piglets). They were randomly divided into 3 groups, and they were set as a low dose group, a high dose group, and a control group. A low dose group containing 0.5 kg of the feed additive of Example 1 per ton of basal feed, a high dose group containing 1 kg of the feed additive of Example 1 per ton of basal feed, complex enzymes and A control group consisted of only the basal diet containing no colistin.

The experimental method was that after the weaned piglets were weaned, the weaned piglets were fed the different groups of diets mentioned above daily for 6 months. To assess piglet growth, daily body weight and feed intake were recorded and body weight gained over time, average daily gain (ADG) and feed conversion ratio (FCR). got The higher the ADG and the lower the FCR value, the better the growth-promoting effect of the diet. The results of the piglets fed the different groups of diets described above for 4 weeks are listed in Table 5 below, and the results of the piglets fed the different groups of diets described above for 6 weeks are listed in Table 6 below.

According to the results in Table 5, the weight gain from the start of feeding to the second week was similar among the low-dose group, high-dose group and control group. When the piglets were continuously fed up to the 4th week, the weight gain from the 2nd to the 4th week was significantly higher in the low- and high-dose groups than in the control group, increasing by approximately 33% and 37.5%, respectively. . Body weight at week 4, ADG from weeks 2 to 4, and FCR from weeks 2 to 4 were better in both the low-dose and high-dose groups compared to the control group relatively high body weight at 4 weeks, relatively high ADG from 2 to 4 weeks, and relatively low FCR from 2 to 4 weeks.

On the other hand, according to the results in Table 6, when the piglets were continuously fed until the 6th week, the body weight at the 6th week was still higher than the control group in both the low dose group and the high dose group. Similarly, regardless of body weight gain by week 6, ADG by week 6, and FCR by week 6, both low-dose and high-dose groups were superior to controls. showed greater weight gain, higher ADG and lower FCR by week 6 of feeding. Therefore, by adding the feed additive prepared by the method of the present invention to the basic animal feed, it was possible to actually exert the effect of promoting the growth of the animal.

<Test Example 4: Evaluation of bacteriostatic action>
In this test example, the feed additive of Example 1 and the unfermented coffee powder of Comparative Example 1 were used to test the bacteriostatic action against different types of pathogens, and the bacteriostatic effects of these groups were evaluated.

(1) Bacteriostatic effect on Escherichia coli, Streptococcus suis and Salmonella choleraesuis

5 g of the feed additive of Example 1 and the unfermented coffee powder of Comparative Example 1 were weighed and added to 25 ml of distilled water, respectively, followed by high speed stirring for 30 minutes and then centrifuging at 10000 rpm for 10 minutes. The supernatant was collected, filtered through a PES membrane with a pore size of 0.45 μm, and then lyophilized to obtain a crude product. Next, 200 mg of the crude product was dissolved in 1 ml of distilled water to prepare test solutions for Example 1 and Comparative Example 1.

Colonies of Escherichia coli, Streptococcus suis and Salmonella choleraesuis were then picked with sterile cotton swabs and attached to appropriate solid media. Here, for Escherichia coli and Salmonella choleraesuis, the international standard test method Mueller Hinton agar (MH agar) is adopted, cultured at 37°C for 16 to 18 hours, and for Streptococcus suis, blood agar containing 5% sheep blood is adopted. and incubated at 37°C for 16-18 hours.

After that, different points (spots) of the solid medium were removed to make holes with a diameter of 8 mm, and 100 μl of the test solutions of Example 1 and Comparative Example 1 were put into the different holes of the solid medium. In addition, 1000 ppm of colicitin and 100 ppm of gentamicin were placed in separate wells of the solid medium as positive controls. After culturing these solid media for 24 hours under appropriate conditions according to the characteristics of individual pathogens, the diameter of the zone of inhibition appearing on the solid media was measured. Table 7 shows the results. The unit is mm.

As shown in the results in Table 7, the Escherichia coli, Streptococcus suis and Salmonella choleraesuis solid media of Comparative Example 1 did not have a zone of inhibition whose diameter could be measured. It shows that it does not have a bacteriostatic effect on For Example 1, the measured diameters of the zones of inhibition on solid media of Escherichia coli and Streptococcus suis were 12 mm and 14 mm, respectively, the same result as gentamicin. In addition, the diameter of the inhibition zone on the solid medium of Salmonella choleraesuis in Example 1 was measured to be 12 mm, which was similar to colistin.

In addition, in order to clearly show whether or not zones of inhibition appear on the solid medium, the bacteriostatic results of the above groups for Escherichia coli, Streptococcus suis and Salmonella choleraesuis are shown in FIGS. 3A to 3C, respectively.

In FIG. 3A showing the results of bacteriostatic action on Escherichia coli, the results for Comparative Example 1, Example 1, and gentamicin are shown from left to right as A1, A2, and A3, respectively. In FIG. 3A, a clear zone of inhibition appeared around the pores of group A3, indicating that the results were not affected by the experimental manipulation. In the results of groups A1 and A2, a clear zone of inhibition also appeared around the pores of group A2, similar to the results of group A3, but no clear zone of inhibition was observed around the pores of group A1. I didn't.

In FIG. 3B, which shows the results of bacteriostasis of Streptococcus suis, the results of Comparative Example 1, Example 1, and gentamicin are indicated from left to right as B1, B2, and B3, respectively. In FIG. 3B, a clear zone of inhibition appeared around the pores of the B3 group, indicating that the results were not affected by the experimental manipulation. In the results of groups B1 and B2, a clear zone of inhibition also appeared around the hole in group B2, which was similar to the result of group B3, but no clear zone of inhibition was observed around the hole in group B1. rice field.

In FIG. 3C showing the results of bacteriostasis of Salmonella choleraesuis, the results of Comparative Example 1, Example 1 and Colistin are indicated from left to right as C1, C2 and C3, respectively. In FIG. 3C, a clear zone of inhibition appeared around the pores in group C3, indicating that the results were not affected by the experimental manipulation. Regarding the results of groups C1 and C2, a clear zone of inhibition appeared around the hole in group C2, similar to the results of group C3, but no clear zone of inhibition was observed around the hole in group C1. I didn't.

According to the results in Table 7 and FIGS. 3A to 3C, the unfermented coffee powder of Comparative Example 1 was treated by the method of the present invention to prepare the feed additive of Example 1. actually had bacteriostatic effects against Escherichia coli, Streptococcus suis and Salmonella choleraesuis, comparable to antibiotics.

(2) Bacteriostatic action against Lactobacillus casei The test solutions of Example 1 and Comparative Example 1 were prepared according to the method shown in (1) Bacteriostatic action against Streptococcus suis, Escherichia coli and Salmonella choleraesuis described above, and were found to be safe for human consumption. The bacteriostatic effect on Lactobacillus casei, a kind of probiotics, was evaluated. Specifically, de Man, Rogosa, Sharpe agar (MRS agar) was used for Lactobacillus casei, the solid medium was placed in an anaerobic incubator, and anaerobically cultured at 37°C for 24 hours. Then, different points of the solid medium were spotted to obtain holes with a diameter of 8 mm, and 100 μl of the test solution of Example 1 and Comparative Example 1 with a concentration of 200 mg/ml, penicillin of 100 U/ml and streptomycin of 100 ppm. , placed in different holes of the solid medium. Next, the solid medium was placed in an anaerobic incubator and anaerobically cultured at 37° C. for 72 hours, after which the diameter of the inhibition zone appearing on the solid medium was measured. The unit is mm.

FIG. 4 shows the results of bacteriostasis of Lactobacillus casei. In the figure, the results for Comparative Example 1, Example 1 and penicillin and streptomycin antibiotics are shown from left to right labeled A1, A2 and A3, respectively. In the results shown in Figure 4, a clear zone of inhibition appeared around the pores of group A3, and the zone of inhibition measured 28 mm, indicating that the results were not affected by the experimental manipulation. The results for groups A1 and A2 showed no obvious zone of inhibition observed in these groups. That is, neither Comparative Example 1 nor Example 1 had the effect of suppressing the growth of Lactobacillus casei. Therefore, when the unfermented coffee powder of Comparative Example 1 was treated by the method of the present invention to prepare the feed additive of Example 1, the feed additive was found to be effective in the growth of probiotics such as Lactobacillus casei. had no effect.

(3) Evaluation of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) The test solution of Example 1 was prepared according to the method shown in (1) Bacteriostatic action experiment above. This experiment was performed according to the broth microdilution test specified by the Clinical and Laboratory Standards Institute (CLSI) to assess MIC and MBC. Common poultry and livestock pathogens were used as pathogens. Poultry pathogens included Riemerella anatipestifer, Gallibacterium anatis, Escherichia coli, Salmonella spp. and Staphylococcus spp., and livestock pathogens included Staphylococcus hyicus, Streptococcus suis, Escherichia coli and Salmonella choleraesuis.

Specifically, the various types of pathogens described above were cultured in tryptone soy broth (TSB) and their concentrations were calculated by subtracting the turbidity value of the optical turbidity test tube (pathogen-free TSB). post), adjusted to 0.5 McFarland, or 1.5×10 ⁸ colonies/milliliter (cfu/ml). The test solution of Example 1 was then serially diluted 2-fold in TSB and 200 μl of the diluted test solution was placed in a sterile 96-well plate. 10 μl of the pathogen-containing solution described above was also placed in a sterile 96-well plate and mixed evenly with the test solution to give a final pathogen concentration of approximately 5×10 ⁵ cfu/ml to 10 ⁶ cfu/ml. The 96-well plates were then cultured under appropriate conditions for 18-24 hours before being removed for observation.

If the test solution has a bacteriostatic action, it inhibits the growth of pathogens, so the broth is clear and transparent. The MIC was the lowest concentration of test solution that allowed the broth to remain clear and transparent. Alternatively, MBC was taken as the minimum concentration at which observable colonies did not form on solid medium after 24 hours incubation of the broth on solid medium. The MIC and MBC for each pathogen in Example 1 are shown in Table 8 below. Each pathogen was tested in 5 or more strains.

According to the results in Table 8, the feed additive of Example 1 had both MIC and MBC below 50 mg/ml for different types of poultry pathogens. The feed additive of Example 1 has particularly good bacteriostasis against Riemerella anatipestifer and Gallibacterium anatis, with MICs of 1.56-6.25 mg/ml and 6.12-12.5 mg/ml respectively, and an MBC of 3.13 respectively. ~12.25 mg/ml and 12.5 mg/ml. For different types of livestock pathogens, both the MIC and MBC of the feed additive of Example 1 were below 50 mg/ml.

According to the results of Test Example 4, the feed additive prepared by the method of the present invention actually inhibited the growth of many kinds of pathogens, but did not affect the growth of Lactobacillus casei at the same time. had the effect of Therefore, this feed additive had a selective bacteriostatic effect against pathogens without affecting probiotics.

<Test Example 5: Evaluation of effect on respiratory diseases>
In Test Example 5, the following experiment was performed using the feed additive of Example 1 and Mycoplasma hyopneumoniae and Mycoplasma pneumonia as pathogens of respiratory diseases. The ATCC numbers for Mycoplasma hyopneumoniae and Mycoplasma pneumonia were ATCC 25934 and ATCC 39342, respectively. In the following experiments, 2 g of the sample additive of Example 1 was weighed and 4 ml of 70% alcohol was added to obtain a solution. After that, ultrasonic extraction was performed at room temperature for 1 hour, followed by centrifugation at 12000 rpm for 10 minutes to collect the supernatant. Thereafter, the solvent of the supernatant was removed by vacuum concentration to obtain an extract of the feed additive of Example 1. The extract served as the test sample for the following experiments. In the following, this extract sample is abbreviated as ME.

(1) Evaluation of pathogen growth inhibitory effect
The ME was dissolved in 50% alcohol by sonication to obtain an ME stock solution with a concentration of 40 mg/ml. Next, after the ME stock solution was filtered through a filtration membrane with a pore size of 0.22 μm, it was added to a medium containing Mycoplasma hyopneumoniae at a concentration of 10 ⁴ cfu/ml and a medium containing Mycoplasma pneumonia at a concentration of 10 ⁶ cfu/ml. A group with different concentrations of 200 μg/ml ME, 400 μg/ml ME, and 800 μg/ml ME was obtained. The medium for Mycoplasma hyopneumoniae was ATCC 1699 medium and the medium for Mycoplasma pneumonia was ATCC 2611 medium.

Next, 1 ml of culture was harvested after 0, 12, 24, 36 and 48 hours of culture time, and the harvested culture was centrifuged to obtain a pathogen pellet. Pathogen pellets were subjected to DNA extraction and the resulting DNA was quantified by qPCR to analyze pathogen load at different incubation times. Results of qPCR for Mycoplasma hyopneumoniae and Mycoplasma pneumonia are shown in Figures 5A and 5B, respectively. A group in which ME was not added to the medium was used as a control group.

In FIG. 5A, for Mycoplasma hyopneumoniae, the pathogen load in the 800 μg/ml ME group was all lower than the control group at each incubation time. Compared to the control group, the pathogen load was clearly lower in the 800 μg/ml ME group, especially at 48 hours of culture time, and was statistically significant with a p-value of less than 0.01. In FIG. 5B, for Mycoplasma pneumonia, the amount of pathogens in the 800 μg/ml ME group was lower than that in the control group after 24 hours of culture, and the amount of pathogens in the 400 μg/ml ME group and the 800 μg/ml ME group were lower than the control group at 36 hours of culture, and at 48 hours of culture, all pathogen levels in the 200 μg/ml ME group, 400 μg/ml ME group and 800 μg/ml ME group were higher than the control group. was low. In addition, compared with the control group, the 200 and 400 μg/ml ME groups were statistically significant with p-values less than 0.05, and the 800 μg/ml ME group was also statistically significant with p-values less than 0.01. was significant.

Thus, the feed additive of Example 1 had the effect of actually inhibiting the growth of Mycoplasma hyopneumoniae and Mycoplasma pneumonia, with the 800 μg/ml ME group having the best effect of inhibition.

(2) Evaluation of pathogen infection suppression effect
Using PK-15 cell line and A549 cell line as cells infected with Mycoplasma hyopneumoniae and Mycoplasma pneumonia, respectively, the inhibitory effect of ME on pathogen infection was evaluated by gentamicin protection assay.

Cell viability experiments were first performed to assess the effect of various concentrations of ME on the proliferation of PK-15 and A549 cell lines. Specifically, DMEM containing 10% fetal bovine serum (FBS) was used as a medium, and the aforementioned cell line was cultured overnight in a 96-well plate. The culture medium was then replaced with culture medium containing different concentrations of ME and treated for 20 hours. Next, after reacting with MTT reagent (0.5 mg/ml) for 4 hours, DMSO was added and treated for 2 hours, and the absorbance at 570 nm was measured to evaluate the cell viability. Cell viability results for the PK-15 and A549 cell lines are shown in Figures 6B and 6D, respectively. A group in which ME was not added to the medium was used as a control group.

After confirming the concentration of ME that does not affect cell viability, experiments were performed to inhibit pathogen infection. Specifically, PK-15 and A549 cell lines were cultured overnight in DMEM containing 10% FBS. The culture medium was then replaced with a culture medium containing ME and treated for 2 hours. Here, 800 μg/ml ME was employed for the treatment of the PK-15 cell line and 1600 μg/ml ME for the treatment of the A549 cell line. Next, for infection, Mycoplasma hyopneumoniae was added to the culture medium of the PK-15 cell line, and Mycoplasma pneumoniae was added to the culture medium of the A549 cell line. rice field. After 24 hours of infection, PBS was used to wash away pathogens that did not adhere to the cells. Culture medium containing 400 μg/ml gentamicin was then added and treated for 4 hours to remove pathogens that adhered to the cells but did not penetrate well. Cells were then harvested for DNA extraction followed by qPCR to measure pathogen load due to successful infection and cell penetration. Here, DNA of pathogens with different pathogen load was extracted and a standard curve measured by qPCR was obtained. FIG. 6A and FIG. 6C show the effect of ME on pathogen infection suppression against Mycoplasma hyopneumoniae and Mycoplasma pneumonia, respectively. A group in which ME was not added to the medium was used as a control group.

According to FIGS. 6A and 6B, in FIG. 6B, culture medium containing 800 μg/ml ME, where >80% cell viability was taken as the standard of no significant effect on cell proliferation. did not affect the proliferation of the PK-15 cell line at all. Still referring to FIG. 6A, compared to the control group, the group treated with 800 μg/ml ME had a significantly lower pathogen load, a statistically significant reduction with a p-value of less than 0.05. Therefore, after treatment of the PK-15 cell line with 800 μg/ml ME, the amount of pathogens entering the cells by infection was significantly reduced.

6C and 6D, in FIG. 6D, culture medium containing 1600 μg/ml ME, where >80% cell viability was taken as the standard of no significant effect on cell proliferation. did not affect the growth of the A549 cell line at all. Still referring to FIG. 6C, compared to the control group, the group treated with 1600 μg/ml ME had a significantly lower pathogen load, a statistically significant reduction with a p-value of less than 0.01. Therefore, after treating the A549 cell line with 1600 μg/ml ME, the amount of pathogens that penetrated the cells by infection was significantly reduced.

As described above, the feed additive of Example 1 does not affect the growth of the PF-15 cell line and the A549 cell line, and reveals the amount of pathogens of Mycoplasma hyopneumoniae and Mycoplasma pneumonia that have entered cells by infection. could be reduced. Therefore, the feed additive of Example 1 actually had the effect of effectively suppressing pathogen infection.

(3) Evaluation of anti-inflammatory effects of pathogens (cell experiments)
Using MH-S cells and Mycoplasma pneumonia, the degree of inflammation was evaluated by measuring the contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

First, cell viability experiments were performed to assess the effect of different ME concentrations on the proliferation of MH-S cells. The specific experimental procedure was the same as that shown in Experiment (2) “Evaluation of effect of inhibiting pathogen infection”. Cell viability results for MH-S cells are shown in FIG. 7C. A group in which ME was not added to the medium was used as a control group.

After confirming concentrations of ME that do not affect cell viability, experiments were performed to suppress pathogen-induced inflammation. Specifically, MH-S cells were cultured overnight in 24-well plates. The culture medium was RPMI containing 10% FBS. The culture medium was then changed to culture medium containing 200 μg/ml ME, 400 μg/ml ME and 800 μg/ml ME and treated for 20 hours.

Mycoplasma pneumonia was then added to the medium for infection. MOI was set to 100. Two hours post-infection, the culture medium was collected and analyzed by enzyme-linked immunosorbent assay (ELISA) to determine the content of TNF-α and IL-6 in the culture medium. The results are shown in FIGS. 7A and 7B, respectively. Control group is culture medium only, ME 800 μg/ml (w/o Mp) is culture medium containing 800 μg/ml ME only, Mp is MH-S cells only, Mp+ME 200 μg/ml, Mp+ME 400 μg /ml and Mp+ME 800 μg/ml are MH-S cells treated with different concentrations of ME.

According to FIG. 7C, the culture medium containing 800 μg/ml ME did not produce any MH-S cells, with a cell viability greater than 80% as the standard for no significant effect on cell proliferation. It can be said that it does not affect proliferation. Further referring to FIG. 7A, for the measurement results of TNF-α, as the concentration of ME increased, TNF-α A trend toward decreasing content was observed. Furthermore, the effect of lowering TNF-α content in the 200 μg/ml ME, 400 μg/ml ME and 800 μg/ml ME groups was statistically significant with p-values less than 0.05, 0.01 and 0.001, respectively. rice field. Thus, the content of TNF-α in the culture medium was effectively reduced after treating MH-S cells with different concentrations of ME.

Further, referring to FIG. 7B, as with TNF-α, the measurement of IL-6 increases the concentration of ME in the group treated with different concentrations of ME compared to the Mp group not treated with ME. A tendency for IL-6 content to decrease with increasing age was observed. Furthermore, the effect of lowering IL-6 content in the 200 μg/ml ME, 400 μg/ml ME and 800 μg/ml ME groups was statistically significant with p-values less than 0.05, 0.01 and 0.001, respectively. rice field. Thus, the content of IL-6 in the culture medium was effectively reduced after treating MH-S cells with different concentrations of ME.

As mentioned above, the contents of TNF-α and IL-6 released from the cells were obviously decreased after the MH-S cells were treated with the feed additive of Example 1. Thus, the feed additive of Example 1 indeed had the effect of effectively inhibiting inflammation induced by respiratory pathogens.

(4) Evaluation of anti-inflammatory effects of pathogens (animal experiments)
BALB/c mice were infected with Mycoplasma pneumonia to induce inflammation, and the number of neutrophils and monocytes infiltrating the alveoli was measured to evaluate the degree of inflammation. Specifically, ME was fed once a day for 7 days at a dosage of 1000 mg/kg, and then BALB/c mice were infected with Mycoplasma pneumonia and continuously fed with ME. After 3 days of infection, BALB/c mice underwent bronchoalveolar lavage to obtain test samples. The samples were then analyzed by flow cytometry to measure total cell counts, neutrophil cell counts, and monocyte cell counts. The results are shown in Figures 8A, 8B and 8C. The control group is PBS-fed BALB/c mice, and the experimental group BALB/c mice are ME-fed mice.

According to FIG. 8A, the total cell counts of the test samples in the control and ME-treated groups were similar, with no statistically significant difference, indicating that these groups are on the same basis for comparison. . Further referring to FIG. 8B, compared with the control group, the cell number of neutrophils in the ME-treated group was clearly decreased, a statistically significant decrease with a p-value of less than 0.01. Regarding FIG. 8C, the number of monocytes in the ME-treated group was slightly decreased compared to the control group.

Therefore, administration of the feed additive of Example 1 to mice actually reduced the number of cells infiltrating the alveoli as a result of infection with respiratory pathogens, and the subsequent degree of inflammation could be improved. rice field.

According to the results of Test Example 5, the feed additive prepared by the method of the present invention actually has the effect of effectively inhibiting the growth and infection of respiratory pathogens, and also has the effect of inhibiting the growth and infection of respiratory pathogens. were able to ameliorate and/or inhibit the inflammation caused by

In conclusion, a feed additive prepared by using coffee powder with a specific content range as a fermentation substrate, controlling the C/N ratio of the fermentation substrate within a specific range, and using Aspergillus oryzae for fermentation, It has the advantage of containing digestive enzymes with relatively high specific activity, selective bacteriostasis and resistance to respiratory pathogens. Therefore, the feed additive can be applied to animal feed, can promote the growth of animals, can exert the effect of preventing and/or treating respiratory diseases, and can properly solve the problem of coffee grounds disposal. solve. It also provides a low cost alternative to animal feed that can promote animal growth and also reduces the risk of respiratory disease transmission in animals. These can further enhance the commercial value of the present invention.

Although many features and advantages of the present invention have been set forth in the foregoing description, along with details of the structure and characteristics of the present invention, what is disclosed herein is merely exemplary. Changes may be made in details, particularly matters of size, within the full scope of the broad general meaning of the terms defined in the claims from the principles of the invention.

In view of the above, in order to solve the environmental protection and waste disposal problems caused by used coffee grounds (hereinafter simply referred to as coffee grounds) , we develop technical means for applying coffee grounds to animal feed. It has become necessary, at the same time, to achieve the objectives of reducing animal feed costs and promoting animal growth.

Claims (15)

A method for preparing a feed additive, comprising:
Step (a): A fermentation substrate containing coffee grounds and auxiliary materials, the content of coffee grounds is 45% by weight or more and 80% by weight or less based on the total weight of the fermentation substrate, and the carbon-nitrogen ratio of the fermentation substrate is 35 or more and 65 or less, and the auxiliary material is corn sand, rice husks, soy flour with husks, broken rice or a combination thereof;
step (b): fermenting the fermentation substrate with Aspergillus oryzae;
A method of adjusting a feed additive containing. 2. The method according to claim 1, wherein the fermentation substrate has a carbon-nitrogen ratio of 35 or more and 60 or less. 3. The method according to claim 1 or 2, wherein the content of said auxiliary material is ≧20% and ≦55% by weight based on said weight of said fermentation substrate. The carbon-nitrogen ratio of the fermentation substrate is 46 or more and 53 or less, the auxiliary material contains rice husk, and the content of the rice husk is 7% or more and 10% or less by weight based on the total weight of the fermentation substrate. The method according to any one of claims 1 to 3, wherein the method is The method according to any one of claims 1 to 4, wherein the content of Aspergillus oryzae is 0.02 wt% or more and 0.2 wt% or less based on the total weight of the fermentation substrate. The method according to any one of claims 1 to 5, wherein the fermentation substrate has a water content of 50% to 75% by weight. 7. The method according to any one of claims 1 to 6, wherein in the step (b), the temperature for fermenting the fermentation substrate with Aspergillus oryzae is 25°C or higher and 40°C or lower, and the fermentation time is 2 days or longer and 12 days or shorter. described method. A feed additive for promoting the growth of animals prepared by the method according to any one of claims 1-7. 9. The feed additive according to claim 8, wherein said feed additive is effective in preventing and/or treating respiratory diseases. A feed additive for preventing and/or treating respiratory diseases, prepared by the method according to any one of claims 1-7. A feed additive for inhibiting pathogen growth, prepared by the method of any one of claims 1 to 7, wherein said pathogens are Streptococcus suis, Escherichia coli, Salmonella spp., Riemerella anatipestifer, A feed additive containing Gallibacterium anatis, Staphylococcus spp., Mycoplasma hyopneumoniae or Mycoplasma pneumoniae. A feed additive for inhibiting pathogen infection, prepared by the method according to any one of claims 1 to 7, wherein said pathogen comprises Mycoplasma hyopneumoniae or Mycoplasma pneumoniae. A feed additive for suppressing inflammation prepared by the method according to any one of claims 1 to 7. 14. The feed additive according to claim 13, wherein the inflammation is pneumonia. 15. The feed additive according to claim 14, wherein pneumonia is induced by Mycoplasma hyopneumoniae or Mycoplasma pneumoniae.

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