KR102169139B1 - Compostion for improving intestinal microflora of poultry comprising Allium hookeri powder as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for improving the intestinal flora of poultry containing the powder of three leaves ( Allium hookeri ) as an active ingredient, and when feeding the three leaf powder of the present invention to broilers with 0.5% of diet, the three leaf powder is broiler It is predicted that it will increase the microbes that are positively correlated with body weight, tibia strength and immunity, which affects the diversity of intestinal microflora of broiler chickens, and enhances functions related to carbohydrate metabolism. It can be usefully used as a composition for improving the flora.

Description

Composition for improving intestinal microflora of poultry comprising Allium hookeri powder as an active ingredient, comprising the powder of three leaves as an active ingredient.

The present invention relates to a composition for improving the intestinal flora of poultry, comprising the powder of three leaves as an active ingredient.

Probiotics are microorganisms that are administered to humans and animals to improve the balance of intestinal microflora, promote growth, increase feed utilization, prevent intestinal fermentation or diarrhea, and remove inhibitors of nutrient intake. The bacteria used in probiotics are mainly resident bacteria that are commonly found in the intestines of healthy people, and Lactobacillus and bifidobacteria are the main species (Fuller, R Probiotics in man and animals, 1989, J Appl Microbiol 66:365-378). Probiotics are showing effects such as enhancing immune function and inhibiting the growth of harmful microorganisms by using the coexistence and coexistence of bacteria. Specifically, Bacillus among the microorganisms present in the intestine is related to feed efficiency, and Clostridiaceae and Clostridium sensu stricto 1 is weight gain, Lachnoclostridium and Ruminococcaceae Groups are known to be associated with immune function against viruses.

In the case of livestock probiotics, various probiotic products such as bacillus, yeast, and lactic acid bacteria are already sold, and it is reported that the supply of probiotics from weaning piglets improves growth and feed efficiency. However, in many cases, when feeding only considering economics, the mechanism of action and efficacy are unclear, and when mixed with antibiotics, the effect is reduced or the stability of probiotics decreases in many cases in the feed processing stage.

Allium hookeri is distributed in southern China, India, Bhutan, and Sri Lanka in East Asia, and grows in forests, wetlands and grasslands of 1400 m to 4200 m above sea level. It is also called root leek, and it is also called samchae because it is similar in taste and shape to ginseng, and it is also called samchae because it has three flavors of bitter, sweet, and spicy. The height of the plant is about 0.6 to 1 m, and it is known that three egg roots, leaves and flowers can be used for edible and medicinal purposes.

The present inventors have searched for a material that can improve the intestinal microbes of livestock while solving the conventional problems of livestock probiotics. As a result, the three leaf powder improves the intestinal microflora of livestock while improving the weight, tibia strength and immunity of livestock. Since it increases the related intestinal microflora, the present invention was completed by confirming that the powder of three leaves can be used as a composition for improving the intestinal flora of poultry.

Korean Patent Registration No. 10-1578982

It is an object of the present invention to provide a composition for improving the intestinal flora of poultry.

In order to achieve the above object, the present invention is three ( Allium hookeri ) Provides a composition for improving the intestinal flora of poultry, including leaf powder as an active ingredient.

When feeding the three leaf powder of the present invention to broilers at 0.5% of the diet, the three leaf powder affects the diversity of intestinal microflora of the broiler, and the positive correlation between body weight, tibia strength, and immunity affecting the productivity of the broiler. Since it increases the microorganisms in the broiler body weight, tibia strength and immunity, it can be usefully used as a composition for improving the intestinal flora of poultry.

1 is a diagram showing the diversity of intestinal microflora of broilers fed with three dry powders:
Control: no treatment control
CS: Positive control, commercial supplement added
Leaf 0.3: Three leaf dry powder added (0.3% of the total weight of the feed);
Leaf 0.5: Three leaf dry powder added (0.5% of the total weight of the feed);
Root 0.3: Three roots dry powder added (0.3% of the total weight of the feed);
Root 0.5: Three roots dry powder added (0.5% of the total weight of the feed)
PC1: principal component 1, the variable that most often describes the distance between samples
PC2: Principal component 2, the second most important variable that describes the distance between samples.
FIG. 2 is a diagram showing microorganisms whose amount varies in the intestine of broilers fed with three dry powders in a phylum unit.
Figure 3 is a comparison of the untreated control (Control) and the positive control (CS), the amount of four microorganisms in the intestine of broilers fed three leaf dry powder (0.5%) varies; Three microbial genera whose amount varies in the intestine of broilers fed with dry leaf powder (0.5%) compared to the untreated control group; And compared with the positive control (CS), a diagram showing one microbial genus whose amount varies in the intestine of broilers fed with dry three leaf powder (0.5%).
Figure 4 is a diagram showing the amount of intestinal microorganisms according to the feeding of three dry powders and the correlation coefficient (Corr) of intestinal microorganisms showing a significant correlation with the body weight of broilers.
Figure 5 is a diagram showing the amount of intestinal microbes according to feeding of three dry powders and a correlation coefficient of intestinal microbes showing a significant correlation with tibia strength of broilers.
Figure 6 is a diagram showing the amount of intestinal microbes according to feeding of three dry powders and a correlation coefficient of intestinal microbes (Corr) showing a significant correlation with the production of IBD (infectious bursal disease) antibodies in broilers.
Figure 7 is a diagram showing the results of predicting the function of the intestinal microorganisms of a broiler (Leaf 0.5) fed with three leaf dry powder at 0.5% of the diet of the untreated control group (Control) and the broiler chicken (U: Unclassified; HD: Human Diseases; GIP: Genetic Information Processing; top graph: function enhancement; bottom graph: function decline).
8 is a diagram showing the weight measurement results of broilers raised by feeding three leaves or dry root powder.
9 is a diagram showing the results of measuring the tibia strength of broilers raised by feeding three leaves or dried root powder.
Figure 10 is a diagram showing the results of measuring the amount of IBD antibody production of broilers raised by feeding dry powder of three leaves or roots.

Hereinafter, the present invention will be described in detail.

The present invention is three ( Allium hookeri ) Provides a composition for improving the intestinal flora of poultry, including leaf powder as an active ingredient.

The powder of the three leaves is preferably prepared by a manufacturing method including drying and powdering, but is not limited thereto. In the above manufacturing method, drying may be hot air drying or freeze drying, but is not limited thereto. In addition, drying may be performed at a temperature of 40 to 70°C, 45 to 70°C, 40 to 65°C, 45 to 65°C, 45 to 60°C, or 50 to 65°C. Drying may be performed for 12 to 36 hours, 12 to 30 hours, 18 to 36 hours, 18 to 30 hours, 20 to 30 hours, 20 to 28 hours, or 22 to 26 hours. If it is outside the range of the drying conditions, it cannot be powdered into a form that is easy to mix with the feed.

The three can be used without limitation, such as cultivated or commercially available.

The composition is for feeding poultry, and the poultry may be chicken, duck or turkey, but is not limited thereto. According to an embodiment of the present invention, the poultry may be chicken, specifically broiler.

The composition can increase the intestinal microbes that are positively correlated with the weight, tibia strength or immunity of poultry. According to an embodiment of the present invention, the intestinal microorganisms correlated positively with the weight of poultry are Eggerthella genus, Christensenellaceae Family, genus Familly XIII AD3011 group or genus Clostridium Can be Further, according to one embodiment of the invention, the intestinal flora in the tibia strength of poultry with positive correlation are Parasutterella may lie, intestinal microorganisms in the poultry immune and positive correlation is Eubacterium genus, Lachnospiraceae UCG-004 Genus, Parasutterella or Ruminococcaceae V9D2013 group can be tricked . Therefore, the composition can enhance the weight, tibia strength or immunity of poultry.

In addition, the powder of the three leaves may be added in an amount of 0.4 to 1.0% by weight of the total poultry feed. Specifically, the powder of the three leaves may be added in an amount of 0.4 to 0.8% by weight, 0.4 to 0.7% by weight, 0.4 to 0.6% by weight, or 0.45 to 0.55% by weight of the total poultry feed.

In a specific embodiment of the present invention, the present inventors produced a dry powder by hot air drying and pulverizing three leaves, and feeding it at 0.5% of the broiler diet affects the diversity of microorganisms in the broiler intestine (Fig. 1 and 3), increase the microorganisms that have a positive correlation with body weight, tibia strength, and IBD antibody production that affect the productivity of broilers (see FIGS. 4 to 6), and functions related to metabolism, especially carbohydrate metabolism. It was confirmed that it is predicted to enhance related functions (see FIG. 7). In addition, it was confirmed that when the dry powder of three leaves was fed at 0.5% of the broiler diet, the broiler's body weight, tibial strength and IBD antibody production increased (see FIGS. 8 to 10).

Therefore, the three leaf powder of the present invention can be used as a composition for improving the intestinal flora of poultry.

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

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example 1> Three Preparation of dry powder

Three leaves and roots were purchased from Golden Tree (Sunchang), washed clean, dried with hot air at 50 to 60° C. for 24 hours, respectively, and then pulverized and powdered.

< Experimental example 1> Three Confirmation of changes in microbiota in broiler gut according to dry powder feeding

When each of the dried three leaves powder and the three root dried powder prepared in Example 1 were fed to broilers, the effect of the three dried powders on the intestinal microflora of the broiler was evaluated.

First, the dried three leaf powder and the three root dried powder prepared in Example 1 were mixed with feed at the level of 0.3% or 0.5% of the diet to broilers, fed daily for 35 days, and the cecum of the broiler was separated. DNA of microorganisms in the appendix was extracted using the AccuPrep Stool DNA Extraction Kit, and regions V3 and V4 of the 16S rRNA gene of the intestinal microorganism were amplified by PCR. As primers, 341F forward primer (5'-CCTACGGGNGGCWGCAG-3', SEQ ID NO: 1) and 805R reverse primer (5'-GACTACHVGGGTATCTAATCC-3', SEQ ID NO: 2) were used. The amplified 16S rRNA was purified using qPCR Quantification Protocol Guide (KAPA Library Quantificatoin kits for Illumina Sequecing platforms) and LabChip GX HT DNA High Sensitivity Kit (PerkinElmer, Massachusetts, USA), and read paired ends of 300bp length with MiSeq™ platform. Was created. The sequenced reads were merged using PEAR and QIIME2 software, and quality control was performed. For the merged read, system classification was performed using the Silva database (Release 128) and the QIIME2 q2-feature-classifier plugin. Diversity indicators, alpha diversity and beta diversity, were analyzed through the QIIME2 q2-diversity plugin, and PERMANOVA was performed with the UniFrac matrix obtained by calculating the distance between the experimental groups to determine how statistically it is distinguished between the experimental groups. (Principal coordinates analysis) plot.

As a result, it was found that the total intestinal microflora of broilers fed dry three leaf powder was different from that of the other experimental groups.In particular, broilers fed with dry three leaf powder at 0.5% of diet were not fed with any additives other than feed. It was also found to be statistically significantly distinguished from the untreated control group (Control). From the above results, it was confirmed that the dry powder of three leaves has an effect on the diversity of intestinal microorganisms in broilers (FIG. 1).

< Experimental example 2> Three Discovery of microorganisms in broiler intestines that have changed depending on dry powder feeding

By feeding the dried powder of three leaves or roots prepared in Example 1, it was attempted to discover microorganisms that change in the broiler intestine.

First, after performing phylogenetic tree analysis using the merged read obtained in Experimental Example 1, the data was classified by genus level, and the amount of microorganisms was corrected with edgeR to correct the amount of data produced between the experimental groups (TMM normalization). . Assuming that the corrected amount of microorganisms follows a negative binomial distribution, statistical analysis was performed using a generalized linear model (GLM) to discover microorganisms (DAM) that are expected to be related to feeding three dry powders. In this case, a false discovery rate (FDR) was used to suggest statistical significance, and statistical significance was determined based on FDR 0.05%.

As a result, when the microorganisms were classified by phylum among the classification units, there was a limit to discovering microorganisms whose amount varies depending on the feeding of dry leaves of three leaves (FIG. 2). Microorganisms were classified into a more subdivided genus, and the effects were affected by feeding dry three leaf powder (0.5%) when compared with the untreated control group (Control) and the positive control group (CS) fed with a commercial feed additive. 8 microorganisms ( Eubacterium nodatum group; Marvinbryantia ; Oscillospira ; Gelria ; Parabacteroides ; Gastranaerophilales ; NB1-n; And Christensenellaceae R-7 group)) was discovered (Fig. 3). Specifically, Fed three leaf dried powder (0.5%) group (Leaf 0.5) is commonly Eubacterium compared to the untreated control group (Control) and a positive control (CS) nodatum group; Marvinbryantia ; Oscillospira ; The amount of microorganisms in the four genus Gelria was significantly changed, compared with the untreated control group (Control), Parabacteroides ; Gastranaerophilales ; The amount of microorganisms in the genus NB1-n was significantly changed, and the amount of microorganisms in the genus of Christensenellaceae R-7 group was significantly changed compared to the positive control (CS). Most of the microorganisms whose amount was significantly changed showed a tendency that the amount of microorganisms decreased as the dried leaves of three leaves were fed, and the strains were identified as pathogenic strains in specific species.

< Experimental example 3> Broiler productivity and intestines Microbial amount Correlation analysis

It was intended to analyze the correlation between the productivity of broilers fed with the three leaves or root dry powder prepared in Example 1 and the amount of microorganisms in the intestine.

Intestinal microflora, which shows a significant correlation with broiler productivity, was expressed by the Spearman correlation coefficient, and the productivity of broilers used three items: body weight, tibia strength, and IBD (infectious bursal disease) antibody production indicating the health of broilers. The amount of microorganisms having a significant correlation was visualized through a heat map.

As a result, intestinal microorganisms showing a significant correlation with broiler productivity were discovered. Among the microorganisms related to body weight, Bacillus has been found to be related to feed efficiency in previous studies, and Clostridium sensu stricto 1 belongs to Clostridiaceae There is also a known association with weight gain. In addition, Lachnoclostridium and Ruminococcaceae, which are known to be associated with immune function against IBD virus. The V9D2013 group showed a strong correlation with IBD antibody production in broilers, and Parasutterella is known to show a strong positive correlation with bone strength and IBD antibody production.

Looking at the correlation coefficient between the found intestinal microbes and the productivity of broilers and the amount of microbes, the microbes that have a positive correlation with the productivity of broilers were found to be relatively abundant in the experimental group that consumed the dried three leaves powder. appear. On the other hand, it was found that microorganisms having a negative correlation with the productivity of broilers were present in a relatively small amount in the experimental group that ingested the dry powder of three leaves (FIGS. 4 to 6). Specifically, when feeding dried three leaves powder at 0.5% of the broiler diet, the amount of intestinal microorganisms Eggerthella genus, Christensenellaceae family, Familly XIII AD3011 group genus, and Clostridium genus that are positively correlated with body weight of broiler chickens increased. (Fig. 4). In addition, the amount of the intestinal microorganism Parasutterella genus, which is positively correlated with the tibia strength of broilers, increased (Fig. 5), and the intestinal microorganisms Eubacterium genus, Lachnospiraceae genus, Parasutterella genus, and Ruminococcaceae positively correlated with IBD antibody production in broiler chickens. The amount in the V9D2013 group increased (Fig. 6). That is, the above results show that the three leaf dry powder can increase the microorganisms that have a positive correlation with the productivity of broiler chickens.

< Experimental example 4> Three Functions of microorganisms in the broiler intestine changed depending on dry powder feeding

By feeding the three leaf root dry powder (0.5%) prepared in Example 1, the function of microorganisms in the broiler intestine was predicted, and statistically analyzed how different the predicted function was compared to the untreated control group (Control).

Specifically, the function of microorganisms was predicted using the Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) program, and at this time, information picked out of the Greengene database was used. The predicted function was compared with the untreated control group (Control) through the Wilcoxon rank sum test and the three leaf dry powder test group (Leaf 0.5) fed 0.5% of the broiler diet.

As a result of dividing the enhanced or degraded function in the experimental group (Leaf 0.5) fed three leaf dry powder compared to the untreated control group (Control), the function related to metabolism was fed three leaf dry powder (0.5%). It was predicted more in one experimental group, and among them, the function related to carbohydrate metabolism was found to be enhanced compared to the untreated control group (FIG. 7). This provides a clue to understanding the biological mechanisms behind broiler weight gain by feeding dry three leaf powder. On the other hand, metabolic functions related to amino acids such as glutamine, alanine, and lysine were shown to be decreased compared to the untreated control group.

< Experimental example 5> Three Evaluation of the productivity of broiler chickens according to dry powder feeding

Three leaf dry powder was fed to broilers to analyze their effects on body weight, tibial strength, blood biochemical properties, and the production of humoral antibodies after live venom vaccination.

<5-1> Experimental animals and breeding management

First, for the broiler used in the experiment, 1,200 male broilers (Arbor Acres, male) of 1 day old were purchased and reared for 35 days in the early stage of broiler (0-3 weeks of age) and late (3-5 weeks of age). Untreated control group (Control), positive control group fed with commercial feed additives (CS), experimental group fed three leaf dry powder to be 0.3% or 0.5% of broiler diet (leaf 0.3 and leaf 0.5, respectively), three root dry powder Was divided into six experimental groups fed to 0.3% or 0.5% of the broiler diet (root 0.3 and root 0.5, respectively), and divided into 4 repetitions per treatment group and 50 numbers per repetition. As for the basic feed, corn-soybean meal-based broiler feed was divided into early (CP (crude protein) 22%, ME (calories) 3,100 kcal/kg) and late (CP 20%, ME 3,150 kcal/kg). . Feed and drinking water were fed freely, and the lighting time was continuously lit for 24 hours. During the test period, the average temperature in the cage was adjusted to 34°C at the time of entry, and the temperature was gradually lowered to 24°C at the age of 5 weeks, and the humidity in the cage was maintained at about 70% in the first half and 60% in the later stages. Other feeding management was carried out in accordance with the procedure of approval (NAAS201707) of the Animal Experimental Ethics Committee of the National Academy of Agricultural Sciences of the Rural Development Administration.

<5-2> weight

The body weight of each experimental animal was measured every week at a certain time.

As a result, all three dry powder intake groups (Leaf 0.3, Leaf 0.5, Root 0.3, and Root 0.5) had significantly higher body weight by 7 to 15% compared to the control and positive control groups, and the dry roots in the dry three leaf powder intake group The body weight was significantly increased at the level of 4 to 8% compared to the powder intake group. Three dried powder, especially three leaf dried powder, was found to have an effect of increasing productivity by helping growth in broilers during the growing season (FIG. 8).

<5-3> Tibia strength

On the 35th day of the end of the test, 12 experimental animals close to the average weight per each experimental group were selected, and the left tibia was separated from the selected experimental animals, and the tibia fracture strength was measured. The tibial fracture strength was 50 mm/min cross-head speed using Instron (Model 3342, Inatron Universal Testing Machine, Instron Corp., USA) equipped with a load cell in the range of 50 kg. ) To measure the fracture strength at the center of the tibia by placing the tibia on a support at 3.35 cm intervals.

As a result, it was found that the growth of the three dried powder intake group improved the tibia strength of broilers as a whole compared to the control group and the positive control group. The three leaf dry powder intake group (Leaf 0.3, Leaf 0.5) not only improved the tibia strength by 7 to 16% compared to the control group and the positive control group, but also improved by 5 to 15% compared to the group ingesting the dry root powder. The above results show that the dry powder of three leaves increases the strength of the tibia in growing broilers, thereby affecting the overall growth and productivity improvement of broilers (FIG. 9).

<5-4> Blood biochemical properties

On the 35th day after the end of the test, 12 experimental animals close to the average weight per each experimental group were selected, the cervical spine of the selected experimental animals was dislocated, and blood was collected from the heart. The collected blood was centrifuged at 1,500 rpm for 15 minutes to obtain serum. Serum albumin, alkaline phosphatase, alanine aminotransferase, calcium (Ca), cholesterol, gamma glutamyltransferase, and globulin , Glucose, phosphorus, total bilirubin, total protein, and albumin/globulin items (Chem 15 CLIP, IDEXX Lab, USA) using an automatic blood analyzer (COBAS MIRA plus, ROCHE diagnostics). , USA).

As a result, there was no significant difference in the biochemical characteristics of blood between the control group and the three dried powder intake group. Three dry powders were found to have no health problems such as liver or kidney when used as a feed additive for 5 weeks (Table 1).

Experimental group Measurement item Control CS Leaf 0.3 Leaf 0.5 Root 0.3 Root 0.5 albumin
(g/dL) 1.12±0.03 ^ns 1.16±0.05 1.10±0.04 1.18±0.06 1.08±0.03 1.10±0.03 Alkaline dephosphorylation enzyme (U/L) 1015±51 ^ns 984±148 1164±157 1169±169 1179±155 1231±137 Alanine aminotransferase (U/L) 14.8±2.0 ^ns 14.9±1.2 11.8±0.8 15.4±2.1 11.8±0.8 13.4±1.2 calcium
(mg/dL) 9.5±0.5 ^ns 9.2±0.3 9.1±0.3 9.3±0.3 8.8±0.2 9.2±0.1 cholesterol
(mg/dL) 81.4±5.1 ^ns 81.8±7.8 89.2±6.5 93.8±7.1 93.5±6.7 78.6±4.9 Gamma-glutamyl transferase (U/L) 15.6±2.0 ^ns 14.3±1.2 13.3±1.3 13.1±0.9 15.8±1.0 13.0±1.9 globulin
(g/dL) 1.6±0.1 ^ns 1.8±0.2 1.5±0.1 1.8±0.1 1.5±0.1 1.7±0.1 Glucose
(mg/dL) 230.1±6.4 ^ns 230.7±4.1 226.2±10.5 247.5±9.5 236.0±9.8 235.5±8.8 sign
(mg/dL) 6.3±0.3 ^ns 6.8±0.3 6.1±0.2 6.9±0.7 6.3±0.2 5.8±0.2 Total bilirubin
(g/dL) 0.21±0.03 ^ns 0.33±0.06 0.25±0.06 0.33±0.06 0.21±0.03 0.26±0.06 Total protein
(g/dL) 2.8±0.1 ^ns 3.0±0.2 2.6±0.1 3.0±0.1 2.6±0.1 2.8±0.1 Albumin/Globulin 0.66±0.04 ^ns 0.65±0.03 0.73±0.03 0.67±0.01 0.70±0.02 0.68±0.03

<5-5> immune response

The humoral immune response was analyzed through the production of humoral antibodies after live venom vaccination. The amount of antibody production was analyzed based on the method of Gelb et al. The amount of IBD (infectious bursal disease) antibody production was measured.

As a result, it was found that the IBD antibody titer was 4 to 10 times higher in the experimental group (Leaf 0.3 and Leaf 0.5) ingesting the dried three leaves powder than the control group or in the group ingesting the dried three root powder. It is shown that the dried powder of three leaves can be helpful in preventing infectious diseases by increasing the production of IBD antibodies in broilers after live single vaccine (FIG. 10).

Claims (7)

Composition for improving the intestinal flora of poultry that increases the intestinal microflora positively correlated with any one or more selected from the group consisting of weight, tibia strength, and immunity of poultry containing powder of allium hookeri leaves as an active ingredient as,
The intestinal microorganisms positively correlated with the weight of the poultry are the genus Eggerthella (genus), the Christensenellaceae family (family), the Familly XIII AD3011 group genus, or the Clostridium genus,
The intestinal microbes that have a positive correlation with the tibia strength of the poultry are of the genus Parasutterella ,
The intestinal microorganisms that are positively correlated with the immunity of the poultry are Eubacterium genus, Lachnospiraceae UCG-004 genus, Parasutterella genus or Ruminococcaceae V9D2013 group genus, a composition for improving the intestinal flora of poultry.
The method of claim 1,
The powder is a composition for improving intestinal flora of poultry prepared by hot air drying and powdering three leaves.
delete delete delete delete The method of claim 1,
The powder of the three leaves is a composition for improving intestinal flora that is added in an amount of 0.4 to 1.0% by weight relative to the total poultry feed.

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