KR20190097502A - animal feed additive for immune activity and animal feed comprising the same - Google Patents

Abstract

The present invention relates to a feed additive for enhancing immunity and a formula feed comprising the same. According to the present invention, the feed additive is manufactured, which comprises a culture prepared by treating K. marxianus yeast (SCHY-1) and S. cerevisiae yeast (SCHY-21), which are isolated from natural environment, under autoclave or with a papain enzyme to yield yeast autolysates and by inoculating a starter strain (SCHY-1+SCHY21) into the yeast autolysates. When fed with formula feed into broiler, dairy cattle and pigs, the feed additive is demonstrated to enhance innate immunity response such as an increase in leukocyte and humoral immune response through the production increase of cytokine IL-2 and immunoglobulin IgG. In addition, formula feed containing the feed additive is demonstrated to significantly increase the number of Lactobacillus among intestinal microorganism of the livestock, and in particular, significantly increase the number of L. farciminis in both dairy cattle and pigs. It is known that the feed additive of the present invention contributes to propagation of beneficial intestinal bacteria in the livestock as well as enhances immunity. Therefore, the feed additive using wild yeast well adapted to natural environments to have excellent growth is expected to be highly applicable to livestock industry.

Description

Animal feed additive for immune activity and animal feed comprising the same}

The present invention relates to a feed additive for immuno-enhancing and a feed composition comprising the same, and more particularly, a feed additive comprising a yeast culture having an immune improvement and enhancement effect on livestock, a feed composition comprising the same, and a method for preparing the same. It is about.

The World Health Organization (WHO) has reported a medical relationship between the use of growth-promoting antibiotics in feeds and human resistance rates based on pathology (WHO, 2000), prohibiting the addition of antibiotics in feeds, It has been reported that an agency should be established to oversee the work. Therefore, since the Korean government is gradually tightening the ban on the use of formulated feed for all antibiotics since July 2011, it is required to develop antibiotic substitutes that can maximize the capacity of livestock without resistance or residual problems unlike antibiotics. It is becoming.

Therefore, as an alternative, various yeast products have been released. For example, the microorganisms are added to the feed to maintain the intestinal flora of the livestock in favor of the host animal or inhibit the growth of pathogenic E. coli, mutagenesis, inhibit carcinogens and enhance immune response, digestibility of nutrients ingested and livestock productivity Has the advantage of improving.

To date, a mutant strain obtained by treating NTG with aspergillus tereus, a yeast fungus capable of mass production of lovastatin, a drug that inhibits blood cholesterol levels, is prepared in solid culture, and it is added to pig and milk feed to blood and meat. Inventions are known which have significantly lowered cholesterol. However, since this is a variant strain rather than using the wild bacteria itself, there is a problem that the effect is reduced as the culture process goes through, there is a problem that the process is complicated and does not obtain a sufficient effect (Patent Document 1. Republic of Korea Patent No. 10-0775134).

In addition, various microorganisms have been developed, but when the microorganism is applied to other fields in addition to the application field according to its characteristics, it is more harmful, and even if it is applied, the cost increases and it is inconvenient to use it.

Recently, in order to solve the above problems, there have been examples of using yeast autolysates having bio components dried through self-extinguishing process, which have been traditionally presented as additives for food and animal feed because of their excellent nutritional properties. However, there are limitations in practical application because they are not significantly improved compared to raw feed in terms of growth and feed efficiency or are expensive in terms of price, and there is a problem of unstable supply of materials, supply of microorganisms for them, and enormous costs for production of yeast autolysates. There is a disadvantage in which this occurs.

Therefore, while solving the above problems, while trying to develop prebiotics that are applicable to various livestock and differentiated from simple active yeasts, the present invention has been completed.

Therefore, the present invention has been made in view of the above problems, an object of the present invention is to improve the problems of feed containing conventional yeast and feed additives comprising a yeast culture to enhance the immune activity and combination comprising the same It is to provide a feed.

The present invention comprises a yeast culture inoculated and cultured with a second yeast in a solid medium containing a yeast autolysate to achieve the above object, wherein the yeast culture is 0.1 to 0.5% by weight in livestock feed based on solids It provides an immune enhancing feed additive, characterized in that the addition.

On the basis of 1 part by weight of the feed additive, Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia kudriavzevii may include one or more parts by weight of one or more raw yeasts.

1 part by weight of the yeast autolysate may be further included based on 1 part by weight of the feed additive.

The yeast autolysate may be prepared by autoclaving the first yeast or by treating the hydrolase. The hydrolase may be a papain enzyme.

The first yeast may be any one or more selected from Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia kudriavzevii .

The second yeast is the same or different from each other , and may be any one or more selected from Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia kudriavzevii .

The present invention provides a compound feed comprising the feed additive for immuno-enhancing to achieve the above object.

The present invention provides a method for producing an immune enhancing feed additive comprising the following steps to achieve the above object.

(Iii) treating the first yeast with autoclave or hydrolase to produce a yeast autolysate; And

(Ii) inoculating and incubating a second yeast in a solid medium containing the yeast autolysate to obtain a yeast culture.

The feed additive for immuno-enhancer according to the present invention can reduce the substrate required for fermentation by improving the digestibility of the nutrients ingested, thereby suppressing the generation of harmful gas produced in the feces. Therefore, feed of yeast feed additives helps to reduce the odor that is a problem in livestock raising. In addition, when feeding the feed for immunosupplement according to the present invention, it is reported that it promotes the growth of livestock, has a positive effect on the composition of intestinal microflora and is effective in reducing the occurrence of odor generated from feces.

Figure 1 shows in detail the place where the sample is taken.
Figure 2 shows the results of analyzing the number of genes analyzed based on the 18S rDNA nucleotide sequence for 44 yeasts isolated from Experimental Example 1. The phylogenetic tree was generated by the neighbor-joining method using MEGA 7, with numbers above or below the branch representing the bootstrap support values.
3 is a growth rate curve for 15 yeasts identified in Example 2 of 2). SCHY-1; Kluyveromyces marxianus SCHY-1, SCHY-21; Saccharomyces cerevisiae SCHY-21, SCHY-82; Pichia kudriavzevii SCHY-82, SCHY-155; P. kudriavzevii SCHY-155, SCHY-159, Meyerozyma caribbica SCHY-159, SCHY-161; S. cerevisiae SCHY-161, SCHY-164; M. caribbica SCHY-164, SCHY-165; P. kudriavzevii SCHY-165, SCHY-167; S. cerevisiae SCHY-167, SCHY-169; Eremothecium coryli SCHY-169, SCHY-170; S. cerevisiae SCHY-170, SCHY-171; W. anomalus SCHY-171, SCHY-172; Candida albicans SCHY-172, SCHY-178; P. kudriavzevii SCHY-178 and SCHY-186; S. cerevisiae SCHY-186.
4 is a SEM photograph of five yeasts among the selected yeasts. 4A and 4B show wild yeast strain SCHY-1, 4C and D show SCHY-161, 4E and F show SCHY-165, and 4G and H show SCHY-171. J is SCHY-21. SEM pictures of A, C, E, G and I are taken at x 10000 magnification, and SEM pictures of B, D, F, H and J are taken at x 1000 magnification.
5 is a graph showing the yeast autolyzate (Examples 3 to 16) prepared with yeast SCHY-1 and SCHY-21 measured at 260 nm.
6 is a graph showing the yeast autolyzate (Examples 3 to 16) prepared with yeast SCHY-1 and SCHY-21 measured at 280 nm.
7 is an SEM photograph of K. marxianus SCHY-1 yeast. 7A and C are enlarged at ㅧ 1000 magnification, and FIGS. 7B and D are enlarged pictures at ㅧ 10,000 magnification.
8 is a SEM photograph of the yeast autolysate obtained by treatment of K. marxianus SCHY-1 yeast by autoclaving. 8A and C are enlarged at ㅧ 1000 magnification, and FIGS. 8B and D are enlarged pictures at ㅧ 10,000 magnification.
9 is a SEM photograph of the yeast autolysate obtained by treating K. marxianus SCHY-1 yeast with papain enzyme. 9A, C, and E are magnified at ㅧ 1000 magnification, and FIGS. 9B, D, and F are photographs enlarged at ㅧ 10,000 magnification.
10 is a graph showing the concentration (ppm) of β-glucan and galactomannan of yeast autolysates prepared according to Examples 3, 5, 7, 9, 11, 13, and 15.
11 is a graph showing the measurement of the protease, α-amylase enzyme activity (U / g) of the yeast autolysate prepared according to Examples 3, 5, 7, 9, 11, 13, 15.
12 is a graph showing the analysis of the gas composition in the powder of the control group (CON), the first experimental group (TRT1) and the second experimental group (TRT2).
FIG. 13A is a chart of the initial control group and the first experimental group (cont-i, T1-i), FIG. 13B is a chart of the control group and the first experimental group (cont-14d, T1-14d) after 2 weeks, 13C is a chart for the control group and the first experimental group (cont-28d, T1-28d) after 4 weeks.

Hereinafter, the present invention will be described in detail.

The present invention is to provide a feed additive applicable to a variety of fields, such as feed for the growth and production of livestock, such as chickens, cattle, pigs, etc. by producing a yeast culture having immuno-enhancing activity, and having such characteristics and advantages Finding microorganisms should be a priority.

Therefore, the present inventors, through numerous repeated experiments, not only has excellent growth rate, but also promotes the growth of beneficial microorganisms in the intestines, increases the quality and quantity of products obtainable from livestock, and improves the immune activity of livestock. I found it. Specifically, by differentiating from the simple active yeast called conventional yeast (Live Yeast), the yeast that optimally generates the beta glucan (β-glucan) and the mannan (animal bioactive material) to livestock environment (chicken farm, pig farm) And yeast) and liquid cultures of these yeasts, followed by a manufacturing process that produces optimum β-glucan / mannan, protease, and α-amylase through a yeast autolysis process. A system capable of mass production of "cultures" was established.

Feed additives containing the above-mentioned yeast culture confirm the possibility of production of new biotic yeast culture (Yeast Culture) non-antibiotic environment-friendly livestock products through broiler and dairy specification test, and changes in the intestinal microflora of broiler, weaning pig and cow It has been confirmed that through the immunity in many ways.

In the present invention, the livestock may be poultry or ruminant, but is not limited thereto, and the poultry may include all poultry that spawn edible eggs such as chicken, duck, quail, turkey or ostrich, and preferably include all of them. Can be broiler. In addition, the ruminant includes cows, cows, horses, goats, sheep, pigs, etc., preferably cows, pigs, most preferably cows.

The feed additive for immune augmentation according to the present invention induces autolysis of a high molecular compound, such as β-glucan / mannan, which is not easily absorbed in an animal, thereby degrading the yeast autolysate into a low molecular compound that is easily absorbed in the body. It is obtained by inoculating and incubating the yeast through the solid phase culture technique as a raw material for the culture, and by developing a yeast culture having an immune enhancing effect, it will have the effect of preventing disease of the livestock.

Immunity-enhancing feed additives according to the present invention has a merit of being able to be used in the field of eco-friendly organic livestock products because it has a remarkably excellent immuno-enhancing effect despite being an antibiotic.

One aspect of the present invention comprises a yeast culture inoculated and cultured with a second yeast in a solid medium containing a yeast autolysate, wherein the yeast culture is added to the animal feed 0.1 to 0.5% by weight based on the solid content It relates to an immune enhancing feed additive.

In addition, when preparing the feed enhancer for immuno-immunity according to the present invention (i) autoclaving the first yeast, or treating with a hydrolytic enzyme to produce a yeast autolysate; And (ii) inoculating and culturing a second yeast in a solid medium comprising the yeast autolysate to obtain a yeast culture.

First, in order to prepare a yeast autolysate, the first yeast is autoclaved or hydrolyzed, specifically, when the autoclave is treated, the first yeast is cultured through a liquid medium, and the culture solution thereof is centrifuged and After drying to obtain a yeast construct, the construct may be autolyzed through autoclaving for 100 to 150 ° C. for 10 to 30 minutes to obtain a yeast autolysate.

In the case of treating papain enzyme, the first yeast is cultured through a liquid medium, and the culture solution is centrifuged and dried to obtain a yeast construct, and then 1 to 3 units / mg hydrolase is added to the construct. It can incubate for 1 to 30 hours at 70 DEG C and autolyze to obtain a yeast autolysate. Most preferably, the hydrolase is papain enzyme.

The liquid medium can be used without limitation so long as it can be used to culture the first yeast. The hydrolase, preferably papain enzyme, inoculated into the yeast construct may be inoculated at 1-10% by weight based on the total medium, but is not limited thereto.

Then, the yeast culture can be prepared by inoculating and incubating the second yeast in a solid medium containing the yeast autolysate. The solid medium can be used without limitation as long as the medium can be used to culture the second yeast, including the yeast autolysate, specifically 1 to 10 parts by weight oat meal, wheat gluten 1 to 10 based on 1 part by weight of water It is preferable that the medium contains a weight part, dry yeast 0.5 to 5 parts by weight, CSL (corn steep liquor) and molasses 0.1 to 1 parts by weight, salt and minerals 0.1 to 0.5 parts by weight, yeast autolysate 0.1 to 0.5 parts by weight, This is not restrictive. The content of components in the medium can be appropriately adjusted by those skilled in the art. In addition, a medium sterilized at a high temperature may be preferable to suppress the growth of various bacteria.

The yeast autolysate contained in the solid medium may be inoculated at 5 to 20% by weight based on the total medium, but is not limited thereto.

The first yeast and the second yeast inoculated into the solid medium may be inoculated in an amount of 1-5% by weight with respect to the whole medium, but are not limited thereto, and the first and second yeasts are the same as each other or different from each other. Strains can be used. In addition, the first and second yeast may use a conventionally known strain, and particularly preferably may be any one selected from Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia kudriavzevii . Preferably yeasts obtained from poultry, pigs and barn, yeasts that can be used in the present invention is usually well propagated at a weak acidity of pH 4.0-6.5, the optimal development temperature may be 25 ℃ -35 ℃. More preferably, the first and second yeast may be any one selected from Kluyveromyces marxianus strain TB7258, Saccharomyces cerevisiae CBS: 6282 and Saccharomyces cerevisiae strain ATCC4098.

Most preferably, the first yeast may be a mixture of Kluyveromyces marxianus strain TB7258, and the second yeast may be a mixture of Kluyveromyces marxianus strain TB7258 and Saccharomyces cerevisiae strain ATCC4098.

Specifically, the yeast culture is preferably a solid phase culture containing 5 x 10 ⁷ to 1 x 10 ⁹ cfu / g of the second yeast.

In addition, based on 1 part by weight of the yeast culture, Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia kudriavzevii may further include any one or more parts by weight of the yeast.

Based on 1 part by weight of the yeast culture, the first yeast may be treated by autoclaving or papain enzyme may be used to prepare yeast autolysate, and may further include 1 part by weight of the yeast autolysate. The yeast autolysate added at this time can increase the absorption of nutrients in the body and at the same time improve immunity, thereby compensating the effect of preventing death from harmful environment and disease. However, although the microorganism other than the yeast of the present invention may be used, in order not to be harmful to other yeast used in the feed additive, it is preferable to use the same yeast as the first yeast described above.

(Iii) lyophilizing the yeast culture may be prepared as a powder.

Feed additive according to the present invention can provide a salary method characterized in that the feed to livestock by blending 0.1 to 0.5% by weight based on the blended feed, the salary may be performed 1 to 10 times a day, It is not limited to this. It can be adjusted appropriately according to the nutritional status of the livestock.

Another aspect of the present invention relates to an animal feed comprising an immuno-enhancing feed additive, wherein the feed additive may be included in an amount of 0.1 to 0.5% by weight, and preferably 0.2% by weight, based on the total feed. . If the feed additive is less than 0.1% by weight based on the total blended feed may cause a problem that the odor reduction effect of the feces, including the immune enhancing effect is not expressed.

In the case of feeding the immune-enhancing feed additive prepared in accordance with the present invention or a compound feed added thereto, the effect of reducing the odor of feces by improving the nutrient digestibility of livestock to suppress the generation of harmful gas (ammonia) in the intestine It was confirmed to have.

In addition, when feeding the feed for the immune-enhanced feed additive prepared in accordance with the present invention or a combination feed added thereto, it has a positive effect on the microorganisms present in the intestine of the livestock, specifically Lactobacillus increases, E. coli decreases It can be seen.

In addition, the feed for additives prepared in accordance with the present invention or the feed compound added thereto does not affect diarrhea and mortality, and does not show toxicity to organs or meat, but rather the quality of milk produced when applied to cows It was confirmed that the amount increased.

In other words, it was confirmed that the feed additive for immunosupplementation prepared in accordance with the present invention or the compounded feed containing the same has other additional effects at the same time. Therefore, it can be seen that the feed additive for immunological enhancement of the present invention or the compound feed added thereto can increase the economic income of the farm.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the scope of the present invention is not limited by the following examples, and those skilled in the art to which the present invention pertains should be within the equivalent scope of the technical concept of the present invention and the claims to be described below. Of course, various modifications and variations are possible.

Example 1: Yeast Isolation and Identification

1) Sample Collection

In order to isolate wild yeasts, samples were collected from various areas around the livestock environment of Chungnam, and livestock houses and zoos in Gyeonggi, Chungbuk, Gangwon, Jeonnam, and Gwangju (Gyeongmyeong, Gyeongsangbuk-do, Nonsan, Asan, Chungnam) , Taean, Seosan, Hongseong, Buyeo and Boryeong). The specific area is shown in Figure 1 below.

Figure 1 shows in detail the place where the sample is taken. 1A shows a sampling place on a national map, and FIG. 1B shows a sampling place on a map of Chungcheongnam-do. The black circles (A to J) in the figure represent the first year (poultry farm and brewery environment) sampling points, and the white circles (K to U) represent the second year (pig and barn environment) sampling points. Specifically, A, Gwangmyeong-si, Gyeonggi-do; B, Taean-gun, C. N .; C, Seosan-si, C. N .; D, Yesan-gun, C. N .; E, Hongseong-gun, C. N .; F, Asan-si, C. N .; G, Boryeong-si, C. N .; H, Buyeo-gun, C. N .; I, Nonsan-si, C. N .; J, Cheongju-si, Chungcheongbuk-do ,; K, Yesan-gun, C. N .; L, Hongseong-gun, C. N .; M, Dangjin-si, C. N .; N, Cheonan-si, C. N .; O, Cheonan-si, C. N .; P, Asan-si, C. N .; Q, Gokseong-gun, Jeollanam-do .; R, Gwangju metropolitan city .; S, Yeongwol, Kangwon-do ,; T, Nonsan-si, C.N. U, Nonsan-si, C.N. C.N. stands for Chungcheongnam-do

2) Isolation of bacteria from the sample

Specifically, wild yeast was to be separated at the sampling area in 1), and samples were collected over a total of two years. 85 samples from poultry farms, 28 samples from barns, 7 from pig houses, 6 from cattle guts, 15 from pig organs, and 25 samples from poultry farms and zoos. A total of 197 samples were collected from 11 crests and grains of livestock, 2 from mushrooms, 9 from drinks and food, and 9 from rice wine. The bacteria isolated from each sample are shown in Table 1 below.

division Sampling location Sample collection time Number of bacteria isolated 1st year
(2016.8-9) 2nd year
(2017.3 ~ 4) One Poultry farm (faces, feed, soil etc) 85 0 85 2 porcine guts (colon, cecum, etc) 0 15 15 3 Swine farm (faces, feed, soil etc) 0 7 7 4 bovine guts (colon, cecum, etc) 0 6 6 5 Cow farm (faces, feed, soil etc) 13 15 28 6 Grains (bean, rice, etc) 0 11 11 7 Mushroom (Ganoderma mushroom, Tibetan Mushrooms) 2 0 2 8 Food (sake, baker) 9 0 9 9 Brewery (Makgeolli) 8 One 9 10 Other animal feces 20 5 25 Sum 148 19 197

3) Yeast Separation Selection  selection

Yeast Peptone Dextrose (YPD) agar and Yeast Mold (YM) agar were selected to select yeast strains from various samples. Specifically, after diluting the sample to 10% with distilled water (DW), aliquots were dispensed by spreading 100 μl, and incubated at 30 ° C. for 48 hours. Feces in the barn samples contain a lot of Candida spp. Thus, excluding this, S. cerevisiae To isolate the strain, the alcohol resistance of S. cerevisiae was determined. That is, ethanol addition medium is used to separate only yeast S. cerevisiae from the bacteria isolated in 2). Specifically, ethanol (Daejung) in YM broth (Becton Dickinson, USA), YM agar (BD, USA) medium , Korea) 5%, 10% 15% was added to the ethanol medium prepared by adding 3%, 7% ethanol (Daejung, Korea) to YPD (BD, USA) medium.

Finally, the ethanol was added medium is effectively S. cerevisiae In order to confirm that the isolates were isolated , C. albicans and S. cerevisiae mixed strains were cultured at 30 ° C. for 48 hours to observe whether S. cerevisiae was selected.

4) Yeast Separation

In order to separate the yeast, a plate medium containing YM agar, 10% ethanol and penicillin-streptomycin was used as described in 3) above. After diluting 1 g of each sample collected from step 1) with 10 ml of distilled water, 100 μl of the sample was dispensed into the prepared plate medium and incubated for 48 hours in an incubator at 30 ° C.

After the incubation was completed, each colony formed in the medium was observed to isolate a strain that is assumed to be yeast while having a unique morphological phenotype. The selected bacteria were separated purely through two passages.

Nine colonies were isolated from 85 samples, including feces, soil, rice straw, and feed in poultry farms. Seven and 28 samples were taken from pig and barn, respectively, and two and six colonies were separated. Other poultry farms including ducks and pheasants. Eleven colonies were isolated from 25 samples, including goats and ostriches in the zoo. In addition, 10 colonies were isolated from 11 grains, and 9 colonies were separated from 9 kinds of food such as 1 colony and fermented bread from 5 kinds of mushrooms and 9 types of makgeolli in the brewery. As a result, a total of 53 yeast colonies were isolated from the final 197 samples, which are summarized in Table 2 below.

division Sampling location Total samples Number of yeasts isolated from the sample One Poultry farm 85 9 2 Porcine guts 15 0 3 Swine farm 7 2 4 bovine guts 6 0 5 Cow farm 28 6 6 Grain 11 10 7 Mushroom 2 One 8 Food 9 5 9 Makgeolli 9 9 10 Other animal feces 25 11 total 197 53

5) Identification of isolated wild yeast

A total of 53 wild yeast 18S rRNAs isolated through step 4) were subjected to PCR (Polymerase Chain Reaction) by primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3 ') and ITS4 (5'-TCCTCCGCTTATT GATATGC-3'). ) And electrophoresis, followed by sequencing by obtaining 18S RNA sequence by ethanol precipitation and searching through NCBI BLAST search (National Center for Biotechnology Information Basic Local Alignment Search Tool) Each wild yeast was identified.

As a result, 44 out of 53 yeasts were identified, which are summarized in Table 3. As shown in Table 3, Kluyveromyces marxianus was isolated from Ganoderma lucidum mushroom, and Saccharomyces cerevisiae YJM1592 was isolated from Makkoli. It was confirmed that S. cerevisiae of different strains was isolated from wine, sake and baker, and Pichia kudriavzevii was mainly isolated from animal feces such as poultry, poultry, cattle and pigs. In addition to S. cerevisiae , Meyerozyma caribbica, Eremothecium coryli and Wickerhamomyces anomalus were isolated from animal feed and grains. In addition, 44 species of yeast isolated and identified from the present experimental example were identified as Texa analyses through the MEGA 7 program, and the results are shown in FIG. 2.

division Isolated and Identified Yeast Names Sampling location Estimated Type
(Putative species) Related Gene Bank Sequences
(Related Genebank sequence) Homology
(Identity) (%) One SCHY-1 Ganoderma lucidum Kluyveromyces marxianus strain TB7258 JQ425345.1 696/697 (99%) 2 SCHY-2 Jangsoo (Draft) Makkoli Saccharomyces cerevisiae YJM1592 CP006433.1 807/807 (100%) 3 SCHY-6 Chicken feces Pichia kudriavzevii strain AUMC10190 KU095862.1 493/493 (100%) 4 SCHY-8 Chicken feces Pichia kudriavzevii strain AUMC10288 KX218263.1 491/491 (100%) 5 SCHY-9 Pool water Candida glabrata strain M356B KP675589.1 844/845 (99%) 6 SCHY-14 Poultry feces Tilletia ayresii strain CBS482.91 KP322985.1 511/511 (100%) 7 SCHY-15 Poultry feces Pichia kudriavzevii strain AUMC10288 KX218263.1 495/495 (100%) 8 SCHY-17 Sake Saccharomyces cerevisiae strain P1 KU131578.1 818/818 (100%) 9 SCHY-19 Baker Saccharomyces cerevisiae strain P1 KU131578.1 776/777 (99%) 10 SCHY-20 Wine Saccharomyces cerevisiae strain ATCC4098 KU729087.1 817/817 (100%) 11 SCHY-21 Wine Saccharomyces cerevisiae strain ATCC4098 KU729087.1 811/813 (99%) 12 SCHY-24 Sake Saccharomyces cerevisiae strain P1 KU131578.1 786/786 (100%) 13 SCHY-35 Chicken feces Pichia kudriavzevii strain ATCC14243 KU729098.1 500/500 (100%) 14 SCHY-59 Chicken feces Pichia kudriavzevii strain ATCC14243 KU729098.1 493/493 (100%) 15 SCHY-69 Bovine feces Pichia manshurica strain PMM10-206L KP132515.1 451/451 (100%) 16 SCHY-120 Chicken feces Pichia kudriavzevii strain ATCC 14243 KU729098.1 492/492 (100%) 17 SCHY-123 Chicken feces Pichia kudriavzevii strain PMM10-138L KP132510.1 494/494 (100%) 18 SCHY-124 Duck feces Pichia kudriavzevii strain AUMC10288 KX218263.1 493/493 (100%) 19 SCHY-125 Duck feces Pichia kudriavzevii strain AUMC10288 KX218263.1 494/494 (100%) 20 SCHY-127 Pheasant feces Pichia kudriavzevii strain AUMC10288 KX218263.1 493/493 (100%) 21 SCHY-128 Pheasant feces Pichia kudriavzevii strain AUMC10288 KX218263.1 491/491 (100%) 22 SCHY-141 Asan Rice Draught Makkoli Saccharomyces cerevisiae YJM1592 CP006433.1 792/796 (99%) 23 SCHY-142 Jangsoo (Draft) Makkoli Saccharomyces cerevisiae YJM1592 CP006433.1 801/801 (100%) 24 SCHY-143 Rice Draught Makkoli Saccharomyces cerevisiae YJM1592 CP006433.1 787/792 (99%) 25 SCHY-82 Porcine feed Pichia kudriavzevii CBS: 5590 KY104596.1 511/512 (99%) 26 SCHY-155 Bovine feces Pichia kudriavzevii strain AUMC10288 KX218263.1 489/489 (100%) 27 SCHY-159 Bovine feed Meyerozyma caribbica CBS: 2022 KY104217.1 584/585 (99%) 28 SCHY-161 Bovine feces Saccharomyces cerevisiae strain GITA14 KY630581.1 815/815 (100%) 29 SCHY-164 Bovine feces Meyerozyma caribbica CBS: 11118 KY104224.1 591/591 (100%) 30 SCHY-165 Feed grains Pichia kudriavzevii CBS: 6799 KY104583.1 504/504 (100%) 31 SCHY-167 Feed grains Saccharomyces cerevisiae strain GITA14 KY630581.1 813/813 (100%) 32 SCHY-168 Feed grains Saccharomyces cerevisiae strain GITA14 KY630581.1 807/807 (100%) 33 SCHY-169 Feed grains Eremothecium coryli CBS: 5749 KY103389.1 648/649 (99%) 34 SCHY-170 Feed grains Saccharomyces cerevisiae strain GITA14 KY630581.1 824/824 (100%) 35 SCHY-171 Feed grains Wickerhamomyces anomalus strain P42B001 JX188245.1 600/600 (100%) 36 SCHY-172 Ostrich feces Candida albicans strain CBS: 6426 KY101918.1 521/521 (100%) 37 SCHY-178 Bovine feces Pichia kudriavzevii CBS: 6799 KY104583.1 505/505 (100%) 38 SCHY-186 Makkoli (pre-sale) Saccharomyces cerevisiae CBS: 6282 KY105061.1 751/752 (99%)

6) Conclusion

As shown in Table 3 and FIG. 2, the yeasts isolated from the barn and brewery environment of the samples belonged to the majority Pichia or Saccharomyces , Meyerozyma, Wickerhamomyces, Candida, Kluyveromyces and Eremothecium genus were Saccharomyces rather than Pichia It was analyzed in close relationship. However, among them, the sequence similarity was as low as 84%, indicating a slight genetic difference.

Genetic homology of the genus Kluyveromyces and Eremothecium showed 97% sequence similarity , and genome homogeneity of the sequence similarity 96% in Meyerozyma and Wickerhamomyces genus. On the other hand, SCHY-172, which is estimated to be Candida albicans , and SCHY-9, which is estimated to be Candida glabrata , were found to have significant genetic distances as they were the same genus. This is thought to be because yeast strains, which are characterized by morphologically forming gastric hyphae, were grouped into Candida genus, rather than molecular genetic classification using sequencing in the past.

Example 2 Selection of Two Yeasts with High Growth Rate

1) Growth Rate Analysis

Among the 38 yeasts isolated from Experimental Example 1, the growth rate of the 20 yeasts of Table 4, in which the active growth is visually confirmed in the solid medium, was evaluated, and the specific growth rates were compared and selected therefrom. Specifically, 20 kinds of yeasts isolated from Example 1 were incubated for 48 hours at 500 ° C. in 500 ml of YM broth, centrifuged to confirm their dry weight, and then, the supernatant was removed, and the weight of the precipitate for each yeast was measured. . The precipitate was diluted with tertiary distilled water, centrifuged again to remove the media components, and the precipitate and tertiary distilled water were mixed at 1: 2 (w / w), frozen at -70 ° C and dried with a lyophilizer to weigh Was measured. The results are shown in Table 4 below. At this time, three repeated experiments were performed for each yeast, and the dry weight obtained in each experiment was expressed as 1st, 2nd, 3rd.

Yeast Number Primary dry weight
(Mg / 500ml) 2nd dry weight
(Mg / 500ml) 3rd dry weight
(Mg / 500ml) Average yield
(Mg) SCHY-1 983 1067 1025 1025 ± 34.29 SCHY-2 884.7 870.3 877.5 877.5 ± 5.88 SCHY-17 750.6 701.1 721.8 724.5 ± 20.30 SCHY-19 756.9 750.6 756.9 755.1 ± 2.97 SCHY-20 856.8 756.9 806.4 806.4 ± 40.78 SCHY-21 998.1 762.3 880.2 880.2 ± 96.26 SCHY-24 884.7 756.9 820.8 820.8 ± 52.17 SCHY-82 818 922 870 870 ± 42.46 SCHY-141 877.5 771.3 828 825.3 ± 43.39 SCHY-142 806.4 721.8 764.1 764.1 ± 34.54 SCHY-143 856.8 785.7 820.8 820.8 ± 29.03 SCHY-152 905 930 918 918 ± 10.21 SCHY-155 848 841 848 845 ± 3.3 SCHY-159 805 870 838 837 ± 26.54 SCHY-161 892 882 887 887 ± 4.08 SCHY-164 811 810 811 811 ± 0.47 SCHY-165 834 745 790 790 ± 36.33 SCHY-167 812 905 859 859 ± 37.97 SCHY-169 762 810 786 786 ± 19.6 SCHY-170 514 425 470 470 ± 36.33 SCHY-171 786 851 819 819 ± 26.54 SCHY-172 1091 910 1,001 1001 ± 73.89 SCHY-178 672 518 595 595 ± 62.87 SCHY-179 1015 940 978 978 ± 30.62 SCHY-182 756 822 789 789 ± 26.94 SCHY-183 512 490 501 501 ± 8.98 SCHY-184 820 858 839 839 ± 15.51 SCHY-185 798 771 785 785 ± 11.03 SCHY-186 921 935 928 928 ± 5.72 total 817.75 815.1 817 817 ± 1.1

As shown in Table 4, it was confirmed that the biomass amount of SCHY-1, SCHY-152, SCHY-172, SCHY-179, SCHY-186 strains isolated from barn or piglet was maintained at 900 mg or more. It was. In addition, SCHY-2, SCHY-21, SCHY-82, SCHY-155, SCHY-159, SCHY-161, SCHY-167, SCHY-171 and SCHY-184 strains showed the highest growth and high yield. It was confirmed.

2) Selection of 15 Yeasts by Measuring Yeast Growth Curves

As described above, 15 strains having a large amount of biomass and excellent growth were inoculated (n = 3) in 15 ml of YM broth to confirm the growth rate at 600 nm after 0, 6, 12, and 24 hours. Absorbance was measured.

3 is a growth rate curve for 15 yeasts selected first from Example 1). SCHY-1; Kluyveromyces marxianus SCHY-1, SCHY-21; Saccharomyces cerevisiae SCHY-21, SCHY-82; Pichia kudriavzevii SCHY-82, SCHY-155; P. kudriavzevii SCHY-155, SCHY-159, Meyerozyma caribbica SCHY-159, SCHY-161; S. cerevisiae SCHY-161, SCHY-164; M. caribbica SCHY-164, SCHY-165; P. kudriavzevii SCHY-165, SCHY-167; S. cerevisiae SCHY-167, SCHY-169; Eremothecium coryli SCHY-169, SCHY-170; S. cerevisiae SCHY-170, SCHY-171; W. anomalus SCHY-171, SCHY-172; Candida albicans SCHY-172, SCHY-178; P. kudriavzevii SCHY-178 and SCHY-186; S. cerevisiae SCHY-186.

As shown in FIG. 3, the yeasts having the absorbance of 2.0 or higher at 24 hours, SCHY-1, SCHY-21, and SCHY-186 were confirmed. -171, SCHY-172, SCHY-179. It can be seen that the trend is similar to the strain selected through the biomass and dry weight measured previously.

3) morphological analysis

First, in order to observe the selected yeast by scanning electron microscopy (SEM), sodium cacodylate trihydrate (Sigma, USA), glutaraldehyde (Daejung, Korea), hexamethyldisilazane (Sigma, USA), paraformaldehyde (Sigma, USA) And osmium tetroxide (Electron microscopy sciences, USA) to prepare a sample. Scanning electron microscopy was taken with FM-SEM (JSM-7401F, JEOL, Japan). Specifically, the lyophilized yeast was 3 hours in Karnosky fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 4M) in 0.1M sodium cacodylate buffer. After fixed for a while, washed three times each 10 minutes with a buffer (0.1M cacodylate buffer, pH 7.4), and then fixed in 1% osmium tetroxide (pH 7.2, 0.1M phosphate buffer, 4 ℃) for 2 hours. The sample is then washed three times with buffer, dehydrated in 50%, 70%, 80%, 90% and 100% of pre-cooled 4 ° C. ethanol solution, and then samples are prepared. The prepared specimens were dried with hexamethyldislazene, coated with platinum sputtering, and observed by FM-SEM.

4 is a SEM photograph of five yeasts among the selected yeasts. 4A and 4B show wild yeast strain SCHY-1, 4C and D show SCHY-161, 4E and F show SCHY-165, and 4G and H show SCHY-171. J is SCHY-21. SEM pictures of A, C, E, G and I are taken at x 10000 magnification, and SEM pictures of B, D, F, H and J are taken at x 1000 magnification.

As shown in FIG. 4, for the morphological analysis of the yeasts selected as having excellent growth rate, three different yeasts and two kinds of S. cerevisiae yeasts were selected from the 15 yeasts, and then, the result of scanning electron microscopy (SEM). The structure of the cell wall, size, microstructure and germination of the external structure was confirmed. S. cerevisiae SCHY-161 and S. cerevisiae SCHY-21 were spherical and about 5 μm in diameter, and K. marxianus SCHY-1 was ellipsoidal about 4 μm. Or less. Pichia kudriavzevii SCHY-165 was about 4 μm and Wickerhamomyces anomalus SCHY-171 was about 2.5 μm.

4) Conclusion

The growth rate was measured in 29 yeasts in total, and the highest growth was achieved. The biomass content was maintained at 900 mg / ml or more, and the two strains, SCHY-1 ( Kluyveromyces marxianus strain), having absorbance of 2.0 or higher in 24 hours of culture. TB7258) and SCHY-21 ( Saccharomyces cerevisiae ATCC4098) were selected.

Examples 3-16. Preparation of Yeast Autolysates of SCHY-1, 21 by Various Methods

In order to confirm the optimal extraction conditions of yeast ( S. cerevisiae ), various physicochemical and enzymatic treatments were carried out using SCHY-17 yeast, and then experiments under the microscope were performed. As a result, autoclaving, homogenization, ultrasound, pH It was confirmed that in the yeast autolysates prepared through the adjustment, hydrolase or papain enzyme treatment, effective autolysis occurred. Therefore, by applying the seven methods to the two types of yeast, respectively, the extraction method to obtain the most optimal yeast autolysate was selected.

First, before the yeast autolysates are prepared in various ways, two kinds of yeasts (SCHY-1 ( Kluyveromyces) selected through the above-described process, which are raw materials thereof, are prepared. marxianus strain TB7258), SCHY-21 ( Saccharomyces cerevisiae ATCC4098)) Prepare each building. Specifically, after yeast culture for 48 hours in YM broth centrifuged for 10 minutes at 1500 ㅧ g and the supernatant was discarded. Thereafter, 30 ml of distilled water was added to remove remaining medium components, vortexed, centrifuged again, and the supernatant was discarded. And 30 ml of water was added again and then lyophilized through a freezing dryer (Hanil, Korea) to obtain a yeast dry matter. Yeast autolysates were prepared by crushing the recovered yeast bodies by the following methods, which are summarized in Table 5 below. Each method is described in more detail below.

division leaven Treatment condition Extraction and Autolysis Conditions pH Temperature
(℃) pressure
(ATM) Amount Processing time Example 3 SCHY-1 physical Autoclaving 5.0 121 1.5 - 80 min Example 4 SCHY-21 Example 5 SCHY-1 Homogenization 5.0 4 One - 15 min Example 6 SCHY-21 Example 7 SCHY-1 ultrasonic wave 5.0 4 One - 10 min Example 8 SCHY-21 Example 9 SCHY-1 Chemical pH 4.0 4.0 50 One - 24 hr Example 10 SCHY-21 Example 11 SCHY-1 pH 5.0 5.0 50 One - 24 hr Example 12 SCHY-21 Example 13 SCHY-1 Biological Papain enzyme 6.0 50 One 0.5% 24 hr Example 14 SCHY-21 Example 15 SCHY-1 Protease 5.0 50 One 0.5% 24 hr Example 16 SCHY-21

1) autoclave

A yeast suspension was prepared by adding 5 ml (pH 5.0) of sterilized deionized water to a total amount of 70 mg of yeast in a yeast dry weight method using an autoclave. At this time, the sterilized deionized water was used by sterilization (AC-12, autoclave, HYSC, Germany) for 20 minutes at 121 ℃. Acidity was adjusted using NaOH or HCl solution. Thereafter, the yeast suspension was autoclaved at 121 ° C. for 15 minutes by sterilization (AC-12, Jeiotech, Korea) to obtain a yeast autolysate.

2) homogenizering

A yeast autolysate using a homogenizer was added to 5 ml (pH 5.0) of sterilized deionized water in a dry yeast mass of 70 mg, and stirred until fully mixed to prepare a yeast suspension. Thereafter, the yeast suspension was homogenized at 15,000 rpm for 15 minutes using Polytron PT 2100 homogenizer (Kinematica AG Co., Switzerland) to obtain a yeast autolysate. At this time, the container containing the yeast suspension was placed soaked about 2.5 cm in an ice bath, and then maintained at a constant temperature (4 ℃).

3) Ultrasound ( ultrasonicating )

A yeast suspension was prepared by adding 5 ml (pH 5.0) of sterile deionized water to a total amount of 70 mg of yeast as a method for preparing a yeast autolysate using an ultrasonic pulverizer. In order to sonicate the yeast suspension, a ULH-700S ultrasonic wave crusher (Jeiotect Co., Korea) and an ultrasonic wave generating titanium terminal (diameter 19.1 mm) were used. Specifically, the yeast suspension was soaked about 2.5 cm in an ice bath to maintain 4 ° C., and then the yeast suspension was exposed to a frequency of 20 kHz through an ultrasonic crusher. The exposure conditions were repeated for 10 minutes with 1 cycle of sonication for 1 second and 9 seconds of suspension.

4) chemical treatment via pH 4.0

PH 4.0 solution was used as a method for preparing yeast autolysates through chemical treatment. Specifically, 5 ml (pH 4.0) of sterile deionized water was added to the yeast dry weight of 70 mg, and stirred until completely mixed to prepare a yeast suspension. The yeast suspension was autolyzed at 50 ° C., 150 rpm, for 24 hours to prepare yeast autolysates.

5) chemical treatment via pH 5.0

A solution of pH 5.0 was used as a method for preparing yeast autolysates by chemical treatment. Specifically, 5 mL (pH 5.0) of sterile deionized water was added to the yeast dry weight of 70 mg, and stirred until completely mixed to prepare a yeast suspension. The yeast suspension was autolyzed at 50 ° C., 150 rpm, for 24 hours to prepare yeast autolysates.

6) hydrolase treatment

A method for preparing yeast autolysates by treatment with protease (Protease, Streptomyces caespitosus- derived proteolytic enzymes (Sigma, USA; Cat no. P-0384)). ) And the mixture was stirred until complete mixing, to prepare a yeast suspension.The yeast suspension was treated with 0.7 Unit / mg of protease to a final concentration of 0.5% (250 mg), 50 ° C 150 rpm, The reaction was carried out for 24 hours to prepare a yeast autolysate.

7) Papain Enzyme Treatment

As a method for preparing yeast autolysates by treating papain enzyme, yeast suspension was prepared by adding 70 mg of yeast dry weight to 5 ml (pH 5.0) of sterile deionized water and stirring until completely mixed. The yeast suspension was treated with 1.8 units / mg of papain enzyme to a final concentration of 0.5% (250 mg), and reacted at 50 ° C. 150 rpm for 24 hours to prepare a yeast autolysate.

Examples 17 to 20. Feed additives (solid yeast culture)

1) Starter-Liquid Medium

In order to develop a starter, the strains were cultured in a liquid medium having the composition of A-F shown in Table 6 below, and the most excellent growth was the liquid medium of the B composition, which was selected as a starter and added to the solid medium.

division
(unit; g) Liquid culture medium
(unit; g) A
(PD Broth) B
(SUN broth) C
(NL broth) D
(ANT broth) E
(AA broth) F
(CM broth) yeast extract 5 10 4 5 2.5 Soluble starch (potato) 4 5 Skim milk powder 10 2.5 peptone 5 10 Glucose 20 10 15 15 20 5 Malts 3 5 Salts 2 2 2 2 2 Minerals 2 2 2 2 2 total 24 22 34 33 39 24 Starter
(SCHY-1, SCHY-21) 5 5 5 5 5 5 Water 1000ml 1000 ml 1000 ml 1000ml 1000 ml 1000 ml

2) starter-solid badge

The composition of the yeast solid medium is EP corn, soybean meal, skim milk powder, corn steep liquor (CSL), molasses, glucose, salt, etc. by appropriately formulating the medium to G ~ M, and then cultured yeast as shown in Table 7 below. As a result, I solid medium was the best growth and was selected as a solid medium for the yeast culture.

division
(unit; g) Solid medium composition G H I J K L M corn EP 800 800 800 800 800 800 800 soybean meal 800 800 800 800 800 800 800 SMP
(Skimm Milk Powder) 25 25 25 CSL - 25 25 25 - 25 25 molasses - - 25 25 - 25 25 glucose - - - 25 - - 25 salts - - - - - - 25 Minerals 10 10 10 10 10 10 10 Starter (SCHY-1, SCHY-21) 700 700 700 700 700 700 700

When the yeast autolysate is added in the composition of the solid fermentation medium selected in Table 7, the optimum composition with the starter was found and cultured in the media composition of 1 to 12 of Table 8, and finally, in Table 9 The solid fermentation medium composition of was selected.

division
(unit; kg) Solid medium composition for yeast culture One 2 3 4 5 6 7 8 9 10 11 12 Yeast Autolysate - 30 - 30 - 30 - 30 - 30 - 30 Oat meal 500 500 500 500 1,000 1,000 1,000 1,000 - - - - Wheat gluten 500 500 500 500 - - - - 1,000 1,000 1,000 1,000 Dry yeast - - 100 100 - - 100 100 - - 100 100 CSL / molasses 50 50 50 50 50 50 50 50 50 50 50 50 Salts / minerals 20 20 20 20 20 20 20 20 20 20 20 20 Steam or water 100 100 100 100 100 100 100 100 100 100 100 100 Starter 200 200 200 200 200 200 200 200 200 200 200 200

Unit (kg) leaven
Autolysate oat
meal wheat
gluten dry
yeast CSL /
molasses salts /
minerals steam or
water *
Starter Example 17 SCHY-1
Autoclaved (Example 1) 500 500 100 50 20 100 200 30 Example 18 Treating SCHY-1 with Papain Enzyme (Example 13) 500 500 100 50 20 100 200 30 Example 19 Autoclaving the SCHY-21 (Example 2) 500 500 100 50 20 100 200 30 Example 20 Treated with SCHY-21 papain enzyme (Example 14) 500 500 100 50 20 100 200 30 * starter; Km ( K. marxianu s SCHY-1), Sc ( S. cerevisiae SCHY-21), complex strain (Km / Sc) was used

Feed additive according to the invention SCHY-1 and SCHY-21 ( Saccharomyces in a grain solid medium added 10% yeast autolysate prepared in Examples 1, 2, 13, 14 cerevisiae ATCC4098) was inoculated with 10% of the starter and incubated at 32 ° C. for 2 days, and a solid culture prepared by drying was used. The specific composition is shown in Table 6 above.

Experimental Example 1. Analysis of autolysis rate of yeast autolysates of Examples 3 to 16

First, in order to analyze the degree of self-degradation of yeast, by measuring the wavelength according to the release of substances in the cell, it is analyzed based on this. Specifically, the absorbances of yeast autolysates of Examples 3 to 16 obtained by different methods were measured at 260 nm and 280 nm, and the absorbances of 260 nm and 280 nm in 1 ml of the yeast suspension before treatment were compared. . At this time, the yeast autolysate was centrifuged (5000 분 g, 5 min, MiniSpin plus, Eppendorf, Hamburg, Germany), the supernatant was separated, diluted 8 times with water or Tris-HCl buffer, and then 260 and 280 nm. Absorbance was measured and recorded at (UV-1800 UV / VIS, Rayleigh, Beijing, China). The greater the difference in absorbance of the control and yeast autolysates at each wavelength can be seen to increase the rate of autolysis. Wherein 260 nm is the absorbance of the nucleic acid and 280 nm is the absorbance of the water soluble protein.

1) 260 nm absorbance analysis

The results of the analysis of the self-degradation method of 8) of the yeast autolysates prepared from Examples 3 to 16 described above are shown in Table 10 and FIG. 5. 5 is a graph showing the yeast autolyzate (Examples 3 to 16) prepared with yeast SCHY-1 and SCHY-21 measured at 260 nm.

division Absorbance division Absorbance Control
(Control) 0.51 ± 0.00 Control
(Control) 0.46 ± 0.00 Example 7 0.81 ± 0.00 Example 8 0.66 ± 0.02 Example 5 1.58 ± 0.01 Example 6 0.60 ± 0.01 Example 3 1.72 ± 0.00 Example 4 1.67 ± 0.01 Example 15 1.76 ± 0.02 Example 16 2.90 ± 0.12 Example 13 2.94 ± 0.10 Example 14 3.42 ± 0.18 Example 11 1.46 ± 0.01 Example 12 1.30 ± 0.00 Example 9 1.70 Example 10 0.69

As shown in Figure 5 and Table 10, through the yeast SCHY-1 or SCHY-21, the yeast autolysates were prepared by the above-described various methods, and analyzed for the self-degradation rate, yeast prepared through autoclaving The absorbance at 260 nm of the autolysates (Examples 3 and 4) was the highest at 1.7, and the yeast autolysates (Examples 13 and 14) prepared through papain enzyme treatment were also found to have very high absorbances at about 3.28. However, although it was mostly similar to the type of yeast, the self-degradation rate of the yeast autolysates (Examples 3, 5, 7, 9, 11, 13, and 15) prepared by treating the SCHY-1 yeast treated the SCHY-21 yeast It was confirmed that higher than the yeast autolysates prepared (Examples 4, 6, 8, 10, 12, 14, 16).

2) 280 nm absorbance (self-decomposition) analysis results

The results of analysis of the yeast autolyzate prepared by the method of Examples 3 to 16 described above by the autolysis rate are shown in Table 11 and FIG. 6. 6 is a graph showing the yeast autolyzate (Examples 3 to 16) prepared with yeast SCHY-1 and SCHY-21 measured at 280 nm.

division Absorbance division Absorbance Control
(Control) 0.31 ± 0.00 Control
(Control) 0.22 ± 0.00 Example 7 0.48 ± 0.00 Example 8 0.27 ± 0.00 Example 5 0.26 ± 0.00 Example 6 0.62 ± 0.00 Example 3 1.41 ± 0.00 Example 4 0.69 ± 0.00 Example 15 1.59 ± 0.00 Example 16 0.71 ± 0.02 Example 13 1.87 ± 0.37 Example 14 2.17 ± 0.14 Example 11 0.53 ± 0.00 Example 12 0.53 ± 0.00 Example 9 0.23 ± 0.01 Example 10 0.23 ± 0.01

As shown in Figure 6 and Table 11, through the yeast SCHY-1 or SCHY-21, the yeast autolysates were prepared by the various methods described above, and the analysis of the self-degradation rate for this, the yeast prepared through autoclaving The absorbance at 280 nm of the autolysates (Examples 3 and 4) was the highest at 1.41, and the yeast autolysates (Examples 13 and 14) prepared through papain enzyme treatment were also found to have very high absorbances at about 2.0. That is, when preparing the yeast autolysates using the SCHY-1 yeast and SCHY-21 yeast selected through the present invention, it can be seen that the yeast autolysates obtained through autoclaving or papain enzyme treatment are most preferred.

Experimental Example  2. SCHY -1 yeast, its yeast On autolysates  Morphological Analysis of

1) Morphological Analysis of Unfragmented SCHY-1 Yeast

After lyophilization, the morphology of K. marxianus SCHY-1 yeast stored at room temperature was observed by scanning electron microscopy (SEM). 7 is a SEM photograph of K. marxianus SCHY-1 yeast, according to which it was confirmed that the SCHY-1 yeast has an irregular shape and size. The SCHY-1 yeast had a size distribution of about 5-7 μm or about 2-4 μm and maintained a round and oval shape when the diameter was relatively large, but the surface was uneven when the diameter was relatively small. When lyophilized, SCHY-1 yeast was found to maintain more than 60% form, and when the yeast was dispersed in an aqueous solution, it was confirmed that the distance and density between cells were kept constant.

2) Morphological Analysis of Yeast in Yeast Autolysates Prepared from Example 3

The yeast autolysates prepared from Example 3 were observed by scanning electron microscopy (SEM). FIG. 8 is an SEM photograph of the yeast autolyzate prepared from Example 3, whereby it was confirmed that SCHY-1 yeast having a regular shape and size existed in the yeast autolyzate obtained by autoclaving. The SCHY-1 yeast size was round and elliptical, about 2-3 μm in diameter. The autoclave treatment of SCHY-1 yeast is believed to be of constant size and shape as the density between yeasts increases due to high pressure.

In addition, the SCHY-1 yeast, the sugar component leaked out by the breakdown of the yeast is located, which fills the cavity and forms a structure similar to the biofilm, which is inferred by the hydrophobic proteoglycan complex in which the cell membrane is partially destroyed. do.

3) Morphological Analysis of Yeast in Yeast Autolysates Prepared from Example 13

The yeast autolysates prepared from Example 13 were observed for morphology by scanning electron microscopy (SEM). FIG. 8 is an SEM photograph of the yeast autolysate prepared from Example 13, whereby it was found to have an irregular shape and size. The crushed SCHY-1 yeast present in the yeast autolysate obtained through papain treatment was about 4-7 μm in diameter, and it was confirmed that the distance between yeasts was close, and that there were many partially recessed portions when compared with undegraded yeast. In addition, the crushed SCHY-1 yeast present in the yeast autolysate obtained by papain treatment confirmed that several hundred yeasts aggregated together to form one cluster. This is because yeast protein is degraded by papain enzyme, and glycoprotein components flow out of the cell wall to induce binding between yeasts.

Experimental Example 3. Component Analysis of Yeast Autolysates of Examples 3 to 16

1) Total-glucan content analysis

The yeast autolysates of Examples 3 to 16 were lyophilized, respectively, and the total-glucan content of each powder was analyzed.

The powder was passed through a 1.0 mm (100 μm) sieve and ground, and then 90 mg of the ground matter was placed in a 20 ㅧ 125 mm test tube (Fisher Brand cμLture tube). 2.0 ml (12 M) of sulfuric acid was added to the test tube, and the test tube was closed. Then, the tube was sufficiently stirred and then left at 4 ° C. for 2 hours. Subsequently, after sufficiently stirring using a vortex apparatus, 4 ml of water was added, stirred, 6 ml of water was added, and the process of stirring was repeated. After heating to high temperature to inactivate papain enzyme and yeast, 6 ml KOH solution (10 M) and 200 mM sodium acetate buffer (pH 5) were added to a total volume of 100 ml. After obtaining a fraction thereof, it was centrifuged at 1500 g for 10 minutes. The supernatant was removed, and the recovered precipitate was fractionated by 0.1 ml. Then, 200 mM sodium acetate buffer (pH 5.0) was added and the first solution [exo-1,3-β-glucanase (20 U / mL) and β-glucosidase (4) was added. U / mL) mixture] 0.1 ml was added and thoroughly stirred and incubated at 40 ° C. for 60 minutes. 3.0 ml of GOPOD reagent was added thereto and further incubated at 40 ° C. for 20 minutes, and then absorbance was measured at 510 nm corresponding to the reagent blank.

2) α-Glucan Content Analysis

The yeast autolysates of Examples 3-16 were lyophilized, respectively, and the α-Glucan content of each powder was analyzed.

After sieving the powder, exactly 100 mg was placed in a 20 x 125 mm Fisher Brand cμLture tube. 2 ml of 2 M KOH was added thereto and stirred using a magnetic stir bar (5 × 15 mm), followed by 8 ml of 1.2 M sodium acetate buffer (pH 3.8). Subsequently, 0.2 ml of a second solution (amyloglucosidase (1,630 U / mL) and invertase (500 U / mL) mixture) was added thereto, mixed well, and maintained at 40 ° C. in a constant temperature bath. After the reaction was completed, after centrifugation at 1500 g for 10 minutes, the supernatant was recovered, and the volume was adjusted so that the final volume was 10.3 ml. To 0.1 ml of the supernatant, 0.1 mL of 200 mM sodium acetate buffer (pH 5.0) and 3.0 mL of GOPOD reagent were added, incubated at 40 ° C. for 20 minutes, and the absorbance was measured at 510 nm corresponding to the reagent base sample. The α-Glucan content from the absorbance was calculated using Megazyme Mega-Calc ™.

3) Glactomannan Content Analysis

The yeast autolysates of Examples 3 to 16 were lyophilized respectively and Glactomannan content was analyzed for each of these powders.

The powder was sieved through a 0.5 mm sieve, and 5 mL of 80% (v / v) ethanol was added to 100 mg and stirred, followed by heating at 85-90 ° C. for 5 minutes. 5 mL of ethanol was added thereto and centrifuged at 1,500 g for 10 minutes, and then the supernatant was discarded and a pellet was recovered. 5 ml of ethanol was added to the recovered precipitate, and the suspension was repeated three times, followed by centrifugation to remove the supernatant. 8 ml of 100 mM sodium acetate buffer (pH 4.5) (Buffer B) was added to the recovered precipitate and resuspended and stirred. The reaction was then placed in boiling water to terminate the reaction and cooled twice at room temperature. After cooling sufficiently, 10 μl of β-mannanase suspension (third solution) was added thereto, stirred for 30 seconds, and reacted at 40 ° C. for 60 minutes. After adjusting the final volume with distilled water, the fractions were collected and centrifuged at 1,500 g for 10 minutes. The obtained supernatant was measured spectrophotometrically at 340 nm wavelength. Blank used DW.

4) Analysis of α-Amylase Content

The yeast autolysates of Examples 3 to 16 were lyophilized, and each of these powders was analyzed for the α-Amylase content.

20 g of buffer A (pH 5.4) was added to 3 g of the powder, followed by intermittent mixing at 40 ° C. for 20 minutes, followed by centrifugation (1,000 g, 10 minutes). At the same time, 100 ml of the first solution was added to 0.5 g of Stansdard sample malt, and stirred at room temperature for about 20 minutes, followed by centrifugation (1,000 g, 10 minutes) to prepare a solution. 0.5 ml of the lysate was added to 9.5 ml of buffer A (pH 5.4) to prepare a # 10 diluent, and a # 1 malt dilution was prepared in the same manner.

Next, 50 ml of buffer A (pH 5.4) was added to 1 g of the sample (precipitate), which was gently stirred for about 15 minutes, and centrifuged (1,000 g, 10 minutes). 1.0 ml was added to 9.0 ml of buffer solution to prepare a # 10 diluent, and in the same manner, a # 1 diluent was prepared.

In order to perform the Amylase HR Reagent solution experiment, 6 ml (ㅧ 1, # 10) of the sample dilutions were pre-incubated at 40 ° C. for 5 minutes, and then 0.2 ml aliquots of the sample dilutions or malt dilutions were prepared. I prepared it. 0.2 ml of Amylase HR Reagent solution was added to each, incubated at 40 ° C. for 10 minutes, and then 3.0 ml of Stopping Reagent was added to record the absorbance (at 400 nm) of the solution by ELISA. A blank absorbance measurement was measured for distilled water as a value (Con.) And calculated through Equation 1 below. α-Amylase activity (Units / g Flour) calculation is as follows.

[Equation 1]

5) Protease Content Analysis

The yeast autolysates of Examples 3 to 16 were each lyophilized and analyzed for Protease content for each of these powders.

100 μl Succinylated casein solution was added to the microplate well, and 100 μl of Assay Buffer was used as a blank. 50 μl of the powder was added to the well to which the Succinylated casein solution was added. After reacting for 20 minutes at room temperature, 50 μl of TNBSA Working solution was added, incubated for 20 minutes, and the absorbance was measured at 450 nm. Absorbance variation (ΔA ₄₅₀ ) was calculated at ₄₅₀ nm minus the measured absorbance and the absorbance of the blank. At this time, all of the reagents were added to the blank in the same manner. In addition, ΔA ₄₅₀ refers to the absorbance value indicated by the proteolytic activity of the protease. The protease content was calculated by substituting the absorbance change into the calibration curve.

6) Conclusion

The content of Total-glucan, β-Glucan, α-Glucan, Glactomannan, α-Amylase and Protease was measured for the yeast autolysates, which are shown in Table 9 and FIGS. 10 and 11.

division -Glucan Glactomannan -Amylase Protease ppm ppm U / g U / g Control
(Control) 155.59 ± 6.79 189.2 ± 17.55 154.3 ± 3.92 595.85 ± 6.08 Example 3 178.57 ± 16.03 174.15 ± 1.76 79.9 ± 7.68 269.1 ± 67.61 Example 5 143.87 ± 1.65 184.9 ± 14.04 140.53 ± 3.45 445.05 ± 50.7 Example 7 147.45 ± 9.36 189.2 ± 3.51 106.22 ± 5.37 445.05 ± 50.7 Example 9 78.67 ± 9.86 204.25 ± 5.27 136.3 ± 3.07 595.13 ± 4.23 Example 11 107.31 ± 8.02 184.9 ± 7.02 168.73 ± 0.38 538.2 ± 16.9 Example 13 66.88 ± 7.4 301.0 ± 14.04 358.5 ± 3.84 1001.37 ± 95.07 Example 15 149.92 ± 9.4 202.1 ± 10.54 213.98 ± 24.25 884.925 ± 88.74

As shown in Table 12 and FIGS. 10 and 11, in the yeast autoclave of Example 3, β-glucan was found to be the most at 178.57 ppm. However, the glactomannan content (174.15 ppm) was lower than that of the control, and the activities of α-amylase and protease were 79.9 and 269.1 U / g.

In contrast, the yeast autolysate (Papain) of Example 13 treated with papain enzyme had a slightly lower yield of β-glucan (66.88 ppm), but yield of glactomannan (301.0 ppm) and protease (358.5 U / g) and α- Amylase (1001.37 U / g) was confirmed to have high activity. That is, it was confirmed that the yeast autolyzate prepared in Example 13 had an excellent efficiency of about 1.5 times or more in terms of the total content except for the content of β-Glucan compared to the control.

Experimental Example 4. Specification Experiment-Broiler

1) Preparation of experimental animals

Specification test was conducted on broiler farm in Anseo-dong, Southeast-gu, Cheonan-si, Chungcheongnam-do. The diet was fed with corn-soybean meal based on NRC (1994) requirements. One-day-old ROSS 308 chicks were published in 768 cages and reared in three-stage cages. The test starting weight was 37 ㅁ 0.51 g was carried out for 35 days. The trial design was fed to the control group (Cont) in the Basal diet, the first experimental group (Experimental group, T1), 0.1% synbiotic yeast culture (feed additive) in the Basal diet, the second experimental group (Experimental group, T2) was fed by the addition of 0.2% synbiotic yeast culture (feed additive) , 12 groups per treatment, 16 numbers per repetition for all three groups. At this time, the synbiotic yeast culture (feed additive) used was prepared according to Example 18.

 Table 13 shows the basic composition of the blended feed according to the intake criteria for chickens by three stages. The following formula 1 feed is a general super feed for chickens 0-7 days old, 2 compound feed is an electrical test feed for chickens 7-21 days old, and 3 compound feed is a late test feed for chickens 21-35 days old. to be.

Mixed ingredients
(Ingredients (% by weight)) Compound Feed 1-7 days old 7 ~ 21 days old 21 ~ 35 days old Corn 51.96 55.64 56.38 Soybean meal 35.96 26.04 18.41 Canola meal 5.00 10.00 15.00 Animal fat 2.50 3.80 5.40 MDCP - 1.20 1.10 DCP 1.50 - - Limestone 1.15 1.30 1.20 L-lysine 0.43 0.50 0.74 DL-Methionine 0.41 0.41 0.51 L-Threonine 0.19 0.20 0.32 L-Tryptophan - 0.01 0.04 NaHCO3 0.10 0.10 0.10 Salt 0.30 0.30 0.30 Vitamin premix 0.20 0.20 0.20 Mineral premix 0.20 0.20 0.20 Choline 0.10 0.10 0.10 Total 100 100 100 Calculated component
(Calculated composition) ME, kcal / kg 3,000 3,100 3,200 CP,% 23.76 21.28 19.98 CF,% 4.82 6.31 8.02 Lys,% 1.5 1.28 1.25 Met,% 0.72 0.66 0.71 AP,% 0.45 0.42 0.40 Ca,% 0.92 0.84 0.80

¹ Serving per kg of a complete diet: 11,025 IU Vitamin A; 1,103 IU vitamin D3; 44 IU Vitamin E; 4.4 mg vitamin K; Riboflavin 8.3 mg; 50 mg niacin; 4 mg thiamine; 29 mg d- pantothenic acid; Choline 166 mg; 33 μg vitamin B12.

² Servings per kilogram of a complete diet: 12 mg Cu (CuSO ₄ ˜5H ₂ O); Zn 85 mg (ZnSO ₄ ); 8 mg Mn (MnO ₂ ); 0.28 mg I (KI); 0.15 mg Se (Na ₂ SeO ₃ ˜5H ₂ O).

2) Weight gain (BWG), feed intake (FI) and feed demand (FCR)

Weight gain was measured at each group at the start of 1-7 days, 7-21 days and 21-35 days of age. Feed intake was calculated by subtracting the remaining amount from feed intake when measuring body weight. Feed demand was calculated by dividing feed intake by weight gain. All data were processed by Duncan's multiple range test (Duncan, 1955) using the General Linear Model procedure of SAS (1996) to test the significance between the means.

Table 14 shows the results of measuring the weight gain, feed intake and feed requirement after administering a compound feed prepared by mixing the feed additive of Example 18 according to the present invention with 0.1% and 0.2% to the broiler, according to 7-21. In the first daily gain, the second experimental group was significantly higher than the control group (P <0.05), and the feed request rate was found to be significantly higher than the control group (P <0.05). In the 21-35 primary daily gain, the second experimental group was significantly higher than the control group (P <0.05), and the overall daily gain was significantly higher in the second experimental group than the control group (P <0.05). The control group was found to be higher than the second experimental group (P <0.05).

division Control
(CON) First experimental group
(TRT1) 2nd experimental group
(TRT2) SEM ² 1-7 days old    BWG, g 90 92 91 2    FI, g 106 103 105 One    FCR 1.183 1.128 1.153 0.025 GF 0.85 0.89 0.87 0.019 7 ~ 21 days old    BWG, g 652 ^b 672 ^ab 678 ^a 9    FI, g 1030 1022 1025 9    FCR 1.583 ^b 1.522 ^b 1.513 ^a 0.016 GF 0.63 ^b 0.66 ^a 0.66 ^a 0.007 21 ~ 35 days old    BWG, g 986 ^b 1009 ^ab 1035 ^a 15    FI, g 1775 1807 1802 22    FCR 1.800 1.794 1.743 0.024 GF 0.57 0.56 0.56 0.008 total    BWG, g 1728 ^b 1774 ^ab 1805 ^a 16    FI, g 2910 2932 2932 26    FCR 1.695 ^a 1.655 ^b 1.625 ^b 0.012 GF 0.59 ^b 0.60 ^ab 0.62 ^a 0.005 Motality,% INO ³ 192 192 192 - FNO ³ 184 189 189 Survival 95.83 ^b 98.44 ^a 98.44 ^a 0.76

² standard error

³ INO is the number of early living spirits in each group

³ FNO is the number of living spirits in each group until the end

^{a, b If} different superscripts exist in the same row, they have different meanings (P <0.05).

3) Odor substances in powder

The broiler's intracellular gas composition was collected from the excreted minute in each group for 35 days and analyzed for ammonia, hydrogen sulfide and total mecaptan. Specifically, the measurement of ammonia, hydrogen sulfide and total mecaptan was measured in a gastec (Model GV ?? 100, Gastec, Japan), stored at room temperature for 5 days, after fermentation for 24 hours after taking 300 g of the powder in a 2,600 ml sealed plastic container. ) Was measured using detection tubes (No. 3L, No. 4LT and No. 70L, Gastec, Japan) for ammonia, hydrogen sulfide and total mecaptan. All data were processed by Duncan's multiple range test (Duncan, 1955) using the General Linear Model procedure of SAS (1996) to test the significance between the means.

When the feed additive of Example 18 is added to the broiler feed, the effect on the malodorous substances in broiler meat is analyzed and shown in FIG. 12. Specifically, FIG. 12 is a graph showing the analysis of the gas composition in the control group CON, the first test group TRT1, and the second test group TRT2, whereby the control group in the ammonia in the rat is significantly higher than the second test group. It was confirmed that the appearance (P <0.05).

4) Microbial composition in powder

On the 35th day of the experiment, the excreted minute was taken from each group for the same time, suspended in sterile saline, homogenized, and then diluted 10-3 to 10-6 to use as a sample for measuring viable cell count. To measure the number of Lactobacillus and E. coli bacteria in broiler meal by experimental treatment, M. agar (Difco, USA) was used for Lactobacillus , and E. coli was incubated at 37 ° C for 38 hours using MacConkey agar (Difco, USA). Was measured and shown in Table 12. All data were processed by Duncan's multiple range test (Duncan, 1955) using the General Linear Model procedure of SAS (1996) to test the significance between the means.

Items, log ₁₀ cfu / g Control
CON First experimental group
TRT1 2nd experimental group
TRT2 SEM ² Lactobacillus 7.08b 7.33ab 7.4a 3.51 E. coil 6.65a 6.42b 6.35b 4.92

² standard error

^{a, b If} different superscripts exist in the same row, they have different meanings (P <0.05).

As shown in Table 15, the second experimental group (TRT2) was significantly higher in the Lactobacillus than the control group (CON) (P <0.05). In E. coli , the control group (CON) was significantly higher than the first and second experimental groups (P <0.05).

5) vent score

On the 35th day of the experiment, the degree of dirt in the anus was visually measured in each group. Specifically, the anal score (Score) is shown in Table 16 measured by the value from very clean 1 to very dirty 3. All data were analyzed by Duncan's multiple range test (Duncan, 1955) using the General Linear Model procedure of SAS (1996) to test the significance between the means.

As shown in Table 16, no significant difference was observed in the anal index indicating diarrhea frequency between the control group and the first and second experimental groups (P> 0.05). This may be said that even if the feed is added to the feed additive according to the present invention does not cause diarrhea or related diseases, it is confirmed the stability.

division Control
CON First experimental group
TRT1 2nd experimental group
TRT2 SEM ² Anal index
vent score 1.37 1.29 1.32 0.05

² standard error

6) Meat quality and long term weight

On the 35th day of the experiment, four groups were selected for each group, and slaughtered by the tibial withdrawal method. The pH was measured using a pH meter (77P, Istek, Korea), and the meat color was measured twice using a colorimeter (Model CR-210. Minolta Co., Japan) twice for each breast meat sample. At this time, the standard color plates were L = 89.2, a = 0.921, and b = 0.783. The water holding capacity was measured by recording the ratio of the total area and the meat area by the method of Hofmann et al. (1982). Drip loss is shown in Table 14 by measuring the loss caused during cold storage for 1 day, 3 days, 5 days and 7 days at 4 ℃. All data were processed by Duncan's multiple range test (Duncan, 1955) using the General Linear Model procedure of SAS (1996) to test the significance between the means.

As shown in Table 17, there was no significant difference in meat quality and organ weight between treatments (P> 0.05). This confirmed that the feed additive according to the present invention does not show toxicity to meat or organs.

division Control
CON First experimental group
TRT1 2nd experimental group
TRT2 SEM ² pH value 5.86 5.76 5.79 0.15 Pectoral muscle color   Lightness (L *) 52.25 53.72 55.23 1.37   Redness (a *) 10.26 10.34 10.45 0.56   Yellowness (b *) 9.17 9.51 9.79 0.57 Cooking loss,% 34.65 33.84 31.41 2.60 WHC,% 54.03 52.48 51.07 2.42 Drip loss,% 1 day storage 2.70 2.64 2.60 0.19 3-day retention 5.58 5.45 5.36 0.10 5-day retention 8.64 8.57 8.44 0.22 7-day retention 10.48 10.22 10.13 0.24 Organ weight,%  Pectoral muscles 18.30 18.42 18.57 0.14 liver 2.88 2.90 2.95 0.13 Fabrizio vesicles
(F bag) 0.10 0.12 0.13 0.01   Abdominal fat 3.39 3.56 3.67 0.31 spleen 0.10 0.13 0.14 0.02 Sandbag 1.22 1.18 1.14 0.07

² standard error

Experimental Example 5. Result of microbial flora

Intestinal microorganisms were isolated from feces of broilers fed feed additives prepared in Example 18, and a total of 44 phylums and 1,690 microorganisms were analyzed. The majority of the sequences were one of Firmicutes, Proteobacteria, Bacterioidetes, Actinobacteria , and genus-level dominant species were Lactobacillus, Arthromitu, and Romboutsia .

Table 18 is a feed additive that has analyzed the effect on intestinal microflora of broilers, in Lactobacillus and E. coli, in Salmonella or decrease in accordance with the specifications broiler experiment Lactobacillus is 41.53% increase (compared to the control group in the primary prepared from Example 18 1st and 2nd experimental group) and 27.68% increase rate (1st and 2nd experimental group compared with control group) in the 2nd time. In contrast, E. coli and Salmonella were not identified. The genus Lactobacillus was significantly higher in the second experimental group than the control group (P <0.05), and E. coli was significantly increased in the control group than the first and second experimental groups (P <0.05). This shows that Lactobacillus inhibits the growth of E. coli .

When fed the feed additive of Example 18 (yeast culture) in the present invention, but is not Salmonella can determine whether increasing or decreasing not detected in both the control and the first and second treatment group, the number of bacteria is E. coli control Compared with the decrease, Lactobacillus was increased, Salmonella was also not detected, it can be seen that the feed additive of the present invention has a positive effect on the intestinal microorganisms of livestock.

Strain % Growth compared to the control Lactobacillaceae ( Lactobacillus ) 1 ^st up 41.53%, 2 ^nd up 27.68% Enterobacteriaceae ( E. coli ) Not detected Salmonella Not detected in all groups

Taken together, the diversity of the intestinal microbial community of the control group Cont and the first and second experimental groups T1 and T2 that did not receive the feed additive of Example 18 was as described above, specifically, the first, Compared with the control group, Firmicutes microbial group was relatively higher than the control group, and Lactobacillus was significantly higher in the first experimental group (95.85 ± 1.71%) and the second experimental group (98.05 ± 0.43%) than the control group. In contrast to the control group that dominates E. coli (18.27 ± 5.95%), the first and second experimental groups confirmed that Bacteroides (22.09 ㅁ 2.91%) and Clostridium (20.51 ㅁ 1.16%) including Lactobacillus were dominant. The second experimental group was found to have the highest proportion of Bacteroides (33.21 ㅁ 5.13%). That is, when the feed additive according to the present invention is fed, it can be seen that it has the effect of promoting the growth of the beneficial microorganisms present in the intestine of the livestock and predominantly.

Experimental Example 5. Specification Experiment-Dairy Cows

1) Preparation of experimental animals

The experiment was carried out for 4 weeks at the livestock farm of the livestock department of Yeonam University (Suhyang-ri, Seonghwan-eup, Seobuk-gu, Cheonan-si, Chungcheongnam-do). Experimental animals used a total of 10 cows (Bos taurus), of which 5 were divided into a control group (Cont) and the remaining 5 animals were divided into the first experimental group (Experimental group, T1) to confirm their age and sex. Table 16. Feed and water were allowed for free feeding. In the experiment, only the Basic diet (TMR) was fed to the control group, and the first experimental group was fed a TMR blended feed mixed with a 0.2% feed additive (Example 18). At this time, the feed additive used was prepared from Example 18.

Ingredients (%) General feed
(Basal diet) Combined feed containing feed additives Control First experimental group Yeast culture ^{1 )} 0 0.2 Wild yeast autolysates ^{2 )} 0 0.2 General feed composition   Yellow corn 53.44 53.24   Soybean meal 33.65 33.65   Corn gluten meal 4.16 4.16   Soybean oil 4.68 4.68   Limestone 1.02 1.02   Tricalcium phosphate 2.01 2.01   Salts 0.25 0.25   DL-Methionine (99%) 0.27 0.27   Lysine-HCl (98%) 0.02 0.02 Vitamin-Mineral mixture ^{3 )} 0.50 0.50   Water 20 20 Total 100.00 100.00 Calculated value  ME (kcal / kg) 3,100 3,100  Crude protein (%) 22.0 22.0  Lysine (%) 1.10 1.10  Methionine (%) 0.87 0.87  Methionine + cystine (%) 0.50 0.50  Ca (%) 1.00 1.00  Available P (%) 0.50 0.50

¹⁾ Yeast culture was obtained by inoculating 10% starter (SCHY-1 + SCHY-2) in a medium containing 10% yeast autolysate (Example 13) and grains, incubated at 32 ° C. for 2 days and dried. It is a feed additive of Example 18.

²⁾ Wild yeast autolysates were obtained by treating SCHY-1 and SCHY-21 with papain enzyme.

³⁾ Vitamin-mineral mixture provided following nutrients per kg of diet: vitamin A, 15,000 IU: vitamin D3, 1,500 IU: vitamin E, 20.0 mg; vitamin K3, 0.70 mg: vitamin B12, 0.02 mg: niacin, 22.5 mg: thiamin, 5.0 mg: folic acid, 0.70 mg: pyridoxin, 1.3 mg: riboflavin, 5 mg: pantothenic acid, 25 mg: choline chloride, 175 mg: Mn, 60 mg: Zn, 45 mg: I, 1.25 mg: Cu, 10.0 mg: Fe, 72 mg: Co, 2.5 mg.

2) mortality and diarrhea

Mortality was calculated by examining the mortality of milking cows during the experimental period. Diarrhea frequency was determined by diarrhea at the time of feeding, and diarrhea index was measured according to feces.

Experiment period Control
Control First experimental group
(T1) Diarrhea frequency
(diarrhea frequency) Diarrhea Index
(Diarrhea index) Diarrhea frequency
(diarrhea frequency) Diarrhea Index
(Diarrhea index) Intial 0 0 0 0 1 week One 2 0 0 2 weeks 0 0 0 0 3 weeks One 3 One 2 4 weeks One 2 One 2 Total 0.6 2.3 0.4 2.0

As shown in Table 20, as a result of measuring the diarrhea frequency and diarrhea index of each group, it was confirmed that there is no diarrhea frequency (two heads) or one head according to the duration of the experiment. Comparing the average diarrhea frequency and diarrhea index it can be seen that the first experimental group is somewhat lower than the control group. In addition, diarrhea occurred in both the control group and the first experimental group during the test time, but this did not indicate severe disease occurrence or mortality of the cow despite some.

3) flow analysis

The flow rate is finished in the morning and afternoon milking and recorded the milking amount by group to calculate the daily milking amount is shown in Table 21.

Experiment period Control (kg) 1st experimental group (kg) Intial 38.7 39.1 1 week 36.5 38.8 2 weeks 35.7 37.8 3 weeks 34.3 36.7 4 weeks 33.9 36.6 Total 35.8 37.8

As shown in Table 21, after feeding the feed of the present invention, the flow rate was confirmed to increase compared to the control over the entire period.

4) Oil component analysis

The milk components were collected before the experiment, 2 weeks after the experiment and 4 weeks after the experiment to analyze the milk components and somatic cell count.

ingredient group Initial 2 weeks 4 weeks Average % Retention Control 3.6 3.2 3.5 3.4 First experimental group 3.6 3.5 3.7 3.6 Milk protein percentage (%) Control 3.0 2.9 3.0 2.9 First experimental group 2.8 2.9 2.9 2.9 Solid free (%) Control 8.8 8.7 8.8 8.8 First experimental group 8.7 8.8 8.8 8.8 MUN (mg / dL) Control 18.9 15.6 14.3 16.3 First experimental group 16.4 12.9 11.6 13.6 Somatic cell numbers
(X 1,000 cells /) Control 124.2 90.8 176.8 130.6 First experimental group 72.8 68.0 115.2 85.3

As shown in Table 22, the maintenance of the control group and the first experimental group after milking was analyzed. As a result, it was confirmed that the first experimental group had a higher retention than the control group. However, there were no differences in milk protein and solids between groups. In the case of milk urea nitrogen (MUN), the optimal level of MUN, the urea nitrogen content in milk, was 12-18 mg / dL. Both the control group and the first experimental group maintained the optimum level, but the first experimental group was relatively lower. It was confirmed that the MUN value was maintained. In other words, it is believed that the feed additive of the present invention exhibits low MUN levels by reducing intestinal odor. After the experiment, it was confirmed that the somatic cell count in milk obtained from the control group and the first experimental group was relatively lower in the first experimental group.

When cows were fed feed additives (yeast glycosides and yeast autolysates obtained by inoculating and incubating yeasts with yeast autolysates) according to the present invention, the yield and maintenance rate of the cows were the same or lower in terms of business. It is an excellent advantage in that it can produce milk of high quality through consumption.

In the present invention, the yeast has a property to bind to organic acids or oxygen, such as lactic acid, and acts as a buffer to increase the pH when ingested in vivo, thereby promoting the growth of anaerobic bacteria, in particular fibrinolytic bacteria, increase the digestibility of feed and produce milk It can be seen that this may help to promote nutrients and improve fat content in dairy components. In addition, yeast autolysates are rich in essential amino acids, B vitamins, minerals, sterols, derived fats and digestive enzymes, so that the digestive enzymes and metabolites released during yeast growth and self-degradation increase animal productivity. It was confirmed that the action to improve the digestibility.

5) Blood component analysis

10 ml in the jugular vein at the same time (11 am to 12 am) in the same subject in each group (n = 2) at the beginning of the experiment for analysis of blood components, after 14 days and after 28 days Blood was collected using a syringe. The collected blood was centrifuged at 4 ° C. for 15 minutes, and only serum was separated and stored at −20 ° C.

The leukocyte counts in the blood were collected in EDTA (K2 EDTA, BD VacutainerTM, USA) anticoagulant tubes, as described above, from the jugular vein at each time period for each group, and the hematology analyze (Advia 120 Bayer, an automated hemocytometer). , Siemens, USA). Some of the collected blood was injected into the CBC bottle containing the anticoagulant EDTA-2K, and then tested using the automated blood analyzer (ADVIA 2120, SIEMENS, USA) for the items listed below in Table 23. Blood analysis items include Red blood cell (RBC), Platelet count (PLT), Hematocrit (HCT), White blood cell (WBC), Red cell distribution width (RDW), Hb conc. distribution width (HDW), Hemoglobin concentration (HGB), Neutrophil (NEUT), Mean corpuscular volume (MCV), Lymphocyte (LYM), Mean cell hemoglobin (MCH), Monocyte (MONO), Mean cell hemoglobin concentration (MCHC), Eosinophil (EOS), Mean platelet volume (MPV), Basophil (BASO), Reticulocytes (RET), Large unstained cells (LUC), Cell hemoglobin concentration mean (CHCM), neutrophil (NEU), eosinophil (EOS), Platelet distribution width ( PDW) was analyzed.

Blood analysis in whole blood (CBC) Blood composition unit Analysis method WBC × 10 ³ / μl Flowcytometry RBC × 10 ⁶ / μl Flowcytometry, Isovolumetry HGB g / dL Modified CN met-Hb method HCT % (RBC × MCV) ÷ 10 MCV fl Histogram MCH Pg (HGB ÷ RBC) × 10 MCHC g / dL [HGB ÷ (RBC × MCV)] × 1000 RDW % Histogram HDW g / dL Histogram PLT × 103 / μl Flowcytometry MPV fl Histogram RET % Flowcytometry, Isovolumetry Leukocyte Differentiation Factor Analysis in WBC Blood composition unit Analysis method NEU % Flow cytometry, peroxidase staining LYM % MONO % EOS % BASO % LUC % Noise-Lymph Histogram

Table 24 summarizes the results of analyzing the change of CBC parameters under the environment for distinguishing the ingested feed.

ingredient Initial 14 day
(2 week) 28 day
(4 week) Control Experimental group 1 Control Experimental group 1 Control Experimental group 1 Red blood cells RBC, x 10 ⁶ cells / 6.46 ± 4.04 6.14 ± 0.41 6.30 ± 0.63 6.40 ± 0.29 6.44 ± 0.51 6.36 ± 0.49  HGB, g / dL 10.9 ± 0.74 10.4 ± 0.87 10.5 ± 1.21 10.4 ± 0.99 10.4 ± 1.22 10.1 ± 1.18  HCT,% 31.4 ± 1.35 29.54 ± 2.42 30.7 ± 3.44 30.9 ± 2.64 30.9 ± 3.06 30.3 ± 3.02  MCV, fl 48.5 ± 3.90 48.1 ± 3.28 48.6 ± 0.84 48.3 ± 3.45 47.9 ± 1.11 47.7 ± 3.45  MCH, pg 16.9 ± 0.44 16.9 ± 1.19 16.6 ± 0.47 16.2 ± 1.38 16.1 ± 0.58 15.9 ± 1.34  MCHC, g / dL 34.7 ± 0.77 35. ± 20.61 34.2 ± 0.76 33.6 ± 0.46 33.6 ± 0.83 33.4 ± 0.60  RDW,% 19.1 ± 0.61 19. ± 20.54 19.5 ± 0.64 19.0 ± 0.38 19.0 ± 0.90 19.2 ± 0.43  HDW,% 2.71 ± 0.22 2.72 ± 0.13 2.81 ± 0.11 2.68 ± 0.16 2.66 ± 0.12 2.6 ± 70.21  CHCM, g / dL 34.3 ± 0.38 34.4 ± 0.40 34.3 ± 0.35 34.3 ± 0.38 34.8 ± 0.23 34.5 ± 0.50  Leukocyte, K / ml WBCB, x10 ³ cells / 10.63 ± 4.04 13.54 ± 5.99 10.94 ± 4.06 13.93 ± 5.68 10.38 ± 3.89 14.3 ± 7.60  NEU 34.8 ± 10.6 25.6 ± 10.5 34.9 ± 11.4 26.68 ± .54 34.8 ± 14.0 30.1 ± 9.03  LYM 55.8 ± 10.2 51.1 ± 7.98 54.4 ± 12.2 59.84 ± 10.76 51.4 ± 10.6 58.3 ± 10.52  MONO 0.2 ± 0.04 0.3 ± 0.15 0.2 ± 0.07 0.38 ± 0.37 0.4 ± 0.22 0.34 ± 0.39  EOS 8.3 ± 1.4 6.6 ± 3.4 9.5 ± 3.35 12.0 ± 6.3 11.9 ± 6.29 9.9 ± 6.0  LUC 0.06 ± 0.08 0.1 ± 0.06 0.08 ± 0.04 0.08 ± 0.04 0.04 ± 0.05 0.14 ± 0.10  BASO 0.8 ± 0.2 0.9 ± 0.3 0.9 ± 0.3 1.0 ± 0.4 1.5 ± 0.4 1.22 ± 0.42 Platelets  PLT, K / ml 355 ± 131 410 ± 63 364 ± 43 389 ± 120 317 ± 61 405 ± 108  MPV, fl 7.5 ± 0.5 7.3 ± 0.2 6.7 ± 0.3 6.9 ± 0.3 8.7 ± 0.9 8.6 ± 0.7  PDW,% 58.7 ± 8.14 51.8 ± 3.24 52.3 ± 3.94 51.24 ± 2.31 62.7 ± 3.80 58.6 ± 4.38  PCT,% 0.26 ± 0.09 0.30 ± 0.04 0.25 ± 0.03 0.26 ± 0.07 0.28 ± 0.05 0.34 ± 0.09

As shown in Table 24, the control group fed the basic diet showed that the RBC and HCT levels decreased initially in the RBC-related items, but did not recover after 2 weeks, but increased slightly after 4 weeks. . In contrast, MCV, RDW, HDW, and CHCM levels increased and decreased in the control group, and HGB, MCH, and MCHC were found to continuously decrease.

In the leukocyte-related category, there was no change in WBC levels in the control group for about 2 weeks, after which the LYM ratio continued to decrease, and the MONO ratio increased more than twice after 4 weeks. The percentage of eosinophils (EOS) continued to increase from 8.3% to 9.5% and 11.9%, while the BASO ratio increased from 0.8% to 0.9% and then to 1.5%.

The platelet item in the control group showed that platelet concentration (PLT) increased from 355 K / ml to 364 K / ml for 2 weeks and then markedly decreased to 317 K / ml after 4 weeks, and the MPV and PDW levels were decreased. Increase in inverse proportion to PLT).

In the first experimental group fed the feed containing 0.2% feed additive, the red blood cell yield increased by 2.4 × 10 ⁵ cells / ml and maintained at 4.0 × 10 ⁴ cells / ml. This suggests that feed with yeast culture has a positive effect on blood production, such as promoting red blood cell production.

When comparing leukocyte concentration (WBC) in the leukocyte-related items, it was confirmed that the first experimental group increased 1.38% more than the control group, and increased 6.72% after 4 weeks. The NEU ratio increased to 4.22% after 2 weeks and 17.58% after 4 weeks, indicating increased immune activity. LYM also increased to 17.1% after 2 weeks and 14.09% after 4 weeks, which means that the first experimental group was continuously enhanced innate and acquired immunity unlike the control group. The EOS rate increased by 5.4%, nearly twice that of the previous two weeks, dramatically increasing the inhibitory response than the control (1.2% increase).

In the platelet category, there was no significant change in the platelet concentration, but the first experimental group showed a significantly higher value than the control group for the entire period.

6) Analysis of IL-2 (Interleukin 2) in Cow Serum

To analyze Bovine IL-2 (Interleukin 2), Mybiosource's IL-2 ELISA Kit (MBS700611, mybiosource, USA) was used. All concentrated reagents in the kit were diluted to 1-fold concentration, Standard was added to the microtubes by 0.5 ml, and then prepared by diluting 500 μl of the standard and diluting it from 2,000 pg / ml to 7 steps twice. Into each well, 100 μl of 7 standard solutions or 10 samples were added and reacted at 37 ° C. for 1 hour. The IL-2 present in the sample or stnadard is bound to the immobilized antibody present on the well, and to detect the bound IL-2, 100 μl of a specific biotin conjugated antibody (biotin-antibody) and detection reagent A are detected. It was added to each well and reacted at 37 degreeC for 1 hour. After aspiration, water washing was performed three times using 200 μl of washing solution, and 100 μl of Avidin conjugated horseradish peroxidase (Avidin-HRP) and detection reagent B were added thereto, reacted at 37 ° C. for 1 hour, and then washed with water five times. It was. When 90 μl of TMB substrate was added and reacted at 37 ° C. for 10-20 minutes, only wells containing biotin-antibody and HRP-avidin bound to Bovine IL-2 components showed color change. 50 μl of the reaction terminating reagent was added, and immediately measured by spectrophotometer at a wavelength of 450 nm.

Initial, μg / ml 14 day, μg / ml
(2 week) Control 53.94 ± 43.59 Control 39.34 ± 24.68 1st group 53.14 ± 54.82 First experimental group 56.75 ± 53.46

As shown in Table 25, the control group confirmed that the blood concentration of Bovine IL-2 decreased by 27.08% for 2 weeks, but increased by 6.79% in the first experimental group, which means that the feed additive according to the present invention enhances innate immunity. It means that there is.

7) Igunoglobulin A (IgA) in bovine serum

Blood was collected from cows of each group, aliquoted using microtubes, and allowed to coagulate for 30 minutes at room temperature. After centrifugation for 15 minutes at 1,000 ㅧ g, the supernatant was aliquoted and stored at -20 ° C or less.

First, Abbexa's IgA ELISA Kit (ABIN2534862, abbexa, USA) was used to analyze the concentration of IgA (Immunoglobulin A) in serum of the first experimental group. All reagents were prepared by diluting to a concentration of ㅧ 1 prior to use, and Standard was pre-loaded into 0.5 ml of microtubes, and 500 µl aliquots were diluted twice from 50 ng / ml to prepare 7 dilutions. Into each well, 100 μl of 7 standard solutions or 10 samples were added and reacted at 37 ° C. for 1 hour. IgA present in the sample or stnadard is bound to the immobilized antibody present on the well, and to detect the bound IgA, 100 μl of a specific biotin conjugated antibody (biotin-antibody) and detection reagent A are added to each well. And reacted at 37 ° C. for 1 hour. After aspiration, the resultant was washed three times with 350 µl of washing solution, and 100 µl of Avidin conjugated horseradish peroxidase (Avidin-HRP) and detection reagent B were added thereto, reacted at 37 ° C for 1 hour, and washed five times after aspiration. It was. When 90 μl of TMB substrate was added and reacted at 37 ° C. for 10-20 minutes, only wells containing biotin-antibody and HRP-avidin bound to Bovine IgA component showed color change. After completion of the reaction, 50 μl of reagent was immediately measured using a spectrophotometer at a wavelength of 450 nm.

Initial, μg / ml 14 day, μg / ml
(2 week) Control 54.97 ㅁ 0.37 Control 51.21 First experimental group 59.71 ㅁ 0.77 First experimental group 72.43 ㅁ 0.41

As shown in Table 26, the control group showed that the concentration of Bovine IgA decreased by 6.84% for 2 weeks, but increased by 21.3% in the first experimental group, indicating that the feed additive according to the present invention enhances humoral immunity. It means.

8) Microbial composition in feces

For each group, feces were collected according to the season, suspended in sterile physiological saline, homogenized, and DNA was extracted for PCR amplification and illumination sequencing. PCR amplification was performed using Primer targeting the V3 to V4 region of the 16S rRNA gene with the extracted DNA. For bacterial amplification, the 341F (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGAC -AGCCTACGGGNGGCWGCAG-3 '; sequence represents the target site primer) and 805R (5'-GTCTCGTGGGCTCGGGATGTGTATAAGAGACAGGACTACHVGGG -TATCTAATCC-3') primers were used. Amplification was performed under the following conditions: initial denaturation at 95 ° C. for 3 minutes, then denaturation at 95 ° C. for 30 seconds, primer annealing at 55 ° C. for 30 seconds, extension at 72 ° C. for 30 seconds, and 5 at 72 ° C. Final stretch for minutes. Secondary amplification was then performed with i5 forward primer (5'-AATGATACGG -CGACCACCGAGATCTACACXXXXXXXXTCGTCGGCAGCGTC-3 '; X represents the barcode region) and i7 reverse primer (5'-CAAGCAGAAGACGGCA -TACGAGATXXXXXXXXAGGGTCTCTCTGG) to attach Illumina NexTera barcodes. The reaction conditions were the same as those of the amplification reaction except that the amplification cycle was set to eight.

PCR products obtained through the above procedure were electrophoresed on 2% agarose gel and visualized under Gel Doc system (BioRad, Hercules, CA, USA). The amplified product was purified by QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). Equal concentrations of purified products were collected together and short fragments (non-targeted products) were removed with an ampure beads kit (Agencourt Bioscience, MA, USA). The quality and product size were evaluated on a Bioanalyzer 2100 (Agilent, Palo Alto, Calif., USA) using a DNA 7500 chip. Pooling and sequencing of the mixed amplicon was performed according to the manufacturer's instructions using the Illumina MiSeq sequencing system (Illumina, USA) from Chunlab, Inc. (Seoul, Korea). The quality was confirmed through raw read processing by the Miseq orthogonal sequencing method (Miseq pipeline method) and the low quality (<Q25) set was filtered by trimming (0.321). After passing Quality Control (QC), the bidirectional sequence data was merged using PandaSeq2. Primer reduced the similarity to 0.8 with ChunLab's in-house program. The sequences were merged using Mothur's3 pre-clustering program, and a unique sequence was extracted that allowed up to two differences between the sequences. The EzTaxon database was used for taxonomic assignment using BLAST 2.2.224 and pairwise alignment 5 was used to calculate the similarity.

Uchime6 and EzTaxon's non-chimeric 16S rRNA databases were used to detect chimeras in reads with similarities with less than 97% similarity. Subsequently, CD-Hit7 and UCLUST8 were used to cluster the sequence data and perform α-diversity analysis to confirm microbial flora change from feces collected from each group. As a result, a total of 29 phylums and 2544 taxa were detected. The majority of sequences belonged to one of Firmicutes, Actinobacteria, Bacterioidetes, Euryarchaeota, Saccharibacteria_TM7, Tenericutes and Proteobacteria . Dominant species in the genus were identified as Paeniclostridium, Romboutsia and Turicibacter .

In addition, for each group (control group (cont-i) at the beginning of the experiment, control group (cont-14d) after 2 weeks of experiment, control group (cont-28d) after 3 weeks of experiment, and first experimental group (T1-14d) after 2 weeks of experiment , 3 weeks after the experiment was confirmed that the microbial community in feces collected from the first experimental group (T1-28d)) is very diverse. Firmicutes was higher in the first experimental group than in the control group, and Lactobacillus was 95.85 ㅁ 1.71% in the first experimental group. In the control group, E. coli was predominantly 18.27 ㅁ 5.95%, whereas in the first experimental group, Lactobacillus and Bacteroides bacteria (22.09 ㅁ 2.91%) and Clostridium bacteria (20.51 ㅁ 1.16%) were dominant, and among them, Bacteroides ( 33.21 ㅁ 5.13%) was confirmed that the highest rate.

Most of the microorganisms of Firmicutes , including various floras such as Bacillus, Lactobacillus and Staphylococcus, were the most common, and the control group increased from 79.76% to 88.03% in 2 weeks, but decreased after 4 weeks and finally increased 3.6% from the beginning. Was observed. In contrast, the first experimental group showed a 8.4% decrease from 2 to 4 weeks. Actinobacteria decreased by 1.7 ~ 3.6% in the control group but increased by 4% in the first experimental group. Proteobacteria increased in the control group, decreased 0.11% after 4 weeks, and decreased 2% in the first experimental group. Saccharibacteria_TM7 decreased 0.16 ~ 0.23% in the control group, but increased 2.7% in the first experimental group. Fibrobacteres decreased by 0.009% to 0.01% in the control group, while that in the first experimental group increased by 0.004%. These measured results are summarized in Table 27 below.

Phylum Control,%
(Basal feed) First experimental group,%
(0.2% yeast additive feed) Cont-i Cont-14d Cont-28d T1-14d T2-28d Firmicutes 79.76 88.03 83.35 69.67 61.28 Actinobacteria 7.61 3.74 5.94 7.30 11.43 Bacteroidetes 6.26 4.81 3.97 10.15 9.18 Euryarchaeota 3.40 0.90 3.90 3.59 7.48 Proteobacteria 0.32 0.56 0.21 2.83 0.84 Sacharibacteria _ TM7 0.93 0.77 0.70 3.52 6.32 Tenericutes 0.93 0.57 1.22 2.09 2.84 Spirochaetes 0.63 0.52 0.49 0.53 0.36 Synergistetes 0.01 - 0.00 0.16 0.11 Cyanobacteria 0.05 0.00 0.03 0.02 0.03 Streptophyta 0.00 0.00 0.05 - 0.00 Fibrobacteres 0.03 0.02 0.02 0.00 0.01 Verrucomicrobia 0.00 0.00 0.02 0.02 0.01 Fusobacteria 0.00 0.00 - 0.01 0.00 Lentisphaerae 0.00 0.01 - 0.01 0.01 Planctomycetes 0.00 0.00 0.01 0.01 0.00 Acidobacteria - 0.00 - - 0.00

9) Species level change

The total flora of the strain was observed and shown in Table 28 below.

Control First experimental group Cont-i Cont-14d Cont-28d T1-14d T1-28d Counts Ratio Counts Ratio Counts Ratio Counts Ratio Counts Ratio Lactobacillaceae_uc 3 0.0058 - - One 0.0019 7 0.0128 2 0.0028 Lactobacillus 161 0.3089 34 0.0918 131 0.2445 482 0.8828 468 0.6575

According to the results, the control group increased in the family, Firmicutes including Lactobacillaceae , and then decreased again after 4 weeks. As a result, the control group was confirmed that there was little change in the strain flora before starting the experiment. The first experimental group began to decrease from 2 weeks.

Lactobacillus showed no significant change in the control group until the beginning of experiment (0.4%), after 2 weeks (0.15%) and after 4 weeks (0.32%). In contrast, in the first experimental group, the ratio was significantly increased after 2 weeks (1.04%) and after 4 weeks (1.42%) in the T1 treatment group. However, it was not observed in Escherichia coli and Salmonella .

A chart showing the ratio in each strain ( Lactobacillaceae_uc ) belonging to the isolated Lactobacillus in the feces collected from each group is shown in FIG. Specifically, Figure 13a is a chart for the initial control group and the first experimental group (cont-i, T1-i), Figure 13b is a chart for the control group and the first experimental group (cont-14d, T1-14d) two weeks later 13C is a chart of the control group and the first experimental group (cont-28d, T1-28d) after 4 weeks. According to the results, each strain belonging to Lactobacillus showed a 5% dominance in the L. farciminis group in the control group , and significantly decreased after 2 weeks (4%) and 4 weeks (6%), whereas the L. hilardii group was initially 26 It was confirmed that the increase from% to 47% and 60%. L. panis group also increased from 5% to 32% and 27%.

In contrast, in the first experimental group , the L. hilardii and L. panis groups decreased by 49% and 25% to 25% and 6%, respectively, to half or less, and the L. farciminis group was 8% to 42%, respectively. It was confirmed that the increase by 5 times.

Claims (14)

And a yeast culture inoculated and cultured with a second yeast in a solid medium containing a yeast autolysate.
The yeast culture is an immune enhancing feed additive, characterized in that added to the animal feed based on solids 0.1 to 0.5% by weight. The method of claim 1,
Based on 1 part by weight of the feed additive, Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia kudriavzevii any one or more raw yeasts selected from Pichia kudriavzevii further comprising a feed additive for immuno-enhancing. The method of claim 1,
The feed additive for immuno-enhancing, characterized in that it further comprises 1 part by weight of yeast autolysate based on 1 part by weight of the feed additive. The method of claim 1,
The yeast autolysate is autoclave sterilized by the first yeast, or a feed additive for immune augmentation, characterized in that it is prepared by treating the hydrolase. The method of claim 4, wherein
The hydrolase is an immune enhancing feed additive, characterized in that the papain enzyme. The method of claim 4, wherein
The first yeast is an immuno-enhanced feed additive, characterized in that any one or more selected from Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia kudriavzevii . The method of claim 1,
The second yeast is the same or different from each other, Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia kudriavzevii characterized in that any one or more of the feed additive for immuno-enhancing. Feed comprising a feed additive for immuno-enhancing according to claim 1. (Iii) treating the first yeast with autoclave or hydrolase to produce a yeast autolysate; And
(Ii) inoculating and culturing a second yeast in a solid medium containing the yeast autolysate to obtain a yeast culture. The method of claim 9,
Wherein the first and second yeast are the same or different from each other, Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia kudriavzevii characterized in that any one or more of the production method of the feed additive for immuno-enhancing. The method of claim 9,
On the basis of 1 part by weight of the feed additive, Kluyveromyces marxianus, Saccharomyces cerevisiae and Pichia kudriavzevii any one or more raw yeasts selected from the production method of the immuno-enhanced feed additive, characterized in that it further comprises. The method of claim 9,
The autoclave treatment of step (iii) is carried out by autolysis through autoclaving for 100 to 150 ° C. for 10 to 30 minutes,
The (iii) step hydrolysis enzyme treatment is a method for producing an immuno-enhanced feed additive, characterized in that is carried out by incubation for 1 to 30 hours at 20 to 70 ℃ by adding 1 to 3 units / mg hydrolase. . The method of claim 12,
The hydrolase is a papain enzyme, characterized in that the method for producing an immune enhancing feed additive. The method of claim 9,
(Iii) a method for producing an immuno-enhanced feed additive, further comprising the step of lyophilizing the yeast culture.

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