KR20180013473A - Novel Lactobacillus casei DK128 strain having probioic activity for animal immune stimulation and method of producing fermented milk containing Opuntia Ficus indica using the same - Google Patents

Abstract

The present invention relates to a novel Lactobacillus casei DK128 strain having probioic activity and to a method for producing Opuntia Humifusa-fermented milk for immunization reinforcement of animals using the same, and in the present invention the Lactobacillus casei DK128 having excellent probioic activity, which is excellent in acid resistance, bile resistance, albumonoid degradation ability, β-glucosidase secretion ability and β-galactosidase secretion ability, was isolated. In addition, the Opuntia Humifusa-fermented milk for immunization reinforcement of animals produced by using the Lactobacillus casei DK128 strain has an effect of reinforcing immunization, and thus the present invention can provide very useful information to related industries.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel Lactobacillus casei strain DK128 having a probiotic activity, and a method for producing the same, which comprises using the Lactobacillus casei strain DK128 for producing an animal immunostimulatory fermented milk. same}

The present invention relates to a novel Lactobacillus casei strain DK128 having a probiotic activity, and a method for producing the fermented milk of animal origin for animal immunity enhancement.

Opuntia ficus indica is a native plant of the country, which is the origin of the Americas. The cactus that grows on the Korean Peninsula, namely Paekyunchozo cactus and Chuncheoncho cactus belongs to the genus Opuntia. The advantage of Cactus cactus is that it is very easy to cultivate. It grows on its own when a single broad stem is placed on the ground. If you give it properly, you do not need chemical fertilizer or pesticide. These cacti are about 15 stalks which can be harvested in one year, and 7 ~ 8 fruits can be harvested. However, there is a disadvantage that there is not much demand yet, but the stem can be used variously in food by removing thorns and bark. According to the results of clinical trials, these craniosaccharides have an excellent effect such as antioxidant, anti-whitening, anti-obesity, anti-inflammatory, dementia inhibition, cerebrovascular system, diabetes and other adult diseases and alcohol metabolism. In particular, flavonoids have been reported as anti-aging and anti-atopic effect, and rich natural vitamin C has been known to provide skin nutrition with a whitening effect due to its melanin pigment inhibiting function and various oils and minerals. As a result of antioxidant tests, the total amount of phenol content of licorice, licorice, cucumber, lentil, citric acid, and citric acid, which contains protein, carbohydrate, ash, fat, calcium, magnesium, iron, chromium, flavonoid, , And EDA (Electron donating ability), respectively. The content of vitamin C was 163.8mg% in the fruit and 71.2% in the stem. The content of flavonoids was higher than that of the native cactus (5%), yulmu (0.19%), mg, and it contains 13 times as much vitamin as oranges, so the weakness of cactus is already well known. It will be necessary to emphasize that food contains many nutrients and flavonoids. However, because of its high nutritional content and flavonoids, it has strong viscosity, and it has sticky and swollen properties when it comes into contact with water even in the state of dry powder.

Accordingly, the present invention provides a method for producing milky-rooted fermented milk having more useful effects by fermenting crude oil or skimmed milk powder with the addition of millet seeds to lactic acid bacteria isolated from the present invention. We want to provide an effective point.

Korean Patent Laid-Open Publication No. 2016-0034009 discloses a method for producing a liquid fermentation product using millennia, and Korean Patent Laid-Open Publication No. 2016-0066260 discloses a method for producing a natural fermented vinegar and a method for producing However, as described in the present invention, a novel Lactobacillus casei strain DK128 having a probiotic activity and a method for producing the fermented milk product for animal immunity enhancement using the same have not been disclosed at all.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a lactic acid bacterium derived from a traditional food, which is excellent in acid resistance, brittle ability, milk protein degradation ability,? -Glucosidase secretion ability and? -Galactosidase secretion ability Lactobacillus casei strain DK128 having probiotic activity was isolated and fermented by inoculating the strain with crude oil or skimmed milk powder added with T.O. seeds. As a result, antioxidant activity, antimicrobial activity and immunity were increased It was confirmed that the preparation of fermented milkshake oil was possible. In addition, the present inventors have completed the present invention by confirming that the immune function is particularly enhanced as a result of feeding the above-mentioned functional teaspoonful of fermented milk to livestock.

In order to solve the above problems, the present invention provides Lactobacillus casei strain DK128 having probiotic activity.

The present invention also provides a fermented milk composition for animal immunity enhancement, comprising the strain, a culture solution thereof, a concentrate of the culture solution or a dried product thereof.

In addition, the present invention provides a method for preparing fermented milk for animal immunity enhancement, comprising fermenting the strain, the culture solution thereof, the concentrate of the culture solution or the dried product thereof in the crude oil or skimmed milk powder to which the millennium seed is added.

The present invention also provides an animal immunomodulating fermented milk produced by the above method.

In addition, the present invention provides a feed additive composition for animal immunity enhancement comprising the fermented milky oil as an active ingredient.

In the present invention, Lactobacillus casei DK128 strain having a probiotic activity excellent in acid resistance, bile resistance, milk protein degradation ability,? -Glucosidase secretion ability and? -Galactosidase secretion ability was isolated . In addition, since the fermented milky oil for animal immunity prepared by using the Lactobacillus casei strain DK128 has an immunity enhancing effect, the present invention can provide very useful information to the related industry.

FIG. 1 shows the results of confirming the activity of? -Glucosidase of lactic acid bacteria isolated in the present invention through Escherichia coli test.
FIG. 2 shows the results of confirming the? -Galactosidase activity of the lactic acid bacteria isolated in the present invention.
FIG. 3 is a graph showing changes in total flavonoid content of fermented milk of animal origin for animal immunity, prepared by adding Lactobacillus casei DK128 isolated from the present invention, before and after fermentation.
FIG. 4 shows the results of analysis of changes in the total polyphenol contents before and after fermentation of Milkweed oil for animal immunity enhancement prepared by adding Lactobacillus casei DK128 isolated in the present invention.
FIG. 5 shows the results of analysis of antioxidant activity changes of fermented milk of animal origin for animal immunity prepared by adding Lactobacillus casei DK128 isolated from the present invention before and after fermentation.
FIG. 6 shows the results of analysis of changes in components (A) and (B) of fermented milk of animal origin for animal immunity enhancement prepared by adding Lactobacillus casei DK128 isolated in the present invention before fermentation.
FIG. 7 is a graph showing the change in total polyphenol content of the animal milk-enhancing fermented milk prepared by adding Lactobacillus casei DK128 isolated according to the present invention to the total amount of tannin seeds. 0.1% (w / v), 0.2%: 0.2% (w / v) addition, 0.3%: 0.3% (w / v) addition of powder at the beginning of the millennium.
FIG. 8 is a graph showing DPPH scavenging activity of antioxidative ability of the animal milk-enhancing fermented milk prepared by adding Lactobacillus casei DK128 isolated according to the present invention, according to the content of the perennial plant. 0.1% (w / v), 0.2%: 0.2% (w / v) addition, 0.3%: 0.3% (w / v) addition of powder at the beginning of the millennium.
FIG. 9 is a graph showing the antioxidative activity of FRP according to the content of TCE in an animal immunity enhancing fermented milk prepared by adding Lactobacillus casei DK128 isolated from the present invention. 0.1% (w / v), 0.2%: 0.2% (w / v) addition, 0.3%: 0.3% (w / v) addition of powder at the beginning of the millennium.
FIG. 10 shows the results of ABTS scavenging ability of the antioxidant activity according to the content of the perennial plant in the animal milker fermented milk prepared by adding Lactobacillus casei DK128 isolated in the present invention. 0.1% (w / v), 0.2%: 0.2% (w / v) addition, 0.3%: 0.3% (w / v) addition of powder at the beginning of the millennium.
FIG. 11 shows the results of analysis of the change in the viable cell counts according to the content of ternary-seed-starch in the animal immunostimulant fermented milk prepared by adding Lactobacillus casei DK128 isolated in the present invention. 0.1% (w / v), 0.2%: 0.2% (w / v) addition, 0.3%: 0.3% (w / v) addition of powder at the beginning of the millennium.

In order to achieve the object of the present invention, the present invention provides Lactobacillus casei strain DK128 having probiotic activity.

The Lactobacillus casei strain DK128 of the present invention is a strain excellent in acid resistance, bile acid resistance, milk protein degradation ability,? -Glucosidase secretion ability and? -Galactosidase secretion ability, and a strain having a probiotic activity Was isolated from traditional foods.

In addition, the present invention provides a composition for preparing animal fermented milk for animal immunity enhancement, comprising the strain, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof.

In the composition according to an embodiment of the present invention, the functionalities may be those having increased antioxidative activity, antimicrobial activity and immunity, but are not limited thereto.

In order to investigate the physiological activity of the milk protein prepared by fermenting crude oil or skimmed milk powder with addition of millet seeds added to the Lactobacillus casei strain DK128, the antioxidant activity, antimicrobial activity, As a result of the immunological test, it was found that the Milkweed fermented milk fermented with the Lactobacillus casei DK128 strain of the present invention has significantly higher antioxidative, antimicrobial and immunological properties than the Milkweed or skim milk powder not fermented by the strain of the present invention Respectively.

Therefore, the composition for preparing animal milkyacid fermented milk for animal immunity enhancement, which comprises the Lactobacillus casei DK128 strain, the culture solution thereof, the concentrate of the culture solution or the dried product thereof, has a remarkable antioxidative, antibacterial and immunological activity And thus can be usefully used for the development of milky oil fermented milk for animal immunity enhancement.

The Lactobacillus casei strain DK128 or Lactobacillus casei strain DK128 contained various substances produced by Lactobacillus casei strain DK128 and thus was included as an active ingredient in the composition for preparing animal milkshake fermented milk for animal immunity enhancement , The same effect as the composition containing the lactic acid bacteria can be exhibited.

The composition of the present invention may further comprise conventional pharmaceutical carriers and excipients in addition to the above-mentioned active ingredients. Such compositions may be prepared by heat drying or freeze-drying according to a conventional method for producing a probiotic composition, . The composition of the present invention may contain the active ingredient in an amount of 10 ⁵ to 10 ¹² cfu / g, preferably 10 ⁷ to 10 ¹¹ cfu / g, of the lactobacillus casei strain DK128, In the case of a culture of Lactobacillus casei strain DK128, it may be contained in an amount of 10 ⁶ to 10 ¹² ml / g, preferably 10 ⁷ to 10 ¹¹ ml / g.

In addition, the present invention provides a method for producing animal milieu fermented milk for animal immunity enhancement, comprising the step of fermenting the strain, the culture solution thereof, the concentrate of the culture solution or the dried product thereof into crude oil or skimmed milk powder to which millennium The present invention provides a fermented milky oil for animal immunity enhancement.

In the present invention, the inoculation and fermentation of the strain may be carried out according to a method commonly used in the art.

 In addition, the present invention provides a feed additive composition for animal immunity enhancement comprising the fermented milky oil as an active ingredient.

The above-mentioned strains are as described above and may be added as a feed additive composition for the purpose of improving immunity. The feed additive of the present invention corresponds to an auxiliary feed in the feed control method.

The feed additive composition of the present invention may be added to the feed and the term "feed" refers to any natural or artificial diet, meal, or the like ingredients for eating, ingesting, digesting, can do. The kind of the feed is not particularly limited, and feeds conventionally used in the art can be used. Non-limiting examples of such feeds include vegetable feeds such as cereals, muscle roots, food processing busines logistics, algae, fibers, pharmaceutical buses, oils, fats, pastes or grain by-products; Animal feeds such as proteins, inorganic substances, oils, fats, mineral oils, fats, single cell proteins, zooplankton or foods.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. For animal immunity enhancement Millennium  For the production of fermented milk Fermentation strain  Selection

1-1. Excellent ability to produce fermented milk for animal immunity enhancement

The 60 isolates from the Korean traditional food were tested for their basic physiological activity after isolation and identified. The acidity test using 36 HCl of the lactic acid bacteria listed in Table 1 below and the bile acid resistance test using the bile salts , Proteolytic activity and β-galactosidase activity, and Esculin agar test were conducted to select eight lactic acid bacteria.

Isolation and Identification of Lactic Acid Bacteria Isolated from Korean Traditional Foods of the Present Invention Sample name Species Sample name Species DK109 Lactobacillus plantarum DK119 Lactobacillus plantarum DK121 Lactobacillus paracasei DK128 Lactobacillus casei DK201 Lactobacillus plantarum DK202 Lactobacillus plantarum DK203 Lactobacillus plantarum DK204 Lactobacillus plantarum DK205 Lactobacillus brevis DK206 Lactobacillus plantarum DK207 Lactobacillus casei DK208 Lactobacillus plantarum DK209 Lactobacillus plantarum DK210 Lactobacillus plantarum DK211 Lactobacillus casei DK212 Lactobacillus casei DK213 Lactobacillus plantarum DK214 Lactobacillus plantarum DK215 Lactobacillus paracasei DK303 Lactobacillus plantarum DK304 Lactobacillus pentosus DK305 Leuconostoc citreum DK306 Weisella cibaria DK307 Weisella cibaria DK308 Weisella cibaria DK309 Weisella cibaria DK310 Lactobacillus sakei DK311 Lactobacillus sakei DK312 Lactobacillus sakei DK313 Lactobacillus sakei DK314 Weissella confusa DK315 Lactobacillus sakei DK316 Lactobacillus sakei DK317 Weisella cibaria DK318 Weisella cibaria

1-2. Confirmation of physiological activity of lactic acid bacteria derived from Korean traditional food and second selection

1-2-1. Acid Resistance of Lactic Acid Bacteria Derived from Korean Traditional Foods

DK101, DK102, DK104, DK107, DK109, DK119, DK121, DK128, DK207, DK211, DK212, DK215, and DK216 were cultured in MRS broth adjusted to pH 2.0 to measure the acid resistance of the isolated and identified lactic acid bacteria. DK301, and DK303, respectively, were maintained at the same level as that of culture at 0 hours.

1-2-2. Bile acid resistance of lactic acid bacteria derived from Korean traditional foods

To determine the bile acid tolerance of lactic acid bacteria, viability was determined after incubation for 2 hours at 37 ° C in MTS broth supplemented with 1.0% oxgall. A total of 32 DK strains, including DK128, DK207, DK211 and DK212, showed viability of 10 ⁷ (CFU / mL) or more in bile acid resistance of lactic acid bacteria.

1-2-3. Β-glucosidase activity through Esculin agar test of lactic acid bacteria derived from Korean traditional foods

In order to confirm β-glucosidase activity, which is used as an index of sugar availability of the selected lactic acid bacteria, Eskurin agar test was conducted by applying Agar well diffusion method as shown in FIG. As a result, β-glucosidase activity of DK109, DK119, DK121, DK128, DK207, DK211, DK212 and DK215 was confirmed by the generated circles around the microorganisms injected from Escherichia agar plates (FIG. 1) Lt; RTI ID = 0.0 > of-glucosidase < / RTI > Therefore, the strains except for the above DK strains were judged to have insufficient glucose utilization ability, and it was expected that the efficacy of TK was low.

1-2-4. The degradation activity of lactic acid bacteria derived from Korean traditional foods

Well diffusion method was applied to measure the lipoprotein resolution of the selected lactic acid bacteria. The activity of the selected lactic acid bacterium on the degradation of milk protein was confirmed by the generated rings around the bacteria injected on the skimmed milk agar medium.

As a result, DK119, DK121 and DK128 were found to be excellent in the degradation of milk protein. DK109 was found to be cultivated from 72 hours after culturing. The other strains except DK were found to have lower milk protein degradation rate than other selected strains.

1-2-5. Β-Galactosidase Activity of Lactic Acid Bacteria Derived from Korean Traditional Foods

To determine β-galactosidase activity of the selected lactic acid bacteria, β-galactosidase activity was measured using ONPG (Sigma, USA) solution after culturing in MRS broth. As a result, DK128, DK207, DK211 and DK212 were 100 unit / mL, and the activity of [beta] -galactosidase of the other strains except for the above DK strains was found to be lower than those of the other strains (FIG. 2).

1-3. 2nd selection of lactic acid bacteria derived from Korean traditional food

DK109, DK119, DK121, DK128, DK207, DK211, DK212, and DK215, which are expected to have superior pharmacological activities of natural products (millennials) due to their excellent physiological activity and β-glucosidase activity among 36 primary lactic acid bacteria A total of 8 species were selected second.

Example  2. Development of Optimal Culture Medium and Selection of Optimal Conditions for Selective Lactic Acid Bacteria

Biochemical characteristics and functionalities of L. acidophilus and DK212, DK211, DK211, and DK212 were investigated by applying JMO medium to DK fermented milk for animal immunity enhancement.

1 mL of each of the strains (DK128, DK207, DK211 and DK212) cultured in 50 mL of JMO medium for 24 hours at 37 ° C. was sampled at 0 h and 24 hours, decanted and counted with MRS agar medium Respectively. As a result, DK128 showed 3.85 × 10 ⁹ CFU / mL, DK207 showed 3.90 × 10 ⁹ CFU / mL, DK211 showed 2.89 × 10 ⁹ CFU / mL and DK212 showed 2.85 × 10 ⁹ CFU / mL after the completion of the culture. All of the 4 strains were found to be suitable for both growth and activity in JMO medium above 10 ⁹ CFU / mL.

Example  3. Millennium  Before and after fermentation Antioxidant ability  Change comparative analysis

After removing the thorns, they were crushed to a level of 10 ~ 20mm, and then lyophilized using a freeze drier, followed by pulverization using an industrial blender to prepare the powder. The preparation method of the fermented milk of the present invention is as described in the following Example 6.

Changes in total flavonoid contents were measured before and after fermentation. The total flavonoid content was measured by setting the absorbance of the UV / VIS spectrophotometer to 415 nm after methanol, 10% AlCl ₃ , 1 M potassium acetate and distilled water were added to the samples before and after fermentation. As a result, the total flavonoid content was higher in the fermented milk added with the perennial plant than in the control (non-fermented milk added at the beginning of the millennium) as described below. In particular, the fermented milk was fermented with DK128, The samples showed a higher flavonoid content than the control and the pre-fermentation samples (Fig. 3).

Changes in total polyphenol contents were measured before and after fermentation. After 0.2 M N-Folin reagent was added to the sample before and after fermentation, the sample was reacted at room temperature for 30 minutes, and 10% Na ₂ CO ₃ was added. After 1 hour at room temperature, the absorbance of the UV / VIS spectrophotometer was set to 700 nm, Phenol content was measured. As a result, there was no difference in the total polyphenol contents of the pre-fermented samples containing the perennials when comparing the pre-fermented control and the pre-fermented samples. However, the total polyphenol contents of the pre-fermented samples containing D- The total polyphenol content of the post-fermentation preparation was higher than that of the pre-fermentation sample (Fig. 4).

The changes of antioxidant ability before and after fermentation were measured. The DPPH reagent was added to the sample before and after the fermentation at the beginning of the millennium, and the reaction was carried out at 37 ° C for 30 minutes. The absorbance was measured by setting the absorbance of the UV / VIS spectrophotometer to 510 nm. As a result, there was no significant difference in the antioxidative activity between the pre-fermentation samples before fermentation and the control group before fermentation. However, the DPPH values after the fermentation with DK128, DK207, DK211 and DK212 were higher than those of the control and fermented samples 5).

In conclusion, TPC, TFC, and antioxidant activity after fermentation showed that antioxidant activity was enhanced by increasing flavonoids and phenolic components.

Example  4. Millennium  Comparative analysis of composition before and after fermentation

The changes of the components before and after fermentation were measured. The HPLC chromatograms of the fermented control and the samples before and after fermentation were compared. 3-O-β-glucoside (1) and isorhamnetin-3-O-β-glucosyl-4'-O-β-glucoside (2) Peaks appeared at 13.7 minutes and 14.5 minutes, respectively.

It was confirmed that the main saccharide before and after fermentation of the millennia early stage was glucose and the amount of glucose after fermentation was smaller than that before fermentation (FIG. 6). It was analyzed that DK128 strain was fermented by glucose during fermentation and fermented by millennia.

Example  5. Millennium  Comparative analysis of antimicrobial activity before and after fermentation

In order to compare the antimicrobial activity before and after the fermentation, four kinds of pathogenic microorganisms were compared with each other. Pathogenic microorganisms include E. coli KCTC 1682, S. aureus KCTC 3881, E. faecalis KCTC 2011, and B. cereus KCTC 3624, which are registered with KCTC And used for the experiment. The cultured strains were inoculated at 1% (v / v) level in TSB medium and cultured at 35 ° C for 24 h. Then, the supernatant of the samples was taken into TSA solid medium before and after fermentation and 200 μl was inoculated in paper well (10 mm) To compare the antimicrobial activity of the samples. As shown in Table 2 below, the antimicrobial activity of DK128 fermented samples was superior to that of the control, and after fermentation, the antibacterial activity against Bacillus cereus and Staphylococcus aureus was superior to that of the control.

Sample E. faecalis
KCTC 2011 B. cereus
KCTC 3624 S. aureus
KCTC 3881 E. coli
KCTC 1682 Control before fermentation + ++ + NE Before fermentation ++ ++ + + Millennium early after fermentation ++ +++ ++ +

The above results were compared with the antimicrobial activity against pathogenic bacteria before and after fermentation, and the antimicrobial activity against pathogenic bacteria was evaluated according to the size (1-2 mm: +, 2-4 mm: ++, 4-8 mm +++) , And NE has no effect on the antimicrobial activity.

Example  6. For animal immunity enhancement Millennium  Production of fermented milk (lactic acid bacteria) products

6-1. Establishment of optimum condition of fermented milk for animal immunity enhancement

6-1-1. Ingredients for animal immunity enhancement

A freeze dryer (OPERON, OPR-FDV-7012) of Dankook University-Industry-Academy Collaboration Center joint equipment room was installed in Petri dishes at 1AH, 12A, 10Hz , And lyophilized at -70 ° C for 24h. After shaking to an industrial blender, samples were stored in a -24 ° C freezer. Skim milk preparation (SMP8515, Schils Food, BV Holland) was used for the experiment. Skim milk powder for the production of lactobacillus was prepared from 'DEAHAN' Were used.

6-1-2. Setting the mixing ratio of Milky way fermented milk for animal immunity enhancement

Fermentation did not occur due to the pre - curd coagulation due to mucopolysaccharide in millennia during fermentation. Therefore, the fermented milk prepared by adding 0.1, 0.2, and 0.3% of milled millet was tested for its functionality and effectiveness. In addition, the fermented milk containing less than 0.2% of pulverized milkshield decreased the viability of the viable cell compared to 0.3%, and the effect thereof was insufficient, and the final compounding ratio was set as shown in Table 3 below. The Lactobacillus casei strain DK128 was inoculated at a concentration of 1 × 10 ⁶ to 1 × 10 ⁷ cfu / ml.

Raw materials Skim milk powder Functional Crystalline Glucose Crushed millennium Purified water Content (% by weight) 15 3 0.3 81.2

6-1-3. Total Polyphenol Contents and Antioxidant Capacity of Milkweed Cheongyangcho Fermented Milk for Animal Immunity Enhancement

The total polyphenol content of the fermented milky oil for animal immunity enhancement was analyzed (Fig. 7). After adding 0.2 M N-Folin reagent to the pretreated fermented milk samples, 10% Na ₂ CO ₃ was added to the sample which was reacted at room temperature for 30 minutes. After 1 hour at room temperature, the absorbance of the UV / VIS spectrophotometer was set to 700 nm, Phenol content was measured. The total polyphenol content tended to increase with increasing of the millenniace of fermented milk for animal immunity enhancement.

6-1-4. Analysis of Antioxidant Capacity (DPPH) according to the Contents of Milk Fermented Milk for Animal Immunity Enhancement

After the addition of 0.02 mM DPPH to the pretreated fermented milk samples, the reaction was allowed to proceed at room temperature for 30 minutes. The DPPH scavenging activity (%) was measured by setting the absorbance of the UV / VIS spectrophotometer to 517 nm. The DPPH scavenging activity (%) tended to increase significantly as the content of millenniace of fermented milk for animal immunity enhancement was increased (Fig. 8).

6-1-5. Analysis of Antioxidant Capacity (FRAP) by the Contents of Fermented Milk of Milk for Animal Immunity Enhancement

After 30min of FRAP solution and dark room in pretreated fermented milk samples, the absorbance of UV / VIS spectrophotometer was set to 593nm, and the FRAP scavenging activity (%) was measured to be a contrast value of troloc concentrate. There was no significant difference according to the content of the millennials of the fermented milk of the animal for the purpose of enhancing animal immunity, but the antioxidant activity was significantly higher than that of the control group without the addition of the millet (Fig. 9).

6-1-6. Analysis of antioxidant activity (ABTS) according to the content of fermented milk

The ABTS stock solution was added to the pretreated fermented milk samples and the reaction was allowed to proceed for 6 min at room temperature. The absorbance of the UV / VIS spectrophotometer was set at 734 nm, and the ABTS scavenging activity (%) was measured. The ABTS scavenging ability of fermented milk for animal immunity enhancement tended to increase with increase of the perennial content (FIG. 10).

6-1-7. Changes of viable cell counts during storage period according to fermented milk content of Milkweed for animal immunity enhancement

During the storage period of the prepared animal immunostimulatory fermented milk, the number of viable cells was measured at intervals of 7 days for 56 days. As a result, it was confirmed that the fermented milk containing 0.3% Respectively. It was analyzed that this would contribute greatly to the deterioration of the product during distribution and the shelf life and stability of the product in the probiotic preparation to be developed in the second year (Fig. 11).

Example  7. Prototype animal milkshake fermented milk for animal immunity enhancement Pigeon  Specification payroll experiment

The experiment was conducted from April 1, 2016 to June 30, 2016, to confirm the animal immunity enhancement effect of fermented milk of Cheonanseoncho. To evaluate the productivity of weaned pigs and the growing pigs by adding fermented millenniums to pig feed, measuring the development of diarrhea and respiratory diseases in pigs, measuring the immune enhancement effect, Respectively.

7-1. Test Animals and Experimental Design

A total of 240 breeding pigs (Yorkshire × Landrace) × Duroc) with an average body weight of 7.22 ± 0.04 kg were randomly assigned to 4 weeks of postweaning (4 weeks) and 8 weeks of experimental design. In total 3 treatments 4 replicates were placed in randomized complete block design (RCBD) according to body weight by 20 rats per pig. The treatments were as follows: 1) Con: Control 2) T1: feed supplemented with 1% pig root 3) T2: feed with 1%

Item No. of replication No. of Pigs Total Con ^* 4 20 80 T1 ^* 4 20 80 T2 ^* 4 20 80 Total 240

1) Con: Control 2) T1: Feed 1% of pig root 3) T2: Feed of 1% fermented milk

7-2. Spec Management

Con was fed with conventional diets, T1 was fed with 1% pig root in control diet, and T2 was fed with 1% of fermented milk. Feeds were fed automatically, and water was freely vegetarianized by an automatic water dispenser. Other specifications were controlled according to the standard specification management method of the poultry farm.

7-3. Survey items and methods

From the beginning of the experiment, we measured body weight and feed intake once a week and calculated ADG, ADFI and G: F ratio. Respectively. Feed intake was calculated by measuring the total amount of feed and residual amount for each measurement, and the daily gain was measured using a pig type (mobile pig weight only scales). The feed conversion ratio was calculated by dividing the feed intake during the test period by the weight gain. For all experimental groups, the clinical symptoms were observed at 10-11 am twice a week, and diarrhea symptoms and respiratory symptoms were measured by observing diarrhea occurrence and respiratory symptoms. For the measurement of harmful substances in the poultry, 10 pcs of feces were collected from 10 pigs per treatment per week while observing the clinical signs at 10-11 am. 100 g of feces were collected from the pigs, and they were dispensed into plastic containers and analyzed using Gastec (Model GV-100, GASTEC , Japan) was used to measure the ammonia concentration and hydrogen sulfide concentration.

7-4. Blood chemistry and IFN-r and TNF-a, IL-6 measurements in pigs

To investigate the effects of fermented milk on the immune formation of porcine pigs, 300 weeds of 24-25 day old were weighed and randomly selected at 20 - week intervals. Blood was collected in EDTA tubes and centrifuged. The serum levels of IFN-r and TNF-a and IL-6 in serum were measured by centrifugation. The results were as follows: And 8 weeks, respectively, to measure blood levels (IDDEX ELISA kit).

7-5. productivity

However, the daily gain of body weight was 523.2g in the treatment of fermented milky oil, which was higher than that of the control and fermented milk treatments. Feed efficiency was the highest in T2 (fermented milk) treatment group, and the control group and pig root treatment group were relatively lower than yogurt treatment group (P <0.05). As a result, the effect of the fermented milk on the growth of the pigs was effective in the growth of the pigs fed the piglets containing the fermented milk containing the fermented milks and the fermented milk, And the feed efficiency was higher than the control group. The body weight at the end of the experiment was also higher in the fermented milk and the kiwifruit compared to the control (Table 5).

Con ^* T1 T2 SE ^** Daily gain (g) 465.7 487.9 523.2 29.00 Daily dietary intake (g) 1321.5 1301.3 1284.5 18.53 Feed efficiency 0.35 0.37 0.41 0.08

1) Con: Control 2) T1: Feed 1% of pig root 3) T2: Feed of 1% fermented milk

** Pooled standard error

In the control group, the body weight gain was significantly lower at 4th and 5th weeks of gestation but tended to recover from 6th week, which is related to the expression of digestive and respiratory symptoms between 4-5 weeks. In the fermented milk treated group, the daily gain of body weight was steady, and it was analyzed that the pigs' disease defense effect was exhibited in the late and late growing period. The effects of dietary supplementation of fermented milk and fermented milk on the productivity of pigs were the highest in the control diet but not in all treatments. The feed efficiency was higher in the fermented milk treatment group and the fermented milk treatment group than in the control group. Especially, in the fermented milk treatment group, And fermented oil treatment group.

7-6. Protective Effect of Piglet on Disease

Table 6 and Table 7 show the results of comparing diarrhea and respiratory symptom incidence for the weaning pigs. As shown in Table 6, the addition of fermented milk and teaspoonful of fermented milk to weaned diets showed the lowest incidence of diarrhea in the fermented milks and the lowest value in fermented milk treatments (P = 0.05). This showed that the growth ability of T2 fermented milk (T2) was the highest in the specification experiment. However, in the case of fermented milk products, there were no consistent results comparable to the specifications. In addition, as shown in Table 7, the incidence of respiratory symptoms was lower than that of digestive symptoms. Although the incidence of respiratory symptoms was lower in both the millennia and the fermented milk treatments than in the control, the feeding of fermented milk at the beginning of the millennium was not enough to block respiratory diseases.

Item No. of pigs No. of Pigs with diarrhea One 2 3 4 5 6 7 8 Con ^* 80 9 7 8 5 4 2 3 3 T1 ^* 80 9 4 2 One 2 One One 2 T2 ^* 80 10 2 One 0 0 0 One 0

1) Con: Control 2) T1: Feed 1% of pig root 3) T2: Feed of 1% fermented milk

Item No. of pigs No. of Pigs with respiratory disease One 2 3 4 5 6 7 8 Con ^* 80 8 7 5 4 7 6 5 6 T1 ^* 80 7 4 5 4 6 5 4 5 T2 ^* 80 8 5 4 3 4 3 3 3

1) Con: Control 2) T1: Feed 1% of pig root 3) T2: Feed of 1% fermented milk

7-7. Effects of Milk Yogurt on the Generation of Toxic Gasses in Feces

Table 8 shows the results of examining the amount of noxious gas generated in pigs by collecting 2 times of feces at intervals of 4 weeks while feeding 8 weeks of diets supplemented with millennium and fermented milk. First, the amount of ammonia (NH ₃ ) produced was 11.2 ppm and 9.6 ppm in fermented milk and T1 and T2 treatments, respectively, compared with the control (p <0.05). Fermented milk was also effective in inhibiting harmful gas .

Item Con ^* T1 T2 medium Standard Deviation Ammonia (ppm) 16.3 13.6 11.2 13.70 0.82 17.6 11.2 9.6 12.80 0.68 Hydrogen sulfide
(ppm) 9.6 7.8 6.7 8.03 0.52 10.3 8.1 5.8 8.07 0.47

1) Con: Control 2) T1: Feed 1% of pig root 3) T2: Feed of 1% fermented milk

On the other hand, hydrogen sulfide (H ₂ S) gas showed 8.1 ppm and 5.8 ppm for T1 and T2 treatments, respectively. As a result of the analysis of noxious gas in the feces of pigs, oral administration of fermented milk of Chunjueseong was evaluated to be effective for suppressing the generation of harmful gas in pig farms by reducing ammonia and hydrogen sulfide in pig manure. Dietary control through dairy products has the potential to help manage and prevent obesity and is believed to be a component of preventive and therapeutic substances that promote health. Therefore, active and continuous research should be conducted so that milk and dairy products can be utilized as functional food and ingredients of this food.

7-7. Effects of Milk Yogurt on Immune Response

In order to investigate the effects of millions of milligrams per day on the immune response of pigs when they were absorbed through fermented milk, blood samples were collected two weeks after feeding the experimental feeds and TNF-a, INF- (Table 9). In addition, it was confirmed that the pigs could effectively inhibit inflammation. TNF-a, INF-γ and IL-6 were measured in blood, and it was confirmed that anti-oxidant activity and anti-inflammatory effect, which inhibits the production of inflammatory mediators, Previous studies have shown that feed efficiency and feed efficiency are improved by feeding probiotics, enzymes, yeast, and antibiotics from weaned pigs to beef cattle and proved as a growth promoter of probiotics. Feeding of the compound probiotics containing millet seeds to the finishing pigs increased the weight gain and feed efficiency as well as decreased the production of NH ₃ and H ₂ S gases, thus improving the breeding environment.

Cytokine Con ^* T1 T2 SE ^** IL-6 (pg / ml) 37.8 42.5 44.2 0.21 TNF-a (pg / ml) 93.7 74.3 51.7 0.24 INF-y (pg / ml) 87.2 76.4 65.7 0.17

1) Con: Control 2) T1: Feed 1% of pig root 3) T2: Feed of 1% fermented milk

** Pooled standard error

7-8. Summary of animal test results

This experiment was carried out to investigate the effect of addition of fermented milk and fermented milk on the productivity of piglets, the defense effect of pigs, and the effect of ammonia and hydrogen sulfide production on manure. Experiments were carried out in three treatments: control (C), control diet (T1) supplemented with 0.1% pig root, and T2 supplemented with 0.1% natural hyper fermented milk. A total of 240 pigs were weighed. The results of the experiment showed that the weight gain and feed intake were significantly (P <0.05) higher in the fermented milk treatments than in the control (P <0.05), and the gain of the fermented milk treatments was slightly improved compared to the control (C). Feed conversion ratio did not show significant difference in all treatments. The incidence of diarrhea in the fermented milk treatment group was the lowest in the fermented milks and the lowest in the fermented milk treatments (P = 0.05). The amount of ammonia gas production was lower in both fermented milk and milled peach fermented milk than in the control, indicating that fermented milk can reduce the rate of ammonia gas production.

In conclusion, the addition of complex enzymes and probiotics improves the productivity of weaned piglets and tends to suppress the production of ammonia gas. As a result, the fermented milk of the present invention has the effect of maintaining the function of the normal digestive organisms that secrete the digestive enzymes, thereby improving the nutrient utilization value of the feed, and increasing the weight gain and feed efficiency of the weaning pigs and growers. In addition, the continuous intake of fermented milk at the beginning of the millennium showed antioxidant and antiinflammatory effects compared to the general fermented milk, thereby further lowering the incidence of diarrhea and respiratory diseases of the weaning pigs and the breeding piglets and reducing the amount of ammonia and hydrogen sulfide gas contained in the feces and urine, It also contributes to solving the environmental pollution problem around the pig farm by showing the effect on the environment improvement.

Claims (7)

Lactobacillus casei strain DK128 with probiotic activity. The strain of claim 1, wherein the strain has acid-fast, brittle, milk protein degrading ability,? -Glucosidase releasing ability and? -Galactosidase releasing ability. A composition for producing animal milky oil for fermented milk for animal immunity, which comprises the strain of claim 1 or 2, a culture thereof, a concentrate of the culture or a dried product thereof. 4. The composition of claim 3, wherein the functionality is increased antioxidant, antimicrobial, and immunity. A method for producing an animal immunity enhancing fermented milk for animal immunization comprising the step of fermenting the strain of claim 1 or 2, the culture liquid thereof, the concentrated liquid of the culture liquid or the dried product thereof into crude oil or skim milk powder to which millenniace is added. 6. An animal immunity enhancing milky fermented milk produced by the method of claim 5. A feed additive composition for animal immunity enhancement comprising the fermented milk of the first aspect of the present invention as an active ingredient.

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