KR20210067850A - Feed additive for preventing diarrhea of calf comprising mixture of Bacillus subtilis, saccharide and mineral as effective component and uses thereof - Google Patents

Abstract

The present invention relates to a feed additive for preventing diarrhea in calves containing a mixture of Bacillus subtilis, saccharides and minerals as an active ingredient, and to uses thereof. The present invention provides a mixture of Bacillus subtilis, saccharides and minerals, which are excellent in preventing diarrhea in calves, as a feed additive, thereby preventing misuse of antibiotics and minimizing growth retardation and death of calves due to diarrhea and being very useful in livestock related fields.

Description

Feed additive for preventing diarrhea of calf comprising mixture of Bacillus subtilis, saccharide and mineral as effective component and uses thereof

The present invention relates to a feed additive for preventing diarrhea in calves containing a mixture of Bacillus subtilis, saccharides and minerals as an active ingredient, and a use thereof.

Due to the recent increase in feed prices and increased sales of imported meat, the difficulties of farmers raising Korean beef are increasing. Among these farms, it can be said that newborn calf mortality causes additional damage on breeding cattle farms. Diarrhea is the most important cause of calf death due to various causes, and calf death and poor growth are considered to be one of the important causes of damage to farms.

Diarrhea is a condition in which the passage time of food (digested) through the digestive tract is shortened or water absorption is decreased, resulting in an increase in the amount of water in the feces and an increase in the amount and frequency of feces. Diarrhea disease is a body's rejection of food, that is, a kind of defense mechanism in the body, but if it persists, dehydration, metabolic disorder, malnutrition, and body temperature drop occur, leading to death.

Diarrhea disease can be divided into infectious diarrhea and non-infectious diarrhea according to the presence or absence of infection with pathogenic microorganisms. Prevention and treatment of infectious diarrhea are important, but prevention of non-infectious diarrhea is also an important factor because non-infectious diarrhea can be converted into infectious diarrhea when it occurs. Non-infectious diarrhea occurs when there is a problem with feeding management. In case of poor quality of the mother cow or when substitute oil is supplied, diarrhea may occur depending on rapid replacement, quality, feeding amount, feeding temperature, etc. A sudden change in the breeding environment causes diarrhea by disrupting the secretion of digestive enzymes and the normal activity of microorganisms in the digestive tract. Prevention of non-infectious diarrhea through microorganisms is thought to have a great effect on preventing the penetration of pathogenic bacteria due to the increase of beneficial microorganisms in the intestines of calves and reducing mortality.

Newborn calves account for 75% of body water, and when diarrhea is severe, calves lose 13-18% of their body weight. In addition to water loss, electrolyte loss occurs, mainly sodium (Na ⁺ ) and potassium (K ⁺ ) loss. Therefore, in order to reduce the mortality rate of calves, proper electrolyte and water supply to calves with diarrhea is very important.

The importance of intestinal microbes in newborn calves is that if antibacterial agents are administered when diarrhea occurs, the primary causative bacteria can be killed, but harmful bacteria that do not die in antibacterial agents existing in the intestinal tract proliferate due to diarrhea, exacerbating symptoms and prolonging diarrhea. Therefore, it is most urgent to normalize the function of the intestine by establishing beneficial microorganisms in the intestine.

Therefore, in the present invention It is intended to prevent dehydration and acidosis by fusion of complex microorganisms and electrolytes for a sufficient supply of electrolytes (minerals) and for the normal settlement of intestinal microbes, and to prevent damage from diarrhea through hydration.

On the other hand, Korean Patent No. 1130989 discloses 'a method for producing a feed additive having antidiarrheal activity in calves containing natural herbal medicines', and Korean Patent No. 0951273 'contains bamboo leaf extract as an inhibitor of livestock diarrhea. It discloses a method for manufacturing a microbial active solution feed additive. However, the 'feed additive for preventing diarrhea in calves containing a mixture of Bacillus subtilis, saccharides and minerals as an active ingredient and use thereof' of the present invention has not yet been disclosed.

The present invention has been derived from the above needs, and the present inventors have isolated and identified Bacillus subtilis SCJMB22 and SCJMB35 strains, which are excellent in enzyme activity and antibacterial activity against harmful bacteria, from traditional foods, and improve diarrhea E. coli, rotavirus, coronavirus, Cryptosporidium and Giardia pathogens that cause diarrhea in calf feces when a mixture of the two strains is added to the complex sugar containing the electrolyte and fed to calves. By confirming that is not detected, the present invention was completed.

In order to solve the above problems, the present invention provides a feed additive for preventing or improving diarrhea in calves containing a mixture of Bacillus subtilis, saccharides and minerals as active ingredients.

In addition, the present invention provides a method for producing a feed additive having antidiarrheal activity in calves, comprising the step of adding Bacillus subtilis to a mixture of sugars and minerals.

The present invention provides a mixture of Bacillus subtilis, saccharides and minerals, which is excellent in preventing diarrhea in calves, as a feed additive, thereby preventing misuse of antibiotics and minimizing growth retardation and death of calves due to diarrhea. can be very useful for

1 is a measurement of the number of viable cells when culturing each of the Bacillus sertilis SCJMB22 (A) and Bacillus sertilis SCJMB35 (B) strains of the present invention in the medium (M1 to M16) prepared for each content of glucose and yeast extract. It is the result.
2 is a result of measuring the number of viable cells when the Bacillus sertilis SCJMB22 strain of the present invention is cultured in the medium (E1 to E10) prepared for each content of other nutrient sources. CO: TSB medium.

In order to achieve the object of the present invention, the present invention provides a feed additive for preventing or improving diarrhea in calves containing a mixture of Bacillus subtilis, saccharides and minerals as active ingredients.

In the feed additive for preventing or improving diarrhea in calves of the present invention, the Bacillus subtilis may have enzyme activity and antibacterial activity against harmful bacteria, preferably protease and amylase activity and Escherichia coli and Salmonella dublin ( salmonella dublin ) may have antibacterial activity, but is not limited thereto.

In addition, the saccharides may be monosaccharides, disaccharides, polysaccharides, or mixtures thereof, preferably a mixture of monosaccharides, disaccharides and polysaccharides, and more preferably glucose, sucrose, beta-glucan and levan, It is not limited thereto.

The feed additive of the present invention contains a mixture of Bacillus subtilis and saccharides having protease and amylase activity and antibacterial activity against Escherichia coli and Salmonella dublin, and mineral components such as potassium and sodium as active ingredients, so it has an effect of improving diarrhea symptoms.

The feed additive of the present invention includes auxiliary components such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and other live microorganism preparations; grains such as milled or crushed wheat, oats, barley, corn and rice; plant protein feeds, such as those based on rape, soybean and sunflower; animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Dry ingredients consisting of sugar and dairy products, for example, various powdered milk and whey powder; lipids, for example main components such as animal fats and vegetable fats optionally liquefied by heating; Additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease prevention agents may be further included.

The feed additive of the present invention may be in the form of a liquid formulation, and may include an excipient for adding feed. Examples of the excipient for feed addition include, but are not limited to, calcium carbonate, wheat flour, zeolite, jade flour, or rice bran.

The feed additive of the present invention may further comprise one or more enzyme agents. For example, lipolytic enzymes such as lipase, phytase that breaks down phytic acid to produce phosphate and inositol phosphate, and alpha-1,4-glycosidic bond (α-1) contained in starch and glycogen Amylase, an enzyme that hydrolyzes 4-glycoside bonds, phosphatase, an enzyme that hydrolyzes organophosphate esters, maltase that hydrolyzes maltose into two molecules of glucose It may contain a converting enzyme, etc. to make a toss mixture.

The feed additive of the present invention may be administered to calves alone or in combination with other feed additives in an edible carrier. Typically, a single daily intake or divided daily intake may be used as is well known in the art.

The present invention also provides a method for preparing a feed additive having antidiarrheal activity in calves, comprising the step of adding Bacillus subtilis to a mixture of sugars and minerals.

Preferably,

1) mixing and heating sucrose, glucose and water;

2) adding potassium chloride and sodium hydrogen carbonate after step 1);

3) after step 2), adding and mixing beta-glucan, levan and glutathione; and

4) adding Bacillus subtilis to the mixture obtained in step 3); may be a method comprising,

more preferably

1) mixing and heating sucrose, glucose and water in a weight ratio of 1-6:1-6:1-6;

2) adding 0.05 to 1% by weight of potassium chloride and 0.05 to 1% by weight of sodium bicarbonate after step 1);

3) after step 2), 0.01 to 0.1 wt% of beta-glucan, 0.05 to 1 wt% of levan, and 0.01 to 0.1 wt% of glutathione are added and mixed; and

^{4) adding 10 6-8} CFU/mL of Bacillus subtilis to the mixture obtained in step 3); may be a method comprising,

most preferably

1) mixing and heating sucrose, glucose and water in a weight ratio of 2:1:1;

2) adding 0.2% by weight of potassium chloride and 0.2% by weight of sodium hydrogen carbonate after step 1);

3) after step 2), adding and mixing 0.05% by weight of beta-glucan, 0.1% by weight of levan and 0.05% by weight of glutathione; and

^{4) adding 10 7} CFU/mL of Bacillus subtilis to the mixture obtained in step 3) above; may be a method comprising, but is not limited thereto.

Hereinafter, an embodiment of the present invention will be described in detail. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Isolation and selection of strains

(1) Isolation of the strain

In order to select food-derived Bacillus with excellent antibacterial activity, 10 g of each sample was diluted in 90 ml of sterile physiological saline using samples such as domestic traditional sauce and fermented kimchi. As a medium for isolating Bacillus, the same sample was smeared on TSB (Tryptic Soya Broth) solid medium supplemented with 1.5% agar, and Bacillus was isolated through shape observation. Candidate strains were cultured by stroke several times, and purified by morphology observation to isolate Bacillus bacteria. For the isolated strains, Bacillus was named with SCJMB numbers, respectively. The purely isolated strains were collected through liquid culture and centrifugation, and Bacillus was preserved through 25% (v/v) glycerol and used in the following examples.

(2) Enzyme activity evaluation

To verify the enzyme activity, an agar well diffusion method was used, and a selection medium containing the components of the substrate reacting specifically with each enzyme was prepared. Minimum medium containing 2% agar and 1% starch soluble to evaluate amylase activity, minimum containing 1.5% agar and 2% skim milk to evaluate protease activity The medium was sterilized at 121° C. for 15 minutes, and 25 ml of each was dispensed in a fixed amount in a Petri dish to prepare a solid medium. Bacillus was cultured in TSB broth at 37° C., shaking at 140 rpm for 24 hours. After centrifugation of the culture medium, the supernatant was taken and filtered with a 0.2 μm syringe filter, and then used for the experiment. 10 μl of the strain supernatant was dispensed into each enzyme activity measurement medium at intervals of 1 cm, and cultured in an incubator at 37° C. for 24 hours. After 24 hours, the clear zone that appeared in the minimal medium containing 2% skim milk and the clear zone that appeared by dispensing the Lugol solution into the minimal medium containing 1% starch were checked. 84 strains of Bacillus were first selected (data not shown).

For the selection of secondary strains with excellent enzyme activity, each of the minimum medium for enzyme activity was punctured to prepare wells with a diameter of 6 mm and used in the experiment. In the same manner as above, 100 μl of the culture supernatant of the primary selection strain was dispensed into perforated wells and cultured in an incubator at 37° C. for 24 hours. After 24 hours, the amylase activity was evaluated by measuring the diameter of the clear zone by dispensing Lugol's solution into a minimal medium containing 1% starch. In addition, after 24 hours, the diameter of the clear zone in the minimal medium containing 2% skim milk was measured to evaluate the protease activity, and according to the result, 40 strains of Bacillus were secondarily selected (Table 1).

Bacillus enzyme activity result SCJMB Enzyme activity (clear zone, diameter mm) SCJMB Enzyme activity (clear zone, diameter mm) protease activity amylase activity protease activity amylase activity 2 22 21 40 25 21 3 23 20 43 25 20 6 23 20 45 22 16 8 22 21 47 24 20 9 23 21 52 24 21 10 22 19 55 21 19 11 21 20 56 25 15 13 24 26 57 23 14 17 21 18 59 19 12 20 21 21 60 25 14 22 25 25 62 27 14 25 11 14 64 19 18 27 24 22 66 20 13 28 20 18 67 18 16 29 22 20 69 19 19 31 20 24 74 22 13 35 28 23 76 21 17 36 26 22 78 19 15 37 22 21 80 18 18 38 24 21 82 19 21

(3) Antimicrobial activity evaluation

Antibacterial activity was evaluated for the secondary selection strain (Bacillus 40 strain) with excellent enzyme activity. For the measurement of antibacterial activity, E. coli K99 strain and Salmonella dublin ATCC14580 strain, which are harmful microorganisms causing intestinal diarrhea in cattle, were subjected to a third selection through Agar Diffusion Assay. . TSB (Tryptic Soy Broth) was used as a growth medium for two harmful microorganisms. A TSB liquid medium was prepared, and inoculated with shaking at 37° C. for 24 hours at 140 rpm. OD (optical density) was measured using an absorbance meter, and ^{diluted to OD 0.4-0.5 (about 10 7} cfu/g). Two diluted harmful microorganisms were smeared on a solid medium, a paper disk was placed, and 50 μl of the culture supernatant of 40 strains of the secondary selection strain Bacillus was absorbed twice twice and cultured in an incubator at 37° C. for 24 hours. After 24 hours, the diameter of the clear zone was measured, and Bacillus 5 strains having antibacterial activity were selected a third time (Table 2).

For the selection of quaternary strains with excellent antibacterial activity, NA (Nutrient Agar) medium was prepared as a bottom medium, and 2 types of harmful microorganisms cultured at an OD value of 0.4~0.5 in TSA soft solid medium with 0.7% agar added were 25ml each. each was busy. 100 μl of the culture supernatant of the perforated and selected strain was injected into the perforated hole and cultured at 37° C. for 12 hours. After 12 hours of incubation, the diameter of the clear zone was measured, and the results were analyzed to finally select 2 strains of Bacillus (SCJMB22, SCJMB35) with excellent enzyme activity and antibacterial activity. The final selected two types of Bacillus (SCJMB22, SCJMB35) were entrusted to Macrogen for identification, and the two types of Bacillus were found to be Bacillus subtilis, and finally Bacillus subtilis SCJMB22, Bacillus It was designated as Sertilis SCJMB35.

Bacillus (SCJMB) antibacterial activity results SCJMB Antibacterial activity (clear zone, diameter mm)
S. dublin E. coli 13 11 11 22 13 12 27 10 11 35 12 11 36 11 11

Example 2. Establishment of the culture medium composition of the selection strain

As a medium for high-concentration culture of strains using industrially applicable raw materials, glucose (hydrous glucose_CJ Cheiljedang) and yeast extract (Angel yeast co.) were selected and used as raw materials, not for reagents. As shown in Table 3 for high-concentration culture of the selection strain, the growth pattern of the bacteria was analyzed by varying the contents of glucose and yeast extract. 1% of the SCJMB22 and SCJMB35 strain cultures were inoculated into 100 mL of TSB liquid medium and used as a pre-culture solution. 100 μl of the culture solution of the two strains was dispensed in a 96-well microplate, and the OD (optical density) corresponding to the culture before incubation at 600 nm (0 hours) was measured using a microplate reader. Cultures of the two strains before sampling were incubated at 37° C. and 140 rpm using a shaking incubator. For the SCJMB22 and SCJMB35 strains, cultured in TSB medium as a control were cultured for each condition, and the number of bacteria was measured through step dilution. As a result, when the content of glucose, the carbon source, was less than 1%, the number of viable cells increased as the content of yeast extract increased, and when the content of the carbon source was less than 1%, the number of viable cells increased. seemed ^{Both strains showed the highest number of viable cells at 1.1×10 9} CFU/mL in M13 medium under the conditions of 3% glucose and 0.5% yeast extract, which was higher than that in TSB medium as a control ( FIG. 1 ).

In addition, as shown in Table 4, the medium composition for other nutrient sources was established and the number of viable cells for each condition was measured. As a result, the highest viable bacteria in the medium composition condition of E10 containing 0.1% each of magnesium sulfate heptahydrate, potassium phosphate monobasic and potassium phosphate dibasic as other nutritional sources. The number was measured ( FIG. 2 ).

Medium composition according to glucose and yeast extract content (%) ingredient content M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 C0 Glucose 0.5% + + + + - - - - - - - - - - - - TSB
One% - - - - + + + + - - - - - - - - 2% - - - - - - - - + + + + - - - - 3% - - - - - - - - - - - - + + + + Yeast
extract 0.5% + - - - + - - - + - - - + - - - One% - + - - - + - - - + - - - + - - 2% - - + - - - + - - - + - - - + - 3% - - - + - - - + - - - + - - - +

Medium composition according to the content of other nutrients (%) ingredient E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 M13 C0 Glucose 3 3 3 3 3 3 3 3 3 3 3 TSB yeast extract 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 sodium chloride 0.5 0.5 Magnesium sulfate heptahydrate 0.1 0.1 0.1 0.1 Ammonium sulfate 0.2 Potassium phosphate monobasic 0.2 0.1 0.1 0.1 Potassium phosphate dibasic 0.2 0.1 0.1 0.2 0.1 Ammonium phosphate dibasic 0.2

Example 3. Diarrhea improvement effect of a mixture of Bacillus subtilis, saccharides and minerals in calves

Sucrose, glucose and purified water were mixed in a ratio of 2:1:1 and heated at 100° C. to a non-browning level, and then the temperature was slowly lowered to prevent coagulation of sugars. Thereafter, 0.2 wt% of potassium chloride and 0.2 wt% of sodium bicarbonate were added for mineral function, and the polysaccharides beta-glucan, levan and glutathione were added to the mixture of monosaccharides, disaccharides and minerals by 0.05 wt%, 0.1, respectively. After mixing by adding and mixing wt% and 0.05 wt%, a complex sugar was prepared. After that, the Bacillus subtilis SCJMB22 and SCJMB35 strains finally selected in Example 1 were cultured in the final medium composition confirmed in Example 2, Bacillus subtilis SCJMB22 and SCJMB35 strain culture medium (each 10 ⁷ CFU/mL) was added to the complex sugar mixture to finally prepare a mixture of Bacillus subtilis, saccharides and minerals. In order to confirm the diarrhea improvement effect of the mixture of Bacillus subtilis, saccharides and minerals of the present invention, after oral administration (20mL) to calves once every 5 days a total of 2 times, a fecal test was performed with 5 diagnostic kits. As a result, as shown in Table 5, it was confirmed that Escherichia coli, rotavirus, coronavirus, Cryptosporidium and Giardia pathogens that cause diarrhea in calf feces were not detected.

fecal test results Bacillus subtilis, a mixture of sugars and minerals before administration after administration coli detection not detected rotavirus detection not detected coronavirus detection not detected Cryptosporidium detection not detected Giardia detection not detected

In addition, after oral administration (20mL) of the mixture of Bacillus subtilis, saccharides and minerals of the present invention to calves once every 5 days, four antibiotics (amoxacillin, azith The frequency of use of mycin, mabofloxacin, buta D) and antidiarrheal drugs (carbofulbit) was measured. As for the number of times of antibiotic use, in the control group that did not feed the mixture of Bacillus subtilis, saccharides and minerals of the present invention, a total of 64 times were used for 2 months, 41 times at 1 month and 23 at 2 months of age, while the mixture of Bacillus subtilis, saccharides and minerals of the present invention was used. In the experimental group fed with , 28 doses at 1 month and 16 doses at 2 months of age were used a total of 44 times over 2 months, confirming that the frequency of use of antibiotics in the experimental group was significantly reduced compared to the control group. In the control group that did not feed the mixture of Bacillus subtilis, saccharides and minerals of the present invention, the antidiarrheal agent was used 98 times at 1 month old and 28 times at 2 months old, a total of 126 times for 2 months, whereas the mixture of Bacillus subtilis, saccharides and minerals of the present invention was used. In the experimental group fed with , 50 doses at 1 month and 6 doses at 2 months of age were used a total of 56 times over 2 months, confirming that the frequency of antidiarrheal use in the experimental group was significantly reduced compared to the control group (Table 6).

Measure the frequency of antibiotic and antidiarrheal use control experimental group Antibiotic 1 month Episode 41 Episode 28 2 months Episode 23 Episode 16 gun Episode 64 Episode 44 antidiarrheal 1 month Episode 98 50 episodes 2 months Episode 28 6 episodes gun Episode 126 Episode 56

Claims (6)

A feed additive for preventing or improving diarrhea in calves containing a mixture of Bacillus subtilis, saccharides and minerals as an active ingredient. The feed additive for preventing or improving diarrhea in calves according to claim 1, wherein the Bacillus subtilis has enzyme activity and antibacterial activity against harmful bacteria. The feed additive for preventing or improving diarrhea in calves according to claim 2, wherein the enzymes are protease and amylase, and the harmful bacteria are Escherichia coli and Salmonella dublin. The feed additive for preventing or improving diarrhea in calves according to claim 1, wherein the saccharides are monosaccharides, disaccharides, polysaccharides, or mixtures thereof. A method for producing a feed additive having antidiarrheal activity in calves, comprising the step of adding Bacillus subtilis to a mixture of sugars and minerals. 6. The method of claim 5,
1) mixing and heating sucrose, glucose and water;
2) adding potassium chloride and sodium hydrogen carbonate after step 1);
3) after step 2), adding and mixing beta-glucan, levan and glutathione; and
4) adding Bacillus subtilis to the mixture obtained in step 3); a method for producing a feed additive having antidiarrheal activity in calves comprising a.

Images