JP5554221B2 - Composition for prevention / treatment of bovine mastitis and method for prevention / treatment of bovine mastitis - Google Patents

Description

  The present invention relates to a composition for preventing and treating bovine mastitis and a method for preventing and treating bovine mastitis.

  Mastitis in dairy cows is a disease directly related to raw milk production and is a problem that causes direct and indirect economic losses to dairymen. In addition, rabies greatly affects the quality of raw milk, such as reducing milk yield and quality. In shipping, if the number of somatic cells in bulk milk exceeds a predetermined value, there is an economic burden that is subject to a penalty. In addition, dairymen change the milking procedure and order, or perform separate squeezing, resulting in increased labor burden due to complicated operations. In the case of shipment stoppage, not only the loss due to the stoppage but also the cost for disposal of raw milk is added.

  It is the disease that causes the most economic damage to dairymen, such as the addition of treatment costs when treated, and the cost of disposing of cattle that are difficult to treat.

  According to the National Institute of Animal Health Research No. 6 (2006), the National Institute of Agricultural and Food Industry Research According to Animal Health Research Results No. 6 (2006), the loss due to mastitis in dairy farmers in Hokkaido is estimated to be 10 billion yen per year Out.

  Furthermore, cows with mastitis suffer from the problem of bovine health damage, such as being susceptible to other diseases. In fact, the number of deaths due to mastitis occupies the top of the total number of deaths due to mastitis, and mastitis accounts for about 30% of the causes of sickness in dairy cows.

  Therefore, prevention and treatment measures for dairy cow mastitis are extremely important problems for dairymen.

  As a treatment method, there is a treatment with an antibiotic, but once it is administered, the shipment of raw milk is restricted due to the problem that the antibiotic remains in the body, so the milk cannot be shipped during the treatment period. In addition, there are many cases of potential illness, and there is still no cure for the fundamental solution.

  Therefore, attempts have been made to prevent and treat mastitis using a composition derived from a natural product that does not use antibiotics. For example, Patent Document 1 discloses a composition for the prevention and treatment of livestock mastitis characterized in that live baker's yeast or a content thereof is an active ingredient. However, the composition for prevention and treatment of mastitis using an active ingredient derived from a natural product has not been sufficiently popular since it has not been sufficiently effective.

JP 2001-224317 A

  It is an object of the present invention to be able to prevent or treat mastitis by providing an improvement (decrease) in the number of somatic cells, which is one of the indicators of mastitis, in a safe and free of side effects. It is an object of the present invention to provide a composition for preventing and treating inflammation and a method for preventing and treating bovine mastitis. And for cattle, there are no side effects, safety and no health damage, and a composition for the prevention and treatment of bovine mastitis, which uses active ingredients derived from natural products for the purpose of preventing deterioration of milk quality, and bovine mastitis. It is to provide a mastitis prevention / treatment method. Another object of the present invention is to provide a composition for preventing and treating bovine mastitis and a method for preventing and treating bovine mastitis, which are effective even after administration. Another object of the present invention is to provide a composition for preventing and treating bovine mastitis and a method for preventing and treating bovine mastitis that are safe and have no side effects, and are more effective in combination with antibiotics. .

The inventors of the present invention have intensively studied to achieve the above object and have completed the present invention. That is, the present invention is as follows.
[1] A composition for prevention and treatment of bovine mastitis, comprising as an active ingredient a culture composition obtained from a culture of microorganisms belonging to the genus Aureobasidium sp.
[2] The culture is a culture obtained by adding lactic acid bacteria or killed microorganisms to a culture medium for cultivating the microorganisms and culturing so as to be assimilated as a nutrient component of the microorganisms. The composition for prevention / treatment of bovine mastitis according to the above [1].
[3] The composition for prevention / treatment of bovine mastitis according to [1] or [2], which is used by being added to bovine feed.
[4] A method for preventing and treating bovine mastitis, which comprises ingesting a culture composition obtained from a culture of microorganisms belonging to the genus Aureobasidium sp.
[5] The culture is a culture obtained by adding lactic acid bacteria or killed microorganisms to a culture medium for cultivating the microorganisms and culturing so as to be assimilated as a nutrient component of the microorganisms. The method for preventing / treating bovine mastitis according to [4] above.
[6] Prevention / treatment of bovine mastitis according to [4] or [5] above, wherein a feed supplemented with a culture composition obtained from a culture of microorganisms belonging to the genus Aureobasidium sp. Method.

  According to the composition for preventing and treating bovine mastitis and the method for preventing and treating bovine mastitis according to the present invention, bovine mastitis can be effectively prevented or treated. Since the active ingredient is a culture composition obtained from a culture of microorganisms belonging to the genus Aureobasidium (Aureobasidium sp.), It is highly safe and does not affect shipping even if administered. In addition, since there is no risk of side effects, it can be used safely from the viewpoint of bovine health management. Above all, by reducing the number of somatic cells, the symptoms of breast inflammation are alleviated to prevent milk yield and quality from being lowered, and productivity is improved.

Changes in the number of somatic cells in milk when administration of Aureobasidium pullulans M-2 culture solution to Holstein lactating cows with mastitis (administration group 1-6) and no mastitis It is a graph which shows the fluctuation | variation (non-administration group 1-13) of the number of somatic cells in the milk | loop of a healthy Holstein lactation cow by a measured value. It is a graph which shows the average value of each month of an administration group or a non-administration group with a standard deviation. It is a graph which shows the result of having statistically processed and compared the result of the fluctuation | variation of the somatic cell number of the administration group 1-6. It is a graph which shows the result of having statistically processed and compared the result of the fluctuation | variation of the somatic cell number of the non-administration group 1-13. It is a graph which shows the result of having statistically processed and compared the somatic cell count of the administration group 1-6 and the non-administration group 1-13, when the August value is set to 1. It is a graph which shows colon_bacillus | E._coli in the milk | gum of August and November of the administration group 1-6 by index display. It is a graph which shows the result of having statistically processed and compared the result of the fluctuation | variation of the number of colon_bacillus | E._coli in milk of the administration group 1-6. It is a chart which shows the number of general viable bacteria in milk of administration group 1-6 by index display. It is a graph which shows the result of having statistically processed and compared the result of the fluctuation | variation of the number of general viable bacteria in the milk of the administration group 1-6. It is a graph which shows transition of the white blood cell count in the blood of the administration group 1-6 with the normal range (thick line of a horizontal axis). It is a graph which shows transition of the number of rod-shaped neutrophils (St) in the blood of the administration group 1-6. It is a graph which shows the result of having statistically processed and compared the fluctuation | variation of the number of rod-shaped neutrophils (St) in the blood of the administration group 1-6. It is a graph which shows the fluctuation | variation of the albumin amount in the blood of the administration group 1-6 with the normal range (thick line of a horizontal axis). It is a graph which shows the fluctuation | variation of the (gamma) GTP value in the blood of the administration group 1-6 with the normal range (thick line of a horizontal axis). It is a graph which shows the fluctuation | variation of the AST / GOT value in the blood of the administration group 1-6 with the normal range (thick line of a horizontal axis). It is a graph which shows the fluctuation | variation of the ALT / GPT value in the blood of the administration group 1-6 with the normal range (thick line of a horizontal axis). It is a graph which shows the fluctuation | variation of the amount of creatinine in the blood of the administration group 1-6 with the normal range (thick line of a horizontal axis). It is a graph which shows transition of the red blood cell count in the blood of the administration group 1-6 with the normal range (thick line of a horizontal axis). It is a graph which shows the fluctuation | variation of the hemoglobin amount in the blood of the administration group 1-6 with the normal range (thick line of a horizontal axis). It is a graph which shows transition of the eosinophil count in the blood of the administration group 1-6 with the normal range (thick line of a horizontal axis). It is a graph which shows the fluctuation | variation of the blood glucose level (glucose amount) in the blood of the administration group 1-6. It is a graph which shows the fluctuation | variation of the total cholesterol level in the blood of the administration group 1-6 with the normal range (thick line of a horizontal axis). It is a graph which shows the fluctuation | variation of the LDL cholesterol level in the blood of the administration group 1-6.

  In the composition for prevention and treatment of bovine mastitis of the present invention, a culture composition obtained from a culture of microorganisms belonging to the genus Aureobasidium (Aureobasidium sp.) (Hereinafter referred to as “Aureobasidium-derived culture composition”) )) As an active ingredient. In the bovine mastitis prevention / treatment method of the present invention, the aureobasidium-derived culture composition is fed to a cow suffering from mastitis or a cow trying to prevent it.

  As this aureobasidium-derived culture composition, a culture itself in which microorganisms belonging to the genus Aureobasidium (Aureobasidium sp.) And having β-glucan production ability (hereinafter referred to as “Aureobasidium microorganisms”) are cultured, Not only the culture solution from which the bacterial cells have been separated and removed by centrifugation, the concentrated solution of the culture solution, the diluted solution of the culture solution, or the solids obtained by removing water from the culture solution, but also desalting them. Those having an increased content of β-glucan, and those obtained by purifying β-glucan from these are also included. Each fraction obtained by fractionation can also be used.

  The above-mentioned aureobasidium-derived culture composition used in the present invention is obtained by converting the β-glucan produced by culturing the above aureobasidium microorganism into a content with respect to 100 g of the culture, 50 to 3 2,000 mg is preferable, and 100 to 2,000 mg is more preferable.

In addition, determination of content of (beta) -glucan can be performed by the following methods, for example. That is, the culture solution is subjected to an enzyme treatment using amylase, amyloglucosidase, protease, or the like, and an α-glucan such as protein or pullulan is removed, followed by ethanol precipitation. Further, it is filtered through a glass filter to obtain a polymer sample. At this time, in order to remove low-molecular substances containing monosaccharides, the cells are thoroughly washed with 80% ethanol. The washed polymer sample is further washed with acetone, and sulfuric acid is added for hydrolysis. After hydrolysis, the solution is neutralized, the filtrate is collected, glucose is quantified by the glucose oxidase method, and the value calculated based on the following Equation 1 is defined as the amount of β-glucan.
Formula 1: β-glucan (g / 100 g) = glucose (g / 100 g) × 0.9

  The content of β-glucan can also be determined as the so-called sugar chain-containing polymer substance (polysaccharide) content. In this case, the culture solution is subjected to enzyme treatment using amylase, amyloglucosidase, protease, etc., and α-glucan such as protein and pullulan is removed, followed by ethanol precipitation. Further, it is filtered through a glass filter to obtain a polymer sample. At this time, in order to remove low-molecular substances containing monosaccharides, the cells are thoroughly washed with 80% ethanol. The washed polymer sample is further washed with acetone, and the weight of the washed polymer sample is measured to determine the amount of the sugar chain-containing polymer substance (polysaccharide).

  The β-glucan quantified in this way is quantified as having a functional group such as a sulfate group or a phosphate group. Therefore, when β-glucan is quantified as a sugar chain-containing polymer substance (polysaccharide) in a broad sense as described above, the content of β-glucan produced by culturing the aureobasidium microorganism is It is preferable that the content is 70 to 5,000 mg, more preferably 140 to 3,000 mg, in terms of the content with respect to 100 g of the mass of the product.

  The Aureobasidium microorganism used in the present invention may be any microorganism that belongs to the genus Aureobasidium and has the ability to produce β-glucan. For example, Aureobasidium pullulans M-1 (Aureobasidium) pullulans M-1, AIST, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center FERM BP-08615), Aureobasidium pullulans M-2, National Institute of Advanced Industrial Science and Technology, Patent Biology Deposit center accession number FERM BP-10014) is preferably used. In addition, β-glucan produced by these strains is obtained from the main chain in which glucose is β-1,3 linked by structural analysis by NMR measurement (13C NMR: Varian's UNITY INOVA500 type, 1HNMR: Varian's UNITY INOVA600 type). It has been clarified that it is β-1,3-1,6-glucan having a structure in which glucose is branched by -1,6 bonds.

  The aureobasidium microorganism can be cultured according to a known method (see Japanese Patent Application Laid-Open No. 57-149301). That is, a medium (pH 5.2 to 2) containing 0.5 to 5.0% by mass of a carbon source (sucrose), 0.1 to 5.0% by mass of an N source, and other trace substances (for example, vitamins and inorganic substances). 6.0) is inoculated with bacteria, and aeration culture is performed at a temperature of 20 to 30 ° C. for 2 to 14 days, preferably aeration and agitation culture. As β-glucan is produced, the viscosity of the culture solution increases and becomes a highly viscous gel. The culture solution thus obtained usually contains 0.6 to 10% by mass of solid content, and the solid content contains 5 to 80% by mass of β-glucan. In addition to β-glucan, other useful components such as phosphorus, potassium, magnesium, and vitamin C are also included.

  The aureobasidium microorganism may be cultured in such a manner that a killed microorganism is added to a culture solution for culturing the microorganism to assimilate it as a nutrient component. In this case, the blending amount of the dead cells in the culture solution is usually about 100 cells / mL (culture solution) to 100 trillion cells / mL (culture solution), although it varies depending on the type of cells used. It is preferable. Microbial killing can be performed by heat sterilization or the like.

  The microorganism used as the killed microorganism is not particularly limited as long as it contains an aureobasidium microorganism nutrient source such as a nitrogen source, and examples thereof include lactic acid bacteria and yeast. Examples of lactic acid bacteria include Enterococcus faecalis, Enterococcus faecalis, Lactobacillus acidphilus, L. gasseri, L. mali, Lactobacillus. Bacillus plantarum (L.plantarum), Lactobacillus buchneri (L.buchneri), Lactobacillus casei (L.casei), Lactobacillus johnsonii (L.johnsonii), Lactobacillus gallinarum (L.gallinarum) , Lactobacillus amylovorus (L.amylovorus), Lactobacillus brevis (L.brevis), Lactobacillus rhamnosus (L.rhamnosus), Lactobacillus kefir (L.kefir), Lactobacillus paracasei (L.paracasei) , Lactobacillus subtypes such as L. crispatus Streptococcus bacteria such as Streptococcus thermophilus, Lactococcus lactis bacteria such as Lactococcus lactis, Bifidobacterium bifidum, Bifidobacterium longum (B. longum), Bifidobacterium addresscentis (B.adolescentis), Bifidobacterium infantis (B.infantis), Bifidobacterium breve (B.breve), Bifidobacterium catenuratum (B Bacteria belonging to the genus Bifidobacterium such as .catenulatum, Bacillus subtilis, Bacillus coagulans such as B. coagulans, Clostridium butilicum such as Clostridium butilicum, etc. be able to.

  Yeasts include imperfect fungi, including Aureobasidium pullans, Saccharomyces cerevisiae, Saccharomyces intemedius, Saccharomyces intemedius, Saccharomyces validus, Saccharomyces validus Saccharomyces ellipsoideus), Saccharomyces mali risler, Saccharomyces mandschuricus, Saccharomyces Vordermannii, Saccharomyces charisom Som (Saccharomyces piriformis), Saccharomyces anamen sis), Saccharomyces cartilaginosus, Saccharomyces Awamori, Saccharomyces Batatae, Saccharomyces Coreanus, robust Saccharomyces Coreanus, Somcharosus Cis (Saccharomyces Carlsbergensis), Saccharomyces Monacensis (Saccharomyces Monacensis), Saccharomyces Marxianus, Zygosaccharomyces (Saccharomyces) Roc Hansenula anomala, etc. can be used.

  In the present invention, the culture obtained by the above culture may be used as it is by heating or pressure-heat sterilization, or after separating and removing the cells by centrifugation or the like, the culture solution may be used after sterilization. Good. Moreover, what was concentrated as needed and also what was dried can also be used. Furthermore, only β-glucan can be extracted and used. Each fraction obtained by fractionation can also be used. A culture of microorganisms belonging to the genus Aureobasidium sp. Is used as a food additive such as a thickening stabilizer and has high safety.

  In the present invention, cattle exhibiting mastitis are targeted for application. Causes of mastitis include infectious microorganisms such as staphylococci, streptococci, and Escherichia coli, abnormal host defense mechanisms due to effects of perinatal period, lactation, stress, nutrition, age, milking management, individual management, feed In addition, there are various breeding environment factors such as the structure of a barn, and the symptom forms include acute mastitis, acute mastitis, chronic mastitis, etc., but in the present invention, any cause or symptom is used. Even mastitis can improve or treat the symptoms of cattle affected by it. Further, in the present invention, even a cow that does not show mastitis is an application target. That is, it can be used for prevention of mastitis.

  The amount to be administered can be appropriately determined depending on the intensity of symptoms, bodily condition, age, administration method, frequency and timing of administration. For example, when a typical effective dose is exemplified, for example, when converted by the amount of β-glucan which is one of the contents of the aureobasidium-derived culture composition, about 0.02 to 200 mg / kg (body weight) per day Take in the amount of. Moreover, it is preferable to make it ingest for a long time and to exhibit an effect continuously. Furthermore, it can also be used in combination with conventionally known methods for treating mastitis, such as in combination with antibiotics.

  In the present invention, as one form thereof, the above-mentioned aureobasidium-derived culture composition can be added to bovine feed and used. According to this, even when using and ingesting continuously for a long period of time without taking a special work time for feeding to cattle, it can be performed with good workability.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

[Production Example 1]
A culture solution of Aureobasidium pullulans M-2 (FERM BP-10014) was prepared as follows.
Pre-culture liquid is inoculated in an appropriate amount into a liquid medium (pH 5.3) containing 1% sucrose, 0.1% ascorbic acid, and 0.1% rice bran, and 25 ° C., 72 to 96 hours (depending on the production batch) Then, aeration stirring culture was performed. After completion of the culture, this culture solution was sterilized at 121 ° C. for 15 minutes. The obtained culture solution after sterilization contained about 1.2% by mass of the solid content, and the β-glucan content in 100 g of the solid content was 32.5 g. Moreover, it converted into content contained in 100g of mass of culture solution itself, and contained 0.39g / 100g. This culture was used for the following tests.

[Example 1]
The white blood cells in milk and the dropped mammary epithelial cells are collectively referred to as somatic cells, but the number of somatic cells increases when they are attacked by mastitis pathogens. Usually, the target value of the number of somatic cells of an individual is 300,000 cells / mL or less, the normal value is 100,000 cells / mL or less, and if it is 300,000 cells / mL or more, it may be infected with mastitis. High nature. Therefore, as the number of somatic cells in mastitis cattle decreases, it can be considered that the condition of bacterial contamination has improved and the symptoms of mastitis have improved, so oral administration of aureobasidium broth is Whether it was effective in improving the number of somatic cells in milk was investigated.

For this purpose, among the Holstein lactating cows that were bred, 6 individuals with a somatic cell count of 300,000 / mL or more in the bovine group test results table were used as test cattle (administration group 1-6). 300 mL of Aureobasidium culture solution prepared in Production Example 1 was continuously given by oral administration once a day in the morning or afternoon for 3 months. The test was conducted from August to November 2010. Milk is collected at the start of the test, and after 1 month, 2 months, and 3 months of the test, and the somatic cell count is automatically analyzed using flow cytometry ("Fossomatic ^TM FC" FOSS) It measured using.

  The results are shown in Table 1. Further, FIG. 1 shows a graph showing the values of the non-administration group of Comparative Example 1 described later, and FIG. 2 shows a graph showing the average value of each month together with the standard deviation. FIG. 3 shows the results of statistical processing of the results of changes in the number of somatic cells in administration groups 1-6 and comparison.

As a result, as shown in Table 1 and FIGS. 1 and 2, a decrease in the number of somatic cells in milk was observed in these administration groups. That is, at the start of the administration at an average of animals treated group 6, 1883 × 10 ³ cells / mL, after administration 1 month, 747.2 × 10 ³ cells / mL, after administration 2 months, 664.2 × 10 ³ After 3 months of administration, the number was 471.8 × 10 ³ cells / mL, which was reduced to a quarter of the value at the start of administration. (Table 1) In addition, when seen individually, administration group 4 showed a decrease of 86%, which was 5221 × 10 ³ cells / mL at the start of administration, but 748 × 10 ³ cells / mL after one month. (Table 1)
As shown in FIG. 3, when the number of somatic cells at the start of administration and after 3 months in the administration group was subjected to a T test, a significant difference was observed with an abstention rate of P <0.05. That is, in the administration group, a decrease in the number of somatic cells was recognized with a significant difference. (Figure 2)
As a result, the average number of somatic cells in bulk milk exceeded 300,000 / mL on the farm, and the penalty was paid about 300,000 yen per month, but the penalty was no longer paid.

[Comparative Example 1]
Example 1 shows the changes in the number of somatic cells in the milk of healthy Holstein lactating cows that have not developed mastitis, for 13 test cows to which no aureobasidium culture solution was given (non-administered group 1-13). It investigated like.

The results are shown in Table 2. FIG. 1 shows a graph showing the values of the administration group of Comparative Example 1 described above, and FIG. 2 shows a graph showing the average value of each month together with the standard deviation.
In FIG. 4, the result of having statistically processed and compared the result of the fluctuation | variation of the somatic cell number of the non-administration group 1-13 is shown. FIG. 5 is a chart showing the results of statistically comparing the increase / decrease values of the administration group 1-6 and the non-administration group 1-13 when the value at the start of the test is 1.

As a result, as shown in Table 2 and FIGS. 1 and 2, in these non-administered groups, an increasing tendency in the number of somatic cells in the milk was observed. That is, the average value of 169 × 10 ³ cells / mL in the non-administered group at the start of the test increased to 557 × 10 ³ cells / mL after 3 months. Individually, in the non-administration group 4, 63 × 10 ³ cells / mL at the start increased to 1048 × 10 ³ cells / mL after 1 month, a 16.6-fold increase. In the non-administration group 8, 329 × 10 ³ cells / mL at the start increased to 1504 × 10 ³ cells / mL, a 4.5-fold increase. In the non-administered group 10, 34 × 10 ³ cells / mL at the beginning was 3276 times after 1 month, 1276 × 10 ³ cells / mL, an increase of 37.5 times. (Table 2)
In addition, as shown in FIG. 4, in the non-administration group, when the number of somatic cells at the start of the test and at the end of the test after 3 months was subjected to a T test, a significant difference was observed with a withdrawal rate of P <0.01. . That is, in the non-administered group, an increase in the number of somatic cells was recognized with a significant difference. (Fig. 4)
In FIG. 5, with respect to the number of somatic cells in the administration group 1-6 and the non-administration group 1-13, the increase / decrease value when the value at the start of the test is set to 1 is calculated using the Mann-Whitney U test. The non-administration group is compared. As a result, a significant difference was observed with an abstention rate of P <0.05. That is, it was found that the increase in the number of somatic cells in the non-administration group was significantly higher than that in the administration group. (Fig. 5)
In general, the environmental factors from summer to autumn are improved naturally and there is an improvement in the number of somatic cells, but here it tends to get worse in almost all cattle, and the environment during the test period of the comparative example It was inferred that the factor was very severe.

[Test Example 1]
About the administration group 1-6 of the said Example 1, the number of colon_bacillus | E._coli in milk was measured by EC culture medium fermentation tube method based on the Food Sanitation Law guideline before administration start and 3 months after administration. The results are shown in Table 3 and FIG. Moreover, the result of the fluctuation | variation of the colon_bacillus | E._coli number of milk of the administration group 1-6 in FIG. 7 was compared using statistical processing.

As a result, the average value of the 6 groups at the start of administration was 781,476 CFU / 100 mL, but decreased to 778 CFU / 100 mL after 3 months. In the administration group 1, the administration group 2 and the administration group 4, the value after 3 months of administration decreased to the detection limit or less. (Table 3)
In addition, as a result of the T test, the number of E. coli after 3 months of administration in the administration group was significantly different from the number of E. coli at the start, with an abstention rate of P <0.05. That is, in the administration group, the number of E. coli decreased with a significant difference. (Fig. 7)

[Test Example 2]
About the administration group 1-6 of the said Example 1, the number of general viable bacteria in milk was measured with the standard agar plate culture method based on the Food Sanitation Law guideline before administration start and 3 months after administration. The results are shown in Table 4 and FIG. Moreover, the result of the fluctuation | variation of the general viable count in milk of the administration group 1-6 in FIG. 9 was compared using statistical processing.

As a result, the average value of 6,875,280 CFU / mL of the 6 groups at the start of administration decreased to 330,650 CFU / mL after 3 months of administration. (Table 4)
In addition, as a result of the T test, a significant difference was observed in the number of general viable bacteria in November in the administration group, with an abstention rate of P <0.05, compared with the number of general viable bacteria in August. That is, in the administration group, the number of general viable bacteria decreased with a significant difference. (Fig. 9)

[Test Example 3]
For administration group 1-6 of Example 1 above, the number of leukocytes and the bloody neutrophils (St) in blood that are said to increase in number among leukocytes due to inflammation are measured every month during the test period. It was measured. The white blood cell count was measured by the DC detection method. The results are shown in Table 5 and FIG. Spider neutrophils (St) were examined by an automatic blood cell sorter and a microscopic examination. The results are shown in Table 6 and FIG.
Moreover, in FIG. 12, the result of having statistically processed and compared the fluctuation | variation of the number of rod-shaped neutrophils (St) in the blood of the administration group 1-6 is shown.

Table 5 and FIG. 10 show fluctuations in the white blood cell count of the administration groups 1-6. The normal range is 5.87 to 9.75 × 10 ³ / μL, and the average value for each month is 7.2 × 10 ³ / μL at the start of administration, and 7.7 × 10 ³ / μL one month after administration. It was within the normal range of μL, 7.1 × 10 ³ / μL after 2 months of administration, and 6.9 × 10 ³ / μL after 3 months of administration. (Fig. 10)
As shown in Table 6 and FIG. 11, the number of rod-shaped neutrophils (St), which is said to increase in value due to inflammation, was an average value of 558 × 10 ³ cells / μL at the start of administration. After 3 months, the average value decreased to 289 × 10 ³ cells / mL. (Table 6)
In addition, as a result of the T test, the number of rod-shaped neutrophils (St) after 3 months of administration in the administration group was significantly different with the abstention rate of P <0.05 compared with the value at the start of administration. It was. That is, in the administration group, the number of rod-shaped neutrophils (St) decreased with a significant difference. (Fig. 12)
As shown in FIG. 10, there was no decrease in the total number of leukocytes, and among the leukocytes, the number of rod-shaped neutrophils (St) that increased in value due to inflammation was decreased. (FIGS. 10 and 11)

[Test Example 4]
About the administration group 1-6 of the said Example 1, in order to confirm the presence or absence of a side effect, the biochemical test item in the blood was investigated every month during the test period. Albumin was measured by the BCG method. γGTP, AST / GOT, and ALT / GPT were measured by the JCCLS-SOP method. Creatinine was measured by the modified Jaffe method and the enzymatic method. Red blood cells were measured by the electric resistance detection method. Hemoglobin was measured by the SLS-Hb method. Eosinophils were measured by the DC method. The blood glucose value (glucose amount) was measured by an enzymatic method. Total cholesterol and LDL cholesterol were measured by enzymatic methods. The results are shown in FIGS. The normal range of each measured value was referred to the value in the “Clinical Pathology Examination Guidelines for Livestock Mutual Aid” (Ministry of Agriculture, Forestry and Fisheries Management Bureau).

  Table 7 below and FIG. 13 show the values of albumin in the blood of the administration groups 1-6.

  Table 8 and FIG. 14 below show the values of blood γGTP in the administration groups 1-6.

  Table 9 and FIG. 15 below show the blood AST / GOT values in the administration groups 1-6.

  Table 10 and FIG. 16 below show the blood ALT / GPT values of the administration groups 1-6.

  Table 11 below and FIG. 17 show the blood creatinine values of administration groups 1-6.

  Table 12 and FIG. 18 below show the values of blood red blood cells in the administration groups 1-6.

  In the following Table 13 and FIG. 19, the values of blood hemoglobin in the administration groups 1-6 are shown.

  Table 14 and FIG. 20 below show the values of blood eosinophils in the administration groups 1-6.

  Table 15 and FIG. 21 below show the blood glucose values of the administration groups 1-6.

  In the following Table 16 and FIG. 22, the total cholesterol values of the administration groups 1-6 are shown.

  Table 17 and FIG. 23 below show the values of LDL cholesterol in the administration groups 1-6.

  In Table 7, Table 8, Table 9, Table 10, FIG. 13, FIG. 14, FIG. 15, and FIG. 16, for each of the administration groups 1-6, albumin, γGTP, AST / GOT, mainly reflecting liver function, It shows the variation of the ALT / GPT value.

Table 7 and FIG. 13 show the amount of albumin in administration group 1-6. The normal value of the amount of albumin is 3.2 to 3.4 g / dL. The average amount of albumin in administration group 1-6 was 3.7 g / dL at the start of administration, and 3.5 g / dL at 1 month after administration, which was higher than the normal range. 3.4 g / dL, 3 months later, 3.2 g / dL and fluctuates in the normal range. (Table 7)
Table 8 and FIG. 14 show the values of γGTP in the administration groups 1-6. The normal value of γGTP is 6.1 to 17.4 U / L. The average value in the administration groups 1-6 was 31.2 IU / L at the start of administration, 27.7 IU / L after 1 month of administration, 27.0 IU / L after 2 months of administration, and 29 after 3 months of administration. 0.0 IU / L. (Table 8)
Table 9 and FIG. 15 show values of the administration group 1-6AST / GOT. AST / GOT was within a normal range of 78 to 132 U / L, and was lower than the normal range except for administration group 6 at the start of administration. The average value of the administration groups 1-6 at this time was 65.0 IU / L. The average value after 1 month of administration was 67.3 IU / L, the average value after 2 months of administration was 69.8 IU / L, and the average value after 3 months of administration was 68.3 IU / L. At 3 months after administration, administration group 4 was in the normal range. (Table 9)
Table 10 and FIG. 16 show the values of ALT / GPT. ALT / GPT has a normal range of 14 to 38 IU / L. (FIG. 16) The average values of ALT / GPT in administration groups 1-6 were 26.8 IU / L at the start of administration, 23.7 IU / L after 1 month of administration, 22.2 IU / L at 2 months of administration, and 3 of administration. After 2 months, it was 25.8 IU / L, which was within the normal range from the start to the start of administration. (Table 10)
In Table 11 and FIG. 17, the fluctuation | variation of the creatinine value reflecting the renal function is shown. As shown in Table 11 and FIG. 17, the average value of creatinine in the administration groups 1-6 was 0.88 mg / dL at the start of administration, 0.93 mg / dL after 1 month of administration, and 0 after 2 months of administration. It was 0.93 mg / dL after 3 months of administration. (Table 11)

  Table 12, Table 13, Table 14, FIG. 18, FIG. 19, and FIG. 20 show fluctuations in red blood cells, hemoglobin, and eosinophil values, which are the main components of blood.

In Table 12 and FIG. 18, the red blood cell count of the administration group 1-6 is shown. The normal range of erythrocytes is 546-674 × 10 ⁴ / μL. The average value of the red blood cells administered group 1-6 is at the start of the administration was below 540.2 × 10 ⁴ / μL and the normal range, after administration 1 month 546.3 × 10 ⁴ / μL, after administration 2 months and it remained within the normal range in average and 573.2 × 10 ⁴ / μL is 560.7 × 10 ⁴ / μL, after 3 months of administration. (Table 12)
In Table 13 and FIG. 19, the amount of hemoglobin in the administration group 1-6 is shown. The normal value of hemoglobin is 8.0-12.6 g / dL. The mean values of hemoglobin in the administration groups 1-6 were 9.1 g / dL at the start of administration, 9.3 g / dL after 1 month of administration, 9.4 g / dL after 2 months of administration, and 9.9 after 3 months of administration. It was within the normal range of 5 g / dL. (Table 13)
Table 14 and FIG. 20 show the number of eosinophils in the administration group 1-6. The normal range of eosinophil count is 0 to 2,400 / μL. The average value of the number of eosinophils in the administration group 1-6 was 1,000 / μL at the start of administration, 760 / μL after 1 month of administration, 608 / μL after 2 months of administration, Three months after administration, the number was 538 / μL. All decreased within the normal range. (Table 14)

  In Table 15 and FIG. 21, the blood glucose concentration (blood glucose level) in the administration group 1-6 is shown. The average glucose concentration in administration group 1-6 was 60.8 mg / dL at the start of administration, 63.5 mg / dL after 1 month of administration, 62.0 mg / dL after 2 months of administration, and 3 months after administration. It was 61.0 mg / dL. (Table 15)

  Table 16, Table 17, FIG. 22, and FIG. 23 show the values of total cholesterol and LDL cholesterol reflecting lipid metabolism.

In Table 16 and FIG. 22, the fluctuation | variation of the total cholesterol level in the blood of the administration group 1-6 is shown. The normal range was 80 to 300 mg / dL, and the average value of administration group 1-6 at the start of administration was 139.0 mg / dL. The concentration was 138.0 mg / dL 1 month after administration, 150.8 mg / dL 2 months after administration, and 153.0 mg / dL 3 months after administration. All were in the normal range. (Table 16)
In Table 17 and FIG. 23, the fluctuation | variation of the blood LDL-cholesterol level of the administration group 1-6 is shown. LDL-cholesterol tends to be higher in late lactation. The average value of administration group 1-6 at the start of administration was 8.8 mg / dL. It was 7.5 mg / dL 1 month after administration, 8.8 mg / dL 2 months after administration, and 9.7 mg / dL 3 months after administration. (Table 17)

[Summary of results]
Speaking of the circumstances surrounding the above test environment, the summer of 2010, which is the test period, was extremely hot and environmentally difficult. Even in the nationwide livestock damage situation (July to August 2010) compiled by the Ministry of Agriculture, Forestry and Fisheries on September 3, 2010, 959 cows were killed and discarded due to heat stroke. In the same barn, even though the number of somatic cells was in the normal range, there was one cow that fell due to heat stroke and was abandoned as it was. In addition, some cows needed to be treated with antibiotics before they became waste cows. Under the influence of such an environment, it was considered that the number of somatic cells in the milk of healthy Holstein lactating cows that did not develop mastitis tended to increase as in Comparative Example 1 above. In other words, it was suggested that environmental factors affecting mastitis were not improved due to seasonal changes.

  On the other hand, in spite of such severe environmental conditions, in the Aureobasidium culture solution administration group, as shown in Example 1 above, improvement in the number of somatic cells in milk was observed. It was. In addition, as shown in Test Example 1 above, it was recognized that the contamination with Escherichia coli in the milk of cows with mastitis tended to decrease by administration of aureobasidium culture medium. Furthermore, as shown in Test Example 2 above, it was recognized that the contamination by viable bacteria in the milk of bovine mastitis also decreased with the administration of aureobasidium culture solution. That is, it was suggested that some inflammation was improved and mastitis was improved.

  In Test Example 3, the number of rod-shaped neutrophils (St), which is said to increase in value due to inflammation, despite the fact that there is no significant change in the total number of leukocytes in bovine mastitis, is the aureobasidium culture. A tendency to decrease with liquid administration was observed.

  Furthermore, in Test Example 4, blood components of mastitis cows were confirmed. As a result of biochemical examination, albumin, γGTP, AST / GOT, ALT / GPT, and creatinine reflecting kidney function are involved in liver function. In addition, red blood cells, hemoglobin, eosinophils, which are the main components of blood, glucose (blood glucose level) as an indicator of health status, total cholesterol and LDL-cholesterol as indicators of lipid metabolism, It was confirmed that none of the normal range was greatly deviated by administration of the sodium culture medium and converged to the normal range.

  Therefore, it was speculated that there was little adverse side effect due to oral administration of the culture composition obtained from the culture of microorganisms belonging to the genus Aureobasidium sp.

  From the above, it is clear that the culture composition obtained from the culture of microorganisms belonging to the genus Aureobasidium sp. Exhibits an improvement effect without causing side effects in the prevention and treatment of bovine mastitis. became.

  All of the above administration groups were administered to cattle having a somatic cell count of 300,000 cells / mL or more, and it was found that the administration was effective after administration. The present invention is effective in the prevention and treatment of bovine mastitis.

Claims (6)

A microorganism belonging to Aureobasidium pullulans is a liquid medium containing at least a carbon source 0.5 to 5.0% by mass , a nitrogen source 0.1 to 5.0% by mass , and vitamins and / or minerals at a temperature of 20 A bovine mastitis characterized by containing as an active ingredient a culture which is cultured by aeration and agitation at -30 ° C for 2-14 days and is cultured to contain 50-3000 mg of β-glucan per 100 g of the culture. Prophylactic / therapeutic composition.   The culture is a culture obtained by adding lactic acid bacteria or killed microorganisms to a culture medium for culturing the microorganisms and cultivating the microorganisms to be assimilated as nutrients of the microorganisms. The composition for prevention and treatment of bovine mastitis as described.   The composition for prevention / treatment of bovine mastitis according to claim 1 or 2, which is used by being added to bovine feed. A microorganism belonging to Aureobasidium pullulans is a liquid medium containing at least a carbon source 0.5 to 5.0% by mass , a nitrogen source 0.1 to 5.0% by mass , and vitamins and / or minerals at a temperature of 20 Prophylactic / therapeutic treatment of bovine mastitis characterized by ingesting the culture cultured at 50 ° C. to 30 ° C. for 2 to 14 days with aeration and agitation and containing 50 to 3000 mg of β-glucan per 100 g of the culture. Method.   The culture is a culture obtained by adding lactic acid bacteria or killed microorganisms to a culture medium for cultivating the microorganisms and culturing so as to be assimilated as a nutrient component of the microorganisms. The prevention / treatment method of bovine mastitis as described.   The method for preventing or treating bovine mastitis according to claim 4 or 5, wherein the feed supplemented with the culture is ingested.