JP2006101784A - New yeast and its utilization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find a microorganism which prevents diseases such as diarrhea and loose stool, the decrease of estrus, has excellent effects, and is useful for probiotec. <P>SOLUTION: The new yeast belongs to Saccharomyces and has an antidiarrhetic activity. When a nutritive composition containing the new yeast is administered to a livestock, effects such as the prevention of diarrhea and loose stool, the shortage of estrus-recovering days, the improvement of conception rates, and the improvement of meat quality are recognized. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel yeast having antidiarrheal activity and use thereof.

  So far, pigs, sheep, horses, cattle, etc. have been raised as livestock, and their meat and milk have been consumed by humans, contributing to the diet. In recent years, due to the increase in demand for livestock meat and milk and the liberalization of meat, further improvement in livestock productivity has become necessary.

  In response to such demands for improving the productivity of livestock, breeding with a large number of animals has been addressed by improving livestock breeds and breeding methods.

  However, breeding with a large number of animals as described above may cause problems in livestock growth due to the deterioration of hygienic environment and stress accompanying this, resulting in diseases such as diarrhea and loose stool, and an increase in the number of days of estrus. In some cases, productivity decreased.

  For example, when diarrhea or loose stool occurs due to infection of livestock with pathogenic microorganisms, the infection spreads quickly when it is reared with a large number of animals, especially fatal for newborns It often becomes a thing. In order to prevent infection by such pathogenic microorganisms, antibiotics and the like have been conventionally administered to livestock, and certain effects have been recognized. However, antibiotics accumulate in meat, and there is concern that humans may be affected by antibiotics by ingesting such meat and seafood, so the amount and type of antibiotics used have been limited. ing.

  Therefore, recently, in order to prevent the occurrence of diarrhea, loose stool, etc. and the increase in days of estrus, attempts have been made to prevent illness without using antibiotics. Probiotics that ingest microorganisms have been performed (Non-Patent Documents 1 to 4). However, the effect was not clear or excellent.

  In addition, in order to breed livestock, it is necessary for livestock to be estrus, pregnant and giving birth at a predetermined time, but it is difficult to estrus with the breeding of multiple heads. Was a problem. For this reason, hormonal agents that promote estrus are also administered, but it is known that hormonal agents flow into the environment and accumulate in fish and shellfish. And there is a concern that humans may be affected by hormones and the like due to ingestion of such seafood and the like, and suppression of the use of hormones and the like is required.

lben and Leibetseder, Tieraerztl. Mschr. 76 (1989), pp. 363-366, OstagVienna publisher Kyriakis et al., Research in Veterinary Science 1999, 67, pp. 223-228 Maenner and Spieler, Microecology and Therapy, 26, 243-256, 1997 O. Siomon, G Breves, 6th Pig and Poultry Nutrition Meeting, 2000, memoir, pp. 45-50

  Accordingly, an object of the present invention is to find a microorganism having excellent effects useful for probiotics, which can prevent diarrhea, loose stool, etc. of livestock and increase in the number of days of estrus resumption even in a normal livestock rearing environment. Is.

  As a result of intensive studies to solve the above problems, the present inventors have found a novel yeast useful for probiotics having the above-described effects. And it discovered that diarrhea, loose stool, etc. were prevented by administering the nutritional composition containing this novel yeast to livestock. Further, the present invention has been completed by finding that livestock to which the nutritional composition is administered grows healthy, shortens the number of days of estrus, improves the pregnancy rate, and has a high quality meat with little fat.

  That is, the present invention provides a novel yeast having antidiarrheal activity and belonging to the genus Saccharomyces.

  The present invention also provides a nutritional composition containing the novel yeast.

  Furthermore, the present invention provides a method for improving meat quality of livestock, a method for preventing diarrhea, and a method for shortening the number of days of estrus, characterized by administering the above nutritional composition.

  By administering a nutritional composition containing the novel yeast of the present invention to livestock, diarrhea, loose stool, etc. are prevented, and the livestock itself grows healthy, shortening the number of days for estrus recurrence, improving the pregnancy rate and improving the meat quality Improvement is observed.

  The novel yeast of the present invention belongs to the genus Saccharomyces and has anti-diarrheal activity.

Here, the anti-diarrheal activity indicates a percentage of the number of heads of diarrhea that have no diarrhea by weaning after administration of 1 ml of a culture solution containing 10 ⁶ to 10 ⁷ novel yeast / 1 ml to a pig within 1 day of life. . The anti-diarrheal activity of the novel yeast of the present invention is 80% or more, preferably 90% or more, more preferably 100%.

As an example of the above-mentioned novel yeast of the present invention, those showing the following mycological properties can be mentioned.
(1) Cell morphology
Oval shape (2μm × 3μm)
(2) Fermentation test (Test results)
Glucose: +
Sucrose: +
Lactose: −
Trehalose: −
Galactose: +
Maltose: −
Raffinose: −
(3) Utilization of carbon source (Test results)
Glucose: +
L-sorbose: +
Maltose: +
Trehalose: +
Melibiose: −
Soluble starch: −
L-arabinose: −
L-rhamnose: +
Ethanol: +
Galactitol: −
Erythritol: −
α-methyl-D-glucoside: +
Lactic acid: +
Citric acid: −
Merezitose: +
Galactose: +
Sucrose: +
Cellobiose: +
Lactose: −
Raffinose: −
D-xylose: +
D-ribose: +
Methanol: −
Glycerin: +
D-mannitol: +
Ribitol: +
Salicin: +
Succinic acid: +
Inositol: −
Inulin: −
(4) Nitrogen source utilization (test results)
Nitrate: −
Cadaverine: +
Ethylamine: +
Nitrite: −
L-lysine: +
(5) Culture conditions (YPD medium) (Test results)
Growth temperature: 10-50 ° C
Optimum culture temperature: 26 ° C
Culturable pH: 3 to 9.5
Optimum culture pH: 6-8
(6) Salt tolerance (YPD medium) (Test results)
94% or more without sodium chloride at a sodium chloride concentration of 1%
Growth

The mycological properties were tested and classified according to the following literature.
Reference 1: Sneath, PHA, Mair, NS, Sharpe, ME and Holt, JG: “
Bergey's Mannual of Systematic Bacteriology ”, Vol. 2 (1986) Williams
& Wilkins.
Reference 2: Holt, JG, Krieg, NR, Sneath, PHA, Staley, JT and
Williams, ST: “Bergey's Mannual of Determinative Bacteriology”,
Ninth Edition (1994) Williams & Wilkins.
Reference 3: Supervision of the Ministry of Health and Welfare, Health Sanitation Bureau: “Food Sanitation Inspection Guidelines-Microorganisms” (1990), Japanese food
Sanitation Association.
Reference 4: Farrow, JAE and Collins, MD: Int. J. Syst. Bacteriol., 35,
73-75 (1985).
Reference 5: Collins, MD Farrow, JAE, Kilpper-Balz, R and Schleifer, KH
: Int. J. Syst. Bacteriol., 34, 220-223 (1984).
Reference 6: Schleifer, KH and Kilpper-Balz, R .: Int. J. Syst. Bacteriol., 34,
31-34 (1984).

  The above mycological properties are more similar to Candida sp than yeast (Saccharomycs). In particular, the utilization of nitrogen sources and the utilization of sugars are different from conventional yeasts. However, for the D2 region of the rDNA of the novel yeast of the present invention, the base sequence was analyzed by the PCR method and cycle sequence using the LSU rDNA mold sequencing kit of Applied Biosystems, and the results were analyzed with microseek D2 ( D2 Fungal Database v.0050c) Part No. The result of library search using 4319240C was 100% consistent with Saccharomycs cerevisiae (FIG. 1).

  And although the novel yeast of this invention has the characteristic that it has anti-diarrhea activity, and the classification | category result by a rDNA analysis does not correspond with the classification | category by a mycological property as mentioned above, it has objectivity by a rDNA analysis. Therefore, it was temporarily identified as Saccharomyces cerevisiae as a new yeast of the genus Saccharomycs.

  The applicant named the above-mentioned new yeast Saccharomyces ST16, and on August 20, 2004, the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1 Chuo 1-chome, Tsukuba City, Ibaraki, 305-8566, Japan) Deposited to No. 6) (deposit number FERM P-20173).

The above-described novel yeast of the present invention is a dried bacterial cell obtained by drying the yeast or a culture solution obtained by culturing the yeast with skim milk or molasses by a conventional method, such as pigs, sheep, horses, cattle and other livestock, more preferably pigs. Can be administered. Moreover, since the novel yeast of this invention has anti-diarrheal activity, diarrhea and loose stool can be prevented by administering this. In this case, the dose of novel yeast is 1 to 10 g, preferably 1 to 1.5 g per head. Moreover, in the case of a culture solution, the dosage is 0.1 to 10 ml, preferably 0.5 to 3 ml, of a culture solution containing 10 ⁶ to 10 ⁷ yeast per ml per head.

In particular, the novel yeast of the present invention can effectively prevent diarrhea and loose stool when administered to young animals within one day of life, and as a result, the survival rate of young animals can be increased. When the novel yeast of the present invention is administered to young animals, 0.1 to 10 ml, preferably 0.5 to 0.5 ml of a culture solution containing 10 ⁶ to 10 ⁷ yeast per ml during the colostrum feeding period. It is preferable to administer 3 ml.

  In addition, since the new yeast of the present invention grows in a healthy manner when continuously fed to livestock, estrus is regularly re-established and the number of estrus is hardly reduced. For example, in the case of a pig that has been continuously ingesting the yeast of the present invention, estrus reappears about 4 days after birth, so it estrus about 2 to 5 times a year, and the pregnancy rate also improves.

  Furthermore, the novel yeast of the present invention can improve meat quality by administering it to livestock in the same manner as described above. For example, in the case of pigs, administration of this increases body weight but decreases fat thickness.

Furthermore, although the novel yeast of the present invention may be administered to livestock as it is as described above, it is preferably combined with other components to make a nutritional composition. Other components include lactic acid bacteria. This lactic acid bacterium synergistically enhances the effect of the yeast. The amount of lactic acid bacteria in the nutritional composition is not particularly limited, but is about 1-2 parts by weight with respect to 1 weight of the culture solution containing 10 ⁶ to 10 ⁷ yeasts.

Although it does not restrict | limit especially as said lactic acid bacteria, 1 type (s) or 2 or more types of lactic acid bacteria chosen from the genus Enterococcus (Enterococcus) genus, Lactococcus (Lactococcus) genus, and Lactobacillus (Lactobacillus) genus are preferable, and among them, the lactic acid bacteria of Enterococcus genus are preferable. preferably, in particular even the present inventors in the lactic acid bacterium Enterococcus have found previously (Japanese Patent Application No. 2003-387622) Enterococcus ST ² are preferred. This Enterococcus ST ² was deposited on November 13, 2003 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1st, 1st, 1st East, 1-chome, Tsukuba City, Ibaraki, 305-8566, Japan). (Deposit number FERM P-19586).

The Enterococcus ST ² is a lactic acid bacteria with bacteriological properties described below.
(Test items) (Test results)
1. Form: Streptococcus 1. Gram staining: +
3. Spore: −
4). Motility: −
5. 5. Attitude toward oxygen: facultative anaerobic Catalase: −
7). Gas production from glucose: −
8). Lactic acid produced: L (+)
9. Growth at 10 ° C: +
Growth at 10.45 ° C: +
11. Growth at 50 ° C:-
Growth in the presence of 12.6.5% NaCl: +
13. Growth at pH 9.6: +
14. Growth in the presence of 40% bile: +
15. Formation of yellow pigment: −
16. Arginine dihydrolase: +
17. Hippuric acid hydrolysis: −
18. Esculin hydrolysis: +
19. Growth with 0.1% methylene blue milk: +
20. Acid generation
D-xylose: −
L-rhamnose: −
Sucrose: +
Lactose: +
Melibiose: +
Raffinose: +
Meletetose: −
Glycerol: −
Adonitol: −
Sorbitol: −
Mannitol: −
21. GC content of intracellular DNA (HPLC method): 38 mol%

The above-mentioned Enterococcus ST ² was identified as belonging to the genus Enterococcus from DNA analysis, but the proportion of double spheres rose to nearly 90%, and slight growth was confirmed in the LB medium adjusted to pH 12 with sodium hydroxide. In addition, it has a particularly strong alkaline resistance, and has a property not found in other Enterococcus microorganisms that emits red fluorescence when exposed to light at 554 nm.

  Moreover, in addition to the said lactic acid bacteria, (beta) -glucan can be mix | blended with the said nutrition composition. Examples of such β-glucan include yeasts such as beer yeast, wine yeast, sake yeast, baker's yeast or marine yeast, or mushrooms such as Agaricus, Meshimakobu, Shiitake, and mycelium derived from these, and beer yeast And / or those derived from mushroom mycelium are preferred, and those derived from brewer's yeast are particularly preferred. By adding these β-glucans, a detoxifying action is exhibited.

These nutritional compositions are preferably 0.1 to 5 parts by weight, preferably 0.5 to 2 parts by weight of β-glucan and 0.01 to 0.5 parts by weight of the lactic acid bacterium with respect to 1 part of the novel yeast of the present invention. It contains 50 parts by weight, preferably 0.1 to 20 parts by weight. The method for producing this nutritional composition is not particularly limited. For example, the yeast of the present invention cultured to 10 ⁶ to 10 ⁹ per ml in skim milk and 10 ⁸ to 10 ⁹ per 1 ml of lactic acid bacteria in animal milk. It is manufactured by mixing the cultivated to the individual in the above amount, adding dry β-glucan to this, and mixing.

  In addition, an excipient such as rice bran, bran or okara can be blended with the above nutritional composition to such an extent that the effects of the present invention are not impaired. Furthermore, these nutritional compositions may be added to commercially available blended samples.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1
Production of nutritional composition:
Nutritional compositions having the compositions described in the following table were produced. Yeast (Saccharomyces ST16) was planted in 10% skim milk, incubated at 37 ° C. for 48 hours, about 5% of which was planted again in 10% skim milk, and incubated at 37 ° C. for 24 hours as an inoculum. Further, lactic acid bacteria (Enterococcus ST ² ) were also cultured in the same manner as seed bacteria. These inoculums were added to dry β-glucan and mixed with stirring to produce a liquid nutritional composition.

Example 2
Administration of nutritional composition to pigs (anti-diarrhea activity):
Each nutritional composition produced in Example 1 was orally administered to each pig (10 heads) within 1 day after birth in each section. Moreover, subsequent breeding was performed until weaning (20 days) using milk. For milk, 1 g of a commercially available mixed feed per piglet was used. Antidiarrheal activity was measured according to the following evaluation criteria. In addition, instead of the nutritional composition, antibiotics were administered, and thereafter, piglets reared in the same manner were used as controls.

(Antidiarrhea activity evaluation criteria)
Evaluation: State of diarrhea ++++: There was no diarrhea before weaning after administration of the nutritional composition ^* .
++: There was rare stool until weaning after administration of the nutritional composition ^* , but it was nourished again
There was no diarrhea before weaning after administration of Composition ^* .
+: There was rare diarrhea before weaning after administration of the nutritional composition ^* , but again nutrition
There was no diarrhea before weaning after administration of Composition ^* .
±: Diarrhea before weaning after administration of nutritional composition ^* , lower after administration of antibiotics
There was no diarrhea.
-: Diarrhea before weaning after administration of nutritional composition ^* and after administration of antibiotics
Child who has died of diarrhea and died
There was a pig.
* Antibiotics in case of control

Example 3
Administration of nutritional composition to pigs (estrus reoccurrence):
A nutritional composition produced in the same manner as in Example 1 (Practical product 1), with 2 kg added per 1000 kg of commercially available mixed feed, was used as a regular feed, and was freely consumed by pigs (50). The estrus rate within 7 days after weaning from July to September for 3 years before administration and 2 years after administration of the diet containing the nutritional composition, and the average pregnancy rate were determined.

  The estrus rate after weaning from July to September for 2 years after ingestion of the diet containing the nutritional composition was 88 to 94%, and the average pregnancy rate was about 95%. On the other hand, the estrus rate after weaning in July-September for 3 years before ingestion of the diet containing the nutritional composition was 68-74%, and the average pregnancy rate was about 60%.

Example 4
Administration of nutritional composition to pigs (improvement of meat quality):
A nutritional composition produced in the same manner as in Example 1 (Practical product 1) with 2 kg added per 1000 kg of commercially available mixed feed is used as a regular feed, which is freely consumed by pigs (50 heads), and until shipment Growing (6-7 months). After shipping, the carcass back fat thickness of each pig was measured (carcass weight average 74 kg). Moreover, the pig which grew only with the compound feed was made into the control.

  As a result, the portion where the carcass back fat thickness of the pigs that had consumed the diet containing the nutritional composition was 25 mm or more was about 8%. On the other hand, the portion where the carcass back fat thickness of pigs grown only with the mixed feed was 25 mm or more was about 24 to 30%.

Example 5
Production of mixed feed containing nutritional composition:
200 g of the nutritional composition produced in Example 1 was mixed with 20 kg of rice bran. Subsequently, this was mixed with 1000 kg of commercially available mixed feed to produce a mixed feed containing a nutritional composition.

  By administering a nutritional composition containing the novel yeast of the present invention to livestock, diarrhea, loose stool, etc. can be prevented. Moreover, livestock grows healthy by administering the nutritional composition, shortening the number of days for estrus reoccurrence, improving pregnancy rate, and improving meat quality.

FIG. 1 is a drawing showing taxonomic positions in a phylogenetic tree based on the nucleotide sequence of the D2 region of the rDNA of the novel yeast of the present invention. FIG. 2 is a photograph showing the form of the novel yeast of the present invention. more than

Claims (15)

  A novel yeast belonging to the genus Saccharomyces and having anti-diarrheal activity. The novel yeast according to claim 1, which has assimilation ability of the following nitrogen source.
(Nitrogen source) (Test results)
Nitrate: −
Cadaverine: +
Ethylamine: +
Nitrite: −
L-lysine: +   The novel yeast according to claim 1 or 2, which is Saccharomyces ST16 (FERM P-20173).   A nutritional composition containing the novel yeast according to any one of claims 1 to 3.   The nutritional composition according to claim 4, further comprising lactic acid bacteria.   The nutritional composition according to claim 5, wherein the lactic acid bacterium is one or more lactic acid bacteria selected from the genus Enterococcus, the genus Lactococcus and the genus Lactobacillus. Lactic acid bacteria, Enterococcus ^ST 2 (FERM P-19586) in a claim 5, wherein or nutritional composition of the sixth Claims.   The nutritional composition according to any one of claims 4 to 7, further comprising β-glucan.   The nutritional composition according to claim 8, wherein the β-glucan is derived from brewer's yeast and / or mushroom mycelium.   The nutritional composition according to any one of claims 4 to 9, which is used for preventing diarrhea and loose stool.   The nutritional composition according to any one of claims 4 to 9, which is used for preventing estrus decline.   The nutritional composition according to any one of claims 4 to 9, which is for livestock.   A method for improving meat quality of livestock, comprising administering the nutritional composition according to any one of claims 4 to 9 to livestock.   A method for preventing diarrhea and loose stool from livestock, comprising administering to the livestock the nutritional composition according to any one of claims 4 to 9. A method for shortening the number of days of estrus recurrence in livestock, comprising administering the nutritional composition according to any one of claims 4 to 9 to livestock.

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