JP7539074B2 - Livestock feed for preventing diarrhea or promoting weight gain - Google Patents

Description

FERM FERM BP-10284

Applicable under Article 30, Paragraph 2 of the Patent Act Sales date: October 7, 2019 to December 25, 2019 Sales location: See Attachments 1 and 2

The present invention relates to livestock feed, feed additives, and diarrhea suppressants or livestock weight gain promoters for inhibiting diarrhea or promoting weight gain.

Postweaning piglet diarrhea (PWD) is a disease caused mainly by infection with enterotoxigenic Escherichia coli (ETEC), which leads to poor weight gain and death due to diarrhea, resulting in economic losses. The use of antibacterial agents is common in PWD control, but the use of live bacteria and organic acid preparations is also attracting attention. Colistin sulfate, which was previously used to control PWD, had its approval as a feed additive revoked in 2018, so there is a demand for new, practical PWD control materials to replace colistin sulfate.

Various materials have been studied to suppress diarrhea and the associated decline in weight gain in suckling to weaning piglets. For example, Bacillus polymyxa strains (Patent Document 1), live Lactobacillus acidophilus and Streptococcus faecalis (Patent Document 2), and killed Enterococcus faecalis EC-12 strain (Accession No. FERM BP-10284) (Patent Document 3, Non-Patent Document 1) are known as diarrhea suppression materials using microorganisms such as lactic acid bacteria. Materials using plant-derived components include tea leaf fiber (Patent Document 4), a material consisting of sugar flavor and licorice extract and/or stevia extract (Patent Document 5), dextran fermentation by-products (Patent Document 6), and a mixture of fine powders of loosestrife, artemisanthemum, and meadowsweet with fine powders of dried banana and locust bean (Patent Document 7). However, there are few diarrhea-suppressing materials in practical use for piglets, and there is still a need for practical materials that have excellent diarrhea-suppressing and weight-gain promoting effects.

International Publication No. 2005/087240 Japanese Patent Publication No. 63-238020 Japanese Patent Publication No. 51-106725 Japanese Patent Application Laid-Open No. 7-16062 JP 2001-95502 A JP 2001-269125 A JP 2004-065068 A

Japanese Society of Swine Disease Research Bulletin, No. 48, p.19-23, 2006

Therefore, the present invention aims to provide a practical means that has excellent effects in suppressing diarrhea and promoting weight gain in livestock such as weaning piglets.

As a result of extensive research, the inventors of the present application discovered that feeding livestock (particularly weaned piglets) with a feed that combines sugar cane extract and killed lactic acid bacteria cells synergistically increases the diarrhea-suppressing effect and the weight-gain-promoting effect, which are significantly more effective than feeding each ingredient alone; therefore, it is possible to obtain sufficiently high diarrhea-suppressing effect and weight-gain-promoting effect while keeping the amount of each ingredient added low, and thus completed the present invention.

That is, the present invention provides a livestock feed for suppressing diarrhea or promoting weight gain, which contains a sugarcane extract at a concentration of 0.0009% to 0.24% in terms of solids concentration excluding sugar, and contains killed lactic acid bacteria cells at a cell count of 5.0 x ¹⁰⁵ cells/g feed to 2.5 x ¹⁰⁸ cells/g feed. The present invention also provides a method for suppressing diarrhea or promoting weight gain in livestock, which comprises feeding the feed of the present invention to livestock. Furthermore, the present invention provides use of the feed of the present invention for suppressing diarrhea or promoting weight gain in livestock .

The feed of the present invention is highly effective in suppressing diarrhea and promoting weight gain in livestock such as weaned piglets. By combining sugar cane extract with killed lactic acid bacteria, the diarrhea suppression effect and weight gain promotion effect are synergistically enhanced, so the amount added to the feed can be kept low, and a feed with excellent diarrhea suppression and weight gain promotion effects can be provided at low cost.

This is the test schedule for "A. Screening of materials to prevent diarrhea in postweaned piglets." This is the relative fecal quality score of the eight test plots in "A. Screening of materials to prevent diarrhea in postweaned piglets." This shows the relative fecal property scores for each test group in "B. Study on the concentration of added sugar cane extract and killed lactic acid bacteria powder 1." This shows the relative fecal property scores for each test group in "C. Study on the concentration of added sugar cane extract and killed lactic acid bacteria powder 2." These are the results of a growth test in "D. The effect on growth of weaned piglets when lactic acid bacteria powder and sugar cane extract are given."

The livestock feed of the present invention is a feed for inhibiting diarrhea or promoting weight gain, and contains sugarcane extract and killed lactic acid bacteria. By combining sugarcane extract and killed lactic acid bacteria, the diarrhea-inhibiting effect and weight-gain promoting effect are synergistically enhanced, and the amount of each component added can be kept low, and in particular the amount of killed lactic acid bacteria added can be kept very low.

In the present invention, diarrhea includes diarrhea caused by various factors, such as diarrhea caused by pathogenic bacteria such as pathogenic E. coli, diarrhea caused by parasites such as coccidia, diarrhea caused by viruses such as rotavirus, and diarrhea caused by stress. In the present invention, diarrhea suppression can be, for example, the suppression of diarrhea in postweaned piglet diarrhea.

The sugarcane extract used in the present invention is a fraction that absorbs light at a wavelength of 420 nm among fractions obtained by fractionating sugarcane juice or sugarcane-derived molasses by cation exchange chromatography. Methods for producing such sugarcane extracts are described, for example, in JP-A-2000-297046, JP-A-2011-32240, JP-A-2012-214481, etc.

As the sugarcane juice, it is preferable to use filtered compressed sugarcane juice. As the sugarcane-derived molasses, it is possible to use the residue obtained after crystallizing and recovering monosaccharides and oligosaccharides from compressed sugarcane juice, which is a by-product of the sugar manufacturing process from sugarcane. The sugarcane-derived molasses may be appropriately diluted to an appropriate Brix (e.g., about 40 to 60), impurities may be coagulated by adding hydrated lime or the like, and then filtered with diatomaceous earth or the like before being subjected to cation exchange chromatography. The term "fractionating sugarcane juice or sugarcane-derived molasses by cation exchange chromatography" includes subjecting sugarcane juice or sugarcane-derived molasses to cation exchange chromatography after appropriate pretreatment such as filtration.

As the cation exchange resin used in the cation exchange chromatography, a strongly acidic type (e.g., sodium salt, potassium salt, or calcium salt of sulfonated polystyrene resin), sodium ion type, or potassium ion type cation exchange resin can be preferably used. In addition, ion exchange resins are classified into gel type resins and porous resins (porous type, microporous type, highly porous type, etc.) based on their form, and it is preferable to use a gel type resin in the production of the sugarcane extract used in the present invention.

The chromatography method may be a single-column batch separation method or a simulated moving bed continuous separation method.

The production of sugarcane extract by the single-column batch separation method is described in detail, for example, in JP-A-2000-297046. Specifically, separation is performed using a column packed with a cation exchange resin having a wet volume of 5 to 500 times the solid content of the raw material, and water as the eluent. The flow rate is preferably SV=0.3 to 1.0 hr ^-1 , the amount of sample provided is 1 to 20% of the resin, and the temperature is preferably 40 to 70°C. The eluate is separated into a plurality of fractions and collected, and the absorbance at a wavelength of 420 nm is measured, and the fraction with the absorbance peak is used as the sugarcane extract. The fraction absorbing light at a wavelength of 420 nm contains a large amount of polyphenols and contains almost no monosaccharides or oligosaccharides.

In the simulated moving bed continuous separation method, the feed rate of the raw material liquid, the eluent flow rate, and the extraction flow rate of each fraction are set according to the composition of the raw material, the type of fixed carrier, and the amount of resin. Specific examples of the production of sugarcane extract using the simulated moving bed continuous separation method are described in, for example, JP 2011-32240 A and JP 2012-214481 A. In the case of the simulated moving bed continuous separation method, a sucrose fraction and a non-sucrose fraction (a fraction containing little or no sucrose) can be separated and recovered, but since the fraction that absorbs light with a wavelength of 420 nm is included in the non-sucrose fraction, the non-sucrose fraction can be used as the sugarcane extract.

The fraction that absorbs light at a wavelength of 420 nm may be used as a liquid sugarcane extract as is or after concentration, or the liquid extract may be adsorbed on a solid support (excipient) such as rice bran oil cake or dextrin, dried, and used as a powder or granule. In addition, such liquid, powder, or granular sugarcane extracts are commercially available (for example, Kibishibori EX and Kibishibori EX-L manufactured by Mitsui Sugar Co., Ltd.), and such commercially available products may be used in the present invention.

The killed lactic acid bacteria used in the present invention are killed bacteria of the Enterococcus faecalis EC-12 strain (accession number FERM BP-10284). The method for preparing the killed bacteria is not particularly limited, and the killed bacteria may be prepared so as to contain almost the entire bacterial components. The EC-12 strain is cultured by a conventional method, the bacteria are collected from the culture, washed, and sterilized, and the resulting product can be used as the killed lactic acid bacteria. The method for the sterilization is not particularly limited, and any method that can sterilize lactic acid bacteria may be used, such as heat treatment, physical treatment such as ultrasonic treatment, and enzyme treatment with a lytic enzyme (lysozyme, etc.). After the sterilization treatment, the bacteria may be concentrated as necessary to increase the concentration of the bacterial components (bacterial density). In addition, the killed lactic acid bacteria may be sterilized and concentrated as necessary, and then dried by spray drying, freeze drying, or the like, and used in a powder form. A method for producing killed bacteria of the Enterococcus faecalis EC-12 strain is also described in, for example, JP 2017-101006 A.

The concentration of sugarcane extract in the feed of the present invention is not particularly limited, but is preferably about 0.0009% to 0.24% in terms of solids concentration excluding sugar (0.005% to 1% in terms of the concentration of the granular product used in the examples below), for example, about 0.0009% to 0.024% (0.005% to 0.1%), about 0.0009% to 0.012% (0.005% to 0.05%), about 0.0009% to 0.0096% (0.005% to 0.04%), about 0.0009% to 0.0072%. (same as 0.005% to 0.03%), about 0.0009% to 0.0048% (same as 0.005% to 0.02%), about 0.0018% to 0.024% (same as 0.01% to 0.1%), about 0.0018% to 0.012% (same as 0.01% to 0.05%), about 0.0018% to 0.0096% (same as 0.01% to 0.04%), about 0.0018% to 0.0072% (same as 0.01% to 0.03%), or about 0.0018% to 0.0048% (same as 0.01% to 0.02%) may also be used.

In the present invention, a concentration of 1% in the feed or an added concentration of 1% refers to a concentration of 1 g per 100 g of feed.

The concentration of killed lactic acid bacteria cells in the feed of the present invention is not particularly limited, but is preferably 5.0× ¹⁰⁵ cells/g feed to 5.0× ¹⁰⁹ cells/g feed (0.00001% to 0.1% in terms of the number of cells) and examples thereof include 5.0× ¹⁰⁵ cells/g feed to 2.5× ¹⁰⁹ cells/g feed (0.00001% to 0.05%), 5.0× ¹⁰⁵ cells/g feed to 5.0× ¹⁰⁸ cells/g feed (0.00001% to 0.01%), 5.0× ¹⁰⁵ cells/g feed to 2.5× ¹⁰⁸ cells/g feed (0.00001% to 0.005%), 5.0× ¹⁰⁵ cells/g feed to 5.0×10 ⁷ pieces/g feed (0.00001% to 0.001%), 5.0×10 ⁵ pieces/g feed to 2.5×10 ⁷ pieces/g feed (0.00001% to 0.0005%), 5.0×10 ⁶ pieces/g feed to 2.5×10 ⁹ pieces/g feed (0.0001% to 0.05%), 5.0×10 ⁶ pieces/g feed to 5.0×10 ⁸ pieces/g feed (0.0001% to 0.01%), 5.0×10 ⁶ pieces/g feed to 2.5×10 ⁸ pieces/g feed (0.0001% to 0.005%), 5.0×10 ⁶ pieces/g feed to 5.0×10 ⁷ pieces/g feed (0.0001% to 0.001%), or 5.0×10 The concentration may be ⁶ to 2.5 x ¹⁰⁷ pieces/g of feed (0.0001% to 0.0005%).

The livestock to which the feed of the present invention is fed is not particularly limited, but preferably the feed of the present invention is a pig feed, for example, a piglet feed. The age or developmental stage of the piglets to which the feed of the present invention is fed is not particularly limited, but the feed can be used as feed for suckling, weaning and early growing piglets, or piglets of about 1 to 50 days of age. By feeding the feed of the present invention to livestock, diarrhea in the livestock can be suppressed or weight gain can be promoted.

The feed additive for inhibiting diarrhea or promoting weight gain of the present invention includes a combination of sugarcane extract and killed lactic acid bacteria cells as defined above. The term "including a combination" means that the feed additive includes a form in which the sugarcane extract and killed lactic acid bacteria cells are mixed as a single agent, and a form in which these two components are provided in combination as separate agents. The feed additive is an additive for livestock feed, and the livestock may be, as described above, pigs, such as piglets, but is not limited thereto. The concentration of the feed additive is not particularly limited, but it is preferable to add each component to the feed at the above-mentioned concentrations, for example. Specifically, the sugarcane extract is preferably added at a solid concentration excluding sugar of about 0.0009% to 0.24% (0.005% to 1% in terms of the concentration of the granular product used in the following examples), and may be, for example, about 0.009% to 0.12% (0.05% to 0.5%) in addition to the above-mentioned concentration range. The killed lactic acid bacteria cells are preferably added at a cell count of 5.0 x ¹⁰ to 5.0 x 10 cells/g feed (0.00001% to ^0.1 % in terms of the concentration of the lactic acid bacteria material used in the Examples below), and may be, for example, in addition to the above-mentioned concentration range, 5.0 x ¹⁰ to 5.0 x ¹⁰ cells/g feed (0.001 to 0.01%).

The diarrhea suppressant or weight gain promoter for livestock of the present invention contains a combination of sugarcane extract and killed lactic acid bacteria as active ingredients. The term "containing a combination" has the same meaning as above. The diarrhea suppressant or weight gain promoter is an agent to be added to feed and given to livestock, and examples of livestock include, but are not limited to, pigs, as above, such as piglets. The agent can be used by adding each component to feed at the concentrations described above.

The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples.

A. Screening of materials to prevent postweaned piglet diarrhea <Objective>
Postweaning piglet diarrhea (PWD) is a disease caused mainly by enterotoxigenic Escherichia coli (ETEC) infection, which leads to poor weight gain and death due to diarrhea, resulting in economic losses. Antimicrobial agents are commonly used to control PWD, but the use of live bacteria and organic acid preparations has also attracted attention. Colistin sulfate, which was previously used to control PWD, was revoked as a feed additive in 2018, and there are concerns that the incidence of PWD will increase. Therefore, we aimed to develop new PWD control materials and investigated the diarrhea prevention effects of various materials.

Materials and Methods
1. Infection test
(1) Test pigs
We used 19-day-old early-weaned piglets. DNA was extracted from the tail and typing of the F4 fimbria receptor-associated gene (MUC4 gene) was performed by the method described in Jensen et al., Veterinary Microbiology 115 (2006) 243-249. We selected individuals that were heterozygous for the gene for the experiment.
(2) Test Feeds Various test feeds were prepared by adding various materials to commercially available feed with a typical composition for young piglet feed (artificial milk).
(3) Test strains The field isolate Rif ETEC88 (accession number NITE P-02895) of enterotoxigenic E. coli (fimbrial genes; F4ab,ac, toxin genes; LT, STa, STb) was used.
(4) Setting of test groups and infection schedule The test schedule is shown in Figure 1. Five piglets were used in group 1 and fed the test feed. On the fifth day of feeding, they were challenged with Rif ETEC88. The challenge bacterial count was 3.0 x ¹⁰⁹ CFU/pig (6.0 x ¹⁰⁸ CFU/ml in 10% sterilized skim milk was orally administered in 5 ml).

2. Evaluation of PWD Fecal characteristics were scored according to the following criteria, and a score was calculated for each test group, with the challenge control group (group not given any materials) being assigned a score of 100.
Score 0 (normal stool): The stool holds its shape well on the cotton swab and has a rough surface.
Score 1 (loose stool): The stool holds its shape on the cotton swab, but is slightly moist and has a shiny surface under light.
Score 2 (Muddy stool): Watery and muddy. Most of the stool falls off when swabbed.
Score 3 (watery stool): The stool is mostly water with some undigested gastrointestinal contents being visible.

<Results>
Figure 2 shows the relative fecal property scores for eight test groups that were fed test feed containing the eight materials shown in Table 1 below. Among the materials shown in Table 1, the sugar cane extract material and killed lactic acid bacteria powder had particularly low fecal property scores, confirming their high diarrhea suppression effect.

The sugarcane extract used was a granule (prepared by the methods described in JP 2000-297046 A, JP 2011-32240 A, JP 2012-214481 A, etc.) obtained by fractionating sugar-depleted squeezed juice from sugarcane by cation chromatography and adsorbing the fraction absorbing light at a wavelength of 420 nm onto rice bran oil cake and drying it. This granule contains 180-240 mg of non-sugar solids per gram. EC-12 (Combi Corporation) used as the killed lactic acid bacteria powder is a lactic acid bacteria material obtained by culturing Enterococcus faecalis EC-12 strain (accession number FERM BP-10284), heat sterilizing it, and then concentrating it, and contains 5 x 10 ¹² or more bacteria per gram.

B. Study on the concentration of sugarcane extract and killed lactic acid bacteria powder 1
＜Objective＞
Based on the results of A above, sugar cane extract and killed lactic acid bacteria powder were selected as candidates for PWD control materials, and the concentration at which they should be added to feed was examined.

Materials and Methods
1. Test animals
18-day-old early weaned piglets were used, 4 or 5 per group. The F4 fimbrial receptor-associated gene (MUC4 gene) was typed by the method described in Jensen et al. 2006 (supra), and individuals heterozygous for the gene were selected for the experiment. The piglets were individually housed in open pens to facilitate the observation of the onset of diarrhea in each individual.

2. Test strains The field isolate Rif ETEC88 (accession number NITE P-02895) of enterotoxigenic E. coli (fimbrial genes; F4ab,ac, toxin genes; LT, STa, STb) was used.

3. Test Feed Test feed was prepared by adding sugarcane extract and killed lactic acid bacteria powder (A-17, Combi Corporation) at the concentrations shown in Tables 2-1 and 2-2 below to commercially available feed with a typical composition for nursery pig feed (artificial milk). A-17 is an equivalent product to EC-12, and is a lactic acid bacteria material obtained by culturing and heat-sterilizing the Enterococcus faecalis EC-12 strain (accession number FERM BP-10284) and concentrating it, containing approximately 5 x ¹⁰¹² bacteria per gram.

4. Exam Schedule
(1) Test animals were introduced and fed the test feed for 5 days.
(2) All piglets were infected with strain 2 by oral administration (challenge bacterial counts ranged from 1.0 × 10 ⁹ CFU/head to 3.0 × 10 ⁹ CFU/head).
(3) The fecal characteristics were observed and scored for 7 days after infection using the same criteria as in A above.
(4) The scores for each test area were calculated so that the challenge control area (area not given the material) was set to 100.

<Results>
The fecal property scores are shown in Figure 3. It was confirmed that the combination of sugar cane extract and killed lactic acid bacteria powder synergistically enhanced the diarrhea suppression effect.

C. Study on the concentration of sugarcane extract and killed lactic acid bacteria powder 2
＜Objective＞
Since a synergistic antidiarrheal effect was observed when sugarcane extract and killed lactic acid bacteria powder were combined, the effectiveness of both ingredients at low concentrations was evaluated.

Materials and Methods
1. Test animals
The rest of the experiment was the same as Test B above, with four horses per section.
2. Test strains: Same as test B above.
3. Test Feed Test feed was prepared by adding sugar cane extract and killed lactic acid bacteria powder (A-17, Combi Corporation) at the concentrations shown in Tables 3-1 and 3-2 below to commercially available feed with a typical composition for young pig feed (artificial milk).

4. Test area setup The test areas were set up as shown in Table 4 below.

5. Exam Schedule
(1) Test animals were introduced and fed the test feed for 5 days.
(2) All piglets were orally infected with strain 2 (challenge bacterial count: 6.3×10 ⁸ CFU/head in groups 1-7 and 8.4×10 ⁸ CFU/head in groups 8-12).
(3) The fecal characteristics were observed and scored for 7 days after infection using the same criteria as in A above.
(4) The scores for each test area were calculated so that the challenge control area (area not given the material) was set to 100.

<Results>
The fecal property scores are shown in Figure 4. No piglets in groups 1 and 5 had watery stools (score 3). Comparing groups 1 to 4 (0.0005% A-17 added), the relative fecal score in group 4 was not different from that in the challenge control group, and the addition of 0.001% sugarcane extract did not have any diarrhea-suppressing effect. On the other hand, comparing groups 2, 6, 9, and 11, which were added with the same amount of sugarcane extract (0.01%), the diarrhea-suppressing effect in groups 2 and 6 increased depending on the amount of A-17 added, but no change was observed in groups 9 and 11. These results show that when sugarcane extract is added at a concentration of 0.01% or less, the addition of 0.00005% or less A-17 does not have a sufficient diarrhea-suppressing effect.

D. Effects of feeding lactic acid bacteria powder and sugar cane extract to weaned piglets on their growth. <Materials and Methods>
(1) Test animals: 24 weaned piglets weighing approximately 6 kg (4 pigs/group x 2 groups x 3 replicates)
(2) Test section Control group: Two types of commercially available artificial pig milk Test group: The control group was supplemented with 0.00025% lactic acid bacteria powder (A-17, Combi Corporation) and 0.0125% sugar cane extract.
(3) Test method Weaned piglets weighing approximately 6 kg were grouped according to sex and weight and subjected to a 19-day growth test (Table 5).

<Results>
The test results are shown in Table 6 and Figure 5. The weight gain was 375g/day in the control group and 398g/day in the test group, and the feed intake was 488g/day in the control group and 527g/day in the test group, showing that the test group was superior to the control group in both weight gain and feed intake. The feed conversion rate was 1.32 in the control group and 1.32 in the test group, with almost no difference between the test groups. This confirmed that the addition of lactic acid bacteria powder and sugar cane extract to pig artificial milk improves the growth of weaned piglets.

Claims (9)

A livestock feed for inhibiting diarrhea or promoting weight gain, comprising a sugar cane extract at a concentration of 0.0009% to 0.24% as a solids concentration excluding sugar, and containing killed lactic acid bacteria cells at a cell count of 5.0 x ¹⁰⁵ cells/g feed to 2.5 x ¹⁰⁸ cells/g feed. The feed according to claim 1, wherein the sugarcane extract is a fraction that absorbs light at a wavelength of 420 nm among fractions obtained by fractionating sugarcane juice or sugarcane-derived molasses by cation exchange chromatography. The feed according to claim 1 or 2, wherein the killed lactic acid bacteria cells are killed cells of Enterococcus faecalis EC-12 strain (accession number FERM BP-10284). The feed according to any one of claims 1 to 3, comprising a sugar cane extract at a concentration of 0.0009% to 0.024% as a solids concentration excluding sugar, and comprising killed lactic acid bacteria cells at a cell count of 5.0 x ¹⁰ cells/g feed to 2.5 x ¹⁰ cells/g feed. The feed according to claim 4, containing sugarcane extract at a solids concentration of 0.0009% to 0.0096%. The feed according to any one of claims 1 to 5, wherein the livestock is a pig. The feed according to claim 6, wherein the livestock is a piglet. A method for suppressing diarrhea or promoting weight gain in livestock, comprising feeding the livestock with the feed according to any one of claims 1 to 7. Use of the feed according to any one of claims 1 to 7 for suppressing diarrhea or promoting weight gain in livestock.