阅读说明：本技术 饲料用添加剂及饲料 (Additive for feed and feed ) 是由 上原章敬 中川一辉 M·前川 于 2019-03-18 设计创作，主要内容包括：通过本发明,提供被覆型的家畜的体增重效果或饲料转化率提高剂,其中,所述提高剂由包含生理活性物质的核、及核的被覆剂构成,所述生理活性物质具有使消化道上皮细胞的膜坚固化的性质。(The present invention provides an agent for improving the body weight gain or feed conversion ratio of a coated livestock, wherein the agent comprises a core containing a physiologically active substance having a property of hardening the membrane of the epithelial cells of the digestive tract and a coating agent for the core.)

1. An agent for improving body weight gain effect or feed conversion ratio of coated livestock, wherein,

the enhancer comprises a core containing a physiologically active substance and a coating agent for the core,

the physiologically active substance has a property of solidifying the membrane of the epithelial cells of the digestive tract.

2. The livestock body weight gain effect or feed conversion ratio improver according to claim 1, wherein said physiologically active substance comprises at least 1 selected from the group consisting of bacteriocins, polyphenols, amino acids or derivatives thereof, organic acids or derivatives thereof, HSP inducer, antioxidants, and polysaccharides.

3. The livestock body weight gain effect or feed conversion ratio improver according to claim 1 or 2, wherein the physiologically active substance is 1 or more bacteriocins selected from nisin, subtilin, phytolaccin, and lactein.

4. The livestock body weight gain effect or feed conversion ratio improving agent according to any one of claims 1 to 3, wherein said physiologically active substance is contained in the form of a culture of a microorganism producing the substance.

5. The livestock body weight gain effect or feed conversion ratio improving agent according to any one of claims 1 to 4, wherein said physiologically active substance is contained in the form of a culture of 1 or more microorganisms selected from the group consisting of Bacillus (Bacillus), Lactococcus (Lactobacillus), Lactobacillus, Leuconostoc (Leuconostoc), Pediococcus (Pediococcus).

6. The livestock body weight gain effect or feed conversion ratio improving agent according to claim 5, wherein said microorganism is Bacillus subtilis, Lactococcus lactis, or Lactobacillus plantarum.

7. The livestock body weight gain effect or feed conversion ratio improving agent according to claim 2, wherein said polyphenol is quercetin or tannin.

8. The livestock body weight gain effect or feed conversion ratio improver according to claim 2, wherein the amino acid is at least 1 selected from the group consisting of glutamine, phenylalanine, tryptophan, valine and tyrosine.

9. The livestock body weight gain effect or feed conversion ratio improving agent according to claim 2, wherein said HSP inducer is polyphosphoric acid or Competence and sporulation factor (conference and sporulation factor).

10. The livestock body weight increasing effect or feed conversion ratio improving agent according to any one of claims 1 to 9, wherein the coating agent contains at least 1 selected from the group consisting of hydrogenated vegetable oil, shellac, zein, hydroxypropylmethylcellulose and maltitol.

11. The livestock body weight gain effect or feed conversion ratio improving agent according to any one of claims 1 to 10, wherein,

the physiologically active substance is more than 1 bacteriocin selected from nisin, subtilin, plant lactobacillin and lactein,

the coating agent is hydrogenated rapeseed oil and/or shellac.

12. The livestock body weight gain effect or feed conversion ratio improver according to claim 11, wherein the coating layer formed by the coating agent has a two-layer structure of a layer formed from hydrogenated rapeseed oil and a layer formed from shellac.

13. The livestock body weight gain effect or feed conversion ratio improver according to claim 12, wherein the layer formed of shellac is attached to the core, on which the layer formed of hydrogenated rapeseed oil is formed.

14. A feed comprising the livestock body weight increasing effect or feed conversion ratio improving agent according to any one of claims 1 to 13.

15. A method of increasing the body weight gain effect or feed conversion ratio of livestock, the method comprising: administering the feed conversion ratio-improving agent according to any one of claims 1 to 13 or the feed according to claim 14 to livestock.

Technical Field

The present invention relates to an additive for feed suitable for livestock, particularly monogastric animals, and a feed containing the additive.

Background

In a report of "white paper of world population" of the united nations, the world population is estimated to reach 70 hundred million people in 2011, and the prediction that 2050 is further increased to 98 hundred million people. The problem of food crisis is also becoming more severe, especially with BRICS as the center, the demand for meat protein is increasing dramatically. Therefore, a technique for improving the productivity (productivity) of livestock meat is required.

As one of techniques for improving the productivity of livestock animals, growth-promoting Antibiotics (AGPs) are known. Antibiotics such as penicillin have been originally developed for therapeutic use mainly for inhibiting the proliferation of pathogenic bacteria, and are widely used as medicines. Thereafter, the use of antibiotics has expanded and has also begun to be widely used for the purpose of promoting the growth of livestock animals since the latter half of the 50 s of the 20 th century. On the other hand, the use for growth promotion was prohibited in europe in 2006 and a strong restriction was determined in the us in 2015, from the viewpoints of problems of drug-resistant live bacteria and environmental pollution. In 2016, it was found that a Colistin (Colistin) -resistant gene was present on a mobile plasmid (non-patent document 1). In 2050, drug-resistant bacteria may have a high risk of exceeding cancer, and if no countermeasures are taken against this problem, the annual mortality rate may exceed cancer and reach 1000 million people, posing a global threat (non-patent document 2). AGP also used for therapy and therapeutic drugs common to humans and animals are also being shifted to the enhancement of restrictions by risk judgment in various countries as an urgent issue. In addition, in the united states, the fast food market is also planning to provide antibiotic-free meat.

One of the candidate alternatives to AGP is bacteriocin. Bacteriocins are a generic term for proteins and peptides produced by bacteria having antibacterial activity mainly against the same species or related species, and Nisin (Nisin) and plant lactobacillin (Plantaricin) are widely used as food preservatives. However, they are not used for feed applications because they are easily decomposed by digestive enzymes. Patent document 1 reports a technique for inhibiting the growth of intestinal pathogenic bacteria and diseases in livestock animals by using a lantibiotic such as nisin, a cell wall-soluble substance such as lysozyme, a sequestering agent such as a chelating agent, and an antibacterial substance such as egg powder in combination. Non-patent document 3 discloses a technique of adding a high-concentration solution obtained by isolating bacteria from a nisin-producing bacterium as a feed.

Regarding substances other than bacteriocins, non-patent document 4 reports that the body weight gain effect (also referred to as body weight gain efficiency) and the feed consumption are not increased and the feed conversion ratio (feed conversion ratio) is hardly or slightly decreased by adding 500 to 1000ppm of quercetin (flavonoid compound) to the feed. Patent document 2 is a technique of incorporating esterified pectin or a mixture thereof into a feed, but it is directed to inflammatory diseases.

On the other hand, the mechanism of action of AGP which exhibits a growth promoting effect in a relatively small amount is not clear. There are various hypotheses as to how antibiotics achieve improvement of feed conversion ratio (non-patent document 5).

Hypothesis 1) AGP prevents potential infections, reducing the metabolic costs required by the immune system;

hypothesis 2) AGP controls intestinal flora, intestinal bacteria suppress metabolites such as ammonia, and thereby promote growth of the host animal;

hypothesis 3) AGP inhibits the proliferation of intestinal bacteria, with a reduction in nutrients utilized by the microorganisms;

hypothesis 4) AGP weakens the digestive tract wall of livestock animals and enhances nutrient absorption.

For these hypotheses, t.a. niewold et al propose the following refutes: 1) AGP exerts antibacterial effect regardless of animal species; 2) even AGP with different antimicrobial spectrum can also play the same growth promoting effect; 3) substances which do not exert a growth promoting effect are also available in therapeutic antibiotics; 4) drug-resistant bacteria can be easily generated by long-term use of a low dosage of AGP with the use concentration below the Minimum Inhibitory Concentration (MIC); 5) the alternative antibacterial substance exhibiting antibacterial action is unstable in its antibacterial effect, although it has a high antibacterial effect (non-patent document 5).

Documents of the prior art

Patent document

Patent document 1: international publication No. 2004/026334

Patent document 2: international publication No. 2017/009257.

Non-patent document

Non-patent document 1: lancet Infect Dis (2016) vol.169p.161-165

Non-patent document 2: modified by Jim O' Neill, Review on adaptive Resistance, May 2016

Non-patent document 3: damian Jozefiak et al, PLoS ONE8(12): e85347,2013

Non-patent document 4: m. Goliomytis et al, Poult Sci (2014) volume 93(8):1957-62

Non-patent document 5: niewold, Poultry Science (2007) vol.86: pp 605-609.

Disclosure of Invention

Technical problem to be solved by the invention

From the above viewpoint, the mechanism of action of AGP is not clear, and thus there is no effective growth promoting substance that can substitute AGP. Accordingly, an object of the present invention is to provide an economical feed additive which exhibits a body-weight increasing effect even in healthy livestock, and a feed containing the additive.

Technical scheme for solving technical problem

The present inventors have conducted extensive studies and, as a result, have found that one of the effects of using AGP at a low concentration is to solidify the membrane of the epithelial cells of the digestive tract. Further, it has been found that the body weight increasing effect and the feed conversion ratio can be improved by coating a physiologically active substance having a property of solidifying the membrane of the epithelial cells of the digestive tract and feeding the coated chicken to healthy chickens. Based on this finding, the present inventors have completed an invention relating to an additive for feed which can improve the body weight gain effect of livestock by coating. That is, the present invention provides the following inventions:

1. an agent for improving body weight gain or feed conversion ratio of a coated livestock, which comprises a core containing a physiologically active substance and a coating agent for the core,

the physiologically active substance has a property of solidifying the membrane of the epithelial cells of the digestive tract;

2. the livestock body weight gain effect or feed conversion ratio improving agent according to the above 1, wherein said physiologically active substance comprises at least 1 selected from the group consisting of bacteriocins, polyphenols, amino acids or derivatives thereof, organic acids or derivatives thereof, HSP inducers, antioxidants, and polysaccharides;

3. the livestock body weight gain effect or feed conversion ratio improving agent according to the above 1 or 2, wherein the physiologically active substance is at least 1 bacteriocin selected from Nisin (Nisin), Subtilin (Subtilin), Plantaricin (Plantaricin), and lactein (Gassericin);

4. the livestock body weight gain effect or feed conversion ratio improving agent according to any one of the above 1 to 3, wherein the physiologically active substance is contained in the form of a culture of a microorganism producing the substance;

5. the livestock body weight gain effect or feed conversion ratio improving agent according to any one of the above 1 to 4, wherein the physiologically active substance is contained in the form of a culture of 1 or more microorganisms selected from the group consisting of Bacillus (Bacillus), Lactococcus (Lactobacillus), Lactobacillus, Leuconostoc (Leuconostoc), Pediococcus (Pediococcus);

6. the livestock body weight gain effect or feed conversion ratio improving agent according to the above 5, wherein said microorganism is Bacillus subtilis, Lactococcus lactis, or Lactobacillus plantarum (Lactbacillus plantarum);

7. the livestock body weight gain effect or feed conversion ratio improving agent according to the above 2, wherein the polyphenol is quercetin or tannin;

8. the livestock body weight gain effect or feed conversion ratio improving agent according to the above 2, wherein the amino acid is at least 1 selected from the group consisting of glutamine, phenylalanine, tryptophan, valine and tyrosine;

9. the livestock body weight gain effect or feed conversion ratio improving agent according to the above 2, wherein the HSP inducer is polyphosphoric acid;

10. the livestock body weight gain effect or feed conversion ratio improving agent according to any one of the above 1 to 9, wherein the coating agent comprises at least 1 selected from the group consisting of hydrogenated vegetable oil, shellac, zein (zein), hydroxypropyl methylcellulose and maltitol;

11. the livestock body weight gain effect or feed conversion ratio improving agent according to any one of the above 1 to 10, wherein the physiologically active substance is 1 or more bacteriocins selected from nisin, subtilin, phytolaccin, and lactein, and the coating agent is hydrogenated rapeseed oil and/or shellac;

12. the livestock body weight gain effect or feed conversion ratio improving agent according to 11, wherein the coating layer formed by the coating agent has a 2-layer structure of a layer formed by hydrogenated rapeseed oil and a layer formed by shellac;

13. the livestock body weight gain effect or feed conversion ratio improving agent according to the above 12, wherein the layer formed of shellac is connected to the core, and a layer formed of hydrogenated rapeseed oil is formed thereon;

14. a feed comprising an agent for improving body weight gain or feed conversion ratio of the livestock according to any one of 1 to 13;

15. a method for increasing body weight gain effect or feed conversion ratio of livestock, which comprises administering the feed conversion ratio-improving agent according to any one of 1 to 13 or the feed according to claim 14 to livestock.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention provides an economical additive for feeds and feeds which can exhibit a body weight increasing effect even in healthy livestock. In addition, the feed additive and the feed of the present invention can exhibit a body weight increasing effect even in a small amount.

Brief description of the drawings

FIG. 1A shows the recovery rate of barrier function at various concentrations of various physiologically active substances (AGP) (Experimental example A);

FIG. 1B shows the recovery rate of barrier function at various concentrations of various physiologically active substances (bacteriocins) (Experimental example A);

FIG. 1C shows the recovery rate of barrier function at various concentrations of various physiologically active substances (polyphenols) (Experimental example A);

FIG. 1D shows the recovery rate of barrier function at various concentrations of various physiologically active substances (amino acids) (Experimental example A);

FIG. 1E shows the recovery rate of barrier function at various concentrations of various physiologically active substances (organic acids) (Experimental example A);

FIG. 1F shows the recovery rates of barrier function at various concentrations of various physiologically active substances (HSP inducer) (Experimental example A);

FIG. 1G shows the recovery of barrier function at various concentrations of various physiologically active substances (antioxidants) (Experimental example A);

FIG. 1H shows the recovery rate of barrier function at various concentrations of various physiologically active substances (polysaccharides) (Experimental example A);

FIG. 2 shows the temporal change in the dissolution rate of quercetin from a two-layer coated feed additive under the conditions of gastric juice and intestinal juice (examples 1, 1-2);

FIG. 3 shows the change with time in the dissolution rate of tyrosine from a two-layer coated feed additive under the conditions of gastric juice and intestinal juice (examples 2, 2-2);

FIG. 4 shows the change with time in the dissolution rate of nisin A from a two-layer coated feed additive under gastric and intestinal juice conditions (example 33-2).

Detailed Description

[ nucleus ]

The present inventors have conducted a thorough investigation of physiologically active substances and have found that at least 1 selected from the group consisting of bacteriocins, polyphenols, amino acids or derivatives thereof, organic acids or derivatives thereof, HSP inducers, antioxidants, and polysaccharides has an excellent membrane-firming ability (membrane-fixing function).

Bacteriocins are classified into two groups, class I and class II, according to the classification by Paul d. cotter (nat. rev. microbiol.2005, vol. 3(10), pp. 777-88).

Bacteriocins belonging to class I, also known as Lantibiotics (Lantibiotics), have in their structure aberrant amino acids resulting from post-translational modifications. Specific examples thereof include nisin A (NisinA), nisin Z (NisinZ), nisin Q (NisinQ), Subtilin (Subtilin), Duramycin (Duramycin), mersacidin, and nisin-481 (lacticin 481).

Class II is a peptide that does not contain an aberrant amino acid in its structure. Class II is further divided into 3 subclasses a-c. Specific examples of bacteriocins belonging to class IIa include pediocin PA-1(pediocin PA-1) and enterocin A (enterocin A). Bacteriocins belonging to class IIb are 2 peptides produced simultaneously, and they are known to synergistically enhance the effects of each other. Specific examples thereof include plant lactocin (Plantaricin: PlnE, PlnF), enterocin X (enterocin X: Xalpha, Xbata), and lactococcus Q (lactococcus Q: Qalpha, Qbeta). Bacteriocins belonging to class IIc have a cyclic structure in which the N-terminus and the C-terminus are linked by a peptide bond. Specific examples thereof include lactein A (Gassercina), cyclosporin A (circulanA), and lactein Q (lactocyclicinQ).

The bacteriocin used in the present invention is preferably a bacteriocin belonging to class I, class IIb or class IIc. The bacteriocins used in the present invention are more preferably Nisin (Nisin), Nisin (Gassericin), Plantaricin (Plantaricin), and Subtilin (Subtilin).

Note that, when a letter is not attached to the name of each bacteriocin, the term indicates a general name of the bacteriocin (for example, the term "nisin" is a concept including nisin a, nisin Z, and the like).

Since it was confirmed by experiments of the present inventors that nisin has membrane-firming ability, an index for screening bacteriocin having membrane-firming ability was examined, and it was found that the membrane-firming ability does not necessarily relate to antibacterial activity or antibacterial spectrum (see experimental example B described later).

Antibiotics used as AGPs are known not to be decomposed by digestive enzymes and therefore remain active in vivo and also have antibacterial activity after excretion as feces. On the other hand, bacteriocins, which are proteins or peptides, are easily decomposed by digestive enzymes and thus lose activity in the digestive tract. When used for feed applications, it is desirable to protect (coat) the feed from inactivation by digestive enzymes, and if it is protected, 10 to 100ppm of the feed can sufficiently exhibit membrane-hardening ability in the small intestine. Then, the bacteriocin entering the feces is inactivated by the enzymes in the feces, and is therefore safe. Therefore, the following advantages are provided: even if an additive for feed or feed containing bacteriocin in place of AGP is administered to livestock, the problem of drug-resistant bacteria is not caused. In the present specification, "ppm" means "mass ppm (ppm by mass)".

Examples of the polyphenol used in the present invention include Quercetin (Quercetin), Curcumin (Curcumin), and the like. Preferably quercetin.

Examples of the amino acid used in the present invention include Glutamine (Gln), Tryptophan (Trp), Valine (Val), tyrosine (Tylosin, Tyr), and phenylalanine (Phe). Examples of the amino acid derivative include Phenyllactate (PLA).

Examples of the organic acid used in the present invention include butyric acid. The total carbon number of the organic acid is about 2 to 5. Examples of the organic acid derivative include esters with alcohols having 1 to 5 carbon atoms.

Examples of the HSP inducer (heat shock protein (HSP) inducer) used in the present invention include polyphosphoric acid (Poly-phospate) and Competence and Sporulation Factors (CSF) derived from Bacillus subtilis).

The antioxidant used in the present invention includes Astaxanthin (Astaxanthin) and the like.

Examples of the polysaccharide used in the present invention include Gum Arabic (Arabic Gum), Pullulan (Pullulan), galactoglucomannan (GGM), and Xanthan Gum (xanthanan Gum, XG).

In the present invention, 1 kind of the physiologically active substance may be used alone, or 2 or more kinds may be used in combination. The physiologically active substance used in the present invention is preferably bacteriocin, polysaccharide, amino acid, or a combination thereof, and more preferably nisin, Tyr, or a combination thereof.

In addition, it is known that polyphosphoric acid enhances barrier function by increasing the production of heat shock protein (HSP inducer) (Shuichi Segawa et al, PLoS ONE,2011,6(8): e 23278). It is thought that if stress response proteins such as heat shock proteins are induced, intercellular barrier function is enhanced.

On the other hand, tight junction (light junction) is a layer formed by the close adhesion of cell membranes of adjacent cells and the fusion of their outer membranes with each other, and is located at the boundary between the basal membrane (basal membrane) and the apical membrane (epithelial membrane) of epithelial cells, and is thought to prevent membrane proteins and lipids from intermixing therebetween (rock wave "biology dictionary" 4 th edition CD-ROM 1998).

It is assumed that the physiologically active substance used in the present invention has a membrane-firming ability resulting from either or both of the function of preventing substance diffusion based on tight junctions and the function of intercellular barrier based on stress response proteins, but this is not limited to any theory. Although it is considered that if the tight junction is solidified, health can be achieved, there has been no feed for delivering a substance capable of solidifying the tight junction to the intestine. Further, it has not been known that an antibacterial agent capable of hardening a tight junction in vitro (in vitro) exhibits a body weight increasing effect when administered to livestock. In the present specification, the film-firming ability is also referred to as a barrier function (barrier function).

The purity of the physiologically active substance used in the present invention is not limited as long as the desired effect can be obtained. For example, a physiologically active substance produced by a microorganism may be used together with the culture. The solid in the fermentation liquor can also be obtained by freeze drying, and the solid can also be obtained as the solid by spray granulation.

Alternatively, the physiologically active substance used in the present invention may be a secretion or extract derived from plants, algae, crustaceans, or fishes as it is. As the physiologically active substance used in the present invention, commercially available products can be used as they are.

Examples of the plant containing polyphenol (polyphenol) in the physiologically active substance include grape, wine, tea, apple, blueberry, persimmon, banana, turmeric, cinnamon, coffee bean, citrus, onion, and the like.

Examples of the microorganism producing polysaccharides in physiologically active substances include galactoglucomannan (GGM) produced by Lipomyces starkeyi (Saccharomyces cerevisiae) and the like, which are obtained by the methods described in Japanese patent laid-open Nos. 7-298873 and 9-131199. Examples of the microorganism producing the polysaccharide in the physiologically active substance include Xanthomonas campestris (Xanthomonas campestris), Flavobacterium furcifolium (Aureobasidium pullulans), Lipomyces starkeye (Lypomomyces Starkey), Laminaria japonica (Phaeophyta) in brown algae, and Eucheuma muricatum (Eucheuma) in algae. Examples of the plant include Acacia senegal (Acacia senegal), guar bean (Cyamopsis tetragonoloba), Hypnea (Hypnea musciformis), Tara (Tara spinosa), and carob bean (Ceratonia siliqua).

Examples of the microorganism producing an amino acid or a derivative thereof in a physiologically active substance include Brevibacterium flavum (Brevibacterium flavum), Corynebacterium glutamicum (Corynebacterium glutamicum), Bacillus subtilis (Bacillus subtilis), and Escherichia coli (Escherichia coli).

Examples of the microorganism producing an organic acid or a derivative thereof in a physiologically active substance include lactobacillus (lactobacillus), Bifidobacterium (Bifidobacterium), Clostridium (Clostridium), and the like.

Examples of the microorganism producing an HSP inducer in a physiologically active substance include Bacillus (Bacillus), Lactococcus (Lactococcus lactis), lactobacillus (lactobacillus), Leuconostoc (Leuconostoc), and Pediococcus (Pediococcus).

Examples of the microorganism producing an antioxidant in a physiologically active substance include Phaffia rhodozyma (Phaffia rhodozyma), Pseudomonas thioparvus (Pseudomonas thiozolinophilum), Haematococcus pluvialis (Hematococcus pluvialis) as an alga, and the like.

The bacteriocin, which is one of the physiologically active substances used in the present invention, is preferably a culture product of 1 or more microorganisms selected from the group consisting of Bacillus (Bacillus), Lactococcus (Lactobacillus), Lactobacillus (Lactobacillus), Leuconostoc (Leuconostoc), and Pediococcus (Pediococcus).

Among them, as the bacteriocin-producing microorganism, Bacillus subtilis, Lactococcus lactis, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus acidovorus, Lactobacillus brevis, Lactobacillus buchneri (cattle) (Lactobacillus buchneri (Lactobacillus), Lactobacillus bulgaricus (Lactobacillus), Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus crispatus (Lactobacillus), Lactobacillus crispus (Lactobacillus), Lactobacillus crispus (Lactobacillus), Lactobacillus crispus (Lactobacillus), Lactobacillus, lactobacillus helveticus (Lactobacillus gallinarum), Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus hilgardii (Lactobacillus hilgardii), Lactobacillus johnsonii (Lactobacillus johnsonii), Lactobacillus malus (Lactobacillus kefiri), Lactobacillus kefiri (Lactobacillus kefiri), Lactobacillus acidophilus (Lactobacillus kefir), Lactobacillus lactis (Lactobacillus lactis), Lactobacillus mucosae (Lactobacillus mucosae), Lactobacillus bakeri (Lactobacillus panis), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus paracasei), Lactobacillus pentosus (Lactobacillus paracasei), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus paracasei), Lactobacillus pentosus (Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus paracasei), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus salivarius), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus plantarum (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus (Lactobacillus plantarum), Lactobacillus (Lactobacillus), Lactobacillus paracasei), Lactobacillus (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus paracasei, Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus paracasei), Lactobacillus (Lactobacillus), Lactobacillus paracasei (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus paracasei (Lactobacillus), Lactobacillus paracasei (Lactobacillus plantarum (Lactobacillus), Lactobacillus paracasei (Lactobacillus), Lactobacillus plantarum (Lactobacillus ), Lactobacillus (Lactobacillus, leuconostoc mesenteroides (Leuconostoc mesenteroides), Pediococcus acidilactici (Pediococcus acidilactici), Pediococcus cerevisiae/Pediococcus catarrhalis (Pediococcus cerevisiae/damnosus), Pediococcus dextrinicus (Pediococcus dextrinicus), Pediococcus pentosaceus (Pediococcus pentosaceus).

Particularly preferred are culture products of Lactococcus lactis (Lactococcus lactis), Bacillus subtilis (Bacillus subtilis), Lactococcus lactis (Lactococcus lactis), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus gasseri (Lactobacillus gasseri), and Clostridium beijerinckii (Clostridium beijerinckii). Among them, a culture product of Lactococcus lactis (Lactococcus lactis) is preferable.

Particularly preferred are culture products of Lactococcus lactis (Lactobacillus lactis) FERM BP-8552 (Streptococcus lactis Z-producing bacterium), Bacillus subtilis (Bacillus subtilis) ATCC6633 (Bacillus subtilis), Lactococcus lactis (Lactobacillus lactis) NCIMB702054 (Streptococcus lactis Z-producing bacterium), Lactobacillus plantarum (Lactobacillus plantarum) JCM1057 (Lactobacillus plantarum-producing bacterium), Lactobacillus gasseri (Lactobacillus gasseri) LA39 JCM11657 (Lactobacillus A-producing bacterium), Clostridium beijerinckii (Clostridium beijijinickii) JCM1390 (Cyclomycin A-producing bacterium). Most preferred are Lactococcus lactis (Lactococcus lactis) FERM BP-8552 (nisin Z producing bacterium), Lactococcus lactis (Lactococcus lactis) NCIMB702054 (nisin Z producing bacterium).

It should be noted that Lactococcus lactis (Lactococcus lactis) FERM BP-8552 was deposited at the International patent organism depositary of the institute for Integrated Industrial science and technology, national institute for independent administrative sciences, 11.19.2003 (east 1-1-1, Nozada 305-. In the present specification, this strain is sometimes referred to as AJ 110212.

Bacillus subtilis ATCC6633 is deposited with American Type Culture Collection, Manassas, Va.A., USA.

Lactococcus lactis NCIMB702054 is deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, Food and Marine Bacteria, NCIMB Ltd., Aberdeen, Scotland, UK., of Able, England, Able, et al, Able, et al.

Lactobacillus plantarum JCM1057, Lactobacillus gasseri LA39 JCM11657 and Clostridium beijerinckii JCM1390 are deposited in the institute of biological resources and research center of national institute of research and development (Postcode 305-.

The core may comprise a protectant. Examples of the protective agent include skim milk powder (skim milk), amino acid salts such as sodium glutamate, sugar alcohols such as sorbitol, trehalose, and disaccharides such as sucrose.

The core may comprise an excipient. The excipient is not particularly limited as long as it is a general excipient for improving moldability, and examples thereof include calcium carbonate, silica, calcium silicate, zeolite, sorbitol, corn starch, talc, yeast bentonite, rice husk, liquid paraffin, polysaccharides other than polysaccharide having a property of aggregating gram-negative bacteria, monosaccharides, disaccharides, and the like. When the core contains an excipient, the amount of the excipient is preferably 0.1 to 100 parts by mass per 100 parts by mass of the core.

The core may also contain any additives conventionally used in feed. When the core contains any additive, the amount of any additive is preferably 0.1 to 100 parts by mass per 100 parts by mass of the core.

[ coating agent ]

The coating agent is a substance capable of forming an enteric coating, and may be used without particular limitation as long as it is safe for livestock to take. The coating agent may be used alone in 1 kind, or 2 or more kinds may be used in combination. From the viewpoint of ease of handling and economy, the coating agent is preferably hydrogenated vegetable oil, or shellac, zein, hydroxypropylmethylcellulose, maltitol, or the like, which is a substance generally used as a coating agent for tablets. Examples of the hydrogenated vegetable oil include rapeseed oil, linseed oil, safflower oil, sunflower seed oil, soybean oil, corn oil, peanut oil, cottonseed oil, sesame oil, rice bran oil, olive oil, palm kernel oil, and hydrogenated oil of coconut oil. The coating agent is preferably hydrogenated rapeseed oil or shellac. The layer of hydrogenated rapeseed oil is preferred because it allows the dissolution of the core in a short time. The layer of shellac is preferred because it dissolves the core when neutral or alkaline (after passing through the stomach).

Alternatively, the coating agent may be a microorganism itself that produces such a physiologically active substance.

The coating agent is preferably contained in an amount of 5 to 90% by mass, more preferably 20 to 30% by mass, based on the total mass of the coated feed additive of the present invention. The coating agent may further contain any additives conventionally contained in feed.

The coating may be a single layer or a multilayer of two or more layers. Since the dissolution in vivo can be easily controlled, a multilayer coating is preferable. In particular, if the outermost layer is a layer of hydrogenated rapeseed oil and the innermost layer in contact with the core is a layer of shellac, the coating agent will not dissolve in the stomach but in the intestine, and therefore it is preferable.

The coated feed additive of the present invention preferably has a dissolution rate in gastric juice of less than 50%, and a difference between the dissolution rate in intestinal juice and the dissolution rate in artificial gastric juice of 10% or more. In order to achieve such dissolution, the dissolution rate can be adjusted by using a double-layer film or a multilayer film, or by controlling the type of coating agent and the thickness of the coating film in each layer.

[ coating method ]

The method for coating the core is not particularly limited, and for example, a coating agent is formed into a liquid state by spraying and heating the core powder or granules to a temperature higher than the melting point while fluidizing the core powder by a commercially available fluidized bed type spray granulator, thereby obtaining a coated feed additive. It is preferable to use a coating-type feed additive obtained from a polysaccharide in the form of powder or granules, since the size of the coating-type feed additive is about 0.05 to 5mm, since handling is easy. The temperature at which the coating agent is heated is not particularly limited as long as it is not lower than the melting point of the coating agent, and is preferably about 5 to 15 ℃.

[ feed ]

The additive for coated feed of the present invention may be administered directly to livestock, or may be formulated together with excipients or diluents such as corn, soybean meal, rice bran, fish meal, brewer's yeast, etc. to make feed. The feed of the invention may also contain any additives that may be contained in the feed. The feed of the invention is suitable for continuous daily intake. The intake amount of the feed varies depending on the size of livestock, and in the case of chickens, for example, the intake amount of the physiologically active substance is preferably about 1 to 200ppm, preferably 10 to 100ppm, based on the feed other than the coated feed additive, on an average of 1 day.

In the present specification, "livestock" means an organism (animal) to which a human is raised. Specifically, ruminants such as cows, sheep and goats, and monogastric animals such as horses, pigs, chickens, dogs and fish are included. It is particularly preferred to administer the feed of the invention to monogastric animals.

The method of feeding the coated feed additive of the present invention is not particularly limited.

Examples

Experimental example A: film hardening test

The membrane-firming ability of the test substances was evaluated as described in J.Nutr. (2009), Vol.139 (5), page 965-974.

Caco-2 cells (human digestive tract epithelial cells (ECACC, No. 86010202)) were seeded on a double-layered Transwell system and cultured in DMEM medium at 37 ℃. On day 12 of culture, TNF-. alpha.was added to decrease the barrier function of Tight junctions (light junctions). On the 14 th day of the culture, the test substance was added, and after 24 hours of the culture at the same temperature, the transmembrane resistance TER (Ω. cm) was measured using Millicell ERS-2 (Millipore corporation)²)，Recovery of barrier function was thus evaluated (% recovery ratio). The results are shown in FIGS. 1A to 1H.

As shown in fig. 1A, it was confirmed that, for example, speltomycin (Avilamycin) and Colistin (Colistin), which are AGP, have a function of hardening the barrier function even at a low concentration. When the group to which only TNF- α was added was used as a control (control), the film-strengthening ability was judged to be exhibited when the Recovery contrast ratio (Recovery ratio/control) was 1.1 or more. Table 1 shows the concentrations of the materials (materials) themselves when the recovery contrast ratio of each physiologically active substance was about 1.1. Quercetin (Qurcetin), Grape Seed Extract (Grape Seed Extract) (manufactured by Meizhisu corporation), nisin A, tyrosine (manufactured by Meizhisu corporation), astaxanthin (manufactured by Disemann (DSM)), and the like.

[ Table 1]

*1: comparison with control (TNF. alpha. only addition group)

*2: lactein (sup): a solution obtained by concentrating the culture supernatant of a producer (Lactobacillus gasseri LA39JCM 11657) 3-fold by an ultrafiltration membrane (MW: 3000)

*3: plant lactobacillin (sup): a solution obtained by concentrating the culture supernatant of a producing bacterium (Lactobacillus plantarum JCM 1057) 3-fold by an ultrafiltration membrane (MW: 3000)

*4: omnivin, manufactured by Aomoto K.K.)^TM10R”。

Example 1: coated Quercetin (Coated-Quercetin)

1-1: preparation of coated Quercetin

As the nucleus, quercetin (a reagent (purity 95%) manufactured by tokyo chemical industry co.); as the coating agent, hydrogenated rapeseed oil (melting point: 67 ℃) and natural resin shellac were used. A coating agent which is formed into a liquid state by heating to a temperature higher than the melting point is sprayed in a predetermined amount onto the powder-or granular-formed cores, thereby obtaining a coated feed additive. The coating is carried out by: 5 parts by mass of shellac as the first layer (inner layer) and 17 parts by mass of hydrogenated rapeseed oil as the second layer (outer layer) were sprayed to 77 parts by mass of the core.

1-2: acid resistance and enteric solubility test

(artificial gastric juice treatment) to pure water produced using a pure water production apparatus manufactured by Merck Millipore (Merck Millipore), 0.2 mass% NaCl and 0.2 mass% pepsin (pepsin) (from pig gastric Mucosa, 1:5000, 2500 units/mg) were added, the pH was adjusted to 2, and then the coated feed additive prepared in 1-1 was added, and enzyme treatment was performed at 37 ℃ for 2 hours. Dissolution was determined by automatic continuous measurement of optical density during this period. "2 hours" is assumed to be the time from when the feed reaches the stomach of the chicken until the feed passes through the stomach of the chicken.

(treatment with Artificial intestinal juice) after the treatment with artificial gastric juice, 0.2% trypsin (trypsin) (from Porcine Pancreas, 1:5000, 4500 units/mg) was added, the pH was adjusted to 6, and then the enzyme treatment was carried out at 37 ℃ for 2 hours. Dissolution was determined by automatic continuous measurement of optical density during this period. Note that "2 hours" is assumed to be the time from when the feed reaches the intestine of the chicken until the feed passes through the intestine of the chicken.

Hydrochloric acid and sodium hydroxide were used for pH adjustment. The optical density was measured as OD660nm using a Biophoto recorder (Biophoto-recorder) TVS062CA manufactured by Advantec corporation.

The results are shown in FIG. 2. Referring to fig. 2, in the case of the two-layer coating, dissolution rate was suppressed to 25% 2 hours after the start of the gastric juice treatment, while dissolution rate was 65% in the intestinal juice treatment. From these results, it was found that the feed additive having the hydrogenated rapeseed oil layer applied as the outer layer and the shellac layer applied as the inner layer had excellent acid resistance and enteric solubility and release control.

1-3: growth test of chickens

The coated quercetin prepared by the two-layer coating method of 1-1 was added to a feed base having the composition shown in table 2 under the condition that the amount of the core material was 20ppm to 200ppm, to obtain a feed composition. In a chicken house raised in open space, newborn chicks of 25 broilers in 1 division were fed with the Feed composition, and the chickens were raised for 22 days at 0 to 21 days of age in 3 replicates (triplicates), and the Body Weight Gain (BWG) and Feed Conversion Ratio (FCR Feed/BWG) were evaluated. As a positive control, a conventional Antibacterial Growth Promoter (AGP) inferior mycin was used. As a product, commercially available product (Surmax 200 (registered trademark) by ELANCO Co., Ltd., no coating) was used as it is.

The results are shown with the negative control as 100. The results are shown in Table 3. Although it is known that uncoated quercetin showed negative effects on intestinal flora (j.nutr.139: 965-974, 2009.), the coated quercetin observed a body weight gain effect, a feed conversion ratio improving effect. Since uncoated quercetin showed negative effects on intestinal flora, no chicken growth test using uncoated quercetin was performed.

[ Table 2]

Feed composition

Raw materials	The mixing proportion is%
		Corn (corn)	45.4
Sorghum grain	10.0
		Bean pulp	30.0
Corn protein powder	4.00
		Fish meal (CP 65%)	3.00
L-lysine hydrochloride	0.31
		DL-methionine	0.35
L-threonine	0.12
		L-arginine	0.16
Animal fat and oil	3.49
		Calcium hydrogen phosphate	1.45
Calcium carbonate	1.06
		Salt	0.30
Vitamin/mineral premix	0.25
		Choline chloride	0.02
L-valine	0.07
		Total up to	100

[ Table 3]

Example 2: coated Phe and coated Tyr

2-1: preparation of coated Phe and coated Tyr

The coated Phe and coated Tyr were prepared as described in examples 1, 1-1.

2-2: acid resistance and enteric solubility test

Acid resistance and enteric test were performed in the same manner as in examples 1, 1 and 2 using the thus obtained coated Tyr. The results are shown in FIG. 3.

2-3: growth test of chickens

The coated Phe and coated Tyr obtained above were added to a feed base having a composition shown in table 2 under the condition that the amount of the core material became 200ppm, and the body weight increasing effect and feed conversion ratio of chickens were evaluated by the method described in example 11-3. The results are shown in Table 4.

Phe and Tyr were confirmed to have a certain body weight increasing effect even when they were not coated, but Phe and Tyr were not detected in intestinal contents and blood as a result of metabolome (metabolome) analysis. Therefore, it is considered that the coated Phe and the coated Tyr are assimilated by intestinal bacteria in feces and are rapidly metabolized in blood. Therefore, it is considered that the coated amino acid exerts an effect more stably than the uncoated amino acid.

[ Table 4]

2-4: evaluation of L-Tyr and coated L-Tyr in Chicken Using Salmonella infection testThe coated Tyr and Tyr prepared in the same manner as in example 2-1 were added to a feed base having a composition shown in table 2 under the condition that the amount of the core material was 200ppm, to prepare a feed. After 1-day-old broiler chickens were put into a feeding facility for infection test (6/replicate, 2 replicates/test group), Salmonella Enterica (SE) was orally administered to the 2-day-old broiler chickens for 21 days, and the body weight gain effect and feed conversion rate of the chickens were evaluated. As a positive control, Enramycin (Enramycin) which is a conventional antibacterial growth-promoting substance was used. As enramycin, a commercially available enramycin (enramycin F-80, manufactured by scientific feed research, Ltd., no coating) was used as it was.

The results are shown with the negative control as 100. The results are shown in Table 5.

[ Table 5]

As a result, tyrosine ("L-Tyr") not coated had no effect, but coated Tyr showed the same body weight increasing effect and feed conversion improving effect as enramycin.

Example 3: coated nisin A (Coated-NisinA)

3-1: preparation of coated nisin A

As the nucleus, nisin a (a reagent manufactured by Sigma Aldrich (Sigma-Aldrich) corporation (nisin content 2.5 mass%, balance sodium chloride and denatured milk solids) was used); as the coating agent, hydrogenated rapeseed oil (melting point: 67 ℃) and natural resin shellac were used. A two-layer coated additive for feed was obtained as described in example 11-1.

3-2: acid resistance and enteric solubility test

Acid resistance and enteric test were performed in the same manner as in example 11-2 using the coated nisin A thus prepared. The results are shown in FIG. 4.

3-3: growth test of chickens

The coated nisin A prepared in 3-1 was added to a feed base having the composition shown in Table 2 under the condition that the amount of the core material was 1ppm to 10ppm, and the body weight increasing effect and feed conversion ratio of chickens were evaluated. In the chicken house raised on the open space, newborn chicks of 25 broilers were raised from 0-day-old to 21-day-old in 3 replicates. The results are shown in Table 6. Although nisin was decreased in activity by gastric acid and completely inactivated by trypsin, a body weight increasing effect and a feed conversion improving effect were observed with coated nisin a.

[ Table 6]

3-4: coated nisin A and coated pullulan in chicken tested for Salmonella infection Evaluation of (2)

The coated nisin a prepared in 3-1 was added to a feed base having the composition shown in table 2, alone or in combination with the coated pullulan, so that the amount of each core material became 10ppm, to prepare a feed. After 0-day-old broiler chickens were put into a feeding facility for infection test (2 replicates/test group, 6 replicates/replicate), Salmonella Enterica (SE) was put into a crop of 2-day-old broiler chickens at 107 probes/replicate, and the test feed was given for 21 days, to evaluate the body weight gain effect and feed conversion rate. The results are shown in Table 7.

[ Table 7]

As a result, both the "coated nisin group" and the "coated nisin + coated pullulan group" showed body weight gain effects and feed conversion rate improvement effects.

3-5: method for testing for nisin A and coated nisin A by Salmonella infection in chickens Evaluation of

The coated nisin a and nisin a prepared in the same manner as in example 3-1 were added to feed in accordance with table 2 to prepare feed. After 0-day-old broiler chickens were put into a feeding facility for infection test (6/replicate, 2 replicates/test group), Salmonella Enterica (SE) was orally administered to the 2-day-old broiler chickens for 21 days, and the body weight gain effect and feed conversion rate were evaluated. As a positive control, Enramycin (Enramycin) which is a conventional antibacterial growth-promoting substance was used. As enramycin, a commercially available enramycin (enramycin F-80, manufactured by scientific feed research, Ltd., no coating) was used as it was.

The results are shown in Table 8.

[ Table 8]

As a result, while the uncoated nisin A had no effect, the coated nisin A showed the same body weight increasing effect and feed conversion rate improving effect as those of enramycin.

Experimental example B: evaluation of antibacterial Activity of bacteriocin and evaluation of antibacterial Spectrum

B-1: determination of minimum inhibitory concentration

The minimum inhibitory concentrations of AGP and nisin were determined and compared.

Lactococcus lactis AJ110212(FERM BP-8552) was used as a nisin Z-producing bacterium. The nisin Z-producing strain was cultured at 30 ℃ and 100rpm in a medium (Lactobacillus) MRS broth 1L manufactured by BD Difco) in a 5L Sakaguchi flask. The culture was carried out for 20 hours, and the Optical density (Optical density) of the culture solution was measured at a wavelength of 610nm by a spectrophotometer (a bio-Optical recorder TVS062CA manufactured by Advantec corporation), and the solution was diluted at 26 times so that the concentration of the solution was 0.1 or more. The resulting culture was centrifuged (6000 G.times.10 min, 4 ℃ C.), and Cell fractions (Wet cells) were separated.

The following strains were used as test bacteria. The medium and the culture temperature are shown in parentheses at the end of the strain. MRS broth of Lactobacillus strain manufactured by Difco was used as MRS medium, products of Nippon pharmaceutical Co., Ltd were used as GAM medium and LB medium, and products of Difco were used as NB medium.

Gram-positive bacteria

Lactobacillus acidophilus (Lactobacillus acidophilus) AJ13778(MRS, 37 ℃, corresponding to the strain deposited under accession number JCM 1132)

Lactobacillus salivarius AJ110152(MRS, 37 ℃, corresponding to the strain deposited under accession number JCM 1231)

Bifidobacterium thermophilum (Bifidobacterium thermophilum) AJ110569(GAM, 37 ℃, corresponding to the strain deposited under accession number JCM 1207)

Bacteroides fragilis (bacteriodes fragilis) JCM11019(GAM, 37 ℃ C.)

Escherichia coli (Escherichia coli) MG1655(LB, 37 ℃, corresponding to the strain deposited under accession number ATCC 700926)

Clostridium perfringens AJ3350(GAM, 37 ℃, corresponding to the strain deposited under accession number ATCC 10873)

Gram-negative bacteria

Enterococcus faecalis (Enterococcus faecalis) AJ110149(MRS, 30 ℃, corresponding to the strain deposited under accession number JCM 5803)

Salmonella enterica AJ2785(NB, 37 ℃, corresponding to the strain deposited under accession number IAM 1648).

Note that the depositary organization for bacteria with a specific accession number, beginning with JCM, is the microbial materials development Room of the institute of Bioresources, national institute of research and development, institute of physical and chemical sciences (postal code 305-. The depository of bacteria identified by accession numbers beginning with the ATCC is the American type culture Collection of Manassas, Va. The IAM-originated Collection of bacteria with specific accession numbers is The Molecular Research Center IAM Culture Collection (IAM Culture Collection, Center for Cellular and Molecular Research, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan) of Molecular bioscience Institute of Molecular biology, Tokyo, Japan (The Collection is transferred to JCM, Molecular transferred to JCM).

The minimum inhibitory concentration was calculated by measuring the antibacterial activity by the Spot-on-lawn method (Spot-on-lawn method) described in Mayr-Harting, A. et al, Methods Microbiol.1972, 7A, page 315-. When the culture solution was used, qualitative judgment was made based on the size of the zone of inhibition. The results are shown in Table 9.

[ Table 9]

Comparison data of minimum inhibitory concentration of AGP and nisin

Minimum inhibitory concentration (mg/mL)

"ND": indicating no effect detected (not detected).

B-2: determination of the antimicrobial Spectrum

For compounds that were difficult to obtain from the reagents, a Bacteriocin (Bacteriocin) -containing culture broth was prepared by culturing the following producing bacteria in a Lactobacillus MRS broth manufactured by Difco at 30 ℃.

Bacteriocins/class I: nisin A and nisin Z used in example 3, 3-1 (the producer was lactococcus lactis NCIMB702054) were used. Bacillus subtilis ATCC6633 is used as a subtilin-producing bacterium. Duramycin was prepared using a reagent (1mg/ml) manufactured by Sigma Aldrich.

Bacteriocin/class IIb: lactobacillus plantarum JCM1057 was used as the Lactobacillus plantarum-producing strain.

Bacteriocin/class IIc: lactobacillus gasseri LA39 JCM11657 was used as the lactic acid bacteria A, and Clostridium beijerinckii JCM1390 was used as the circulans A producing bacteria.

The antimicrobial spectrum was evaluated by the Spot-on-lawn method (Spot-on-lawn method) using a 10-fold concentrated culture supernatant. The results of the examination are shown in Table 10.

[ Table 10]

Antibiogram of bacteriocins

Nisin A (1mg/m1), duramycin (1mg/ml), others: bacteriocin producing bacteria

"+" to "++++": indicating the strength of the antibacterial effect. The more "+" indicates a stronger effect.

"ND": indicating no effect detected (not detected).

As is clear from a comparison between tables 9 and 10 and table 1, the film-strengthening function is independent of the presence or absence of the antibacterial activity or the strength thereof.

Example 4: coated bacteriocin-producing probiotic (Probiotics)

4-1: cultivation of bacteriocin-producing bacteria

In example 3, nisin A was used, but in example 4, lactococcus lactis FERM BP-8552 was used as a nisin Z-producing bacterium. The nisin Z-producing bacteria were cultured in 5L sakaguchi flask (1L Lactobacillus MRS broth manufactured by BD Difco) at 30 ℃ and 100 rpm. The culture solution was prepared by measuring the optical density at a wavelength of 610nm by a spectrophotometer (a bio-optical recorder TVS062CA manufactured by Advantec corporation) based on 20 hours, and diluting the solution at 26-fold so that the concentration of the solution was 0.1 or more.

Likewise, bacterial fractions of subtilin and of plantaricin were obtained. Bacillus subtilis ATCC6633 was used as a subtilin-producing bacterium. As the lactobacillus plantarum producing bacterium, lactobacillus plantarum JCM1057 was used.

4-2: preparation of bacteriocin-producing bacteria powder

The cell fraction of nisin Z obtained in 4-1 was added to 120ml of a protective agent and powdered by a spray dryer (inlet temperature 80 ℃ C., outlet temperature 50 ℃ C.) or by lyophilization under reduced pressure. The protective agents are as follows.

(A) Skim milk (Skim milk)10 mass% (BD Co., Ltd.) + glutamic acid sodium salt 3 mass% (MSG, AJICO Co., Ltd.)

(B) MSG 3% by mass, sorbitol 10% by mass, trehalose 10% by mass, sucrose 10% by mass (MSG manufactured by AJICO Co., Ltd., except MSG manufactured by Wako pure chemical industries Co., Ltd.)

The viable cell count of the obtained nisin Z-producing bacteria powder was measured. The viable cell count was measured as follows: 0.01g of the powder sample was suspended in 1ml of physiological saline, and sequentially diluted with 10-fold physiological saline, 0.1ml was applied to an MRS agar plate, cultured at 30 ℃ for 24 hours, and the colony forming unit (cfu)/g was measured based on the number of generated colonies.

Similarly, powders of subtilin-producing bacteria and lactobacillus plantarum producing bacteria were obtained, and the producing bacteria were determined.

4-3: coated nisin Z-producing bacterium powder, coated subtilin-producing bacterium powder, and coated plant Preparation of powder of lactobacillus producing bacterium

The nisin Z-producing bacteria powder prepared in 4-2 was coated in the same manner as described in example 11-1 to obtain a coated nisin Z-producing bacteria powder.

Similarly, a coated surfactin-producing bacterial powder and a coated plant lactobacillus powder were prepared.

4-4: acid resistance and enteric solubility test

The coated bacteriocin-producing strain powder obtained in 4-3 was subjected to the artificial gastric juice treatment and the artificial intestinal juice treatment described in example 11-2, respectively.

Acid resistance was evaluated by measuring the number of viable bacteria and antimicrobial activity of each bacteriocin after the treatment with artificial gastric juice and the treatment with artificial intestinal juice. The viable cell count was measured according to the 4-2. The antibacterial activity was measured as described in Experimental example B.B-2. The results are shown in Table 11. The meanings of "+" and the like and "ND" in the table are the same as those described in table 10.

[ Table 11]

4-5: chicken growth test Using coated Nisin Z-producing bacteria powder

The coated nisin Z-producing bacteria powders prepared in 4-3 were added to the feed bases shown in Table 2 under conditions such that the viable cell counts shown in the following table were reached, and the body weight increasing effect and feed conversion ratio of chickens were evaluated by the methods described in examples 11-3.

[ Table 12]

Example 5: growth test of chickens

Three conditioned feeds were prepared, namely: the standard feed was supplemented with a drug-containing feed (PC) containing 0.05 mass% of antibiotics (lasalocid) and 0.01 mass% of throughout), a PRB-added feed (nisin (Lc)) containing 2 mass% of a nisin a culture obtained by culturing lactococcus lactis NCIMB8780 in the same manner as in example 4-1, and a drug-free feed (standard feed only) (NC), and then the standard feed was administered to newborn chicks. The experiment was repeated 3 parts of the feed under 1 condition using 10 male newborn chicks of cobo (Cobb) broiler chickens, and the body weight gain effect and feed conversion ratio of the chickens were evaluated. The bulk drug (NC) used standard feed (ME3160kcal, CP 22 mass%, no antibiotics). For the PC and Nisin (Nisin) addition groups, 2 mass% of antibiotics (lasalomycin and throughout) or Nisin Z-containing solutions were added to standard feed (ME3160kcal, CP 22 mass%), respectively.

[ Table 13]

*: antibiotics: 0.05% of lasalocid and 0.01% of throughout are added.

**: for nisin, 2% lactococcus lactis culture medium was added.