WO2022102753A1 - Agent microbien viable contenant lactococcus lactis, microorganisme appartenant au genre lactobacillus ou mélange associé, et son procédé de production - Google Patents

Agent microbien viable contenant lactococcus lactis, microorganisme appartenant au genre lactobacillus ou mélange associé, et son procédé de production Download PDF

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WO2022102753A1
WO2022102753A1 PCT/JP2021/041767 JP2021041767W WO2022102753A1 WO 2022102753 A1 WO2022102753 A1 WO 2022102753A1 JP 2021041767 W JP2021041767 W JP 2021041767W WO 2022102753 A1 WO2022102753 A1 WO 2022102753A1
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viable
agent
microorganism
agent according
lactobacillus
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Japanese (ja)
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まゆみ 前川
章敬 上原
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味の素株式会社
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry

Definitions

  • the present invention relates to a live bacterial agent containing Lactococcus lactis, a microorganism belonging to the genus Lactobacillus, or a mixed microorganism thereof, and a method for producing the same.
  • Lactic acid bacteria have been used in the field of fermented foods for a long time. Going back in history, Louis Pasteur discovered lactic acid bacteria in 1857, after which Ilya Ilyich Mechnikov wrote his book, The Prolongation of Life (non-Life). In Patent Document 1), he advocated the theory of immortality and longevity. With this as an opportunity, the usefulness of lactic acid bacteria has attracted attention in the fields of health food and medicine. Since then, there has been increasing interest in the establishment of lactic acid bacteria in the human intestine and the healthy state of the intestinal flora, and many studies have been conducted on the functions of lactic acid bacteria.
  • Non-Patent Document 2 oligosaccharides selectively used by microorganisms having beneficial effects are called “prebiotics” (Non-Patent Document 2). Furthermore, studies on synergistic effects by combining “probiotics” and “prebiotics” have also been reported (Non-Patent Document 3). As described above, lactic acid bacteria have various physiological functions, have been widely studied in a wide range of applications, have high expectations for industrial use, and many products have been developed. However, the substance itself that induces physiological functions and its mechanism of action have not yet been fully elucidated.
  • lactic acid bacteria is being studied not only in the field of human health and nutrition, but also in livestock animals and pets.
  • the reason is the problem of drug resistance genes caused by antibiotic growth promoter (AGP), which is one of the techniques for improving the productivity of livestock animals.
  • AGP antibiotic growth promoter
  • Antibiotics were originally developed for therapeutic purposes mainly in inhibiting the growth of pathogens, but after that, their uses expanded, and from the latter half of the 1950s, their use for the purpose of promoting the growth of livestock animals became widespread.
  • AGP antibiotic growth promoter
  • Non-Patent Document 4 the Colistin resistance gene, which was used as one of the AGPs, was found in a pig farm in China, and it was found that this gene exists on a mobile plasmid. If AGP continues to be used as it is, drug-resistant bacteria will become a greater risk than cancer in 2050, and if no measures are taken to address this issue, the annual mortality rate will exceed cancer and reach 10 million people. It is predicted that this may reach the limit and pose an international threat (Non-Patent Document 5).
  • regulations on AGP which is also used for therapeutic purposes in each country, and therapeutic drugs common to humans and animals have been tightened.
  • Non-Patent Document 6 There are many studies on probiotics and prebiotics as candidates for alternative materials to AGP (Non-Patent Document 6). Studies using the genus Bacillus (Non-Patent Document 7, Patent Document 1) and studies using lactic acid bacteria (Non-Patent Document 8, Non-Patent Document 9, Patent Document 2) as examples of feeding probiotics to monogastric animals. There is. However, there is still no definitive solution that surpasses the benefits of AGP and meets market expectations. In ruminants, monensin is widely used as an antibiotic. Methane released from ruminants is one of the major greenhouse gases from livestock, and the energy loss of host animals is equivalent to 2-12% of dietary energy, only from the economic point of view of livestock production.
  • Non-Patent Document 10 As a material alternative to monensin, a method of feeding fumaric acid (Patent Document 3) and a method of feeding cysteine (Patent Document 4) are known, but their effects are weak.
  • nisin which is one of the bacteriocins, has an effect of suppressing methane production, but the effect is not sustained because it is decomposed in the rumen by a bacterial protease (Non-Patent Document 11).
  • Non-Patent Document 12 There is also a report suggesting suppression of methane generation using lactic acid bacteria, but it has not yet been embodied as a viable bacterial agent imparted with acid resistance.
  • the active ingredients secreted by lactic acid bacteria include organic acids such as lactic acid and bacteriocins.
  • Bacteriocin is a general term for proteins and peptides that have antibacterial activity mainly against the same species and related species, and nisin and plantaricin are widely used as food preservatives.
  • Antibiotics are persistent substances that are not decomposed by digestive enzymes, but bacteriocins are proteins and are easily decomposed by digestive enzymes.
  • there are many studies on antibacterial activity and antibacterial spectrum of antibiotics and bacteriocins but there are very few reports that these substances have other functions. As a study investigating the possibility of having various functions, a function of enhancing the toughness of the intestinal epithelium has been reported (Patent Document 5).
  • Oligosaccharides have been commercialized as prebiotics, but it is known to promote the growth of so-called "good bacteria” present in the intestinal flora (Non-Patent Document 13). However, the effect on the entire intestinal flora is not large, and the effect of this addition has not been elucidated. One of the reasons is that oligosaccharides are easily metabolized, and even if they have an effective function, they cannot exert the function. On the other hand, by using persistent polysaccharides instead of oligosaccharides, so-called "bad bacteria” such as salmonella, E. coli, and Campylobacter, which are gram-negative bacteria that are not easily decomposed by intestinal bacteria and induce enteritis, are selected.
  • so-called "bad bacteria” such as salmonella, E. coli, and Campylobacter, which are gram-negative bacteria that are not easily decomposed by intestinal bacteria and induce enteritis
  • Patent Document 6 A technique for aggregating and excreting from the intestine has been reported (Patent Document 6). There is no report of a combination of the "bad bacteria” aggregating function due to polysaccharides and the intestinal colonization of "good bacteria” and the function of the active ingredient secreted by "good bacteria”.
  • Non-Patent Document 14 Non-Patent Document 14
  • lipoteichoic acid lipoteichoic acid
  • McPatent Document 15 there is Moonlighting protein as a substance involved in the interaction between the host and bacteria.
  • This protein is a general term for proteins having a plurality of different actions, and one of them is translation growth factor (Translation elongation factor Tu, EF-Tu).
  • Tu Translation elongation factor
  • EF-Tu exists inside the cell, it has also been clarified that it is a factor that partially moves extracellularly and colonizes the mucin layer (Non-Patent Document 16).
  • Non-Patent Document 17 a method of adding silica as a method of imparting acid resistance to microorganisms.
  • the present invention provides the following viable bacterial agent and a method for producing the same.
  • the above-mentioned viable agent 2.
  • the viable agent according to 1 above, wherein the microorganism is Lactococcus lactis.
  • 3. The viable agent according to 2 above, wherein the microorganism is a microorganism that produces Nisin. 4.
  • the viable bacterial agent according to 4 above, wherein the microorganism has a gene for biosynthesizing an intestinal colonization factor. 10. 9. The viable bacterial agent according to 9 above, wherein the intestinal colonization factor is a lectin, lipoteichoic acid, or EF Tu. 11. The viable agent according to any one of 4 to 10 above, wherein the microorganism is Lactbacillus plantarum. 12. The microorganism is at least one selected from the group consisting of Lactococcus lactis FERM BP-8552, Lactobacillus plantarum TUA1478L, Lactobacillus plantarum TUA1490L, and Lactobacillus plantarum TUA2424L, according to the above 1.
  • Viable fungus agent 13. The viable bacterial agent according to any one of 1 to 12 above, wherein the protective agent further comprises glutamine. 14. The viable bacterial agent according to any one of 1 to 13 above, wherein the protective agent further comprises arginine hydrochloride. 15. The viable bacterial agent according to any one of 1 to 14 above, wherein the protective agent further comprises cellulose. 16. The viable bacterial agent according to any one of 1 to 12 above, wherein the protective agent comprises silica, skim milk, sodium glutamate, and glutamine. 17. 16. The viable cell agent according to 16 above, wherein the protective agent contains 1 to 10 parts by mass of silica, 10 to 50 parts by mass of sodium glutamate, and 10 to 50 parts by mass of glutamine with respect to 100 parts by mass of skim milk.
  • the protective agent contains 1 to 10 parts by mass of silica, 10 to 50 parts by mass of sodium glutamate, 10 to 50 parts by mass of glutamine, and 10 to 50 parts by mass of arginine hydrochloride with respect to 100 parts by mass of skim milk. 18.
  • A Microorganisms belonging to the genus Lactococcus lactis, Lactococcus lactis, or a mixture thereof are cultivated.
  • B After completion of the culture, a protective agent containing at least silica and monosodium glutamate is added to the obtained culture and mixed.
  • C A viable bacterial agent is obtained by leaving the obtained mixture to stand and incorporating the protective agent into the cells. Manufacturing method of live bacterial agent. 23. 22. The production method according to 22 above, further comprising a step of spray-drying, freeze-drying, or stirring freeze-drying after the step (c).
  • 24 A method for increasing the body weight-increasing effect and feed efficiency of livestock, which comprises administering the livestock agent according to any one of 1 to 19 to livestock.
  • a viable cell agent capable of delivering viable cells to the intestine while maintaining the viable cell count.
  • FIG. 1 shows the repair rate of barrier function at various concentrations of Quercetin, NisinA and Lactococcus lactis FERM BP-855.
  • FIG. 2 shows a transmission electron micrograph of Lactococcus lactis FERM BP-8552 treated with a protective agent containing silica. The gourd-shaped black shadow present in the center of FIG. 2A shows the bacterial cells themselves.
  • FIG. 2B shows that silicon is present in the cells. Multiple white dots indicate the presence of elemental silicon.
  • FIG. 3 shows the growth rate of Bacillus subtilis (C-3102) and Lactobacillus plantarum strains under acidic conditions.
  • FIG. 4 shows the repair rate of barrier function at various concentrations of Quercetin and Lactobacillus plantarum strains.
  • FIG. 5 shows the bacteriocin biosynthetic genes present in Lactobacillus plantarum strain, TUA1490L, TUA2424L and TUA1478L.
  • FIG. 6 shows a transmission electron micrograph of Lactobacillus plantarum treated with a protective agent containing silica.
  • FIG. 6A shows the whole picture of the bacterial cells.
  • FIG. 6B is a diagram showing the presence or absence of Si in the horizontal direction from the left end to the right end along the arrow of FIG. 6C. Peaks near 40-45 points indicate the presence of Si.
  • FIG. 6C is an enlarged view in the arrow direction of FIG. 6A.
  • FIG. 7 shows the time course of the elution rate of Arabic Gum (AG) from the two-layer coated feed additive under the conditions of gastric juice and intestinal juice.
  • AG Arabic Gum
  • living agent includes living microorganisms that have a beneficial effect on the host when ingested in sufficient amounts.
  • the microorganism contained in the viable agent of the present invention is Lactococcus lactis, a lactic acid bacterium belonging to the genus Lactobacillus, or a mixture thereof.
  • the Lactococcus lactis that can be used in the present invention the FERM BP-8552 strain is preferable because Nisin is highly secreted.
  • the FERM BP-8552 strain was released on November 19, 2003 at the Patent Organism Depositary Center, Industrial Technology Research Institute (1-1-1, Higashi, Tsukuba City, Ibaraki Prefecture, Japan, postal code 305-8566, now independent. It has been deposited with the Japan Product Evaluation Technology Infrastructure Organization, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan, postal code 292-0818).
  • the genus Lactobacillus that can be used in the present invention includes Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus alactosus, and Lactobacillus.
  • alimentarius Lactobacillus amylophilus, Lactobacillus amylovorans, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus animalis, Lactobacillus animalis, Lactobacillus animalis Lactobacillus animalis Lactobacillus animalis Lactobacillus animalis Lactobacillus animalis Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus lactobacillus buch bulgaricus), Lactobacillus catenaforme, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus cellobiosus, Lactobacill
  • Lactobacillus plantarum is preferable because it has abundant eating experience and the bacteriocin produced by it, Plantaricin, is widely used all over the world.
  • the Lactobacillus plantarum is preferably at least one selected from the group consisting of the TUA1478L strain, the TUA1490L strain (FERM P-21709), and the TUA2424L strain.
  • the TUA2424L strain capable of preventing intestinal inflammation is preferable because of its high acid resistance and ability to harden the membrane of gastrointestinal epithelial cells (synonymous with barrier repair ability).
  • the TUA1478L strain was commissioned by the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (post code 292-0818, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan).
  • the TUA1490L strain (FERM P-21709) was deposited internationally under the number NITE BP-03351, the accession number NITE BP-03352, and the TUA2424L strain under the accession number NITE BP-03353.
  • the microorganism used in the present invention preferably produces bacteriocin.
  • Bacteriocin is a general term for proteins and peptides produced by bacteria that have antibacterial activity mainly against the same species and related species.
  • Lactococcus lactis those capable of producing nisin, which is a kind of bacteriocin, are preferable.
  • lactobacillus plantarum those capable of producing bacteriocins such as Plantaricin-EF, Plantaricin-JK, Plantaricin-NC8 ⁇ , and Plantaricin-J51 are preferable.
  • TUA1478L strain capable of producing plantaricin-JK, plantaricin-N, plantaricin-A, and plantaricin-EF, plantaricin-NC8 ⁇ , plantaricin-A, and TUA1490L strain capable of producing plantaricin-EF is preferred.
  • pheromones such as plantaricin A and NC8-IF; those capable of producing other peptides such as PlnN and OrfZ2 are also preferable.
  • Lactobacillus plantarum which produces plantaricin A, is preferable because it has a membrane hardening effect.
  • the microorganism contained in the viable agent of the present invention has a gene that biosynthesizes an intestinal colonization factor because it can colonize the intestinal tract and exert an effect for a long period of time.
  • Intestinal colonization factors include lectins, lipoteichoic acid and EF-Tu (Translation elongation factor Tu).
  • EF-Tu Translation elongation factor Tu
  • the present inventors have confirmed that the EF-Tu synthetic gene is present in the TUA1478L strain, the TUA1490L strain, and the TUA2424L strain. Therefore, since these three strains are considered to be colonizable in the intestinal tract, they are proliferative in the intestinal flora and are expected to exert their effects for a longer period of time.
  • the genus Lactobacillus is preferable, Lactobacillus plantarum is more preferable, and Lactobacillus plantarum TUA2424L strain is particularly preferable.
  • the viable agent of the present invention is treated with a protective agent containing at least silica and monosodium glutamate (MSG).
  • MSG monosodium glutamate
  • the acid resistance of the microorganism can be improved.
  • higher acid resistance can be obtained by incorporating silicon atoms into the cells.
  • Monosodium glutamate is also taken up into the cells depending on the treatment conditions.
  • Sodium glutamate is also considered to play a role in suppressing the decrease in the viable cell count during drying.
  • the amounts of silica and monosodium glutamate are not particularly limited as long as they are sufficient to protect the microorganisms.
  • the amount of silica is preferably 0.01 to 5.0 g with respect to 1 g of dried cells, from the viewpoint of intracellular permeation amount, osmotic pressure and the like. When it is 0.1 to 1.0 with respect to 1 g of dried cells, it is more preferable from the viewpoint of stability and economy.
  • the amount of sodium glutamate is preferably 0.01 to 10.0 g with respect to 1 g of dried cells, from the viewpoint of intracellular permeation amount, osmotic pressure and the like. The amount is 0.15 g to 3.5 g with respect to 1 g of dried cells, which is more preferable from the viewpoint of stability and economy.
  • the protective agent further contains glutamine because the acid resistance is further improved. Depending on the treatment conditions, glutamine is also taken up into the cells.
  • the amount of glutamine is preferably 0.01 to 2.5 g with respect to 1 g of dried cells, from the viewpoint of intracellular permeation amount, osmotic pressure and the like. The amount is 0.2 to 2.5 g with respect to 1 g of dried cells, which is more preferable from the viewpoint of stability and economy.
  • the protective agent further contains arginine hydrochloride because the acid resistance is further improved. Depending on the treatment conditions, arginine hydrochloride is also taken up into the cells.
  • the amount of arginine hydrochloride is preferably 0.01 to 10.0 g with respect to 1 g of dried cells, from the viewpoint of intracellular permeation amount, osmotic pressure and the like.
  • the amount is 0.1 to 3.5 g with respect to 1 g of dried cells, which is more preferable from the viewpoint of stability and economy.
  • the protective agent further contains cellulose because a dry powder can be prepared more stably.
  • the amount of cellulose is 1 g to 20 g with respect to 1 g of dried cells, it is particularly preferable because a more stable dry powder can be prepared.
  • the protective agent further contains glutamine and arginine hydrochloride because the acid resistance is further improved.
  • a ratio of glutamine to arginine hydrochloride in a weight ratio of 2: 1 to 2: 3 is preferable from the viewpoint of stability and economy.
  • the protective agent further contains glutamine and cellulose because a dry powder can be prepared more stably.
  • the protective agent further contains arginine hydrochloride and cellulose because a dry powder can be prepared more stably.
  • the protective agent further contains skim milk because it can suppress a decrease in the viable cell count during drying.
  • the amount of skim milk is preferably 0.5 g to 20 g with respect to 1 g of dried cells, from the viewpoint of intracellular permeation amount, osmotic pressure and the like. 1 g to 10 g with respect to 1 g of dried cells is more preferable from the viewpoint of stability and economy.
  • the protective agent contains silica, sodium glutamate, glutamine and skim milk, it is preferable from the viewpoint of intracellular permeation amount and osmotic pressure that impart acid resistance.
  • the protective agent consists only of silica, monosodium glutamate, glutamine and skim milk.
  • the protective agent contains 1 to 10 parts by mass of silica, 10 to 50 parts by mass of sodium glutamate, and 10 to 50 parts by mass of glutamine with respect to 100 parts by mass of skim milk, from the viewpoint of stability and economy. Is preferable.
  • the protective agent contains silica, sodium glutamate, glutamine, arginine hydrochloride and skim milk
  • the protective agent consists only of silica, monosodium glutamate, glutamine, arginine hydrochloride and skim milk.
  • the protective agent contains 1 to 10 parts by mass of silica, 10 to 50 parts by mass of sodium glutamate, 10 to 50 parts by mass of glutamine, and 10 to 50 parts by mass of arginine hydrochloride with respect to 100 parts by mass of skim milk.
  • Including is preferable from the viewpoint of stability and economic efficiency.
  • Cellulose can also be used as a protective agent for treating bacterial cells together with silica and monosodium glutamate, and as an excipient, it constitutes a viable bacterial agent together with the dried bacterial cells. May be.
  • the viable bacterial agent of the present invention may contain bacteriocin, polysaccharide, oligosaccharide and the like as further active ingredients.
  • the viable agent of the present invention is in the form of a supplement, feed, etc. for animals other than humans, if the viable agent of the present invention contains bacteriocin and / or polysaccharide, the effect of exterminating bad bacteria of bacteriocin and It is possible to exert a synergistic effect of reducing inflammation induction by improving the intestinal flora through aggregation of bad bacteria.
  • Bacteriocins are classified into two classes, Class I and Class II, according to Paul D. Cotter's classification (Nat. Rev. Microbiol. 2005 Volume3 (10), pp777-88).
  • Examples of the bacteriocin that can be used in the present invention include bacteriocins belonging to ClassI, ClassIIb and ClassIIc. Specifically, at least one selected from the group consisting of nisin, gassericin, plantaricin, and subtilin can be preferably used.
  • each bacteriocin When the name of each bacteriocin is not followed by an alphabet, it means a general term for the bacteriocin (for example, the term “nisin” is a concept including NisinA, NisinZ, etc.). Of these, nisin and plantaricin are preferable because they have abundant eating experience and are widely used all over the world.
  • the antibacterial effect of exterminating bad bacteria is set to about 0.001 to 0.1% by mass with respect to the total mass of the viable bacterial agent. It is preferable from the viewpoint of. 0.01 to 0.05% by mass is more preferable from the viewpoint of stability and economy.
  • polysaccharide that can be used in the present invention those having a property of aggregating Gram-negative bacteria such as Eschericha coli are preferable. Whether or not it has the property of aggregating Gram-negative bacteria can be specified by the method disclosed in International Publication No. 2019/177172. Specifically, purulan, xanthan gum, guar gum, carrageenan, arabic gum, pectin, carboxymethyl cellulose, chondroitin, tara gum, locust bean gum, alginate (sodium salt, potassium salt, calcium salt, or ammonium salt), alginate ester and these. At least one selected from the group consisting of a mixture of the above can be preferably used.
  • arabic gum, pullulan, and xanthan gum are preferable from the viewpoint of cost effectiveness.
  • the content of the polysaccharide is about 10 to 1000 ppm with respect to the feed addition amount of the viable bacterial agent in that a stable body-increasing effect can be exhibited. 10 to 200 ppm is more preferable from the viewpoint of cost effectiveness.
  • the oligosaccharide at least one selected from xylooligosaccharide, fructooligosaccharide, and galactooligosaccharide can be preferably used. Of these, fructooligosaccharides are preferable from the viewpoint of cost effectiveness.
  • the content of the oligosaccharide is about 0.1 to 50% by mass with respect to the total mass of the viable agent from the viewpoint of stability and economy. .. 10 to 20% by mass is more preferable from the viewpoint of cost effectiveness.
  • the viable agent of the present invention includes various antioxidants such as citric acid, ascorbic acid and vitamin E; vitamins such as vitamins A, B1 and B2; minerals such as calcium, magnesium and manganese; flavors and the like. It may contain an ingredient.
  • the content of such an optional component is usually about 0.01 to 10% by mass based on the total mass of the viable agent.
  • the viable bacterial agent of the present invention was obtained by culturing (a) a microorganism belonging to the genus Lactococcus lactis, Lactococcus lactis, or a mixed microorganism thereof, and (b) after completion of the culture. It can be produced by adding and mixing a protective agent containing at least silica and sodium glutamate to the culture, and (c) leaving the obtained mixture to stand to allow the protective agent to be incorporated into the cells.
  • a medium for lactic acid bacteria such as a general MRS medium, GYP medium, BLB medium, or an improved medium thereof can be used.
  • the medium may further contain a safe substance that does not interfere with the growth of the microorganism, but is preferably free of animal-derived materials from the viewpoint of preventing bovine spongiform encephalopathy.
  • the culture conditions are not particularly limited, but for Lactococcus lactis, the temperature is generally preferably 30 to 39 ° C, the pH of the medium is preferably 4.5 to 7.5, and the culture time is preferably 5 to 30 hours.
  • the temperature is generally preferably 35 to 37 ° C.
  • the pH of the medium is preferably 5.5 to 6.5
  • the culture time is preferably 10 to 25 hours. Even under other conditions, it can be carried out in combination with other conditions such as a medium. Culturing should be carried out until a sufficient viable cell count is obtained. As a guide, it is about 10 ⁇ 10 cfu / mL. Whether or not a sufficient viable cell count was obtained can be determined, for example, by confirming that the turbidity of the appropriately serially diluted culture solution at a wavelength of 610 nm is 0.1 or more.
  • the culture cultured in this way can be filtered, centrifuged, or membrane-separated to separate the cells, and the recovered cells can be treated with a protective agent to obtain a viable cell agent.
  • the cultured culture itself can be treated with a protective agent described later to obtain a viable bacterial agent.
  • the conditions for separating the cells from the culture can be appropriately determined by those skilled in the art.
  • the protective agent is added to the culture or the collected cells.
  • the protective agent may be added in a total amount at a time, or may be added in small portions.
  • the protective agent may be added as it is, or may be dissolved or dispersed in water and added in a liquid form. It is preferable to add it in a liquid form because it can be evenly distributed throughout the cells.
  • the protective agent is preferably added in an amount of 1 to 20 g as a solid content with respect to 1 g of dried cells, and more preferably in an amount of 1 to 10 g. After adding the entire amount of the protective agent, stir and mix to bring the protective agent into contact with the cells.
  • the stirring can be performed mildly without foaming.
  • the protective agent can be taken into the cells. That is, in general, when producing a viable cell agent, the viable cell count is obtained by storing the cells at about 10 ° C. during the waiting time until the next drying step is started after the step of fermenting the viable cells. To maintain. At this time, by bringing the cells into contact with the protective agent, the protective agent is taken into the cells, and the cells obtain stronger acid resistance. However, the protective agent is not taken into the cells simply by adding the protective agent.
  • the protective agent was incorporated into the cells by storing the protective agent for several hours, at least for 2 hours or more for amino acids, and for 10 hours or more for Si.
  • the viable cell agent of the present invention can maintain a high viable cell count in the living body to which it is administered. Therefore, if the temperature is 10 ° C., it is preferable to keep the contact for about 10 to 18 hours, and more preferably about 10 to 16 hours.
  • the growth and metabolism of cells and the rate of uptake of protective agents decrease as the temperature drops from the optimum temperature. Therefore, if the storage temperature is lowered to, for example, 4 ° C., it is preferably about 12 to 22 hours, more preferably about 20 hours.
  • the storage temperature is raised to, for example, 20 ° C., it is preferably about 10 to 16 hours, more preferably about 14 hours. From the viewpoint of cost effectiveness, it is particularly preferable to store at 10 ° C. for about 10 to 16 hours. Whether or not the protective agent has been incorporated into the cells can be confirmed by observing with a transmission electron microscope or by measuring the change in the supernatant concentration of the protective agent before and after taking in the protective agent.
  • the cells may be dried. Drying may be carried out by spray drying (Spray Dryer (SD)), freeze drying (Freeze Dry (FD)), stirring freeze drying (Freeze Granulation-Freeze Dry (FG)) or room temperature vacuum drying. It is still better to perform by spray drying, freeze drying, or stirring freeze drying. Of these, freeze-drying is preferable from the viewpoint of cost effectiveness. Stirring freeze-drying is preferable because it can be produced more efficiently.
  • the drying temperature is mainly determined by the type of microorganism, but in the case of Lactococcus lactis, it is preferably ⁇ 20 ° C.
  • the water content of the viable cell agent after drying is preferably 5% by weight or less, more preferably 3% by weight or less. In the present specification, the water content can be measured by drying at 105 ° C. for 240 minutes using a constant temperature dryer and measuring the water content from the weight difference before and after drying.
  • the optional component may be added before the step (c) or in the middle of the step (c). Alternatively, it may be added after the step (c). For example, it may be added separately before and after the step (c). From the viewpoint of cost effectiveness that can simplify the sterilization step of the excipient, it is preferable to add it after the step (c). When the step (d) is carried out, it is preferable to add it after the step (d).
  • an optional component such as an excipient (for example, cellulose
  • the optional component may be added before the step (c) or in the middle of the step (c). Alternatively, it may be added after the step (c). For example, it may be added separately before and after the step (c). From the viewpoint of cost effectiveness that can simplify the sterilization step of the excipient, it is preferable to add it after the step (c).
  • the step (d) is carried out, it is preferable to add it after the step (d).
  • the viable bacterial agent of the present invention can also be administered to humans and animals in the form of supplements. It can also be ingested in yogurt or the like.
  • the above-mentioned microorganism is viable in an amount of 0.1 mg to 10 g, preferably 1 mg to 1 g, per 1 kg of the body weight of the subject to be administered. It is better to include it in the agent.
  • the ingestion amount, use amount or dose of the viable bacterial agent of the present invention at one time is appropriately adjusted according to the body weight of the subject, etc., but the raw material of the present invention is, for example, 3 to 5 g / kg body weight. It is preferable to ingest, use or administer the fungal agent.
  • the daily intake, use or dose of the viable bacterial agent of the present invention also varies depending on the body weight of the subject and the like, but is preferably 18 to 30 g / adult (assuming a body weight of 60 kg), for example.
  • the term "supplement” refers to a supplement taken by humans or animals for the purpose of maintaining, recovering or promoting health, or preventing or ameliorating a disease.
  • Examples of animals that can take the supplement of the present invention include ruminants such as cows, sheep and goats, and monogastric animals such as horses, pigs, chickens, dogs and fish.
  • ruminants such as cows, sheep and goats
  • monogastric animals such as horses, pigs, chickens, dogs and fish.
  • the antibiotic containing the viable bacterial agent of the present invention can avoid the problem of drug-resistant bacteria and can be used safely.
  • the amount of the microorganism is 0.001 g to 1 g per 1 kg of the body weight of the administration subject, preferably 0. It is preferable to include it in the viable cell agent in an amount of 01 g to 1 gcfu.
  • ⁇ Livestock growth promoter> As described in the background technology column, antibiotics have been mainly used as growth promoters for livestock, but in some countries the use is prohibited due to drug-resistant bacteria and environmental pollution. .. Since the viable bacterial agent of the present invention has antibacterial activity, it can also be used as a growth promoting agent for livestock in place of the conventional growth promoting antibiotic for livestock. According to the growth promoter of the present invention, problems such as drug-resistant bacteria and environmental pollution can be avoided. At this time, it is preferable to include the microorganism in the viable cell agent in an amount of 10 ⁇ 7 to 10 ⁇ 10 cfu, preferably 10 ⁇ 8 to 10 ⁇ 9 cfu, per 1 kg of the body weight of the administration target.
  • Lactococcus lactis contained in the viable agent of the present invention produces nisin, which is a kind of bacteriocin.
  • Nisin is known to have a methanogenic inhibitory effect on ruminant lumens (Non-Patent Document 9).
  • the effective dose depends on the type and body weight of the animal, but for example, when administered to cattle, the microorganism is preferably 10 ⁇ 6 to 10 ⁇ 9 cfu, more preferably 10 ⁇ 6 to 10 ⁇ 9 cfu, based on 1 kg of body weight per day. It is 10 ⁇ 7 to 10 ⁇ 8 cfu.
  • the viable bacterial agent of the present invention can be given to animals as it is, or can be given as a feed together with excipients or diluents such as corn, soybean flour, rice bran, fish meal and brewer's yeast.
  • the feed of the present invention may also contain any additives that may be included in the feed. It is appropriate that the feed of the present invention is continuously ingested every day. Examples of animals to which the feed of the present invention can be administered include ruminants such as cows, sheep and goats, and monogastric animals such as horses, pigs, chickens, dogs and fish. It is particularly preferable to feed the feed of the present invention to monogastric animals.
  • the effective dose depends on the type and body weight of the animal, but for example, when administered to chickens, the microorganism is preferably 10 ⁇ 7 to 10 ⁇ 10 cfu, more preferably 10 ⁇ 7 to 10 ⁇ 10 cfu, based on 1 kg of body weight per day. It is 10 ⁇ 8 to 10 ⁇ 9 cfu.
  • Test Example 1 Antibacterial spectrum of Lactococcus lactis, which is a Nisin-producing strain, The antibacterial spectra of the culture supernatants of the reagent products Nisin A and Nisin Z-producing bacteria were compared.
  • NisinA used a reagent manufactured by Sigma-Aldrich (Nisin content 2.5% by mass, balance sodium chloride and denatured milk solids).
  • As the NisinZ-producing bacterium Lactococcus lactis AJ110212 (FERM BP-8552) was used.
  • NisinZ-producing strains were cultured at 30 ° C. at 100 rpm for 20 hours by a conventional method using Lactobacillus MRS medium manufactured by BD Difco.
  • a sufficient amount of cells was obtained from the culture solution by measuring the optical density at a wavelength of 610 nm with a spectrophotometer (Biophoto-recorderTVS062CA manufactured by ADVANTEC) and confirming that it was 0.1 or more after 26-fold dilution. It was judged. Centrifuge the obtained culture solution (6,000 G x 10 min, 4 ° C) to remove the bacterial cell fraction, filter the supernatant fraction (ADVANTEC DISMIC-25CS, 0.20 ⁇ L filter unit), and aseptically. The supernatant was obtained. The following strains were used as the test bacteria. The medium and culture temperature are described in parentheses at the end of the strain.
  • the MRS medium used was Lactobacillus MRS Broth manufactured by Difco
  • the GAM medium and LB medium used were those manufactured by Nissui Pharmaceutical Co., Ltd.
  • the NB medium used was those manufactured by Difco.
  • Lactobacillus sakei JCM1157 (MRS, 37 °C) Lactobacillus acidophilus JCM1132 (MRS, 37 °C)
  • Lactobacillus salivarius JCM1231 MRS, 37 °C
  • Bifidobacterium thermophilum JCM1207 (GAM, 37 °C)
  • Enterococcus faecalis JCM5803 (MRS, 30 °C) Escherichia coli ATCC700926 (LB, 37 °C)
  • Salmonella enterica IAM1648 (NB, 37 °C) were used.
  • the minimum growth inhibition intensity is qualitative in the size of the inhibition circle using the Spot-on-lawn method described in Mayr-Harting, A. et al., Methods Microbiol. 197 2, 7A, pp315-422. I decided. The results are shown in Table 1. As a result, it was confirmed that there was almost no difference in the antibacterial spectra of the reagent products NisinA and NisinZ-producing bacteria. Therefore, it is considered that NisinZ-producing bacterium Lactococcus lactis FERM BP-8552 can be used as a viable agent.
  • Test Example 2 Membrane fastening test of Lactococcus lactis using Caco-2 cells According to the description of J. Nutr. (2009) volume 139 (5), pp965-974, the membrane fastening ability of the culture supernatant of lactic acid bacteria was increased. evaluated. Nisin A and Quercetin were used as reagent products, and Lactococcus lactis FERM BP-8552, a Nisin Z-producing bacterium, was used as a lactic acid bacterium.
  • Lactic acid bacteria are cultivated in MRS medium at 30 ° C for 20 hours, then the culture solution is centrifuged (6,000 G x 10 min, 4 ° C) to remove the bacterial cell fraction, and the supernatant fraction is filtered (filtered). ADVANTEC DISMIC-25CS, 0.20 ⁇ L filter unit) was used to obtain a sterile supernatant.
  • Caco-2 cells human gastrointestinal epithelial cells (ECACC, Code86010202)
  • DMEM medium 37 ° C.
  • TNF- ⁇ was added to reduce the barrier function of Tight Junction.
  • Example 3 Preparation (culture) of a viable bacterial agent containing Lactococcus lactis Lactococcus lactis FERM BP-8552 was cultured in Lactobacilli MRS Broth medium manufactured by BD Difco as follows, and then pulverized. That is, the main culture (fermenter, 20 L) was carried out through pre-seed culture (Sakaguchi flask, 50 ml) and seed culture (Sakaguchi flask, 1 L). NisinZ-producing bacterium Lactococcus lactis FERM BP-8552 was cultured at 30 ° C. at 100 rpm. The culture should be performed for about 20 hours.
  • the turbidity (Optical density, wavelength 610 nm) is measured with a spectrophotometer UVmini-1240 manufactured by Shimadzu Corporation, and the 26-fold dilution of the culture solution is 0.1 or more. By confirming that, it was judged that a sufficient amount of bacterial cells were obtained.
  • the cell fraction of the culture solution (20 L) was separated by a centrifuge. The centrifuge was carried out using a Beckman Coulter Avanti JE at a temperature of 15 ° C., a centrifugal speed of 6,750 G, and an hour of 15 minutes.
  • the cell fraction of the culture solution (20 L) obtained here was an amount equivalent to 20 g (10 ⁇ 12 cfu / g) as dried cells.
  • (Preparation liquid before powder) 3 g of silica (Carplex CS-7 manufactured by EVONIC), 100 g of Skim milk (manufactured by BD), and 30 g of monosodium glutamate (manufactured by Ajinomoto Co., Inc.) (0.15 g of silica, 5.0 g of Skim milk, and 5.0 g of dried cells per 1 g of dried cells).
  • Test Example 4 Acid resistance test of live bacterial agent containing Lactococcus lactis (artificial gastric juice treatment) 1 ml of pure water containing 0.2% NaCl and 0.2% pepsin (from Porcine stomach Mucosa, 1: 5,000, 2,500 unit / mg) was adjusted to pH 1.5 to prepare an acid-resistant treatment liquid. Then, 0.02 g of the lactic acid bacterium powder obtained by drying by the FD method in Example 3 was added, and acid resistance treatment was carried out at 37 ° C. for 2 hours.
  • Test Example 5 Incorporation of protective agent into cells Obtained by drying by the FD method in Example 3 by EDX analysis (Energy dispersive X-ray spectroscopy) using a transmission electron microscope (TEM).
  • EDX analysis Energy dispersive X-ray spectroscopy
  • TEM transmission electron microscope
  • the Si element in the cells was analyzed.
  • the TEM image is shown in FIG.
  • the gourd-shaped black shadow present in the center of FIG. 2A shows the bacterial cells themselves.
  • FIG. 2B shows that silicon is present in the cells.
  • Multiple white dots indicate the presence of elemental silicon.
  • the shade of color has nothing to do with the amount of uptake. From this result, it was confirmed that Si, which normally does not exist in the cells, is incorporated into the cells. This phenomenon suggests that the viable bacterial agent of Example 3 may be imparted with acid resistance.
  • Test Example 6 Feeding test of live bacterial agent containing Lactococcus lactis to Salmonella-infected chickens 3.0 x 10 ⁇ 10 cfu (0.63 g) of cells dried by the FD method in Example 3 in a feed matrix of 12 kg having the composition shown in Table 4. ) And 5.63 g of viable cell agent mixed with 5 g of the excipient Cellulose was added to prepare a test feed. The final concentration of viable bacteria in the feed was 3 x 10 ⁇ 6 cfu / g, and after the 1-day-old broiler was introduced into the breeding facility for infection testing (6 birds / repeat, 2 repeats / test plot), Salmonella enterica (SE).
  • SE Salmonella enterica
  • Test Example 7 Growth experiment of Lactobacillus plantarum under acidic conditions (strain selection) Lactobacillus plantarum TUA1478L, TUA1490L, and TUA2424L were statically cultured in MRS medium at 37 ° C. As a control, the Bacillus subtilis C3102 strain used as a viable agent in Patent Document 1 was cultured with shaking (70 rpm) at 30 ° C. using Biophoto-recorder TVS062CA manufactured by ADVANTEC in LB medium.
  • the optical density of each cell was measured with a spectrophotometer at a wavelength of 610 nm, and it was confirmed that it was 0.1 or more after 26-fold dilution, and it was judged that a sufficient amount of cells was obtained. Then, the bacterial cell fraction obtained by centrifugation was suspended in physiological saline prepared at pH 2, and allowed to stand at 37 ° C. for 0.5 hour, 1 hour, 2 hours, 4 hours, and an acid resistance test was carried out. ..
  • Test Example 8 Membrane fastening test of Lactobacillus plantarum using Caco-2 cells According to the description of J. Nutr. (2009) volume 139 (5), pp965-974, the membrane fastening ability of the culture supernatant of lactic acid bacteria was increased. evaluated. Quercetin was used as a reagent product, and Lactobacillus plantarum JCM1057, Lactobacillus plantarum TUA1490L, and TUA2424L, which produce Plantaricin A, were used as lactic acid bacteria.
  • the test substance On the 14th day of culturing, the test substance was added, and after culturing at the same temperature for 24 hours, the transepithelial electrical resistance value TER ( ⁇ * cm2) was measured using Millicell ER S-2 (manufactured by Millipore). , The recovery ratio% of the barrier function was evaluated. The results are shown in FIG.
  • Test Example 9 Confirmation of bacteriocin biosynthetic gene of Lactobacillus plantarum (genome analysis) Whole-genome data of Lactobacillus plantarum TUA1478L, TUA1490L, TUA2424L were acquired. Using the Plantaricin gene cluster (amino acid sequence) of 9 strains registered in NCBI as a query, tBLASTn search was performed on the genomes (base sequences) of Lactobacillus plantarum TUA1478L, TUA1490L, and TUA2424L.
  • NCBI registered strains are Lactobacillus plantarum V90, YM 4-3, YM 5-2, 8P-A3, C11, J51, NC8, PCS20, 423.
  • the homology (% identity) of the alignment region is 90% or more and the ratio of the region length obtained to be aligned (query coverage) in the total length of the query sequence is 90% are extracted.
  • the operon structure was manually organized using information from records with longer alignment regions and higher homology among the 9 strains. The results are shown in FIG.
  • the TUA1478L strain has Plantaricin-JK, Plantaricin-N, Plantaricin-A, and Plantaricin-EF biosynthetic genes
  • the TUA 2424L strain has Plantaricin-EF biosynthetic genes
  • the TUA 1490L strain has Plantaricin-NC8 ⁇ , Plantaricin. It was confirmed that the biosynthetic genes of -A and Plantaricin-EF were present.
  • Test Example 10 Confirmation of adhesion factor EF Tu gene in Mucin layer of Lactobacillus plantarum (genome analysis) Whole-genome data of Lactobacillus plantarum 3 strains TUA1478L, TUA1490L, TUA2424L were obtained. The total length and homology of the Translation elongation factor Tu (EF-Tu) synthetic gene were evaluated for these three strains with reference to the WCFS1 strain registered at NCBI at the following site. That is, a BLAST search was performed on the genome sequences of the three strains using the base sequence of the target gene of the NCBI reference strain as a query, and the region (putative gene region) in which the alignment was obtained was performed on the genome sequences of the three strains.
  • EF-Tu Translation elongation factor Tu
  • Example 11 Preparation (culture) of a viable bacterial agent containing Lactobacillus plantarum Lactobacillus plantarum TUA1478L, TUA1490L, TUA2424L were cultured using BD Difco's Lactobacilli MRS Broth medium as follows, and then pulverized. That is, the main culture (fermenter, 20 L) was carried out through pre-seed culture (falcon tube, 50 ml) and seed culture (medium bottle, 1 L). The culture was carried out at a temperature of 37 ° C. for 20 hours.
  • the cell fraction of the culture solution (20 L) was separated by a centrifuge.
  • the centrifuge was a Beckman Coulter Avanti JE, and the centrifuge was centrifuged at a temperature of 15 ° C., a centrifugal speed of 6,750 G, and an hour of 15 minutes.
  • the cell fraction of the obtained culture solution (20 L) was an amount equivalent to 20 g (10 ⁇ 12 cfu / g) as dried cells.
  • (Preparation liquid before powder) A bacterial cell fraction (equivalent to 20 g as dried cells) was added to 1,000 ml of a protective agent solution in which the amount of the protective agent (unit g) shown in Table A was mixed with water, and the powder prepreparation solution was 1,200 ml. was prepared and allowed to stand in a refrigerator at 4 ° C. for 20 hours.
  • each protective agent component is 0.35 g of silica (Carplex CS-7 manufactured by EVONIC), 5.0 g of Skim milk (manufactured by BD), and 1.5 g of arginine hydrochloride (Ajinomoto Co., Inc.) per 1 g of dried cells. Equivalent to 1.5 g of monosodium glutamate (manufactured by Ajinomoto Co., Inc.) and 1.0 g of glutamine (manufactured by Ajinomoto Co., Inc.).
  • Pre-powder preparation is sprayed into liquid nitrogen while stirring with a stirrer to form frozen particles, and cellulose equivalent to half the amount of dry substance in the protective agent solution.
  • the powder (Table A) was mixed and pulverized by freezing and vacuum drying. (Evaluation of viable cell count) To measure the viable cell count, 0.02 g of the powder sample obtained by the three powdering methods was suspended in 1 ml of physiological saline, diluted 10-fold with physiological saline, and 0.1 ml of the diluted solution was placed on an MRS agar plate.
  • Test Example 12 Incorporation of a protective agent into the cells Lactobacillus plantarum obtained in Example 11 by EDS (Energy Dispersive X-ray Spectrometer) analysis using a scanning transmission electron microscope (STEM). TUA2424L strain The elements in the cells were analyzed. The results are shown in Figure 6.
  • FIG. 6A shows the whole picture of the bacterial cells. An enlarged view in the direction of the arrow in FIG. 6A is FIG. 6C.
  • FIG. 6B analyzes the presence or absence of Si in the horizontal direction from the left end to the right end along the arrow in FIG. 6C. Peaks near 40-45 points indicate the presence of Si. From this result, it was confirmed that Si was present in a high concentration in the central part of the bacterial cell. This phenomenon suggests that the viable cell agent of Example 11 may be imparted with acid resistance.
  • Test Example 13 Confirmation of acid resistance gene of Lactobacillus plantarum Similar to Example 10, WCFS1 strain registered in NCBI was referred to, and the genes involved in acid resistance Glutamate decarboxylase "gadB" and Glutamine synthetase synthesis were performed for these three strains. The full length and homology of the genes were evaluated. It was confirmed that the above two enzyme synthesis genes were present in the two strains TUA1478L and TUA1490L, and the homology was 99% or more. It was also confirmed that the TUA 2424L strain had a Glutamate decarboxylase synthetic gene and had a homology of 99% or more.
  • Test Example 14 Acid resistance test of live bacterial agent containing Lactobacillus plantarum (artificial gastric juice treatment) 0.2% NaCl and 0.2% pepsin (from Porcine stomach Mucosa, 1: 5,000, 2,500unit / mg) were added to pure water produced using a pure water production device manufactured by Merck Millipore to adjust the pH to 1.5. It was artificial gastric juice.
  • Lactobacillus plantarum was cultured, the cell fraction was separated, and the cells were added to 1000 mL of a protective agent solution prepared by mixing the amount of the protective agent shown in Table B (unit g) below with water.
  • a fraction was added to prepare 1200 mL of a pre-powder preparation solution for Lactobacillus plantarum, and the pre-powder preparation solution was pulverized by the FD method to prepare a viable agent for Lactobacillus plantarum.
  • the concentration of each protective agent component is 0.35 g of silica (Carplex CS-7 manufactured by EVONIC), 5.0 g of Skim milk (manufactured by BD), and 1.5 g of arginine hydrochloride (Ajinomoto Co., Inc.) per 1 g of dried cells. Equivalent to 1.5 g of monosodium glutamate (manufactured by Ajinomoto Co., Inc.) and 1.0 g of glutamine (manufactured by Ajinomoto Co., Inc.).
  • the viable cell preparation thus prepared was put into the artificial gastric juice, subjected to enzyme treatment at 37 ° C. for 2 to 4 hours, and the viable cell count was measured.
  • the results are shown in Table 9. "+” In the table means that each component is included.
  • “2 to 4 hours” assumes the time from when the viable bacterial agent reaches the stomach of the chicken to when it passes through the intestine.
  • Reference Example 15 Preparation of coated Arabic Gum (Coated-AG)
  • Preparation of additive for coated feed Arabic Gum (manufactured by Wako Pure Chemical Industries, Ltd.) is used as the core material, and rapeseed hydrogenated oil (melting point 67) is used as the coating agent. °C) was used.
  • a coated feed additive was obtained by spraying a predetermined amount of a coating agent liquefied by heating to a temperature higher than the melting point onto the powdered or granulated core. In this coating, 75 parts by mass of the core was coated with 25 parts by mass of hydrogenated rapeseed oil.
  • Reference example 16 Enteric solubility test of Coated-AG (Artificial gastric juice treatment) 0.2% NaCl and 0.2% pepsin (from Porcine stomach Mucosa, 1: 5,000, 2,500unit / mg) were added to pure water produced using a pure water production device manufactured by Merck Millipore, adjusted to pH 2, and then a reference example.
  • the coated feed additive (core is Arabic Gum) prepared in No. 15 was added and subjected to enzyme treatment at 37 ° C. for 2 hours. In addition, "2 hours” assumes the time from when the feed reaches the stomach of the chicken to when it passes through.
  • Test Example 17 Feeding test of live bacterial agent containing Lactobacillus plantarum to Salmonella-infected chickens
  • the live bacterial agent prepared by powdering by the FG method in Example 11 was put into a feed matrix having the composition shown in Table 4 and live bacteria.
  • a test feed was prepared by adding the agent so that the concentration of the agent was 1.0 x 10 ⁇ 6 cfu / g.
  • Salmonella enterica (SE) was orally administered to the 2-day-old broiler, and the test feed was fed for 21 days.
  • the body-increasing effect and feed efficiency of Salmonella enterica were evaluated. The effect was weak when the viable bacterial agent was used alone, but the body-building effect was enhanced when used in combination with Coated-AG.
  • Reference Example 18 Donation test of Coated-NisinA and Coated pullulan to Salmonella-infected chickens (preparation of Coated-NisinA and Coated pullulan) Nisin A (a reagent manufactured by Sigma-Aldrich (Nisin content 2.5% by mass, balance sodium chloride and denatured milk solids) was used as the core, and rapeseed hardening oil (melting point 67 ° C.) and natural resin Shellac were used as the coating agent.
  • a coated feed additive was obtained by spraying a powdered or granulated core with a predetermined amount of a coating agent liquefied by heating to a temperature higher than the melting point. The coating was applied to 77 parts by mass of the core.
  • Reference example 19 Lactobacillus plantarum TUA2424L cells were cultured at 37 ° C. using MRS medium. After culturing, centrifugation (6000 rpm, 10 min) was performed using a centrifuge SS-1500X (Sakuma Seisakusho), and the supernatant fraction was removed to obtain a bacterial cell fraction. After dispersion in physiological saline in which MSG 3% was dissolved, the cells were left to stand so that the amount of dried cells was 10 g / L. Table 12 shows the amount of glutamic acid (Glu) uptake for each elapsed time under each condition of temperature (10, 20, 30 ° C.) and glucose (Glc, 0.6%) addition.
  • Glu glutamic acid
  • the amount of Glu uptake was calculated according to the following formula after measuring the glutamic acid concentration in the supernatant for each elapsed time as described below. That is, the supernatant fraction obtained by centrifuging (6000 rpm, 10 min) with a centrifuge SS-1500X (Sakuma Seisakusho) was diluted 50-fold with pure water and manufactured by Oji Measuring Instruments Co., Ltd. The measurement was performed using the offline biosensor BF-7D, and the glutamate concentration in the supernatant fraction was calculated from the dilution ratio to obtain the glutamate concentration for each elapsed time.
  • A Glu concentration g / L (0 hours)-Glu concentration g / L (each elapsed time)
  • B bacterial cell mass 10 g / L From Table 12, it was found that the amount of glutamic acid taken up during low-temperature storage was large, and the amount of glutamic acid taken up by the culture medium containing glucose and having active metabolism was large.

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Abstract

La présente invention concerne un agent microbien viable contenant Lactococcus lactis, un micro-organisme appartenant au genre Lactobacillus ou un mélange de ces micro-organismes, les micro-organismes susmentionnés ayant été traités avec un agent protecteur comprenant au moins de la silice et du glutamate de sodium.
PCT/JP2021/041767 2020-11-13 2021-11-12 Agent microbien viable contenant lactococcus lactis, microorganisme appartenant au genre lactobacillus ou mélange associé, et son procédé de production WO2022102753A1 (fr)

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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0286769A (ja) * 1988-09-26 1990-03-27 Kubota Ltd 粉末ラクトバチルス・プランタラムの製法
JPH08205857A (ja) * 1995-02-01 1996-08-13 Morinaga Milk Ind Co Ltd 微生物保護剤及び該保護剤を用いた凍結又は凍結乾燥微生物の製造法
JP2005500267A (ja) * 2001-05-23 2005-01-06 ソシエテ デ プロデユイ ネツスル ソシエテ アノニム 乳酸菌からのリポテイコ酸、およびグラム陰性菌、潜在的病原性グラム陽性菌によって媒介される免疫応答を調節するためのその使用
WO2006067940A1 (fr) * 2004-12-01 2006-06-29 Meiji Dairies Corporation Bacteries de l’ acide lactique se liant a des types sanguins abo humains
JP2008212006A (ja) * 2007-02-28 2008-09-18 Meiji Milk Prod Co Ltd 腸管バリア機能の機能回復剤及び腸管バリア透過性の亢進阻害剤
JP2010259341A (ja) * 2009-04-30 2010-11-18 Hiroshima Univ 細胞表面にケイ酸層を備える微生物、およびその作製方法
JP2011148703A (ja) * 2008-03-19 2011-08-04 Morishita Jintan Co Ltd 血中リン濃度上昇抑制剤
JP2012056921A (ja) * 2010-09-13 2012-03-22 Kaneka Corp 生菌製剤
JP2019523219A (ja) * 2016-05-18 2019-08-22 スキンバイオセラピューティクス ピーエルシー 組成物およびその使用
WO2019177171A1 (fr) * 2018-03-16 2019-09-19 味の素株式会社 Additif alimentaire pour animaux et aliment pour animaux

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0286769A (ja) * 1988-09-26 1990-03-27 Kubota Ltd 粉末ラクトバチルス・プランタラムの製法
JPH08205857A (ja) * 1995-02-01 1996-08-13 Morinaga Milk Ind Co Ltd 微生物保護剤及び該保護剤を用いた凍結又は凍結乾燥微生物の製造法
JP2005500267A (ja) * 2001-05-23 2005-01-06 ソシエテ デ プロデユイ ネツスル ソシエテ アノニム 乳酸菌からのリポテイコ酸、およびグラム陰性菌、潜在的病原性グラム陽性菌によって媒介される免疫応答を調節するためのその使用
WO2006067940A1 (fr) * 2004-12-01 2006-06-29 Meiji Dairies Corporation Bacteries de l’ acide lactique se liant a des types sanguins abo humains
JP2008212006A (ja) * 2007-02-28 2008-09-18 Meiji Milk Prod Co Ltd 腸管バリア機能の機能回復剤及び腸管バリア透過性の亢進阻害剤
JP2011148703A (ja) * 2008-03-19 2011-08-04 Morishita Jintan Co Ltd 血中リン濃度上昇抑制剤
JP2010259341A (ja) * 2009-04-30 2010-11-18 Hiroshima Univ 細胞表面にケイ酸層を備える微生物、およびその作製方法
JP2012056921A (ja) * 2010-09-13 2012-03-22 Kaneka Corp 生菌製剤
JP2019523219A (ja) * 2016-05-18 2019-08-22 スキンバイオセラピューティクス ピーエルシー 組成物およびその使用
WO2019177171A1 (fr) * 2018-03-16 2019-09-19 味の素株式会社 Additif alimentaire pour animaux et aliment pour animaux

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DE SOUZA DAUREA, GREENE LEWIS: "Intestinal permeability and systemic infections in critically ill patients: Effect og glutamine", CRITICAL CARE MEDICINE, vol. 33, no. 5, 1 January 2005 (2005-01-01), US , pages 1125 - 1135, XP009536752, ISSN: 0090-3493, DOI: 10.1097/01.ccm.0000162680.52397.97 *

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