Formulation for intestinal organic acid enhancement
A yeast-based intestinal organic acid promotion formulation addresses the lack of understanding of yeast's impact on intestinal flora by increasing organic acid production, benefiting the health of non-human mammals.
Patent Information
- Application Number
- JP2023115687
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2023-07-14
- Publication Date
- 2025-01-24
AI Technical Summary
The effects of yeast on intestinal flora and the impact on the living body have not been fully elucidated, particularly for non-human mammals such as livestock, necessitating the development of new preparations using yeast for their production and nurturing.
An intestinal organic acid promotion formulation containing yeast cell wall components, nucleic acids, and optionally ligninulfonic acid, with specific molecular weight ranges and concentrations, is developed for oral administration to promote organic acids in the intestine, particularly in livestock.
The formulation increases the production of organic acids, such as lactic and propionic acid, enhancing intestinal flora and promoting healthy growth in non-human mammals.
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Abstract
Description
[Technical field]
[0001] The present disclosure relates to an intestinal organic acid enhancing preparation, and more particularly to an orally administered preparation used to enhance a specific organic acid in the intestine of a non-human mammal. [Background technology]
[0002] Dietary fiber is not digested by human digestive enzymes and passes through the small intestine to reach the large intestine. Ingested dietary fiber is fermented by the intestinal flora present in the human large intestine, thereby increasing the production of short-chain fatty acids, lowering the pH in the intestine, and promoting the growth of beneficial bacteria (Non-Patent Document 1). These changes are known to contribute to improving the intestinal environment, such as by inhibiting the growth of bacteria that produce putrefactive metabolites (Non-Patent Document 2) and improving intestinal immune function (Non-Patent Document 3).
[0003] In food manufacturing, yeast is used in the production of bread, alcoholic beverages, etc., which require a fermentation process. Yeast is also used in a wide range of fields, such as biofuels, chemicals, and pharmaceuticals, and research on yeast is ongoing (Non-Patent Document 4). The cell wall, which is a component of yeast, is composed of 29 to 64% β-glucan, 31% mannan, 13% protein, 9% lipid, and 1 to 2% chitin (Non-Patent Document 5), and is rich in dietary fiber. The yeast cell wall protects cells from osmotic shock and mechanical stress, and plays an important role in maintaining cell morphology and vital activity (Non-Patent Document 6). In addition, it has been reported that yeast cell walls bind carcinogenic mycotoxins, and that adding yeast cell wall to livestock feed suppresses the impact of mycotoxins remaining in the feed on the health of livestock (Non-Patent Document 7).
[0004] β-glucan is a dietary fiber in which D-glucose is bound by β-glycosidic bonds. Yeast-derived β-glucan has a structure in which a linear β-1,3 bond of 30 residues is mixed with long branches via β-1,6 bonds, and is water-insoluble (Non-Patent Document 8). In a previous study, administration of yeast β-glucan to a high-fat diet-induced obese mouse model improved the intestinal flora, suppressed the increase in proinflammatory cytokines due to obesity, and reduced postprandial blood glucose levels (Non-Patent Document 9). In addition, long-term administration of yeast β-glucan to high-fat diet-induced obese rats changed the intestinal flora and increased short-chain fatty acid production. Furthermore, it was suggested that obesity may have been prevented through changes in the intestinal flora (Non-Patent Document 10). Yeast cell walls also contain mannan, a water-soluble dietary fiber, and yeast mannan has a structure in which a linear α-1,6 mannoside backbone has branches of α-1,2 and α-1,3 mannoside bonds (Non-Patent Document 11). In an in vitro intestinal fermentation study using human feces, yeast mannan showed an increase in short-chain fatty acid production (Non-Patent Document 12).
[0005] Yeast is a rich source of protein and has been attracting attention as an alternative livestock feed in areas where soybean production is difficult (Non-Patent Document 13). Torula yeast (Cyberlindnera jadinii) is sold as a nutritional food for animal feed. In addition to its function as a protein source, it has been reported that torula yeast improves the balance of intestinal flora, improves the digestibility of crude protein at high doses, and heightens the intestinal villi, contributing to nutrient absorption and intestinal health (Non-Patent Document 14).
[0006] Nucleic acids are composed of nucleotides and play important roles in many biological processes. Nucleotides can be synthesized either endogenously or from diet, but endogenous synthesis alone is considered insufficient to perform physiological functions. Nucleotides absorbed from the diet are considered essential nutrients for regulating immune and gastrointestinal functions and optimizing the intestinal microbiota. Previous studies have shown that feeding yeast-derived nucleotides to chickens increases the height of the ileal villi, suggesting that yeast nucleotides are useful for intestinal development (Non-Patent Document 15).
Prior technical literature
Non-licensed literature
[0007] [Non-licensed document 1] Topping, DL & Clifton, PM Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol Rev 81, 1031-1064 (2001).
Non-licensed Document 2
Non-licensed Document 4
Non-licensed Document 5
Non-licensed Document 6
Non-Patent Document 12
Non-Patent Document 13
Non-Patent Document 14
Non-Patent Document 15
[0008] As mentioned above, various studies have been conducted on the effective use of yeast. However, many aspects of the physiological effects on the body and the effects on the intestinal flora of yeast preparations, which are prepared by first extracting yeast nucleic acids from the cell wall and then mixing them with the yeast cell wall, have not yet been fully elucidated.
[0009] In view of the above circumstances, one of the problems to be solved is to provide a new useful preparation using yeast. Another problem to be solved is to provide a new preparation that utilizes yeast and is useful for producing or raising non-human mammals such as livestock. [Means for solving the problem]
[0010] The invention presented in this disclosure can be understood in multiple aspects and may include, for example, the following embodied aspects as a means for solving the problems. Note that in this disclosure, the invention presented in this disclosure is also referred to simply as "the present invention" either comprehensively or according to each aspect.
[0011] [1] An intestinal organic acid enhancing preparation, wherein the organic acid has 2 or 3 carbon atoms, the preparation contains a yeast cell wall component and a nucleic acid having a molecular weight of 5,000 to 100,000, and is orally administered to a non-human mammal. [2] The formulation according to [1] above, wherein the organic acid is lactic acid and / or propionic acid. [3] The formulation according to [1] or [2] above, wherein the yeast cell wall component comprises β-glucan. [4] The preparation according to any one of [1] to [3] above, wherein the content of the yeast cell wall component in the preparation is 3 to 40 wt %. [5] The formulation according to any one of [1] to [4] above, wherein the nucleic acid is a ribonucleic acid derived from yeast. [6] The formulation according to [5] above, wherein the content of the ribonucleic acid in the formulation is 3 to 50% by weight. [7] The preparation according to [5] or [6] above, wherein the yeast-derived ribonucleic acid is attached to the surface of the yeast cell wall component. [8] The formulation according to any one of the above [1] to [7], further comprising lignosulfonic acid. [9] The preparation according to any one of the above-mentioned [1] to [8], wherein the intestine is the cecum.
[10] The formulation according to any one of [1] to [9] above, wherein the non-human mammal is a livestock animal.
[11] The formulation according to any one of [1] to [9] above, wherein the non-human mammal is an animal belonging to the family Bovidae, Suidae, Equidae, Camelidae, Ursidae, Cervidae, Felidae, Canidae, Primates other than humans, Rodentia, Lagomorpha, or Marine Mammals. Effect of the Invention
[0012] In one or more aspects of the invention presented in this disclosure, yeast can be utilized to provide new useful formulations. In one or more aspects of the invention provided in this disclosure, yeast can be utilized to provide new formulations useful in the production or raising of non-human mammals, such as livestock. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0013] Hereinafter, an embodiment of the present invention will be described. In this disclosure, the term "one embodiment" in relation to the present invention refers to any one embodiment in describing the present invention in detail, unless otherwise specified, and does not deny or limit the existence of other or multiple embodiments. As described below, the present invention may have multiple embodiments within its scope. The multiple embodiments may also be provided as modified forms, for example, by various combinations of components (or technical features) shown in this disclosure. In addition, in this disclosure, when the term "embodiment" is simply stated, it includes one or multiple embodiments, unless otherwise specified.
[0014] In this disclosure, unless otherwise specified, the expression "AA-BB" in relation to a numerical range means "AA or more and BB or less" (here, "AA" and "BB" indicate arbitrary numerical values). In addition, the units of the lower limit and upper limit are the same as the units immediately following the latter (i.e., "BB" here) unless otherwise specified. In addition, in this disclosure, the combination of the lower limit and upper limit of the numerical range can be arbitrarily selected from the numerical group of the lower limit or upper limit values exemplified as preferred numerical values, etc. In addition, the expression "X and / or Y" means both X and Y, or either one of them.
[0015] As one embodiment of the present invention, an intestinal organic acid enhancing preparation is provided. In the present disclosure, the intestinal organic acid enhancing preparation refers to a preparation used to enhance organic acid in the intestine. In addition, in the present disclosure, "enhancing organic acid" (or "enhancing organic acid") means increasing the amount of organic acid in some form, and can be embodied in the form of, for example, increasing the amount of organic acid produced, increasing the concentration of organic acid, etc.
[0016] The intestinal organic acid enhancer according to one embodiment of the present invention can be suitably used to enhance organic acids in the cecum, particularly in the intestine.
[0017] The term "organic acid" is used in a broad sense as a general term for organic compounds that exhibit acidity, but in the present disclosure, in a narrow sense, it means a low molecular weight organic compound that has a carboxy group (-COOH) and exhibits acidity. Here, "low molecular weight" means that it is not a so-called "polymer compound", and as a guideline, it means a compound with a molecular weight of approximately 10,000 or less. In a preferred embodiment of the present invention, the organic acid is an organic acid with 2 or 3 carbon atoms, or an organic acid with one carboxy group and 2 or 3 carbon atoms, and specific examples include acetic acid, lactic acid, and propionic acid. A more preferred embodiment of the present invention is a preparation for promoting lactic acid and / or propionic acid.
[0018] In one embodiment of the present invention, the intestinal organic acid enhancing preparation contains a yeast cell wall component and a nucleic acid having a molecular weight of 5,000 to 100,000.
[0019] The nucleic acid may be not only a nucleic acid as a polymer but also a nucleotide, which is a structural unit of a nucleic acid, but a relatively large molecule is preferable. In a preferred embodiment, the nucleic acid may be, for example, a nucleic acid molecule having a weight molecular weight (Mw) in the range of 5,000 to 100,000. More specifically, it is as follows. The lower limit of the weight average molecular weight of the nucleic acid can be preferably 5,000 or more, more preferably 6,000, 7,000, 8,000, or 9,000 or more, and even more preferably 10,000, 15,000, or 20,000 or more. The upper limit of the weight average molecular weight of the nucleic acid can be preferably 100,000 or less, more preferably 80,000 or less, and even more preferably 70,000, 60,0000, or 50,000 or less. The molecular weight distribution of the nucleic acid can be determined, for example, by GPC (gel permeation chromatography).
[0020] The type of sugar constituting the nucleic acid may be either deoxyribose or ribose. That is, the nucleic acid may be either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The types of bases constituting the nucleic acid mainly include adenine, guanine, thymine, cytosine, and uracil, that is, the types of nucleosides constituting the nucleic acid include adenosine, guanosine, cytidine, uridine, and thymidine. The phosphate constituting the nucleotide may be monophosphate or may be composed of multiple phosphates. As the nucleic acid, a commercially available product may be used. As the nucleic acid, one type may be blended alone, or multiple types may be blended in a mixture. As the nucleic acid, ribonucleic acid and nucleotides are preferable, and ribonucleic acid is more preferable.
[0021] The origin of the nucleic acid is not particularly limited, and may be artificially synthesized or may be derived from a natural product. In a preferred embodiment of the present invention, nucleic acid extracted or purified from a microorganism such as yeast may be used. The nucleic acid extracted or purified from the inside of a microorganism such as yeast is exposed to the outside of the body, and is easily absorbed when ingested by a target organism such as an animal that is given an intestinal organic acid enhancing preparation.
[0022] The yeast-derived component may be obtained by obtaining the yeast itself or by culturing the yeast. There is no particular limitation on the culturing of the yeast, and it may be cultured by a general method. In a preferred embodiment of the present invention, for example, microorganisms such as yeast may be grown using waste biological resources, such as wood sugars contained in waste wood. By obtaining nucleic acids from the yeast cultured in this way and using them in a yeast preparation, waste materials can be converted into useful substances, which can contribute to the formation of a sustainable recycling-based society.
[0023] The content of nucleic acid in the intestinal organic acid enhancing formulation may be appropriately adjusted. The content of nucleic acid in the intestinal organic acid enhancing formulation may be preferably 3 to 50% by weight. More specifically, it is as follows. The lower limit of the nucleic acid content in the intestinal organic acid enhancer can be preferably 3% by weight or more, more preferably 5, 6, or 7% by weight or more, and even more preferably 8, 9, or 10% by weight or more. The upper limit of the nucleic acid content in the intestinal organic acid enhancing preparation is preferably 50% by weight or less, and may be appropriately adjusted below this upper limit, for example, 40, 30, 25, or 20% by weight or less.
[0024] A preferred embodiment of the present invention is an intestinal organic acid enhancing preparation containing the nucleic acid having the preferred molecular weight (e.g., 5,000 to 100,000) shown above in the preferred content shown above. In this case, it is not necessary that the intestinal organic acid enhancing preparation does not contain any nucleic acid molecule having a molecular weight other than the preferred molecular weight shown above, but the content is preferably small, specifically, preferably less than the content of the nucleic acid molecule having the preferred molecular weight shown above, and more preferably 1% by weight or less.
[0025] In one embodiment of the present invention, the intestinal organic acid enhancing preparation contains a yeast cell wall component. The term "yeast cell wall component" means a part or the whole of a cell wall derived from yeast, or a fibrous component. The yeast cell wall component may be an enucleated yeast obtained by enucleating nucleic acid from yeast cells, a cell wall that retains the shape of the yeast's outer shell, or a cell wall that has been pulverized to such an extent that the shape of the outer shell is no longer retained. The cell wall component used in the present invention may be a part of the yeast cell wall, but it is preferable that at least the fibrous material remains. By retaining the fibrous material, the mycotoxin adsorption property is excellent. On the other hand, if the decomposition proceeds to such an extent that no fibrous material remains, the mycotoxin adsorption property tends to decrease.
[0026] The content of the yeast cell wall component in the intestinal organic acid enhancing preparation may be appropriately adjusted. The content of the yeast cell wall component in the intestinal organic acid enhancing preparation may be preferably 3 to 40% by weight. More specifically, it is as follows. The lower limit of the content of the yeast cell wall component in the intestinal organic acid enhancing preparation is preferably 3% by weight or more, more preferably 5% by weight or more, and even more preferably 10% by weight or more. The upper limit of the yeast cell wall component content in the intestinal organic acid enhancing preparation is preferably 40% by weight or less, more preferably 35% by weight or less, and even more preferably 30% by weight or less.
[0027] Yeast can be preferably used as a raw material for nucleic acid and yeast cell wall components in the intestinal organic acid enhancing preparation. The type of yeast that can be used may be spore-forming yeast or non-spore-forming yeast. Specific examples of yeast include the following types:
[0028] Examples of spore-forming yeasts include yeasts of the genera Shizosaccharomyces, Saccharomyces, Kluyveromyces, Hansenula, Pichia, Debaryomyces, and Lipomyces, and more specifically, Shizosaccharomyces pombe, Shizosaccharomyces octosporus, Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces rouxii, and Saccharomyces spp. rouxii); Kluyveromyces fragilis, Kluyveromyces lactis; Hansenula anomala; Pichia membranaefaciens; Debaryomyces hansenii; and Lipomyces starkeyi.
[0029] Examples of asporogenous yeasts include yeasts of the genera Torulopsis, Candida, and Rhodotorula, more specifically, Torulopsis versatilis, Candida tropicalis, Candida lipolytica, Candida utilis, and Rhodotorula glutinis. In addition, yeasts of the genus Candida are also called torula yeasts in terms of taxonomy, and are sometimes classified as yeasts of the genus Cyberlindnera.
[0030] Preferred examples of yeast that can be used include brewer's yeast, wine yeast, baker's yeast, and torula yeast, and more specifically, examples of yeast that can be used include Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces rouxii, Kluyveromyces fragilis, Torulopsis versatilis, Candida tropicalis, Candida lipolytica, Candida utilis, and Rhodotorula glutinis. In addition, Candida utilis is sometimes classified as a species of torula yeast (Cyberlindnera jadinii) in taxonomic terms.
[0031] In another preferred embodiment of the present invention, in addition to the nucleic acid and the yeast cell wall component, lignosulfonic acid can be further added to the intestinal organic acid enhancing preparation. By adding lignosulfonic acid, spoilage of the intestinal organic acid enhancing preparation made from yeast can be suppressed.
[0032] Lignosulfonic acid is a compound having a skeleton in which the carbon at the α-position of the side chain of the hydroxyphenylpropane structure of lignin is cleaved to introduce a sulfo group. Lignosulfonic acid may be in the form of a salt. In this disclosure, unless otherwise specified, the term "lignosulfonic acid" may include the form of a lignosulfonate. In order to add lignosulfonic acid to an intestinal organic acid enhancing formulation, a lignosulfonate may be added. Examples of lignosulfonate include calcium salts, magnesium salts, sodium salts, calcium-sodium mixed salts, ammonium salts, and organic ammonium salts. Lignosulfonic acid can be obtained, for example, from sulfite pulp waste liquor generated in the papermaking industry. In addition, the lignosulfonic acid used in the present invention may be lignosulfonic acid modified with a polyelectrolyte having functional groups such as a sulfonic group, a carboxyl group, and a phenolic hydroxyl group.
[0033] When lignosulfonic acid is added, the content of lignosulfonic acid in the intestinal organic acid enhancing formulation may be appropriately adjusted. The content of lignosulfonic acid in the intestinal organic acid enhancing formulation may be preferably 1 to 50% by weight. More specifically, it is as follows. The lower limit of the content of lignosulfonic acid in the intestinal organic acid enhancing preparation is preferably 1% by weight or more, more preferably 3% by weight or more, and even more preferably 5% by weight or more. The upper limit of the lignin sulfonic acid content in the intestinal organic acid enhancing preparation is preferably 50% by weight or less, more preferably 40% by weight or less, and even more preferably 30, 20, or 10% by weight or less.
[0034] In another preferred embodiment of the intestinal organic acid enhancing preparation, a sulfite may be further added. The incorporation of a sulfite can contribute to the inhibition of oxidation or the inhibition of the proliferation of unwanted bacteria.
[0035] Furthermore, the intestinal organic acid enhancing preparation may contain other optional ingredients, such as moisture, oils, pH adjusters, antioxidants, preservatives, coloring materials, fragrances, excipients, vitamins, hormones, amino acids, antibiotics, and antibacterial agents, as necessary.
[0036] The intestinal organic acid enhancing preparation is preferably in a form that can be administered orally, and may be in a form generally adopted as a feed or feed additive. Examples of the form of the intestinal organic acid enhancing preparation include powder, granule, mash, pellet, crumble, and flake. The form of the intestinal organic acid enhancing preparation may be a single form, or a mixture of two or more of the above forms, such as a mixture of pellet and flake, or a mixture of mash and pellet.
[0037] The preparation of the intestinal organic acid enhancing preparation according to one embodiment of the present invention can be prepared by mixing nucleic acid, yeast cell wall components, and other optional components such as lignin sulfonic acid, and each component may be mixed one by one, or each component may be added and mixed at the same time. The nucleic acid, yeast cell wall components, and lignin sulfonic acid are as described above. Each component may be prepared individually, or yeast may be cultured and the nucleic acid and yeast cell wall components may be prepared using the yeast as a raw material.
[0038] As one embodiment of the present invention, for example, yeast may be subjected to a nucleation treatment to separate nucleic acid and enucleated yeast, which are then recovered, and nucleic acid having a molecular weight of 5,000 to 100,000 and yeast cell wall components may be prepared separately. The thus-prepared nucleic acid and yeast cell wall components may be mixed to obtain an intestinal organic acid enhancing preparation.
[0039] The nucleation treatment can be carried out, for example, by contacting the yeast with an alkaline chemical, saline solution, cell wall-dissolving enzyme, etc., to dissolve the yeast cell wall, elute the yeast contents into a medium, and separate the cell wall components from other components. The separated components may be purified and powdered as necessary. In one embodiment of the present invention, it is more preferable to separate the nucleic acid and the yeast cell wall components and then recombine them in terms of enhancing the effect of immune activation.
[0040] The alkaline treatment for the nucleation treatment may be carried out by a method generally used for obtaining yeast extracts. That is, an alkaline agent may be used to dissolve or destroy part or all of the yeast cell wall, so that the components within the yeast cell are eluted. Preferred alkaline agents include, for example, sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, and sodium carbonate.
[0041] In one embodiment of the present invention, the nucleic acid extracted from yeast may be attached to the surface of the yeast cell wall component to prepare a preparation. In a preferred embodiment, the yeast cell wall component may be the yeast shell (i.e., the shell composed of the yeast cell wall) remaining after dissolving a part of the yeast cell wall to extract the nucleic acid from within the yeast cell body. The cell wall shell remaining after the nucleic acid is extracted is used as a carrier for the nucleic acid, resulting in a yeast preparation with excellent nucleic acid absorption and easy handling as a preparation.
[0042] In the present disclosure, the term "non-human mammal" refers to a mammal other than humans. Examples of non-human mammals include Bovidae, Suidae, Equidae, Camelidae, Ursidae, Cervidae, Felidae, Canidae, Primates other than humans, Rodentia, Lagomorpha, and marine mammals. In an embodiment of the present invention, the subject of the non-human mammal is preferably, for example, livestock, more preferably, cattle, sheep, goats, and pigs. The intestinal organic acid-enhancing preparation according to one embodiment of the present invention can contribute to the healthy growth of livestock, and therefore can be suitably used in livestock production.
[0043] The intestinal organic acid enhancing preparation may be (1) added to a basic feed and fed as a mixed feed, or (2) the intestinal organic acid enhancing preparation and other feed ingredients may not be directly mixed but may be prepared separately, and the intestinal organic acid enhancing preparation and the basic feed may be fed separately and used in combination. In this disclosure, when the term "combined use" is used without any particular limitation, it is used as a term that includes both of the above (1) and (2) forms.
[0044] In the present disclosure, the term "basal feed" refers to a feed that is fed to provide major nutritional components to non-human mammals. Ingredients of the basic feed that contain major nutritional components for non-human mammals include vegetable feed and / or animal feed. The vegetable feed is a feed derived from plants, such as corn, milo, barley, wheat, cassava, rice bran, bran, soybean meal, rapeseed meal, rice, rice bran, and beet, as well as processed products thereof. The animal feed is a feed derived from animals, such as fish meal, pork meal, chicken meal, skim milk powder, and concentrated whey. These may be used alone or in combination of two or more.
[0045] As described above, in a preferred embodiment of the present invention, the intestinal organic acid enhancer and basic feed may be fed in combination to the target non-human mammal. Although it depends on various conditions such as the type and growth stage of the target non-human mammal, when used in combination with basic feed, the intestinal organic acid enhancer may be fed at a ratio of preferably 0.4 to 20.0% by weight based on the total amount of the intestinal organic acid enhancer and basic feed. More specifically, it is as follows. The lower limit of the intestinal organic acid enhancing preparation relative to the total amount of the intestinal organic acid enhancing preparation and the basic feed is preferably 0.4 or 0.5% by weight or more, more preferably 0.8, 1.0, 1.5, 1.8, or 2.0% by weight or more, and even more preferably 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0% by weight or more. By setting the amount to be equal to or more than the above lower limit, it is easy to enhance a specific organic acid in the intestine. The upper limit of the amount of the intestinal organic acid enhancing preparation relative to the total amount of the intestinal organic acid enhancing preparation and the basic feed can be arbitrary, but from the standpoint of balance with other nutritional components, it is preferably 20.0 wt% or less, more preferably 15 wt% or less, and even more preferably 10 wt% or more. EXAMPLES
[0046] The present invention will be described in more detail below with reference to examples. However, the technical scope (or technical reach) of the invention presented in this disclosure is not limited to the following examples.
[0047] 1. Sample Torulaplus (registered trademark, manufactured by Nippon Paper Industries Co., Ltd.) was used as the yeast preparation to be used as a sample. Torulaplus contains ribonucleic acid with a weight-average molecular weight (Mw) of approximately 20,000, enucleated yeast, and lignosulfonic acid. Enucleated yeast is yeast from which the cellular contents have been removed, and is composed almost entirely of cell walls. In the dried Torulaplus, ribonucleic acid is attached to the outer shell of the enucleated yeast. The content of each component in Torulaplus is approximately 8% ribonucleic acid, approximately 10% cell wall product derived from enucleated yeast, and approximately 15% lignosulfonic acid or its salt.
[0048] The moisture content (normal pressure heat drying method), crude protein (combustion method), crude fat (Soxhlet extraction method), crude ash (direct ashing method), and dietary fiber (Prosky method) of Torula Plus were measured by the Japan Food Research Laboratories. RNA was measured using the simplified STS method, and β-glucan was measured using a β-glucan analysis kit (yeast mushroom type; Megazyme, Wicklow, Ireland). The measurement results are shown in Table 1.
[0049] [Table 1]
[0050] 2. Animal experiments and test diets Animal experiments were performed with the approval of the Obihiro University of Agriculture and Veterinary Medicine Animal Experiment Committee (approval number: No. 22-159). Thirty-six male Fischer 344 rats (7 weeks old, 130-160 g) were purchased from Charles River Japan. All rats were kept in individual plastic cages at room temperature of 23 ± 1°C, humidity of 60 ± 5%, and a 12-h light-dark cycle. Rats were treated in accordance with the “Guide for the Care and Use of Laboratory Animals.” After a 7-day acclimation period, rats were randomly divided into six groups (six rats per group) and fed the test diet conforming to AIN-93G (manufactured by Oriental Yeast Co., Ltd.) for four weeks. The test diets were prepared by adding 0.2% (T0.2), 0.5% (T0.5), 1% (T1), 2% (T2), and 5% (T5) of Torula Plus to the AIN-93G-based diet as the control (CON) diet. The amounts of cellulose and casein in the test diets were adjusted according to the amounts of dietary fiber and protein in Torula Plus. The composition of the test diets is shown in Table 2.
[0051] [Table 2]
[0052] During the study period, rats had free access to food and water, and food intake was measured daily and body weight was measured once a week. Feces were collected for the last two days of the study, freeze-dried, and weighed. On the final day of the study, rats were euthanized under isoflurane inhalation anesthesia, and the liver, fat, and cecum were removed and weighed. A portion of the cecal contents was stored at -80°C until bacterial DNA extraction. A suspension was prepared by adding distilled water to the cecal contents (1 g contents + 5 g distilled water), and the pH was measured. The remaining suspension was stored at -30°C until other analyses.
[0053] 3. Cecal microbiota analysis Cecal bacterial DNA was extracted from cecal contents by the "phenol-free repeated beads beating plus column method" (Yu, Z. & Morrison, M. Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques 36, 808-812 (2004).) and purified with the Maxwell® RSC Blood DNA kit (Promega, Madison, WI, USA). DNA concentration and purity were measured using a NanoDrop 2000c (Thermo Fischer Scientific).
[0054] Paired-end sequencing was performed using the Illumina MiSeq system (Illumina, San Diego, CA, USA). 16S rRNA gene amplicons (V3-V4 regions) were prepared for the MiSeq system (Illumina) according to the 16S Metagenomic Sequencing Library Preparation Guide (Part#15044223Rev.B).
[0055] 16S rRNA gene sequence data analysis was performed using QIIME2 ver. 2022.08 according to the method of Tamura et al. (Tamura, M. et al. Effects of a high-γ-polyglutamic acid-containing natto diet on liver lipids and cecal microbiota of adult female mice. Biosci Microbiota Food Health 40, 176-185 (2021).). Bacterial classification was performed using the Silva database. α and β diversity were analyzed standardized by the number of sequences, 8 and 168 (not shown). β diversity was calculated by the “weighted UniFrac distance metric”, and principal coordinate analysis plots were created using R software (version 4.1.1; R Core Teams 2021, Vienna, Austria). The occupancy of bacterial taxa at the phylum, family, and genus levels in the cecal microbiota of rats is shown in Table 3.
[0056] 4. Cecal organic acid analysis The organic acid concentration in the rat cecum was measured using HPLC (LC-10AD, Shimadzu Corporation). HPLC samples were prepared with some modifications based on a previous method (Han, KH et al. Spent turmeric reduces fat mass in rats fed a high-fat diet. Food Funct 7, 1814-1824 (2016)). 50 μL of 5% perchloric acid solution was added to 450 μL of the supernatant of the cecal content suspension and stirred. The supernatant was collected by centrifugation (12,000 g, 4°C, 10 minutes) and passed through a cellulose acetate membrane filter (0.45 μm, Toyo Roshi Co., Ltd.) to prepare the HPLC sample. The analytical conditions were as previously reported (Nagata, R. et al. Chemical modification of cornstarch by hydroxypropylation enhances cecal fermentation-mediated lipid metabolism in rats. Starch / St▲a▼rke 72, 1900050 (2020).). Column: RSpakKC-811 (8.0 mm x 300 mm, Shodex) Column temperature: 47°C, Mobile phase: 2 mM perchloric acid aqueous solution 1.0 mL / min Reaction reagent: ST3-R (Shodex) 0.5mL / min Measurement wavelength 430nm
[0057] The results of measuring the organic acid concentrations in the rat cecum are shown in Table 4.
[0058] 5.Statistical analysis Data are expressed as mean ± SEM. Significant differences among the six groups were compared with the CON group using one-way analysis of variance followed by Dunnett's multiple test (SPSS version 17, SPSS Inc., Chicago, IL, USA). Correlations between data were evaluated by Pearson's correlation analysis. In all statistical analyses, p<0.05 was set as the statistical significance level.
[0059] [Table 3]
[0060] [Table 4]
[0061] As shown in Table 3, ingestion of Torulaplus increased the number of bacteria in the cecal flora, including the Oscillospiraceae UCG-005 genus and the Lachnospiraceae NK4A136 genus.
[0062] As shown in Table 4, in the organic acids in the cecum, intake of Torulaplus was confirmed to increase the concentrations of lactic acid, propionic acid, and total short-chain fatty acids. In addition, although no significant difference was observed, the T5 group also showed a high acetate concentration. The bacteria of the Oscillospiraceae UCG-005 and Lachnospiraceae NK4A136 genera, which were particularly increased by intake of Torulaplus, are considered to be short-chain fatty acid producing bacteria. In this disclosure, Pearson's correlation analysis showed that the Oscillospiraceae UCG-005 genus showed a significant positive correlation with acetic acid (r=0.47, p<0.01) and total short-chain fatty acids (r=0.43, p<0.01), and the Lachnospiraceae NK4A136 genus showed a positive correlation with lactic acid (r=0.37, p<0.05), propionic acid (r=0.35, p<0.05), n-butyric acid (r=0.48, p<0.01) and total short-chain fatty acids (r=0.30, p=0.073). Therefore, it is possible that the Oscillospiraceae UCG-005 and Lachnospiraceae NK4A136 genera contributed greatly to organic acid production in this disclosure.
Claims
1. A preparation for increasing an intestinal organic acid, the organic acid having 2 or 3 carbon atoms, the preparation containing a yeast cell wall component and a nucleic acid having a molecular weight of 5,000 to 100,000, and the preparation being orally administered to a non-human mammal.
2. The formulation of claim 1 , wherein the organic acid is lactic acid and / or propionic acid.
3. The formulation of claim 1 , wherein the yeast cell wall component comprises β-glucan.
4. The preparation according to claim 1, wherein the content of the yeast cell wall component in the preparation is 3 to 40% by weight.
5. The formulation of claim 1 , wherein the nucleic acid is a ribonucleic acid derived from yeast.
6. The preparation according to claim 5, wherein the content of said ribonucleic acid in said preparation is 3 to 50% by weight.
7. The preparation according to claim 5 , wherein the yeast-derived ribonucleic acid is attached to the surface of the yeast cell wall component.
8. 10. The formulation of claim 1 further comprising lignosulfonic acid.
9. The formulation of claim 1 , wherein the intestine is the cecum.
10. The formulation according to claim 1 , wherein the non-human mammal is a livestock animal.
11. 2. The formulation of claim 1, wherein the non-human mammal is an animal belonging to the family Bovidae, Suidae, Equidae, Camelidae, Ursidae, Cervidae, Felidae, Canidae, non-human Primates, Rodentia, Lagomorpha, or Marine Mammals.