Agent for reducing concentration of protein in blood

Abstract

The present invention provides: an agent for reducing the concentration of a protein in blood, the agent containing Pediococcus acidilactici; and a food, a feed, and a medicine each of which is for reducing the concentration of a protein in blood and contains the agent for reducing the concentration of a protein in blood or Pediococcus acidilactici.

Description

Blood protein concentration reducer

[0001] The present invention relates to an agent for reducing blood protein concentration.

[0002] Proteins are important molecules that play a vital role in biological functions. However, excessive protein translation is known to induce intracellular stress and cause a decline in protein quality due to protein misfolding, which may accelerate cellular aging. However, there are still limited reports on methods to suppress protein translation.

[0003] Overtranslated proteins can increase blood protein concentrations (total protein concentrations) and lead to hyperproteinemia. For example, in multiple myeloma, specific plasma cells in the bone marrow become malignant and abnormally proliferate, resulting in the unlimited production of large amounts of monoclonal immunoglobulins called M proteins. As a result, hyperproteinemia occurs when blood globulin and protein concentrations increase abnormally. Hyperproteinemia due to increased blood globulin and protein concentrations is often observed in patients with autoimmune diseases, chronic infectious diseases, and other diseases, as well as in elderly individuals. Non-Patent Document 1 reports that blood protein and globulin concentrations increase with age in dogs.

[0004] Hyperproteinemia increases blood viscosity and can cause hyperviscosity syndrome, which is accompanied by symptoms such as dizziness, headache, and dehydration. It is also known that hyperproteinemia can cause organ damage, such as kidney damage, due to a large amount of immunoglobulin in the blood. Therefore, it is undesirable for blood protein concentrations to increase above the normal range. It is believed that blood protein concentrations can be reduced to treat or prevent hyperproteinemia, for example, by inhibiting protein translation.

[0005] However, protein translation inhibition may have harmful side effects. Blood proteins are primarily composed of albumin and globulin. Blood albumin concentration is an indicator of overall nutritional status, and a decrease in blood albumin concentration indicates a deterioration in nutritional status. When reducing blood protein concentration through protein translation inhibition, a decrease in blood albumin concentration can lead to nutritional deficiencies and weight loss, which is particularly undesirable in patients with diseases or elderly individuals. Therefore, it is desirable to develop a method that can reduce blood protein concentration without causing a decrease in blood albumin concentration or weight loss.

[0006] Lactic acid bacteria are known to have various health-beneficial functions. Patent Document 1 discloses that administration of the lactic acid bacteria Pediococcus acidilactici strain improves mitochondrial function and has the effect of extending lifespan.

[0007] Non-Patent Document 2 discloses that administration of lactic acid bacteria Pediococcus spp. increased serum protein concentration, serum albumin concentration, and serum globulin concentration in growing sheep, and improved nutrient digestibility.

[0008] However, there have been no reports yet on the reduction of blood protein and blood globulin concentrations by administering lactic acid bacteria.

[0009] International Publication No. WO2020 / 067368

[0010] Chang YM, et al., (2016) PLoS ONE 11(2): e0149650El-Katcha MI, et al., (2016) Alexandria Journal of Veterinary Sciences, 49(1): 44-54

[0011] An objective of the present invention is to provide an agent for reducing blood protein concentration.

[0012] As a result of extensive research to solve the above problems, the present inventors discovered that administration of a Pediococcus acidilactici culture can reduce blood protein concentrations in animals, and that in doing so, it is possible to avoid a decrease in blood albumin concentrations and suppress protein translation, thereby completing the present invention.

[0013] That is, the present invention includes the following embodiments.

[0014] [1] A blood protein concentration reducer comprising Pediococcus acidilactici.

[0015] [2] The blood protein concentration-reducing agent according to [1] above, wherein the reduction in blood protein concentration is accompanied by an increase in the albumin / globulin ratio.

[0016] [3] The blood protein concentration reducer according to [1] or [2] above, wherein the Pediococcus acidilactici is Pediococcus acidilactici OB7260 strain having accession number NITE BP-02634.

[0017] [4] The blood protein concentration-reducing agent according to any one of [1] to [3] above, wherein the reduction of blood protein concentration is accompanied by protein translation inhibition.

[0018] [5] The blood protein concentration-reducing agent according to [4] above, wherein the protein translation inhibition is due to decreased expression of a ribosomal subunit gene.

[0019] [6] The blood protein concentration-reducing agent according to any one of [1] to [5] above, which does not have a weight-reducing effect.

[0020] [7] A food or feed for reducing blood protein concentration, comprising Pediococcus acidilactici or the blood protein concentration reducer according to any one of [1] to [6] above.

[0021] [8] A pharmaceutical for reducing blood protein concentration, comprising Pediococcus acidilactici or the blood protein concentration reducer according to any one of [1] to [6] above.

[0022] [9] The food or feed described in [7] above, wherein the reduction in blood protein concentration is accompanied by an increase in the albumin / globulin ratio.

[0023]

[10] The pharmaceutical according to the above [8], wherein the reduction in blood protein concentration is accompanied by an increase in the albumin / globulin ratio.

[0024]

[11] The food or feed according to [7] above, wherein the Pediococcus acidilactici is the Pediococcus acidilactici OB7260 strain having the accession number NITE BP-02634.

[0025]

[12] The pharmaceutical according to the above-mentioned [8], wherein the Pediococcus acidilactici is Pediococcus acidilactici strain OB7260 having accession number NITE BP-02634.

[0026] The present invention also encompasses the following embodiments:

[13] A method for reducing a blood protein concentration in a subject, comprising administering to the subject the blood protein concentration-reducing agent according to any of [1] to [6] above, Pediococcus acidilactici, or a food, feed, or pharmaceutical product containing the blood protein concentration-reducing agent or Pediococcus acidilactici.

[0027]

[14] The method according to

[13] above, wherein the Pediococcus acidilactici is the Pediococcus acidilactici OB7260 strain having the accession number NITE BP-02634.

[0028]

[15] Use of Pediococcus acidilactici in the manufacture of a blood protein concentration lowering agent, or a food, feed, or pharmaceutical for lowering blood protein concentration.

[0029]

[16] The use according to the above-mentioned

[15] , wherein the Pediococcus acidilactici is the Pediococcus acidilactici strain OB7260 having the accession number NITE BP-02634.

[0030] This specification includes the disclosure of Japanese Patent Application No. 2024-032560, from which the present application claims priority.

[0031] According to the present invention, it is possible to bring about a reduction in blood protein concentrations in animals while avoiding a decrease in blood albumin concentrations.

[0032] Figure 1 shows the effect of a lactic acid bacteria preparation on plasma protein concentrations in dogs. The vertical axis represents the change in plasma protein concentration (g / dL) at each time point after administration compared to immediately before administration. 2m: 2 months after administration (at the end of the 2-month administration period), 3.5m: 3.5 months after administration (1.5 months after the end of the administration period), 8m: 8 months after administration (6 months after the end of the administration period) (same for the following figures). Statistical analysis was performed using the Mann-Whitney test. ＊＊ : p < 0.01, ＃ : p<0.1. Figure 2 shows the effect of the lactic acid bacteria preparation on plasma globulin concentrations in dogs. The vertical axis represents the change in plasma globulin concentration (g / dL) at each time point after the start of administration compared to just before the start of administration. Statistical analysis was performed using the Mann-Whitney test. ＊＊ : p<0.01. Figure 3 shows the effect of a lactic acid bacteria preparation on plasma albumin concentrations in dogs. The vertical axis represents the change in plasma albumin concentration (g / dL) at each time point after the start of administration of the lactic acid bacteria preparation compared to immediately before the start of administration. Statistical analysis was performed using the Mann-Whitney test. ns: p≧0.1. Figure 4 shows the effect of a lactic acid bacteria preparation on the A / G ratio in dogs. The vertical axis represents the change in A / G ratio at each time point after the start of administration of the lactic acid bacteria preparation compared to immediately before the start of administration. Statistical analysis was performed using the Mann-Whitney test. ＊: p<0.05. Figure 5 shows the effect of a lactic acid bacteria preparation on dog body weight. The vertical axis represents the change in body weight (kg) at each time point after the start of administration compared to immediately before the start of administration of the lactic acid bacteria preparation. Statistical analysis was performed using the Mann-Whitney test. ns: p≧0.1. Figure 6 shows the changes in gene expression levels in the active group (administered with a lactic acid bacteria preparation) compared to the placebo group for each gene (vertical axis) belonging to the gene group of the large ribosomal subunit (RPL), as shown by transcriptome analysis in dogs. Statistical analysis was performed using the Wald test. ＊＊ : adjusted p<0.05, ＊ : adjusted p<0.1, ＃ : p (unadjusted) < 0.05. Figure 7 shows the changes in gene expression levels of each gene (vertical axis) belonging to the small ribosomal subunit (RPS) gene group in the active group (administered a lactic acid bacteria preparation) compared to the placebo group, as shown by transcriptome analysis in dogs. Statistical analysis was performed using the Wald test. ＊＊ : adjusted p<0.05, ＃ p (unadjusted) <0.05. Figure 8 shows the changes in gene expression levels of each gene (vertical axis) belonging to the mitochondrial ribosomal large subunit (MRPL) gene group in the active group (administered a lactic acid bacteria preparation) compared to the placebo group, as shown by transcriptome analysis in dogs. Statistical analysis was performed using the Wald test. ＊＊ : adjusted p<0.05, ＊ : adjusted p<0.1, ＃ : p (unadjusted) < 0.05. Figure 9 shows the change in gene expression levels of each gene (vertical axis) belonging to the mitochondrial small ribosomal subunit (MRPS) gene group in the active group (administered a lactic acid bacteria preparation) compared to the placebo group, as shown by transcriptome analysis in dogs. Statistical analysis was performed using the Wald test. ＊＊ : adjusted p<0.05, ＃p (unadjusted)<0.05. Figure 10 shows the change in gene expression levels of each gene (vertical axis) belonging to the gene group of the large ribosomal subunit (RPL) in the lactic acid bacteria-treated group compared to the medium-treated group, as revealed by transcriptome analysis in mice. Statistical analysis was performed using the Wald test. ＊ : adjusted p<0.1, ＃ p (unadjusted)<0.05. Figure 11 shows the change in gene expression levels of each gene (vertical axis) belonging to the small ribosomal subunit (RPS) gene group in the lactic acid bacteria-treated group compared to the medium-treated group, as determined by transcriptome analysis in mice. Statistical analysis was performed using the Wald test. ＊ : adjusted p<0.1, ＃ p (unadjusted)<0.05. Figure 12 shows the change in gene expression levels of each gene (vertical axis) belonging to the mitochondrial large ribosomal subunit (MRPL) gene group in the lactic acid bacteria-treated group compared to the medium-treated group, as revealed by transcriptome analysis in mice. Statistical analysis was performed using the Wald test. ＃ p (unadjusted)<0.05. Figure 13 shows the change in gene expression levels of each gene (vertical axis) belonging to the mitochondrial small ribosomal subunit (MRPS) gene group in the lactic acid bacteria-treated group compared to the medium-treated group, as determined by transcriptome analysis in mice. Statistical analysis was performed using the Wald test. ＃ : p (unadjusted) < 0.05. Figure 14 shows the change in mouse body weight over time during the lactic acid bacteria administration period. The vertical axis represents the mouse body weight (g), and the horizontal axis represents the number of weeks after the start of lactic acid bacteria administration.

[0033] The present invention is described in detail below. The present invention is based on the new finding that administration of a lactic acid bacteria culture, particularly a Pediococcus acidilactici culture, has the effect of reducing blood protein levels. The present invention relates to the use of the lactic acid bacteria Pediococcus acidilactici for reducing blood protein levels. The present invention relates to a formulation or composition containing Pediococcus acidilactici.

[0034] The Pediococcus acidilactici used in the present invention is not limited to a specific strain, but a preferred example is the Pediococcus acidilactici OB7260 strain. The Pediococcus acidilactici OB7260 strain was deposited on February 14, 2018, at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) under the Budapest Treaty under accession number NITE BP-02634. The Pediococcus acidilactici used in the present invention may be a live or dead cell. The Pediococcus acidilactici used in the present invention may be in any form of cells, such as a culture (for example, a culture solution), diluted cells, concentrated cells, heated cells, dried cells, or freeze-dried cells.

[0035] The present invention relates to an agent for reducing blood protein concentration, comprising Pediococcus acidilactici. In one embodiment, the present invention relates to an agent for reducing blood protein concentration, comprising the Pediococcus acidilactici strain OB7260.

[0036] In one embodiment, a culture (e.g., culture broth) of Pediococcus acidilactici or a processed product thereof (including but not limited to, for example, a culture supernatant, a diluted product, a concentrated product, a heated product, a dried product, a freeze-dried product, or any combination thereof) may be used for reducing blood protein concentrations in the present invention. The present invention also provides a blood protein concentration-reducing agent comprising a culture (e.g., culture broth) or a processed product thereof (including but not limited to, for example, a culture supernatant, a diluted product, a concentrated product, a heated product, a dried product, a freeze-dried product, or any combination thereof) of Pediococcus acidilactici, for example, Pediococcus acidilactici strain OB7260.

[0037] The blood protein concentration-reducing agent according to the present invention may be a composition. In one embodiment, the blood protein concentration-reducing agent according to the present invention may be a composition containing Pediococcus acidilactici. The blood protein concentration-reducing agent according to the present invention may contain Pediococcus acidilactici as an active ingredient. The blood protein concentration-reducing agent according to the present invention may contain an effective amount of Pediococcus acidilactici cells. The blood protein concentration-reducing agent according to the present invention may also contain other components derived from a Pediococcus acidilactici culture (e.g., a culture solution), such as water, a medium, medium-derived components, and cell-derived components (exopolysaccharides (EPS), metabolites, etc.). The blood protein concentration-reducing agent according to the present invention may further contain additives that are acceptable for incorporation in the food, feed, or pharmaceutical fields, such as, but not limited to, carriers, excipients, stabilizers, preservatives, antioxidants, anti-adherents, disintegrants, binders, humectants, thickeners, buffers, surfactants, suspending agents, solubilizers, emulsifiers, coating agents, flavorings, fragrances, colorants, etc. The blood protein concentration-reducing agent according to the present invention may be in any form, such as a liquid, semi-solid, solid (powder, granules, tablet, etc.), or encapsulated form.

[0038] The blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention can be used to reduce blood protein concentrations in animals to which it is administered. Blood protein concentration refers to the total protein concentration in blood and may also be simply referred to as "total protein concentration." Blood protein concentration may be plasma protein concentration, serum protein concentration, or whole blood protein concentration.

[0039] In the present invention, "reduction in blood protein level" means a reduction in blood protein level in a subject animal after administration of the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention. The reduction in blood protein level in the present invention refers to a reduction in blood protein level compared to a control subject not administered the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention. The blood protein concentration in a subject animal after administration of the blood protein concentration-lowering agent or Pediococcus acidilactici according to the present invention at least one time point (for example, at least one time point between the start of administration and 6 months after completion of administration, typically 2 months after completion of administration) is reduced (preferably statistically significantly) compared to the blood protein concentration before administration (control), or the amount of change in the blood protein concentration compared to the blood protein concentration before administration (a reduction is represented by a positive (+) numerical value, and an increase is represented by a negative (-) numerical value) is indicative of the blood protein concentration-lowering agent or Pediococcus acidilactici according to the present invention. When the change in blood protein concentration at the same time point in a subject animal (control) raised under the same conditions without administration of Diococcus acidilactici is statistically significantly greater than the blood protein concentration before the start of the test (the time point corresponding to the time before administration) (a reduction is represented by a positive (+) value, and an increase is represented by a negative (-) value), it can be determined that the blood protein concentration-lowering agent or Pediococcus acidilactici has a blood protein concentration-lowering effect (which can also be referred to as an effect of suppressing an increase in blood protein concentration) compared to control subjects not administered the agent. The blood protein concentration-lowering effect of administering the blood protein concentration-lowering agent or Pediococcus acidilactici according to the present invention can be determined, for example, but not limited to, from blood samples taken at the end of a two-month administration period, 1.5 months, and / or 6 months after the administration period. Reduction of blood protein concentration is expected to lead to, for example, treatment or prevention of hyperproteinemia, prevention of increased blood viscosity and resulting organ damage, as well as suppression of accelerated cellular aging.

[0040] In a preferred embodiment, the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention not only reduces blood protein concentrations, but also increases the albumin / globulin ratio (A / G ratio) in a subject animal after administration. The blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention can reduce blood protein concentrations accompanied by an increase in the albumin / globulin ratio (A / G ratio). The increase in the albumin / globulin ratio in the present invention refers to an increase in the albumin / globulin ratio compared to a control subject not administered the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention. The albumin / globulin ratio in a subject animal after administration of the blood protein concentration-reducing agent according to the present invention or Pediococcus acidilactici at at least one time point (for example, at least one time point between the start of administration and 6 months after completion of administration, typically 2 months after completion of administration) is increased (preferably statistically significantly) compared to the albumin / globulin ratio before administration (control), or the amount of change (an increase is represented by a positive (+) value, and a decrease is represented by a negative (-) value) in the albumin / globulin ratio before administration is indicative of the blood protein concentration-reducing agent according to the present invention or Pediococcus acidilactici. If the change in the albumin / globulin ratio at the same time point in a subject animal (control) raised under the same conditions without administering Pediococcus acidilactici is statistically significantly greater than the albumin / globulin ratio before the start of the test (the time point corresponding to the time before administration as described above) (an increase is represented by a positive (+) value, and a decrease is represented by a negative (-) value), it can be determined that the blood protein concentration-reducing agent or Pediococcus acidilactici brings about an increase in the albumin / globulin ratio (which can also be referred to as suppression of a decrease in the albumin / globulin ratio) compared to control subjects not administered the agent.

[0041] Blood proteins are primarily composed of albumin and globulin. For example, plasma proteins contain a wide variety of proteins, of which approximately 60% is albumin and approximately 40% is globulin. Blood albumin concentration is an indicator of overall nutritional status, and a decrease in blood albumin concentration indicates a deterioration in nutritional status. The majority of blood globulins are various immunoglobulins. Generally, blood globulin concentration is calculated as the value (approximate value) obtained by subtracting blood albumin concentration from blood protein concentration. The albumin / globulin ratio is calculated as the ratio of blood albumin concentration to blood globulin concentration and is used as one of the indicators of health abnormalities in blood biochemistry tests. A decrease in the albumin / globulin ratio occurs due to a decrease in albumin and / or an increase in globulin. A low albumin / globulin ratio is typically observed in liver disease, kidney disease, infection, nutritional deficiency, cachexia, malignant tumors, multiple myeloma, autoimmune diseases, etc. Conversely, an increase in the albumin / globulin ratio occurs mainly due to a decrease in globulin, and is observed in hypogammaglobulinemia or agammaglobulinemia, etc. Therefore, the ability of the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention to increase the albumin / globulin ratio means that it can bring about a reduction in blood globulin concentration without causing a decrease in blood albumin concentration. Such an increase in the albumin / globulin ratio based on maintaining blood albumin concentration and reducing blood globulin concentration is extremely useful for health, as it can reduce blood protein concentration while maintaining nutritional status (without causing a deterioration in nutritional status).

[0042] In a preferred embodiment, the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention does not have a weight-reducing effect. In the present invention, a change in body weight, such as a weight loss, refers to a change in body weight, such as a weight loss, compared to a control subject not administered with the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention. If the body weight of a subject animal after administration of the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention is not reduced (preferably statistically significantly) compared to the body weight before administration (control), or if the change in body weight compared to the body weight before administration does not show a statistically significant difference compared to the change in body weight of a subject animal (control) raised under the same conditions except for not being administered the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention, it can be determined that the blood protein concentration-reducing agent or Pediococcus acidilactici does not have a weight-reducing effect compared to a control subject not administered the agent or Pediococcus acidilactici. The fact that the blood protein concentration-reducing agent or Pediococcus acidilactici of the present invention does not have a weight loss effect further supports the excellent advantage of being able to reduce blood protein concentration while maintaining nutritional status.

[0043] Administration of the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention preferably does not cause abnormalities in the albumin and globulin values ​​of test items in blood biochemistry tests (e.g., plasma biochemistry tests, whole blood biochemistry tests, or serum biochemistry tests). In one embodiment, administration of the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention preferably does not cause abnormalities in the values ​​of at least one or more, preferably all, test items in blood biochemistry tests (e.g., plasma biochemistry tests, whole blood biochemistry tests, or serum biochemistry tests). Test items include, but are not limited to, total protein, albumin, globulin, albumin / globulin ratio (A / G ratio), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), amylase, lipase, urea nitrogen, creatinine, total cholesterol, triglycerides, sodium, potassium, chloride, calcium, inorganic phosphorus, glucose (blood sugar), total bile acids, and total bilirubin.

[0044] For example, in a plasma biochemistry test for dogs, the reference ranges for total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), amylase, lipase, urea nitrogen, creatinine, total cholesterol, triglycerides, sodium, potassium, chloride, calcium, inorganic phosphorus, glucose (blood sugar), total bile acids, and total bilirubin are as shown in Table 5 below.

[0045] Furthermore, for example, the reference ranges for total protein, albumin, globulin, albumin / globulin ratio (A / G ratio), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), amylase, lipase, urea nitrogen, creatinine, total cholesterol, triglycerides, sodium, potassium, chloride, calcium, inorganic phosphorus, glucose (blood sugar), total bile acids, and total bilirubin in human plasma biochemistry tests are shown in Table 1.

[0046]

[0047] In the present invention, "abnormal" values ​​of test items in blood biochemistry tests mean that the values ​​deviate from the reference range (normal range) of each test item in the biological species of the target animal to which the blood protein concentration-reducing agent or Pediococcus acidilactici of the present invention is administered. "Causing abnormalities" in the value of a test item means that the value of a test item that was within the reference range before administration of the blood protein concentration-reducing agent or Pediococcus acidilactici of the present invention changes so that it deviates from the reference range due to said administration. "Doing not cause abnormalities" in the value of a test item means that the value of a test item that was within the reference range before administration of the blood protein concentration-reducing agent or Pediococcus acidilactici of the present invention does not deviate from the reference range (remains within the reference range) due to said administration.

[0048] Furthermore, the reduction in blood protein concentration by the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention may be accompanied by protein translation inhibition (inhibition of translation from mRNA to protein). The protein translation inhibition brought about by administration of the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention refers to protein translation inhibition (a decrease in protein translation level) compared to a control subject not administered the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention. In a preferred embodiment, the protein translation inhibition brought about by administration of the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention is protein translation inhibition due to reduced expression of ribosomal subunit genes. In a preferred embodiment, administration of the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention reduces the expression of at least one ribosomal subunit gene in each of the gene group for the large subunit of ribosomes (intranuclear ribosomes) RPL (e.g., the genes shown in Figures 6 and 10 described below, or their homologs such as human homologs), the gene group for the small subunit of ribosomes (intranuclear ribosomes) RPS (e.g., the genes shown in Figures 7 and 11 described below, or their homologs such as human homologs), the gene group for the large subunit of mitochondrial ribosomes MRPL (e.g., the genes shown in Figures 8 and 12 described below, or their homologs such as human homologs), and the gene group for the small subunit of mitochondrial ribosomes MRPS (e.g., the genes shown in Figures 9 and 13 described below, or their homologs such as human homologs), resulting in a reduction in the overall protein translation level.

[0049] The present invention also provides a food product containing the blood protein concentration-reducing agent or Pediococcus acidilactici of the present invention. In the present invention, "food" refers to food manufactured for human consumption. The food product of the present invention may be a food product for reducing blood protein concentrations. The food product of the present invention may further contain additives acceptable in the food industry, such as, but not limited to, carriers, excipients, stabilizers, preservatives, antioxidants, anti-adherents, disintegrants, binders, humectants, thickeners, buffers, surfactants, suspending agents, solubilizers, emulsifiers, coating agents, flavorings, fragrances, and colorants. The food product of the present invention may further contain other food ingredients. The food product of the present invention may be of any type and in any form, such as, but not limited to, dairy products, confectioneries, prepared foods, noodles, processed meat products, processed vegetable products, breads, soups, seasonings, beverages, etc. The food product of the present invention may also be a functional food product, such as a food for specified health uses, a food with functional claims, a food with nutrient function claims, or a supplement. Functional foods encompass all health foods, including health foods to which a health claim based on the Codex Alimentarius (Joint FAO / WHO Food Standards Commission) is applied. Functional foods may be in any dosage form, such as solid preparations such as tablets, granules, powders, pills, or capsules; liquid preparations such as solutions, suspensions, or syrups; or preparations having a typical food form (e.g., beverages, confectioneries, etc.). The food of the present invention may be a food composition.

[0050] The present invention also provides a feed containing the blood protein concentration-reducing agent or Pediococcus acidilactici of the present invention. In the present invention, "feed" refers to food prepared for feeding to non-human animals for ingestion. The feed of the present invention may be a feed for reducing blood protein concentrations. The feed of the present invention may further contain additives acceptable in the feed industry, for example, but not limited to, feed additives such as carriers, excipients, stabilizers, preservatives, antioxidants, anti-adherents, disintegrants, binders, humectants, thickeners, buffers, surfactants, suspending agents, solubilizers, emulsifiers, coating agents, flavoring agents, fragrances, and colorants. The feed of the present invention may further contain other feed ingredients. The feed of the present invention may be in any form, such as solid, semi-solid, or liquid. The feed of the present invention may be a feed composition.

[0051] The present invention also provides a pharmaceutical comprising the blood protein concentration-reducing agent or Pediococcus acidilactici of the present invention. The "pharmaceutical" of the present invention may be for humans or for non-human animals (veterinary pharmaceuticals). The pharmaceutical of the present invention may be a pharmaceutical for reducing blood protein concentrations. The pharmaceutical of the present invention may further comprise pharmaceutical additives acceptable in the pharmaceutical field, such as, but not limited to, carriers, excipients, stabilizers, preservatives, antioxidants, anti-adherents, disintegrants, binders, humectants, thickeners, buffers, surfactants, suspending agents, solubilizers, emulsifiers, coating agents, flavoring agents, fragrances, and colorants. The pharmaceutical of the present invention may further comprise other physiologically active ingredients. The pharmaceutical of the present invention may be of any type and in any form, including, but not limited to, solid formulations such as tablets, granules, powders, pills, and capsules; semi-solid formulations such as gels; and liquid formulations such as solutions, suspensions, and syrups. The pharmaceutical product according to the present invention may be a pharmaceutical composition.

[0052] The foods, feeds, and pharmaceuticals of the present invention can be effectively used as foods, feeds, and pharmaceuticals for reducing blood protein concentrations, respectively, to achieve the above-mentioned blood protein concentration-reducing effect. The foods, feeds, and pharmaceuticals of the present invention can be intended to achieve the above-mentioned effects achieved by the blood protein concentration-reducing agent or Pediococcus acidilactici of the present invention, i.e., an increase in the albumin / globulin ratio (A / G ratio), protein translation inhibition, etc. The protein translation inhibition achieved by the foods, feeds, and pharmaceuticals of the present invention can be protein translation inhibition achieved by the blood protein concentration-reducing agent or Pediococcus acidilactici of the present invention through reduced expression of ribosomal subunit genes. In a preferred embodiment, the foods, feeds, and pharmaceuticals of the present invention do not have a weight loss effect, as described above. In a preferred embodiment, the foods, feeds, and pharmaceuticals of the present invention do not cause abnormalities in the values ​​of each test item in blood biochemistry tests (e.g., plasma biochemistry tests, whole blood biochemistry tests, or serum biochemistry tests), as described above.

[0053] In the present invention, the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention can be administered to a target animal to reduce the blood protein concentration in the target animal. The present invention also provides a method for reducing the blood protein concentration in a target animal, which comprises administering the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention to the target animal.

[0054] Similarly, in the present invention, blood protein concentrations in a subject can be reduced by administering or feeding the food, feed, or pharmaceutical product of the present invention to the subject animal. The present invention also provides a method for reducing blood protein concentrations in a human subject, comprising administering the food of the present invention to the subject. The present invention also provides a method for reducing blood protein concentrations in a non-human animal subject, comprising feeding the feed of the present invention to the subject animal. The present invention also provides a method for reducing blood protein concentrations in a subject, comprising administering the pharmaceutical product of the present invention to the subject animal. In one embodiment, the method for reducing blood protein concentrations can be a method for treating or preventing increased blood protein concentrations or hyperproteinemia.

[0055] In the present invention, the subject to which the blood protein concentration-reducing agent or Pediococcus acidilactici of the present invention, or the food, feed, or pharmaceutical of the present invention is administered may be any animal, including humans and non-human animals. The subject may be, for example, a primate such as a human, chimpanzee, or gorilla; a rodent such as a mouse, rat, guinea pig, or hamster; or a mammal such as a dog, cat, rabbit, horse, cow, pig, sheep, goat, camel, or donkey. The subject may also be a bird such as a chicken, quail, duck, parrot, parakeet, or pigeon. The subject is preferably one in need of a reduction in blood protein concentration. In one embodiment, the subject is preferably a middle-aged or elderly animal. In the present invention, "middle-aged or elderly" refers to 45 years or older in humans and the equivalent age in non-human animals. In the present invention, "elderly" refers to 60 years or older in humans and the equivalent age in non-human animals. In one embodiment, the subject may be an animal that exhibits an abnormally elevated or increasing trend in blood protein concentration, for example, compared to a reference range for a biochemical test, hi one embodiment, the subject may be a middle-aged or elderly animal that exhibits such an abnormally elevated or increasing trend in blood protein concentration.

[0056] "Administration" in the present invention may be by any route of administration. "Administration" in the present invention is preferably, but not limited to, oral administration or enteral administration such as tube administration into the digestive tract. "Administration" in the present invention may be by ingestion of food or feeding of feed. "Feeding" in the present invention means ingestion of feed by oral administration.

[0057] The blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention, or the food, feed, or pharmaceutical according to the present invention may be administered to a subject only once, but preferably twice or more times. The administration is preferably carried out repeatedly or periodically over at least a certain period of time. The administration may be carried out, for example, once, twice, or three or more times per day. The administration may be carried out for any period of time, for example, 1 to 7 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year, or longer.

[0058] The amount of the blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention, or the food, feed, or pharmaceutical according to the present invention administered to a subject is an amount equivalent to Pediococcus acidilactici cells, and may be, for example, 0.1 mg to 10 g / kg body weight per administration, but is not limited thereto. The blood protein concentration-reducing agent or Pediococcus acidilactici according to the present invention, or the food, feed, or pharmaceutical according to the present invention, can be administered to a subject in an effective amount, more specifically, in an amount equivalent to an effective amount of Pediococcus acidilactici for reducing blood protein concentrations (an amount containing the effective amount of Pediococcus acidilactici).

[0059] In the present invention, the effects of reducing blood protein concentration, increasing the albumin / globulin ratio (A / G ratio), suppressing protein translation, and the like in a subject receiving the above-mentioned administration are brought about after the start of administration, and may be observed, for example, during the administration period and / or after the end of the administration period (for example, up to 6 months, typically at least 2 months, after the end of the administration period).

[0060] The present invention also provides use of Pediococcus acidilactici in the production of the blood protein concentration-lowering agent of the present invention, or the food, feed, or pharmaceutical product of the present invention for reducing blood protein concentrations.

[0061] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0062] Example 1: Examination of the Effect of Lactic Acid Bacteria Administration in Dogs 1) Preparation of Formulation A medium having the composition shown in Table 2 was prepared as follows and used for culturing lactic acid bacteria. Whey protein concentrate (Fonterra) and water were mixed and dissolved in a ratio of 2.5:45, and protease (Amano Enzyme) was added in the ratio shown in Table 2. Enzyme treatment was carried out at 48°C for 3 hours to obtain a whey hydrolysate. Subsequently, yeast extract (Asahi Group Foods), sodium acetate (Mitsubishi Chemical), manganese-rich water-soluble yeast extract (Oriental Yeast Co., Ltd.), and glucose (Sanei Saccharification) were dissolved in water in the ratio shown in Table 2 and then mixed with the whey hydrolysate obtained above. The pH of the resulting mixture was adjusted to 6.7 with sodium hydroxide, and the mixture was then heat-sterilized at 115°C for 12 minutes to prepare a medium.

[0063]

[0064] To prepare the active formulation, a lactic acid bacteria preparation, the medium prepared as described above was cooled to 30°C. Then, 280 L of the medium (Table 2) was inoculated with the lactic acid bacteria Pediococcus acidilactici OB7260 strain and cultured at 30°C. Culture was performed under anaerobic conditions using nitrogen gas, and the pH of the culture was maintained at 6 with potassium carbonate solution during the culture. 24 hours after inoculation with the lactic acid bacteria, the culture was cooled to below 10°C to terminate the culture. After the culture was completed, 280 L of the culture was centrifuged, 266 L of culture supernatant was removed, and the pellet was suspended in the remaining liquid to obtain a 20-fold concentrated bacterial solution. To this concentrated bacterial solution, 1.44 kg of 50% sucrose solution and 3.52 kg of 28.9% dextrin solution were added, mixed, and then flash-frozen at -40°C. The frozen product was freeze-dried and then pulverized. Silicon dioxide was added to the resulting powder (freeze-dried powder) at a ratio of 98.2:1.8 (freeze-dried powder: silicon dioxide) and mixed. The dry weight ratios (%) of the components contained in the active formulation thus prepared are shown in Table 3. The bacterial cell concentration of the OB7260 strain in the freeze-dried powder was 6.6 x 10 １１ The number was 1 / g.

[0065]

[0066] To prepare a placebo formulation, a 50% sucrose solution and a dextrin solution were added to the medium prepared as described above and cooled to 30°C (Table 2), mixed, and then rapidly frozen at -40°C. The frozen product was freeze-dried and then pulverized, and silicon dioxide was added to the resulting freeze-dried powder in a ratio of 98.2:1.8 (freeze-dried powder:silicon dioxide) and mixed. The dry weight ratios (%) of the components contained in the placebo formulations prepared in this manner are shown in Table 4.

[0067]

[0068] 2) Effect of formulation administration on canine blood protein concentrations. Sixteen middle-aged toy poodles (8-12 years old at the start of administration) kept as pet dogs at the same facility in Ibaraki Prefecture, Japan, were used as test subjects to receive either the active formulation (containing the OB7260 strain) or a placebo formulation. The test dogs were divided into two groups of eight dogs each. One group (placebo group) received regular chow mixed with 1.5 g of placebo formulation (powder), and the other group (active group) received regular chow mixed with 1.5 g of active formulation (powder) once daily for two months. This formulation administration study was conducted under the supervision of a veterinarian. No abnormalities were observed during the formulation administration study, and all dogs completed the study.

[0069] Blood samples were taken from each test dog immediately before the start of the formulation administration study (0 m), at the end of the 2-month formulation administration period (2 m), 1.5 months after the end of the formulation administration period, and 6 months after the end of the formulation administration period (3.5 months (3.5 m) and 8 months (8 m) from the start of the formulation administration study, respectively), and plasma biochemistry tests were performed. The body weight of each test dog was also measured on the day of blood collection.

[0070] The results of the plasma biochemistry tests are shown in Table 5. The values ​​for each item (except total bilirubin) represent the mean ± standard deviation.

[0071]

[0072] The change in plasma protein concentration (total protein concentration) was calculated by subtracting the value at 0 m from the values ​​at 2 m, 3.5 m, and 8 m. The change in plasma protein concentration (mean value) at each time point for each group is shown in Figure 1.

[0073] In the active group, the change in plasma protein concentration after the start of administration was negative at 2 m, 3.5 m, and 8 m, indicating a decrease in total protein concentration compared to the baseline (0 m) (Figure 1). In contrast, in the placebo group, plasma protein concentration did not change significantly after administration (Figure 1). Plasma protein concentrations in the active group were lower than those in the placebo group at 2 m, 3.5 m, and 8 m, with a statistically significant decrease at 3.5 m (p<0.05; Mann-Whitney test). They also showed a tendency for a decrease at 8 m, 6 months after the end of administration (p<0.1).

[0074] The change in plasma globulin concentration was calculated by subtracting the 0 m measurement value from the 2 m, 3.5 m, and 8 m measurement values. The change (mean) in plasma globulin concentration at each time point for each group is shown in Figure 2. The plasma globulin concentration was calculated by subtracting the plasma albumin concentration measurement value from the plasma protein concentration measurement value according to conventional methods.

[0075] In the active group, the change in plasma globulin concentration after the start of administration was negative at 2 m, 3.5 m, and 8 m, indicating a decrease in globulin concentration compared to the value immediately before administration (0 m) (Figure 2). In contrast, in the placebo group, the change in plasma globulin concentration was small even after administration began (Figure 2). Plasma globulin concentrations in the active group were lower than those in the placebo group at 2 m, 3.5 m, and 8 m, and were statistically significantly lower at 3.5 m.

[0076] On the other hand, as shown in Table 5, the plasma albumin concentrations in the active and placebo groups did not show significant changes at 2 m, 3.5 m, and 8 m after the start of administration compared to the values ​​immediately before the start of administration (0 m). The changes were calculated by subtracting the plasma albumin concentration at 0 m from the plasma albumin concentrations at 2 m, 3.5 m, and 8 m, and the changes (mean values) in plasma albumin concentration at each time point for each group are shown in Figure 3. There was also no statistically significant difference (significance level p<0.1; Mann-Whitney test) between the active and placebo groups in the change in plasma albumin concentration. Therefore, it is believed that the decrease in plasma protein concentration in the active group was largely due to a decrease in plasma globulin concentration.

[0077] Because blood albumin concentration is used as an indicator of overall nutritional status, the maintenance of blood albumin concentration indicates that administration of the lactic acid bacteria preparation did not adversely affect nutritional status. Table 6 shows the albumin / globulin ratio (A / G ratio) at each time point in the active and placebo groups. The A / G ratio at 0 m was subtracted from the A / G ratios at 2 m, 3.5 m, and 8 m to calculate the change, and the mean A / G ratio change at each time point for each group is shown in Figure 4. The active group showed an increase in the A / G ratio and its change after the start of administration compared to the placebo group (Table 6, Figure 4), indicating that administration of the lactic acid bacteria preparation reduced blood globulin concentration without causing a decrease in blood albumin concentration.

[0078]

[0079] The results of the plasma biochemistry tests at each time point showed that the values ​​of each item were within the reference range at all time points in both the active and placebo groups, except for triglyceride, which exceeded the upper limit of the reference range at 8 m in the placebo group (Table 5). These results indicate that administration of the lactic acid bacteria preparation does not cause blood biochemistry abnormalities.

[0080] The weight change was calculated by subtracting the weight at 0 m from the weight at 2 m, 3.5 m, and 8 m. The weight change (mean) for each group at each time point is shown in Figure 5. No statistically significant differences in weight change were observed between the groups at 2 m, 3.5 m, and 8 m (Figure 5). These results indicate that administration of the lactic acid bacteria preparation does not result in weight loss or any adverse health effects.

[0081] 3) Whole blood transcriptome analysis Whole blood transcriptome analysis was performed on the blood collected at the end (2 m) of the two-month administration period of the formulation in 2) above.

[0082] For this analysis, the collected blood was immediately placed in RNAprotect Animal Blood Tubes (QIAGEN) and stored, and then analyzed using RNeasy. （Ｒ） RNA was extracted using the Protect Animal Blood System (QIAGEN). Sequence data were obtained using TruSeq Stranded Total RNA with Ribo-Zero Globin (Illumina) and a NovaSeq 6000 system (Illumina). Trimming, mapping, read count calculation, and log ２ (Fold Change) [log ２ The software used for calculation of [fold change] and significance testing was Trimmomatic, HISAT2, featureCounts, and DESeq2, respectively. Correction for multiple comparisons was performed using the Benjamini-Hochberg method, and adjusted p-values ​​were calculated.

[0083] Using transcriptome analysis based on the acquired data, we investigated the expression of genes for the four ribosomal subunits responsible for protein synthesis as a factor related to protein concentration. Specifically, we extracted genes for various ribosomal subunits belonging to the large subunit RPL and small subunit RPS of the ribosome (intranuclear ribosome), as well as the large subunit MRPL and small subunit MRPS of the mitochondrial ribosome. We then calculated the ratio (expression level ratio) of the expression levels of each extracted gene between the active group and the placebo group and performed a significance test.

[0084] The results are shown in Figures 6 to 9. In all four gene groups, namely, the RPL gene group (Figure 6), the RPS gene group (Figure 7), the MRPL gene group (Figure 8), and the MRPS gene group (Figure 9), the expression level ratio between the active group and the placebo group (active group / placebo group (log ２ The number of genes whose expression was decreased in the active group was calculated by multiplying the expression level ratio between the active group and the placebo group (active group / placebo group (log ２ The number of genes with positive Fold Change values ​​(genes whose expression increased in the active group) was greater than the number of genes with positive Fold Change values ​​(genes whose expression increased in the active group).

[0085] In the active group, among the genes belonging to the RPL gene group, the RPS gene group, the MRPL gene group, and the MRPS gene group, the numbers of genes whose expression levels were statistically significantly (adjusted p-value < 0.1) decreased (indicated by ** and * in the negative area of ​​the horizontal axis in the figure) were 13, 4, 8, and 3, respectively, while the numbers of genes whose expression levels were statistically significantly (adjusted p-value < 0.1) increased (indicated by ** and * in the positive area of ​​the horizontal axis in the figure) were 0, 1, 1, and 0, respectively (Figures 6 to 9).

[0086] These results suggest that the lactic acid bacteria preparation reduces the expression of many ribosomal subunit genes, thereby suppressing protein translation and resulting in a decrease in plasma protein concentration. As described above, the lactic acid bacteria preparation was shown to be able to reduce protein translation levels and plasma protein concentration without causing adverse health effects.

[0087] Example 2: Examination of the effect of administration of a lactic acid bacteria suspension to mice The lactic acid bacteria Pediococcus acidilactici OB7260 strain was inoculated into an MRS medium and anaerobically cultured overnight at 30° C. 50 mL of the resulting culture solution was centrifuged (15,000 rpm, 4° C., 10 minutes), 47.5 mL of the culture supernatant was removed, and the pellet was suspended in the remaining liquid to prepare a concentrated bacterial suspension of the OB7260 strain.

[0088] Sixty female, 20-month-old C57BL6 / J mice were divided into three groups. The first group (the lactic acid bacteria-treated group) received the concentrated bacterial solution prepared above, while the second group (the control group) received MRS medium. Each was orally administered 500 μL / day using a mouse probe, five days a week for two months. The third group (the non-treated group) received neither the concentrated bacterial solution nor MRS medium, but was otherwise maintained under the same conditions as the OB7260-treated group and the medium-treated group.

[0089] At the end of the two-month treatment period, blood was collected from the tail vein of each mouse. Whole blood transcriptome analysis was performed on the collected blood. The collected blood was dissolved in DNA / RNA Shield (Zymo Research), and RNA was extracted using a spin column method. 240 ng of the resulting RNA was used for library construction. rRNA was removed using the method described by Bogdanova et al. (2011) with some modifications. A library (RNA-Seq library) was prepared using the Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research). The resulting RNASeq library was sequenced using a NovaSeq 6000 system (Illumina) to a sequencing depth of at least 30 million read pairs (150 bp paired-end sequencing) to obtain sequence data.

[0090] Based on the obtained data, gene expression changes of four gene groups of ribosomal subunits were analyzed in the same manner as in Example 1. That is, genes of various ribosomal subunits belonging to the large subunit RPL and small subunit RPS of the ribosome (intranuclear ribosome), and the large subunit MRPL and small subunit MRPS of the mitochondrial ribosome were extracted, and the ratio (expression level ratio) of the expression level of each extracted gene between the lactic acid bacteria-administered group and the medium-administered group was calculated, and a significance test was performed.

[0091] FIG. 10 shows the results for the RPL gene group (rpl group), FIG. 11 for the RPS gene group (rps group), FIG. 12 for the MRPL gene group (mrpl group), and FIG. 13 for the MRPS gene group (mrps group).

[0092] Similar to the results of the dog analysis in Example 1, the gene expression levels of many nuclear ribosomal subunit genes in the lactic acid bacteria-administered group were statistically significantly lower than those in the culture medium-administered group (Figures 10 and 11). Furthermore, while the gene expression levels of many mitochondrial ribosomal subunit genes in the lactic acid bacteria-administered group were statistically significantly lower than those in the culture medium-administered group, no mitochondrial ribosomal subunit genes were statistically significantly higher than those in the culture medium-administered group (Figures 12 and 13). These results indicate that administration of lactic acid bacteria solution also reduced the expression of many ribosomal subunit genes in mice, suppressing protein translation. It is believed that administration of this lactic acid bacteria solution also reduced plasma protein concentrations in mice by suppressing protein translation.

[0093] After blood sampling at the end of the two-month administration period, the mice were kept until death. The date of death was recorded, and their body weight was measured weekly from the start of administration. The changes in body weight over time are shown in Figure 14. No weight loss was observed, indicating that administration of the lactic acid bacteria solution did not have any adverse effects on health.

[0094] According to the present invention, protein translation can be effectively inhibited without adverse health effects, and blood protein concentrations, particularly blood globulin concentrations, can be reduced to within the healthy reference range typically measured in blood biochemistry tests. The reduction in blood protein and globulin concentrations can prevent, for example, increased blood viscosity and organ damage associated with excessive increases in blood protein concentrations, thereby maintaining or improving health, and therefore the present invention is useful in the fields of medicine and healthcare (maintenance and promotion of health).

[0095] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A blood protein concentration reducing agent containing Pediococcus acidilactici.

2. The blood protein concentration-reducing agent according to claim 1, wherein the reduction in blood protein concentration is accompanied by an increase in the albumin / globulin ratio.

3. The blood protein concentration-reducing agent according to claim 1, wherein the Pediococcus acidilactici is Pediococcus acidilactici strain OB7260 having accession number NITE BP-02634.

4. The blood protein concentration-reducing agent according to claim 1, wherein the reduction in blood protein concentration is accompanied by inhibition of protein translation.

5. The blood protein concentration-reducing agent according to claim 4, wherein the protein translation inhibition is due to a decrease in the expression of a ribosomal subunit gene.

6. The blood protein concentration reducing agent according to claim 1, which does not have a weight loss effect.

7. A food or feed for reducing blood protein concentration, comprising Pediococcus acidilactici or the blood protein concentration-reducing agent according to any one of claims 1 to 6.

8. A pharmaceutical for reducing blood protein concentration, comprising Pediococcus acidilactici or the blood protein concentration-reducing agent according to any one of claims 1 to 6.

9. The food or feed according to claim 7, wherein the reduction in blood protein concentration is accompanied by an increase in the albumin / globulin ratio.

10. The pharmaceutical product according to claim 8, wherein the reduction in blood protein concentration is accompanied by an increase in the albumin / globulin ratio.

11. The food or feed according to claim 7, wherein the Pediococcus acidilactici is the Pediococcus acidilactici strain OB7260 having the accession number NITE BP-02634.

12. The pharmaceutical product according to claim 8, wherein the Pediococcus acidilactici is Pediococcus acidilactici strain OB7260 having accession number NITE BP-02634.

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