AGENT FOR IMPROVING INTESTINAL FLORA

Abstract

The purpose of the present invention is to provide an agent for improving intestinal flora, the agent being capable of improving intestinal flora by increasing the number of bacteria belonging to the genus Bifidobacterium and/or the genus Lactobacillus in the intestines. In the present invention, an enzyme-containing substance derived from fungi belonging to the genus Penicillium increases the number of both bacteria belonging to the genus Bifidobacterium and bacteria belonging to the genus Lactobacillus in the intestines, and is capable of exhibiting the excellent effect of improving the intestinal flora.

Claims

1-7. (canceled)

8. A method for improving intestinal flora, comprising orally applying, to an individual in need of improvement of the intestinal flora, a substance containing an enzyme derived from a fungus of genus Penicillium.

9-11. (canceled)

12. The method according to claim 8, wherein the enzyme derived from a fungus of genus Penicillium is a lipase and/or a nuclease derived from the fungus of genus Penicillium.

13. The method according to claim 8, wherein the enzyme derived from a fungus of genus Penicillium is a lipase derived from Penicillium camemberti and/or a nuclease derived from Penicillium citrinum.

14. The method according to claim 8, wherein the substance contains the lipase which is used for increasing the number of bacteria of genus Bifidobacterium and/or genus Lactobacillus in the intestine.

15. The method according to claim 8, wherein the substance is blended into a pharmaceutical preparation.

16. The method according to claim 8, wherein the substance is blended into food or beverage.

17. The method according to claim 12, wherein the nuclease derived from a fungus of genus Penicillium is a member selected form the group consisting of: an exonuclease and an endonuclease.

18. The method according to claim 12, wherein the nuclease derived from a fungus of genus Penicillium includes 5-phosphodiesterase.

19. The method according to claim 8, wherein an amount of the substance containing the enzyme derived from genus Penicillium blended into the supplement may be 0.001 to 0.9 g, preferably from 0.01 to 0.5 g, and more preferably from 0.02 to 0.2 g in terms of the amount of the enzyme derived from genus Penicillium per g of the supplement.

20. The method according to claim 8, wherein the amount of substance containing lipase derived from genus Penicillium blended into the supplement may be 3,000 to 1,000,000 U/g, preferably 10,000 to 600,000 U/g, and more preferably 15,000 to 300,000 U/g in terms of the amount of the lipase derived from genus Penicillium.

21. The method according to claim 12, wherein the amount of substance containing nuclease derived from genus Penicillium blended into the supplement may be 300 to 50,000 U/g, preferably 1,000 to 30,000 U/g, and more preferably 1,500 to 15,000 U/g in terms of the amount of the nuclease derived from genus Penicillium.

22. The method according to claim 8, wherein the enzyme derived from a fungus of genus Penicillium is derived from a genus selected from the group consisting of: Penicillium camemberti (P. camemberti), Penicillium citrinum (P. citrinum), Penicillium islandicum (P. islandicum), Penicillium verrucosum (P. verrucosum), Penicillium glabrum (P. glabrum), Penicillium chrysogenum (P. crysogenum), Penicillium digitatum (P. digitatum), Penicillium dodgei (P. dodgei), Penicillium expansum (P. expansum), and Penicillium multicolor (P. multicolor).

23. The method according to claim 8, wherein the enzyme derived from a fungus of genus Penicillium is a member selected from the group consisting of: lipase, nuclease, protease, amylase and cellulase.

Description

DESCRIPTION OF EMBODIMENTS

[0025] An agent for improving intestinal flora of the present invention is characterized by comprising, as an active ingredient, a novel substance containing an enzyme derived from a fungus of genus Penicillium. Hereinafter, the agent for improving intestinal flora of the present invention will be described in detail.

[Active Ingredient]

[0026] An agent for improving intestinal flora of the present invention comprises, as an active ingredient, a substance containing an enzyme derived from a fungus of genus Penicillium. By selecting and using the substance containing an enzyme derived from genus Penicillium, the numbers of both bacteria of genus Bifidobacterium and bacteria of genus Lactobacillus in the intestine can be increased to improve the intestinal flora effectively.

[0027] In the present invention, the wording substance containing an enzyme derived from a fungus of genus Penicillium refer to a material containing an enzyme derived from a fungus of genus Penicillium, and includes not only a product obtained by purifying one or more enzymes derived from a fungus (fungi) of genus Penicillium but also a mixture of one or more enzymes derived from a fungus (fungi) of genus Penicillium with other proteins, excipients or the like. In addition, in the present invention, the wording substance containing an enzyme derived from a fungus of genus Penicillium also includes a purified product or a crude product of culture in which a fungus (fungi) of genus Penicillium is cultured to produce an enzyme(s).

[0028] A microorganism from which the enzyme used in the present invention is derived is a fungus of genus Penicillium, and specific examples thereof include Penicillium camemberti (P. camemberti), Penicillium citrinum (P. citrinum), Penicillium islandicum (P. islandicum), Penicillium verrucosum (P. verrucosum), Penicillium glabrum (P. glabrum), Penicillium chrysogenum (P. crysogenum), Penicillium digitatum (P. digitatum), Penicillium dodgei (P. dodgei), Penicillium expansum (P. expansum), or Penicillium multicolor (P. multicolor). Among these Penicillium fungi, suitable examples include Penicillium camemberti or Penicillium citrinum.

[0029] In addition, the kind of the enzyme derived from a fungus of genus Penicillium as used in the present invention is not particularly limited, and examples thereof include a lipase, nuclease, protease, amylase, and cellulase. Among these enzymes, lipase and nuclease are preferred from the viewpoint of more effectively increasing the number of bacteria of genus Bifidobacterium and bacteria of genus Lactobacillus in the intestine.

[0030] Among the lipases derived from a fungus of genus Penicillium, especially the lipase derived from Penicillium camemberti is excellent in the action of increasing the number of bacteria of genus Bifidobacterium or bacteria of genus Lactobacillus in the intestine, and is suitably used in the present invention.

[0031] Meanwhile, the nuclease derived from a fungus of genus Penicillium may be a nuclease capable of hydrolyzing either RNA or DNA, or may be a nuclease capable of hydrolyzing both RNA and DNA. In addition, the nuclease derived from a fungus of genus Penicillium may be either an exonuclease or an endonuclease, but is preferably an exonuclease. A suitable example of the nuclease derived from a fungus of genus Penicillium includes 5-phosphodiesterase. The 5-phosphodiesterase refers to 5-exonuclease (EC. 3.1.4.1), which is a nuclease that hydrolyzes 5-nucleotides sequentially from the 3-end of nucleic acid (RNA and DNA). Among the nucleases derived from Penicillium fungi, in particular, the nuclease derived from Penicillium citrinum, especially the 5-exonuclease derived from Penicillium citrinum is excellent in the action of increasing the number of bacteria of genus Bifidobacterium or bacteria of genus Lactobacillus in the intestine, and is suitably used in the present invention.

[0032] The enzyme derived from genus Penicillium can be produced by a known production method using, for instance, a microorganism culture or a genetic engineering technique. Since the enzyme derived from genus Penicillium is commercially available, a commercially available product may also be used. For example, a lipase preparation derived from Penicillium camemberti and a 5-exonuclease preparation derived from Penicillium citrinum can be obtained from Amano Enzyme Inc.

[0033] The substance containing an enzyme derived from a fungus of genus Penicillium as used in the present invention may comprise one kind of enzyme derived from a fungus of genus Penicillium or may comprise two or more kinds of enzymes derived from a fungus (fungi) of genus Penicillium.

[Dose]

[0034] The dose of the agent for improving intestinal flora (substance containing an enzyme derived from a fungus of genus Penicillium) of the present invention may be determined appropriately according to, for example, the type of active ingredient used, the type of product in which the agent is used, the use, the expected effect, and the form of application.

[0035] For example, the amount of the substance containing an enzyme derived from a fungus of genus Penicillium as ingested or administered per day for an adult is 0.01 to 10 g, preferably 0.05 to 5 g, and more preferably 0.1 to 2.5 g in terms of the amount of enzyme derived from a fungus of genus Penicillium. As used herein, the wording in terms of the amount of enzyme derived from a fungus of genus Penicillium refers to a value obtained by converting the amount of substance containing an enzyme derived from a fungus of genus Penicillium into the amount of enzyme derived from a fungus of genus Penicillium as contained in the substance containing an enzyme derived from a fungus of genus Penicillium.

[0036] For example, when a substance containing lipase derived from a fungus of genus Penicillium is used, the daily dose for an adult human as an amount of the substance containing lipase derived from a fungus of genus Penicillium ingested or administered may be 10,000 to 2,000,000 U, preferably 30,000 to 1,000,000 U, and more preferably 50,000 to 600,000 U in terms of the amount of lipase derived from a fungus of genus Penicillium. As used herein, the wording in terms of the amount of lipase derived from a fungus of genus Penicillium refers to a value obtained by converting the amount of the substance containing lipase derived from a fungus of genus Penicillium into the amount of lipase derived from a fungus of genus Penicillium as contained in the substance containing lipase. In addition, as used herein, the lipase activity (U) is a value determined using the following method:

(Method of Measuring Lipase Activity)

[0037] Vinyl laurate is added to 2 w/v % aqueous polyvinyl alcohol solution so as to have a final concentration of 3%, and the mixture is emulsified at 14,200 to 14,800 rpm for 10 minutes while being cooled to 10 C. or less. The sample is stored in a refrigerator, left for 1 hour, and then immediately used.

[0038] First, 4 mL of 0.1 mol/L McIlvaine buffer (pH 5.6) and 5 mL of vinyl laurate emulsion are each accurately weighed, and placed in a flat-bottom test tube (30120 mm), shaken, and left at 400.5 C. for 10 to 15 minutes. Then, 1 mL of sample solution containing a sample whose activity is to be measured is accurately added, and immediately shaken.

[0039] This solution is left standing at 400.5 C. for precisely 30 minutes, and 10 mL of liquid obtained by mixing ethanol and acetone at a volume of 1:1 is then added and shaken.

[0040] Next, 10 mL of 0.05 mol/L aqueous sodium hydroxide solution is precisely added, 10 mL of liquid obtained by mixing ethanol and acetone at a volume of 1:1 is further added and shaken, and an excessive amount of sodium hydroxide is then titrated with 0.05 mol/L hydrochloric acid (T.sub.30 mL) (indicator: 2 drops of phenolphthalein test liquid).

[0041] Separately, as a blank, 4 mL of 0.1 mol/L McIlvaine buffer (pH 5.6) and 5 mL of vinyl laurate emulsion are each precisely weighed, and put in a flat-bottom test tube (30120 mm) and shaken, and left at 400.5 C. for 30 minutes or more. Then, 10 mL of liquid obtained by mixing ethanol and acetone at a volume of 1:1 is added, and 1 mL of sample solution containing a sample whose activity is to be measured is then accurately added and shaken.

[0042] Next, 10 mL of 0.05 mol/L aqueous sodium hydroxide solution is precisely added, and titration is performed in the same manner as follows (To mL).

[0043] The titration is performed by adding 0.05 mol/L hydrochloric acid to pH 10.00 while using a pH meter. Under these conditions, the amount of enzyme that results in an increase of 1 mol of fatty acid per minute is defined as 1 U. The following equation is used for the calculation.

[00001]$\begin{array}{cc}\mathrm{Fat}\mathrm{digestive}\mathrm{powder}\left(U/g,U/\mathrm{mL}\right)=50\left(T_0-T_{30}\right)1/30fn& \left[\mathrm{Mathematical}\mathrm{Formula}1\right]\end{array}$ [0044] T.sub.0: titer value (mL) of blank [0045] T.sub.30: titer value (mL) of reaction solution [0046] 50: equivalent amount of fatty acids (mol) per mL of 0.05 mol/L hydrochloric acid (for quantification) [0047] 30: reaction time (min) [0048] f: factor of 0.05 mol/L hydrochloric acid (for quantification) [0049] n: dilution factor per g or mL of a sample

[0050] Meanwhile, for example, when a substance containing nuclease derived from a fungus of genus Penicillium is used, the daily dose for an adult human as an amount of the substance containing nuclease derived from a fungus of genus Penicillium ingested or administered may be 1,000 to 100,000 U, preferably 3,000 to 60,000 U, and more preferably 5,000 to 30,000 U in terms of the amount of nuclease derived from a fungus of genus Penicillium. As used herein, the wording in terms of the amount of nuclease derived from a fungus of genus Penicillium refers to a value obtained by converting the amount of the substance containing nuclease derived from a fungus of genus Penicillium into the amount of nuclease derived from a fungus of genus Penicillium as contained in the substance containing nuclease. Note that as used herein, the nuclease activity of 1 U is the amount of enzyme that releases 1 mol of phosphoric acid per minute when acts on the AMP which is a substrate. For example, when the nuclease is a phosphodiesterase, the activity can be measured in accordance with Method 1 of phosphodiesterase activity test method as specified in Ninth Edition of Japan's Specifications and Standards for Food Additives 2018 (the Ministry of Health, Labour and Welfare the Consumer Affairs Agency Japan).

[Use]

[0051] The agent for improving intestinal flora of the present invention can increase the number of beneficial bacteria such as bifidobacteria and lactic acid bacteria in the intestine, by the effect of the substance containing an enzyme derived from genus Penicillium, to form a beneficial bacteria-dominated flora, and thus is used for the purpose of forming a healthy intestinal environment, maintaining a healthy intestinal environment, and the like. In particular, the agent for improving intestinal flora of the present invention has an excellent effect of increasing the numbers of both bacteria of genus Bifidobacterium and bacteria of genus Lactobacillus in the intestine, and thus can also be used as an agent for increasing the numbers of bacteria of genus Bifidobacterium and bacteria of genus Lactobacillus in the intestine.

[0052] Additionally, the agent for improving intestinal flora of the present invention can form a beneficial bacteria-dominated flora to form a healthy intestinal environment, and thus can also be used for the purpose of preventing or treating a disease and/or a symptom due in part to an unhealthy intestinal environment. Examples of the disease and/or symptom include decreased immunity, colorectal cancer, allergic disease, nonalcoholic steatohepatitis, obesity, and inflammatory bowel disease.

[Form of Use]

[0053] The agent for improving intestinal flora of the present invention is orally applied by oral ingestion or oral administration. The agent for improving intestinal flora of the present invention is orally ingested or orally administered to exert the effect of improving intestinal flora after arriving at the intestine, and thus can be blended for use with various products such as foods and beverages, pharmaceutical preparations for oral administration, animal feed, and pet foods.

[0054] When the agent for improving intestinal flora of the present invention is blended with the various products, the products may contain probiotics and/or prebiotics as required together with the agent for improving intestinal flora of the present invention.

[0055] Examples of microorganisms used as the probiotics include lactic acid bacteria, bifidobacteria, and Bacillus subtilis var natto. Specific examples of the lactic acid bacteria include lactic acid bacteria of genus Lactobacillus, such as Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus plantarum; lactic acid bacteria of genus Enterococcus, such as Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae; and lactic acid bacteria of genus Streptococcus, such as Streptococcus bovis and Streptococcus thermophilus. Specific examples of the bifidobacteria include Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium pseudolongum, and Bifidobacterium thermophilum. These probiotics may be used alone or in combination.

[0056] Examples of the prebiotics include xylooligosaccharides, fructooligosaccharides, soybean oligosaccharides, isomaltooligosaccharides, lactosucrose, galactooligosaccharides, and lactulose. These prebiotics may be used alone or in combination.

[0057] The dosage form of a product with which the agent for improving intestinal flora of the present invention is blended may be any of a solid form, a semi-solid form, a liquid form, and the like, and is selected appropriately according to the type and use of the product.

[0058] When the agent for improving intestinal flora of the present invention is used for foods and beverages, the substance containing an enzyme derived from genus Penicillium is provided as a food or a beverage that exerts the effect of improving intestinal flora, either as is or in combination with other food materials or additive ingredients to be prepared in a desired form. Examples of the food and beverage include foods for specified health uses, nutritional supplements, functional foods, and foods for patients, as well as common foods and beverages. Forms of these foods and beverages include, but are not limited to, supplements, such as tablets, granules, powders, capsules, and soft capsules; and beverages, such as energy drinks, fruit beverages, carbonated beverages, and lactic acid beverages.

[0059] When the agent for improving intestinal flora of the present invention is used for foods and beverages, the amount of the agent blended into the food and beverages may be determined appropriately according to, for example, the type of active ingredient used, and the form of the food and beverage, within a range satisfying the above-described dose.

[0060] For example, when the agent for improving intestinal flora of the present invention is provided in the form of a supplement, the amount of the substance containing an enzyme derived from genus Penicillium blended into the supplement may be 0.001 to 0.9 g, preferably from 0.01 to 0.5 g, and more preferably from 0.02 to 0.2 g in terms of the amount of the enzyme derived from genus Penicillium per g of the supplement.

[0061] More specifically, when the substance containing lipase derived from genus Penicillium is used as the agent for improving intestinal flora of the present invention and is provided in the form of a supplement, the amount of substance containing lipase derived from genus Penicillium blended into the supplement may be 3,000 to 1,000,000 U/g, preferably 10,000 to 600,000 U/g, and more preferably 15,000 to 300,000 U/g in terms of the amount of the lipase derived from genus Penicillium.

[0062] In addition, when the substance containing nuclease derived from genus Penicillium is used as the agent for improving intestinal flora of the present invention and is provided in the form of a supplement, the amount of substance containing nuclease derived from genus Penicillium blended into the supplement may be 300 to 50,000 U/g, preferably 1,000 to 30,000 U/g, and more preferably 1,500 to 15,000 U/g in terms of the amount of the nuclease derived from genus Penicillium.

[0063] For example, when the agent for improving intestinal flora of the present invention is provided in the form of a beverage, the amount of the substance containing an enzyme derived from genus Penicillium blended into the beverage may be 0.00001 to 0.02 g/mL, preferably 0.0001 to 0.01 g/mL, and more preferably 0.0002 to 0.005 g/mL in terms of the amount of the enzyme derived from genus Penicillium.

[0064] More specifically, when the substance containing lipase derived from genus Penicillium is used as the agent for improving intestinal flora of the present invention and is provided in the form of a beverage, the amount of the substance containing lipase derived from genus Penicillium blended into the beverage may be 300 to 10,000 U/mL, preferably 100 to 6,000 U/mL, and more preferably 150 to 3,000 U/mL in terms of the amount of the lipase derived from genus Penicillium.

[0065] In addition, when the substance containing nuclease derived from genus Penicillium is used as the agent for improving intestinal flora of the present invention and is provided in the form of a beverage, the amount of the substance containing nuclease derived from genus Penicillium blended into the beverage may be 3 to 500 U/mL, preferably 10 to 300 U/mL, and more preferably 15 to 150 U/mL in terms of the amount of the nuclease derived from genus Penicillium.

[0066] Further, when the agent for improving intestinal flora of the present invention is used in the field of foods and beverages, the substance containing an enzyme derived from genus Penicillium can be provided as a food additive for improving intestinal flora, either alone or in combination with other ingredients.

[0067] When the agent for improving intestinal flora of the present invention is used for a pharmaceutical preparation for oral administration, the agent for improving intestinal flora of the present invention is provided as a pharmaceutical preparation for oral administration that exert the effect of improving intestinal flora, either alone or in combination with, for example, other pharmacologically active ingredients, pharmaceutically acceptable bases, or additives to be prepared in a desired form. Forms of the pharmaceutical preparation specifically include, but are not limited to, preparations for oral administration, such as tablets, granules, powders, capsules, soft capsules, syrups, and liquids.

[0068] Examples of the bases and additives used for manufacturing the pharmaceutical preparation for oral administration include aqueous bases such as water and alcohols, oily bases, excipients, binders, filling agents, disintegrants, lubricants, algefacients, pH-adjusting agents, thickeners, antioxidants, sequestering agents, surfactants, emulsifiers, solubilizers, solubilizing agents, flavoring agents, and antiseptics.

[0069] When the agent for improving intestinal flora of the present invention is used as a pharmaceutical preparation for oral administration, the proportion of the agent blended into the pharmaceutical preparation for oral administration may be determined appropriately according to, for example, the type of active ingredient used and the form of the pharmaceutical preparation for oral administration, within a range satisfying the above-described dose.

[0070] For example, when the agent for improving intestinal flora of the present invention is provided in the form of a solid or semi-solid pharmaceutical preparation for oral administration, the amount of the substance containing an enzyme derived from genus Penicillium blended into the solid or semi-solid pharmaceutical preparation for oral administration may be 0.001 to 0.5 g, preferably from 0.01 to 0.3 g, and more preferably from 0.02 to 0.15 g in terms of the amount of the enzyme derived from genus Penicillium per g of the pharmaceutical preparation for oral administration.

[0071] More specifically, when the substance containing lipase derived from genus Penicillium is used as the agent for improving intestinal flora of the present invention and is provided in the form of a solid or semi-solid pharmaceutical preparation for oral administration, the amount of the substance containing lipase derived from genus Penicillium blended into the solid or semi-solid pharmaceutical preparation for oral administration may be 3,000 to 1,000,000 U/g, preferably 10,000 to 600,000 U/g, and more preferably 15,000 to 300,000 U/g in terms of the amount of the lipase derived from genus Penicillium.

[0072] In addition, when the substance containing nuclease derived from genus Penicillium is used as the agent for improving intestinal flora of the present invention and is provided in the form of a solid or semi-solid pharmaceutical preparation for oral administration, the amount of the substance containing nuclease derived from genus Penicillium blended into the solid or semi-solid pharmaceutical preparation for oral administration may be 300 to 50,000 U/g, preferably 1,000 to 30,000 U/g, and more preferably 1,500 to 15,000 U/g in terms of the amount of the nuclease derived from genus Penicillium.

[0073] More specifically, when the substance containing nuclease derived from genus Penicillium is used as the agent for improving intestinal flora of the present invention and is provided in the form of a liquid pharmaceutical preparation for oral administration, the amount of the substance containing nuclease derived from genus Penicillium blended into the liquid pharmaceutical preparation for oral administration may be 30 to 10,000 U/mL, preferably 100 to 6,000 U/mL, and more preferably 150 to 3,000 U/mL in terms of the amount of the lipase derived from genus Penicillium.

[0074] In addition, when the substance containing nuclease derived from genus Penicillium is used as the agent for improving intestinal flora of the present invention and is provided in the form of a liquid pharmaceutical preparation for oral administration, the amount of the substance containing nuclease derived from genus Penicillium blended into the liquid pharmaceutical preparation for oral administration may be 3 to 500 U/mL, preferably 10 to 300 U/mL, and more preferably 15 to 150 U/mL in terms of the amount of the nuclease derived from genus Penicillium.

[0075] When the agent for improving intestinal flora of the present invention is used for animal feed or a pet food, the agent for improving intestinal flora of the present invention is provided as animal feed or a pet food that exerts the effect of improving intestinal flora, either alone or in combination with other animal feed ingredients to be prepared in a desired form. Examples of the animal feed ingredients used for the animal feed or pet food include cereal crops, such as corn, wheat, barley, and rye; bran, such as wheat bran and rice bran; dregs, such as corn gluten meal and corn germ meal; animal-derived feed, such as skimmed milk powder, whey, fish flour, and powdered bone; yeasts, such as brewer's yeast; calcium, such as calcium phosphate and calcium carbonate; vitamins; amino acids; and saccharides.

[0076] When the agent for improving intestinal flora of the present invention is used as animal feed or a pet food, the proportion of the agent blended into the animal feed or pet food may be determined appropriately according to, for example, the form and type of the animal feed or pet food, and the type of the animal to which the agent is applied.

[0077] For example, the amount of the substance containing an enzyme derived from genus Penicillium blended into the animal feed or per food may be 0.00001 to 0.02 g, preferably from 0.0001 to 0.01 g, and more preferably 0.0002 to 0.005 g in terms of the amount of the enzyme derived from genus Penicillium per g of the feed or pet food.

[0078] More specifically, when the substance containing lipase derived from genus Penicillium is used as the agent for improving intestinal flora of the present invention, the amount of the substance containing lipase derived from genus Penicillium blended into the animal feed or pet food may be 10 to 2,000 U/g, preferably 20 to 1000 U/g, and more preferably 50 to 500 U/g in terms of the amount of the lipase derived from genus Penicillium.

[0079] In addition, when the substance containing nuclease derived from genus Penicillium is used as the agent for improving intestinal flora of the present invention, the amount of the substance containing nuclease derived from genus Penicillium blended into the animal feed or pet food may be 1 to 200 U/g, preferably 2 to 150 U/g, and more preferably 5 to 100 U/g in terms of the amount of the nuclease derived from genus Penicillium.

EXAMPLES

[0080] The present invention is described more specifically below with reference to examples, although the present invention should not be construed to be limited to these examples.

Test Example 1: Effect of Enzyme Derived from Genus Penicillium on Intestinal Flora

[0081] Using SD rats (male, 4 weeks old, from Hiroshima Institute for Experimental Animals), influence of the ingestion of an enzyme preparation containing a lipase derived from Penicillium camemberti (Amano Enzyme Inc.) (lipase activity: 50 U/mg) or an enzyme preparation containing a nuclease derived from Penicillium citrinum (5-phosphodiesterase) (Amano Enzyme Inc.) (nuclease activity: 7U/mg) on the intestinal flora was investigated. In the experiment, the rats were divided into two groups of a control group and a working group, with eight rats per group. The rats of each group were fed ad libitum for 14 days with the animal feed shown in Table 1.

TABLE-US-00001 TABLE 1 Control Example 1 Example 2 Example 3 Enzyme preparation 0.2 0.4 containing a lipase derived from Penicillium camemberti Enzyme preparation 0.2 containing a nuclease derived from Penicillium citrinum Beef tallow 30.0 30.0 30.0 30.0 Casein 20.0 20.0 20.0 20.0 L-Cysteine 0.3 0.3 0.3 0.3 Vitamin mixture.sup.# 1.0 1.0 1.0 1.0 Mineral mixture.sup.# 3.5 3.5 3.5 3.5 Cellulose 5.0 5.0 5.0 5.0 Sucrose 20.0 20.0 20.0 20.0 Corn starch 20.2 20.0 19.8 20.0 Total 100.0 100.0 100.0 100.0 The unit of the content of each component in the table is % by weight. .sup.#refers to AIN-93 (a standard purified diet composition for mice and rats formulated by the American Institute of Nutrition (AIN)).

[0082] After 14 days of feeding with the animal feed, the body weights of the rats in each group were determined, and, after extraction of DNA from the cecal contents of the rats, the 16S rRNA gene sequencing analysis (Mishima et al., Am J Physiol Renal Physiol, 2018, 315: F824-F833) was performed to quantify numbers of bacteria of genus Bifidobacterium and genus Lactobacillus in the cecal contents.

[0083] The results are shown in Table 2. In Examples 1 to 3, the numbers of bacteria of genus Bifidobacterium and bacteria of genus Lactobacillus in the intestine were both higher than those in the control. That is, it was revealed that ingestion of each enzyme derived from a fungus of genus Penicillium promoted the proliferation of bacteria of genus Bifidobacterium and bacteria of genus Lactobacillus in the intestine, leading to improvement of the intestinal flora.

TABLE-US-00002 TABLE 2 Average body weight and 16S rRNA sequencing analysis results Control Example 1 Example 2 Example 3 Average body weight (g) 203 4 202 3 204 2 198 3 Bacteria of genus 0.06 6.19 7.58 14.27 Bifidobacterium 0.06 0.79 2.21 3.16* (% of total bacteria) Bacteria of genus 7.26 25.15 46.03 56.56 Lactobacillus 1.87 4.59 8.94* 8.94* (% of total bacteria) The measurement results in the table are expressed as mean standard deviation (SE), and * indicates p < 0.05 by Student's t test.