阅读说明：本技术 植物化学物质吸收促进用组合物 (Composition for promoting absorption of phytochemicals ) 是由 森藤雅史 北出晶美 市川聪美 于 2019-08-07 设计创作，主要内容包括：公开了一种难水溶性植物化学物质吸收促进用组合物,其能够显著提高植物化学物质向体内的摄入、特别是向血液中的转移速度和/或转移量。由德氏乳杆菌保加利亚亚种OLL1251产生的、含有多糖的乳酸菌产生物能够显著提高植物化学物质向体内的摄入、特别是向血液中的转移速度和/或转移量,因此,本发明的植物化学物质吸收促进用组合物将含有多糖的乳酸菌产生物作为有效成分。进而,该植物化学物质吸收促进用组合物作为食品添加用组合物是有用的,另外,添加有这些的饮食品或饮食品组合物增加了促进植物化学物质吸收的效果。(Disclosed is a composition for promoting the absorption of a poorly water-soluble phytochemical, which can significantly improve the rate and/or amount of the phytochemical taken into the body, particularly the transfer into the blood. Since the polysaccharide-containing lactic acid bacterium product produced by lactobacillus delbrueckii subsp. bulgaricus OLL1251 can significantly improve the rate of and/or the amount of the transfer of a phytochemical into the body, particularly into the blood, the phytochemical absorption-promoting composition of the present invention contains the polysaccharide-containing lactic acid bacterium product as an active ingredient. Further, the phytochemical absorption-promoting composition is useful as a composition for adding food, and foods and drinks or food compositions containing the same have an increased effect of promoting the absorption of phytochemicals.)

1. A composition for promoting the absorption of a hardly water-soluble phytochemical, which comprises a polysaccharide-containing lactic acid bacterium-producing substance as an active ingredient,

the lactic acid bacteria-producing substance is produced by Lactobacillus delbrueckii subsp.bulgaricus OLL 1251.

2. The composition for promoting absorption of poorly water-soluble phytochemicals according to claim 1, wherein the lactic acid bacteria producer is produced by a combination of Lactobacillus delbrueckii subsp.

3. The composition for promoting absorption of a poorly water-soluble phytochemical according to claim 1 or 2, wherein a dissolution rate of the poorly water-soluble phytochemical in water is 88% or less.

4. The composition for promoting absorption of a poorly water-soluble phytochemical according to claim 1 or 2, wherein a dissolution rate of the poorly water-soluble phytochemical in water is 50% or less.

5. The composition for promoting absorption of a poorly water-soluble phytochemical according to any one of claims 1 to 4, wherein the poorly water-soluble phytochemical is selected from the group consisting of a polyphenol, an organosulfur compound, and a terpenoid compound.

6. The composition for promoting absorption of a poorly water-soluble phytochemical according to claim 5, wherein the polyphenol is a flavonoid compound.

7. The composition for promoting absorption of a poorly water-soluble phytochemical according to claim 6, wherein the flavonoid is selected from the group consisting of flavones, isoflavones, flavonols, flavanones, flavan-3-ols, and anthocyanidins, and analogues thereof.

8. The composition for promoting absorption of a poorly water-soluble phytochemical according to claim 7, wherein the flavonoid is a flavone, an isoflavone, a flavonol, a flavanone, a flavan-3-ol, or an analog thereof.

9. The poorly water-soluble phytochemical absorption-promoting composition according to claim 5, wherein the terpenoid is tetraterpene and an analog thereof.

10. The composition for promoting absorption of a poorly water-soluble phytochemical according to any one of claims 1 to 9, wherein the poorly water-soluble phytochemical is selected from quercetin, genistein, epicatechin, luteolin, naringenin, hesperidin, β -carotene, α -carotene, β -cryptoxanthin, capsanthin, lutein, lycopene and the like thereof.

11. The composition for promoting absorption of a poorly water-soluble phytochemical according to any one of claims 1 to 10, wherein the lactic acid bacterium-producing substance is fermented milk.

12. A food additive comprising the composition for promoting absorption of a poorly water-soluble phytochemical according to any one of claims 1 to 11.

13. A food or drink or a food or drink composition comprising the composition for promoting absorption of a hardly water-soluble phytochemical according to any one of claims 1 to 11.

14. A method for promoting the intake of a poorly water-soluble phytochemical into the body of a human or an animal, which comprises administering or allowing the intake of a polysaccharide-containing lactic acid bacterium-producing substance to the human or the animal.

15. The method according to claim 14, wherein the polysaccharide-containing lactic acid bacterium productis in the form of the composition for promoting absorption of a poorly water-soluble phytochemical according to any one of claims 1 to 11.

16. Use of a polysaccharide-containing lactic acid bacterium-producing substance for promoting the intake of a poorly water-soluble phytochemical into the human or animal body.

17. Use of a lactic acid bacterium-derived product containing a polysaccharide for producing the composition for promoting absorption of a poorly water-soluble phytochemical according to any one of claims 1 to 11.

18. Lactobacillus delbrueckii subspecies bulgaricus OLL1251(NITE BP-02703).

Technical Field

The present invention relates to a phytochemical absorption-promoting composition having an action of promoting absorption of hardly water-soluble Phytochemicals (Phytochemicals) into the body.

Background

Phytochemicals are generally not plant-derived compounds that are required for maintaining normal body function, but that have a positive impact on health. For example, isoflavone, which is a polyphenol, is rich in soybean and shows the function of improving climacteric disorder/preventing osteoporosis, and quercetin is rich in onion and shows the function of improving blood flow and reducing body fat. In addition, the beta-carotene, which is a terpenoid, is rich in carrot and pumpkin, and shows the function of maintaining visual function, mucous membrane in vivo, skin and immune function, and the lycopene is rich in tomato, and shows the function of reducing blood cholesterol and blood pressure.

It is known that: many phytochemicals are decomposed and lost by processing and cooking, and further, have low transferability into the living body because of a large amount of components that are difficult to be water-soluble. As a method for promoting the absorption of a hardly water-soluble phytochemical, the following methods are generally used: a method for improving solubility and dispersibility by emulsification preparation; a method of reducing particle size; a method of amorphization is performed.

Further, as a method for promoting absorption of a poorly water-soluble phytochemical, for example, japanese patent application laid-open No. 2016-216440 (patent document 1) discloses a method for promoting absorption of catechins, which comprises a step of selecting a catechin-based absorption promoter selected from the group consisting of resveratrol, hesperetin, momordica grosvenori (Siraitia grosvenorii) extract, Zizyphus jujuba (Zizyphus jujuba var. inermis) extract, Citrus aurantifolia (Citrus aurantifolia) extract, lemon (Citrus limon) extract, pineapple (anas comosus) extract, apigenin, glucose, difructose-1, 2'; 2, 3' bisanhydride III, sucralose, aspartame or a salt thereof, erythritol, inositol, citric acid or a salt thereof, phytic acid or a salt thereof, and gallic acid or a salt thereof.

Further, japanese patent laid-open No. 2016 and 93143 (patent document 2) discloses: by adding polyphenols such as catechin having low bioavailability to fats and carbohydrates of a specific formulation, absorption and accumulation of polyphenols such as catechin in blood plasma can be improved.

In addition, Japanese patent application laid-open No. 2016-506381 (patent document 3) discloses a catechin bioavailability enhancer comprising cyclodextrin as an active ingredient.

However, these documents do not disclose that a lactic acid bacterium product containing a polysaccharide as an active ingredient has an effect of promoting absorption of a phytochemical.

On the other hand, a combination of lactic acid bacteria and a phytochemical is disclosed in, for example, japanese patent laid-open No. 8-322464 (patent document 4). This publication discloses: fermented milk containing about 0.1 to 2000ppm of catechins and tocopherols is contained in yogurt containing lactic acid bacteria and bifidobacteria, respectively, and the survival rate of bifidobacteria is said to be improved. In addition, Japanese patent laid-open No. 2015-527076 (patent document 5) describes a method for producing a milk product-based nutritional composition having a thick texture, and describes that phytochemicals can be contained. However, none of these patent documents discloses that the lactic acid bacteria product has an effect of promoting the absorption of phytochemicals.

In addition, some of the present inventors have previously found that a polysaccharide-containing lactic acid bacterium product significantly increases the rate and/or amount of transfer of a poorly water-soluble phytochemical into the body, particularly into the blood, and proposed a composition having an action of promoting absorption of a poorly water-soluble phytochemical into the body (PCT/JP 2018/010366).

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2016-216440

Patent document 2: japanese patent laid-open publication No. 2016-93143

Patent document 3: japanese patent laid-open publication No. 2016-506381

Patent document 4: japanese laid-open patent publication No. 8-322464

Patent document 5: japanese laid-open patent publication No. 2015-527076

Disclosure of Invention

Problems to be solved by the invention

The inventors of the present invention found that: the polysaccharide-containing lactic acid bacterium product produced by a specific strain further significantly improves the rate and/or amount of the transfer of a poorly water-soluble phytochemical into the body, particularly into the blood.

Further, it has been found that a lactic acid bacterium product containing a polysaccharide can be efficiently produced by combining a specific strain with the previously proposed phytochemical absorption-promoting composition. The present invention is based on these findings.

Accordingly, an object of the present invention is to provide a phytochemical absorption-promoting composition having an action of promoting absorption of a phytochemical that is hardly soluble in water into the body.

Further, an object of the present invention is to provide a food additive comprising the hardly water-soluble phytochemical absorption-promoting composition, and a food or drink or a food or drink composition to which the composition is added.

Means for solving the problems

The hardly water-soluble phytochemical absorption-promoting composition of the present invention is characterized by comprising a polysaccharide-containing lactic acid bacterium product as an active ingredient, wherein the lactic acid bacterium product is produced by lactobacillus delbrueckii subsp.

The food additive, food, drink, or drink composition of the present invention contains the hardly water-soluble phytochemical absorption-promoting composition of the present invention.

Further, the present invention relates to a method for promoting the intake of a poorly water-soluble phytochemical into the body of a human or an animal, which comprises administering or causing the intake of a polysaccharide-containing lactic acid bacterium-producing substance to the human or the animal.

The present invention also relates to the use of a polysaccharide-containing lactic acid bacterium-producing substance for promoting the intake of a poorly water-soluble phytochemical into the human or animal body.

The present invention also relates to the use of a polysaccharide-containing lactic acid bacterium-producing substance for producing the composition for promoting absorption of a poorly water-soluble phytochemical substance.

Drawings

Fig. 1 is a graph showing changes in the concentration of β cryptoxanthin in serum when rats were administered β cryptoxanthin alone or β cryptoxanthin and yogurt together.

Fig. 2 is a graph showing changes in the concentration of β cryptoxanthin in serum when rats were administered capsanthin alone or both capsanthin and yogurt.

Fig. 3 is a graph showing changes in the concentration of hesperetin conjugate in serum when only hesperidin is administered to rats, or hesperidin and yogurt are administered simultaneously.

Detailed Description

Phytochemicals

In the present invention, "phytochemicals" means: natural chemical substances present in plants, modified forms thereof, and compositions containing these substances are compounds or compositions which are not required for maintaining normal body functions but are taken as substances having a positive effect on maintaining/improving health or which are taken in the future. Thus, in the present invention, the phytochemical includes, in addition to a compound of plant origin which is pure or has a certain purity, a substance in the form of a composition of plant origin, such as a fraction, containing such a compound as a main component.

According to a preferred embodiment of the present invention, the phytochemical is, for example, a polyphenol, an organosulfur compound, a terpenoid compound, or the like.

In the present invention, preferable examples of the polyphenol include flavonoids, diketones, tetraterpenes, and the like, and specific examples thereof include flavonoids such as flavones (e.g., apigenin, luteolin, and the like), isoflavones (e.g., genistein, daidzein, and the like), flavonols (e.g., quercetin, myricetin, kaempferol, and the like), flavanones (e.g., hesperetin, naringenin, and the like), flavan-3-ols (e.g., catechin, epicatechin, and the like), anthocyanidins (e.g., cyanidin, delphinidin, and the like). Further, as the diketone, curcumin is exemplified.

Further, preferable examples of the organic sulfur compound include isothiocyanates (e.g., sulforaphane), cysteine sulfoxides (e.g., methyl cysteine sulfoxide), and sulfonium compounds (e.g., allicin).

Examples of the terpenoid include tetraterpenes, and specific examples thereof include carotenoids (e.g., β -carotene, α -carotene, β -cryptoxanthin, lycopene, lutein, capsanthin, astaxanthin, and the like).

In the present invention, the above-mentioned phytochemicals also include their analogs, and examples of preferred examples thereof include glycosides (genistin) and conjugates (glucuronic acid conjugate, sulfuric acid conjugate) for genistein, methylated compounds (isorhamnetin), glycosides (rutin, quercetin glucoside), conjugates (glucuronic acid conjugate, sulfuric acid conjugate) for quercetin, and glycosides (hesperidin) and conjugates (glucuronic acid conjugate, sulfuric acid conjugate) for kaempferol. Further, as epicatechin and catechin, isomers (catechin), polymers (procyanidin B1, procyanidin B2, procyanidin B5, procyanidin C1, etc.), conjugates (glucuronic acid conjugate, sulfuric acid conjugate), and gallic esters (epicatechin gallate, epigallocatechin gallate) can be cited. For hesperetin, mention may be made of glycosides (hesperidin) and conjugates (glucuronic acid conjugate, sulfuric acid conjugate), and for naringenin, mention may be made of glycosides (kaempferol, astragaloside) and conjugates (glucuronic acid conjugate, sulfuric acid conjugate). Examples of β -carotene include isomers (α -carotene, γ -carotene) and metabolites (retinol palmitate, apo-10-carotenal, retinol), and examples of lycopene include metabolite (apo-10-lycopene aldehyde).

Furthermore, in the present invention, the phytochemicals also include extracts and concentrates derived from plants. Preferred examples of the genistein include extracts and concentrates derived from soybean, adzuki bean, pea and broad bean, extracts and concentrates derived from onion and apple, extracts and concentrates derived from kaempferol, extracts and concentrates derived from tea and broccoli, extracts and concentrates derived from cacao bean and tea, extracts and concentrates derived from orange peel, extracts and concentrates derived from grapefruit and orange, extracts and concentrates derived from beetroot, perilla, chrysanthemum coronarium and green pepper, extracts and concentrates derived from cacao bean, extracts and concentrates derived from beta-carotene, The concentrate includes, for alpha-carotene, carrot extract and concentrate, lycopene extract and concentrate, and xanthophyll extract and concentrate, for yellow carrot, spinach, and marigold, and for capsanthin, red pepper, and green pepper extract and concentrate.

In the present invention, the phytochemical is a substance that is hardly soluble in water. According to a preferred embodiment of the present invention, the poorly water-soluble phytochemicals are: the dissolution rate in water is 88% or less, more preferably 50% or less, still more preferably 20% or less, and most preferably 1% or less. Here, the "dissolution rate" in the present invention is an index indicating that a compound is easily dissolved in water, and is a parameter as follows: the concentration (w/v) of the supernatant obtained by shaking and dissolving the compound in pure water and then centrifuging the solution was divided by the concentration (w/v) before shaking and dissolving the solution, and the value was expressed as a percentage (%). The concentration can be determined using a spectrophotometer. In the present invention, a solution prepared by adding 33.3mg of the phytochemical compound to 10mL of pure water is preferably used for the measurement of the "dissolution rate". In the case of a phytochemical that is less soluble in water, the "dissolution rate" can be measured using a solution prepared by adding 3.3mg to 10mL of pure water. The temperature conditions for measuring the "dissolution rate" were 21. + -. 2 ℃. The poor water solubility of phytochemicals can be expressed by using as an index "the concentration (w/v) of a supernatant obtained by shaking and dissolving a compound in pure water and then performing centrifugal separation" obtained every time the "dissolution rate" is measured, and in this case, the dissolution rate in water is 88% or less, which corresponds to 293mg/100g or less. It is considered that the phytochemicals to be the absorption-promoting object in the present invention are not necessarily dissolved in water. Namely, it is considered that: the effect of promoting absorption according to the present invention can also be obtained for phytochemicals ingested in a solid state or suspended in water.

Composition for promoting absorption

In the present invention, the absorption promotion of phytochemicals means: the intake of phytochemicals into the body, particularly the transfer rate and/or amount into the blood, was significantly increased as compared to the control group that was ingested without the polysaccharide-containing lactic acid bacteria production. Specifically, either or both of a high blood concentration as compared to the control group or a large area under the blood concentration-time curve (AUC) as compared to the control group are produced after the administration. Thus, the effect of the intake of phytochemicals can be obtained in a smaller amount or in a shorter time, while the raw material cost can be reduced. In addition, by adding the phytochemical absorption-promoting composition of the present invention to foods and beverages, or food and beverage compositions, the added value of foods and beverages, or the like, and the commercial value can be improved.

Polysaccharide-containing lactic acid bacterium product

In the present invention, the "lactic acid bacterium product" containing a polysaccharide broadly includes not only a lactic acid bacterium fermentation product but also a composition containing a polysaccharide obtained by fermentation of a lactic acid bacterium such as a lactic acid bacterium culture or a lactic acid bacterium metabolite.

In the present specification, "polysaccharide" refers to a sugar chain polymer composed of saccharides such as galactose, glucose, rhamnose, mannose, and N-acetylglucosamine. The polysaccharide may contain an acidic polysaccharide having a phosphate group bonded thereto, in addition to a neutral polysaccharide. In addition, the molecular weight thereof is usually in the range of 5000 to 50 ten thousand.

In the present invention, as the "lactic acid bacterium-producing substance", those produced by Lactobacillus delbrueckii subsp. bulgaricus OLL1251 (hereinafter, sometimes abbreviated as "Lactobacillus bulgaricus OLL 1251" or "OLL 1251") are used. The "lactic acid bacteria product" produced by the OLL1251 has a more excellent effect of promoting the absorption of phytochemicals than the similar strains.

In addition, according to a preferred embodiment of the present invention, it is preferable to use "lactic acid bacteria-producing material" produced by combining OLL1251 and streptococcus thermophilus OLS3290 (hereinafter, sometimes abbreviated as "OLS 3290"). By combining the OLL1251 and the OLS3290, the following advantages can be obtained: can efficiently produce a "lactic acid bacterium product", and can obtain a "lactic acid bacterium product" having an excellent absorption-promoting effect on phytochemicals in a particularly short fermentation time.

The aforementioned Lactobacillus delbrueckii subspecies Bulgaria OLL1251 has undergone international collection based on the Budapest treaty at the national institute of technology and evaluation (Collection) in 2018, 4/25.4, patent microorganism Collection (NPMD) (Chamber 2-5-8122 of Gentianjin, Qianye county, Japan) under the deposit number NITE BP-02703.

Furthermore, Streptococcus thermophilus OLS3290 was deposited internationally under the Budapest treaty at 2004 on 19.1.2004 (Collection date) under the deposit number FERM BP-19638 at the national institute of technology and evaluation, patent deposit of microorganisms (NPMD).

In the present specification, the term "lactic acid bacteria fermentation product" means: a culture obtained by fermentation using lactobacillus delbrueckii subsp bulgaricus OLL1251 or, as a preferred embodiment, a combination of lactobacillus delbrueckii subsp bulgaricus OLL1251 and streptococcus thermophilus OLS3290, a composition containing the same, and a composition treated with the same. Therefore, the lactic acid bacteria fermented product includes a fermented product of lactobacillus delbrueckii subsp bulgaricus OLL1251 or a combination of lactobacillus delbrueckii subsp bulgaricus OLL1251 and streptococcus thermophilus OLS3290, and a treated product thereof, for example, includes: a culture filtrate or a culture supernatant obtained by sterilizing a culture (lactic acid bacteria fermentation product) by filtration/centrifugation, membrane separation or the like; a concentrate, a paste, a diluted product, or a dried product (for example, one obtained by freezing, heating, or reducing pressure) obtained by concentrating the culture filtrate/culture supernatant, lactic acid bacteria fermented product, or the like using a rotary evaporator or the like. The treatment may be carried out by combining 1 or more of the above-mentioned treatment steps such as filtration, centrifugation, sterilization such as membrane separation, precipitation, concentration, pasting, dilution, and drying. Examples of the culture medium include a skim milk powder medium containing a yeast extract, and an MRS medium.

According to a preferred embodiment of the present invention, the lactic acid bacterium product is particularly preferably a lactic acid bacterium lactobacillus delbrueckii subsp bulgaricus OLL1251, or a milk fermentation product, a milk culture, or a milk metabolite, which combines lactobacillus delbrueckii subsp bulgaricus OLL1251 with streptococcus thermophilus OLS 3290. Examples of the milk fermentation product, the milk culture product, and the milk metabolite include fermented milk (yogurt). In the present invention, the fermented milk (yogurt) may preferably be provided as a supernatant. The fermented milk can be added with culture solution such as skimmed milk powder and whey decomposition product, thickener such as pectin, guar gum, xanthan gum, carrageenan and processed starch, and gelling agent.

Examples of the milk in the present invention include animal milk such as cow milk, processed products thereof (e.g., skim milk, whole milk powder, skim milk powder, condensed milk, casein, whey, fresh cream, compound cream, butter, buttermilk powder (butter milk powder), cheese, etc.), and vegetable milk such as soybean milk. The milk may be sterilized or not.

According to one embodiment of the present invention, a material called a fermented milk material mixture can be used as a material for fermented milk (yogurt). The fermented milk raw material mixture is a mixture containing raw milk and other ingredients. The fermented milk raw material mixture can be obtained by, for example, heating and dissolving raw materials that are commonly used in the production of fermented milk, such as raw milk, water, and other optional ingredients (e.g., granulated sugar, sugars, sweeteners, sour agents, minerals, vitamins, and flavors), and mixing them. The raw milk may also contain water, raw milk, sterilized milk, skim milk, whole milk powder, skim milk powder, whole concentrated milk, skim concentrated milk, buttermilk, butter, cream, cheese, etc. The raw milk may contain Whey Protein Concentrate (WPC), Whey Protein Isolate (WPI), α -lactalbumin (α -La), β -lactoglobulin (β -Lg), and the like.

In the present invention, fermented milk (yogurt) can be prepared by a method that is conventional in the art. That is, the fermented milk (yogurt) can be produced through the steps of preparing a raw material mixture, sterilizing (heating) the raw material mixture, cooling the raw material mixture, adding a fermentation agent, fermenting, cooling the fermented milk, and the like. In these steps, the usual conditions used in the production of fermented milk (yogurt) can be suitably employed. It is preferable to carry out the (heating) sterilization step of the raw material mixture, the cooling step of the raw material mixture, the addition step of the fermentation agent, the fermentation step, and the cooling step of the fermented milk in this order.

In the present invention, as a medium for culturing the lactic acid bacteria lactobacillus delbrueckii subsp bulgaricus OLL1251 or the combination of lactobacillus delbrueckii subsp bulgaricus OLL1251 and streptococcus thermophilus OLS3290, a medium generally used in the art for culturing lactic acid bacteria can be used. That is, any medium may be used as long as it contains a suitable amount of nutrients such as nitrogen sources and inorganic substances in addition to the main carbon source. As the carbon source, lactose, glucose, sucrose, fructose, powder hydrolysate, molasses and the like can be used depending on the assimilation of the bacteria used. As the nitrogen source, organic nitrogen-containing substances such as casein hydrolysate, whey protein hydrolysate, α -lactalbumin, β -lactoglobulin, glycomacropeptide and soybean protein hydrolysate can be used. In addition, as the proliferation promoter, meat extract, fish meat extract, yeast extract, and the like can be used.

In the present invention, the lactic acid bacterium Lactobacillus delbrueckii subspecies bulgaricus OLL1251 or the combination of Lactobacillus delbrueckii subspecies bulgaricus OLL1251 and Streptococcus thermophilus OLS3290 may be cultured in an anaerobic state or in a microaerobic state used for liquid static culture or the like. The culture under anaerobic conditions may be carried out by a known method such as a method of culturing in a carbon dioxide gas phase, but other methods may be used. The culture temperature is usually preferably in the range of 30 ℃ to 47 ℃, more preferably 35 ℃ to 46 ℃, and still more preferably 37 ℃ to 45 ℃. The pH of the medium during the culture of lactic acid bacteria is preferably maintained in the range of 6 to 7, but may be in other pH ranges as long as the bacteria grow. The culture time of the lactic acid bacteria and the like is preferably in the range of usually 1 hour to 48 hours, more preferably 1.5 hours to 36 hours, and still more preferably 2 hours to 24 hours.

According to one embodiment of the present invention, the fermented milk (yogurt) typically has a non-fat milk solid content of 8 wt% or more and a lactic acid bacteria count or yeast count of 10⁶More than 10 per ml¹¹In the range of less than or equal to one/ml.

Composition, form, and optional ingredients of phytochemical absorption-promoting composition

In the present invention, the "composition for promoting absorption" can be used as the "lactic acid bacterium product" in the form of a fermentation product, culture, metabolite, or the like of the lactic acid bacterium lactobacillus delbrueckii subspecies bulgaricus OLL1251, or a combination of the lactobacillus delbrueckii subspecies bulgaricus OLL1251 and streptococcus thermophilus OLS3290, but it is preferably formulated and used. Therefore, in the present invention, the term "composition for promoting absorption" includes, for example, a composition provided in the form of a pharmaceutical, a preparation to be taken directly, preferably orally, that is, a so-called nutritional supplement, and a composition added as a food additive to other foods and drinks to provide the foods and drinks with a phytochemical absorption promoting effect.

In addition, according to the present invention, a food or drink, a processed food or drink, and a food or drink composition containing the phytochemical absorption-promoting composition of the present invention are also included in the present invention.

In the invention, the preparation refers to: acceptable additives are used in combination for formulation, and the oral formulation is preferably prepared by a conventional method. The preparation can be in the form of solid preparation such as tablet, powder, fine granule, capsule, pill, sustained release agent, etc., or liquid preparation such as solution, suspension, emulsion, etc. Examples of additives acceptable for formulation include excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, colorants, flavors, buffers, antioxidants, pH adjusters, and the like. Specific examples of the food additive include a processed seasoning, a flavoring, a cooking mixture, and the like.

In the present invention, the food or drink and the food or drink composition are processed for human or animal diet, and are not particularly limited as long as they are in a form that can be orally ingested, such as a solution, a suspension, an emulsion, a powder, a solid molded product, and the like. Examples of the food, drink and drink composition include milk products such as milk beverages (including processed milk), yogurt, lactic acid bacteria beverages, fermented milk, ice cream, butter-type products, and cheese-type products; beverages such as refreshing beverages, fruit juice beverages, vegetable beverages, soybean milk beverages, coffee beverages, tea beverages, jelly beverages, nutritional beverages, beauty beverages, powdered beverages such as cocoa and milkshakes, powdered sports beverages, nutrition-enhanced powdered beverages, beauty powdered foods, powdered soups, cake materials, concentrated beverages, and alcoholic beverages; wheat flour products such as bread, macaroni, flour, cake mix, fried flour, bread flour, etc.; snacks such as chocolate, chewing gum, candy, biscuit, fruit gum, snack, Japanese snack, jelly, pudding, etc.; curry, pasta, canned Japanese food, stewed Japanese dish, and steamed food of Japanese food; processing oil and fat such as butter, margarine, butter, mayonnaise, etc.; food such as freeze-dried food; canned agricultural products such as agricultural products, jam/fruit jam, pickles, boiled beans, grains, porridge and the like; processed aquatic products; processed livestock products; frozen foods such as pizza, baked rice (Doria), baked foods, side dishes, and fried foods; liquid diet, semi-liquid diet, and animal feed, tablet, and oral cosmetic.

In the present invention, the food, drink, and food composition may contain classified foods such as functional foods, health nutritional foods, health foods, foods for specified health use, functional foods, nutritional functional foods, foods for patients, milk powder for infants, milk powder for pregnant women or nursing women, or foods and drinks with a disease risk reduction indication. Here, the indication of reduction in disease risk is an indication of food or drink having a possibility of reducing disease risk, and is an indication or approved indication based on or made with reference to a standard established by the food standards committee (commission on the law) under the FAO/WHO contract.

In the present invention, any component may be added to the food or drink and the food or drink composition as necessary. Such optional ingredients are not particularly limited, and examples thereof include sweeteners, souring agents, juices of vegetables, fruits and extracts thereof, nutrients such as vitamins, minerals and amino acids, lactic acid bacteria (excluding essential lactic acid bacteria according to the embodiment of the present invention), useful microorganisms and fermentation products thereof such as bifidobacterium and propionibacteria, functional sugars such as oligosaccharide, conventional functional materials such as royal jelly, glucosamine, astaxanthin, collagen and polyphenol, flavors, pH adjusters, excipients, souring agents, coloring agents, emulsifiers, storage agents, and the like, which are generally blended in foods and drinks.

As is clear from the above, according to one embodiment of the present invention, there is provided use of a lactic acid bacterium product containing a polysaccharide for producing the composition for promoting absorption of a poorly water-soluble phytochemical of the present invention.

Method for taking composition for promoting absorption of phytochemicals

In the present invention, the intake amount of the phytochemical absorption-promoting composition can be determined as appropriate, and according to one embodiment of the present invention, the intake amount of the polysaccharide is an amount of about 200 μ g or more per day, preferably in a range of 200 μ g or more per day and 60000 μ g or less per day, more preferably in a range of 300 μ g or more per day and 45000 μ g or less per day, further preferably in a range of 400 μ g or more per day and 30000 μ g or less per day, and particularly preferably in a range of 500 μ g or more per day and 15000 μ g or less per day (it should be noted that the expression "mass/day or more" is the same as the expression of "mass or more per day" and the expression of "mass/day or less" is the same as the expression of "mass or less per day"). The period of ingestion is also not particularly limited, and for example, it is preferably ingested at least 1 time orally.

According to another embodiment of the present invention, the required amount can be converted from the amount necessary for administration in an animal experiment (e.g., a mouse experiment) to the amount necessary for administration to a human body using the following formula based on the data of the food safety committee.

(necessary dose (converted value) to human body) × (female body weight lower limit value: 40kg) ÷ (safety factor: 100))

As apparent from the above, according to one embodiment of the present invention, there is provided a method for promoting the intake of a poorly water-soluble phytochemical in the human or animal body, which comprises administering or allowing the human or animal to take a polysaccharide-containing lactic acid bacterium-producing substance. In addition, according to another aspect of the present invention, there is provided use of a polysaccharide-containing lactic acid bacterium-producing substance for promoting intake of a poorly water-soluble phytochemical into the human or animal body.

Examples

In the following examples, the following strains were used for comparison.

Lactobacillus delbrueckii subspecies bulgaricus OLL1247

This strain was deposited internationally under the Budapest treaty at the national institute for technical assessment, patent microorganism Collection (NPMD) (2-5-8122, Gentianjin, Kyowa prefecture, Japan) at 3/6 (depository) 2014 under the deposit number NITE BP-01814.

Lactobacillus delbrueckii subspecies bulgaricus OLL1224

The strain was deposited internationally under the budapest treaty at 2009, day 7 and 2 (deposit date) under deposit number NITE BP-778 at the national institute of technical and assessment, the patent and microbiological collection center (NPMD).

Streptococcus thermophilus OLS3078

The strain was deposited under the Budapest treaty at 23.8.2013 (on deposit), national institute of technical and evaluation, patent microorganism Collection (NPMD), with deposit number NITE BP-01697.

In the following examples, the following measurement methods were used.

Determination of polysaccharide content in yogurt

The content of polysaccharides in yoghurt was determined according to the phenol-sulfuric acid method (Hodge et al, "Methods in carbohydrate chemistry", volume 1, page 338 (1962)). Specifically, the following is described.

First, 1g of trichloroacetic acid was added to 10g of yogurt and sufficiently stirred. Then, the yogurt added with trichloroacetic acid was subjected to a centrifugal separation treatment at 10000rpm, 10 minutes and 4 ℃, and then the supernatant was transferred to another test tube. Next, 99.5% ethanol was added to the supernatant in a volume of 2 times, and then the ethanol-added supernatant was left in a freezing chamber overnight, resulting in a precipitate in a test tube. The precipitate was centrifuged at 10000rpm for 10 minutes at 4 ℃ and then 3mL of ultrapure water was added to the obtained precipitate as a polysaccharide extract. Furthermore, 500. mu.L of a phenol reagent (5% (w/v)) was added to 500. mu.L of the polysaccharide extract solution, and the mixture was stirred, and then 2.5mL of concentrated sulfuric acid was further added to the mixture, and immediately the mixture was stirred for 10 seconds. Then, the mixture was left at room temperature for 20 minutes or more, and then the absorbance of the mixture at 490nm was measured with a spectrophotometer. A control solution was prepared as follows and its absorbance at 490nm was measured in the same manner as described above. To 500. mu.L of a standard glucose solution, 500. mu.L of a phenol reagent (5% (w/v)) was added and the mixture was stirred, and then 2.5mL of concentrated sulfuric acid was further added to the mixture and the mixture was immediately stirred for 10 seconds. Then, the mixture was left at room temperature for 20 minutes or more.

Measurement of quercetin metabolite

The quercetin conjugate and isorhamnetin conjugate as quercetin metabolites were determined as follows. To 50. mu.L of serum were added 45. mu.L (10000U/mL, manufactured by Sigma-Aldrich Co., Ltd.) of glucuronidase solution dissolved in 0.1M sodium acetate buffer solution (pH5.0) and 5. mu.L of ascorbic acid solution dissolved in 0.1M sodium acetate buffer solution (pH5.0), and the mixture was heated at 37 ℃ for 2 hours. The enzyme reaction was stopped by adding 300. mu.L of methanol, and the mixture was centrifuged (12000rpm, 10 minutes, 4 ℃). The supernatant was transferred to another tube and the solvent was removed by concentration by centrifugation. Samples for HPLC were prepared by dissolving 300. mu.L of a 50% acetonitrile solution containing 0.1% formic acid.

Nexera XR (manufactured by Shimadzu corporation) was used for HPLC, and 4500QTRAP (manufactured by SCIEX corporation) was used for MS/MS detector. As the column, ACQUITY UPLC HSST 31.8 μm (2.1X 50mm) (manufactured by Waters corporation) was used, and the column temperature was set to 40 ℃. For the mobile phase, a solution containing 0.1% formic acid was prepared as the a liquid, and an acetonitrile solution containing 0.1% formic acid was prepared as the B liquid. The solution B was held at 30% for 1 minute and then decreased to 45% in 4.5 minutes, thereby eluting the target substance. Then, the column was washed with 99% of solution B for 2 minutes and held with 30% of solution B for 3 minutes. The flow rate was set to 0.3 mL/min. MS/MS analysis was performed using ESI negative ion mode. The analysis conditions for MS/MS were set as follows: the gas curtain gas flow is 30psi, the collision gas flow is 9psi, the ion spray voltage is-4500V, the turbine gas temperature is 600 ℃, and the ion source gas is 70 psi.

Determination of beta-carotene

To 50 μ L of serum, 90 μ L of physiological saline was added, and 300 μ L of dichloromethane methanol solution (dichloromethane: methanol ═ 1: 2) was added. Further, 150. mu.L of hexane was added to extract β -carotene. The upper layer was transferred to another tube and the solvent was removed by nitrogen reflux. Samples for HPLC were prepared by dissolving 150. mu.L of 30% ethyl acetate 70% methanol solution containing 0.1% ammonium acetate.

The HPLC used was 1200 series (manufactured by Agilent Technologies, Inc.). The column used was TSKgel ODS-80TsQA (5.0X 250mm) (manufactured by Tosoh corporation), and the column temperature was set to 40 ℃. For the mobile phase, a 30% ethyl acetate 70% methanol solution containing 0.1% ammonium acetate was prepared. The flow rate was set to 0.3 mL/min, and the absorption wavelength at 450nm was measured.

Evaluation of solubility of phytochemicals

(1) Experimental methods

Use of: epicatechin (manufactured by Sigma-Aldrich Co.), catechin (manufactured by Tokyo Kasei Kogyo Co., Ltd.), quercetin (manufactured by Wako pure chemical industries, Ltd.), genistein (manufactured by Tokyo Kasei Kogyo Co., Ltd.), rutin (manufactured by Wako pure chemical industries, Ltd.), alpha glucosyl rutin (manufactured by Wako pure chemical industries, Ltd.), hesperidin (manufactured by Wako pure chemical industries, Ltd.), naringin (manufactured by Sigma-Aldrich Co., Ltd.), naringenin (manufactured by Sigma-Aldrich Co.), kaempferol (manufactured by Extra Synthase Co., Ltd.), beta-carotene (manufactured by Wako pure chemical industries, Ltd.), beta-cryptoxanthin (manufactured by Wako pure chemical industries, Ltd.), capsanthin (manufactured by Tokyo Kasei Kogyo Co., Ltd.), lycopene (manufactured by Wako pure chemical industries, Ltd.), and luteolin (manufactured by Tokyo Kasei Kogyo Co., Ltd.). 10mL of ultrapure water was added to each 33.3mg of epicatechin, catechin, quercetin, genistein, naringenin, kaempferol, and luteolin in accordance with the administration amounts used in the experimental examples. As for the phytochemical substance as glycoside, 10mL of ultrapure water was added to 67.3mg (33.3 mg in terms of quercetin), 89.6mg (33.3 mg in terms of quercetin), 67.3mg (33.3 mg in terms of hesperetin), 71.0mg (33.3 mg in terms of naringenin) of alpha glucosyl rutin, to the extent that the amount of aglycone (the part other than glucurone obtained by hydrolyzing glycosides) was 33.3 mg. As terpenoids, 10mL of ultrapure water was added to 3.3mg of each of beta-carotene and lycopene in an amount corresponding to the amount used in the experimental examples. The resulting solution was shaken for 3 hours, and then centrifuged at 2000 Xg for 10 minutes. The centrifugation supernatant was filtered using a 0.45 μ L filter. The absorbance of the centrifuged supernatant was measured using a spectrophotometer (epicatechin, catechin, hesperidin, naringin, naringenin, luteolin at 280nm, genistein at 260nm, quercetin, rutin, alpha-glucosylrutin, kaempferol at 360nm, beta-carotene, alpha-carotene, beta-cryptoxanthin at 450nm, lycopene, capsanthin at 470 nm). The respective compounds were dissolved in 80% methanol or methanol to prepare a calibration curve, and the concentration of the centrifuged supernatant was determined. The above series of operations was carried out at a temperature of 21. + -. 2 ℃. Further, the dissolution rate was calculated according to the following formula.

Dissolution rate (%) ((concentration of centrifugal supernatant after shaking dissolution (w/v) ÷ (concentration of solution before shaking dissolution (w/v)) × 100)

(2) Results

The results are shown in Table 1. The dissolution rate of catechin and alpha glucosyl rutin is above 89%, and the product is water-soluble phytochemical. On the other hand, epicatechin, genistein, quercetin, rutin, kaempferol, hesperidin, naringin, naringenin, β -carotene, α -carotene, lycopene, luteolin, β -cryptoxanthin, and capsanthin have a dissolution rate of 88% or less, and are hardly soluble in water. These results imply that: the intake of a polysaccharide-containing lactic acid bacterium product promotes the absorption of a poorly water-soluble phytochemical having a dissolution rate of 88% or less. The "concentration (w/v) of the centrifugal supernatant after the shaking and dissolution" obtained when the dissolution rate was measured is referred to as "saturation solubility", and is also shown in table 1. The phytochemicals as glycosides are described as aglycone equivalent values.

[ Table 1]

TABLE 1 dissolution rates of phytochemicals

Experimental example 1: absorption promotion of beta-carotene (OLL1251 vs. other strains)

(1) Preparation of yogurt

A culture medium containing 10% by mass of skimmed milk powder and 0.5mM sodium formate was inoculated with Lactobacillus bulgaricus OLL1251, Lactobacillus bulgaricus OLL1224, and Lactobacillus bulgaricus OLL1247, respectively, and then the culture medium was fermented at 43 ℃ to pH4.6 and heated. The yogurt thus obtained contained 136. mu.g/g, 88. mu.g/g, and 68. mu.g/g of polysaccharide, respectively.

(2) Experimental methods

After acclimating 32 rats (SD, male, 8 weeks old, Japan SLC, Inc.) for 7 days, the rats were divided into 8 groups. After a 16-hour fast, the rats in each group were given β -carotene, β -carotene and yogurt (lactobacillus bulgaricus OLL1251), β -carotene and yogurt (lactobacillus bulgaricus OLL1224), β -carotene and yogurt (lactobacillus bulgaricus OLL1247), respectively. Here, beta-carotene was administered at 5mg/kg body weight and yogurt was administered at 11.3g/kg body weight. Blood was collected from the tail vein before administration, 60 minutes, 120 minutes, 240 minutes, and 480 minutes after administration, and serum was obtained. The serum concentration of beta-carotene was determined according to the method described above.

Hereinafter, a rat group (control) to which β -carotene was administered is referred to as "β -carotene group", a rat group (example) to which β -carotene and yogurt (lactobacillus bulgaricus OLL1251) were administered is referred to as "β -carotene + OLL1251 group", a rat group (comparative example) to which β -carotene and yogurt (lactobacillus bulgaricus OLL1224) were administered is referred to as "β -carotene + OLL1224 group", and a rat group (comparative example) to which β -carotene and yogurt (lactobacillus bulgaricus OLL1247) were administered is referred to as "β -carotene + OLL1247 group".

(3) Results

The results are shown in Table 2. For the area under the plasma concentration-time curve (AUC), the amounts of β -carotene + OLL1251, β -carotene + OLL1224, and β -carotene + OLL1247 were significantly increased compared to the β -carotene group. The results indicate that ingestion of yogurt promotes the absorption of beta-carotene.

[ Table 2]

TABLE 2 plasma Carotene concentration-area under time Curve (AUC)

Mean. + -. standard deviation of the mean

P < 0.05 was significantly different from the beta carotene group

Experimental example 2: absorption promotion of beta-carotene (Streptococcus thermophilus OLS3290 alone)

(1) Preparation of yogurt

After inoculating Streptococcus thermophilus OLS3290 and Streptococcus thermophilus OLS3078 to a medium containing 10 mass% of skimmed milk powder and 0.1 wt% of casein peptide (manufactured by DOMO), the medium was fermented to pH4.6 at 43 ℃ and heated. The yogurt thus obtained contained 76.3. mu.g/g and 45.8. mu.g/g of polysaccharide, respectively.

(2) Experimental methods

After acclimating 24 rats (SD, male, 8 weeks old, Japan SLC, Inc.) for 7 days, the rats were divided into 8 groups. After a 16-hour fast, the rats in each group were given β -carotene, β -carotene and yogurt (Streptococcus thermophilus OLS3290), β -carotene and yogurt (Streptococcus thermophilus OLS3078), respectively. Here, beta-carotene was administered at 5mg/kg body weight and yogurt was administered at 11.3g/kg body weight. Blood was collected from the tail vein before administration, 60 minutes, 120 minutes, 240 minutes, and 480 minutes after administration, and serum was obtained. The serum concentration of beta-carotene was determined according to the method described above.

Hereinafter, a group of rats (control) to which β -carotene was administered is referred to as "β -carotene group", a group of rats (reference example) to which β -carotene and yogurt (streptococcus thermophilus OLS3290) were administered is referred to as "β -carotene + OLS3290 group", and a group of rats (reference example) to which β -carotene and yogurt (streptococcus thermophilus OLS3078) were administered is referred to as "β -carotene + OLS3078 group".

(3) Results

The results are shown in Table 17. For the area under the plasma concentration-time curve (AUC), there was a significant increase in the β -carotene + OLS3290 group compared to the β -carotene group. The results indicate that ingestion of yogurt promotes the absorption of beta-carotene.

[ Table 3]

TABLE 3 plasma Carotene concentration-area under time Curve (AUC)

Mean. + -. standard deviation of the mean

P < 0.05 was significantly different from the beta carotene group

Experimental example 3: absorption promotion of beta-carotene (Lactobacillus bulgaricus OLL1251 and Streptococcus thermophilus OLS3290) In combination with (1)

(1) Preparation of yogurt

A commercially available starter (a sea yogurt, Fujicco co., ltd. manufactured), lactobacillus bulgaricus OLL1247 and streptococcus thermophilus OLS3078, lactobacillus bulgaricus OLL1251 and streptococcus thermophilus OLS3290 were inoculated into a medium containing 10 mass% of skim milk powder, and the medium was fermented at 43 ℃ until ph4.6 was reached and heated. The yogurt thus obtained contained 15. mu.g/g, 54. mu.g/g, and 67. mu.g/g of polysaccharides.

(2) Experimental methods

After acclimating 32 rats (SD, male, 8 weeks old, Japan SLC, Inc.) for 7 days, the rats were divided into 8 groups. After a 16-hour fast, the rats in each group were given β -carotene, β -carotene and yogurt (lihain yogurt), β -carotene and yogurt (lactobacillus bulgaricus OLL1247 and streptococcus thermophilus OLS3078), β -carotene and yogurt (lactobacillus bulgaricus OLL1251 and streptococcus thermophilus OLS3290), respectively. Here, beta-carotene was administered at 5mg/kg body weight and yogurt was administered at 11.3g/kg body weight. Blood was collected from the tail vein before administration, 60 minutes, 120 minutes, 240 minutes, and 480 minutes after administration, and serum was obtained. The serum concentration of beta-carotene was determined according to the method described above.

Hereinafter, a rat group (control) to which β -carotene was administered is referred to as "β -carotene group", a rat group (comparative example) to which β -carotene and yogurt (lihain yogurt) were administered is referred to as "β -carotene + lihain YG group", a rat group (comparative example) to which β -carotene and yogurt lactobacillus bulgaricus OLL1247 and streptococcus thermophilus OLS3078 were administered is referred to as "β -carotene + OLL1247 × OLS 3078", and a rat group (example) to which β -carotene and yogurt (lactobacillus bulgaricus OLL1251 and streptococcus thermophilus OLS3290) were administered is referred to as "β -carotene + OLL1251 × OLS 3290".

(5) Results

The results are shown in Table 4. For the area under the plasma concentration-time curve (AUC), the β -carotene + OLL1247 × OLS3078 group and the β -carotene + OLL1251 × OLS3290 group were significantly increased compared to the β -carotene group. The results indicate that ingestion of yogurt promotes the absorption of beta-carotene.

[ Table 4]

TABLE 4 plasma Carotene concentration-area under time Curve (AUC)

Mean. + -. standard deviation of the mean

P < 0.05 was significantly different from the beta carotene group

Experimental example 4: absorption of quercetin (Lactobacillus bulgaricus OLL1251 and Streptococcus thermophilus OLS 329)Group 0 In combination)

(1) Yogurt for use

The yoghurt prepared in experimental example 3 was used.

(2) Experimental methods

After acclimating 24 rats (SD, male, 8 weeks old, Japan SLC, Inc.) for 7 days, the rats were divided into 8 groups. After a 16 hour fast, quercetin and yogurt were administered to each group of rats, respectively. Here, quercetin was administered at 50mg/kg body weight, and yogurt was administered at 11.3g/kg body weight. Blood was collected from the tail vein before administration, 60 minutes, 120 minutes, 240 minutes, and 480 minutes after administration, and serum was obtained. The serum concentrations of quercetin conjugate and isorhamnetin conjugate, which are metabolites of quercetin, were determined according to the above-described method.

Hereinafter, the rat group to which quercetin was administered (control) is referred to as "quercetin group", the rat group to which quercetin and yogurt (lactobacillus bulgaricus OLL1247 and streptococcus thermophilus OLS3078) were administered (comparative example) is referred to as "quercetin + OLL1247 × OLL3078 group", and the rat group to which quercetin and yogurt (lactobacillus bulgaricus OLL1251 and streptococcus thermophilus OLS3290) were administered (example) is referred to as "quercetin + OLL1251 × OLL3290 group".

(3) Results

The results are shown in Table 5. Area under the blood concentration-time curve (AUC) for quercetin conjugate is significantly increased in the quercetin + OLL1247 × OLS3078 group, and quercetin + OLL1251 × OLS3290 group, as compared with the quercetin group. This result suggests that lactic acid bacteria-based fermentation promotes the absorption of quercetin.

[ Table 5]

TABLE 5 area under the blood concentration-time curve (AuC) for quercetin and isorhamnetin conjugates

Mean. + -. standard deviation of the mean

P < 0.05 with significant difference to quercetin group

Experimental example 5: absorption promotion of beta-carotene (in comparison with other absorption promoters)

(1) Preparation of yoghurt-derived polysaccharide concentrates

A part of the yogurt (Lactobacillus bulgaricus OLL1251 and Streptococcus thermophilus OLS3290) of example 4 was taken out, and 3 times the amount of ethanol was added to the supernatant and stored in a frozen state. Then, this supernatant was subjected to a centrifugal separation treatment, resulting in a precipitate. The precipitate was then freeze dried to give a polysaccharide concentrate. Note that 70mg of the polysaccharide concentrate was contained in 11.3g of yogurt. Hereinafter, the obtained polysaccharide concentrate is referred to as "a polysaccharide concentrate derived from lactic acid bacteria".

(2) Experimental methods

After acclimating 48 rats (SD, male, 8 weeks old, Japan SLC, Inc.) for 7 days, the rats were divided into 8 groups. After a 16-hour fasting, rats in each group were administered β -carotene, β -carotene and polysaccharide concentrate derived from lactic acid bacteria, β -carotene and cyclodextrin (manufactured by CycloChem co., ltd.), β -carotene and starch (rice-derived) (manufactured by SIGMA corporation), β -carotene and pectin (apple-derived) (manufactured by wako pure chemical industries), β -carotene and indigestible dextrin (manufactured by pine-grain chemical industries), respectively. Beta-carotene was administered at 5mg/kg body weight, and polysaccharide was administered at 70mg/kg body weight. Blood was collected from the tail vein before administration, 60 minutes, 120 minutes, 240 minutes, and 480 minutes after administration, and serum was obtained.

Hereinafter, for convenience of explanation, a group of rats to which β -carotene was administered (control) is referred to as a "β -carotene group", a group of rats to which β -carotene and a polysaccharide concentrate derived from lactic acid bacteria (example) are administered is referred to as a "β -carotene + polysaccharide concentrate derived from lactic acid bacteria group", a group of rats to which β -carotene and cyclodextrin were administered (comparative example) is referred to as a "β -carotene + polysaccharide concentrate derived from lactic acid bacteria group", a group of rats to which β -carotene and starch (rice source) were administered (comparative example) is referred to as a "β -carotene + starch (rice source) group", a group of rats to which β -carotene and pectin (apple source) were administered (comparative example) is referred to as a "β -carotene + pectin (apple source) group", and a group of rats to which β -carotene and indigestible dextrin were administered (comparative example) is referred to as a "β -ion-enriched food group Carotene + indigestible dextrin group ".

(5) Results

The results are shown in Table 6. For the area under the concentration-time curve (AUC) in serum, there was a significant increase in the β -carotene + lactobacillus-derived polysaccharide concentrate group compared to the β -carotene group. On the other hand, the beta-carotene + cyclodextrin group was significantly reduced compared to the beta-carotene group. This result means that the intake of the polysaccharide derived from lactic acid bacteria promotes the absorption of β -carotene as compared with other polysaccharides.

[ Table 6]

TABLE 6 plasma Carotene concentration-area under time Curve (AUC)

Mean. + -. standard deviation of the mean

P < 0.05 was significantly different from the beta carotene group

The # P < 0.05 is significantly different from the cyclodextrin group, the starch (rice-derived) group and the indigestible dextrin group

Experimental example 6: absorption promotion of beta-carotene in human body (added to carrot juice)

(1) Preparation of yogurt

After inoculating Lactobacillus bulgaricus OLL1251 and Streptococcus thermophilus OLS3290 to a medium containing 10 mass% of skimmed milk powder, the medium was fermented at 43 ℃ until pH4.6 was reached.

(2) Experimental methods

After 5 subjects were fasted for 12 hours, 180g of commercially available carrot juice (containing 11mg of. beta. -carotene) was taken, and serum was collected 4 hours after the intake. After 1 week of removal, 180g of the carrot juice and 100g of yogurt were mixed and taken, and serum was collected 4 hours after the intake. The obtained serum was covered with physiological saline, and immediately centrifuged by ultracentrifugation (155000 Xg, 30 minutes) to separate chylomicron.

(3) Results

The results are shown in Table 7. The amount of change in plasma β -carotene concentration in the chylomicron fraction before and after the intake of carrot juice was significantly increased in the carrot juice + yogurt group as compared with the carrot juice group. This result means that lactic acid bacteria-based fermentation in the human body promotes the absorption of beta-carotene contained in carrot juice.

[ Table 7]

TABLE 7 amount of change in plasma beta-carotene concentration of chylomicron fraction before and after intake of carrot juice

Mean. + -. standard deviation of the mean

P < 0.05 was significantly different from carrot juice group

Experimental example 7: absorption promotion of carotenoid in human body (added to vegetable juice)

(1) Preparation of yogurt

Lactobacillus bulgaricus OLL1251 and Streptococcus thermophilus OLS3290 were inoculated into a medium containing 10 mass% of skim milk powder, and then the medium was fermented at 40 ℃ until pH4.2 was reached. The yoghurt thus obtained contained 90. mu.g/g of polysaccharide.

(2) Experimental methods

The BMI is 18.5kg/m and is more than 20 years old and less than 35 years old²Above and below 25.0kg/m²The 10 male subjects in (a) were subjected to the test. After fasting for 16 hours, a single intake of a beverage containing 100g of vegetable juice (commercially available spinach puree, commercially available tomato puree, commercially available carrot juice, 5mg of α -carotene, 10mg of β -carotene, 3mg of lutein, and 10mg of lycopene) and 100g of water, or a beverage containing 100g of the aforementioned vegetable juice and 100g of yogurt was carried out. After 2 weeks of washout, a single intake of the drink not ingested in the 1 st intake trial was allowed. Before intake of the test foodBlood was collected 2 hours, 4 hours, 6 hours, and 8 hours after ingestion to obtain plasma.

Hereinafter, for convenience of explanation, the case of taking vegetable juice (control) will be referred to as "vegetable juice group", and the case of taking vegetable juice and yogurt (example) will be referred to as "vegetable juice + yogurt group".

(3) Results

The results are shown in Table 8. The area under the serum concentration-time curve (AUC) for alpha-carotene, beta-carotene, lutein, lycopene was significantly increased in the vegetable juice + yogurt group compared to the vegetable juice group. This result means that yoghurt promotes the absorption of carotenoids contained in vegetable juices in the human body.

[ Table 8]

TABLE 8 blood concentration of vegetable juice-derived carotenoids area under the time curve (AUC)

Mean. + -. standard deviation of the mean

P < 0.05 with significant difference compared to the vegetable juice group

Experimental example 8: absorption promotion of carotenoids in human body (added to a composition containing beta-carotene and lycopene) In preparation)

(1) Yogurt for use

The yoghurt prepared in experimental example 7 was used.

(2) Experimental methods

The BMI is 18.5kg/m and is more than 20 years old and less than 35 years old²Above and below 25.0kg/m²The 9 male subjects in (a) were subjected to the test. After a 16-hour fasting, a single intake of a beverage containing 25g of a carotenoid preparation (β -carotene 10mg (San-Ei Gen f.f.i., manufactured by inc., New Carotin Base 250), 10mg of lycopene (manufactured by Lycored corporation, TOMATO-O-Red 2% SG)) and 100g of water, or a beverage containing 25g of a carotenoid preparation and 100g of yogurt was performed. After 2 weeks of washout, the sheets were allowed to standSub-ingestion of beverages not ingested in the 1 st ingestion trial. Blood was collected before, 2 hours after, 4 hours after, 6 hours after, and 8 hours after ingestion of the test food, and plasma was obtained.

Hereinafter, for convenience of explanation, the case of taking vegetable juice (control) will be referred to as "carotenoid preparation group", and the case of taking a carotenoid preparation and yogurt (example) will be referred to as "carotenoid preparation + yogurt group".

(3) Results

The results are shown in Table 9. The area under the serum concentration-time curve (AUC) for β -carotene, lycopene was significantly increased in the carotenoid formulation + yoghurt group compared to the carotenoid formulation group. This result means that yoghurt promotes the absorption of carotenoids contained in carotenoid preparations in the human body.

[ Table 9]

TABLE 9 Carotenoid formulation blood concentration of carotenoid from blood-level area under time curve (AUC)

Mean. + -. standard deviation of the mean

P < 0.05 significant differences compared to the carotenoid preparation group

Experimental example 9: absorption promotion of beta cryptoxanthin

(1) Yogurt for use

Lactobacillus bulgaricus OLL1251 and Streptococcus thermophilus OLS3290 were inoculated into a medium containing 10 mass% of skim milk powder, and then the medium was fermented at 43 ℃ until pH4.2 was reached and heated. The yoghurt thus obtained contained 105.8. mu.g/g of polysaccharide.

(2) Experimental methods

After acclimating 16 rats (SD, male, 8 weeks old, Japan SLC, Inc.) for 7 days, the rats were divided into 8 groups. After a 16 hour fast, the rats in each group were given β -cryptoxanthin, β -cryptoxanthin and yogurt, respectively. Beta-cryptoxanthin at 3mg/kg body weight and yogurt at 11.3g/kg body weight were administered. Blood was collected from the tail vein before administration, 60 minutes, 120 minutes, 240 minutes, and 480 minutes after administration, and serum was obtained. Hereinafter, for convenience of explanation, the group of rats to which β cryptoxanthin was administered (control) is referred to as "β cryptoxanthin group", and the group of rats to which β cryptoxanthin and yogurt were administered (example) is referred to as "β cryptoxanthin + yogurt group".

(3) Determination of beta cryptoxanthin

To 50 μ L of serum, 90 μ L of physiological saline was added, and 300 μ L of dichloromethane methanol solution (dichloromethane: methanol ═ 1: 2) was added. Further, 150. mu.L of hexane was added to extract β -carotene. The upper layer was transferred to another tube and the solvent was removed by nitrogen reflux. Samples for HPLC were prepared by dissolving 150. mu.L of 30% ethyl acetate 70% methanol solution containing 0.1% ammonium acetate.

(4) Analytical conditions of HPLC

The HPLC was performed using 1200 series (Agilent Technologies). The column used was TSKgel ODS-80TsQA (5.0X 250mm) (manufactured by Tosoh corporation), and the column temperature was set at 40 ℃. For the mobile phase, a 30% ethyl acetate 70% methanol solution containing 0.1% ammonium acetate was prepared. The flow rate was set to 0.3 mL/min, and the absorption wavelength at 450nm was measured.

(5) Results

The results are shown in table 10 and fig. 1. The blood levels of β cryptoxanthin after 60 minutes, 120 minutes, and 240 minutes of administration were significantly increased in the β cryptoxanthin + yogurt group, as compared to the β cryptoxanthin group. In addition, for the area under the curve (AUC) in blood, there was a significant increase in the β -cryptoxanthin + yoghurt group compared to the β -cryptoxanthin group. The results indicate that ingestion of yogurt promotes the absorption of β cryptoxanthin. Note that in the figure, the symbol "+" indicates that there is a significant difference from the β cryptoxanthin group at P < 0.05.

[ Table 10]

Mean. + -. standard deviation of the mean

P < 0.05 with significant difference to the beta cryptoxanthin group

Experimental example 10: absorption promotion of capsanthin

(1) Yogurt for use

The yogurt used in experimental example 9 was used.

(2) Experimental methods

After acclimating 16 rats (SD, male, 8 weeks old, Japan SLC, Inc.) for 7 days, the rats were divided into 8 groups. After a 16 hour fast, each group of rats was given capsanthin, capsanthin and yogurt, respectively. Capsanthin was administered at 5mg/kg body weight and yogurt was administered at 11.3g/kg body weight. Blood was collected from the tail vein before administration, 60 minutes, 120 minutes, 240 minutes, and 480 minutes after administration, and serum was obtained. Hereinafter, for convenience of explanation, the group of rats to which capsanthin was administered (control) is referred to as "capsanthin group", and the group of rats to which capsanthin and yogurt were administered (example) is referred to as "capsanthin + yogurt group".

(3) Measurement of capsanthin

To 50 μ L of serum, 90 μ L of physiological saline was added, and 300 μ L of dichloromethane methanol solution (dichloromethane: methanol ═ 1: 2) was added. Further, 150. mu.L of hexane was added to extract β -carotene. The upper layer was transferred to another tube and the solvent was removed by nitrogen reflux. Samples for HPLC were prepared by dissolving 150. mu.L of 30% ethyl acetate 70% methanol solution containing 0.1% ammonium acetate.

(4) Analytical conditions of HPLC

The HPLC was performed using 1200 series (Agilent Technologies). The column used was TSKgel ODS-80TsQA (5.0X 250mm) (manufactured by Tosoh corporation), and the column temperature was set at 40 ℃. For the mobile phase, a 30% ethyl acetate 70% methanol solution containing 0.1% ammonium acetate was prepared. The flow rate was set to 0.3 mL/min, and the absorption wavelength at 470nm was measured.

(5) Results

The results are shown in table 11 and fig. 2. Blood levels of capsanthin at 60 minutes, 120 minutes, 240 minutes, and 480 minutes after administration were significantly increased in the capsanthin + yogurt group, as compared to the capsanthin group. In addition, for the area under the curve (AUC) in blood, there was a significant increase in the capsanthin + yogurt group compared to the capsanthin group. The results indicate that ingestion of yogurt promotes capsanthin absorption. In the figure, the symbol "+" indicates that the difference is significant at P < 0.05 relative to the capsanthin group.

[ Table 11]

Mean. + -. standard deviation of the mean

P < 0.05 was significantly different from the capsanthin group

Experimental example 11: absorption of hesperidin is promoted

(1) Yogurt for use

The yogurt used in experimental example 9 was used.

(2) Experimental methods

After acclimating 16 rats (SD, male, 8 weeks old, Japan SLC, Inc.) for 7 days, the rats were divided into 8 groups. After a 16 hour fast, the rats in each group were given hesperidin, hesperidin and yoghurt, respectively. Hesperidin was administered at 162mg/kg body weight, and yogurt was administered at 11.3g/kg body weight. Blood was collected from the tail vein before administration, 60 minutes, 120 minutes, and 240 minutes after administration, and serum was obtained. Hereinafter, for convenience of explanation, the group of rats to which hesperidin was administered (control) is referred to as "hesperidin group", and the group of rats to which hesperidin and yogurt were administered (example) is referred to as "hesperidin + yogurt group".

(3) Measurement of hesperetin metabolite

Hesperetin conjugates as hesperidin metabolites were determined as follows. To 50. mu.L of serum were added 45. mu.L (10000U/mL, manufactured by Sigma-Aldrich Co.) of glucuronidase solution dissolved in 0.1M sodium acetate buffer solution (pH5.0) and 5. mu.L of 0.1M ascorbic acid solution dissolved in 0.1M sodium acetate buffer solution (pH5.0), and the mixture was heated at 37 ℃ for 2 hours. The enzyme reaction was stopped by adding 300. mu.L of methanol, and the mixture was centrifuged (12000rpm, 10 minutes, 4 ℃). The supernatant was transferred to another tube and the solvent was removed by concentration by centrifugation. Samples for HPLC were prepared by dissolving 300. mu.L of a 50% acetonitrile solution containing 0.1% formic acid.

(4) Analytical conditions of HPLC

HPLC was performed using Nexera XR (manufactured by Shimadzu corporation) and 4500QTRAP (manufactured by SCIEX) was used as an MS/MS detector. As the column, ACQUITY UPLC HSST 31.8 μm (2.1X 50mm) (manufactured by Waters corporation) was used, and the column temperature was set to 40 ℃. For the mobile phase, a solution containing 0.1% formic acid was prepared as the a liquid, and an acetonitrile solution containing 0.1% formic acid was prepared as the B liquid. The solution B was held at 30% for 1 minute and then decreased to 45% in 4.5 minutes, thereby eluting the target substance. Then, the column was washed with 99% of solution B for 2 minutes and held with 30% of solution B for 3 minutes. The flow rate was set to 0.3 mL/min. MS/MS analysis was performed in ESI negative ion mode. The analysis conditions of MS/MS are as follows: the gas curtain gas flow is 30psi, the collision gas flow is 9psi, the ion spray voltage is-4500V, the turbine gas temperature is 600 ℃, and the ion source gas is 70 psi.

(5) Results

The results are shown in table 12 and fig. 3. The plasma concentration of hesperetin conjugate 60 minutes after administration was significantly increased in the hesperidin + yoghurt group compared to the hesperidin group. In addition, for the area under the curve (AUC) in blood, the hesperidin + yoghurt group was significantly increased compared to the hesperidin group. The results indicate that ingestion of yogurt promotes the absorption of hesperetin conjugate. Note that in the figure, the symbol "+" indicates that there is a significant difference from the hesperidin group at P < 0.05.

[ Table 12]

Mean. + -. standard deviation of the mean

The P < 0.05 has significant difference relative to the hesperidin group

Experimental example 12: transition of pH in lactic acid bacteria fermentation

(1) Experimental methods

The change in pH was confirmed by inoculating only lactobacillus bulgaricus OLL1251, lactobacillus bulgaricus OLL1251 and streptococcus thermophilus OLS3290 into a medium containing 10 mass% of skim milk powder, and then fermenting the medium at 43 ℃.

(2) Results

The results are shown in Table 13. The reduction of pH was rapidly performed by combining lactobacillus bulgaricus OLL1251 with streptococcus thermophilus OLS3290 and fermenting it, compared with fermentation with lactobacillus bulgaricus OLL1251 alone. This result means that the activity of lactic acid bacteria is active and further means that much polysaccharide is produced. From the results, it was confirmed that: the fermentation time for producing polysaccharides can be shortened by the combination of bacteria.

[ Table 13]

Experimental example 13: absorption promotion of carotenoid in human body (added to carrot juice)

(1) Preparation of yogurt

Lactobacillus bulgaricus OLL1251 and Streptococcus thermophilus OLS3290 were inoculated into a medium containing 10 mass% of skim milk powder, and then the medium was fermented at 40 ℃ until pH4.2 was reached. The yoghurt thus obtained contained 90. mu.g/g of polysaccharide.

(2) Experimental methods

The BMI is 18.5kg/m and is more than 20 years old and less than 35 years old²Above and below 25.0kg/m²The male subject 16 was subjected to the test as a subject. After fasting for 16 hours, a single intake of 100g of carrot juice (commercially available carrot juice containing 6mg of α -carotene and 10mg of β -carotene) and 100g of water, or a single intake of 100g of carrot juice and 100g of yogurt was carried out. After 2 weeks of washout, a single intake of the drink not ingested in the 1 st intake trial was allowed. Collecting blood before ingestion of the test food, 2 hours after ingestion, 4 hours after ingestion, 6 hours after ingestion, or 8 hours after ingestionPlasma was obtained.

Hereinafter, for convenience of explanation, the case of taking carrot juice (control) will be referred to as "carrot juice group", and the case of taking carrot juice and yogurt (example) will be referred to as "carrot juice + yogurt group".

(3) Results

The results are shown in Table 14. The area under the plasma concentration-time curve (AUC) for alpha-carotene, beta-carotene was significantly increased in the carrot juice + yogurt group compared to the carrot juice group. This result means that the yogurt promotes the absorption of carotenoids contained in carrot juice in the human body.

[ Table 14]

Mean. + -. standard deviation of the mean

P < 0.05 was significantly different from the carrot juice group

Experimental example 14: absorption promotion of carotenoids in human body (added to tomato juice)

(1) Preparation of yogurt

Lactobacillus bulgaricus OLL1251 and Streptococcus thermophilus OLS3290 were inoculated into a medium containing 10 mass% of skim milk powder, and then the medium was fermented at 40 ℃ until pH4.2 was reached. The yoghurt thus obtained contained 90. mu.g/g of polysaccharide.

(2) Experimental methods

The BMI is 18.5kg/m and is more than 20 years old and less than 35 years old²Above and below 25.0kg/m²The male subject 15 was subjected to the test as a subject. After a 16-hour fasting, the user took a single drink of a beverage containing 100g of tomato juice (commercially available tomato juice (containing 10mg of lycopene) and 100g of water, or a beverage containing 100g of the aforementioned tomato juice and 100g of yogurt, and after 2 weeks of elimination, the user took a single drink that was not taken in the 1 st intake test, and blood was collected before intake of the test food, 2 hours after intake, 4 hours after intake, 6 hours after intake, and 8 hours after intake, and plasma was obtained。

Hereinafter, for convenience of explanation, the case of taking tomato juice (control) will be referred to as "tomato juice group", and the case of taking tomato juice and yogurt (example) will be referred to as "tomato juice + yogurt group".

(3) Results

The results are shown in Table 15. For the area under the plasma concentration-time curve (AUC) of lycopene, there was a significant increase in the tomato juice + yoghurt group compared to the vegetable juice group. This result means that yoghurt promotes the absorption of carotenoids contained in tomato juice in humans.

[ Table 15]

Mean. + -. standard deviation of the mean

P < 0.05 was significantly different from tomato juice group

Experimental example 15: absorption of carotenoid in human body is promoted (added into spinach juice)

(1) Preparation of yogurt

Lactobacillus bulgaricus OLL1251 and Streptococcus thermophilus OLS3290 were inoculated into a medium containing 10 mass% of skim milk powder, and then the medium was fermented at 40 ℃ until pH4.2 was reached. The yoghurt thus obtained contained 90. mu.g/g of polysaccharide.

(2) Experimental methods

The BMI is 18.5kg/m and is more than 20 years old and less than 35 years old²Above and below 25.0kg/m²The male subject 16 was subjected to the test as a subject. After a 16-hour fasting, a single intake of a beverage containing 100g of spinach juice (commercially available spinach juice containing 4.0mg of lutein and 2.4mg of β -carotene) and 100g of water, or a beverage containing 100g of the aforementioned spinach juice and 100g of yogurt was carried out. After 2 weeks of washout, a single intake of the drink not ingested in the 1 st intake trial was allowed. Blood was collected before, 2 hours after, 4 hours after, 6 hours after, and 8 hours after ingestion of the test food, and plasma was obtained.

Hereinafter, for convenience of explanation, the case of taking spinach juice (control) is referred to as "spinach juice group", and the case of taking spinach juice and yogurt (example) is referred to as "spinach juice + yogurt group".

(3) Results

The results are shown in Table 16. The area under the plasma concentration-time curve (AUC) of lutein and beta-carotene was significantly increased in the spinach juice + yogurt group compared to the spinach juice group. This result means that yoghurt promotes the absorption of carotenoids contained in spinach juice in the human body.

[ Table 16]

Mean. + -. standard deviation of the mean

P < 0.05 has significant difference compared with the spinach juice group

Experimental example 16: absorption of carotenoid in human body is promoted (added to Wenzhou orange juice)

(1) Preparation of yogurt

Lactobacillus bulgaricus OLL1251 and Streptococcus thermophilus OLS3290 were inoculated into a medium containing 10 mass% of skim milk powder, and then the medium was fermented at 40 ℃ until pH4.2 was reached. The yoghurt thus obtained contained 90. mu.g/g of polysaccharide.

After 5 subjects fasted for 12 hours, they ingested commercially available orange juice (100 g, containing β -cryptoxanthin 3mg), and serum was collected 4 hours after ingestion. After 1 week of removal, 100g of the orange juice and 100g of yogurt were mixed and taken, and serum was collected 4 hours after the intake.

(3) Results

The results are shown in Table 17. The plasma β cryptoxanthin concentration variation before and after orange juice intake was significantly increased in the orange juice + yogurt group compared to the orange juice group. This result means that lactic acid bacteria-based fermentation in humans promotes the absorption of carotenoids contained in orange juice.

[ Table 17]

Mean. + -. standard deviation of the mean

P < 0.05 had significant differences relative to the orange group.