JP2022096542A - Chocolate containing galactooligosaccharide and lactobacillus brevis species of lactic acid bacteria - Google Patents
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Abstract
Description
特許法第３０条第２項適用申請有り １．ホームページのアドレス ｈｔｔｐｓ：／／ｗｗｗ．ｌｏｔｔｅ．ｃｏ．ｊｐ／ｐｒｏｄｕｃｔｓ／ｂｒａｎｄ／ｎｙｕｓａｎｋｉｎ－ｃｈｏｃｏｌａｔ／ 掲載日 ：令和２年 ９月２９日 ２．テレビコマーシャルの放送局 （全国）フジテレビ、テレビ朝日、テレビ東京 （地方）メーテレ、朝日放送 テレビコマーシャルの放送日：令和２年 ９月２９日 ３、販売した場所 株式会社山星屋（大阪本社 大阪府大阪市中央区南船場１丁目１５番１４号 堺筋稲畑ビル３階、東京本社 東京都港区海岸３丁目９番１５号 ＬＯＯＰ－Ｘ ４階） 販売日 令和２年 ９月１４日There is an application for application of Article 30, Paragraph 2 of the Patent Law. Homepage address https: // www. lotte. co. jp / products / brand / nyusankin-chocolat / Publication date: September 29, 2nd year of Reiwa 2. TV commercial broadcasting stations (nationwide) Fuji TV, TV Asahi, TV Tokyo (regional) Metere, Asahi Broadcasting TV commercial broadcasting date: September 29, 2nd year of Reiwa 3, Place of sale Yamaboshiya Co., Ltd. (Osaka Headquarters Osaka Prefecture) 1-15-14 Minamisenba, Chuo-ku, Osaka Sakaisuji Inahata Building 3rd floor, Tokyo Headquarters 3-9-15 Kaigan, Minato-ku, Tokyo LOOP-X 4th floor)
本発明は、乳酸菌ラクトバチルスブレビス種とガラクトオリゴ糖とを含有した便通促進用もしくは便通改善用チョコレートに関する。 The present invention relates to chocolate for promoting or improving bowel movements containing a lactic acid bacterium Lactobacillus brevis species and galactooligosaccharide.
ヒトの腸管内には多種・多様な細菌が生息し、複雑な腸内菌叢を形成している。近年、腸内環境と全身の健康との関係が多く報告され、腸内環境が健康の維持・増進に重要であることが示唆されている。更に、腸と脳が密接に関係していることも報告されており、非便秘気味の方に比べて便秘気味の方で心の健康、睡眠など、多岐にわたってＱＯＬが低いことが報告されている。日本においては２０１９年度に行われた国民生活基礎調査によると、便秘の有訴者率は３４．８人（／１０００人）（男性２５．４人（／１０００人）、女性４３．７人（／１０００人））であり、約４４０万人近くが便秘に悩んでいることが報告されている。このような症状を改善する手段として、プロバイオティクスやプレバイオティクスがある。プロバイオティクスは一般的に賞味期限が短く、また、胃酸に弱いため健康効果を期待する上で乳酸菌を生きて腸まで届けることが重要とされている。これまでに乳酸菌ラクトバチルスブレビス種をチョコレートに配合し、経時安定性、耐酸性を向上させた品質の製品が開発されている（特許文献１）。また、乳酸菌ラクトバチルスブレビス種とガラクトオリゴ糖とを開示する文献もある（特許文献２、３）。 A wide variety of bacteria inhabit the human intestine, forming a complex intestinal flora. In recent years, many reports have been made on the relationship between the intestinal environment and general health, suggesting that the intestinal environment is important for maintaining and improving health. Furthermore, it has been reported that the intestine and the brain are closely related, and it has been reported that the QOL of constipated people is lower than that of non-constipated people in a wide range of areas such as mental health and sleep. .. In Japan, according to the National Lifestyle Survey conducted in FY2019, the rate of constipation complaints is 34.8 (/ 1000) (male 25.4 (/ 1000)) and female 43.7 (female). / 1000 people)), and it is reported that nearly 4.4 million people are suffering from constipation. There are probiotics and prebiotics as means for improving such symptoms. Probiotics generally have a short best-by date and are vulnerable to gastric acid, so it is important to deliver lactic acid bacteria alive to the intestines in order to expect health benefits. So far, quality products have been developed in which lactic acid bacterium Lactobacillus brevis seeds are blended with chocolate to improve stability over time and acid resistance (Patent Document 1). There is also a document that discloses a lactic acid bacterium Lactobacillus brevis species and a galactooligosaccharide (Patent Documents 2 and 3).
ラクトバチルスブレビスＮＴＴ００１は京都の伝統的な漬物『すぐき漬け』より単離された植物由来の乳酸菌であり、生きたまま腸に到達することが報告されており、宿主に有益な機能を発揮する可能性が期待される。１．０×１０９ｃｆｕ／日、２週間の継続摂取により、糞便中のラクトバチルス、糞便中の酢酸が増加し、腸内腐敗産物であるインドールが低下するなど腸内環境改善機能を有することが報告されている（非特許文献１，２）。 Lactobacillus brevis NTT001 is a plant-derived lactic acid bacterium isolated from the traditional pickles "Suguki-zuke" in Kyoto, and it has been reported that it reaches the intestines alive and can exert beneficial functions for the host. Sex is expected. 1.0 × 10 9 cfu / day, continuous ingestion for 2 weeks has the function of improving the intestinal environment, such as increasing lactobacillus in feces and acetic acid in feces, and decreasing indole, which is an intestinal putrefactive product. Has been reported (Non-Patent Documents 1 and 2).
ガラクトオリゴ糖はガラクトースを主成分とするオリゴ糖で、難消化性の糖質である。ヒトにおいては、一日当たり４．０ｇのガラクトオリゴ糖を３週間摂取することで糞便中ビフィドバクテリウムが増加することが報告されている（非特許文献３） Galactooligosaccharide is an oligosaccharide containing galactose as a main component and is an indigestible sugar. In humans, it has been reported that ingestion of 4.0 g of galactooligosaccharide per day for 3 weeks increases fecal bifidobacteria (Non-Patent Document 3).
しかし、特許文献１のチョコレートでは便通改善効果が十分とは言えない。また、特許文献２、３は、食用油を含むプレバイオティック組成物、またはプレバイオティクスを含む油性懸濁液に関する発明であり、便通促進もしくは便通改善を目的とした発明ではない。
非特許文献１、２では、腸内腐敗産物であるインドールが低下するなど腸内環境改善機能を有することが報告されているものの、１．０×１０９ｃｆｕ／日の摂取量では便通促進機能を有していないことが報告されている。非特許文献３では、ガラクトオリゴ糖単独の効果が示されているが、一日当たり４．０ｇより少ない量のガラクトオリゴ糖を摂取することで便通促進作用効果が認められるかは判明していない。
However, it cannot be said that the chocolate of Patent Document 1 has a sufficient effect of improving bowel movements. Further, Patent Documents 2 and 3 are inventions relating to a prebiotic composition containing edible oil or an oily suspension containing prebiotics, and are not inventions for the purpose of promoting or improving bowel movements.
Although it is reported in Non-Patent Documents 1 and 2 that it has an intestinal environment improving function such as reduction of indole, which is an intestinal putrefactive product, it has a bowel movement promoting function at an intake of 1.0 × 10 9 cfu / day. It is reported that it does not have. Non-Patent Document 3 shows the effect of galactooligosaccharide alone, but it is not clear whether the effect of promoting bowel movement is recognized by ingesting a amount of galactooligosaccharide less than 4.0 g per day.
本発明の目的は、乳酸菌ラクトバチルスブレビス種とガラクトオリゴ糖とを含有したチョコレートにより、お腹の調子を整える機能を提供することである。 An object of the present invention is to provide a function of adjusting the condition of the abdomen by chocolate containing lactic acid bacterium Lactobacillus brevis species and galactooligosaccharide.
本発明の目的は、乳酸菌ラクトバチルスブレビス種であるラクトバチルスブレビスＮＴＴ００１（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ ＮＴＴ００１）とガラクトオリゴ糖とを含有した便通促進用もしくは便通改善用チョコレートにより達成される。 An object of the present invention is achieved by a chocolate for promoting or improving stools containing lactobacillus brevis NTT001 (Lactobacillus brevis NTT001), which is a species of lactic acid bacterium Lactobacillus brevis, and galactooligosaccharides.
本発明では、ラクトバチルスブレビスＮＴＴ００１とガラクトオリゴ糖の併用作用を詳細に検証するため、ランダム化プラセボ対照二重盲検クロスオーバー試験を実施した。また本発明では、関与成分の適正量を検証するため、２つのヒト試験を実施した。被験食品であるラクトバチルスブレビスＮＴＴ００１およびガラクトオリゴ糖含有チョコレート（ラクトバチルスブレビスＮＴＴ００１ １．３×１０８ｃｆｕ、ガラクトオリゴ糖０．３８ｇ含有）を、ヒト試験（実施例１）では１日当たり４枚、ヒト試験（比較例１）では１日当たり３枚摂取させた。その他の試験概要は実施例１と比較例１で同様であり、便秘傾向の健常成人を対象にラクトバチルスブレビスＮＴＴ００１・ガラクトオリゴ糖含有チョコレートの２週間継続摂取試験を行い、糞便中のラクトバチルス及びビフィドバクテリウム、ラクトバチルスブレビスＮＴＴ００１の腸管到達性、糞便中の有機酸や腐敗産物、便通ならびに便性状への影響を検討した。 In the present invention, a randomized placebo-controlled, double-blind crossover study was performed to examine in detail the combined action of Lactobacillus brevis NTT001 and galactooligosaccharides. Further, in the present invention, two human tests were carried out in order to verify the appropriate amount of the components involved. Lactobacillus brevis NTT001 and galactooligosaccharide-containing chocolate (lactobacillus brevis NTT001 1.3 × 10 8 cfu, containing 0.38 g of galactooligosaccharide), which are the test foods, were used in a human test (Example 1) with 4 sheets per day. In (Comparative Example 1), 3 sheets were ingested per day. The outline of other tests is the same in Example 1 and Comparative Example 1. Lactobacillus brevis NTT001 / galactooligosaccharide-containing chocolate was continuously ingested for 2 weeks in healthy adults with constipation tendency, and lactobacillus and bi in feces. The effects of Fidobacterium and Lactobacillus brevis NTT001 on the intestinal reach, organic acids and putrefactive products in feces, bowel movements and fecal properties were investigated.
（実施例１）
１ 試験食品
試験食品として、ラクトバチルスブレビスＮＴＴ００１・ガラクトオリゴ糖含有チョコレート（被験食品）及びラクトバチルスブレビスＮＴＴ００１とガラクトオリゴ糖を含まないプラセボチョコレート（対照食品）を用いた。
具体的なチョコレートの配合を、以下の表１に示す。
(Example 1)
1 Test food As the test food, lactobacillus brevis NTT001 / galactooligosaccharide-containing chocolate (test food) and lactobacillus brevis NTT001 and galactooligosaccharide-free placebo chocolate (control food) were used.
Specific chocolate formulations are shown in Table 1 below.
ラクトバチルスブレビスＮＴＴ００１およびガラクトオリゴ糖配合チョコレートは、チョコレートにラクトバチルスブレビスＮＴＴ００１粉末及びガラクトオリゴ糖を添加・混合し（３０℃前後／数分）、成形、冷却（７－１０℃／３０－５０分）して製造した。
すなわち、ラクトバチルスブレビスＮＴＴ００１およびガラクトオリゴ糖配合チョコレートは、一般的なチョコレートの製法を用いて製造することができる。原料であるカカオ豆を、選別、分離、焙炒、磨砕してカカオマスとし、カカオマスと、砂糖、粉乳、植物油脂、ココアバターおよび乳化剤の一部を原料混合機で混合し、リファイナーにより粒子が所定の大きさになる様に均一に微細化し、精練（コンチング）し、精練の後半の段階で、香料、ラクトバチルスブレビスＮＴＴ００１菌末と、残りのココアバターおよび乳化剤を添加してチョコレート生地を調製する。その後、テンパリングを行い、成型用の型に充填し、冷却・固化させ、型抜し、得られたチョコレートを包装する。ラクトバチルスブレビスＮＴＴ００１菌末を含んだチョコレート生地は、別途テンパリングしたチョコレート生地に添加しても良い。ガラクトオリゴ糖は原料の混合時に添加しても良い。
Lactobacillus brevis NTT001 and galactooligosaccharide-blended chocolate are prepared by adding and mixing Lactobacillus brevis NTT001 powder and galactooligosaccharide (around 30 ° C./several minutes), molding and cooling (7-10 ° C./30-50 minutes). Manufactured.
That is, Lactobacillus brevis NTT001 and galactooligosaccharide-containing chocolate can be produced by using a general chocolate production method. The raw material cocoa beans are sorted, separated, roasted and ground to make cocoa mass, and sugar, powdered milk, vegetable oil, cocoa butter and a part of the emulsifier are mixed with the raw material mixer, and the particles are formed by the refiner. The chocolate dough is prepared by adding fragrance, lactobacillus brevis NTT001 bacterial powder, and the remaining cocoa butter and emulsifier in the latter half of the scouring. do. After that, it is tempered, filled in a molding mold, cooled and solidified, die-cut, and the obtained chocolate is packaged. The chocolate dough containing Lactobacillus brevis NTT001 bacterial powder may be added to the separately tempered chocolate dough. Galactooligosaccharides may be added when the raw materials are mixed.
被験食品は、チョコレート１枚（４ｇ）あたりにラクトバチルスブレビスＮＴＴ００１が１．３×１０８ｃｆｕ、ガラクトオリゴ糖が０．３８ｇ含まれるよう設計した。 The test food was designed to contain 1.3 × 10 8 cfu of Lactobacillus brevis NTT001 and 0.38 g of galactooligosaccharide per piece of chocolate (4 g).
実施例１では被験食品を１日あたり４枚（ラクトバチルスブレビスＮＴＴ００１として５．０×１０８ｃｆｕ、ガラクトオリゴ糖として１．５ｇ）と設定した。 In Example 1, the number of test foods was set to 4 per day (5.0 × 108 cfu as Lactobacillus brevis NTT001 and 1.5 g as galactooligosaccharide).
なお、摂取開始時と終了時にそれぞれ分析を行い、被験食品は１枚（４ｇ）当たりラクトバチルスブレビスＮＴＴ００１を１．３×１０８ｃｆｕ以上、ガラクトオリゴ糖０．３８ｇ以上含有していた。また、被験者には被験食品と対照食品は、味・外観による区別ができないようにした。 Analysis was performed at the start and end of ingestion, and the test food contained 1.3 × 108 cfu or more and 0.38 g or more of galactooligosaccharide per sheet (4 g) of Lactobacillus brevis NTT001. In addition, the subjects were unable to distinguish between the test food and the control food by taste and appearance.
試験食品に用いたラクトバチルスブレビスＮＴＴ００１（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ ｓｕｂｓｐ．ｃｏａｇｕｌａｎｓ; 特許微生物寄託番号ＦＥＲＭ ＢＰ－４６９３）はＮｏｓｔｅｒ株式会社より提供された。ガラクトオリゴ糖はサンブライト社のものを使用した。 Lactobacillus brevis subsp. Coagulans (patented microorganism deposit number FERM BP-4693) used for the test food was provided by Noster Co., Ltd. The galactooligosaccharide used was that of Sunbright.
２ 被験者
本試験では、下記の選択基準を満たし、かつ除外基準に抵触しない健常成人２２名（年齢４４．７±１０．８歳、ＢＭＩ２１．４±３．０ｋｇ・ｍ－２）、を被験者とした。
2 Subjects In this study, 22 healthy adults (age 44.7 ± 10.8 years, BMI 21.4 ± 3.0 kg · m -2 ) who met the following selection criteria and did not violate the exclusion criteria were used as subjects. did.
選択基準は、（ｉ）２０歳以上６５歳未満の男女、（ii）スクリーニング検査時に持参した２週間の被験者日誌の排便回数が週３～５回の者、（iii）試験の目的・内容について十分な説明を受け、同意能力があり、よく理解した上で自発的に参加を志願し、書面で本試験参加に同意した者、とした。除外基準は、（ｉ）整腸剤や便秘薬（下剤を含む）を常用している者、（ii）スクリーニング検査時に便秘改善によいとされる健康食品類を常用している者、（iii）スクリーニング検査時に、抗生物質など消化吸収に影響を与える薬剤を服用している者、（iv）試験期間中に乳酸菌・ビフィズス菌・納豆菌などの生菌類含有食品、オリゴ糖・食物繊維を強化した食品、便秘改善によいとされる健康食品類（特定保健用食品、機能性表示食品を含む）、及び糖アルコール多量含有食品の摂取を止めることが出来ない者、（ｖ）食物アレルギーを有する者、（vi）日常的に多量のアルコールを摂取している者、（vii）緊急に治療を要する疾患に罹患している者、または重篤な合併症を有する者、（viii）消化吸収や排便に影響を与える消化器疾患、または手術歴がある者、（ix）被験者背景アンケートの回答から被験者として不適当であると判断された者、（ｘ）妊娠している者、試験期間中妊娠の意思がある者、授乳中の者、（xi）薬物依存、アルコール依存の既往歴あるいは現病歴がある者、（xii）他の食品の摂取や薬剤を使用する試験、化粧品及び薬剤などを塗布する試験に参加中の者、参加の意思がある者、（xiii）その他、試験責任医師が被験者として不適当と判断した者、とした。 The selection criteria are (i) men and women between the ages of 20 and 65, (ii) those who defecate 3 to 5 times a week in the subject diary brought during the screening test, and (iii) the purpose and content of the study. Those who received sufficient explanation, had the ability to consent, voluntarily volunteered to participate after understanding well, and agreed to participate in this test in writing. Exclusion criteria are (i) those who regularly use intestinal regulators and constipation drugs (including laxatives), (ii) those who regularly use health foods that are said to be good for improving constipation at the time of screening tests, (iii) screening. Those who are taking drugs that affect digestion and absorption such as antibiotics at the time of inspection, (iv) Foods containing live bacteria such as lactic acid bacteria, bifidus bacteria, natto bacteria, and foods enriched with oligosaccharides and dietary fiber during the test period , Health foods that are said to be good for improving constipation (including foods for specified health use and foods with functional claims), and those who cannot stop eating foods containing a large amount of sugar alcohol, (v) Those who have food allergies, (Vi) Those who consume large amounts of alcohol on a daily basis, (vii) those who suffer from urgently-needed diseases, or those who have serious complications, (viii) for digestion, absorption and defecation Those who have a history of affected gastrointestinal disorders or surgery, (ix) those who are judged to be inappropriate as subjects based on the answers to the subject background questionnaire, (x) those who are pregnant, and those who intend to become pregnant during the study period. Those who have, are breastfeeding, (xi) those who have a history of drug dependence, alcohol dependence or current medical history, (xii) tests of ingesting other foods or using drugs, tests of applying cosmetics and drugs, etc. Those who are participating in, those who are willing to participate, (xiii) and others who are judged by the investigator to be inappropriate as subjects.
また、試験期間中、（ｉ）試験食品は試験責任医師及び試験分担医師の指示通りに摂取すること、（ii）試験食品は、被験者本人以外には摂取させないこと、（iii）試験以前と同様の生活を送ること（暴飲・暴食、ダイエット、海外旅行等での食生活の大幅な変更、今までしていた運動を急に止める、新しく運動を始める、ことはしないこと）、（iv）多量のアルコールの摂取を控えること（１日最大量：１日あたり平均で純アルコール４０ｇ相当まで）、（ｖ）整腸剤・便秘薬等の便秘改善に影響を及ぼす可能性がある医薬品はできる限り使用を避けること、（vi）乳酸菌・ビフィズス菌・納豆菌などの生菌類含有食品、オリゴ糖・食物繊維を強化した食品、便秘改善によいとされる健康食品類（特定保健用食品、機能性表示食品を含む）、及び糖アルコール多量含有食品の摂取を避けること、（vii）他の食品の摂取や薬剤を使用する試験、化粧品及び薬剤などを塗布する試験に参加しないこと、を指示した。 In addition, during the test period, (i) the test food should be taken as instructed by the investigator and the test coordinator, (ii) the test food should not be taken by anyone other than the subject, and (iii) the same as before the test. (Drinking / eating, dieting, drastic changes in eating habits due to overseas travel, suddenly stopping the exercise that you have been doing, starting a new exercise, do not do it), (iv) Large amount Refrain from ingesting alcohol (maximum daily dose: up to 40 g of pure alcohol on average per day), (v) Use as much as possible foods that may affect constipation improvement such as intestinal regulators and constipation drugs. Avoid, (vi) Foods containing live bacteria such as lactic acid bacteria, bifidus bacteria, natto bacteria, foods fortified with oligosaccharides and dietary fiber, health foods that are said to be good for improving constipation (foods for specified health use, foods with functional claims) (Including), and avoid ingestion of foods containing a large amount of sugar alcohol, (vii) instructed not to participate in the ingestion of other foods, the test using drugs, the test to apply cosmetics and drugs, etc.
３ 試験スケジュール
本試験はランダム化プラセボ対照二重盲検クロスオーバー試験で実施し、統計解析責任者がスクリーニング検査時の年齢、性別、糞便中ラクトバチルス、糞便中酢酸、糞便中インドールに偏りを示さないように、ランダムに割り当てられた乱数種を用いて２群に層別ランダム化した（統計解析ソフトＪＭＰ１０使用）。
3 Study Schedule This study was conducted in a randomized, placebo-controlled, double-blind, crossover study, and the statistical analyst showed bias in age, gender, fecal lactobacillus, fecal acetic acid, and fecal indole at the time of the screening test. Randomized into two groups using randomly assigned random number species (using statistical analysis software JMP10).
試験実施に関与しないコントローラーが２群を被験食品先行摂取群、対照食品先行摂取群に割り付けた。なお、割付表はコントローラーが封緘し、割付表開封時まで密封保管した。試験は、前観察期を２週間設定した後、摂取Ｉ期、休止期、摂取II期をそれぞれ２週間の計８週間で行った。 Controllers not involved in the study assigned the two groups to the test food pre-ingestion group and the control food pre-ingestion group. The allocation table was sealed by the controller and kept sealed until the allocation table was opened. In the test, after setting the pre-observation period for 2 weeks, the intake I period, the rest period, and the intake II period were each performed for 2 weeks, for a total of 8 weeks.
被験者には、摂取期間中に試験食品を１６ｇ（４ｇ×４枚）／日を、摂取時間を指定せず自由摂取させた。 The subjects were allowed to freely ingest 16 g (4 g × 4 sheets) / day of the test food during the ingestion period without specifying the ingestion time.
４ 評価項目
ラクトバチルスブレビスＮＴＴ００１とガラクトオリゴ糖との併用摂取による腸に対する影響を検証するため、主要評価項目は試験食品摂取期間における糞便中ラクトバチルス（生菌）数とし、副次評価項目は糞便中ラクトバチルスブレビス（生菌）数、糞便中ビフィドバクテリウム、糞便中有機酸含量、糞便中腐敗産物・アンモニア含量、便通・便性（排便回数、排便日数、排便量、便形状、便の色、便の臭い、排便後の感覚）とした。
4 Evaluation item In order to verify the effect on the intestine by the combined intake of lactobacillus brevis NTT001 and galactooligosaccharide, the primary endpoint is the number of lactobacillus (live bacteria) in feces during the test food intake period, and the secondary endpoint is fecal. Number of lactobacillus brevis (live bacteria), stool bifidobacteria, stool organic acid content, stool spoilage product / ammonia content, stool / fecal properties (number of stools, number of stool days, stool volume, stool shape, stool color) , Smell of stool, sensation after stool).
５ 糞便解析
前観察期、摂取Ｉ期、休止期、摂取II期の計４回、採便させた。便検体は、被験者が自宅で採便し、便が出たタイミングで便全量を専用容器に採取し、蓄冷剤と共に嫌気条件下で、測定施設に直送した。
5 Fecal analysis Feces were collected four times in total, including the pre-observation period, intake I period, rest period, and intake II period. The stool sample was collected by the subject at home, and the entire amount of stool was collected in a special container at the timing when the stool was released, and sent directly to the measurement facility together with the ice pack under anaerobic conditions.
５－１ 糞便中ラクトバチルス、ラクトバチルスブレビス、ビフィドバクテリウムの測定
糞便中ラクトバチルス（生菌数）の測定は光岡らの方法を一部改変して培養法により測定した。ラクトバチルス選択培地として、変法ＬＢＳ寒天培地を用いた。培地ごとにコロニーを観察し、コロニー形態と菌数を記載した。糞便中ラクトバチルスブレビスの生菌数測定は既報を参考に実施した。なお、糞便中ラクトバチルスブレビス測定は被験食品先行摂取群、対照食品先行摂取群から糞便中ラクトバチルスの菌数が少ない人から優先的に５名ずつ選抜してＰＣＲ解析対象とした。糞便中ビフィドバクテリウム数の測定はリアルタイムＰＣＲにより測定した。
5-1 Measurement of lactobacillus, lactobacillus brevis, and bifidobacteria in feces The measurement of lactobacillus (viable cell count) in feces was measured by a culture method with a partial modification of the method of Mitsuoka et al. A modified LBS agar medium was used as the Lactobacillus selective medium. Colonies were observed for each medium, and the colony morphology and the number of bacteria were described. The viable cell count of Lactobacillus brevis in feces was measured with reference to the previous report. For the measurement of Lactobacillus brevis in feces, 5 persons were preferentially selected from the test food pre-ingestion group and the control food pre-ingestion group from those with a small number of Lactobacillus in feces and used for PCR analysis. The number of bifidobacteria in feces was measured by real-time PCR.
５－２ 糞便中有機酸含量、糞便中腐敗産物含量及び糞便中アンモニア含量の測定
有機酸は乳酸、コハク酸、ギ酸、酢酸、プロピオン酸、ｉｓｏ－酪酸、ｎ－酪酸、ｉｓｏ－吉草酸、ｎ－吉草酸についてイオン排除ＨＰＬＣにより分析した。糞便中の腐敗産物はインドール、フェノール、スカトール、パラエチルフェノール、パラクレゾールについてＧＣ－ＭＳを用いて分析した。また、糞便中アンモニア含量については、アンモニアテストワコー（ＷＡＫＯ）を用いて測定した。
5-2 Measurement of stool organic acid content, stool spoilage product content and stool ammonia content Organic acids are lactic acid, succinic acid, formic acid, acetic acid, propionic acid, iso-fatty acid, n-fatty acid, iso-valeric acid, n -Valeric acid was analyzed by ion exclusion HPLC. The spoilage products in feces were analyzed using GC-MS for indole, phenol, skatole, paraethylphenol, and paracresol. In addition, the ammonia content in feces was measured using Ammonia Test Wako (WAKO).
６ 日誌による調査項目
被験者に対しては、試験期間中毎日（前観察期、摂取Ｉ期、休止期、摂取II期の計８週間）、試験食品摂取の有無（摂取期のみ）、排便回数、排便量、便形状、便の色、便の臭い、排便後の感覚、整腸剤・便秘薬を含む医薬品使用の有無、生理の有無、その他胃腸症状中心に体調の変化の有無、生活状況の変化の有無を「被験者日誌」に記載させた。被験者日誌の排便量については、鶏卵（Ｍサイズ１個）の大きさに換算した個数で記録させた。便形状は見本を参考に（ｉ）コロコロ状、（ii）カチカチ状、（iii）バナナ状、（iv）半練状、（ｖ）泥状、（vi）水状の６段階、便の色は見本色を見て、（ｉ）黄褐色、（ii）茶褐色、（iii）黒褐色の３段階、便の臭いは（ｉ）全く気にならない、（ii）ほとんど気にならない、（iii）普通、（iv）臭い、（ｖ）とても臭い、の５段階評価をさせた。排便後の感覚は（ｉ）スッキリ感がある、（ii）普通、（iii）残便感がある、の３段階評価をさせた。
6 Survey items by diary For subjects, daily during the test period (pre-observation period, intake I period, rest period, intake II period, total 8 weeks), presence / absence of test food intake (intake period only), number of defecations, Defecation volume, stool shape, stool color, stool odor, post-defecation sensation, presence / absence of medications including intestinal regulators / constipation agents, presence / absence of physiology, presence / absence of changes in physical condition centered on other gastrointestinal symptoms, changes in living conditions The presence or absence was recorded in the "subject diary". The amount of defecation in the subject's diary was recorded by the number converted to the size of a chicken egg (1 M size). Refer to the sample for the stool shape: (i) roller-like, (ii) tick-like, (iii) banana-like, (iv) semi-kneaded, (v) muddy, (vi) water-like 6 levels, stool color Looking at the sample color, there are three stages: (i) yellowish brown, (ii) brownish brown, (iii) blackish brown, the stool odor is (i) not at all, (ii) almost unnoticeable, (iii) normal. , (Iv) Smell, (v) Very Smell, 5 grades. The sensation after defecation was evaluated on a three-point scale: (i) refreshing, (ii) normal, and (iii) residual stool.
また、被験者には試験期間中毎日、食事・間食・禁止食品・サプリメント・健康食品類・ドリンク剤・アルコール等の摂取内容をすべて食事日誌に記載させた。各摂取期終了前３日間は、記載内容をもとに栄養管理ソフト（株式会社建帛社：エクセル栄養君ｖｅｒ．８）を使用して栄養摂取量を概算した。また、同期間は飲酒量を算出した。 In addition, the subjects were asked to record all the intake contents of meals, snacks, prohibited foods, supplements, health foods, drinks, alcohol, etc. in the meal diary every day during the test period. For 3 days before the end of each intake period, the nutritional intake was estimated using nutritional management software (Kenjo Co., Ltd .: Excel Nutrition-kun ver.8) based on the description. In addition, the amount of alcohol consumed was calculated during the same period.
７ 統計解析方法
主要評価項目の糞便中ラクトバチルス（生菌数）について一般化線形モデル（ＧＬＭ）により持ち越し効果を検証した。時期効果を消去した上で対照食品先行摂取群と被験食品先行摂取群で対応のないｔ検定を実施した。前後比較については試験食品摂取期ごとに対応のあるｔ検定で実施した。なお、糞便中のラクトバチルスブレビス（生菌数）については、各試験食品の摂取前後で対応のあるｔ検定で実施した。有意水準は両側検定で５％とし、糞便中の細菌数の統計解析においては、検出限界値以下の場合には検出限界値を採用した。なお、統計解析ソフトはＩＢＭ ＳＰＳＳ Ｓｔａｔｉｓｔｉｃｓ ２４を使用した。
7 Statistical analysis method The carry-over effect of lactobacillus (live cell count) in feces, which is the primary endpoint, was verified by a generalized linear model (GLM). After eliminating the timing effect, an unpaired t-test was performed between the control food pre-ingestion group and the test food pre-ingestion group. The comparison before and after was performed by the t-test corresponding to each test food intake period. Lactobacillus brevis (live cell count) in feces was tested by a paired t-test before and after ingestion of each test food. The significance level was set to 5% by the two-sided test, and in the statistical analysis of the number of bacteria in feces, the detection limit was adopted when it was below the detection limit. IBM SPSS Statistics 24 was used as the statistical analysis software.
（結果）
１ 被験者
試験参加者２２名のうち中止症例は生じなかったため、試験完了被験者は２２名であった。試験食品摂取率は２１名の被験者で１００％、１名の被験者で９８．２％であった。食事記録から禁止食を複数回にわたり摂取した被験者１名を解析から除外し、被験食品先行摂取群１０名、対照食品先行摂取群１１名、合計２１名（男性４名、女性１７名、平均年齢４４．３±１０．９歳）のデータを解析した。
(result)
1 Subjects Of the 22 participants in the study, there were no discontinued cases, so 22 subjects completed the study. The test food intake rate was 100% for 21 subjects and 98.2% for 1 subject. One subject who ingested the prohibited diet multiple times was excluded from the analysis from the dietary record, and 10 subjects in the test food pre-ingestion group and 11 subjects in the control food pre-ingestion group, a total of 21 subjects (4 males, 17 females, average age). The data of 44.3 ± 10.9 years old) was analyzed.
割付後の被験者および解析対象被験者の年齢、身長、体重、ＢＭＩ、糞便中ラクトバチルス（生菌数）、糞便中酢酸、糞便中インドールは被験食品先行摂取群、対照食品先行摂取群の両群間で差は認められなかった（表２）。 The age, height, weight, BMI, fecal lactobacillus (viable cell count), fecal acetic acid, and fecal indole of the subjects after allocation and the subjects to be analyzed were between the test food pre-ingestion group and the control food pre-ingestion group. No difference was observed in (Table 2).
被験食品摂取期と対照食品摂取期の間に、食事調査（エネルギー、タンパク質、脂質、炭水化物、食物繊維総量）、飲酒量のすべての項目で有意な差は認められなかった。また、試験期間中に認められた有害事象は２２例中１例２件（感冒からの副鼻腔炎、食欲不振）であったが、試験責任医師により試験食品との因果関係は”関連なし”と判定され、試験食品に起因する副作用は認められなかった。 No significant difference was observed in all items of dietary survey (energy, protein, lipid, carbohydrate, total amount of dietary fiber) and drinking amount between the test food intake period and the control food intake period. In addition, adverse events observed during the study period were 1 in 22 cases (sinusitis from the common cold, loss of appetite), but the causal relationship with the test food was "not related" by the investigator. No side effects caused by the test food were observed.
数値は、平均値±標準偏差を表す。
Numerical values represent mean ± standard deviation.
２ 糞便中ラクトバチルス
主要評価項目である糞便中ラクトバチルス（生菌数）について時期効果、順序効果の検定を行ったところ、時期効果ｐ＝０．５７２、順序効果ｐ＝０．８１４であり、いずれも有意な差は認められなかった。このことから、持ち越し効果はなくクロスオーバー試験は妥当であると判断し、有効性の解析を実施した。各試験食品摂取による糞便中ラクトバチルスへの影響を表３に示した。糞便中ラクトバチルス（ｌｏｇ ｃｆｕ／ｇ）は、摂取前と比較して被験食品摂取後に有意に増加（摂取前：４．５±０．４、摂取後：５．２±０．３、ｐ＜０．０５）したのに対し、対照食品摂取期では有意な変化は認められなかった（摂取前：５．０±０．４、摂取後：４．６±０．４）。また被験食品摂取後の糞便中ラクトバチルスは対照食品摂取後よりも有意に高かった（ｐ＜０．０５）。
2 Lactobacillus in feces The primary endpoints of lactobacillus in feces (viable cell count) were tested for timing effect and order effect, and the timing effect p = 0.572 and order effect p = 0.814. No significant difference was observed in either case. Based on this, it was judged that there was no carry-over effect and the crossover study was appropriate, and the effectiveness was analyzed. Table 3 shows the effects of ingestion of each test food on lactobacillus in feces. Fecal lactobacillus (log cfu / g) increased significantly after ingestion of the test food compared to before ingestion (before ingestion: 4.5 ± 0.4, after ingestion: 5.2 ± 0.3, p < In contrast to 0.05), no significant change was observed during the control food intake period (before ingestion: 5.0 ± 0.4, after ingestion: 4.6 ± 0.4). In addition, lactobacillus in feces after ingestion of the test food was significantly higher than that after ingestion of the control food (p <0.05).
３ 糞便中ラクトバチルスブレビス、糞便中ビフィドバクテリウム
糞便中ラクトバチルスブレビス（生菌）のＰＣＲ検出率について、どの被験者においても試験食品摂取前は検出限界未満であった。被験食品摂取時には糞便中でラクトバチルスブレビスが検出された被験者は１０名中７名であったのに対し、対照食品摂取時では１０名中０名であった。糞便中ラクトバチルスブレビス数の推移を表２に示した。糞便中ラクトバチルスブレビス数（ｌｏｇ ｃｆｕ／ｇ）について、被験食品摂取前は検出限界未満、被験食品摂取後は４．１±０．５であった。糞便中ビフィドバクテリウム（ｌｏｇ ｃｆｕ／ｇ）については、被験食品摂取期では摂取前８．２±０．２、摂取後８．２±０．２、対照食品摂取期では摂取前８．３±０．２、摂取後８．２±０．２であった（表３）。
3 Lactobacillus brevis in feces, bifidobacteria in feces The PCR detection rate of Lactobacillus brevis (live bacteria) in feces was below the detection limit before ingestion of the test food in all subjects. Lactobacillus brevis was detected in feces in 7 out of 10 subjects when the test food was ingested, whereas 0 out of 10 subjects ingested the control food. Table 2 shows changes in the number of Lactobacillus brevis in feces. The number of Lactobacillus brevis (log cfu / g) in feces was below the detection limit before ingestion of the test food and 4.1 ± 0.5 after ingestion of the test food. For bifidobacteria (log cfu / g) in feces, 8.2 ± 0.2 before ingestion, 8.2 ± 0.2 after ingestion during the test food ingestion period, and 8.3 before ingestion during the control food ingestion period. It was ± 0.2 and 8.2 ± 0.2 after ingestion (Table 3).
＃摂取前と比較して有意差（ｐ＜０．０５）あり
＊対照食品摂取期と比較して有意差（ｐ＜０．０５）あり
# There is a significant difference (p <0.05) compared to before ingestion
* There is a significant difference (p <0.05) compared to the control food intake period.
４ 糞便中有機酸
糞便中有機酸のうち、乳酸、コハク酸、ギ酸、酢酸、プロピオン酸、ｉｓｏ－酪酸、ｎ－酪酸、ｉｓｏ－吉草酸含量は、試験実施期間中に有意な変化は認められなかった（表４）。糞便中ｎ－吉草酸含量については、対照食品摂取期において摂取前と比較して摂取後に有意に減少したが（ｐ＜０．０５）、被験食品摂取期と比較して有意な違いは認められなかった。
4 Organic acids in feces Among the organic acids in feces, the contents of lactic acid, succinic acid, formic acid, acetic acid, propionic acid, iso-fatty acid, n-buty acid, and iso-valeric acid showed significant changes during the test period. There was no (Table 4). The fecal n-valeric acid content was significantly decreased after ingestion compared to before ingestion during the control food ingestion period (p <0.05), but a significant difference was observed compared with the test food ingestion period. I didn't.
＃摂取前と比較して有意差（ｐ＜０．０５）あり
＊対照食品摂取期と比較して有意差（ｐ＜０．０５）あり
# There is a significant difference (p <0.05) compared to before ingestion
* There is a significant difference (p <0.05) compared to the control food intake period.
５ 糞便中腐敗産物・アンモニア
糞便中腐敗産物（インドール、パラクレゾール、フェノール、スカトール、パラエチルフェノール）およびアンモニア含量の解析結果を表５に示した。糞便中パラクレゾール含量（ｎｍｏｌ／ｇ）については、摂取前と比較して被験食品摂取後に有意に減少（摂取前：７４７．１±１３１．５、摂取後：５７９．４±９９．９、ｐ＜０．０５）したのに対し、対照食品摂取期では有意な変化は認められなかった（摂取前：８１８．４±１１６．５、摂取後：７９４．６±２１４．５）。しかし、被験食品摂取後の糞便中パラクレゾール含量は対照食品摂取後と比較して有意な違いは認められなかった。糞便中インドール、フェノール、スカトール、パラエチルフェノール、およびアンモニア含量については、試験実施期間中に有意な変化は認められなかった。
5 Fecal putrefactive products / ammonia Table 5 shows the analysis results of fecal putrefactive products (indole, paracresol, phenol, skatole, paraethylphenol) and ammonia content. The content of paracresol in feces (nmol / g) was significantly reduced after ingestion of the test food compared to before ingestion (before ingestion: 747.1 ± 131.5, after ingestion: 579.4 ± 99.9, p. In contrast to <0.05), no significant change was observed during the control food intake period (before ingestion: 818.4 ± 116.5, after ingestion: 794.6 ± 214.5). However, the fecal paracresol content after ingestion of the test food was not significantly different from that after ingestion of the control food. No significant changes were observed in fecal indole, phenol, skatole, paraethylphenol, and ammonia content during the study period.
＃摂取前と比較して有意差（ｐ＜０．０５）あり
# There is a significant difference (p <0.05) compared to before ingestion
６ 便通および便性に対する影響
試験食品摂取による便通・便性状への影響を表６に示した。被験食品摂取期、対照食品摂取期ともに摂取前と比較して摂取後に排便回数が有意に増加し（ｐ＜０．０５）、被験食品摂取期の排便回数は対照食品摂取期と比較して有意に高い傾向が認められた（ｐ＝０．０７）。排便日数、排便量においても被験食品摂取期に摂取前と比較して摂取後に有意に増加したが（ｐ＜０．０５）、群間差は認められなかった。また、便性状（便形状、便の色、便の臭い、排便後の感覚）については、試験実施期間中に有意な変化は認められなかった。
6 Effects on bowel movements and bowel movements Table 6 shows the effects of ingestion of test foods on bowel movements and bowel movements. In both the test food intake period and the control food intake period, the number of defecations was significantly increased after ingestion compared to before intake (p <0.05), and the number of defecations during the test food intake period was significant compared to the control food intake period. A high tendency was observed (p = 0.07). The number of days of defecation and the amount of defecation also increased significantly after ingestion compared to before ingestion during the test food ingestion period (p <0.05), but no difference was observed between the groups. In addition, no significant changes were observed in the stool properties (stool shape, stool color, stool odor, sensation after defecation) during the test period.
＃摂取前と比較して有意差（ｐ＜０．０５）あり
# There is a significant difference (p <0.05) compared to before ingestion
被験者選定の際にスクリーニング前の排便日誌をもとに選定を行ったが、前観察期において排便回数が便秘傾向の定義よりも多い被験者（２週間の被験者日誌の排便回数が週３～５回の者）が２名含まれていた。本試験の目的である、便秘傾向者に対する被験食品の整腸効果を正確に把握するため、上記２名を除く１９名で排便回数の層別解析を実施した（表７）。その結果、被験食品摂取期、対照食品摂取期ともに摂取前と比較して摂取後に排便回数が有意に増加し（ｐ＜０．０５）、被験食品摂取期の排便回数は対照食品摂取期と比較して有意に高かった（ｐ＜０．０５）。また、上記便秘傾向者１９名において被験食品摂取後の糞便中ラクトバチルスは対照食品摂取後よりも有意に高かった（ｐ＜０．０５）。 When selecting the subjects, the selection was made based on the defecation diary before screening, but the subjects who had more defecations than the definition of constipation tendency in the pre-observation period (the number of defecations in the subject diary for 2 weeks was 3 to 5 times a week). 2 people were included. In order to accurately understand the intestinal regulation effect of the test food on constipation-prone persons, which is the purpose of this study, a stratified analysis of the number of defecations was performed by 19 persons excluding the above 2 persons (Table 7). As a result, the number of defecations after ingestion was significantly increased (p <0.05) in both the test food ingestion period and the control food ingestion period compared to before ingestion, and the defecation frequency in the test food ingestion period was compared with the control food ingestion period. It was significantly higher (p <0.05). In addition, the fecal lactobacillus after ingestion of the test food was significantly higher than that after ingestion of the control food in the 19 constipation-prone persons (p <0.05).
ラクトバチルスは、糞便１ｇあたりの当該菌数の常用対数を表す
＃前観察期と比較して有意差（ｐ＜０．０５）あり
＊対照食品摂取群と比較して有意差（ｐ＜０．０５）あり
Lactobacillus represents the common logarithm of the number of bacteria per gram of feces.
# There is a significant difference (p <0.05) compared to the previous observation period
* Significant difference (p <0.05) compared to the control food intake group
実施例１において被験食品を一日当たり４枚、２週間の継続摂取により、便秘傾向者における排便回数の増加が確認でき、被験食品摂取後の糞便中ラクトバチルスの数が増加することも確認できた。 In Example 1, it was confirmed that the number of defecations in the constipated person increased and the number of lactobacillus in the feces after ingesting the test food increased by continuously ingesting 4 test foods per day for 2 weeks. ..
（比較例１）
一日当たりの摂取量を３枚に減らした試験設計でヒト試験を実施し、同様の作用が確認できるか検証した。
(Comparative Example 1)
A human test was conducted with a test design in which the daily intake was reduced to 3 sheets, and it was verified whether the same effect could be confirmed.
１ 試験食品
実施例１と同様にして、試験食品として、ラクトバチルスブレビスＮＴＴ００１・ガラクトオリゴ糖含有チョコレート（被験食品）及びラクトバチルスブレビスＮＴＴ００１とガラクトオリゴ糖を含まないプラセボチョコレート（対照食品）を用いた。
具体的なチョコレートの配合を、以下の表８に示す。
1 Test food In the same manner as in Example 1, lactobacillus brevis NTT001 / galactooligosaccharide-containing chocolate (test food) and lactobacillus brevis NTT001 and galactooligosaccharide-free placebo chocolate (control food) were used.
Specific chocolate formulations are shown in Table 8 below.
比較例１のラクトバチルスブレビスＮＴＴ００１およびガラクトオリゴ糖配合チョコレートも、実施例１記載のチョコレートと同様に、チョコレートにラクトバチルスブレビスＮＴＴ００１粉末及びガラクトオリゴ糖を添加・混合し（３０℃前後／数分）、成形、冷却（７－１０℃／３０－５０分）して製造した。 Lactobacillus brevis NTT001 and galactooligosaccharide-blended chocolate of Comparative Example 1 were also molded by adding / mixing Lactobacillus brevis NTT001 powder and galactooligosaccharide (around 30 ° C./several minutes) to chocolate in the same manner as the chocolate described in Example 1. , Cooled (7-10 ° C./30-50 minutes) to produce.
被験食品は、チョコレート１枚（４ｇ）あたりにラクトバチルスブレビスＮＴＴ００１が１．３×１０８ｃｆｕ、ガラクトオリゴ糖が０．３８ｇ含まれるよう設計した。 The test food was designed to contain 1.3 × 10 8 cfu of Lactobacillus brevis NTT001 and 0.38 g of galactooligosaccharide per piece of chocolate (4 g).
比較例１では１日あたり３枚（ラクトバチルスブレビスＮＴＴ００１として３．８×１０８ｃｆｕ、ガラクトオリゴ糖として１．１ｇ）と設定した。 In Comparative Example 1, 3 sheets per day (3.8 × 10 8 cfu as Lactobacillus brevis NTT001 and 1.1 g as galactooligosaccharide) were set.
２ 被験者
実施例１と同様に採用したが、健常成人２２名（年齢４４．０±１２．４歳、ＢＭＩ２１．２±２．６ｋｇ・ｍ－２）を被験者とし、比較例１の被験者選定にあたっては、実施例１に参加していた被験者も被験者候補（合計３３名）として組込み、その中から最終的に２２名の被験者を選定した。
2 Subjects The subjects were selected in the same manner as in Example 1, but 22 healthy adults (age 44.0 ± 12.4 years, BMI 21.2 ± 2.6 kg · m -2 ) were selected as subjects in Comparative Example 1. Incorporated the subjects who participated in Example 1 as subject candidates (33 subjects in total), and finally 22 subjects were selected from them.
３ 試験スケジュール
実施例１と同様に行った。
被験者には、摂取期間中に試験食品を１２ｇ（４ｇ×３枚）／日を、摂取時間を指定せず自由摂取させた。
４ 評価項目、５ 糞便解析、５－１ 糞便中ラクトバチルス、ラクトバチルスブレビス、ビフィドバクテリウムの測定、５－２ 糞便中有機酸含量、糞便中腐敗産物含量及び糞便中アンモニア含量の測定、６ 日誌による調査項目、７ 統計解析方法は、実施例１と同様とした。
3 Test schedule The same as in Example 1 was performed.
The subjects were allowed to freely ingest 12 g (4 g × 3 sheets) / day of the test food during the ingestion period without specifying the ingestion time.
4 Evaluation items, 5 Fecal analysis, 5-1 Measurement of lactobacillus, lactobacillus brevis, and bifidobacteria in feces 5-2 Measurement of organic acid content in feces, putrefactive product content in feces, and ammonia content in feces, 6 Survey items by diary, 7 Statistical analysis method was the same as in Example 1.
（結果）
１ 被験者
試験参加者２２名のうち中止症例は生じなかったため、試験完了被験者は２２名であった。試験食品摂取率は２０名の被験者で１００％、残り２名の被験者ではそれぞれ９６．５、９２．９％であった。棄却基準、解析対象除外基準に該当した被験者は認められなかったため、有効性の解析対象者は２２名であった。割付後の被験者の年齢、身長、体重、ＢＭＩ、糞便中ラクトバチルス（生菌数）、糞便中酢酸、糞便中インドールは被験食品先行摂取群、対照食品先行摂取群の両群間で差は認められなかった（表９）。被験食品摂取期と対照食品摂取期の間に食事調査、飲酒量のすべての項目で有意な差は認められなかった。また、試験期間中に有害事象の発現は認められなかった。
(result)
1 Subjects Of the 22 participants in the study, there were no discontinued cases, so 22 subjects completed the study. The test food intake rate was 100% for 20 subjects and 96.5 and 92.9% for the remaining 2 subjects, respectively. No subjects met the rejection criteria and the analysis target exclusion criteria, so the number of subjects analyzed for efficacy was 22. Differences in the age, height, weight, BMI, fecal lactobacillus (viable cell count), fecal acetic acid, and fecal indole of the subjects after allocation were observed between the test food pre-ingestion group and the control food pre-ingestion group. Not possible (Table 9). No significant difference was observed in all items of dietary survey and alcohol consumption between the test food intake period and the control food intake period. No adverse events were observed during the study period.
２ 糞便中ラクトバチルス
主要評価項目である糞便中ラクトバチルス（生菌数）について時期効果、順序効果の検定を行ったところ、いずれも有意な差は認められなかったことから、持ち越し効果はなくクロスオーバー試験は妥当であると判断し、有効性の解析を実施した。糞便中ラクトバチルス（ｌｏｇ ｃｆｕ／ｇ）は、摂取前と比較して被験食品摂取後に有意に増加（摂取前：３．９±０．３、摂取後：５．３±０．３、ｐ＜０．０５、表１０）したのに対し、対照食品摂取期では有意な変化は認められなかった（摂取前：４．５±０．５、摂取後：４．５±０．３）。また被験食品摂取後の糞便中ラクトバチルスは対照食品摂取後よりも有意に高かった（ｐ＜０．０５）。
2 Lactobacillus in feces Lactobacillus in feces (live cell count), which is the primary endpoint, was tested for timing effect and order effect, and no significant difference was observed in either case. The overtest was judged to be appropriate, and the efficacy was analyzed. Fecal lactobacillus (log cfu / g) increased significantly after ingestion of the test food compared to before ingestion (before ingestion: 3.9 ± 0.3, after ingestion: 5.3 ± 0.3, p < In contrast to 0.05, Table 10), no significant change was observed during the control food intake period (before ingestion: 4.5 ± 0.5, after ingestion: 4.5 ± 0.3). In addition, lactobacillus in feces after ingestion of the test food was significantly higher than that after ingestion of the control food (p <0.05).
＃摂取前と比較して有意差（ｐ＜０．０５）あり
＊対照食品摂取期と比較して有意差（ｐ＜０．０５）あり
# There is a significant difference (p <0.05) compared to before ingestion
* There is a significant difference (p <0.05) compared to the control food intake period.
３ 糞便中ラクトバチルスブレビス、糞便中ビフィドバクテリウム
糞便中ラクトバチルスブレビス（生菌）のＰＣＲ検出率について、どの被験者においても試験食品摂取前は検出限界未満であった。被験食品摂取時には糞便中でラクトバチルスブレビスが検出された被験者は１０名中８名であったのに対し、対照食品摂取時では１０名中０名であった。糞便中ラクトバチルスブレビス数および糞便中ビフィドバクテリウム数の推移を表１０に示した。糞便中ビフィドバクテリウムについては、試験実施期間中に有意な変化は認められなかった。
3 Lactobacillus brevis in feces, bifidobacteria in feces The PCR detection rate of Lactobacillus brevis (live bacteria) in feces was below the detection limit before ingestion of the test food in all subjects. Lactobacillus brevis was detected in feces at the time of ingestion of the test food in 8 out of 10 subjects, whereas it was 0 out of 10 at the time of ingestion of the control food. Table 10 shows changes in the number of Lactobacillus brevis in feces and the number of bifidobacteria in feces. No significant changes were observed in fecal bifidobacteria during the study period.
４ 糞便中有機酸・腐敗産物・アンモニア
糞便中有機酸のうち酢酸、プロピオン酸、ｉｓｏ－酪酸では、被験食品摂取期において摂取前と比較して摂取後で有意に高い値が認められたが、対照食品摂取期と比較して有意な違いは認められなかった。その他の有機酸については試験実施期間中に有意な変化は認められなかった（表１１）。糞便中腐敗産物・アンモニアについては、試験実施期間中に有意な変化は認められなかった（表１２）。
4 Organic acids in feces, spoilage products, and ammonia Of the organic acids in feces, acetic acid, propionic acid, and iso-butyric acid showed significantly higher values after ingestion than before ingestion during the test food ingestion period. No significant difference was observed compared to the control food intake period. No significant changes were observed in other organic acids during the test period (Table 11). No significant changes were observed in fecal putrefactive products / ammonia during the test period (Table 12).
＃摂取前と比較して有意差（ｐ＜０．０５）あり
# There is a significant difference (p <0.05) compared to before ingestion
５ 便通および便性に対する影響
試験食品摂取による便通・便性状への影響を表１３に示した。被験食品摂取期において摂取前と比較して摂取後に排便回数、排便日数、排便量が有意に増加したが（ｐ＜０．０５）、対照食品摂取期と比較して有意な違いは認められなかった。また、便性状（便形状、便の色、便の臭い、排便後の感覚）については、試験実施期間中に有意な変化は認められなかった。
5 Effects on bowel movements and bowel movements Table 13 shows the effects of ingestion of test foods on bowel movements and bowel movements. The number of defecations, the number of days of defecation, and the amount of defecation significantly increased after ingestion compared with before ingestion during the test food ingestion period (p <0.05), but no significant difference was observed compared with the control food ingestion period. rice field. In addition, no significant changes were observed in the stool properties (stool shape, stool color, stool odor, sensation after defecation) during the test period.
＃摂取前と比較して有意差（ｐ＜０．０５）あり
# There is a significant difference (p <0.05) compared to before ingestion
以上のように、実施例１（試験食品の摂取枚数が４枚／日）では対照食品摂取時と比較して被験食品摂取時に糞便中ラクトバチルス（生菌数）の有意な増加が認められた。対照食品摂取時と被験食品摂取時で糞便中有機酸含量や腐敗産物含量について有意な違いは認められなかったものの、排便回数については対照食品摂取時と比較して被験食品摂取時に高くなる傾向が認められた。更に、排便回数が便秘傾向の定義よりも多い被験者（２週間の被験者日誌の排便回数が週３～５回の者）が２名含まれていたことから、便秘傾向者で層別解析（ｎ＝１９）を実施したところ、対照食品摂取時と比較して被験食品摂取時に糞便中ラクトバチルス（生菌数）や排便回数が有意に高かった。一方、比較例１の試験食品の摂取枚数を３枚／日に減らして試験を実施したところ、対照食品摂取時と比較して被験食品摂取時に糞便中ラクトバチルス（生菌数）の有意な増加は認められたものの、排便回数については群間差が認められなかった。 As described above, in Example 1 (the number of test foods ingested was 4 / day), a significant increase in lactobacillus (live cell count) in feces was observed when the test food was ingested as compared with the time when the control food was ingested. .. Although there was no significant difference in fecal organic acid content or spoilage product content between the control food intake and the test food intake, the number of defecations tended to be higher when the test food was ingested than when the control food was ingested. Admitted. Furthermore, since two subjects with more defecation frequency than the definition of constipation tendency (those who defecate 3 to 5 times a week in the subject diary for 2 weeks) were included, stratified analysis (n) for constipation tendency subjects. = 19) When the test food was ingested, the fecal lactobacillus (live cell count) and the number of defecations were significantly higher than those in the control food. On the other hand, when the test was carried out by reducing the number of test foods ingested in Comparative Example 1 to 3 sheets / day, the fecal lactobacillus (live cell count) was significantly increased when the test food was ingested as compared with the control food ingestion. However, there was no difference in the number of defecations between the groups.
本発明では、それぞれ単独では便通促進作用が期待できない摂取量ではあるものの、ラクトバチルスブレビスＮＴＴ００１（５．０×１０８ｃｆｕ／日）、ガラクトオリゴ糖（１．５ｇ／日）を併用することで、便秘傾向者に対する便通促進作用が認められた。
すなわち、ラクトバチルスブレビスＮＴＴ００１を一日当たり５．０×１０８ｃｆｕ以上、ガラクトオリゴ糖を一日当たり１．５ｇ以上併用して摂取することで、便秘傾向者に対する便通促進作用が認められることが判明した。なお、本発明におけるチョコレート中のガラクトオリゴ糖は、全て砂糖と代替置換して配合することも可能だが、一日当たり摂取量として１．５ｇ以上を担保していれば、風味の面を考慮して自由に調整して配合できる。ラクトバチルスブレビスＮＴＴ００１は、他のラクトバチルスブレビス種細菌と置換して配合しても同様の効果が得られるが、一日当たり摂取量としては５．０×１０８ｃｆｕ以上が望ましい。なお、風味や製造適性の面を考慮して、チョコレート１ｇ当たり１．０×１０１２ｃｆｕ以下とするのが望ましい。
In the present invention, although the intake amount cannot be expected to promote bowel movements by themselves, by using Lactobacillus brevis NTT001 (5.0 × 108 cfu / day) and galactooligosaccharide (1.5 g / day) in combination, A bowel movement promoting effect was observed for people with constipation tendency.
That is, it was found that by ingesting Lactobacillus brevis NTT001 in combination of 5.0 × 108 cfu or more per day and galactooligosaccharide in combination of 1.5 g or more per day, a bowel movement promoting effect is observed for constipation-prone persons. The galactooligosaccharides in chocolate in the present invention can all be substituted with sugar and blended, but if the daily intake is guaranteed to be 1.5 g or more, it is free in consideration of flavor. Can be adjusted and blended. The same effect can be obtained by substituting Lactobacillus brevis NTT001 with other Lactobacillus brevis species bacteria, but the daily intake is preferably 5.0 × 108 cfu or more. In consideration of flavor and manufacturing aptitude, it is desirable that the amount is 1.0 × 10 12 cfu or less per 1 g of chocolate.
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