TWI304340B - Use of probiotic lactic acid bacteria for preparing a carrier for normalizing the skin's immune function - Google Patents

Description

1304340 A7 B7 五、發明説明（1 本發明係關於使用益生乳酸菌來製備平衡皮膚之免疫功 能的載體。特定而言，本發明係關於在緊迫的狀況下，像 是暴露在紫外線照射下，使用益生乳酸菌之微生物來改善 皮膚的免疫功能，導致免疫抑制，特別是用來使皮膚的免 疫活性正常化’並降低在這類狀況下發展出過度·反應的傾 向。 大氣中臭氧層的持續降低，同時增加了到達植物表面的 紫外線照射，這已經引起其對人類健康之可能影響的極大 興趣。雖然暴露在紫外線照射下對於人類產生維他命D是 必要的，但逐漸增加的證據暗示廣泛暴露在陽光下，尤其 是在紫外線照射下，引起皮膚的各種問題，包括引起某些 皮膚癌，並引起加速的皮膚老化（光老化）。 目前假定產生皮膚癌的主要因素是紫外線照射引起在皮 膚中生殖細胞之DNA中的突變傷害，而UV-光對皮膚免疫 系統的傷害顯然似乎支援其進一步發展所需的第二個因素 。在健康的系統中，藉著皮膚免疫系統的正常功能排除早 期的惡性細胞。但是在紫外線照射時，土膚的免疫系統似 乎經歷壓抑，且不能再進行其正常的監督。結果未排除極 早期的皮膚癌症細胞，該狀態最後將導致惡性細胞逃脱免 疫系統，並發展成腫瘤。 除了這些已建立的健康關連之外，研究亦提供證據，暗 示暴露在紫外線照射下，可能對活體中的各種免疫反應， 對局部的，在UV-照射的皮膚中，以及全身的，也就是遠 離照射之皮膚的地方有不利影響。已經發現使老鼠暴露在 -4 - 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公爱)_ 装 訂1304340 A7 B7 V. INSTRUCTIONS (1) The present invention relates to the use of probiotic lactic acid bacteria to prepare a carrier for balancing the immune function of the skin. In particular, the present invention relates to the use of probiotics under urgent conditions, such as exposure to ultraviolet radiation. Microorganisms of lactic acid bacteria improve the immune function of the skin, leading to immunosuppression, especially to normalize the immune activity of the skin' and reduce the tendency to develop excessive reactions in such conditions. The ozone layer in the atmosphere continues to decrease while increasing Ultraviolet exposure to the surface of plants has caused great interest in the possible effects of human health. Although exposure to ultraviolet light is necessary for humans to produce vitamin D, increasing evidence suggests widespread exposure to sunlight, especially It is caused by various problems caused by ultraviolet rays, including causing certain skin cancers and causing accelerated skin aging (photoaging). It is currently assumed that the main cause of skin cancer is ultraviolet radiation caused by DNA in germ cells of the skin. Mutation damage, while UV-light is free of skin The damage of the system clearly seems to support the second factor needed for its further development. In healthy systems, early malignant cells are excluded by the normal function of the skin's immune system. But in the case of ultraviolet radiation, the immune system of the skin seems to experience Repressed, and can no longer perform its normal supervision. The results did not rule out very early skin cancer cells, which will eventually lead to malignant cells escape the immune system and develop into tumors. In addition to these established health concerns, the study also provides Evidence suggests that exposure to UV radiation may have adverse effects on various immune responses in the living, local, in UV-irradiated skin, and throughout the body, that is, away from the irradiated skin. Exposed to -4 - This paper scale applies to China National Standard (CNS) A4 specification (210X297 public) _ binding

線 1304340 A7 B7 五、發明説明（2 ) UV-B照射下’干擾了對uv_引起之皮膚癌的排斥，並在未 經照射的地方，開始引起延遲型和接觸性過敏（DTH，CHS) 反應。認爲在紫外線照射時發現的這些形式之免疫抑制， 與抗原-專一之抑制者T_淋巴細胞的謗導作用有關連。DTH 反應是特別重要的，因爲該Τ -淋巴細胞-調節的免疫反應， 係負責保護對抗許多慢性的傳染病。 目前的實驗證據暗示衍生自經過U V -照射之角化細胞的 可溶性物質，可能是UV-引起之DTH-和CHS-反應的全身性 抑制的介體。以在活體内，在老鼠中CHS之全身性抑制的 作用範圍爲基礎，已經提出出現在角質層中的尿刊酸，組 胺酸的脱胺產物，是該型UV-引起之免疫抑制的一種光受 體。 除了抑制皮膚免疫系統之外，已經發現紫外線照射亦對 皮膚引起炎症性和應激性的影響，最後可能導致皮膚之紅 斑、水腫及/或皮屑或鱗屑（過度角化）的發展。這些炎症性 及/或應激性反應是完全分離的，並可與首先提及的分離， 因爲與免疫系統的抑制相反，這些反應寧可與其刺激有關。 在此項技藝中，已經數次嘗試緩和紫外線照射對皮膚的 有害影響，像是藉著使用防曬劑或其他特殊的藥學製劑。 在 J· Invest. Dermatol·，97(1991)，624-628 中，報告吸收紫外 線照射之化合物（防曬劑）的局部應用，在預防紫外線照射-Line 1304340 A7 B7 V. Description of invention (2) UV-B irradiation interferes with the rejection of skin cancer caused by uv_ and begins to cause delayed and contact allergy (DTH, CHS) in unexposed areas. reaction. It is believed that these forms of immunosuppression found during UV irradiation are associated with the targeting of antigen-specific suppressor T-lymphocytes. The DTH response is particularly important because the sputum-lymphocyte-regulated immune response is responsible for protecting against many chronic infectious diseases. Current experimental evidence suggests that soluble substances derived from U V -irradiated keratinocytes may be mediators of systemic inhibition of UV-induced DTH- and CHS-reactions. Based on the range of action of systemic inhibition of CHS in mice in vivo, it has been proposed that urinary acid present in the stratum corneum, a deamination product of histidine, is a type of UV-induced immunosuppression. Photoreceptor. In addition to inhibiting the skin's immune system, it has been found that ultraviolet radiation also causes inflammatory and stress effects on the skin, which may eventually lead to the development of erythema, edema and/or dandruff or scales (over-keratinization) of the skin. These inflammatory and/or stress responses are completely isolated and can be separated from the first mentioned, as opposed to inhibition of the immune system, these reactions are rather related to their stimulation. In this art, attempts have been made to mitigate the deleterious effects of ultraviolet radiation on the skin, such as by the use of sunscreens or other special pharmaceutical agents. In J. Invest. Dermatol, 97 (1991), 624-628, the topical application of UV-irradiated compounds (sunscreens) is reported to prevent UV exposure -

I 引起之紅斑和水腫上是有效的，但不能預防U V -光引起的 免疫-抑制。藉著數個其他的研冗證實該發現，因此該防曬 劑似乎可預防炎症反應及/或刺激，但不能提供對抗紫外線 -5- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 裝 訂I is effective in causing erythema and edema, but does not prevent immuno-inhibition caused by U V - light. The discovery was confirmed by several other studies, so the sunscreen seems to prevent inflammation and/or irritation, but it does not provide protection against UV-5- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297) Binding

線 1304340 A7 B7 五、發明説明（3 ) 照射之免疫抑制影響的完整預防性保護。 此外，發現已知經常用來治療應激性和炎症性皮膚的藥 學製劑，像是皮質類固醇、啕哚美辛或乙醯水楊酸，當在 造成傷害之後使用，並沒有治療UV-光引起之皮膚免疫系 統之抑制狀況的效果。如同由Bergstresser等人在 Immunology 46(1989)，219-245中提出的，皮質類固醇的局 部應用通常會降低皮膚的免疫反應。雖然已經由Reeve等人 在 Cancer Letters 95(1995)，213-219 中證實了 Θ丨嗓美辛抑制 光-致癌作用，但該效果顯然涉及腫瘤發展的開始期和促進 期，並因此認爲其功能爲產生抗-致癌作用的效果，而非對 皮膚免疫系統的效果。因此，藉以使製劑能夠抑制炎症反 應和刺激的模式，可能無法保護皮膚的免，疫系統。 換句話説，證實可有效對抗紫外線照射引起之免疫抑制 的製劑，在改善由於皮膚暴露在紫外線照射下而引起之炎 症性和應激性影響方面，並未顯示任何效果。在美國專利 第5,302,389號中，提出經由微脂粒包膠之DNA修補酵素的 應用，來治療或預防UV-引起之免疫-抑制的製劑和方法。 然而，該製劑對於治療皮膚的炎症性和應激性反應是無效 的0 因此，在此項技藝中需要可在皮膚經歷緊迫狀況時，降 低皮膚發展出過高-反應的製劑，且其能夠降低紫外線照射Line 1304340 A7 B7 V. INSTRUCTIONS (3) Complete preventive protection of the immunosuppressive effects of irradiation. In addition, it has been found that pharmaceutical preparations that are often used to treat stressful and inflammatory skin, such as corticosteroids, indomethacin or acetaminophen, are used after causing harm and are not treated by UV-light. The effect of the inhibition of the skin's immune system. As suggested by Bergstresser et al., Immunology 46 (1989), 219-245, local application of corticosteroids generally reduces the skin's immune response. Although indomethacin has been shown to inhibit photo-carcinogenesis by Reeve et al., Cancer Letters 95 (1995), 213-219, this effect clearly relates to the onset and promotion phases of tumor development and therefore The function is to produce an anti-carcinogenic effect, not an effect on the skin's immune system. Therefore, in order to enable the preparation to inhibit the pattern of inflammatory reactions and irritation, it may not be able to protect the skin from the disease system. In other words, it has been confirmed that the preparation which is effective against the immunosuppression caused by ultraviolet irradiation does not exhibit any effect in improving the inflammatory and stress effects caused by the exposure of the skin to ultraviolet rays. In U.S. Patent No. 5,302,389, the use of a DNA repair enzyme for microlipid encapsulation to treat or prevent UV-induced immuno-inhibition formulations and methods is proposed. However, the formulation is ineffective for treating inflammatory and stressful responses to the skin. Thus, there is a need in the art to reduce the skin's development of over-reactive formulations when the skin experiences an urgent condition, and which can be reduced Ultraviolet radiation

I 對抑制皮膚之免疫系統的影響。 在過去，各種公告描述了對個體適切地發揮有利影響的 微生物。根據 R. Fuller 在 Journal of Applied Bacteriology -6- 本纸張尺度適用中國國家標準(CNS) A4規格(210 x 297公釐） 1304340 A7 B7 五、發明説明（4 ) 66(1989)，365-378中，將這些微生物稱爲”益生乳酸菌"，並 將其定義爲藉著改善其腸道内的微生物平衡，有利地影響 宿主的活微生物。 益生乳酸菌是非-病原性和非-產毒素的生物，其在通過 胃和小腸的通路内存活。當由宿主連續攝食時，它們最後 可移居腸道，至相當大的程度，因此與其他可能具有病原 性的細菌競爭營養及/或在胃-腸壁上的附著位置，並降低 其數量，以及降低或預防感染。 直到現在，已經發現許多不同的益生乳酸菌微生物，報 告它們全部在腸道中，經由毒素、代謝副產物、短鏈脂防 酸及其類似物的產生，而發揮其效用。 亦已經證實在宿主中，胃腸道的微生物區可影響黏膜的 免疫力。根據 Schiffrin等人在 Am. J. Clin. Nutr. 66(1997)， 520中，腸上皮細胞、血液白血球、B -和T -淋巴細胞，還 s 有免疫系統的輔助細胞，已經全部涉及上文提及的免疫力 至某種程度。因此，認爲益生乳酸菌生物在許多層面與免 疫系統產生交互作用，包括細胞***素的產製、單核細胞 的增殖、巨噬細胞的呑噬作用，以及殺死細菌和原蟲病原 的免疫力，及其類似者。 因爲益生乳酸菌之生物活性主要是發生在個體的腸道黏 膜，或相鄰的組織内/上，所以認爲這類微生物的影響主I influence the immune system that inhibits the skin. In the past, various announcements have described microorganisms that have a beneficial effect on individuals. According to R. Fuller in Journal of Applied Bacteriology -6- This paper scale applies Chinese National Standard (CNS) A4 specification (210 x 297 mm) 1304340 A7 B7 V. Description of invention (4) 66 (1989), 365-378 These microorganisms are referred to as "probiotic lactic acid bacteria" and are defined as beneficially affecting the living microorganisms of the host by improving the balance of microorganisms in the intestinal tract. Probiotic lactic acid bacteria are non-pathogenic and non-toxin-producing organisms, It survives through the passage of the stomach and small intestine. When continuously fed by the host, they can eventually migrate to the intestines to a considerable extent, thus competing with other potentially pathogenic bacteria for nutrition and/or in the stomach-intestinal wall. The attachment position on the top, and the reduction in its number, as well as reducing or preventing infection. Until now, many different probiotic lactic acid bacteria have been discovered, all reported in the intestine, via toxins, metabolic by-products, short-chain lipid acid and similar The production of the substance, and its utility. It has also been confirmed that in the host, the microbial area of the gastrointestinal tract can affect the immunity of the mucosa. According to Schiffrin et al. Am. J. Clin. Nutr. 66 (1997), 520, intestinal epithelial cells, blood leukocytes, B- and T-lymphocytes, and s-assisted cells of the immune system, all involving the aforementioned immunity To some extent, it is believed that probiotic lactic acid organisms interact with the immune system at many levels, including the production of cytokinins, the proliferation of monocytes, the phagocytosis of macrophages, and the killing of bacteria and The immunity of the worm pathogen, and the like. Because the biological activity of the probiotic lactic acid bacteria mainly occurs in the intestinal mucosa of the individual, or in the adjacent tissue, it is considered that the main influence of such microorganisms

I 要是在身體的該部份。在這方面，文獻提供了由個體攝食 益生乳酸菌，可在胃腸道中顯示出某些影響的足夠證據。 WO 00/41707揭示催 口延乳酸桿菌（Lactobacillus salivarius) 本紙張尺度適用中國國家標準(CNS) A4規格(210 x 297公釐) 1304340 A7 B7 五、發明説明（5 ) ， 品系’在不想要的胃腸炎症性活性的預防或治療中是有用 的，像是炎症性腸病或應激性腸徵候群。此外，在w〇 00/35465中，揭示乳酸桿菌及/或其他益生乳酸菌生物的口 服組合物，以供建立及維持健康的泌尿生殖菌叢。 在引導本發明的廣泛研究期間，目前驚人地發現益生乳 酸菌亦在個體的體内，在遠離其移居的地方發揮作用。特 定而言，已經發現益菌助生質微生物亦在個體皮膚中的免 疫系統上，發揮其活性，因此，已經發現當個體攝食時， 其可平衡皮膚之免疫系統暴露在緊迫下固有的抑制作用， 像是物理、化學或生物學的緊迫，而它們亦可在暴露於這 類緊迫狀況下時，降低個體發展出炎症性及/或應激性反應 的傾向。 因此’根據本發明最廣泛的觀點，提供益生乳酸菌或其 培養物上清液，在製備平衡皮膚之免疫功能的載體上的用 途0 爲了本發明，”平衡皮膚之免疫功能”一詞應解釋爲在導 致免疫系統之抑制作用的緊迫狀況下，使免疫功能正常化 ，也就是説，即使是在暴露於這類緊迫狀況下時，仍將免 疫功犯或免疫狀況維持在皮膚中主要的正常水準下。緊迫 狀況應解釋爲包括物理緊迫’例如皮膚的紫外線职射，化 學緊迫，像是暴露在化學製劑，或過敏原物質下，或生物 學緊迫’像是接觸或被微生物感染，像是病原性細菌、病 毒、眞菌等等。在這類狀況下，一方面是抑制免疫系統， 例如當暴露在UV-光的刺激下時，另一方面則是過度-刺激 • 8 - 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 1304340I should be in this part of the body. In this regard, the literature provides sufficient evidence that a probiotic lactic acid bacterium is ingested by an individual and that it exhibits some effects in the gastrointestinal tract. WO 00/41707 discloses Lactobacillus salivarius. This paper scale applies to Chinese National Standard (CNS) A4 specification (210 x 297 mm) 1304340 A7 B7 V. Invention description (5), strain 'in unwanted It is useful in the prevention or treatment of gastrointestinal inflammatory activity, such as inflammatory bowel disease or stressful intestinal syndrome. Further, in WO 00/35465, an oral composition of Lactobacillus and/or other probiotic lactic acid bacteria is disclosed for establishing and maintaining a healthy genitourinary flora. During the extensive research that led to the present invention, it has now surprisingly been found that probiotic lactic acid bacteria also function in individuals' bodies, away from where they migrate. In particular, it has been found that probiotic microbes also exert their activity on the immune system in the skin of an individual, and therefore it has been found that when an individual ingests food, it balances the skin's immune system to an underlying inhibition of stress, Like physical, chemical or biological urgency, they can also reduce the propensity of an individual to develop an inflammatory and/or stress response when exposed to such stressful conditions. Thus, in accordance with the broadest aspect of the present invention, the use of a probiotic lactic acid bacterium or a culture supernatant thereof for the preparation of a carrier for balancing the immune function of the skin is used. For the purposes of the present invention, the term "balanced immune function of the skin" should be interpreted as Under the urgent conditions that lead to the inhibition of the immune system, the immune function is normalized, that is, the immune normality or immune status is maintained at the main normal level in the skin even when exposed to such urgent conditions. under. Urgent conditions should be interpreted to include physical stresses such as UV exposure to the skin, chemical stress, such as exposure to chemicals, or allergens, or biological urgency such as exposure or infection by microorganisms, such as pathogenic bacteria. , viruses, sputum, etc. In such cases, on the one hand, the immune system is suppressed, for example when exposed to UV-ray stimuli, and on the other hand, it is over-stimulated. 8 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1304340

免疫系統’像是暴露在刺激劑和過敏原下時。此外，在本 發明的前後文中，並择春物卜、，主 · 八培餐物上清;夜應指足益生乳酸菌本身 的培養物上清液，或從装φ厶祐a、曲，、 4仗具Τ为離之濃縮形式，或具有活性 的代謝產物（們）。 目前已經發現在對個體投與可移居至個體之腸道的益生 乳酸菌，或對個體投與其培養物上清液的案例中，發現某 二緊d兄的免疫抑制影響，像是例如紫外線照射，明顯 是較低的’或實f上降低，同時炎症性及/或應激性反應僅 以縮減的程度發生，或完全不發生。 在圖中， 圖1顯示在各種老鼠模式中，以判定耳朵腫脹來檢查與接 觸性過敏反應有關的炎症性反應； 圖2顯π利用對抗ICAM-1之抗體，在各種指示之動物模 式中獲得的結果； 圖3顯示在各種受試的動物模式中，TGF_卢的含量； 圖4顯示在各種受試的動物模式中，表皮IL_i()的含量； 圖5顯示益生乳酸菌對uv照射引起之免疫系統抑制的影 響，根據以耳朵腫脹判定的接觸性過敏反應來顯示之； 圖6顯示益生乳酸菌對uv照射引起之免疫系統抑制的影 響，以(：^18抑制％來顯示之。 本發明先暫時提供腸道菌叢組合物，亦可在遠離益生乳 酸菌微生物在個體中移居之處的區域，也就是皮膚，對某 些保護性功能具有有利的影響。此外，已經顯示益生乳酸 菌可在皮膚中控制分歧的處理，像是在免疫_抑制狀況的期 -9- T紙張尺度適财g @家標準(CNS) M規格(训χ撕公董)— 1304340 A7 B7 五、發明説明（7 ) 間，向上-調節免疫系統，並在同時向下-調節過度-反應， 像是炎症性或過敏性反應，像是濕療或異位性皮膚炎。隻 影響對於较年長的個體是特別眞實的，其正常便具有降低 的免疫能力0 、 根據較佳的具體實施例，包括在載體内的益生乳酸菌係 選自包括乳酸菌，尤其是乳酸桿菌（Lactobacilli)及/或雙歧 样菌（Bifidobac-teria)，且更佳的是強森乳桿菌 (Lactobacillus johnsonii)、路特乳桿菌（Lactobacillus reuteri) 、鼠李乳桿菌（Lactobacillus rhamnosus)、副乾赂乳样菌 (Lactobacillus paracasei)、乾酪乳桿菌（Lactobacillus casei) 、兩歧雙歧桿菌（Bifidobacterium bifidum)、短小雙歧桿菌 (B. breve)、動物雙歧桿菌（B. animalis)、嬰兒雙歧桿菌（B. infantis)、青春雙歧样菌（B· dolescentis)、，假鏈雙歧桿菌（B. pseudocatemxlatum)。因爲它們不同的氧需求，故乳酸桿菌 和雙歧桿菌移居在腸道的不同地方，可使用其混合物，以 便提供廣效的範圍。 根據最佳的具體實施例，所使用的品系爲強森乳桿菌 (Lai)其在布達佩斯條約下存放在巴斯德協會（Institute Pasteur)，並接受存放編號CNCM 1- 1225，或副乾酪乳桿菌 (ST11)品系，根據布達佩斯條約存放在巴斯德協會，並接 受存放編號CNCM 1-2116。 i 載體可以是任何可在其中包括益生乳酸菌微生物或其培 養物上清液的食品或藥學產品，或可供口服或局部應用的 化粧產品。食品或藥學載體的實例爲牛乳、優格、凝乳、 -10- 本紙張尺度適用中國國家標準(CNS) A4規格(21〇x 297公釐） 1304340 A7 __B7 五、發明説明（8 ) 乾酪、發酵乳、以牛乳爲基礎的發酵產品、冰淇淋、以發 酵毅類爲基礎的產品、以牛乳爲基礎的粉末、嬰兒配方或 寵物食品，或錠劑、液態的細菌懸浮液、脱水的口服添加 物、濕的口服添加物、脱水的管式飼料或濕的管式飼料。 至於化粧產品，預見洗劑、洗髮精、乳霜，像是保濕乳霜 、防釀劑、日fe後用的乳霜或抗老化乳霜，及/或款膏，可 以活形式、半-活性或除活性之形式，將微生物包含在其中 ，例如以冷凍乾燥的散劑。在化粧產品中亦可包含微生物 的培養物上清液，可視需要以濃縮的形式。 應了解當以微生物之形式投與個體時，益生乳酸菌的平 衡活性將是劑量依賴性的。因此，預見包括從1〇5至1〇12個 生物/克這麼多的產物，該生物可以是活潑的或已死亡的。 當使用益生乳酸菌之培養物的上清液時，可使用在納入產 品内之前，先經歷一或多個純化步驟的上清液，而得以濃 縮或分離活性成份（們）/代謝產物（們）。純化化合物，以及 在所獲得的溶離份中檢測其活性的方法‘技術，爲熟諳此 藝者已熟知的。 雖然本發明適用於所有的活生物，像是人類和動物，特 別是寵物，但益生乳酸菌最好是供較年長的個體使用，其 通常具有降低的免疫能力〇 在引導本發明的遠大實驗期間，利用無菌動物模式，集The immune system' appears to be exposed to irritants and allergens. In addition, in the context of the present invention, the selection of the spring material, the main · Ba Pei meal supernatant; the night should refer to the probiotic lactic acid bacteria itself culture supernatant, or from the installation of φ 厶 a a, Qu, 4 The cockroach is a concentrated form or an active metabolite (s). It has been found that in the case of administering to a subject a probiotic lactic acid bacterium that can migrate to the intestinal tract of an individual, or injecting a supernatant to the culture of the individual, it is found that the immunosuppressive effect of a certain two brothers, such as ultraviolet radiation, Significantly lower 'or lower', while inflammatory and/or stressful responses occur only to a reduced extent, or not at all. In the figure, Figure 1 shows the inflammatory response associated with contact allergic reactions in various mouse models to determine ear swelling; Figure 2 shows π using antibodies against ICAM-1, obtained in various indicator animal models Figure 3 shows the content of TGF_Lu in various animal models tested; Figure 4 shows the content of epidermal IL_i() in various animal models tested; Figure 5 shows the probiotic lactic acid bacteria caused by UV irradiation The effect of immune system inhibition is shown by the contact allergic reaction determined by ear swelling; Figure 6 shows the effect of probiotic lactic acid bacteria on immune system inhibition caused by uv irradiation, which is shown by (%18% inhibition). Providing a composition of the intestinal flora for a while, or in a region away from where the probiotic lactic acid bacteria migrate in the individual, that is, the skin, has a beneficial effect on certain protective functions. In addition, it has been shown that probiotic lactic acid bacteria can be in the skin. Controlling the divergence of treatment, such as in the period of immune_inhibition status -9-T paper scale suitable for wealth g @家标准(CNS) M specification (training tearing Dong) - 1304340 A7 B7 5. Description of the invention (7), up-regulating the immune system, and simultaneously down-regulating excessive-response, such as inflammatory or allergic reactions, such as wet therapy or atopic dermatitis. Older individuals are particularly sturdy, which normally have reduced immunity. 0. According to a preferred embodiment, the probiotic lactic acid bacteria included in the carrier are selected from the group consisting of lactic acid bacteria, especially Lactobacilli and/or Bifidobac-teria, and more preferably Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus Paracasei), Lactobacillus casei, Bifidobacterium bifidum, B. breve, B. animalis, B. infantis B. dolescentis, B. pseudocatemxlatum. Lactobacillus and Bifidobacteria migrated because of their different oxygen requirements. In different parts of the intestine, mixtures thereof may be used in order to provide a wide range of effects. According to a preferred embodiment, the strain used is Lactobacillus johnsonii (Lai) which is deposited under the Budapest Treaty with the Pasteur Association ( Institute Pasteur), and accepts the storage number CNCM 1- 1225, or the Lactobacillus paracasei (ST11) strain, deposited under the Budapest Treaty at the Pasteur Association, and accepts the storage number CNCM 1-2116. The i carrier may be any food or pharmaceutical product in which the probiotic lactic acid bacteria microorganism or its culture supernatant can be included, or a cosmetic product for oral or topical application. Examples of food or pharmaceutical carriers are milk, yogurt, curd, -10-. The paper size applies to the Chinese National Standard (CNS) A4 specification (21〇x 297 mm) 1304340 A7 __B7 V. Description of invention (8) Cheese, Fermented milk, milk-based fermentation products, ice cream, fermented products, milk-based powders, infant formulas or pet foods, or lozenges, liquid bacterial suspensions, dehydrated oral supplements , wet oral supplements, dehydrated tubular feed or wet tubular feed. As for makeup products, anticipation lotions, shampoos, creams, such as moisturizing creams, anti-glace, creams or anti-aging creams, and/or creams, can be live, half- The active or in addition to the active form, the microorganism is included therein, for example, as a lyophilized powder. The culture supernatant of the microorganism may also be included in the cosmetic product, and may be in a concentrated form as needed. It will be appreciated that when administered to an individual in the form of a microorganism, the balanced activity of the probiotic lactic acid bacteria will be dose dependent. Therefore, it is foreseen to include so many products from 1〇5 to 1〇12 organisms/gram, which may be lively or dead. When the supernatant of the culture of probiotic lactic acid bacteria is used, the supernatant which has undergone one or more purification steps before being incorporated into the product can be used to concentrate or separate the active ingredients (men) / metabolites (men) . Methods for purifying compounds, as well as detecting their activity in the obtained fractions, are well known to those skilled in the art. Although the invention is applicable to all living organisms, such as humans and animals, particularly pets, probiotic lactic acid bacteria are preferably used by older individuals, which generally have reduced immunity, during the ambitious experiment leading to the present invention. Using sterile animal models, sets

I 中於微生物菌叢結合特徵（MAC)對無菌動物特徵（GAC)， 將其特徵視爲重要的値，特別是顯露在皮膚上的過敏性/炎 症性疾病。 -11 - 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐）I. In the microbial flora binding characteristics (MAC) versus sterile animal characteristics (GAC), its characteristics are considered to be important defects, especially allergic/inflammatory diseases that are exposed on the skin. -11 - This paper size applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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1304340 A7 B7 五、發明説明（9 下列的實例進一步解釋本發明，而非對其加以限制。 藉著數種參數評估對DNCB發展出之過敏反應的程度（參 見下文）。 關於實驗用的雄性老鼠C3H(lps+)，以四組每組八隻老 鼠集合由 Research Center 0rUans(CNRS 〇rUans，France)供 應的抶菌老。各組反映腸道的不同狀態，並命名爲"C，，（ 帶有傳統（正常）微生物菌叢的組）、” L v ”（帶有僅由活乳酸 菌組成之微生物菌叢的組）、”LD”（餵食死的益生乳酸菌（ 死的乳酸桿菌）的組）和” A，，（無菌的（無菌的）老鼠）；所有的 組均維持在分開的蘢子中，而c組維持在正常的狀況下。 投與老鼠的应生乳酸菌是乳酸桿菌ST1丨（獲自Institute Pasteur，以CNCM 1-2116之登錄編號），在1〇8 cfu/毫升 (c fu =菌落形成單位）的溶液中以活的形式提供之，或是使 用經由照射的已死或失活形式，當應用集中餵食時，以在 水中之2.1克/25¾升的溶液，或是以33克/公升放在水中 (下文）。 欲獲得具有基本上僅由活的乳酸菌組成之微生物菌叢的 老鼠，在第7和第8天時分別以含有108 cfu/毫升的〇 . 5毫升 ;谷液集中銀食典囷動物（在前）兩次（LV组）。或者，爲了獲 得具有益生乳酸菌品系（藉著照射使其失活）之腸道微生物 菌叢的老鼠，從第36至38天，從第41至45天，從第48至 49天和從第52天，在指定之日分別以含有21克/2 5毫升失 活微生物的0 · 2毫升溶液集中餵食無菌動物。從第5 5天開 始，將上文的溶液換成含有3 . 3克/公升失活微生物的溶液 -12- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐)1304340 A7 B7 V. INSTRUCTIONS (9 The following examples further illustrate the invention without limiting it. Several parameters are used to assess the extent of allergic reactions to DNCB development (see below). About experimental male mice C3H (lps+), with four groups of eight mice in each group collected by the Research Center 0rUans (CNRS 〇rUans, France). Each group reflects the different states of the intestine and is named "C,, (with a group of traditional (normal) microbial flora), "L v " (group with microbial flora consisting only of live lactic acid bacteria), "LD" (group of probiotic lactic acid bacteria (dead lactobacilli) fed with) "A, (sterile (sterile) mice); all groups are maintained in separate scorpions, while group c is maintained under normal conditions. The lactic acid bacteria that are administered to mice are Lactobacillus ST1 丨 (obtained) From Institute Pasteur, in the registration number of CNCM 1-2116, in live solution in a solution of 1〇8 cfu/ml (c fu = colony forming unit), or using dead or inactivated via irradiation Form, when For concentrated feeding, use a solution of 2.1 g / 253⁄4 liter in water, or 33 g / liter in water (below). To obtain a mouse with a microbial flora consisting essentially of only live lactic acid bacteria, On the 7th and 8th day, respectively, containing 105 cfu/ml of 〇. 5 ml; the gluten concentrate of silver ritual animals (before) twice (LV group). Or, in order to obtain a probiotic lactic acid strain (by The mice that inactivated the intestinal microflora from the 36th to the 38th day, from the 41st to the 45th, from the 48th to the 49th and the 52nd day, respectively, on the designated day, containing 21 grams /2 5 ml of inactivated microorganisms in 0.2 ml of concentrated solution for feeding sterile animals. From the 5th day, replace the above solution with a solution containing 3.3 g / liter of inactivated microorganisms-12 - paper scale Applicable to China National Standard (CNS) A4 specification (210 X 297 mm)

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’在飲水中供應該溶液直到實驗結束爲止（LD組）。欲獲得 傳統（正常）的腸道微生物菌叢，在到達之後三天，將^菌 動物移至正常餵食的普通動物籠中，使其發展出正常的$ 生物菌叢。在A、L V和L D組中的所有動物，均按規律的基 礎檢查其糞便的微生物組成，以確保維持其特定的腸道狀 態。 藉著在第4 5和5 0天時，分別在每側局部塗抹5 〇微升i % 一硝基氯苯（DNCB)，將在不同組中的動物敏感化。在最後 的敏感化之後5天，連續3天（第55、56和57天）藉著在所 有老鼠組別的右耳上塗抹25微升1% DNCB，並在左耳上塗 抹25微升載體（橄欖油），來激發動物。 在第58天時，檢查由於上文以dncb處理而引起之過敏 反應的影響。關於此點，在第5 8天測量每隻動物右耳和左 耳的厚度’並計算在相同動物的兩耳之間，在厚度上的差 異。在圖1中出示結果，並概述於下文表: 表1 組別 △耳厚度10·2毫米+ s//"n A 22.9 + 2.2 LD 19.0 + 3.6 LV 1 17.0 + 3.3 C 12.1 + 2.8 從上文中’顯然根據耳厚度測量到的過敏反應，按照 -13- 本紙張尺度適用中國國豕標準(CNS) A4規格(21〇X 297公袭) 1304340 A7 B7 五、發明説明（11 ) C<LV<LD<A的順序增加。 從這些結果，可斷定傳統的腸道菌叢，與缺乏這類微生 物菌叢的老鼠相比較，容許調節炎症反應。此外，亦可看 到單獨益生乳酸菌微生物ST11的實行，引起由DNCB引起 之炎症反應的降低，也就是降低除了乳酸腸菌叢以外的動 物之耳水種的傾向。該發現是驚人的，因爲腸道通常由不 同微生物的集合所組成，其全部滿足不同的工作。 此外，爲了對於在分子層面上的免疫相關反應獲得生化 資料，製備右耳的緊迫狀況低溫切片，並檢查ICAM-1和 TGF-卢的含量。ICAM-1是前-炎症（pro-inflammatory)標記 ’而TGF- 則是抗-炎症標記。因此，預期引起緊迫狀況 將導致ICAM_ 1含量變高，而TGF-/3含量變低。 爲了實驗，使用1/25之大鼠抗·老鼠ICAM-1抗體（純種系 KAT-l)(Caltag Laboratories，CA, USA)，此時兔子抗-大鼠的 FITC爲 l/700(Dako, Ca，USA)。爲 了判定 TGF_/?，使用 1/20 的兔子抗-人類 / 老鼠 TGF- (V)(Santa Cruz Biotechnology， Ca，USA)，此時豬抗-兔-FITC 爲 l/1000(Dako, Ca，USA)。 如同可從圖2中看到的，與在具有傳統的，也就是正常的 微生物菌叢之動物中發現到的相比較，在無菌動物中可檢 測到高含量的炎症性細胞***素IC A Μ - 1，同時在以活益 生乳酸菌處理的動物中，可觀察到降低含量的1(： AM-1。The solution was supplied in drinking water until the end of the experiment (LD group). To obtain a traditional (normal) intestinal microflora, three days after arrival, the bacteria were transferred to a normal feeding common animal cage to develop a normal $bio-cluster. All animals in the A, L V and L D groups were examined for their microbial composition on a regular basis to ensure that their specific intestinal status was maintained. Animals in different groups were sensitized by topical application of 5 〇 microliters of i% mononitrochlorobenzene (DNCB) on each side on days 45 and 50, respectively. 5 days after the last sensitization, for 3 consecutive days (days 55, 56 and 57) by applying 25 microliters of 1% DNCB to the right ear of all mouse groups and applying 25 microliters of carrier on the left ear (Olive oil) to stimulate animals. On day 58, the effects of the allergic reaction caused by the above treatment with dncb were examined. In this regard, the thickness of the right ear and the left ear of each animal was measured on day 58 and the difference in thickness between the ears of the same animal was calculated. The results are presented in Figure 1 and are summarized in the following table: Table 1 Group △ ear thickness 10 · 2 mm + s / / " n A 22.9 + 2.2 LD 19.0 + 3.6 LV 1 17.0 + 3.3 C 12.1 + 2.8 From above In the article 'obviously the allergic reaction measured according to the thickness of the ear, according to the scale of the paper - China National Standard (CNS) A4 specification (21〇X 297 public attack) 1304340 A7 B7 5, invention description (11) C<LV&lt The order of LD<A increases. From these results, it can be concluded that the traditional intestinal flora allows to regulate the inflammatory response as compared with mice lacking such microflora. Further, it can be seen that the practice of the probiotic lactic acid bacteria microorganism ST11 alone causes a decrease in the inflammatory reaction caused by DNCB, that is, a tendency to reduce the ear water species other than the lactic acid enterobacteria. The finding is striking because the intestines are usually composed of a collection of different microorganisms, all of which satisfy different tasks. In addition, in order to obtain biochemical data for immune-related reactions at the molecular level, cryosections of the immediate condition of the right ear were prepared, and the contents of ICAM-1 and TGF-Lu were examined. ICAM-1 is a pro-inflammatory marker and TGF- is an anti-inflammatory marker. Therefore, it is expected that an urgent situation will result in an increase in the ICAM-1 content and a decrease in the TGF-/3 content. For the experiment, 1/25 of rat anti-mouse ICAM-1 antibody (Pure Line KAT-1) (Caltag Laboratories, CA, USA) was used, at which time the rabbit anti-rat FITC was 1/700 (Dako, Ca, USA). To determine TGF_/?, 1/20 rabbit anti-human/mouse TGF- (V) (Santa Cruz Biotechnology, Ca, USA) was used, at which time the pig anti-rabbit-FITC was 1/1000 (Dako, Ca, USA) ). As can be seen from Figure 2, high levels of inflammatory cytokinin IC A can be detected in sterile animals compared to those found in animals with conventional, normal microbial flora. - 1 At the same time, a reduced content of 1 (: AM-1) was observed in animals treated with live probiotic lactic acid bacteria.

I 以失活之益生乳酸菌處理之動物，亦顯示降低含量的 ICAM-1。根據該發下，可建立下列的順jc=LV>LD〉A。 再者’以與該炎症性標記有關的發現爲基礎，具有僅由 -14- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐） -裝 訂I Animals treated with inactivated probiotic lactic acid bacteria also showed reduced levels of ICAM-1. According to this issue, the following cisjc=LV>LD>A can be established. Furthermore, based on the findings related to the inflammatory marker, it has a Chinese National Standard (CNS) A4 specification (210 X 297 mm) only for the -14- paper scale - binding

線 1304340 A7 B7 五、發明説明（12 乳酸菌組成之腸道微生物菌叢的動物，顯示出與具有傳統 微生物菌叢一樣低含量的ICAM- 1。 與其相反，圖3顯示與具有傳統微生物菌叢之動物相比較 ’在播囷動物中降低了抗-炎症標記TGF-/?的含量。且具 有活微生物菌叢之動物，與無菌動物相比較，清楚地顯示 出增加的TGF-卢含量。建立該標記的順序爲〇LV>LD>A。 此外’從老鼠的令部取彳寸皮膚試樣’並根據在Peguet等人 ，Br. J. Dermatol. 133(1995)，661中描述的方法，接受萃取 作用，將該文件併入本文中以作爲參考。在取得皮膚試樣 之後’將它們放在冰上’移至萃取緩衝溶液（10 mM Tris HC1，pH 7·4; 2 mM MgCl; 150 mM NaCl; 1%三通X100; 2 mM PMSF)中，並經歷超音波振盪處理。亦從動物中取得血清 試樣。檢查血清試樣和皮膚萃取物的IL-10含量。 爲了進行實驗，使用得自Biosource International，C A USA的超敏感ELISA試驗Cytoscreen®。根據標準曲線（0.625 至80微克/毫升）來計算IL-10的含量。， 關於萃取物，以細胞物質的總量爲基礎，根據萃取物中 所含有的蛋白質之含量（Lowry法）來報告結果。以微微克 IL-10來表示結果，並出示在圖4和下文的表II中。 -15- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 装 訂Line 1304340 A7 B7 V. Description of the invention (12 animals of the intestinal microflora consisting of lactic acid bacteria, showing the same low content of ICAM-1 as with traditional microbial flora. Conversely, Figure 3 shows that it has a traditional microbial flora. Animals compared 'in the sowing animals, the anti-inflammatory marker TGF-/? was reduced. Animals with viable microflora clearly showed increased TGF-lu content compared to sterile animals. The order of the markers is 〇LV>LD>A. In addition, 'take the skin sample from the mouse's order' and accept it according to the method described in Peguet et al., Br. J. Dermatol. 133 (1995), 661. Extraction, which is incorporated herein by reference. After the skin samples were taken, 'put them on ice' and transfer to an extraction buffer solution (10 mM Tris HC1, pH 7.4; 2 mM MgCl; 150 mM) NaCl; 1% tee X100; 2 mM PMSF), and subjected to ultrasonic oscillation treatment. Serum samples were also obtained from animals. Check the IL-10 content of serum samples and skin extracts. From Biosource International CA USA's ultra-sensitive ELISA test Cytoscreen®. The IL-10 content is calculated from the standard curve (0.625 to 80 μg/ml). The extract is based on the total amount of cellular material, based on the extract. The protein content (Lowry method) is used to report the results. The results are expressed in picograms of IL-10 and are shown in Figure 4 and Table II below. -15- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) binding

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〜___表II 一^ IL-10微微克/毫克_ _____6.3+2.2__ ^ 一___6·8+1·7__ ------11.1 + 1.8__ L^^ _____2L2+3.2_____ ^ "清楚看到的，IL-10的含量在具有傳統微生物菌叢 中的老^tb^r ^ 疋取咼的’而在以活益生乳哮菌處理的組別中 優於热菌的動物。可建立下列的順序C>LV>LD>A。 乂在本實驗中的發現爲基礎，可清楚地看到不同的受試 參數（TGF-/3、KAMq、IL_1〇)，證實在耳厚度上發現差 二的結果’也就是説，藉著移植乳酸菌微生物菌叢而調節 炎症反應。 一在本實驗中，uv_光在皮膚中引起的免疫系統抑制，可 藉著過敏反應的減少發展而察知，使用它來研究益生乳酸 因在本質上反應緊迫狀況而修補免疫系統之能力的潛力， 像是以正常之方式，暴露在過敏原物質下。 建互3組受試動物。第一組動物是以已知可謗發過敏反應 的化合物，二硝基氟苯（DNFB)處理，並對其判定皮膚之免 疫系統的反應。第二組在暴露於上文的化合物之前，先接 受U V照射，並評估在這些條件下該暴露對免疫反應的影響 。第二組接受活的和失活的益生乳酸菌，或其培養物上清 -16- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐)~___Table II A ^ IL-10 picogram / mg _ _____6.3 + 2.2__ ^ A ___6·8+1·7__ ------11.1 + 1.8__ L^^ _____2L2+3.2_____ ^ &quot It is clear that the content of IL-10 is superior to that of the thermobacteria in the group treated with the active probiotics in the traditional microbial flora, which is the same as the 'tb^r^ in the traditional microbial flora. The following sequence C>LV>LD>A can be established. Based on the findings in this experiment, different test parameters (TGF-/3, KAMq, IL_1〇) can be clearly seen, confirming the result of finding a difference in the thickness of the ear. That is, by transplantation The lactic acid bacteria microbial flora regulates the inflammatory response. In this experiment, the immune system inhibition caused by uv_light in the skin can be detected by the reduction of allergic reactions, and it is used to study the potential of probiotic lactic acid to repair the immune system due to the inherently urgent reaction. , as in the normal way, exposed to allergens. Establish three groups of test animals. The first group of animals were treated with a compound known to be an allergic reaction, dinitrofluorobenzene (DNFB), and reacted to the immune system of the skin. The second group received U V exposure prior to exposure to the above compounds and assessed the effect of the exposure on the immune response under these conditions. The second group accepts live and inactivated probiotic lactic acid bacteria, or its culture supernatant -16- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

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1304340 A7 B7 五、發明説明（14 ) 液，並暴露在UV-光下。在這一組中，評估益菌助生質對 免疫反應之修補的影響。爲了進行該實驗，使用在8至10 週齡之間的雌性Skhl/hr老鼠（獲.自Charles River Laboratories(France))。使用在表III中列舉之產物，作爲投 與不同組的益生乳酸菌：1304340 A7 B7 V. INSTRUCTIONS (14) Liquid and exposed to UV-light. In this group, the effect of probiotics on the repair of immune responses was assessed. For this experiment, female Skhl/hr mice between 8 and 10 weeks of age (obtained from Charles River Laboratories (France)) were used. The products listed in Table III were used as probiotic lactic acid bacteria in different groups:

表III 產物1 培養基 產物2 活的Lai(獲自 Institute Pasteur, CNCM 1-1225) 產物3 失活的Lai 產物4 Lai的上清液 產物5 活的 ST 11 (獲自 Institute Pasteur，CNCM 1-2116) 產物6 失活的ST11 ' 產物7 ST11的上清液 以含有109 cfu/毫升的1 · 5毫升冷凍等份提供益生乳酸菌 試樣。使用培養基作爲對照組。 集合32組每組10老鼠。在第〇天，使動物暴露在uv-光 下的那一天之前1 〇天，開始餵食動物個別的產物，並持續 這類餵食直到第1 2天，此時以DNFB開始激發。因此，總 共餵食動物該產物23天。產物1至7(在煎），以100微升/動 物之,劑量集中餵食5-32組，相當於大約108 cfu/動物/天。 表IV列舉產物和接受它們的組別。 -17- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐） 1304340 A7 B7 五、發明説明（15 )Table III Product 1 Media product 2 Live Lai (obtained from Institute Pasteur, CNCM 1-1225) Product 3 Inactivated Lai product 4 Lai supernatant product 5 Live ST 11 (obtained from Institute Pasteur, CNCM 1-2116 The supernatant of product 6 inactivated ST11 'product 7 ST11 was supplied as a sample of probiotic lactic acid bacteria in a 1.5 cc frozen aliquot containing 109 cfu/ml. The medium was used as a control group. Collect 32 groups of 10 mice per group. On the third day, one day before the day when the animals were exposed to uv-light, the individual products of the animals were started to feed, and this type of feeding was continued until the first day, at which time the stimulation was initiated with DNFB. Therefore, the animal was fed a total of 23 days. Products 1 to 7 (at the frying) were fed in a concentration of 100 microliters per animal at a dose of 5-32 groups, equivalent to approximately 108 cfu/animal per day. Table IV lists the products and the groups that accepted them. -17- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1304340 A7 B7 V. Invention description (15)

表IVTable IV

在第〇天，以異氟烷/氧輕度地麻醉老鼠，並接著以含有 氣1000瓦（Oriel)燈的太陽-模擬器照射，其包括裝設有口WG 320Π毫米厚之濾光片和UG11濾光片n亳米厚（8仏〇抝的二 色鏡（Onel，Statford，US A)。該過濾源提供了模擬的太陽u § 光譜（29^=00毫微米），幾乎排除了所有的可見光和紅外線 輻射。藉著放射計ARCC 1600(〇sram)判定照射量，在UVB 處爲1.95¾瓦/平方公分，在UVA處爲9·35毫瓦/平方公分。 應用的光譜符合根據SPF COLIPA的標準。 照射第 3、4、7、8、9、1 1、12、1 5、1 6、1 9、2 0、 23、24、27、28、31、32組的老鼠，但保護耳朵（第〇天 )。其接受2 · 5 Μ E D的單獨劑量。 在，第5和第6天，藉著分別在腹部局部塗抹5〇微升丙酮（ 第 1、3、5、7、9、11、13、15、17、19、21、23、25 、27、29、31組）和DNFB(第2、4、6、l8、10、12、14 、16、18、20、22、24、26、28、30、32 組），引起過 -18- 本紙張尺度適用中國國家標準(CNS) Α4規格(210X297公釐) A7 B7 1304340 五、發明説明（16 ) 敏反應。 在第12天時，在所有老鼠的右耳上塗抹5微升在丙酮中 之0.2%的DNFB溶液。在試驗開始（第0天）和結束（第13天） 時將動物稱重。 在暴露於UV-光下之後24小時，也就是在第1天時，進行 炎症反應的評估。評估兩個參數，也就是藉著紅斑和水腫 的臨床判定，評估在背上紅斑的強度，計算每組的平均値 ，以及背部皮膚厚度的增加，計算未照射和照射動物的平 均値。 在第1 3天時，進行右和左耳之皮膚厚度的判定。計算相 同動物之耳朵皮膚厚度的差異，以及每組的平均値。在下 文表V中出示的結果，在圖5和6中以圖解説明之。On day 13, mice were lightly anesthetized with isoflurane/oxygen and then irradiated with a sun-simulator containing a 1000 watt Oriel lamp, which included a filter with a mouth WG 320 mm thick and The UG11 filter is n亳m thick (8 仏〇拗 dichroic mirror (Onel, Statford, US A). This filter source provides a simulated solar § spectrum (29^=00 nm), almost eliminating all Visible and infrared radiation. The amount of exposure was determined by radiometer ARCC 1600 (〇sram), 1.952⁄4 W/cm 2 at UVB and 9.35 mW/cm 2 at UVA. The applied spectrum is in accordance with SPF. COLIPA criteria. Irradiation of mice in groups 3, 4, 7, 8, 9, 1 1 , 12, 15 , 16 , 19 , 2 0 , 23 , 24 , 27 , 28 , 31 , 32 , but protected Ear (day )). It received a single dose of 2 · 5 Μ ED. On days 5 and 6, the 5 liters of acetone (1, 3, 5, 7, 9 were applied locally on the abdomen). , 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31) and DNFB (2, 4, 6, l8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 groups), cited -18- The paper size is applicable to China National Standard (CNS) Α4 specification (210X297 mm) A7 B7 1304340 V. Invention description (16) Sensitive reaction. On the 12th day, apply 5 micros on the right ear of all mice. Raise 0.2% DNFB solution in acetone. Weigh the animals at the beginning (Day 0) and end (Day 13) of the test. 24 hours after exposure to UV-light, ie on Day 1. To evaluate the inflammatory response. Evaluate the two parameters, that is, the clinical judgment of erythema and edema, evaluate the intensity of erythema on the back, calculate the average sputum of each group, and increase the thickness of the back skin, calculate the unirradiated and irradiated Average 动物 of animals. On day 13th, the skin thickness of the right and left ears was determined. The difference in skin thickness of the ears of the same animal was calculated, as well as the average enthalpy of each group. The results shown in Table V below, This is illustrated in Figures 5 and 6.

表V 藉著耳朵厚度判定的過敏性反應 照射 MED 處理 謗導 △耳朵厚度 1CT2毫米土 s//"n 藉著照射引起 的HSC抑制％ 1 0 猶 丙嗣 1·1±0·2 2 0 麵 DNFB 0.3% 19·3±0.4 3 2.5 • 丙嗣 1.0土0.2 4 2.5 DNFB 0.3% 7·5±0·2 61.1 5 j 0 培養基 丙酮 1·0±0·1 6 0 培養基 DNFB 0.3% 19·0 土 0.2 0 7 2.5 培養基 丙_ 1·0 土 0.2 -19- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 1304340 A7 B7 五、發明説明（17 ) 8 2.5 培養基 DNFB 0.3% 7.4 ±0:1 61.6 9 0 Lai活的 丙嗣 1·1±0·3 10 0 Lai活的 DNFB 0.3% 18.9土0.2 0 11 2.5 Lai活的 丙酮 1·3±0·2 12 2.5 Lai活的 DNFB 0.3% 15.4±0.2 20.2 13 0 Lai死的 丙酮 1.3 土 0.2 14 0 Lai死的 DNFB 0.3% 19.4 土 0·3 0 15 2.5 Lai死的 丙酮 1·4±0·1 16 2.5 Lai死的 DNFB 0.3% 9.6 土 0.3 50.3 17 0 Lai的培養 物上清液 丙酮 1·1±0·2 18 0 Lai的培養 物上清液 DNFB 0.3% 19·5±0·4 0 19 2.5 Lai的培養 物上清液 丙酮 1·1±0·3 20 2.5 Lai的培養 物上清液 DNFB 0.3% 7·4±0·1 61.7 21 0 ST11活的 丙酮 0.6 ±0:2 22 0 ST11活的 DNFB 0.3% 18.8±0·2 0 23 2.5 ST11活的 丙嗣 1·1±0.2 24 .2.5 ST11活的 DNFB 0.3% 13.2±0·3 31.6 25 0 ST11死的 丙酮 1.4±0.2 26 0 ST11死的 DNFB 0.3% 19.4 土 0·4 0 27 2.5 ST11死的 丙酮 1·1±0·3 ·； 裝 訂Table V Allergic reaction determined by ear thickness Irradiation MED treatment 谤 Guide △ Ear thickness 1CT2 mm soil s//"n HSC inhibition by irradiation % 1 0 犹 嗣 嗣 1·1±0·2 2 0 DNFB 0.3% 19·3±0.4 3 2.5 • Propionate 1.0 soil 0.2 4 2.5 DNFB 0.3% 7·5±0·2 61.1 5 j 0 Medium acetone 1·0±0·1 6 0 Medium DNFB 0.3% 19· 0 Soil 0.2 0 7 2.5 Medium C _ 1·0 Soil 0.2 -19- This paper scale is applicable to China National Standard (CNS) A4 specification (210X 297 mm) 1304340 A7 B7 V. Invention description (17) 8 2.5 Medium DNFB 0.3 % 7.4 ±0:1 61.6 9 0 Lai live 嗣1·1±0·3 10 0 Lai live DNFB 0.3% 18.9 soil 0.2 0 11 2.5 Lai live acetone 1·3±0·2 12 2.5 Lai live DNFB 0.3% 15.4±0.2 20.2 13 0 Lai dead acetone 1.3 soil 0.2 14 0 Lai dead DNFB 0.3% 19.4 soil 0·3 0 15 2.5 Lai dead acetone 1·4±0·1 16 2.5 Lai dead DNFB 0.3% 9.6 Soil 0.3 50.3 17 0 Lai culture supernatant A1.1±0·2 18 0 Lai culture supernatant DNFB 0.3% 19·5±0·4 0 19 2.5 Lai culture Clear acetone 1·1±0·3 20 2.5 Lai culture supernatant DNFB 0.3% 7·4±0·1 61.7 21 0 ST11 live acetone 0.6 ±0:2 22 0 ST11 live DNFB 0.3% 18.8±0· 2 0 23 2.5 ST11 live 嗣1·1±0.2 24 .2.5 ST11 live DNFB 0.3% 13.2±0·3 31.6 25 0 ST11 dead acetone 1.4±0.2 26 0 ST11 dead DNFB 0.3% 19.4 soil 0· 4 0 27 2.5 ST11 dead acetone 1·1±0·3 ·; binding

-20- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐） 1304340 A7 B7 五、發明説明（18 ) 28 2.5 ST11死的 DNFB 0.3% 10.6±0.3 45.1 29 0 ST11的培養 物上清液 丙酮 1·0±0·2 30 0 ST11的培養 物上清液 DNFB 0.3% 19.3土0.4 0 31 2.5 ST11的培養 物上清液 丙酮 1.2±0.2 32 2.5 ST11的培養 物上清液 DNFB 0.3% 11·0±0·2 43 如同預期，在所有未接受處理和在僅接受培養基的對照 組老鼠中，暴露在UV-光下全身性地抑制對DNFB的過敏性 反應。那些接受活益生乳酸菌微生物或其培養物上清液（條 件培養基）的老鼠，清楚地顯示出免疫系統的恢復，並顯示 對DNFB的過敏性反應。從這些發現中，顯然益生乳酸菌， 及其出現在培養物上清液中的部份代謝產物，顯然能夠在 UV-光引起之抑制條件下，調節免疫系統，在本質上得以 修補免疫反應。 -21 - 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐） 申請日期 Q〇 in 96 ____090126606 土上各櫚由本局填註） ：]： 槪 發明 SS名稱 中 文 A4 C4 中文說明書替換頁(96年3月） 專利説明書 益生乳酸菌於製備使皮膚免疫功能正常化之載體之 用途 英 文-20- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1304340 A7 B7 V. Invention description (18) 28 2.5 ST11 dead DNFB 0.3% 10.6±0.3 45.1 29 0 ST11 culture Supernatant acetone 1·0±0·2 30 0 ST11 culture supernatant DNFB 0.3% 19.3 soil 0.4 0 31 2.5 ST11 culture supernatant acetone 1.2±0.2 32 2.5 ST11 culture supernatant DNFB 0.3% 11·0±0·2 43 As expected, allergic reactions to DNFB were systemically inhibited by exposure to UV-light in all control mice that were not treated and in medium-only control. Those mice that received live probiotic lactic acid bacteria or their culture supernatants (conditional medium) clearly showed recovery of the immune system and showed an allergic reaction to DNFB. From these findings, it is apparent that probiotic lactic acid bacteria, and some of the metabolites present in the culture supernatant, are apparently capable of modulating the immune system under UV-light-induced inhibition, thereby substantially repairing the immune response. -21 - This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) Application date Q〇in 96 ____090126606 Each palm of the soil is filled by this bureau) :]: 槪Invention SS name Chinese A4 C4 Chinese manual Replacement page (March 1996) Patent Description Probiotic Lactobacillus for the preparation of a carrier for normalizing skin immune function

USE OF PROBIOTIC LACTIC ACID BACTERIA FOR PREPARING A CARRIER FOR NORMALIZING THE SKIN’S IMMUNE FUNCTION 姓 名 國 人 1. 馬庫斯巴爾 MARKUS BAUR 2. 萊爾布來頓 LIONEL BRETON 3. 法蘭克斯庫斯FRANCOIS COUZY 4. 安德利吉妮奇 AUDREY GUENICHE 1.德國 2. 3.4.法國 # 住、居所 1 ·德國斯特葛特市康妮·卡爾路53號 2. 法國維斯利斯市史託利市14號 3. 瑞士拉克羅斯/魯特利市魯特蒙特斯路498號 4. 法國魯利馬莫森市雷盟路29號 訂 姓 么 (名稱? 國 瑞士商雀巢製品股份有限公司 SOCIETE DES PRODUITS NESTLE S. Α· 線 三、申請人 (、事矛節 瑞士 瑞士威威市(郵政信箱353號) S表名USE OF PROBIOTIC LACTIC ACID BACTERIA FOR PREPARING A CARRIER FOR NORMALIZING THE SKIN'S IMMUNE FUNCTION Name of the country 1. Markus BAUR 2. LIONEL BRETON 3. FRANCOIS COUZY 4. Andre Ginich AUDREY GUENICHE 1.Germany 2. 3.4.France# Residence, Residence 1 · Connie Calpe 53 in Stuttgart, Germany 2. No. 14, Stolli, Vislis, France 3. Lacros/Lu, Switzerland No. 498, Rutmonts Road, Terry City 4. No. 29, Leimeng Road, Russoma, France. Name (Name? National Swiss Business Nestle Products Co., Ltd. SOCIETE DES PRODUITS NESTLE S. Α· Line III. Application People (, the spear festival Swissôtel Switzerland (postal box 353) S table name

諾曼.維利 ROMAN VUILLENorman Villier ROMAN VUILLE

7408 ^-960319.DOC 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) Ι30434Θ>〇ι266〇6號專利申請案 μ、 中文說明書替換頁(96年3月） ^ 五、發明説明（19 ) 圖式簡單說明 圖1顯示在各種老鼠模式中，以判定耳朵腫脹來檢查與接 觸性過敏反應有關的炎症性反應； 圖2顯示利用對抗ICAM- 1之抗體，在各種指示之動物模 式中獲得的結果； 圖3顯示在各種受試的動物模式中，TGF-/3的含量； 圖4顯示在各種受試的動物模式中，表皮IL-1 0的含量； 圖5顯示益生乳酸菌對UV照射引起之免疫系統抑制的影 響，根據以耳朵腫脹判定的接觸性過敏反應來顯示之； 圖6顯示益生乳酸菌對UV照射引起之免疫系統抑制的影 響，以CHS抑制％來顯示之。 74089-960319.DOC -22- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐)7408 ^-960319.DOC This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm) Ι30434Θ>〇ι266〇6 Patent Application μ, Chinese Manual Replacement Page (March 96) ^ V. INSTRUCTION (19) Schematic description of the figure Figure 1 shows the inflammatory response associated with contact allergic reactions in various mouse models to determine ear swelling; Figure 2 shows the use of antibodies against ICAM-1 in various indicator animal models Results obtained in Figure 3; Figure 3 shows the content of TGF-/3 in various animal models tested; Figure 4 shows the content of epidermal IL-1 0 in various animal models tested; Figure 5 shows the probiotic lactic acid bacteria pair The effect of suppression of the immune system by UV irradiation is shown by the contact allergic reaction determined by ear swelling; Fig. 6 shows the effect of probiotic lactic acid bacteria on immune system inhibition by UV irradiation, which is shown by % inhibition of CHS. 74089-960319.DOC -22- This paper size applies to China National Standard (CNS) A4 specification (210X 297 mm)

Claims (1)