TWI458438B - Fermentation and culture methods, plant fermentation extracts, plant fermentation extract powders, and plant fermentation extract complexes - Google Patents

Fermentation and culture methods, plant fermentation extracts, plant fermentation extract powders, and plant fermentation extract complexes Download PDF

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TWI458438B
TWI458438B TW100105235A TW100105235A TWI458438B TW I458438 B TWI458438 B TW I458438B TW 100105235 A TW100105235 A TW 100105235A TW 100105235 A TW100105235 A TW 100105235A TW I458438 B TWI458438 B TW I458438B
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fermentation
fermentation extract
wheat
plant
extract
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TW201117728A (en
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Genichiro Soma
Chie Kohchi
Hiroyuki Inagawa
Takashi Nishizawa
Yukinori Takahashi
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Genichiro Soma
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發酵及培養方法、植物發酵萃取物、植物發酵萃取物粉末以及該植物發酵萃取物配合物Fermentation and culture method, plant fermentation extract, plant fermentation extract powder, and plant fermentation extract complex

本發明係關於既使添加於含人類的哺乳動物(具體為家畜、寵物等)、鳥類(具體為飼養雞、賞鳥類等)、兩生類、爬蟲類、魚類(具體為水產養殖魚、寵物魚類等)、無脊椎動物及醫藥品、動物用醫藥品、醫藥部外品、化妝品、食品、功能性食品、飼料及浴用劑等亦可得到安全且免疫賦活物質之發酵及培養方法、植物發酵萃取物之製造方法、含有發酵及培養方法所得之免疫賦活物質的植物發酵萃取物、由該植物發酵萃取物所得之免疫賦活物質含有粉末、及該植物發酵萃取物所調製的植物發酵萃取物配合物。The present invention relates to the addition of mammals (specifically livestock, pets, etc.), birds (specifically, raising chickens, bird watching, etc.), biogenics, reptiles, fish (specifically aquaculture fish, pet fish). , etc., invertebrates and pharmaceuticals, animal medicines, pharmaceutical products, cosmetics, foods, functional foods, feeds and baths, etc., can also be obtained by fermentation and culture methods for safe and immunogenic substances, plant fermentation extraction a method for producing a substance, a plant fermentation extract containing an immunostimulating substance obtained by fermentation and a culture method, an immunostimulating substance obtained by fermenting the plant extract, a powder, and a plant fermentation extract complex prepared by the plant fermentation extract .

有關含人類的哺乳動物(具體為家畜、寵物等)、鳥類(具體為飼養雞、賞鳥類等)、兩生類、爬蟲類、魚類(具體為水產養殖魚、寵物魚類等)、無脊椎動物，確立其含感染防除技術之疾病預防、治療法係為重要的課題。且要達到此目的時，被要求使用無須使用化學物質、無環境污染、無產生耐性菌、不會累積於人體中的方法。本發明者對於上述的課題，發現小麥水萃取物等的來自植物的免疫賦活物質可安全地達成疾病預防與治療效果(專利文獻1、專利文獻2)。又，欲達成以上目的，發現可使用由小麥共生細菌的稻穀病原細菌(Pantoea agglomerans)所得之低分子量脂多醣(非專利文獻2)。另一方面，由最近的研究得知，脂多醣以外的種種物質可顯示免疫賦活效果，含有這些複數的免疫賦活物質之天然物質原料受到注目。For mammals containing humans (specifically livestock, pets, etc.), birds (specifically for raising chickens, bird watching, etc.), biogenics, reptiles, fish (specifically aquaculture fish, pet fish, etc.), invertebrates, Establishing a disease prevention and treatment system with infection control technology is an important issue. In order to achieve this, it is required to use a method that does not require the use of chemicals, environmental pollution, no resistance to bacteria, and does not accumulate in the human body. In the above-mentioned problem, the present inventors have found that a plant-derived immunostimulating substance such as a wheat water extract can safely achieve disease prevention and treatment effects (Patent Document 1 and Patent Document 2). Further, in order to achieve the above object, it has been found that a low molecular weight lipopolysaccharide obtained from a rice pathogenic bacterium (Pantoea agglomerans) can be used (Non-Patent Document 2). On the other hand, it has been known from recent studies that various substances other than lipopolysaccharide can exhibit an immunostimulating effect, and natural material materials containing these plural immunostimulating substances are attracting attention.

然而，使用微生物的發酵技術不僅使用於食品領域中，亦廣泛地使用於其他領域中。例如以葡萄酒為主的酒類製造、醬油或味噌之製造、乳酪等發酵乳製品之製造、醫藥品的製造等及廣泛領域。使用於這些發酵的微生物之範圍極為廣泛，以麯(真菌)酵母、乳酸菌等為主者，但幾乎未有使用革氏陰性菌之報告。一般發酵為有機物經微生物作用之分解作用現象，廣義為藉由微生物之有用物質的生產(非專利文獻3)。作為使用微生物的發酵技術，可舉出作為代表性的葡萄酒。葡萄酒為使用附著於葡萄皮上的葡萄酒酵母菌之發酵技術，生產物為醇類。又，使用微生物的發酵技術中，使用革氏陰性菌者可舉出使用甲烷菌進行甲烷發酵，使用乙酸菌進行乙酸發酵，使用運動發酵單胞菌(Zymomonas mobilis)由龍舌蘭的根莖之乙醇發酵(龍舌蘭酒製造)等，但以食用植物作為原料，使用與該植物作特殊共生為特徵之微生物進行發酵培養的技術為未知，作為發酵產物免疫賦活物質並未受到注目。However, fermentation techniques using microorganisms are not only used in the food field, but are also widely used in other fields. For example, the production of wines based on wine, the manufacture of soy sauce or miso, the manufacture of fermented dairy products such as cheese, the manufacture of pharmaceuticals, and the like. The range of microorganisms used in these fermentations is extremely wide, and it is mainly composed of koji (fungi) yeast, lactic acid bacteria, etc., but there is almost no report of the use of gram-negative bacteria. In general, fermentation is a phenomenon in which an organic substance is decomposed by a microorganism, and is broadly produced by a useful substance of a microorganism (Non-Patent Document 3). As a fermentation technique using a microorganism, a representative wine is mentioned. The wine is a fermentation technique using a wine yeast attached to the grape skin, and the product is an alcohol. Further, in the fermentation technique using microorganisms, those using the Gram-negative bacteria include methane fermentation using methanogens, acetic acid fermentation using acetic acid bacteria, and ethanol from the rhizome of agave using Zymomonas mobilis. Fermentation (made of tequila), etc., but the use of edible plants as raw materials, and the use of microorganisms characterized by special symbiosis with the plants for fermentation culture is unknown, and the immunostimulating substances as fermentation products have not attracted attention.

另一方面，藉由微生物發酵時，必須具有滿足一般微生物生長時的發酵基質之營養條件。即，作為碳源，必須存在含有充分的葡萄糖、果糖等單糖類等作為微生物的營養素而可被利用的物質。因此原先已含有多量的果糖之如葡萄的水果，無須進行任何加工步驟即可作為發酵基質利用，其他則必須進行加熱或酵素處理等之藉由微生物的發酵前階段處理。例如前述運動發酵單胞菌(Zymomonas mobilis)為使用於製造龍舌蘭酒上之微生物，此為由非食用植物之龍舌蘭的根莖所得之多醣類經加熱分解發酵性單糖後，藉由該微生物的發酵得到發酵產物的酒類。因此使用一般微生物進行發酵培養時，澱粉等多醣類並非適當的發酵基質。例如有文獻指出稻穀病原細菌(Pantoea agglomerans)無法分解澱粉(非專利文獻4)。On the other hand, when fermenting by microorganisms, it is necessary to have nutritional conditions that satisfy the fermentation substrate at the time of general microbial growth. In other words, as a carbon source, it is necessary to use a nutrient which is sufficient as a microorganism such as a monosaccharide such as glucose or fructose. Therefore, a fruit such as a grape which originally contains a large amount of fructose can be used as a fermentation substrate without any processing steps, and others must be subjected to a pre-fermentation stage of microorganisms by heating or enzyme treatment. For example, the aforementioned Zymomonas mobilis is a microorganism used in the manufacture of tequila, and the polysaccharide obtained from the rhizome of agave of a non-edible plant is decomposed by fermenting the fermentable monosaccharide. A wine obtained by fermentation of the microorganism to obtain a fermentation product. Therefore, when fermentation is carried out using a general microorganism, a polysaccharide such as starch is not a suitable fermentation substrate. For example, it has been reported that rice pathogenic bacteria (Pantoea agglomerans) cannot decompose starch (Non-Patent Document 4).

本發明者已確立小麥粉之水萃取物中含有可賦活化免疫的有效成分(非專利文獻5)。又，穀物(小麥、米)、海草(海帶芽、昆布、洋栖菜、海苔等)、豆類(大豆、紅豆)中已含有有效成分(非專利文獻6)。作為該生物活性，具有人類或老鼠之各疾病(糖尿病、高脂質血症、異位性皮膚炎、癌症)等預防效果，故可有效地預防魚類、甲殼類、鳥類的感染(專利文獻1、非專利文獻1)。然而小麥粉水萃取物要達到所期待的上述效果必須攝取大量的小麥粉。The present inventors have established that an aqueous extract of wheat flour contains an active ingredient capable of activating immunity (Non-Patent Document 5). Further, the cereal (wheat, rice), seaweed (kelp bud, kelp, yam, seaweed, etc.), and beans (soybean, red bean) already contain an active ingredient (Non-Patent Document 6). As a biological activity, it has preventive effects such as diseases of humans or mice (diabetes, hyperlipidemia, atopic dermatitis, cancer), and thus can effectively prevent infection of fish, crustaceans, and birds (Patent Document 1 Non-patent document 1). However, in order to achieve the above-mentioned effects, the wheat flour water extract must ingest a large amount of wheat flour.

另一方面，稻穀病原細菌(Pantoea agglomerans)為共生於小麥的細菌，因供給磷、氮於小麥故對於小麥栽培為有用的菌(非專利文獻7)。又，於歐洲已知稻穀病原細菌(Pantoea agglomerans)不僅於小麥、亦附著於梨或蘋果等果實的表皮上，有該菌附著時可預防因霉菌所引起的腐爛病，故開發該菌利用於無毒下對自然環境佳之防霉劑已被進行(非專利文獻8)。且，所謂共生即為「一般表示相異種的生物一起生活的現象，此時彼此的行動或生理維持一定的密切關係。因此，不僅是生活於相同地方之概念，對於共生者之生活上的意義及必須性、關係的持續性、共生者的空間位置關係等，共生可分成各式各樣的類型、區別。一般為共生者的生活上利益、非利益之有無做為基準下，共生分為共利共生、片利共生、寄生之三大類」(非專利文獻9)。稻穀病原細菌(Pantoea agglomerans)的情況為，已知可於任意產地、任意種類的小麥中分離出(非專利文獻5)，又亦可由果實分離出(非專利文獻10、11)。稻穀病原細菌(Pantoea agglomerans)可產生抗生素(非專利文獻12、13)而由真菌或其他細菌保護植物，及進行磷、氮固定(非專利文獻7)。因此，稻穀病原細菌(Pantoea agglomerans)常在於植物上，擔任植物的有益角色，有時此並非稱為「共生」而稱為「寄生」。且，本發明者已知稻穀病原細菌中含有可賦活化免疫的有效成分。又，由該菌所得之低分子量脂多醣具有對人類或老鼠之各疾病(糖尿病、高脂質血症、異位性皮膚炎、癌症)等預防效果，對魚類及甲殼類、鳥的感染預防亦有效(專利文獻3、非專利文獻2)。On the other hand, the rice bacterium (Pantoe agglomerans) is a bacterium that is symbiotic with wheat, and is useful for cultivating wheat by supplying phosphorus and nitrogen to wheat (Non-Patent Document 7). In addition, it is known in Europe that Pantoea agglomerans is not only attached to wheat, but also to the epidermis of fruits such as pears or apples, and when the bacteria adheres, it can prevent rot caused by mold, so the bacteria are used for development. A fungicide which is good for the natural environment without toxicity has been carried out (Non-Patent Document 8). Moreover, the so-called symbiosis is a phenomenon in which the living creatures of different species are generally living together. At this time, the actions or physiology of each other are closely related. Therefore, not only the concept of living in the same place, but also the meaning of the life of the symbiotic. And the necessity, the continuity of the relationship, the spatial positional relationship of the symbiotics, etc., the symbiosis can be divided into various types and differences. Generally, the symbiotic life is divided into the interests of the symbiotic, and the symbiosis is divided into There are three major categories of symbiosis, symbiosis, and parasitism (Non-Patent Document 9). In the case of the rice phytopathogenic bacteria (Pantoe agglomerans), it is known that it can be isolated from any type of wheat in any place (Non-Patent Document 5) or separated from fruits (Non-Patent Documents 10 and 11). The rice pathogenic bacteria (Pantoea agglomerans) can produce antibiotics (Non-Patent Documents 12 and 13), and plants are protected by fungi or other bacteria, and phosphorus and nitrogen are fixed (Non-Patent Document 7). Therefore, the rice pathogenic bacteria (Pantoea agglomerans) often lie on plants and serve as a beneficial role for plants. Sometimes this is not called "symbiosis" and is called "parasitic". Further, the present inventors have known that rice pathogenic bacteria contain an active ingredient capable of activating immunity. In addition, the low molecular weight lipopolysaccharide obtained from the bacterium has preventive effects against various diseases of humans or mice (diabetes, hyperlipidemia, atopic dermatitis, cancer), and infection prevention for fish, crustaceans, and birds. Effective (Patent Document 3, Non-Patent Document 2).

如此狀況下，本發明者考慮到作為製造安全且便宜的免疫賦活物質之方法，確立出使用稻穀病原細菌之植物發酵萃取物的製造方法。換言之，(1)使用含於培養液的蛋白質之主成分由來自植物者取代之培養基，使稻穀病原細菌於低成本下培養的同時使植物成分發酵、(2)調製出含有大量含於植物的稻穀病原細菌或發酵生產物之原料、利用此開發出含人類的哺乳動物(具體為家畜、寵物等)、鳥類(具體為飼養雞、賞鳥類等)、兩生類、爬蟲類、魚類(具體為水產養殖魚、寵物魚類等)、無脊椎動物及醫藥品、動物用醫藥品、醫藥部外品、化妝品、功能性食品、食品、飼料及浴用劑受到注目。然而，並非意味著共生於植物的微生物為例如來自食用植物的原料之植物成分可作為發酵基質被利用。例如，小麥粉為存在於小麥粒之澱粉質等複合有機物質，但與小麥共生微生物之稻穀病原細菌為隔著外皮而並非直接接觸，故稻穀病原細菌是否可使用於小麥粉的發酵培養並非單由微生物與小麥共生之關連性即可瞭解，事實上至今未知稻穀病原細菌可資化小麥粉，完全無相關報導。相反地，基於至今的公知事實，稻穀病原細菌不能利用小麥澱粉作為發酵基質。Under the circumstances, the inventors of the present invention have established a method for producing a plant fermentation extract using rice pathogenic bacteria as a method for producing a safe and inexpensive immunostimulating substance. In other words, (1) the main component of the protein contained in the culture solution is replaced by a plant-derived medium, the rice pathogenic bacteria are cultured at a low cost, and the plant components are fermented, and (2) the plant contains a large amount of plants. Raw materials for rice pathogenic bacteria or fermentation products, using this to develop human-containing mammals (specifically livestock, pets, etc.), birds (specifically for raising chickens, bird watching, etc.), biogenic, reptilian, and fish (specifically Aquaculture fish, pet fish, etc., invertebrates and pharmaceuticals, animal medicines, pharmaceutical products, cosmetics, functional foods, food, feed and bath agents have attracted attention. However, it does not mean that a microorganism which is symbiotic to a plant is a plant component such as a raw material derived from a edible plant, and can be utilized as a fermentation substrate. For example, wheat flour is a complex organic substance such as starch which is present in wheat granules, but the rice pathogenic bacterium of wheat symbiotic microorganism is not directly in contact with the outer skin, so whether the rice pathogenic bacterium can be used for fermentation of wheat flour is not a single It can be understood from the relationship between microorganisms and wheat symbiosis. In fact, it is unknown that rice pathogenic bacteria can be used to chemically wheat flour, and there is no relevant report. Conversely, based on the well-known facts to date, rice pathogenic bacteria cannot utilize wheat starch as a fermentation substrate.

因此，含於植物的糖質以澱粉的狀態保持之情況較為多，此於食用植物、特別為穀類時特別顯著。一般微生物並未具有高澱粉資化功能。對於此已知一部份的通性革氏陰性菌可發酵澱粉。例如已知Erwinia可資化澱粉。該發酵技術為，發酵澱粉時另外添加適當培養基中進行大量培養的微生物，其為利用微生物所具有的澱粉酶活性，但對於培養時直接使用澱粉且同時發酵的型態為至今無法想像到的。公知技術為單有效地利用微生物所具有的澱粉酶活性為目的之發酵，但並非可推想到澱粉可作為基質而使微生物增殖。另一方面，本發明的實施例中揭示因澱粉作為唯一碳源故使微生物增殖以外亦產生發酵產物，本案實施例並非僅為單純的發酵，其所揭示的發酵培養亦與公知技術有著相當大的差異。Therefore, the saccharide contained in the plant is kept in a state of starch, which is particularly remarkable in the case of edible plants, particularly cereals. Generally, microorganisms do not have high starch utilization functions. For this known part of the universal Gram-negative bacteria fermentable starch. For example, Erwinia is known to be a starch. This fermentation technique is a microorganism in which a large amount of culture is carried out by adding a suitable medium in the case of fermenting starch, which is an amylase activity which is utilized by a microorganism, but a form in which starch is directly used for culture and fermented at the same time is unimaginable. The known technique is a fermentation for the purpose of effectively utilizing the amylase activity of the microorganism, but it is not conceivable that the starch can be used as a matrix to proliferate the microorganism. On the other hand, in the examples of the present invention, since the starch is used as the sole carbon source, the fermentation product is produced in addition to the proliferation of the microorganisms. The present embodiment is not merely a simple fermentation, and the disclosed fermentation culture is also quite large with the known technology. The difference.

另一方面，相反地某些微生物既使保持分解澱粉的功能，並非表示此微生物為直接以澱粉作為基質進行增殖。培養時微生物的增殖亦為目的時，培養開始時所加入的微生物量極少，此時既使微生物具有少量的澱粉酶活性，該活性因過於薄弱亦無法充分地分解基質，而無法達到微生物的增殖。事實上，多數微生物無法以澱粉作為唯一的碳源而進行增殖。On the other hand, on the contrary, some microorganisms maintain the function of decomposing starch, and do not mean that the microorganism proliferates directly with starch as a substrate. When the growth of microorganisms during culture is also for the purpose, the amount of microorganisms added at the beginning of the culture is extremely small, and at this time, even if the microorganism has a small amount of amylase activity, the activity is too weak to sufficiently decompose the matrix, and the microorganism cannot be proliferated. . In fact, most microorganisms cannot proliferate with starch as the sole source of carbon.

然而，使用稻穀病原細菌於含有以小麥粉為主成分的培養基中進行發酵及培養時，或可低成本下製造出富含免疫賦活物質的植物發酵萃取物(以下將使用稻穀病原細菌以含有小麥粉為主成分的培養基中發酵及培養所得之植物發酵萃取物稱為小麥發酵萃取物)，則可提供於例如人類、對畜產及水產養殖領域下的環境佳、安全下預防感染有效之醫藥品、動物用醫藥品、醫藥部外品、化妝品、食品、功能性食品、飼料及浴用劑等。本發明係以上述背景下，發現稻穀病原細菌可於以小麥粉作為基質下進行生長，而進行種種重複實驗而完成本發明。However, when rice pathogenic bacteria are used for fermentation and culture in a medium containing wheat flour as a main component, or a plant fermented extract rich in immunostimulating substances can be produced at a low cost (hereinafter, rice pathogenic bacteria are used to contain wheat) The plant fermentation extract obtained by fermentation and culture in a medium containing powder as a main component is called a wheat fermentation extract), and can be provided, for example, in humans, in the field of livestock and aquaculture, and in a safe and safe way to prevent infection. , animal drugs, pharmaceutical products, cosmetics, food, functional foods, feed and bath agents. In the above background, the present inventors have found that rice pathogenic bacteria can be grown under wheat flour as a substrate, and various repeated experiments are carried out to complete the present invention.

本發明所提供的植物發酵萃取物係為，由發酵及培養所得之培養液，經固液分離所得之液體成分、及固液分離後所得之固體成分經萃取過程後所得之液體成分等之總稱。即，植物發酵萃取物係由本發明的發酵及培養方法所得之培養液，及含有使用其培養液的全部或一部份可調製出的所有萃取物。理所當然地，植物發酵萃取物可乾燥後作為植物發酵萃取物粉末利用，或植物發酵萃取物粉末溶解於任意濃度的適當溶液中，例如含有生理食鹽水之磷酸緩衝液等中利用。The plant fermentation extract provided by the present invention is a general term for a liquid composition obtained by fermentation and culture, a liquid component obtained by solid-liquid separation, and a liquid component obtained by extraction after solid-liquid separation. . That is, the plant fermentation extract is a culture liquid obtained by the fermentation and culture method of the present invention, and all the extracts which can be prepared by using all or a part of the culture liquid using the same. As a matter of course, the plant fermentation extract can be dried and used as a plant fermentation extract powder, or the plant fermentation extract powder can be dissolved in a suitable solution of any concentration, for example, a phosphate buffer containing physiological saline.

專利文獻1：特開平3-218466號公報Patent Document 1: JP-A-3-218466

專利文獻2：特開平8-198902號公報Patent Document 2: Japanese Patent Publication No. 8-198902

專利文獻3：WO 00/57719Patent Document 3: WO 00/57719

專利文獻4：特開平6-78756號公報Patent Document 4: Japanese Laid-Open Patent Publication No. Hei 6-78756

專利文獻5：特開平4-187640號公報Patent Document 5: Japanese Patent Publication No. 4-187640

專利文獻6：特開平4-49240號公報Patent Document 6: Japanese Patent Publication No. 4-49240

專利文獻7：特開平4-99481號公報Patent Document 7: Japanese Patent Publication No. 4-99481

專利文獻8：特開平5-155778號公報Patent Document 8: Japanese Patent Publication No. 5-155778

非專利文獻1：稻川　裕之　其他8名，〝Biotherapy〞，1991年，第5卷，4號，p617-621Non-Patent Document 1: Inagawa Yuki Other 8 people, 〝Biotherapy〞, 1991, Vol. 5, No. 4, p617-621

非專利文獻2：Soma,G-I.其他1名，〝Tumor Necrosis Factor: Molecular and Cellular Biology and Clinical Revenvance〞，1993年，p203-220Non-Patent Document 2: Soma, G-I. The other one, 〝Tumor Necrosis Factor: Molecular and Cellular Biology and Clinical Revenvance〞, 1993, p203-220

非專利文獻3：山田　常雄　其他6名，〝生物學事典第3版〞，1983年，p1021Non-Patent Document 3: Yamada Toshio Other 6th, 〝Biology Book 3rd Edition, 1983, p1021

非專利文獻4：Gavini,F.其他6名，〝Int. J. Syst. Bacteriol.〞，1989年，第39卷，p337-345Non-Patent Document 4: Gavini, F. Other 6th, 〝Int. J. Syst. Bacteriol.〞, 1989, Vol. 39, p337-345

非專利文獻5：Nishizawa,T.其他7名，〝Chem. Pharm. Bull.〞，1992年，第40卷，2號，p479-483Non-Patent Document 5: Nishizawa, T. Other 7th, 〝Chem. Pharm. Bull.〞, 1992, Vol. 40, No. 2, p479-483

非專利文獻6：Inagawa,H.其他8名，〝Chem. Pharm. Bull.〞，1992年，第40卷，4號，p994-997Non-Patent Document 6: Inagawa, H. Other 8th, 〝Chem. Pharm. Bull.〞, 1992, Vol. 40, No. 4, p994-997

非專利文獻7：Neilson,A. H.〝J. Appl. Bacteriol.〞，1979年，第46卷，3號，p483-491Non-Patent Document 7: Neilson, A. H. 〝 J. Appl. Bacteriol. 〞, 1979, Vol. 46, No. 3, p483-491

非專利文獻8：Nunes,C.其他3名，〝Int. J. Food Microbiol.〞，2001年，第70卷，1，2合併號，p53-61Non-Patent Document 8: Nunes, C. Other 3, 〝Int. J. Food Microbiol.〞, 2001, Vol. 70, 1, 2 merger number, p53-61

非專利文獻9：山田常雄其他6名，〝生物學事典第3版〞，1983年，p287-288Non-Patent Document 9: Other 6 of Yamada's Chang Xiong, 3rd Edition of the Biology Book, 1983, p287-288

非專利文獻10：Nunes,C.其他4名，〝J. Appl. Microbiol.〞，2002年，第92卷，2號，p247-255Non-Patent Document 10: Nunes, C. Other 4, 〝J. Appl. Microbiol.〞, 2002, Vol. 92, No. 2, p247-255

非專利文獻11：Asis,C. A. Jr.其他1名，〝Lett. Appl. Microbiol.〞2004年，第38卷，1號，p19-23Non-Patent Document 11: Asis, C. A. Jr. 1 other, 〝Lett. Appl. Microbiol. 〞 2004, Vol. 38, No. 1, p19-23

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非專利文獻13：Kearns,L. P.其他1名，〝Appl Environ Microbiol.〞1998年，第64卷，5號，p1837-1844Non-Patent Document 13: Kearns, L. P. 1 other, 〝Appl Environ Microbiol.〞 1998, Vol. 64, No. 5, p1837-1844

如上述，免疫賦活物質為植物本身所含有或與植物共生之微生物的構成成分或生產物的情況為多。因此，欲得到可攝取且安全的來自天然物之免疫賦活物質，可由食用植物本身萃取出成分(例如limulus陽性糖脂質、專利文獻1)或效率地培養於食用植物共生的微生物後取得其構成成分或生產物(例如低分子量脂多醣，專利文獻2)。然而，含於食用植物的免疫賦活物質的含量較少，若希望藉由食用後之免疫賦活效果必須攝取極多量的食品，或若要保持適當的免疫賦活物質攝取量並不容易，效果無法達成。且由植物萃取作為食品或藥劑利用時必須花費高成本缺乏實用性。As described above, the immunostimulating substance is often a constituent component or a product of a microorganism contained in the plant itself or symbiotic with the plant. Therefore, in order to obtain an ingestible and safe immunostimulating substance derived from a natural product, the component can be extracted from the edible plant itself (for example, limulus-positive glycolipid, Patent Document 1) or efficiently cultured in a symbiotic microorganism of the edible plant to obtain a constituent component. Or a product (for example, a low molecular weight lipopolysaccharide, Patent Document 2). However, the content of the immunostimulating substance contained in the edible plant is small, and if it is desired to ingest a very large amount of food by the immune activating effect after eating, or if it is not easy to maintain an appropriate amount of the immune activating substance, the effect cannot be achieved. . Moreover, it is necessary to use high cost and lack of practicality when using plant extract as a food or a medicament.

另一方面，受到注目的與植物共生之微生物，例如小麥共生細菌之稻穀病原細菌(Pantoea agglomerans)含有構成免疫賦活有效成分之低分子量脂多醣成分。然而至今對於低分子量脂多醣的萃取，必須使用含於培養液之蛋白質為主成分為來自動物者，例如必須使用NZ胺或胰化蛋白或酪蛋白胺基酸等高價的培養液進行稻穀病原細菌的培養。因此，作為廣用性高的免疫賦活物質難以便宜價格下提供，無法避免同時混入來自BSE其他動物之未知有害物質。On the other hand, microorganisms that are symbiotic with plants, such as wheat symbiotic bacteria, Pantoea agglomerans contain a low molecular weight lipopolysaccharide component constituting an active ingredient for immunization. However, to date, for the extraction of low molecular weight lipopolysaccharide, it is necessary to use the protein contained in the culture solution as the main component for the animal, for example, it is necessary to use the high-priced culture medium such as NZ amine or trypsin or casein amino acid for the rice pathogenic bacteria. Cultivation. Therefore, it is difficult to provide a highly versatile immunostimulating substance at a low price, and it is unavoidable to simultaneously mix unknown harmful substances from other animals of BSE.

鑑於上述問題點，本發明為提供一種使用安全原料且便宜價格下有效率地得到免疫賦活物質之發酵及培養方法，藉由該方法所得之植物發酵萃取物、該植物發酵萃取物所得之植物發酵萃取物粉末及添加該植物發酵萃取物粉末的植物發酵萃取物配合物為目的。In view of the above problems, the present invention provides a fermentation and culture method for obtaining an immunostimulating substance efficiently and inexpensively using a safe raw material, and the plant fermentation extract obtained by the method, the plant fermentation obtained by the plant fermentation extract The extract powder and the plant fermentation extract complex to which the plant fermentation extract powder is added are for the purpose.

本發明的發酵及培養方法為，將來自食用植物的原料藉由於特定植物中共生的兼性厭氧性革氏陰性菌進行發酵，同時培養該兼性厭氧性革氏陰性菌為桿菌為特徵者。The fermentation and culture method of the present invention is characterized in that a raw material derived from an edible plant is fermented by a facultative anaerobic Gram-negative bacterium which is symbiotic in a specific plant, and the facultative anaerobic Gram-negative bacterium is cultured as a bacterium. By.

又，作為碳源的澱粉藉由該兼性厭氧性革氏陰性菌發酵下，可經由單純的過程進行發酵及培養。Further, the starch as a carbon source can be fermented and cultured through a simple process by fermentation of the facultative anaerobic Gram-negative bacteria.

該兼性厭氧性革氏陰性菌以兼性厭氧性桿菌為佳。The facultative anaerobic Gram-negative bacteria are preferably facultative anaerobic bacilli.

該兼性厭氧性革氏陰性菌屬於腸內細菌科為佳。The facultative anaerobic Gram-negative bacteria is preferably an enterobacteriaceae.

又，該兼性厭氧性桿菌屬於團泛菌屬(pantoea)、沙雷氏菌屬(serratia)、或腸內桿菌屬(enterbacter)者為佳。Further, the facultative anaerobic bacterium is preferably of the genus Pantoea, serratia, or enterbacter.

又，該兼性厭氧性桿菌為稻穀病原細菌(Pantoea agglomerans)，而碳源可為澱粉。Further, the facultative anaerobic bacterium is a rice pathogenic bacterium (Pantoea agglomerans), and the carbon source may be starch.

又，該食用植物為穀物、海草、或豆類、或這些混合物為佳。Further, the edible plant is preferably cereal, seaweed, or beans, or a mixture thereof.

又，該穀物的原料為小麥粉、米粉、小麥糠粉、米糠、或酒粕為佳，特別為小麥粉含有作為蛋白質源之麵筋，故既使不使用來自動物的原料亦可效率良好下進行發酵及培養。Further, the raw material of the cereal is wheat flour, rice flour, wheat bran flour, rice bran, or wine cellar, and in particular, the wheat flour contains gluten as a protein source, so that fermentation can be carried out efficiently without using raw materials derived from animals. And training.

又，來自該海草的原料為海帶芽粉、和布蕪、或昆布粉為佳。Further, the raw material derived from the seaweed is preferably kelp bud powder, cloth crepe or kelp powder.

又，來自該豆類的原料為***，故含有大量的蛋白質而無須來自動物的原料，既使不使用來自動物的原料亦可效率良好下進行發酵及培養。Further, since the raw material derived from the beans is tofu, it contains a large amount of protein and does not require raw materials derived from animals, and can be efficiently fermented and cultured without using raw materials derived from animals.

又，本發明的植物發酵萃取物，其特徵為由前述發酵及培養方法所得者。Further, the plant fermentation extract of the present invention is characterized by being obtained by the above fermentation and culture method.

又，本發明的植物發酵萃取物粉末，其特徵為如前述植物發酵萃取物所得者。Further, the plant fermentation extract powder of the present invention is characterized by being obtained from the above-mentioned plant fermentation extract.

又，本發明的植物發酵萃取物配合物，其特徵為添加如前述植物發酵萃取物或植物發酵萃取物粉末者。Further, the plant fermentation extract complex of the present invention is characterized in that a plant fermentation extract or a plant fermentation extract powder is added as described above.

又，前述植物發酵萃取物配合物，其中該植物發酵萃取物配合物可為醫藥品、動物用醫藥品、醫藥部外品、化妝品、食品、功能性食品、飼料、或沐浴劑。Further, the plant fermentation extract complex, wherein the plant fermentation extract complex may be a pharmaceutical, an animal medicine, a pharmaceutical product, a cosmetic, a food, a functional food, a feed, or a body wash.

又，前述植物發酵萃取物，其為存在多黏菌素B下亦顯示巨噬細胞活化能之物理化學性質者為佳。又，前述植物發酵萃取物具有免疫賦活活性。Further, the plant fermentation extract is preferably a physicochemical property which exhibits macrophage activation energy in the presence of polymyxin B. Further, the aforementioned plant fermentation extract has an immunostimulating activity.

本發明因以未含來自動物成分之培養基進行培養，故提供一種不會有混入來自動物成分的雜質之問題，例如不會有BSE或未知有害物質混入的危險，可高安全性且便宜等多方面用途下提供植物發酵萃取物的製造方法，且安全、便宜下製造出免疫賦活物質含有植物發酵萃取物或植物發酵萃取物，以及該培養液、免疫賦活物質、萃取物及萃取物粉末、更提供添加該萃取物或萃取物粉末的醫藥品、動物用醫藥品、醫藥部外品、化妝品、食品、功能性食品、飼料及浴用劑等。Since the present invention is cultured in a medium which does not contain animal components, it provides a problem that impurities from animal components are not mixed, for example, there is no risk of BSE or unknown harmful substances being mixed, and it is highly safe and inexpensive. Provided in a method for producing a plant fermentation extract, and safely and inexpensively producing an immunostimulating substance comprising a plant fermentation extract or a plant fermentation extract, and the culture liquid, the immunostimulating substance, the extract and the extract powder, and more A pharmaceutical product, an animal medical product, a pharmaceutical external product, a cosmetic, a food, a functional food, a feed, a bathing agent, and the like which are added with the extract or the extract powder are provided.

將來自食用植物的原料藉由與特定植物共生的兼性厭氧性革氏陰性菌進行發酵，同時可培養該兼性厭氧性革氏陰性菌之單純過程下發酵及培養的技術，至今未被想到，且由至今的發酵技術之常識亦無法輕易地推想得知。The raw material from the edible plant is fermented by a facultative anaerobic Gram-negative bacterium that is symbiotic with a specific plant, and the fermentation and culture technique of the facultative anaerobic Gram-negative bacteria can be cultured. It is thought that the routine of fermentation technology up to now cannot be easily imagined.

又，作為顯示物質的免疫賦活效果的指標，可使用由巨噬細胞是否產生TNF(TNF衍生活性)之方法。且藉由TNF產生量可定量化免疫賦活效果。其中使用來自小麥的limulus陽性植物糖脂質、來自稻穀病原細菌的低分子量脂多醣，對由巨噬細胞的TNF產生作檢討時，來自小麥的limulus陽性植物糖脂質、來自稻穀病原細菌的低分子量脂多醣皆以多黏菌素B處理時，不會由巨噬細胞產生TNF，但由本發明的實施例顯示本發明的植物發酵萃取物進行如上處理時由巨噬細胞產生大量的TNF。此為進行發酵培養結果所得之本發明的植物發酵萃取物，其為具有與成為其原料的植物本身、及使用於發酵的微生物本身的構成成分所造成的免疫賦活效果於質地上相異的效果。Further, as an indicator of the immunostimulating effect of the substance, a method of whether or not TNF (TNF-derived activity) is produced by macrophages can be used. And the immunostimulating effect can be quantified by the amount of TNF produced. Among them, limulus-positive plant glycolipids derived from wheat and low-molecular-weight lipopolysaccharides derived from rice pathogenic bacteria are used to review TNF production from macrophages, limulus-positive plant glycolipids from wheat, and low molecular weight lipids from rice-pathogenic bacteria. When the polysaccharide is treated with polymyxin B, TNF is not produced by macrophages, but it is shown by the examples of the present invention that the plant fermentation extract of the present invention produces a large amount of TNF from macrophages when subjected to the above treatment. This is a plant fermentation extract of the present invention obtained as a result of fermentation culture, and has an effect of having an immunostimulating effect different from that of a plant itself which is a raw material thereof and a constituent component of the microorganism itself used for fermentation. .

實施發明的最佳型態The best form of implementing the invention

以下對本發明的較適實施型態作詳細說明。The preferred embodiments of the present invention are described in detail below.

I：使用稻穀病原細菌(Pantoea agglomerans)之植物發酵萃取物的製造方法重點I: Focus on the manufacturing method of plant fermentation extract using rice pathogenic bacteria (Pantoea agglomerans)

本案中，本發明者首先發現稻穀病原細菌可將澱粉作為直接碳源生長，發明使用稻穀病原細菌進行發酵及便宜地製造出富含作為培養生成物的免疫賦活物質之小麥發酵萃取物之方法。藉由此，作為具體例子為可提供人類、畜產、水產養殖之領域下對環境溫和且安全下可預防感染的有效醫藥部外品、化妝品、食品、功能性食品、飼料。In the present invention, the present inventors first discovered that rice pathogenic bacteria can grow starch as a direct carbon source, and invented a method of fermenting using rice pathogenic bacteria and inexpensively producing a wheat fermentation extract rich in an immunostimulating substance as a culture product. As a specific example, it is an effective pharmaceutical product, cosmetics, food, functional food, and feed that can provide an environment-friendly and safe preventable infection in the fields of humans, livestock, and aquaculture.

1：稻穀病原細菌的分離1: Separation of rice pathogenic bacteria

將小麥粉懸浮於水中，將澄清液體塗佈於L-broth洋菜培養基中進行培養後出現微生物的菌落。這些菌落以一定方法進行微生物的鑑定。例如選擇出革氏染色陰性、葡萄糖厭氣性代謝反應陽性、氧化酶活性陰性的菌落，且使用ID測試‧EB-20(日水製藥)等，選擇出與標準稻穀病原細菌具有相同性質者。作為標準的稻穀病原細菌可由理化學研究所生物基盤研究不微生物系統保存設施獲得(非專利文獻4)。以下說明中，百分率的表示若無特別說明則表示重量。The wheat flour was suspended in water, and the clear liquid was applied to the L-broth canola medium for cultivation, and microbial colonies appeared. These colonies were identified by microorganisms in a certain way. For example, colonies which are negative for Gram stain, positive for glucose anaerobic metabolism, and negative for oxidase activity are selected, and those having the same properties as standard rice pathogenic bacteria are selected using ID test ‧ EB-20 (Nissui Pharmaceutical Co., Ltd.). The rice pathogenic bacteria which are standard can be obtained by the Institute of Physical Chemistry, Bio-Based Research, Non-Microbial System Preservation Facility (Non-Patent Document 4). In the following description, the percentage indicates the weight unless otherwise specified.

2：免疫賦活活性之評估2: Evaluation of immune activating activity

本實施的型態中，作為小麥發酵萃取物顯示免疫賦活作用的指標，將巨噬細胞活性化能由巨噬細胞的TNF產生作評估。In the form of the present embodiment, as an indicator of the immunostimulating action of the wheat fermentation extract, activation of macrophages can be evaluated by TNF production by macrophages.

3：來自稻穀病原細菌的低分子脂多醣3: Low molecular weight lipopolysaccharide from rice pathogenic bacteria

又，藉由使用稻穀病原細菌之發酵及培養，可期待其中含有作為免疫賦活之有效成分之一的來自稻穀病原細菌之低分子量脂多醣。低分子量脂多醣與廣泛被使用的高分子量型脂多醣(以下稱為脂多醣)相比其安全性極高，且其生物活性亦與一般的脂多醣相比較為優良。因此，測定低分子量脂多醣含量。對於低分子量脂多醣於專利文獻2中已詳細說明。且，本實施例雖與小麥發酵萃取物相關，但本發明並未將植物限定為小麥、將免疫賦活物質限定為低分子量脂多醣。Further, by using fermentation and culture of rice pathogenic bacteria, it is expected to contain a low molecular weight lipopolysaccharide derived from a rice pathogenic bacterium which is one of active ingredients for immunization. The low molecular weight lipopolysaccharide is extremely safe compared to the widely used high molecular weight lipopolysaccharide (hereinafter referred to as lipopolysaccharide), and its biological activity is superior to that of the general lipopolysaccharide. Therefore, the low molecular weight lipopolysaccharide content was determined. The low molecular weight lipopolysaccharide has been described in detail in Patent Document 2. Further, although this example relates to wheat fermented extracts, the present invention does not limit plants to wheat, and the immunostimulating substance is limited to low molecular weight lipopolysaccharides.

於是稻穀病原細菌可使用公知方法進行培養(專利文獻2、非專利文獻8)，但公知培養液中所含有的蛋白質主要成分為來自動物者，其培養基的成本較為高。且，動物給予例如功能性食品或功能性飼料、或經皮性投與時，會有如BSE的來自動物的雜質混入而造成食物上安全性問題，且由製造成本更高，由實用性來看並非好方法。因此本發明者欲得到具有安全且便宜的免疫賦活作用之天然物進行詳細研究結果，欲取得小麥發酵萃取物依據實施例所示使用稻穀病原細菌進行發酵及培養方法而完成。含於培養液的蛋白質主成分為於過去為使用來自動物者，本發明則使用來自植物者。一般培養液體中添加由消化酵素分解來自牛奶的酪蛋白等蛋白質之產物。此時1L的培養基原價約250日圓，但由小麥粉取代時原價為約16日圓。至今並未進行植物及與其共生的微生物雙方之免疫賦活活性使其高濃度下會融合化之相乘作用為目的的發酵。Therefore, the rice pathogenic bacteria can be cultured by a known method (Patent Document 2 and Non-Patent Document 8). However, it is known that the main component of the protein contained in the culture solution is derived from an animal, and the cost of the culture medium is relatively high. Moreover, when an animal is administered, for example, a functional food or a functional feed, or is administered transdermally, there is a problem of food safety caused by the incorporation of impurities from the animal such as BSE, and the manufacturing cost is higher, and from the viewpoint of practicality Not a good way. Therefore, the present inventors intend to obtain a natural product having a safe and inexpensive immunostimulating effect, and carry out detailed research results, and the wheat fermentation extract is obtained by performing fermentation and culture methods using rice pathogenic bacteria as shown in the examples. The main component of the protein contained in the culture solution is used in the past for the animal, and the present invention uses the planter. A product of a protein such as casein derived from milk which is decomposed by digestive enzymes is added to the culture liquid. At this time, the original price of the 1 L medium was about 250 yen, but when the wheat flour was replaced, the original price was about 16 yen. Up to now, there has been no fermentation for the purpose of multiplying the fusion activity of plants and their symbiotic microorganisms at a high concentration.

以下以實施例對本發明內容作詳細說明，但作為本發明於實施例中所記載的微生物並未限定為稻穀病原細菌，作為食用植物的小麥或原料亦未限定為小麥粉，亦可使用含有大量免疫賦活物質的其他食用植物，經過一般步驟所得之原料、例如可適用於海帶芽、穀物(來自穀物的原料含有小麥粉、米粉、小麥糠粉、米糠、或酒粕等)、海草(來自海草的原料含有海帶芽粉、和布蕪、或昆布粉)、豆類(來自該豆類的原料含有***)等。已知這些植物中含有蛋白質、醣類，適用於使用稻穀病原細菌之發酵及培養上。又，已知這些植物一般與常在性細菌，例如與沙雷氏菌屬(serratia)、或腸內桿菌屬(enterbacter)共生(非專利文獻4)、使用於發酵的微生物當然亦可使用於這些植物共生的兼性厭氧性革氏陰性菌。Hereinafter, the contents of the present invention will be described in detail by way of examples. However, the microorganisms described in the examples of the present invention are not limited to rice pathogenic bacteria, and wheat or raw materials used as edible plants are not limited to wheat flour, and may be used in large amounts. Other edible plants that immunize the active material, the raw materials obtained through the general steps, for example, can be applied to kelp buds, cereals (the raw materials from cereals contain wheat flour, rice flour, wheat bran flour, rice bran, or wine cellar, etc.), seaweed (from seaweed The raw material contains kelp bud powder, and cloth crepe or kelp powder), beans (the raw materials from the beans contain bean curd), and the like. These plants are known to contain proteins and sugars and are suitable for fermentation and culture using rice pathogenic bacteria. Further, it is known that these plants are generally symbiotic with a common bacterium, for example, serratia or enter bacteria (Non-Patent Document 4), and microorganisms used for fermentation can of course be used. These plants are symbiotic facultative anaerobic Gram-negative bacteria.

II：綜合發明的重點II: The focus of comprehensive invention

(1)小麥、其共生細菌之稻穀病原細菌、及組合這些之發酵產物的融合而具有免疫賦活作用之物質的小麥發酵萃取物為新穎物，但本發明並未限定於此。(1) The wheat fermentation extract of wheat, the commensal bacteria of the commensal bacteria, and the substance having the immunostimulating action in which the fermentation products are combined is a novel, but the present invention is not limited thereto.

(2)使用革氏陰性菌的稻穀病原細菌製造植物發酵萃取物係為新的技術。但本發明並未限定於此。(2) The production of plant fermentation extracts using rice pathogenic bacteria of Gram-negative bacteria is a new technique. However, the invention is not limited thereto.

III：小麥發酵萃取物的具體製造方法III: Specific manufacturing method of wheat fermentation extract

(1)稻穀病原細菌依據一般方法由小麥粉中分離(非專利文獻1)。且，僅一次分離鑑定後，此菌可保存於50%甘油等中。(1) Rice pathogenic bacteria are isolated from wheat flour according to a general method (Non-Patent Document 1). Moreover, the strain can be stored in 50% glycerol or the like after isolation and identification only once.

(2)調製出0.05～5%的食鹽、0.005～1莫耳的磷酸緩衝液、或混合鹽類溶液(0.5～10%的磷酸第二鈉、0.05～5%的磷酸第一鉀、0.05～5%的氯化鈉、0.05～5%的氯化銨)等。(2) Preparing 0.05 to 5% of salt, 0.005 to 1 mol of phosphate buffer, or mixed salt solution (0.5 to 10% of second sodium phosphate, 0.05 to 5% of potassium phosphate, 0.05 to 0.05) 5% sodium chloride, 0.05 to 5% ammonium chloride).

(3)將小麥粉懸浮於水中至0.05～10%濃度。(3) The wheat flour is suspended in water to a concentration of 0.05 to 10%.

(4)調製出0.2～3莫耳的氯化鎂溶液。(4) A 0.2 to 3 molar magnesium chloride solution was prepared.

(5)調製出0.2～3莫耳的氯化鉀溶液。(5) A 0.2 to 3 molar potassium chloride solution was prepared.

(6)2至5則依據所需可進行高壓滅菌釜之滅菌操作。(6) 2 to 5 The sterilization operation of the autoclave can be carried out according to the requirements.

(7)將2至5作適量混合，加入水後成為含有0.1～5%的小麥粉之懸浮液。依據所需加入鹼溶液或酸性溶液後pH成為中性。(7) 2 to 5 are mixed in an appropriate amount, and after adding water, a suspension containing 0.1 to 5% of wheat flour is obtained. The pH becomes neutral depending on the desired addition of an alkaline solution or an acidic solution.

(8)藉由7的情況，於每1L的培養基中添加10～50000單位的澱粉酶，於10℃～80℃下保溫1～24小時，亦可消化部份的小麥澱粉。(8) In the case of 7, 10 to 50,000 units of amylase may be added per 1 L of the medium, and the mixture may be incubated at 10 to 80 ° C for 1 to 24 hours to digest part of the wheat starch.

(9)7至8的步驟中添加於1中分離之稻穀病原細菌。(9) The steps of 7 to 8 are added to the rice pathogenic bacteria isolated in 1.

(10)9於1～40℃下進行發酵。依情況可靜置或震盪。又，亦可進行數小時之攪拌。(10) 9 Fermentation is carried out at 1 to 40 °C. It can be placed or shaken depending on the situation. Further, it is also possible to carry out stirring for several hours.

(11)將10進行6小時至一星期的發酵。發酵進行下小麥粉的水溶液會著色成黃色。(11) Fermenting 10 for 6 hours to one week. The aqueous solution of the wheat flour under fermentation is colored yellow.

(12)11的發酵途中可添加適當的鹼溶液，使pH成為中性、或可添加小麥粉懸浮液或無機鹽類。An appropriate alkali solution may be added during the fermentation of (12)11 to make the pH neutral, or a wheat flour suspension or an inorganic salt may be added.

(13)發酵終了後，藉由離心分離(1000～5000 rpm，10～60分鐘)等操作回收沈澱物的固體成分。沈澱物作為小麥粉發酵物，可直接作為飼料或混合於飼料的原料使用。(13) After the completion of the fermentation, the solid content of the precipitate is recovered by centrifugation (1000 to 5000 rpm, 10 to 60 minutes). The precipitate is used as a fermented wheat flour and can be used directly as a feed or as a raw material mixed with feed.

(14)製造小麥發酵萃取務實，將13懸浮於水或鹽類緩衝液等中，將此於80～140℃下進行10分鐘至6小時的熱處理。且經由離心分離或過濾可除去固體成分。所除去的沈澱物中再次加入水或緩衝液進行數次重複的加熱萃取。(14) Production of Wheat Fermentation Extraction is practicable, and 13 is suspended in water or a salt buffer or the like, and this is heat-treated at 80 to 140 ° C for 10 minutes to 6 hours. The solid component can be removed by centrifugation or filtration. The removed precipitate was again added with water or a buffer for several repeated heat extractions.

(15)依據於14所製造的小麥發酵萃取之用途，可再進行簡單的純化步驟。即，14的萃取物中加入氯化鈉等鹽類使其成為最終濃度為0.05～1莫耳/L，其後添加萃取物的1～3倍量的乙醇等溶劑使其沈澱。將此可使用離心分離機等進行回收。將此沈澱可再以乙醇等溶劑洗淨。將此乾燥後可做出粉末。(15) A simple purification step can be carried out depending on the use of the 14 wheat fermentation extracts produced. In other words, a salt such as sodium chloride is added to the extract of 14 to have a final concentration of 0.05 to 1 mol/L, and then a solvent such as ethanol is added in an amount of 1 to 3 times the amount of the extract to precipitate. This can be recovered using a centrifugal separator or the like. This precipitate can be washed again with a solvent such as ethanol. This can be dried to make a powder.

A.　有關小麥發酵萃取物之製造方法的實施例A. Example of a method for producing a wheat fermentation extract 實施例1Example 1 稻穀病原細菌的小麥培養基中之生長試驗Growth test in rice medium of rice pathogenic bacteria

欲確認以小麥粉作為碳源時是否可使小麥常在性共生菌之稻穀病原細菌增殖，對小麥粉固體培養基之稻穀病原細菌的生長作調查。To determine whether wheat flour is often propagated in the rice commensal bacteria of the symbiotic bacteria when wheat flour is used as a carbon source, the growth of rice pathogenic bacteria in wheat flour solid medium is investigated.

(1)作成含有0.5%小麥粉作為碳源之M9洋菜培養基。(1) An M9 agar medium containing 0.5% wheat flour as a carbon source was prepared.

(2)經LB洋菜培養基，取出稻穀病原細菌的1個菌落懸浮於1 ml的PBS中。將此再以PBS作10倍至10000倍之階段式稀釋，再將各0.1 ml接種於1的M9洋菜培養基。(2) One colony of the rice pathogenic bacteria was taken out in 1 ml of PBS via LB agar medium. This was further diluted with PBS from 10 to 10,000 times, and each 0.1 ml was inoculated into 1 M9 agar medium.

(3)於37℃下培養6天後，觀察菌落的出現。其結果於10000倍稀釋的0.1 ml所播種的培養皿中觀察到約300個菌落。(3) After 6 days of culture at 37 ° C, the appearance of colonies was observed. As a result, about 300 colonies were observed in a 10,000-fold diluted 0.1 ml seeded petri dish.

由此可知，確認出稻穀病原細菌可利用小麥作為碳源。From this, it was confirmed that rice pathogenic bacteria can utilize wheat as a carbon source.

實施例2Example 2 小麥發酵萃取物的製造Manufacture of wheat fermentation extract

(1)0.5 g的小麥粉中加入5 ml的蒸餾水使其懸浮，澄清液0.1 ml添加於L-broth洋菜培養基中，於37℃下經一晚培養。(1) 0.5 g of wheat flour was suspended by adding 5 ml of distilled water, and 0.1 ml of a clarified liquid was added to L-broth agar medium, and cultured overnight at 37 °C.

(2)分離黃色的菌落，以一般方法進行菌的鑑定，分離出稻穀病原細菌，將此懸浮於50%甘油溶液中，於冷凍庫中保存。將此保存液的一部份塗抹於LB洋菜培養基，放置於37℃下作成稻穀病原細菌之獨立菌落。(2) The yellow colonies were separated, and the bacteria were identified by a general method, and the rice pathogenic bacteria were isolated, suspended in a 50% glycerin solution, and stored in a freezer. A portion of this preservation solution was applied to LB agar medium, and placed at 37 ° C to form an independent colony of rice pathogenic bacteria.

(3)2L的三角錐形瓶中加入64 g的磷酸第二鈉‧7結晶水、15 g的磷酸第一鉀、2.5 g的氯化鈉、5 g的氯化銨，加入純水使全量成為1L(無機鹽類混合溶液)；取13.1 g的氯化鎂‧2結晶水，加入純水使全量成為100 mL(氯化鈣溶液)；將4L的純水放入5L的三角錐形瓶中(純水)。上述溶液及純水皆放入高壓滅菌釜(TOMY BS-325，120℃，20分鐘)進行滅菌處理。(3) Add 2 g of triangular sodium phosphate ‧7 crystal water, 15 g of first potassium phosphate, 2.5 g of sodium chloride, 5 g of ammonium chloride, and add pure water to make a full amount 1L (inorganic salt mixed solution); take 13.1 g of magnesium chloride ‧ 2 crystal water, add pure water to make the whole amount into 100 mL (calcium chloride solution); put 4L of pure water into a 5L triangular conical flask ( Pure water). The above solution and pure water were placed in an autoclave (TOMY BS-325, 120 ° C, 20 minutes) for sterilization.

(4)於1L的三角錐形瓶中放入24 g的小麥粉(日清製粉)，加入純水使全量成為600 ml。將此進行相同高壓釜滅菌後，加入3 mg的α-澱粉酶(SIGMA,Bacillus，1 mg蛋白質中1500～3000單位的酵素活性)，65℃的水浴下加熱4～12小時(小麥粉澱粉酶處理液)。(4) Put 24 g of wheat flour (Nisshin powder) in a 1 L triangular conical flask, and add pure water to make the whole amount 600 ml. After the same autoclave sterilization, 3 mg of α-amylase (SIGMA, Bacillus, 1500-3000 units of enzyme activity in 1 mg protein) was added, and heated in a water bath at 65 ° C for 4 to 12 hours (wheat starch amylase). Treatment solution).

(5)將各如表1所示量之調整的溶液放入經滅菌之3L之Sakaguchi錐形瓶作為小麥粉培養基。(5) Each of the adjusted solutions as shown in Table 1 was placed in a sterilized 3 L Sakaguchi Erlenmeyer flask as a wheat flour medium.

(6)種菌的調製。與上述相同組成所調製之5的10 ml小麥粉培養基中，放入2中由小麥粉所分離出的1個稻穀病原細菌菌落，於37℃下緩緩攪拌1晚(12～15小時)使其發酵，調製出小麥粉發酵用種菌。(6) Modulation of inoculum. In a 10 ml wheat flour medium prepared in the same composition as above, one rice bacterial colony isolated from wheat flour was placed in 2, and slowly stirred at 37 ° C for 1 night (12 to 15 hours). The fermentation is carried out to prepare an inoculum for fermentation of wheat flour.

(7)於5中加入6全量並於37℃下攪拌的同時，經20～30小時使其發酵。測定發酵液的pH，加入氨水後將pH調整為7。於此無菌下加入150 ml的小麥粉澱粉酶處理液與無機鹽類混合溶液37.5 ml，進行相同的20～30小時之發酵。相同操作在重複一次後總計發酵時間為65～80小時。(7) A total amount of 6 is added to 5 and stirred at 37 ° C while fermenting for 20 to 30 hours. The pH of the fermentation broth was measured, and the pH was adjusted to 7 after the addition of ammonia water. Under this sterility, 150 ml of a mixture of wheat starch amylase treatment solution and inorganic salt mixed solution of 37.5 ml was added, and the same fermentation was carried out for 20 to 30 hours. The same operation was repeated for one time and the total fermentation time was 65 to 80 hours.

(8)7的小麥粉發酵溶液經離心分離(日立，高速冷卻離心機SCR-20B，5000 rpm，20分鐘，4℃)，回收沈澱物。The wheat flour fermentation solution of (8) 7 was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 5000 rpm, 20 minutes, 4 ° C), and the precipitate was recovered.

(9)8的沈澱物中加入磷酸緩衝液使其成為全量100 ml的懸浮狀態，取出33 ml移入50 ml的離心管中，於沸騰水浴中進行30分鐘的熱萃取。加熱終了後，冷卻至室溫，離心分離本液體(日立，高速冷卻離心機SCR-20B，10000 rpm，20分鐘，20℃)。經離心後將82 ml的淡黃色澄清液傾析回收至另外容器中。Phosphate buffer was added to the precipitate of (9) 8 to make it a suspension of 100 ml in total. 33 ml was taken out and transferred to a 50 ml centrifuge tube, and subjected to hot extraction in a boiling water bath for 30 minutes. After the end of the heating, the mixture was cooled to room temperature, and the liquid was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 10000 rpm, 20 minutes, 20 ° C). After centrifugation, 82 ml of the pale yellow clear liquid was decanted and recovered into another container.

(10)9的80 ml澄清液中加入8.9 ml的5莫耳氯化鈉溶液，於此加入178 ml的乙醇使其成為白濁狀。將此放置於冷凍庫(-90℃)中一晚後，本液體經離心分離(日立，高速冷卻離心機SCR-20B，10000 rpm，20分鐘，4℃)後除去澄清液得到沈澱物。沈澱物中加入經冷卻的10 ml 70%乙醇使其懸浮後，離心分離本液體(日立，高速冷卻離心機SCR-20B，1000 rpm，20分鐘，20℃)，洗淨沈澱物。風乾沈澱物後溶解於蒸餾水中，得到11 ml的小麥發酵萃取物溶液。8.9 ml of a 5 molar sodium chloride solution was added to 80 ml of the clear solution of (10) 9, and 178 ml of ethanol was added thereto to make it cloudy. After leaving this in a freezer (-90 ° C) for one night, the liquid was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 10000 rpm, 20 minutes, 4 ° C), and the clear liquid was removed to obtain a precipitate. After the precipitate was suspended by adding 10 ml of 70% ethanol, the liquid was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 1000 rpm, 20 minutes, 20 ° C), and the precipitate was washed. The precipitate was air-dried and dissolved in distilled water to obtain 11 ml of a wheat fermentation extract solution.

(11)乾燥重量的測定：預先秤取0.3 ml後移至1.5 ml的塑膠試管爭，冷凍後，以冷凍乾燥機進行冷凍乾燥得到7.45 mg。因此10的小麥發酵萃取物之乾燥重量為1 ml的溶液中為24.8 mg，全量11 ml中為273 mg。(11) Determination of dry weight: 0.3 ml was weighed beforehand and transferred to 1.5 ml of plastic test tube. After freezing, freeze-drying was carried out in a freeze dryer to obtain 7.45 mg. Therefore, the dry weight of the wheat fermentation extract of 10 is 24.8 mg in a 1 ml solution and 273 mg in a total amount of 11 ml.

(12)以相同方法製造出獨立8次的小麥發酵萃取物，各以Bradford蛋白濃度測定法，將蛋白質定量BSA作為標準蛋白質，測定各樣品的蛋白質質量。作為比較對象，使用經純化的limulus陽性糖脂質(專利文獻1)與低分子脂多醣(專利文獻2)。測定結果如表2所示。有關表2～5、7的小麥發酵萃取物之數值為，上述10所得之小麥發酵萃取物經乾燥所得之1g重量中的含有量mg。(12) The wheat fermentation extracts were independently produced eight times in the same manner, and the protein quality of each sample was determined by using Bradford protein concentration measurement method to quantify protein BSA as a standard protein. As a comparison object, purified limulus-positive glycolipid (Patent Document 1) and low-molecular lipopolysaccharide (Patent Document 2) were used. The measurement results are shown in Table 2. The value of the wheat fermentation extract of Tables 2 to 5 and 7 is the content of mg in 1 g of the obtained wheat fermentation extract obtained by the above-mentioned 10.

(13)糖含量的測定：藉由酚硫酸法將葡萄糖作為標準糖進行測定。測定結果如表3所示。(13) Measurement of sugar content: Glucose was measured by phenol sulfuric acid method as a standard sugar. The measurement results are shown in Table 3.

(14)核酸含有量測定：將經100倍稀釋的樣品進行210～340 nm的吸光度測定。減去260 nm吸光度至320 nm的吸光度所得值、與DNA的吸光度1OD算出50μg的最大含有量。測定結果如表4所示。(14) Measurement of nucleic acid content: The 100-fold diluted sample was measured for absorbance at 210 to 340 nm. The maximum absorbance of 50 μg was calculated by subtracting the absorbance at 260 nm absorbance to 320 nm and the absorbance at DNA 1OD. The measurement results are shown in Table 4.

(15)藉由limulus測定之limulus活性物質含有量的測定：limulus活性物質量為使用生化學工業的toxycolor system，作為標準limulus活性物質使用生化學工業Et-1。測定結果如表5。(15) Determination of the content of the limulus active substance measured by limulus: the mass of the limulus active substance is a tooxycolor system using the biochemical industry, and the biochemical industry Et-1 is used as a standard limulus active substance. The measurement results are shown in Table 5.

(16)碘-澱粉反應：使用碘試藥1N(12.7 g的碘中及25 g的碘化鉀中加入10 ml的水，仔細混合後，加入水使其成為100 ml)時，以水稀釋200倍，將5μl加入預先以1 mg/ml的濃度溶解之小麥發酵萃取物0.1 ml中，仔細攪拌。小麥發酵萃取物馬上由淡紫色轉換成濃紫色(陽性)。Limulus陽性糖脂質、低分子量脂多醣於相同條件下操作時並無相同發色現象(陰性)。以上結果歸納於表6中。(16) Iodine-starch reaction: dilute 200 times with water using iodine test reagent 1N (12.7 g of iodine and 25 g of potassium iodide added with 10 ml of water, carefully mixed, and added with water to make it 100 ml) 5 μl was added to 0.1 ml of the wheat fermentation extract previously dissolved at a concentration of 1 mg/ml, and carefully stirred. The wheat fermentation extract is immediately converted from lavender to rich purple (positive). Limulus-positive glycolipids and low-molecular-weight lipopolysaccharides did not have the same color development (negative) when operated under the same conditions. The above results are summarized in Table 6.

由上述結果得知，小麥發酵萃取物與limulus陽性糖脂質、低分子量脂多醣於蛋白質含量、唐含量、核酸含量(limulus陽性糖脂質並無數據故除去)、limulus活性物質含量、碘-澱粉反應所有項目中皆顯示相異性，故本發明係為新穎之物質。以上的結果簡化歸納於如表7表示。即，本實施例的植物發酵萃取物因顯示如下各理化學性質，故與limulus陽性糖脂質低分子量脂多醣為相異的新穎者。含有5～15%的蛋白質、20～45%的糖、10～35%的核酸、及10～40%的limulus陽性物質，於碘-澱粉反應時為陽性，於存在多黏菌素B下亦顯示巨噬細胞活化能。According to the above results, the wheat fermentation extract and limulus-positive glycolipid, low-molecular-weight lipopolysaccharide in protein content, Tang content, nucleic acid content (limulus-positive glycolipid has no data removed), limulus active substance content, iodine-starch reaction The dissimilarity is shown in all items, and the present invention is a novel substance. The above results are simplified and summarized as shown in Table 7. That is, the plant fermentation extract of the present example is novel in that it differs from the limulus-positive glycolipid low molecular weight lipopolysaccharide by exhibiting the following physicochemical properties. Containing 5 to 15% protein, 20 to 45% sugar, 10 to 35% nucleic acid, and 10 to 40% limulus positive substance, positive in iodine-starch reaction, in the presence of polymyxin B Shows macrophage activation energy.

過小：比小麥發酵萃取物之數值範圍(平均±標準差)少許多之值。Too small: much less than the numerical range (mean ± standard deviation) of the wheat fermentation extract.

過小：比小麥發酵萃取物之數值範圍(平均±標準差)多許多之值。Too small: much more than the numerical range (mean ± standard deviation) of the wheat fermentation extract.

實施例3Example 3 小麥發酵萃取物之免疫賦活作用Immune activation of wheat fermentation extract

於48格培養皿中放入作為人類巨噬細胞使用的急性骨髓性白血病細胞株之THP-1(1×106 個/250μl：放有10%牛胚胎血清之RPMI 1640培養基)，進行30分鐘的預培養。使各樣品的最終濃度成為1～10000 ng/ml加入250μl(最終量500μl)。調製出樣品中含有多黏菌素B(12.5μg/ml)之群。經4小時培養後，回收培養澄清液及細胞。澄清液的TNF活性使用L-929細胞障礙試驗進行測定。結果如表8所示。小麥發酵萃取物於多黏菌素B存在下亦可由巨噬細胞中產生TNF，但低分子量脂多醣及limulus陽性糖脂質於多黏菌素B存在下並不會由巨噬細胞中產生TNF。因此可得知小麥發酵萃取物與低分子量脂多醣或limulus陽性糖脂質相比具有相異的生物活性。THP-1 (1×10 6 cells/250 μl: RPMI 1640 medium containing 10% bovine embryo serum) was used as a human myeloid leukemia cell line in a 48-well culture dish for 30 minutes. Pre-culture. The final concentration of each sample was added to 250 μl (final amount 500 μl) at 1 to 10000 ng/ml. A group containing polymyxin B (12.5 μg/ml) in the sample was prepared. After 4 hours of culture, the culture supernatant and cells were recovered. The TNF activity of the clarified liquid was determined using the L-929 cell disorder test. The results are shown in Table 8. Wheat fermentation extract can also produce TNF from macrophages in the presence of polymyxin B, but low molecular weight lipopolysaccharide and limulus positive glycolipids do not produce TNF from macrophages in the presence of polymyxin B. It is therefore known that wheat fermentation extracts have different biological activities compared to low molecular weight lipopolysaccharides or limulus positive glycolipids.

B.　小麥發酵萃取物對飼料的應用實施例B. Application examples of wheat fermentation extracts for feed 實施例4Example 4

放入小麥發酵萃取物之養雞飼料(大規模試驗之broiler飼養之暴斃抑制效果)Feeding chicken feed with wheat fermentation extract (burst suppression effect of large-scale trial of broiler feeding)

製造出含有430μg/kg之實施例2所製造出的小麥發酵萃取物的飼料。所提供的雞為一群約5500～6000隻之broiler commercial雞。對照組為未含小麥發酵萃取物之飼料。對於孵化後3星期齡投與含有小麥發酵萃取物之飼料，每日投與至7週齡。每日測定死亡數。廢棄未達到出場基準的雞。結果如表9所示。試驗組(含有小麥發酵萃取物之飼料)的除去率為較低之1.9%，對照組為3.3%。成長率於試驗組為98.1%，於對照組為96.7%，故顯示提高1.4%的成長率。進行試驗組與對照組之間的出場實際數目與除去數目之顯著差檢定時，發現於χ2 檢定下有P＜0.0001之顯著差。由此可知，含有小麥發酵萃取物之飼料的broiler飼養顯示感染防除效果。A feed containing 430 μg/kg of the wheat fermentation extract produced in Example 2 was produced. The chickens provided are a group of about 5,500 to 6,000 brailer commercial chickens. The control group was a feed that did not contain wheat fermentation extract. For the 3 weeks old after hatching, the feed containing the wheat fermentation extract was administered daily to 7 weeks of age. The number of deaths was measured daily. Dispose of chickens that do not meet the benchmark. The results are shown in Table 9. The removal rate of the test group (feed containing wheat fermentation extract) was 1.9% lower, and that of the control group was 3.3%. The growth rate was 98.1% in the test group and 96.7% in the control group, which showed an increase of 1.4%. A significant difference between the actual number of exits and the number of removals between the test group and the control group was observed, and it was found that there was a significant difference of P < 0.0001 under the χ 2 test. From this, it can be seen that the broiler feeding of the feed containing the wheat fermentation extract shows an infection control effect.

實施例5Example 5 放入小麥發酵萃取物之養殖魚用飼料(鰤魚的野外試驗之感染防除效果)Feeding of farmed fish with wheat fermented extract (infection control effect of wild fish in squid)

放入實施例2所製造的小麥發酵萃取物之飼料的感染防禦效果以餵食野外試驗的一群約5200尾的鰤魚進行。結果如表10所示。因鏈球菌所引起的死亡於非投與對照組時到達4.8%。攝取100μg/kg/日(體重1 kg，每天)的小麥發酵萃取物之群(試驗組)與非投與群(對照組)中，小麥發酵萃取物投與群之死亡率(P＜0.00001)有顯著地降低。The infection-preventing effect of the feed which was placed in the wheat fermented extract produced in Example 2 was carried out by feeding a group of about 5,200 squid in the field test. The results are shown in Table 10. Death due to streptococcus reached 4.8% when not administered to the control group. Mortality of wheat fermented extracts in the group of wheat fermented extracts (test group) and non-administered group (control group) at 100 μg/kg/day (body weight 1 kg per day) (P<0.00001) There is a significant reduction.

實施例6Example 6 小麥發酵萃取物(對於錦鯉疱疹病毒症(Koi herpesvirus disease)之感染防除效果)Wheat Fermentation Extract (for infection control effect of Koi herpesvirus disease)

(1)鯉魚：體重70 g的黑鯉魚。試驗為一群20條。(1) Squid: Black squid weighing 70 g. The test was a group of 20.

(2)錦鯉疱疹病毒之調整：因錦鯉疱疹病毒感染死亡的鯉魚之鰓1g中加入10 ml的Hanks'緩衝食鹽溶液(HBSS)後進行均質，過濾0.45 micron的過濾器，將該濾液作為病毒溶液。(2) Adjustment of koi herpes virus: 10 ml of Hanks' buffered saline solution (HBSS) was added to the squid 1 g of the koi herpes virus infection, homogenized, and a 0.45 micron filter was filtered, and the filtrate was used as a virus solution. .

(3)錦鯉疱疹病毒感染：將上述濾液以600μl/100 g體重，注射於鯉魚的腹腔。(3) Koi herpes virus infection: The above filtrate was injected into the abdominal cavity of the squid at 600 μl/100 g body weight.

(4)含有小麥發酵萃取物的飼料製作：將購得之飼料以0、5、10、20 mg/kg飼料比率混合於實施例2所製造出的小麥發酵萃取物中而製成。(4) Feed preparation containing wheat fermentation extract: The purchased feed was prepared by mixing the feed of 0, 5, 10, 20 mg/kg in the wheat fermentation extract produced in Example 2.

(5)給餌方法：以體重的1%重量之各飼料進行1日1次的給餌。將此換算成小麥發酵萃取物量時分別相當於0、50、100、200μg/kg體重/日。(5) Method of feeding the bait: feeding the bait once a day for each feed having a weight of 1% by weight. This is equivalent to 0, 50, 100, 200 μg/kg body weight/day, respectively, when converted to the amount of wheat fermentation extract.

(6)實驗：將一星期的含有小麥發酵萃取物之飼料的給餌後，使其感染病毒，再經10天之含有小麥發酵萃取物之飼料的餵食。觀察病毒感染操作後10天間鯉魚的生存率。其結果如圖1所示。(6) Experiment: After one week of feeding the feed containing the wheat fermentation extract, the virus was infested, and the feed containing the wheat fermentation extract was fed for 10 days. The survival rate of squid was observed 10 days after the virus infection operation. The result is shown in Figure 1.

以無添加小麥發酵萃取物餵食之鯉魚於第6天全死亡。另一方面以含有小麥發酵萃取物的飼料餵食的試驗組，於感染第10天，生存率皆有顯著增加(Kaplan-Meier法、Logrank試驗下危險率為0.01%以下)。特別為以100μg/kg體重/日進行餵食的群有65%的生存率。The carp fed without the added wheat fermentation extract died on the sixth day. On the other hand, in the test group fed with the feed containing wheat fermentation extract, the survival rate was significantly increased on the 10th day of infection (the Kaplan-Meier method, the risk rate under the Logrank test was 0.01% or less). In particular, the group fed at 100 μg/kg body weight/day had a 65% survival rate.

C.　放入小麥發酵萃取物之手部乳液的製造C. Manufacture of hand lotion in wheat fermented extract

如表11所記載的脂溶性基材1的軟膏中加入1成左右之實施例2所製造的小麥發酵萃取物後混合成為軟膏。As the ointment of the fat-soluble base material 1 described in Table 11, about 10% of the wheat fermentation extract produced in Example 2 was added and mixed to form an ointment.

實施例8Example 8 放入小麥發酵萃取物之保濕霜的製造Manufacture of moisturizing cream with wheat fermented extract 1.　放有小麥發酵萃取物之保濕霜處方1. Formulated with moisturizing cream for wheat fermented extract

使用成分如表12所示。A組於70℃下加熱溶解，於此加入以1/4量的純水溶解並於70℃下加熱溶解的B組、及以1/4量的純水溶解並於70℃下加熱溶解的C組，經均質充分混合後冷卻至40℃，加入D組後將pH調整治6.8後，加入剩餘的純水與以實施例2製造的適量小麥發酵萃取物，充分混合後得到乳液。且小麥發酵萃取物預先溶解於純水中至5 mg/ml，對於100g的乳液添加0.1 ml。The ingredients used are shown in Table 12. The group A was dissolved by heating at 70 ° C, and the group B dissolved in 1/4 amount of pure water was heated and dissolved at 70 ° C, dissolved in 1/4 amount of pure water, and dissolved at 70 ° C by heating. Group C, after homogenization and thorough mixing, cooled to 40 ° C. After adding the D group and adjusting the pH to 6.8, the remaining pure water and the appropriate amount of the wheat fermentation extract prepared in Example 2 were added and thoroughly mixed to obtain an emulsion. And the wheat fermentation extract was previously dissolved in pure water to 5 mg/ml, and 0.1 ml was added to 100 g of the emulsion.

2.　放有小麥發酵萃取物之保濕霜的效果2. The effect of moisturizing cream with wheat fermentation extract

本乳霜讓男女43人使用後作問卷調查。其結果對於保濕效果確實有效果的答案者為18名，稍有效果的答案者為18名，毫無效果的答案者僅為2人，無回答者為5名(一標本符號檢定：P＜0.0001)。對於乾燥肌膚的改善效果，確實有效果的答案者為6名，稍有效果的答案者為13名，無效果的答案者為0名，無回答者為24名(一標本符號檢定：P＜0.0001)。對於使用後肌膚症狀惡化狀況認為有的為0名。又，讓4名輕度擬異位性症狀者使用本乳霜後進行問卷調查，對於異位性皮膚炎的改善，認為確實有效果的答案者為3名，稍有效果的答案者為1名(一標本符號檢定：P＜0.125)。其他認為對於青春痘的治療有提早痊癒的回答者為1名。又，本乳霜讓9名男性於刮鬍子後使用並進行問卷調查的結果，8名認為刮鬍子後的疼痛感減輕、具有粗糙感防止、刮傷提早痊癒等效果(一標本符號檢定：P＜0.01)。且對於2名有五十肩症狀的患者塗佈於肩膀上，嘗試疼動減輕效果時，認為具有效果者為1名。This cream was used by a survey of 43 men and women. The result is 18 for the moisturizing effect, 18 for the less effective answer, 2 for the unresponsive answer, and 5 for the unanswered one (a standard symbol check: P< 0.0001). For the improvement effect of dry skin, there are 6 respondents who have an effective effect, 13 who have a slightly effective answer, 0 who have no effect answer, and 24 who have no answer (a specimen symbol check: P< 0.0001). It is considered to be 0 in the case of deterioration of skin condition after use. In addition, four people with mild ectopic symptoms were investigated with this cream, and for the improvement of atopic dermatitis, the number of respondents who thought it was effective was 3, and the answer with a slight effect was 1 Name (a specimen symbol check: P <0.125). Other respondents who believe that the treatment of acne has early recovery is one. In addition, this cream allowed 9 men to use after shaving and conducted a questionnaire survey. Eight people thought that the pain after shaving was reduced, the effect of roughness was prevented, and the scratch was cured early (a specimen symbol check: P) <0.01). In addition, when two patients with symptoms of fifty shoulders were applied to the shoulders and tried to reduce the pain, the effect was considered to be one.

且，該乳霜使用於火傷患者時，對於兩手皮膚皆相同程度的火傷患者，單手以實施例2所製造的含有小麥發酵萃取物之乳霜塗抹，另一手則以未含小麥發酵萃取物的乳霜塗抹後，含有小麥發酵萃取物之乳霜塗抹的手明顯有提早復原的現象。將此對於10名的火傷患者進行塗抹時，任一人塗膜含有小麥發酵萃取物之乳霜的火傷處皆有明顯的提早復原之現象(Fisher直接概率法檢驗：P＜0.0001)。由上述得知本小麥發酵萃取物對於火傷具有治療效果。Moreover, when the cream is used in a fire-injured patient, the fire-injured patient with the same degree of skin on both hands is applied by one hand with the cream containing the wheat fermentation extract prepared in Example 2, and the other hand is the wheat-free fermentation extract. After the application of the cream, the hand coated with the cream of the wheat fermented extract obviously has early recovery. When this was applied to 10 fire-injured patients, there was a significant early recovery of the fire wounds of the cream containing the wheat fermentation extract of any one of the films (Fisher direct probabilistic test: P < 0.0001). From the above, it is known that the wheat fermentation extract has a therapeutic effect on fire injury.

實施例9Example 9 放入小麥發酵萃取物之化妝水的製造Manufacture of lotion for wheat fermented extract 1.　放入小麥發酵萃取物之化妝水處方1. Put a lotion prescription for wheat fermented extract

使用成分如表13所示。小麥發酵萃取物為，5 mg/ml的濃度預先溶解於純水的實施例2所製造的小麥發酵萃取物溶液，對於100 g的化妝水而言添加0.1 ml。The ingredients used are shown in Table 13. The wheat fermentation extract was a wheat fermentation extract solution prepared in Example 2 in which a concentration of 5 mg/ml was previously dissolved in pure water, and 0.1 ml was added for 100 g of the lotion.

2.　放入小麥發酵萃取物之化妝水的效果2. Effect of putting on the lotion of wheat fermentation extract

讓5名女性使用本化妝水後進行問卷調查。其結果對於保濕認為良好者為5名，認為普通為2名。皆無發生肌膚不適者。Five women were asked to use the lotion to conduct a questionnaire survey. As a result, it was considered to be 5 in the case of moisturizing, and it was considered to be 2 in general. No skin discomfort occurred.

實施例10Example 10 放有小麥發酵萃取物的沐浴劑之製造Manufacture of body wash containing wheat fermentation extract

以活體功能改善為目的添加小麥發酵萃取物製作成沐浴劑。沐浴劑的基本成分如表14所示。A wheat fermentation extract is added for the purpose of improving the function of the living body to prepare a body wash. The basic components of the body wash are shown in Table 14.

作為放入小麥發酵萃取物之沐浴劑，係為添加實施例2所製造出的110μg小麥發酵萃取物所製造者。讓102人被驗者於隱蔽下使用放有萃取物者與未放萃取物之對照組，進行使用於洗澡時的一般浴缸(160～200公升)中時的問卷調查((1)身體保溫程度、(2)水不易冷卻的程度、(3)疲勞恢復效果、(4)易入睡程度、(5)肩膀酸痛減輕程度、(6)肌肉疼痛治療效果、(7)神經痛治療效果、(8)腰痛治療效果、(9)虛寒體質治療效果、(10)香港腳改善效果、(11)肌膚粗糙改善效果、(12)異位皮膚炎治療效果)。其結果，與對照組相比發現有7%以上改善的有(1)身體保溫程度(10%)、(2)水不易冷卻的程度(7.9%)、(6)肌肉疼痛治療效果(13%)、(8)腰痛治療效果(16%)、(9)虛寒體質治療效果(10%)、(11)肌膚粗糙改善效果(7.3%)等(Mantel-Haenszel檢定：P＜0.04)。由上述結果得知，將小麥發酵萃取物作為沐浴劑使用時，對於疼痛的緩和效果與身體保溫確實有改善。The bathing agent in which the wheat fermentation extract was placed was prepared by adding 110 μg of the wheat fermentation extract produced in Example 2. A questionnaire survey was conducted in a general bathtub (160 to 200 liters) used for bathing in a concealed control group with a mixture of extracts and unexposed extracts (1) (2) the degree of difficulty in cooling the water, (3) the effect of fatigue recovery, (4) the degree of easy sleep, (5) the degree of shoulder pain reduction, (6) the treatment effect of muscle pain, (7) the therapeutic effect of neuralgia, (8) ) Low back pain treatment effect, (9) Debilitating physique treatment effect, (10) Hong Kong foot improvement effect, (11) Skin roughness improvement effect, (12) Heterotopic dermatitis treatment effect). As a result, compared with the control group, it was found that there were more than 7% improvement (1) degree of body heat preservation (10%), (2) degree of difficulty in cooling water (7.9%), and (6) treatment effect of muscle pain (13%). ), (8) low back pain treatment effect (16%), (9) debilitating physique treatment effect (10%), (11) skin roughness improvement effect (7.3%), etc. (Mantel-Haenszel test: P < 0.04). From the above results, it was found that when the wheat fermentation extract was used as a body wash, the pain-relieving effect and the body heat retention were indeed improved.

實施例11Example 11 放入小麥發酵萃取物之糖果的製造Manufacture of candy into wheat fermented extract

(1)於原料的細砂糖、水飴、水中以5：5：5：1的比率添加實施例2所製造的小麥發酵萃取物並混合、加熱煮沸至120～160℃。(1) The wheat fermentation extract prepared in Example 2 was added in a ratio of 5:5:5:1 in fine sugar, leeches, and water of the raw material, and mixed, and heated and boiled to 120 to 160 °C.

(2)由1所得者於冷卻用鐵板上冷卻後，以棒狀延伸再成型為1g左右的粒狀糖。(2) The obtained one is cooled on a cooling iron plate, and then stretched in a rod shape to form about 1 g of granular sugar.

將適量的本粒狀糖放入20 ml的水中，使其加熱溶解。作為該溶液中的小麥發酵萃取物有效成分，測定脂多醣量為4.6μg/g。將該糖給因感冒而有喉嚨痛的男女患者6名攝取。其後直接對於喉嚨疼痛的改善作問卷調查。對於喉嚨痛的改善有6名覺得有減輕(一標本符號檢定：P＜0.03)。Place an appropriate amount of this granulated sugar in 20 ml of water and dissolve it by heating. As the active ingredient of the wheat fermentation extract in the solution, the amount of lipopolysaccharide was determined to be 4.6 μg/g. The sugar was given to 6 male and female patients who had a sore throat due to a cold. A questionnaire survey was then conducted directly on the improvement of sore throat. For the improvement of sore throat, 6 people felt that there was a reduction (a specimen symbol check: P < 0.03).

實施例12Example 12 放入小麥發酵萃取物的醇類分解功能增強食品之製造Alcohol decomposition function of wheat fermentation extract enhances food manufacturing

現今作為醇類分解功能增強食品之購得製品中混合實施例2所製造的小麥發酵萃取物，對於新效果之咽喉痛緩和是否有效作檢討。In the case of the commercially available product of the alcohol decomposition-enhancing food-enhancing food, the wheat-fermented extract produced in the second embodiment was examined for the effectiveness of the new effect of throat soreness.

購得商品：商標「飲酒息」Purchased goods: trademark "drinking interest"

成分如表15所示The composition is shown in Table 15.

現今的「飲酒息」中含有七葉膽(Gynostemma pentaphyllum)萃取物及綠茶萃取物，但對於作為植物萃取物的有效成分之一的脂多醣僅為1包中0.002μg程度。因此添加適量的脂多醣含有量較高的小麥發酵萃取物時，可期待獲得新功能。小麥發酵萃取物的有效成分之一的脂多醣之1包2g中添加1～30μg為佳(作為小麥發酵萃取物為5～150μg)，故製造出1包中含有50μg的小麥發酵萃取物之製品。飲酒息的製造步驟中每100 g的製品中，添加2.5 mg的實施例2所製造的小麥發酵萃取物。其結果，製造出每2g的製品中含有50μg的小麥發酵萃取物之新製品。The current "drinking" contains Gynostemma pentaphyllum extract and green tea extract, but the lipopolysaccharide which is one of the active ingredients of the plant extract is only about 0.002 μg in one pack. Therefore, when an appropriate amount of wheat fermentation extract having a high content of lipopolysaccharide is added, a new function can be expected. It is preferable to add 1 to 30 μg of 1 g of lipopolysaccharide, which is one of the active ingredients of the wheat fermentation extract, (5 to 150 μg as a wheat fermentation extract), thereby producing a product containing 50 μg of wheat fermentation extract in one package. . In the manufacturing step of drinking alcohol, 2.5 mg of the wheat fermentation extract prepared in Example 2 was added per 100 g of the product. As a result, a new product containing 50 μg of the wheat fermentation extract per 2 g of the product was produced.

以邊喝酒邊唱卡拉OK時會有咽喉痛的成人男女20名作為對象，各10人服用原先的「飲酒息」，另10名服用含有小麥發酵萃取物的「飲酒息」。對於公知效果的醇類分解能強化效果、及咽喉痛緩和效果作檢討。其後直接對於咽喉疼痛緩和效果作問卷調查。其結果，認為「放有小麥發酵萃取物之飲酒息」對於咽喉痛有減輕效果者為10名中8名，對於「飲酒息」的10名中2名認為有減輕效果作比較，於統計上有顯著差(Fisher直接概率法檢驗：P＜0.012)。In order to drink karaoke, there are 20 adults and men with sore throat, and each of them takes the original "drinking interest", and the other 10 take "drinking interest" containing wheat fermented extract. The effect of alcohol decomposition and the effect of sore throat on the well-known effect are reviewed. Thereafter, a questionnaire survey was conducted directly on the effect of sore throat relief. As a result, it is considered that the "drinking of the fermented wheat extract" has a reduction effect on the sore throat, and 8 of the 10 people have a reduction effect on the "drinking interest rate". There was a significant difference (Fisher direct probability test: P < 0.012).

E.　小麥發酵萃取物的藥效實施例E. Pharmacodynamic examples of wheat fermentation extracts 實施例13Example 13 放入小麥發酵萃取物的甘油溶液之製造(對於異位性皮膚炎的治療效果)Manufacture of glycerin solution for wheat fermentation extract (therapeutic effect on atopic dermatitis)

對於臉部、手腳、身體、脖子、手腕、背部等觀察到皮疹、自覺症狀為中等程度至重度症狀之難治性異位性皮膚炎患者男女9名(25歲至34歲)，將含有實施例2所製造的小麥發酵萃取物50μg/ml的50%甘油溶液以1天2～3次，1次2～3 ml的程度讓患者服用。對有自覺狀態(搔癢感)的患者分類成輕度、中度、重度。使用2星期至2個月後再度受診，評估效果。結果為顯著有效者(皮疹的顯著改善與自覺狀態幾乎消失)為4名(44%)，有效(皮疹的輕度改善與自覺狀態的減少)為4名(44%)，不變為1名(11%)，惡化為0名(一標本符號檢定：P＜0.03)，由上述得知有效率為89%。For the face, hands, feet, body, neck, wrist, back and other rashes, consciously symptomatic moderate to severe symptoms of refractory atopic dermatitis, 9 males and females (25 to 34 years old), will contain examples The 50 mg/ml 50% glycerin solution of the wheat fermentation extract produced by the two was administered to the patient 2 to 3 times a day, and 2 to 3 ml once. Patients with a conscious state (itching sensation) were classified as mild, moderate, and severe. The patient was re-examined after 2 weeks to 2 months to evaluate the effect. The results were significantly more effective (significant improvement in rash and almost disappeared from the conscious state) of 4 (44%), effective (light improvement in rash and reduction in conscious state) of 4 (44%), unchanged to 1 (11%), the deterioration to 0 (one specimen symbol test: P < 0.03), from the above, the effective rate was 89%.

實施例14Example 14 小麥發酵萃取物的止痛作用Analgesic effect of wheat fermentation extract

將實施例2所製得的小麥發酵萃取物溶解於蒸餾水中，對每匹老鼠使用探針器，進行0.2 ml的經口投予。經90分鐘後以腹腔內投予0.7%的乙酸，經5分鐘後觀察其情況，測定30分鐘後引起身體抽動的數目。結果表示表6中可阻斷蒸餾水投予對照組織身體抽動數30%之各試料必要量。The wheat fermentation extract obtained in Example 2 was dissolved in distilled water, and a probe was used for each mouse to carry out oral administration of 0.2 ml. After 90 minutes, 0.7% acetic acid was intraperitoneally administered, and after 5 minutes, the condition was observed, and the number of body twitches caused after 30 minutes was measured. The results indicate that the necessary amount of each sample in which the distilled water was administered to the control tissue 30% of the body twitch was blocked in Table 6.

來自大腸的低分子量脂多醣之有效成分作為1時小麥發酵萃取物為7，顯示小麥發酵萃取物具有優良的止痛作用。The active ingredient of the low molecular weight lipopolysaccharide from the large intestine was 7 as the wheat fermentation extract at 1 hour, indicating that the wheat fermentation extract had an excellent analgesic effect.

實施例15Example 15 小麥發酵萃取物的異位性皮膚炎抑制效果Inhibition effect of atopic dermatitis on wheat fermentation extract

欲對於小麥發酵萃取物的異位性皮膚炎之效果作調查，導入I型過敏模型。對於一群3～4匹的雄性BALB/c老鼠進行1μg/老鼠之抗硝基苯、老鼠單株抗體的靜脈投予。一小時後將實施例2所製得之小麥發酵萃取物(4μg/老鼠)進行腹部皮內投予或經口投予(100μg/老鼠)，再經1小時後，魚老鼠耳殼表面上塗佈20μl含有0.25%的二硝基氟苯之丙酮-橄欖油混合溶液(4：1)作為抗原。塗佈後於第1、2、24及48小時以厚度量測器測定耳殼厚度，與塗佈前的厚度之差(△)作為浮腫程度。藥劑投予效果為抗原投予1小時後被確認的早期反應、與24小時後被誘導出的遲發反應，各抑制情況由以下式子求得抑制率並評估。{抑制率=(1-藥劑投予後的△耳殼之浮腫/對照的△耳殼之浮腫)×100}結果如表17所示。由表得知，小麥發酵萃取物於皮內投予、或經口投予皆可抑制過敏反應。To investigate the effect of atopic dermatitis on wheat fermentation extracts, a type I allergy model was introduced. For a group of 3 to 4 male BALB/c mice, 1 μg/mouse of anti-nitrobenzene and mouse monoclonal antibodies were administered intravenously. One hour later, the wheat fermentation extract (4 μg/mouse) prepared in Example 2 was intradermally administered intradermally or orally (100 μg/mouse), and after 1 hour, the surface of the ear shell of the fish mouse was coated. 20 μl of cloth containing 0.25% dinitrofluorobenzene in acetone-olive oil mixed solution (4:1) as an antigen. The thickness of the ear shell was measured by a thickness gauge at 1, 2, 24, and 48 hours after coating, and the difference (Δ) from the thickness before coating was used as the degree of edema. The drug administration effect was an early reaction confirmed after the antigen was administered for 1 hour, and a delayed reaction which was induced 24 hours later, and the inhibition rate was determined by the following formula for each inhibition. {Inhibition rate = (1 - edema of △ ear shell after administration of drug / edema of △ ear shell of control) × 100} The results are shown in Table 17. It is known from the table that the wheat fermentation extract can inhibit allergic reactions by intradermal administration or oral administration.

實施例16Example 16 小麥發酵萃取物的感染預防效果Infection prevention effect of wheat fermentation extract

欲對小麥發酵萃取物之感染預防效果作調查，導入耐甲氧西林金黃色葡萄球菌(MRSA)感染模型。對於一群10匹雄性BALB/c老鼠(6～8週齡)以200 mg/kg的環磷酵胺(CY)進行腹腔內投予，經5天後進行實施例2所製得之小麥發酵萃取物之皮內投予。3小時後進行MRSA(3×107 菌落形成單位(CFU))的靜脈內投予，調查其生存日數。結果如表18所示。由表得知小麥發酵萃取物與生理食鹽水(對照組)群相比，於統計學上具有顯著差(χ2 檢定，P＜0.001)，顯示對於MRSA具有感染預防效果。In order to investigate the infection prevention effect of wheat fermentation extract, a model of methicillin-resistant Staphylococcus aureus (MRSA) infection was introduced. For a group of 10 male BALB/c mice (6-8 weeks old), 200 mg/kg cyclophosphamide (CY) was intraperitoneally administered, and after 5 days, the wheat fermentation extraction obtained in Example 2 was carried out. Intradermal administration of the substance. After 3 hours, MRSA (3 × 10 7 colony forming units (CFU)) was administered intravenously, and the number of days of survival was examined. The results are shown in Table 18. It was found from the table that the wheat fermentation extract was statistically significantly inferior to the physiological saline (control group) group (χ 2 assay, P < 0.001), indicating an infection prevention effect on MRSA.

實施例17Example 17 小麥發酵萃取物的癌細胞轉移治療效果Cancer metastasis treatment effect of wheat fermentation extract

欲調查小麥發酵萃取物之癌細胞轉移治療效果，導入MethA癌細胞之肺轉移模型。對於一群10匹雄性BALB/c老鼠(6～8週齡)進行1×105 細胞之MethA細胞的靜脈內投予，12天後4天連續，進行實施例2所製造的小麥發酵萃取物之皮內投予。細胞移植20天後剖開檢查，摘出肺，進行福馬林固定。肺由肉眼觀察，測定結節數。結果如表19所示。由表得知，小麥發酵萃取物與生理食鹽水(對照組)相比，於統計學上具有顯著差(t-檢定，P＜0.001)，顯示對於MethA之肺癌細胞轉移具有治療效果。To investigate the effect of cancer cell transfer treatment of wheat fermentation extracts, a lung metastasis model of MethA cancer cells was introduced. For a group of 10 male BALB/c mice (6-8 weeks old), 1×10 5 cells of MethA cells were administered intravenously, and 12 days later, 4 days in a row, the wheat fermentation extract prepared in Example 2 was subjected to Intradermal administration. After 20 days of cell transplantation, the rats were dissected and the lungs were removed for formalin fixation. The lungs were observed by the naked eye and the number of nodules was determined. The results are shown in Table 19. It is known from the table that the wheat fermentation extract has a statistically significant difference (t-test, P < 0.001) compared with physiological saline (control group), indicating a therapeutic effect on metastasis of lung cancer cells of MethA.

F.　***發酵萃取物之相關實施例F. Related examples of bean curd fermentation extract 實施例18Example 18 ***發酵萃取物的製造Manufacture of bean curd fermentation extract

(1)2公升的三角錐形瓶中放入1.0 L的水、與0.2 g的磷酸第一鉀、1.15 g的磷酸第二鉀、8 g的食鹽、0.2 g的氯化鉀。(1) In a 2 liter triangular conical flask, 1.0 L of water, 0.2 g of potassium phosphate, 1.15 g of potassium phosphate, 8 g of salt, and 0.2 g of potassium chloride were placed.

(2)於1中加入20g的乾燥***。(2) 20 g of dried bean curd residue was added to 1.

(3)將2以高壓滅菌釜滅菌。(3) Sterilize 2 in an autoclave.

(4)種菌的調製。如前組成所調製的2%***培養基5 ml中放入由小麥粉分離出的稻穀病原細菌之1個菌落，於37℃下緩緩攪拌1晚(15小時)使其發酵，完成***用種菌的準備。(4) Modulation of inoculum. One colony of rice pathogenic bacteria isolated from wheat flour was placed in 5 ml of the 2% bean curd medium prepared in the previous composition, and slowly stirred at 37 ° C for 1 night (15 hours) to ferment to complete the bean curd residue. Prepare with inoculum.

(5)於3加入4全量，於37℃下緩緩攪拌下發酵48小時。(5) 4 whole amount was added to 3, and fermentation was carried out for 48 hours while stirring slowly at 37 °C.

(6)5的***發酵溶液以高壓滅菌釜進行120℃20分鐘的加熱萃取。將此進行離心分離(窪田8800，2000 rpm，10分鐘)，回收澄清液，作為***發酵萃取物。The tofu residue fermentation solution of (6) 5 was subjected to heating extraction at 120 ° C for 20 minutes in an autoclave. This was centrifuged (Putian 8800, 2000 rpm, 10 minutes), and the clear liquid was recovered as a fermentation extract of bean curd residue.

(7)乾燥重量的測定：將預先稱取0.3 ml後移至1.5 ml的塑膠試管，冷凍後，以冷凍乾燥機進行冷凍乾燥時為5.97 mg。因此，6的***發酵萃取物之乾燥重量為每1 ml的溶液中為19.9 mg，全量為1000 ml時為19.9 g。(7) Measurement of dry weight: 0.3 ml was preliminarily weighed and transferred to a 1.5 ml plastic test tube, and after freezing, it was 5.97 mg when freeze-dried by a freeze dryer. Therefore, the dry weight of the fermented extract of the bean curd residue of 6 was 19.9 mg per 1 ml of the solution, and the total amount was 19.9 g when it was 1000 ml.

(8)以Bradford蛋白濃度測定法，將蛋白質定量BSA作為標準蛋白質，測定10倍稀釋之樣品的蛋白質質量。結果如表15。(8) Protein quality of a 10-fold diluted sample was determined by Bradford protein concentration assay using protein quantification BSA as a standard protein. The results are shown in Table 15.

(9)核酸含有量測定：將經100倍稀釋的樣品進行210～340 nm的吸光度測定。減去260 nm吸光度至320 nm的吸光度所得值、與DNA的吸光度1OD算出50μg的最大含有量。(9) Measurement of nucleic acid content: The 100-fold diluted sample was subjected to absorbance measurement at 210 to 340 nm. The maximum absorbance of 50 μg was calculated by subtracting the absorbance at 260 nm absorbance to 320 nm and the absorbance at DNA 1OD.

(10)糖含量的測定：藉由酚硫酸法將葡萄糖作為標準精進行測定。(10) Measurement of sugar content: Glucose was measured by phenol sulfuric acid method as a standard fine.

(11)藉由limulus測定之limulus活性物質含有量的測定：limulus活性物質量為使用生化學工業的toxycolor system，作為標準limulus活性物質使用生化學工業Et-1。(11) Determination of the content of the limulus active substance measured by limulus: the mass of the limulus active substance is a tooxycolor system using the biochemical industry, and the biochemical industry Et-1 is used as a standard limulus active substance.

實施例19Example 19 ***萃取物之免疫賦活作用Immunostimulating effect of bean curd extract

於48格培養皿中放入作為人類巨噬細胞使用的急性骨髓性白血病細胞株之THP-1(1×106 個/250μl：放有10%牛胚胎血清之RPMI 1640培養基)，進行30分鐘的預培養。使各樣品的最終濃度成為100～10000 ng/ml加入250μl(最終量500μl)。調製出樣品中含有多黏菌素B(12.5μg/ml)之群(成為僅含100 ng/ml的多黏菌素B的群)。經4小時培養後，回收培養澄清液及細胞。澄清液的TNF活性使用L-929細胞障礙試驗進行測定。結果如表21所示。***發酵萃取物於多黏菌素B存在下亦可由巨噬細胞中產生TNF，但低分子量脂多醣及limulus陽性糖脂質於多黏菌素B存在下並不會由巨噬細胞中產生TNF。因此可得知***發酵萃取物與低分子量脂多醣或limulus陽性糖脂質相比具有相異的生物活性。THP-1 (1×10 6 cells/250 μl: RPMI 1640 medium containing 10% bovine embryo serum) was used as a human myeloid leukemia cell line in a 48-well culture dish for 30 minutes. Pre-culture. The final concentration of each sample was added to 250 μl (final amount 500 μl) at 100 to 10000 ng/ml. A group containing polymyxin B (12.5 μg/ml) in the sample (to be a group containing only 100 ng/ml of polymyxin B) was prepared. After 4 hours of culture, the culture supernatant and cells were recovered. The TNF activity of the clarified liquid was determined using the L-929 cell disorder test. The results are shown in Table 21. The fermentation extract of bean curd can also produce TNF from macrophages in the presence of polymyxin B, but low molecular weight lipopolysaccharide and limulus positive glycolipid do not produce TNF from macrophages in the presence of polymyxin B. . Therefore, it can be known that the bean curd fermentation extract has a different biological activity than the low molecular weight lipopolysaccharide or the limulus positive glycolipid.

N.D.：未測定N.D.: not determined

G.　米粉發酵萃取物之相關實施例G. Related examples of rice flour fermentation extract 米粉發酵萃取物的製造Manufacture of rice flour fermentation extract 實施例20Example 20

(1)2公升的三角錐形瓶中放入1.0 L的水、與0.2 g的磷酸第一鉀、1.15 g的磷酸第二鉀、8 g的食鹽、0.2 g的氯化鉀。(1) In a 2 liter triangular conical flask, 1.0 L of water, 0.2 g of potassium phosphate, 1.15 g of potassium phosphate, 8 g of salt, and 0.2 g of potassium chloride were placed.

(2)於1中加入20 g的乾燥米粉。(2) Add 20 g of dry rice flour to 1.

(3)將2以高壓滅菌釜滅菌。(3) Sterilize 2 in an autoclave.

(4)種菌的調製。如前組成所調製的2%乾燥後培養基5 ml中放入由小麥粉分離出的稻穀病原細菌之1個菌落，於37℃下緩緩攪拌1晚(15小時)使其發酵，完成米粉用種菌的準備。(4) Modulation of inoculum. One colony of rice pathogenic bacteria isolated from wheat flour was placed in 5 ml of the 2% dried medium prepared in the previous composition, and slowly stirred at 37 ° C for 1 night (15 hours) to ferment the rice flour. Preparation of inoculum.

(5)於3加入4全量，於37℃下緩緩攪拌下發酵72小時。(5) 4 whole amount was added to 3, and fermentation was carried out for 72 hours with gentle stirring at 37 °C.

(6)5的米粉發酵溶液以高壓滅菌釜進行120℃ 20分鐘的加熱萃取。將此進行離心分離(窪田8800，2000 rpm，10分鐘)，回收澄清液，作為米粉發酵萃取物。The rice flour fermentation solution of (6) 5 was subjected to heating extraction at 120 ° C for 20 minutes in an autoclave. This was centrifuged (Putian 8800, 2000 rpm, 10 minutes), and the clear liquid was recovered as a rice flour fermentation extract.

(7)藉由limulus測定之limulus活性物質含有量的測定：limulus活性物質量為使用生化學工業的toxycolor system，作為標準limulus活性物質使用生化學工業Et-1。測定米粉發酵萃取物中的limulus活性物質含有量為1.7μl/mg。(7) Determination of the content of the limulus active substance measured by limulus: the mass of the limulus active substance is a tooxycolor system using the biochemical industry, and the biochemical industry Et-1 is used as a standard limulus active substance. The content of the limulus active substance in the rice flour fermentation extract was determined to be 1.7 μl/mg.

實施例21Example 21 米粉萃取物之免疫賦活作用Immune activation of rice flour extract

於48格培養皿中放入作為人類巨噬細胞使用的急性骨髓性白血病細胞株之THP-1(1×106 個/250μl：放有10%牛胚胎血清之RPMI 1640培養基)，進行30分鐘的預培養。使各樣品的最終濃度成為1～10000 ng/ml加入250μl(最終量500μl)。調製出樣品中含有多黏菌素B(12.5μg/ml)之群。經4小時培養後，回收培養澄清液及細胞。澄清液的TNF活性使用L-929細胞障礙試驗進行測定。結果如表22所示。米粉發酵萃取物於多黏菌素B存在下亦可由巨噬細胞中產生TNF，但低分子量脂多醣及limulus陽性糖脂質於多黏菌素B存在下並不會由巨噬細胞中產生TNF。因此可得知米粉發酵萃取物與低分子量脂多醣或limulus陽性糖脂質相比具有相異的生物活性。THP-1 (1×10 6 cells/250 μl: RPMI 1640 medium containing 10% bovine embryo serum) was used as a human myeloid leukemia cell line in a 48-well culture dish for 30 minutes. Pre-culture. The final concentration of each sample was added to 250 μl (final amount 500 μl) at 1 to 10000 ng/ml. A group containing polymyxin B (12.5 μg/ml) in the sample was prepared. After 4 hours of culture, the culture supernatant and cells were recovered. The TNF activity of the clarified liquid was determined using the L-929 cell disorder test. The results are shown in Table 22. Rice flour fermentation extract can also produce TNF from macrophages in the presence of polymyxin B, but low molecular weight lipopolysaccharide and limulus positive glycolipid do not produce TNF from macrophages in the presence of polymyxin B. Therefore, it can be known that the rice flour fermentation extract has a different biological activity than the low molecular weight lipopolysaccharide or the limulus positive glycolipid.

H.　海帶芽發酵萃取物之相關實施例H. Related examples of kelp bud fermentation extract 實施例22Example 22 海帶芽和布蕪發酵的製造Manufacture of kelp buds and fabric fermentation

(1)2公升的三角錐形瓶中放入1.0 L的水、與0.2 g的磷酸第一鉀、1.15 g的磷酸第二鉀、8 g的食鹽、0.2 g的氯化鉀。(1) In a 2 liter triangular conical flask, 1.0 L of water, 0.2 g of potassium phosphate, 1.15 g of potassium phosphate, 8 g of salt, and 0.2 g of potassium chloride were placed.

(2)於1中加入20 g的海帶芽和布蕪米粉。(2) Add 20 g of kelp buds and cloth glutinous rice flour to 1.

(3)將2以高壓滅菌釜滅菌。(3) Sterilize 2 in an autoclave.

(4)種菌的調製。如前組成所調製的2%乾燥後培養基5 ml中放入由海帶芽和布蕪分離出的稻穀病原細菌之1個菌落，於37℃下緩緩攪拌1晚(15小時)使其發酵，完成海帶芽和布蕪用種菌的準備。(4) Modulation of inoculum. One colony of rice pathogenic bacteria isolated from kelp buds and cloth buds was placed in 5 ml of the 2% dried medium prepared in the previous composition, and slowly stirred at 37 ° C for 1 night (15 hours) to ferment. Preparation of kelp buds and fabrics for inoculum.

(5)於3加入4全量，於37℃下緩緩攪拌下發酵72小時。(5) 4 whole amount was added to 3, and fermentation was carried out for 72 hours with gentle stirring at 37 °C.

(6)5的海帶芽和布蕪發酵溶液以高壓滅菌釜進行120℃ 20分鐘的加熱萃取。將此進行離心分離(窪田8800，2000 rpm，10分鐘)，回收澄清液，作為海帶芽和布蕪發酵萃取物。The kelp bud and cloth fermentation solution of (6) 5 was heated and extracted in an autoclave at 120 ° C for 20 minutes. This was centrifuged (Putian 8800, 2000 rpm, 10 minutes), and the clear liquid was recovered as a fermentation extract of kelp buds and cloth.

(7)藉由limulus測定之limulus活性物質含有量的測定：limulus活性物質量為使用生化學工業的toxycolor system，作為標準limulus活性物質使用生化學工業Et-1。測定海帶芽和布蕪發酵萃取物中的limulus活性物質含有量為132μl/mg。(7) Determination of the content of the limulus active substance measured by limulus: the mass of the limulus active substance is a tooxycolor system using the biochemical industry, and the biochemical industry Et-1 is used as a standard limulus active substance. The content of the limulus active substance in the kelp bud and the fabric extract was determined to be 132 μl/mg.

實施例23Example 23 海帶芽和布蕪萃取物之免疫賦活作用Immune activation of kelp buds and fabric extracts

於48格培養皿中放入作為人類巨噬細胞使用的急性骨髓性白血病細胞株之THP-1(1×106 個/250μl：放有10%牛胚胎血清之RPMI 1640培養基)，進行30分鐘的預培養。使各樣品的最終濃度成為1～10000 ng/ml加入250μl(最終量500μl)。調製出樣品中含有多黏菌素B(12.5μg/ml)之群。經4小時培養後，回收培養澄清液及細胞。澄清液的TNF活性使用L-929細胞障礙試驗進行測定。結果如表22所示。海帶芽和布蕪發酵萃取物於多黏菌素B存在下亦可由巨噬細胞中產生TNF，但低分子量脂多醣及limulus陽性糖脂質於多黏菌素B存在下並不會由巨噬細胞中產生TNF。因此可得知海帶芽和布蕪發酵萃取物與低分子量脂多醣或limulus陽性糖脂質相比具有相異的生物活性。THP-1 (1×10 6 cells/250 μl: RPMI 1640 medium containing 10% bovine embryo serum) was used as a human myeloid leukemia cell line in a 48-well culture dish for 30 minutes. Pre-culture. The final concentration of each sample was added to 250 μl (final amount 500 μl) at 1 to 10000 ng/ml. A group containing polymyxin B (12.5 μg/ml) in the sample was prepared. After 4 hours of culture, the culture supernatant and cells were recovered. The TNF activity of the clarified liquid was determined using the L-929 cell disorder test. The results are shown in Table 22. The kelp bud and fabric extract can also produce TNF from macrophages in the presence of polymyxin B, but low molecular weight lipopolysaccharide and limulus positive glycolipid in the presence of polymyxin B are not in macrophages. Produce TNF. Therefore, it can be known that the kelp bud and the cloth extract fermentation extract have different biological activities compared to the low molecular weight lipopolysaccharide or the limulus positive glycolipid.

產業上可利用性Industrial availability

本發明係關於可便宜地製造出安全免疫賦活物質的植物發酵萃取物，所得之植物發酵萃取物可利用於含人類的哺乳動物(具體為家畜、寵物等)、鳥類(具體為飼養雞、賞鳥類等)、兩生類、爬蟲類、魚類(具體為水產養殖魚、寵物魚類等)、無脊椎動物及醫藥品、動物用醫藥品、醫藥部外品、化妝品、食品、功能性食品、飼料及浴用劑等。The invention relates to a plant fermentation extract which can manufacture a safe immune activating substance inexpensively, and the obtained plant fermentation extract can be utilized for a mammal containing humans (specifically, livestock, pets, etc.), birds (specifically, raising chicken, rewarding Birds, etc., biogenic, reptilian, fish (specifically aquaculture fish, pet fish, etc.), invertebrates and pharmaceuticals, animal medicines, pharmaceutical products, cosmetics, food, functional foods, feed and Bathing agents, etc.

[圖1]圖1表示藉由添加小麥發酵萃取物之飼料所顯示的錦鯉疱疹發病抑制效果圖。Fig. 1 is a graph showing the effect of suppressing the incidence of koi herpes by adding a feed of wheat fermented extract.

Claims (7)

一種發酵及培養方法，其特徵為將含有來自食用植物，且主成分為多糖的糖質之原料，藉由稻穀病原細菌(Pantoea agglomerans)，在單獨下使其發酵，同時在單獨下培養該稻穀病原細菌。 A fermentation and culture method characterized in that a raw material containing a saccharide derived from a food plant and having a main component of a polysaccharide is fermented by a rice pathogenic bacterium (Pantoe agglomerans) alone, and the rice is cultivated separately under the same conditions. Pathogenic bacteria. 一種植物發酵萃取物，其特徵為將含有來自食用植物，且主成分為多糖的糖質之原料，藉由稻穀病原細菌，在單獨下使其發酵，同時在單獨下培養該稻穀病原細菌。 A plant fermentation extract characterized in that a raw material containing a saccharide derived from a food plant and having a main component of a polysaccharide is fermented by a rice pathogenic bacterium alone, and the rice pathogenic bacterium is cultured separately. 一種植物發酵萃取物粉末，其特徵係由如申請專利範圍第2項之植物發酵萃取物所得者。 A plant fermentation extract powder characterized by being obtained from a plant fermentation extract according to item 2 of the patent application. 一種植物發酵萃取物配合物，其特徵為添加如申請專利範圍第2項之植物發酵萃取物或如申請專利範圍第3項之植物發酵萃取物粉末者。 A plant fermentation extract complex characterized by adding a plant fermentation extract according to item 2 of the patent application or a plant fermentation extract powder according to claim 3 of the patent application. 如申請專利範圍第4項之植物發酵萃取物配合物，其中前述植物發酵萃取物配合物為醫藥品、動物用醫藥品、醫藥部外品、化妝品、食品、功能性食品、飼料、或浴用劑。 The plant fermentation extract complex according to claim 4, wherein the plant fermentation extract complex is a pharmaceutical, an animal medicine, a pharmaceutical product, a cosmetic, a food, a functional food, a feed, or a bathing agent. . 如申請專利範圍第2項之植物發酵萃取物，其為顯示即使在多黏菌素B存在下亦表示巨噬細胞活化能的物理化學性質者。 The plant fermentation extract of claim 2, which is a physicochemical property showing the activation energy of macrophages even in the presence of polymyxin B. 如申請專利範圍第2項之植物發酵萃取物，其為具有免疫賦活活性。 A plant fermentation extract according to claim 2 of the patent application, which has an immunostimulating activity.
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