TWI730719B - Plant fermented product, method of manufacturing the same, composition of plant fermented product and use thereof - Google Patents
Plant fermented product, method of manufacturing the same, composition of plant fermented product and use thereof Download PDFInfo
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本發明係有關於一種植物醱酵物及其製造方法、含彼之組成物暨其用途，特別是有關於一種利用多菌種乳桿菌醱酵的植物醱酵物及其製造方法、含彼之組成物暨其用途。 The present invention relates to a plant ferment and its manufacturing method, a composition containing it and its use, in particular to a plant ferment using multi-strain Lactobacillus ferment and its manufacturing method, and a composition containing it Cum its use.
高血糖是指身體無法有效利用或儲存所攝取的碳水化合物，使得血液中的葡萄糖濃度高於標準值。當有高濃度的葡萄糖長期存在血液中，可能引發糖尿病、心血管疾病、腦部萎縮、慢性腎臟病、眼睛病變、神經病變及失智等等疾病，且傷口不容易癒合，容易導致感染而惡化。 Hyperglycemia refers to the inability of the body to effectively use or store the carbohydrates it has taken, making the blood glucose concentration higher than the standard value. When a high concentration of glucose exists in the blood for a long time, it may cause diabetes, cardiovascular disease, brain atrophy, chronic kidney disease, eye disease, neuropathy, dementia and other diseases, and the wound is not easy to heal, which may easily lead to infection and worsening .
目前常用於降血糖的藥物包含雙胍(Biguanides)類、磺醯尿素(Sulfonylurea)類、美格替耐(Meglitinides)類、唑烷二酮(Thiazolidinedione，TZD)類、葡萄糖苷酶抑制劑(α-glucosidase inhibitor)類、二基胜肽酶-4抑制劑(Dipeptidyl peptidase-4 inhibitor，DPP-4 inhibitor)及鈉-葡萄糖共同運輸蛋白抑制劑(sodium glucose co-transporters 2 inhibitor，SGLT2 inhibitor)類。然而，前述類型的藥物常有副作用發生，例如腸胃不適、食慾降低、疲勞、體重減輕/增加、低血糖(頭暈、心悸、冒冷汗、暈倒)、水腫、脹氣、腹瀉、腹痛、頭痛、肌肉痛、關節痛、泌尿道感染、利尿、脫水、低血壓、血脂異常及/或低血糖。 Currently commonly used drugs for lowering blood sugar include Biguanides, Sulfonylurea, Meglitinides, Thiazolidinedione (TZD), Glucosidase inhibitors (α- glucosidase inhibitor) class, Dipeptidyl peptidase-4 inhibitor (Dipeptidyl peptidase-4 inhibitor, DPP-4 inhibitor) and sodium glucose co-transporters 2 inhibitor (SGLT2 inhibitor). However, the aforementioned types of drugs often have side effects such as gastrointestinal upset, decreased appetite, fatigue, weight loss/increase, hypoglycemia (dizziness, palpitations, cold sweats, fainting), edema, flatulence, diarrhea, abdominal pain, headache, muscles Pain, joint pain, urinary tract infection, diuresis, dehydration, hypotension, dyslipidemia and/or hypoglycemia.
其次，目前雖有使用益生菌及/或益生菌醱酵物調節血糖，然而，益生菌調節血糖的效果，根據使用益生菌菌種的不同，而有截然不同的結果。以往多菌種益生菌及/或益生菌醱酵物在調節血糖的研究，大多著重於嗜酸乳桿菌(Lactobacillus acidophilus)與其他益生菌的組合。有研究顯示，嗜酸乳桿菌與乾酪乳桿菌(Lactobacillus casei)的組合物可以降低大鼠的血糖濃度。另有研究顯示，嗜酸乳桿菌與雙叉乳酸桿菌屬(Bifidobacterium sp.)的乳製品醱酵物可改善糖尿病患者的空腹血糖濃度。然而，使用特定多菌種乳桿菌的植物醱酵物於調節血糖的效果，則少有研究。 Secondly, although probiotics and/or probiotic ferments are currently used to regulate blood sugar, the effect of probiotics in regulating blood sugar varies greatly depending on the type of probiotic used. In the past, researches on multi-strain probiotics and/or probiotic ferment in regulating blood sugar mostly focused on the combination of Lactobacillus acidophilus and other probiotics. Studies have shown that the combination of Lactobacillus acidophilus and Lactobacillus casei (Lactobacillus casei) can reduce the blood glucose concentration of rats. Other studies have shown that lactobacillus acidophilus and Bifidobacterium sp. (Bifidobacterium sp.) dairy fermented products can improve fasting blood glucose levels in diabetic patients. However, the effect of using specific multi-strain Lactobacillus plant ferment in regulating blood sugar has been seldom studied.
有鑑於此，亟須提供一種植物醱酵物及其製造方法、含彼之組成物暨其用途，以解決上述問題。 In view of this, it is urgent to provide a plant ferment and its manufacturing method, a composition containing it, and its use to solve the above-mentioned problems.
因此，本發明之一態樣係提供一種植物醱酵物的製造方法，其係將含有多菌種乳桿菌菌液加入植物基質中， 以進行醱酵步驟。 Therefore, one aspect of the present invention is to provide a method for manufacturing plant ferment, which involves adding a multi-strain Lactobacillus strain into a plant matrix, To carry out the fermentation step.
本發明之另一態樣係在提供一種植物醱酵物，其係藉由上述方法所製得，此植物醱酵物包含特定總活菌數的多菌種乳桿菌。 Another aspect of the present invention is to provide a plant ferment, which is prepared by the above method, and the plant ferment contains a specific total number of multi-strain lactobacilli.
本發明之又一態樣係在提供一種植物醱酵物的組成物，其係包含上述之植物醱酵物作為有效成分。 Another aspect of the present invention is to provide a composition of a plant ferment, which contains the above-mentioned plant ferment as an effective ingredient.
本發明之再一態樣係在提供一種植物醱酵物於製備調節血糖之組成物的用途，此組成物包含植物醱酵物，可減緩及/或預防高血糖及/或其併發症。 Another aspect of the present invention is to provide a use of a plant ferment for preparing a composition for regulating blood sugar. The composition includes a plant ferment, which can slow down and/or prevent hyperglycemia and/or its complications.
根據本發明之上述態樣，提出一種植物醱酵物的製造方法。首先，對植物漿液進行殺菁步驟，以獲得植物基質，其中殺菁步驟係以70℃至100℃進行30分鐘至60分鐘。然後，提供多菌種乳桿菌，其中多菌種乳桿菌包含胚芽乳桿菌(Lactobacillus plantarum)、乾酪乳桿菌(Lactobacillus casei)、洛德乳桿菌(Lactobacillus reuteri)、短乳桿菌(Lactobacillus brevis)及鼠李糖乳桿菌(Lactobacillus rhamnosus)。 According to the above aspects of the present invention, a method for producing plant ferment is proposed. First, a cyanolysis step is performed on the plant slurry to obtain a plant substrate, wherein the cyanolysis step is performed at 70° C. to 100° C. for 30 minutes to 60 minutes. Then, a multi-strain Lactobacillus is provided, wherein the multi-strain Lactobacillus includes Lactobacillus plantarum , Lactobacillus casei , Lactobacillus reuteri , Lactobacillus brevis and murine Lactobacillus rhamnosus (Lactobacillus rhamnosus).
上述多菌種乳桿菌寄存於台灣新竹食品路331號財團法人食品工業發展研究所生物資源中心(BCRC)，其中胚芽乳桿菌的寄存編號為BCRC 11697，乾酪乳桿菌的寄存編號為BCRC 17942，洛德乳桿菌的寄存編號為BCRC 16091，短乳桿菌的寄存編號為BCRC 11196，鼠李糖乳桿菌的寄存編號為BCRC 80663。前述多菌種乳桿菌於申請日已處於可分讓之狀態。多菌種乳桿菌的總 活菌數為至少107菌落形成單位(colony-forming unit，CFU)/毫升(mL)。 The above-mentioned multi-strain Lactobacillus is deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan. The deposit number of Lactobacillus embryos is BCRC 11697, and the deposit number of Lactobacillus casei is BCRC 17942. The deposit number of Lactobacillus deer is BCRC 16091, the deposit number of Lactobacillus brevis is BCRC 11196, and the deposit number of Lactobacillus rhamnosus is BCRC 80663. The aforementioned multi-strain Lactobacillus has been in a divisible state on the application date. Total number of viable cells of Lactobacillus strains multiple forming units (colony-forming unit, CFU) / milliliter (mL) of at least 107 colonies.
接著，將多菌種乳桿菌之菌液加入植物基質以進行醱酵步驟，藉此獲得植物醱酵物，其中醱酵步驟係於35°C至40℃進行36小時至60小時。 Then, the bacterial liquid of the multi-strain Lactobacillus is added to the plant substrate to perform the fermentation step, thereby obtaining the plant ferment, wherein the fermentation step is performed at 35°C to 40°C for 36 hours to 60 hours.
依據本發明之一實施例，上述植物漿液係由植物性材料與水經均質化步驟後所得。 According to an embodiment of the present invention, the above-mentioned plant slurry is obtained by homogenizing plant materials and water.
依據本發明之一實施例，上述胚芽乳桿菌、乾酪乳桿菌、洛德乳桿菌、短乳桿菌及鼠李糖乳桿菌的之任兩者的細胞數比為3：1至1：3。 According to an embodiment of the present invention, the cell number ratio of any two of the above-mentioned Lactobacillus embryo, Lactobacillus casei, Lactobacillus lordella, Lactobacillus brevis, and Lactobacillus rhamnosus is 3:1 to 1:3.
依據本發明之一實施例，上述植物醱酵物之酸鹼值為小於pH 4。 According to an embodiment of the present invention, the acid-base value of the above-mentioned plant ferment is less than pH 4.
依據本發明之一實施例，上述植物醱酵物包含總活菌數為至少107菌落形成單位(colony-forming unit，CFU)/毫升(mL)，其中胚芽乳桿菌、乾酪乳桿菌、洛德乳桿菌、短乳桿菌與鼠李糖乳桿菌之任兩者的含量比例為5：3至3：5。 According to an embodiment of the present invention, the above-mentioned plant ferment contains at least 10 7 colony-forming units (CFU)/ml (mL) of total viable bacteria, among which Lactobacillus embryo, Lactobacillus casei, and Loder's milk The content ratio of any two of bacillus, Lactobacillus brevis and Lactobacillus rhamnosus is 5:3 to 3:5.
根據本發明之另一態樣，提出一種植物醱酵物，其係如上所述之植物醱酵物的製造方法所獲得，其中植物醱酵物含有總活菌數至少107菌落形成單位(colony-forming unit，CFU)/毫升(mL)之多菌種乳桿菌。 According to another aspect of the present invention, a plant is proposed a starter Po, Po fermentation method for producing a plant which was obtained by the system described above, wherein the plant comprises a starter Po total number of viable cells of at least 107 CFU (colony-forming unit , CFU)/ml (mL) of multi-strain Lactobacillus.
依據本發明之一實施例，上述植物醱酵物包含植物醱酵液及/或植物醱酵凍乾粉。 According to an embodiment of the present invention, the above-mentioned plant ferment includes plant ferment liquid and/or plant ferment freeze-dried powder.
根據本發明之又一態樣，提出一種含植物醱酵物的組成物，其係包含上述植物醱酵物作為有效成分。 According to another aspect of the present invention, a composition containing a plant ferment is provided, which contains the above-mentioned plant ferment as an active ingredient.
根據本發明之再一態樣，提出一種植物醱酵物於製備調節血糖之組成物的用途，其中組成物包含如上所述的植物醱酵物。 According to another aspect of the present invention, a use of a plant ferment for preparing a composition for regulating blood sugar is proposed, wherein the composition comprises the plant ferment as described above.
依據本發明之一實施例，當組成物投予大鼠時，植物醱酵物於組成物之有效劑量為每天給予每千克體重0.1g至2.5g。 According to an embodiment of the present invention, when the composition is administered to rats, the effective dose of the plant ferment in the composition is 0.1 g to 2.5 g per kilogram of body weight per day.
依據本發明之一實施例，當組成物投予成人時，植物醱酵物於組成物之有效劑量為每天給予每千克體重0.015g至0.5g。 According to an embodiment of the present invention, when the composition is administered to an adult, the effective dose of the plant ferment in the composition is 0.015 g to 0.5 g per kilogram of body weight per day.
應用本發明之植物醱酵物及其製造方法、含彼之組成物暨其用途，可利用多菌種乳桿菌菌液加入植物基質中進行醱酵，可得到植物醱酵物。另外，向一對象投予含有植物醱酵物的組成物後，可減緩及/或預防高血糖及/或其併發症。 By applying the plant fermented product, its manufacturing method, the composition containing it, and its application, the multi-strain Lactobacillus bacteria liquid can be added to the plant substrate for fermenting, and the plant fermented product can be obtained. In addition, after administering a composition containing plant ferment to a subject, it can slow down and/or prevent hyperglycemia and/or its complications.
為讓本發明之上述和其他目的、特徵、優點與實施例能更明顯易懂，所附圖式之詳細說明如下： In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious and understandable, the detailed description of the attached drawings is as follows:
〔圖1〕係繪示根據本發明一實施例之血漿葡萄糖濃度的折線圖。 [Figure 1] is a line graph showing the plasma glucose concentration according to an embodiment of the present invention.
〔圖2〕係繪示根據圖1之血漿葡萄糖濃度的折線下之面積的直條圖。 [Figure 2] is a bar graph showing the area under the broken line of the plasma glucose concentration according to Figure 1.
〔圖3〕係繪示根據本發明一實施例之胰島素阻抗程度的直條圖。 [Figure 3] is a bar graph showing the degree of insulin resistance according to an embodiment of the present invention.
承上所述，本發明提供一種植物醱酵物的製造方法。首先，對植物漿液進行殺菁步驟，以獲得植物基質，其中殺菁步驟係以70℃至100℃進行30分鐘至60分鐘。倘殺菁步驟的溫度低於70℃或時間少於30分鐘，則無法有效軟化植物漿液中的組織、消滅部分微生物及排除植物漿液內的空氣。倘殺菁步驟的溫度高於100℃或時間多於60分鐘，則將破壞植物漿液中的營養物質，導致後續醱酵步驟時，無法提供充足的養分。 Based on the above, the present invention provides a method for manufacturing plant ferment. First, a cyanolysis step is performed on the plant slurry to obtain a plant substrate, wherein the cyanolysis step is performed at 70° C. to 100° C. for 30 minutes to 60 minutes. If the temperature of the cyanine-killing step is lower than 70°C or the time is less than 30 minutes, the tissues in the plant slurry cannot be effectively softened, some microorganisms are eliminated, and the air in the plant slurry is eliminated. If the temperature of the cyanine-killing step is higher than 100°C or the time is more than 60 minutes, the nutrients in the plant slurry will be destroyed, resulting in the failure to provide sufficient nutrients during the subsequent fermentation step.
在一實施例中，上述植物漿液係由植物性材料與水經均質化步驟後所得。植物性材料的種類及部位不拘，可例如但不限於南瓜、山苦瓜、小黃瓜、紅蘿蔔、菠菜、蘋果、番石榴、木瓜、藍莓及/或薑黃之至少一者的根、莖、葉、花及/或果實。 In one embodiment, the above-mentioned plant slurry is obtained by homogenizing plant materials and water. The types and parts of plant materials are not limited, such as but not limited to roots, stems, leaves of at least one of pumpkin, bitter gourd, cucumber, carrot, spinach, apple, guava, papaya, blueberry and/or turmeric, Flowers and/or fruits.
在本實施例中，提供多菌種乳桿菌(Lactobacillus sp.)的菌液，以於後續醱酵步驟中使用。多菌種乳桿菌包含胚芽乳桿菌(Lactobacillus plantarum)、乾酪乳桿菌(Lactobacillus casei)、洛德乳桿菌(Lactobacillus reuteri)、短乳桿菌(Lactobacillus brevis)及鼠李糖乳桿菌(Lactobacillus rhamnosus)。前述多菌種乳桿菌寄存 於台灣新竹食品路331號財團法人食品工業發展研究所生物資源中心(BCRC)，其中胚芽乳桿菌的寄存編號為BCRC 11697，前述乾酪乳桿菌的寄存編號為BCRC 17942，前述洛德乳桿菌的寄存編號為BCRC 16091，前述短乳桿菌的寄存編號為BCRC 11196，前述鼠李糖乳桿菌的寄存編號為BCRC 80663。 In this embodiment, a bacterial liquid of Lactobacillus sp. is provided for use in the subsequent fermentation step. The multi-strain Lactobacillus includes Lactobacillus plantarum , Lactobacillus casei , Lactobacillus reuteri , Lactobacillus brevis , and Lactobacillus rhamnosus . The aforementioned multi-strain Lactobacillus is deposited at the Biological Resource Center (BCRC), Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan. The deposit number of Lactobacillus embryo is BCRC 11697, and the deposit number of Lactobacillus casei is BCRC 17942. The deposit number of the aforementioned Lactobacillus lordella is BCRC 16091, the deposit number of the aforementioned Lactobacillus brevis is BCRC 11196, and the deposit number of the aforementioned Lactobacillus rhamnosus is BCRC 80663.
在一實施例中，多菌種乳桿菌的總活菌數為至少107菌落形成單位(colony-forming unit，CFU)/毫升(mL)。 In one embodiment, the total number of viable cells of Lactobacillus strains plurality of at least 107 CFU (colony-forming unit, CFU) / milliliter (mL).
在一實施例中，胚芽乳桿菌、乾酪乳桿菌、洛德乳桿菌、短乳桿菌及鼠李糖乳桿菌之任兩者的細胞數比為3：1至1：3。倘上述多菌種乳桿菌的細胞數比落於前述範圍之外，則於後續醱酵步驟時，將導致乳桿菌無法達到較佳的生長。 In one embodiment, the cell number ratio of any two of Lactobacillus embryo, Lactobacillus casei, Lactobacillus lordella, Lactobacillus brevis, and Lactobacillus rhamnosus is 3:1 to 1:3. If the cell number ratio of the above-mentioned multi-strain Lactobacillus falls outside the aforementioned range, the subsequent fermentation step will cause the Lactobacillus to fail to achieve better growth.
接著，將多菌種乳桿菌之菌液加入植物基質，以進行醱酵步驟，藉此獲得植物醱酵物。醱酵步驟係於35℃至40℃進行36小時至60小時。倘醱酵步驟的溫度落於前述範圍之外，則乳桿菌的生長不佳。倘醱酵步驟的時間少於36小時，則無法得到足夠的醱酵產物。倘醱酵步驟的時間多於60小時，則將影響植物醱酵物的味道及口感。 Then, the bacterial liquid of the multi-strain Lactobacillus is added to the plant substrate to carry out the fermentation step, thereby obtaining the plant fermentation product. The fermentation step is carried out at 35°C to 40°C for 36 hours to 60 hours. If the temperature of the fermentation step falls outside the aforementioned range, the growth of Lactobacillus is poor. If the time for the fermentation step is less than 36 hours, sufficient fermentation product cannot be obtained. If the fermentation step takes more than 60 hours, it will affect the taste and texture of the plant fermented product.
本發明另提供一種植物醱酵物，其係如上所述之植物醱酵物的製造方法所獲得，其中植物醱酵物含有總活菌數為至少107菌落形成單位(colony-forming unit，CFU)/毫升(mL)之多菌種乳桿菌，其中胚芽乳桿菌、乾酪 乳桿菌、洛德乳桿菌、短乳桿菌與鼠李糖乳桿菌之任兩者的含量比例為5：3至3：5。 The present invention further provides a plant Po fermentation thereof, the method for producing a plant Po fermentation product which system described above, the obtained, wherein the plant Po yeast containing the total number of viable cells forming units (colony-forming unit, CFU) of at least 107 cfu / ml (mL) of the multi-strain Lactobacillus, in which the content ratio of any two of Lactobacillus embryo, Lactobacillus casei, Lactobacillus lordrea, Lactobacillus brevis and Lactobacillus rhamnosus is 5:3 to 3:5.
所獲得的植物醱酵物之酸鹼值為小於pH 4。倘酸鹼值為至少pH 4，則將無法維持植物醱酵物中的總活菌數為至少107CFU/mL。 The acid-base value of the obtained plant ferment is less than pH 4. If the acid-base value is at least pH 4, the total viable cell count in the plant ferment will not be maintained at at least 10 7 CFU/mL.
在一實施例中，植物醱酵物可選擇為植物醱酵液及/或植物醱酵凍乾粉。植物醱酵液是指在醱酵步驟後，所獲得的液態植物醱酵物。植物醱酵凍乾粉是指將植物醱酵液進行凍乾後，所獲得無水的固態植物醱酵凍乾粉。 In one embodiment, the plant ferment can be selected as plant ferment liquid and/or plant ferment freeze-dried powder. Plant fermented liquid refers to the liquid plant fermented product obtained after the fermenting step. The freeze-dried plant fermented powder refers to the anhydrous solid plant fermented freeze-dried powder obtained after freeze-drying the plant fermented liquid.
本發明又提供一種植物醱酵物的組成物，其係包含植物醱酵物作為有效成分。組成物可以是口服組成物，經口投予本發明之組成物。 The present invention also provides a composition of plant ferment, which contains plant ferment as an effective ingredient. The composition may be an oral composition, and the composition of the present invention is administered orally.
組成物亦可以是食品組成物或醫藥組成物等，舉例來說，食品組成物可例如為穀物類製品、水果類製品、蔬菜類製品、肉類製品、魚類製品、蛋類製品、奶類製品、飲品、健康食品、保健食品、機能性食品、營養補充食品或特殊營養食品。 The composition can also be a food composition or a pharmaceutical composition. For example, the food composition can be, for example, cereal products, fruit products, vegetable products, meat products, fish products, egg products, milk products, Drinks, health foods, health foods, functional foods, nutritional supplements or special nutritional foods.
醫藥組成物可選擇性地包含藥學上可接受的成分混合而成，可包含但不限於溶劑、乳化劑、懸浮劑、分解劑、結合劑、賦形劑、安定劑、螫合劑、稀釋劑、膠凝劑、防腐劑、潤滑劑、吸收延緩劑及/或脂質體。 The pharmaceutical composition may optionally include a mixture of pharmaceutically acceptable ingredients, and may include, but is not limited to, solvents, emulsifiers, suspending agents, decomposing agents, binding agents, excipients, stabilizers, chelating agents, diluents, Gelling agents, preservatives, lubricants, absorption delaying agents and/or liposomes.
在一實施例中，上述食品組成物及醫藥組成物之劑型不限於特定形式，可為液體及/或固體，可包含但不限於錠劑、顆粒劑、粉末劑、膠囊、溶液、懸浮液及/或分散液 等。 In one embodiment, the dosage form of the above-mentioned food composition and medical composition is not limited to a specific form, and may be liquid and/or solid, and may include, but is not limited to, tablets, granules, powders, capsules, solutions, suspensions, and / Or dispersion Wait.
本發明再提供一種植物醱酵物於製備調節血糖之組成物的用途，其中組成物包含如上所述的植物醱酵物。 The present invention further provides a use of a plant ferment for preparing a composition for regulating blood sugar, wherein the composition comprises the plant ferment as described above.
本發明所述之調節血糖係指，透過測定空腹血糖值(fasting blood glucose)、血清胰島素(serum insulin)、葡萄糖耐受性(glucose tolerance)、胰島素阻抗(insulin resistance)、醣化血色素(glycosylated Hb)、血漿脂肪(blood lipid)及脂蛋白(lipoprotein)等指標，判斷血糖的代謝狀況。當血糖無法被正常代謝時，有可能引起高血糖及/或其併發症。 The regulation of blood glucose in the present invention refers to the measurement of fasting blood glucose, serum insulin, glucose tolerance, insulin resistance, and glycosylated Hb. , Plasma fat (blood lipid) and lipoprotein (lipoprotein) and other indicators to determine the metabolic status of blood sugar. When blood sugar cannot be metabolized normally, it may cause hyperglycemia and/or its complications.
在應用上述組成物時，有關使用量、使用型態、使用次數可視需求彈性調整。在一實施例中，當植物醱酵物包含107CFU/mL的乳桿菌總活菌數時，可投予大鼠本發明之植物醱酵物於組成物的有效劑量為每天給予每千克體重0.1g至2.5g，或者可投予成人本發明之植物醱酵物於組成物的有效劑量為每天給予每千克體重0.015g至0.5g〔根據2005年美國食品藥物管理局所公告之實驗初期估算方法(Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers)，以人體每日每公斤體重之建議攝取量(g/kg b.w./d)的6.2倍為大鼠1倍劑量換算〕。使用次數可於一天1次或一天數次。 When applying the above composition, the amount of use, the type of use, and the number of uses can be adjusted flexibly according to demand. In one embodiment, when the plant ferment contains 10 7 CFU/mL of total viable lactobacillus, the effective dose of the plant ferment of the present invention in the composition that can be administered to rats is 0.1 g per kilogram of body weight per day. To 2.5g, or the effective dose that can be administered to adults of the plant ferment of the present invention in the composition is 0.015g to 0.5g per kilogram of body weight per day (According to the 2005 U.S. Food and Drug Administration announced the early experimental estimation method (Estimating the The maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers) is calculated based on 6.2 times the recommended daily intake per kilogram of body weight (g/kg bw/d) of the human body as 1 times the dose of rats]. It can be used once a day or several times a day.
以下利用數個實施例以說明本發明之應用，然其並非用以限定本發明，本發明技術領域中具有通常知識者，在不脫離本發明之精神和範圍內，當可作各種之更動與潤飾。 Several embodiments are used below to illustrate the application of the present invention, but they are not used to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various modifications and changes without departing from the spirit and scope of the present invention. Retouch.
1.植物醱酵物的製備 1. Preparation of plant ferment
(1)製備植物醱酵液 (1) Preparation of plant fermented liquid
混合預先製備好的植物基質及多菌種乳桿菌菌液，於37℃厭氧培養48小時，以獲得植物醱酵液。植物醱酵液的酸鹼值為pH 3.57±0.07。另利用標準平板計數法(standard plate count method)測得植物醱酵液含有(1.8±0.3)×108CFU/mL的乳桿菌。關於標準平板計數法是屬本發明所屬技術領域具有通常知識者所熟知，在此不另贅述。 Mix the pre-prepared plant substrate and the multi-strain Lactobacillus liquid, and anaerobic culture at 37°C for 48 hours to obtain the plant fermentation liquid. The acid-base value of the plant fermented liquid is pH 3.57±0.07. In addition, the standard plate count method was used to determine that the plant fermented liquid contained (1.8±0.3)×10 8 CFU/mL Lactobacillus. The standard plate counting method is well-known to those with ordinary knowledge in the technical field of the present invention, and will not be repeated here.
上述植物基質係將南瓜、山苦瓜、小黃瓜、紅蘿蔔、菠菜、蘋果、番石榴、木瓜、藍莓及薑黃切塊後，以體積比例為1：1的份量與水混合，再以95℃殺菁40分鐘獲得。 The above-mentioned plant substrate is to cut pumpkin, bitter gourd, cucumber, carrot, spinach, apple, guava, papaya, blueberry, and turmeric, then mix it with water in a volume ratio of 1:1, and then kill at 95℃ Jing obtained in 40 minutes.
上述多菌種乳桿菌菌液包含1%的胚芽乳桿菌(BCRC 11697)、1%的乾酪乳桿菌(BCRC 17942)、3%的洛德乳桿菌(BCRC 16091)、1%的短乳桿菌(BCRC 11196)及1%的鼠李糖乳桿菌(BCRC 80663)，其均可自BCRC分讓獲得。 The above-mentioned multi-species Lactobacillus bacterium liquid contains 1% Lactobacillus embryo (BCRC 11697), 1% Lactobacillus casei (BCRC 17942), 3% Lactobacillus lordrea (BCRC 16091), 1% Lactobacillus brevis ( BCRC 11196) and 1% Lactobacillus rhamnosus (BCRC 80663), which can be obtained from BCRC distribution.
(2)測定植物醱酵液中的乳桿菌菌種比例 (2) Determine the proportion of Lactobacillus strains in the plant fermented liquid
利用逢機擴增多型性核酸-聚合酶連鎖反應(Random Amplified Polymorphic DNA-Polymerase chain reaction，RAPD-PCR)測定植物醱酵液中各種乳桿菌的含量比例。利用逢機引子(Random primer)進行PCR，將乳桿菌DNA的片段擴增後，使用電泳分析PCR產物，藉由PCR產物所呈現之片段多寡、位置差異、長度不同以及訊號強弱，而區分乳桿菌菌種及相對菌數百分比。 Use random amplified polymorphic nucleic acid-polymerase chain reaction (Random Amplified Polymorphic DNA-Polymerase chain reaction, RAPD-PCR) was used to determine the content ratio of various lactobacilli in plant ferment. Use Random primers to perform PCR. After amplifying the fragments of Lactobacillus DNA, the PCR products are analyzed by electrophoresis, and the number of fragments, position differences, length differences, and signal strengths presented by the PCR products are used to distinguish Lactobacilli Strains and relative bacterial count percentage.
本發明藉由引子〔如序列辨識編號(SEQ ID NO)：1、SEQ ID NO 2、SEQ ID NO 3、SEQ ID NO 4、SEQ ID NO 5及SEQ ID NO 6的任一條或前述之任意組合〕擴增植物醱酵液中，多菌種乳桿菌的部分序列。關於萃取DNA、PCR及電泳的方法是屬本發明所屬技術領域具有通常知識者所熟知，在此不另贅述。 The present invention uses primers (such as SEQ ID NO: 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 or any combination of the foregoing ] Amplify the partial sequence of the multi-strain Lactobacillus in the plant fermented liquid. The methods for DNA extraction, PCR, and electrophoresis are well known to those with ordinary knowledge in the technical field to which the present invention belongs, and will not be repeated here.
結果顯示，植物醱酵液包含25±3%的胚芽乳桿菌、25±2%的乾酪乳桿菌、15±1%的洛德乳桿菌、17±3%的短乳桿菌以及18±2%的鼠李糖乳桿菌。 The results show that the plant ferment contains 25±3% of Lactobacillus embryo, 25±2% of Lactobacillus casei, 15±1% of Lactobacillus lordella, 17±3% of Lactobacillus brevis and 18±2% of Lactobacillus brevis Lactobacillus rhamnosus.
(3)製備植物醱酵凍乾粉 (3) Preparation of freeze-dried plant fermented powder
將植物醱酵液以-40℃、4pa的條件，真空冷凍乾燥24小時後，可獲得植物醱酵凍乾粉。利用市售紅外線水分測定儀測定植物醱酵凍乾粉的水分含量為8.83%±0.10%。另利用標準平板計數法測得植物醱酵凍乾粉含有(2.6±0.6)×108CFU/g的乳桿菌。 After the plant fermented liquid is freeze-dried in a vacuum at -40°C and 4pa for 24 hours, the plant fermented freeze-dried powder can be obtained. The moisture content of the freeze-dried plant fermented powder measured by a commercially available infrared moisture meter was 8.83%±0.10%. In addition, a standard plate counting method was used to determine that the plant fermented lyophilized powder contained (2.6±0.6)×10 8 CFU/g of Lactobacillus.
2.植物醱酵液於抗氧化的功效 2. The anti-oxidant effect of plant fermented liquid
(1)總酚(total phenolic contents，TPC)含量的測定 (1) Determination of total phenolic contents (TPC)
於25μL的植物醱酵液中加入1mL 2%的碳酸鈉(Na2CO3)溶液，均勻混合後避光靜置2分鐘，再加入250μL 50%的福林酚試劑(Folin-Ciocalteu’s phenol reagent)，均勻混合後避光靜置30分鐘，以市售分光光度計測定波長750nm的吸光值。對照沒食子酸(Galic acid)標準品製作校正曲線，換算每毫升植物醱酵液中所含的沒食子酸當量(gallic acid equivalents，GAE)，以計算植物醱酵液的總酚量，其中，結果係使用平均值±標準差(mean±SD)(n=3)表示。 Add 1 mL of 2% sodium carbonate (Na 2 CO 3 ) solution to 25 μL of plant fermentation solution, mix well and let stand for 2 minutes in the dark, then add 250 μL of 50% Folin-Ciocalteu's phenol reagent After uniformly mixing, let it stand for 30 minutes in the dark, and measure the absorbance at 750 nm with a commercially available spectrophotometer. Make a calibration curve against the standard galic acid (Galic acid), convert the gallic acid equivalents (GAE) contained in each milliliter of plant fermented liquid, to calculate the total phenolic content of the plant fermented liquid, Among them, the results are expressed using mean±SD (n=3).
結果顯示，植物醱酵液的總酚量相當於17.47±0.29mg/mL之沒食子酸當量(mg GAE/mL)。 (2)DPPH自由基清除活性(DPPH free radical scavenging activity)測定 The results showed that the total phenol content of the plant fermented liquid was equivalent to the gallic acid equivalent (mg GAE/mL) of 17.47±0.29 mg/mL. (2) DPPH free radical scavenging activity measurement
DPPH自由基在甲醇中呈藍紫色，且於波長517nm有最大吸光值。當自由基清除劑存在時，DPPH自由基的顏色由藍紫轉為淡黃色，吸光值也隨之降低。 The DPPH radical is blue-violet in methanol, and has a maximum absorbance at a wavelength of 517nm. When the free radical scavenger is present, the color of DPPH free radical changes from blue-violet to light yellow, and the light absorption value also decreases.
取50μL植物醱酵液以蒸餾水稀釋至400μL，加入1mL 0.2mM的2,2-二苯基-1-苦味胼基(2,2-Diphenyl-1-picryl-hydrazyl，DPPH)溶液(秤取3.9mg DPPH溶於50mL甲醇)，混合均勻後避光靜置50分鐘，以市售分光光度計測量波長517nm的吸光值。吸光值愈低，DPPH自由基之清除能力愈強，抗氧化 能力越佳。 Dilute 50μL of plant fermentation solution with distilled water to 400μL, add 1mL 0.2mM 2,2-Diphenyl-1-picryl-hydrazyl (2,2-Diphenyl-1-picryl-hydrazyl, DPPH) solution (weigh 3.9 mg DPPH is dissolved in 50 mL methanol), mix well and stand for 50 minutes in the dark, and measure the absorbance at 517 nm with a commercially available spectrophotometer. The lower the light absorption value, the stronger the scavenging ability of DPPH free radicals and the anti-oxidation The better the ability.
對照L-抗壞血酸(L-Ascorbic acid，Vitamin C)標準品製作標準曲線，換算每毫升植物醱酵液相當於L-抗壞血酸當量(Vitamin C equivalents)之DPPH自由的基清除能力。結果顯示，植物醱酵液的DPPH自由之基清除能力相當於437.02±14.47μg/mL之L-抗壞血酸當量。 Comparing L-Ascorbic acid (Vitamin C) standard products to make a standard curve, convert the DPPH free radical scavenging capacity of L-ascorbic acid equivalents (Vitamin C equivalents) per milliliter of plant ferment. The results show that the DPPH free radical scavenging ability of the plant fermented liquid is equivalent to the L-ascorbic acid equivalent of 437.02±14.47μg/mL.
另以蒸餾水取代植物醱酵液作為空白組(blank)，以下式(I)計算DPPH自由基之清除率。結果顯示，植物醱酵液具有90%以上的DPPH自由基之清除率。 In addition, distilled water was used to replace the plant fermented liquid as a blank group, and the following formula (I) was used to calculate the scavenging rate of DPPH free radicals. The results show that the plant fermented liquid has a scavenging rate of DPPH free radicals of more than 90%.
DPPH自由基之清除率(%)=(1-A517nmsample/A517nmblank)×100% (I) DPPH free radical scavenging rate (%)=(1-A517nm sample /A517nm blank )×100% (I)
(3)還原力的測定 (3) Determination of reducing power
藉由還原物質，鐵氰化鉀〔potassium ferricyanide，K3Fe(CN)6〕可被還原成亞鐵氰化鉀〔potassium ferrocyanide，K4Fe(CN)6〕，亞鐵氰化鉀再與三價鐵離子(Fe3+)作用生成亞鐵氰化鐵{ferric ferrocyanide，Fe4〔Fe(CN)6〕3}，亞鐵氰化鐵於波長700nm下具有最大吸光值。當還原物質的還原能力越強時，生成亞鐵氰化鐵的越多，吸光值也隨之增加。 By reducing substances, potassium ferricyanide [potassium ferricyanide, K 3 Fe(CN) 6 ] can be reduced to potassium ferrocyanide [potassium ferrocyanide, K 4 Fe(CN) 6 ], and potassium ferricyanide is then combined with Ferric ions (Fe3 + ) react to produce ferric ferrocyanide {ferric ferrocyanide, Fe4〔Fe(CN) 6 〕 3 }, which has a maximum absorbance at a wavelength of 700nm. When the reducing ability of the reducing substance is stronger, the more ferrous ferrocyanide is produced, and the light absorption value also increases.
取50μL的植物醱酵液以蒸餾水稀釋至500μL，加入500μL 0.2M磷酸鈉緩衝溶液(pH 6.6)及500μL之1% K3Fe(CN)6溶液，混合均勻後，於50℃水浴20 分鐘。再移至於冰上快速冷卻5分鐘後，加入500μL 10% TCA溶液，混合均勻後，以3000rpm離心10分鐘。取上清液500μL並加入500μL的去離子水及100μL 0.1%的FeCl3．6H2O溶液，混合均勻後避光靜置10分鐘，以市售分光光度計測定波長700nm的吸光值，吸光值愈高，代表植物醱酵液的還原力愈強。對照L-抗壞血酸標準品製作標準曲線，換算每毫升植物醱酵液相當於L-抗壞血酸當量(Vitamin C equivalents)之還原力，其中，結果係使用平均值±標準差(mean±SD)(n=3)表示。 Dilute 50μL of plant fermentation solution with distilled water to 500μL, add 500μL of 0.2M sodium phosphate buffer solution (pH 6.6) and 500μL of 1% K 3 Fe(CN) 6 solution, mix well, and incubate in a water bath at 50°C for 20 minutes. Move to ice and quickly cool for 5 minutes, add 500 μL of 10% TCA solution, mix well, and centrifuge at 3000 rpm for 10 minutes. Take 500μL of supernatant and add 500μL of deionized water and 100μL of 0.1% FeCl 3 . After mixing the 6H 2 O solution, let it stand for 10 minutes in the dark. Use a commercially available spectrophotometer to measure the absorbance at 700 nm. The higher the absorbance, the stronger the reducing power of the plant fermented liquid. Comparing L-ascorbic acid standard products to make a standard curve, convert the reducing power of vitamin C equivalents per milliliter of plant ferment liquid, where the result is the mean ± standard deviation (mean ± SD) (n= 3) Representation.
結果顯示，植物醱酵液的還原力相當於13.76±0.01mg/mL之L-抗壞血酸當量。 The results showed that the reducing power of the plant fermented liquid was equivalent to 13.76±0.01 mg/mL of L-ascorbic acid equivalent.
(4)螯合亞鐵離子能力測定 (4) Determination of the ability to chelate ferrous ions
亞鐵離子(Fe2+)與3-(2-吡啶基)-5,6-二苯基-1,2,4-三嗪-4’,4”-二磺酸鈉鹽〔3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4’,4”-disulfonic acid sodium salt，菲洛嗪(FerroZine)〕的複合物，複合物在波長562nm下有最大吸光值，若抗氧化物質與亞鐵離子螯合，則亞鐵離子無法與菲洛嗪(FerroZine)產生複合物，則吸光值降低。 Ferrous ion (Fe 2+ ) and 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4',4”-disulfonic acid sodium salt [3-( 2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4',4”-disulfonic acid sodium salt, FerroZine (FerroZine)) complex, the complex has a maximum absorbance at a wavelength of 562nm If the antioxidant is chelated with ferrous ions, the ferrous ions cannot form a complex with FerroZine, and the light absorption value will decrease.
取400μL的植物醱酵液，加入1480μL的甲醇及40μL 2mM的氯化亞鐵四水合物(FeCl2．4H2O)溶液，混合均勻後靜置30秒。再加入80μL 5mM的菲洛嗪(FerroZine)溶液，混合均勻後避光靜置10分鐘，以市售分光光度計測定波長562nm的吸光值，吸光值愈低， 代表植物醱酵液螯合亞鐵離子能力愈強。 Take 400μL plant Po fermentation broth, and methanol was added 40μL 2mM 1480μL ferrous chloride tetrahydrate (FeCl 2 .4H 2 O) solution was mixed uniformly for 30 seconds. Then add 80μL of 5mM FerroZine (FerroZine) solution, mix evenly and let stand for 10 minutes in the dark. Use a commercially available spectrophotometer to measure the absorbance at 562nm. The lower the absorbance, it means that the plant fermented liquid chelates ferrous iron. The stronger the ion capacity.
對照乙二胺四乙酸(ethylene diamine tetraacetic acid，EDTA)標準品製作標準曲線，換算每毫升植物醱酵液相當於EDTA當量之螯合亞鐵離子能力，其中，結果係使用平均值±標準差(mean±SD)(n=3)表示。 Comparing ethylene diamine tetraacetic acid (EDTA) standard products to make a standard curve, convert the ferrous ion chelating capacity equivalent to EDTA equivalent per milliliter of plant fermented liquid, and the result is the average value ± standard deviation ( mean±SD)(n=3) means.
結果顯示，植物醱酵液的螯合亞鐵離子能力相當於199.07±16.15μg/mL之EDTA當量。 The results show that the chelating capacity of ferrous ferrous ions is equivalent to EDTA equivalent of 199.07±16.15μg/mL.
另以蒸餾水取代植物醱酵液作為空白組(blank)，以下式(II)計算亞鐵離子的螯合率。結果顯示，植物醱酵液具有相當於30%的亞鐵離子螯合率。 In addition, distilled water was used to replace the plant fermented liquid as a blank group, and the chelating rate of ferrous ions was calculated by the following formula (II). The results show that the plant fermented liquid has a ferrous ion chelating rate equivalent to 30%.
亞鐵離子螯合率(%)=(1-A562nmsample/A562nmblank)×100% (II) Ferrous ion chelation rate (%)=(1-A562nm sample /A562nm blank )×100% (II)
3.建立糖尿病大鼠的動物模式 3. Establish an animal model for diabetic rats
(1)糖尿病大鼠的誘發 (1) Induction of diabetic rats
將6週齡的Sprague-Dawley(SD)系雄性大鼠飼養於恆溫22±2℃，12小時照光及12小時黑暗之循環光照之環境，並給予充足飼料及飲水。大鼠經1週馴養適應後，於7週齡後開始進行試驗。試驗係依循農業委員會實驗動物照護及使用指引及台灣動物保護法的相關規定進行。 6-week-old Sprague-Dawley (SD) male rats were raised in a constant temperature of 22±2°C, 12 hours of light and 12 hours of darkness in a circulating light environment, and they were given adequate feed and drinking water. After 1 week of domestication and adaptation, the rats started the experiment after 7 weeks of age. The experiment was conducted in accordance with the guidelines for the care and use of laboratory animals of the Agriculture Committee and the relevant regulations of the Taiwan Animal Protection Law.
將大鼠隨機分成5組，每組10隻，分別健康組(控制組，其係每天管餵生理食鹽水)、實施例1至實施例3與 比較例1。實施例1至實施例3與比較例1依照台灣衛生福利部公告現行的「健康食品之調節血糖功能評估方法」誘發大鼠糖尿病，其係給予大鼠腹腔注射單一劑量之230mg/kg的菸鹼醯胺(Nicotinamide，NA)，15分鐘後再給予大鼠腹腔注射單一劑量之鏈佐黴素(Streptozotocin，STZ)藥劑。鏈佐黴素藥劑係溶於生理食鹽水中，含有65mg/kg的鏈佐黴素及10mM的檸檬酸鈉，pH4.5。於注射鏈佐黴素藥劑一周後，採血測量隔夜空腹(進食後17~24小時)的血糖值，血糖值若達或超過13mM(230+10mg%)時，則視為有糖尿病。接著，每天管餵0.1g/kg bw/d的植物醱酵凍乾粉(實施例1)、0.5g/kg bw/d的植物醱酵凍乾粉(實施例2)、2.5g/kg bw/d的植物醱酵凍乾粉(實施例3)或生理食鹽水(糖尿病控制組，比較例1)。 The rats were randomly divided into 5 groups, each with 10 rats, and the healthy group (control group, which was fed with physiological saline every day), Example 1 to Example 3 and Comparative example 1. Example 1 to Example 3 and Comparative Example 1 induced diabetes in rats according to the current "Evaluation Method for Blood Glucose Regulation Function of Healthy Food" announced by the Ministry of Health and Welfare of Taiwan, which was administered to rats by intraperitoneal injection of a single dose of 230 mg/kg nicotine Nicotinamide (NA), 15 minutes later, rats were intraperitoneally injected with a single dose of streptozotocin (STZ). The streptozotocin medicament is dissolved in physiological saline, containing 65 mg/kg streptozotocin and 10 mM sodium citrate, pH 4.5. One week after the injection of streptozotocin, blood is collected to measure the blood glucose level on an overnight fasting (17-24 hours after eating). If the blood glucose level reaches or exceeds 13mM (230+10mg%), it is considered to have diabetes. Then, feed 0.1g/kg bw/d plant ferment freeze-dried powder (Example 1), 0.5g/kg bw/d plant ferment freeze-dried powder (Example 2), 2.5g/kg bw per day /d of plant fermented freeze-dried powder (Example 3) or physiological saline (Diabetes control group, Comparative Example 1).
4週後將試驗大鼠以CO2迷昏，以肝門靜脈採血方式收集血液樣本，進行以下評估。 After 4 weeks, the test rats were stunned with CO 2 and blood samples were collected by blood sampling from the hepatic portal vein for the following evaluations.
(2)試驗大鼠的各項基本生理參數 (2) The basic physiological parameters of experimental rats
在試驗期間，對大鼠的飼料、飲水量及體重，每週紀錄兩次，觀察大鼠各項基本生理參數的變化。 During the experiment, the feed, drinking water and body weight of the rats were recorded twice a week to observe the changes in the basic physiological parameters of the rats.
表1係在試驗期間，大鼠飲水量之變化，單位為毫升(mL)。表1數據係使用杜凱多重分析法(Tukey's multiple analysis method)，當樣本為每組10隻大鼠時(n=10)，結果以「平均值±標準差(standard deviation，SD)」表示。此外，不同上標字母的數值間，代表組間在統計上具顯著性差異(P<0.05)。以下所列的表2~表7亦同。 Table 1 shows the changes in the amount of water the rats drink during the test period, in milliliter (mL). The data in Table 1 are based on Tukey's multiple analysis method. When the sample is 10 rats per group (n=10), the results are expressed as "mean ± standard deviation" (standard deviation). deviation, SD)" said. In addition, the values of different superscript letters represent statistically significant differences between groups (P<0.05). The same applies to Tables 2 to 7 listed below.
表1顯示實施例3的大鼠之飲水量於第2週開始有顯著性下降，而實施例1與實施例2的大鼠於第3週開始也有顯著性下降的情形。 Table 1 shows that the water consumption of the rats of Example 3 decreased significantly from the second week, and the rats of Examples 1 and 2 also decreased significantly from the third week.
表1
表2係在試驗期間，大鼠攝食量之變化，單位為克(g)。表2顯示實施例3的大鼠之攝食量，於第2週開始有顯著性下降的現象。 Table 2 shows the changes in food intake of rats during the test period, in grams (g). Table 2 shows that the food intake of the rats in Example 3 decreased significantly from the second week.
表2
表3係在試驗期間，大鼠體重之變化，單位為克(g)。表3顯示實施例3的大鼠之體重，於第3週開始有顯著性上升的現象。 Table 3 shows the changes in the body weight of rats during the test period, in grams (g). Table 3 shows that the body weight of the rats in Example 3 increased significantly from the third week.
表3
4.植物醱酵凍乾粉於調節血糖的效果 4. The effect of plant ferment freeze-dried powder in regulating blood sugar
(1)植物醱酵凍乾粉於調節空腹血糖值(Fasting blood glucose)之效果 (1) The effect of plant fermented freeze-dried powder in regulating fasting blood glucose (Fasting blood glucose)
依照上述「健康食品之調節血糖功能評估方法」中的己糖激酵素法(hexokinase assay)，測定大鼠血糖濃度。使用市售血糖機，利用葡萄糖受己糖激酵素(hexokinase)催化生成葡萄糖-6-磷酸鹽(Glucose 6-phosphate，G-6-P)，而G-6-P與NADP+反應生成NADPH。再經由測定NADPH於波長340nm的最大吸光度，即可定量出血糖濃度。 The blood glucose concentration of rats was measured according to the hexokinase assay in the above-mentioned "Methods for the Evaluation of Blood Glucose Regulation Function of Health Foods". Using a commercially available blood glucose machine, glucose is catalyzed by hexokinase to generate glucose-6-phosphate (G-6-P), and G-6-P reacts with NADP+ to generate NADPH. Then by measuring the maximum absorbance of NADPH at a wavelength of 340nm, the blood glucose concentration can be quantified.
表4係在試驗期間，大鼠空腹血糖值之變化，單位為毫克/分升(mg/dL)。表4顯示，相較於比較例1，實施例1~3的大鼠之空腹血糖值於第4週開始有顯著性的下降。 Table 4 shows the changes in fasting blood glucose levels of rats during the test period, in milligrams per deciliter (mg/dL). Table 4 shows that compared with Comparative Example 1, the fasting blood glucose values of the rats of Examples 1 to 3 have a significant decrease starting from the 4th week.
表4
(2)植物醱酵凍乾粉於調節血清胰島素(Serum insulin)之效果 (2) The effect of plant fermented freeze-dried powder on regulating serum insulin (Serum insulin)
使用市售大鼠胰島素酵素免疫吸附分析試劑組(Rat Insulin ELISA，Mercodia，Sweden)進行胰島素含量測定。分別吸取10μL血清和標準溶液分別至96孔微量多孔盤中，然後加入100μL、1倍之酵素共軛溶液(enzyme conjugate solution)，置於搖擺震盪器上室溫下搖擺2小時。再以1倍之清洗緩衝溶液(wash buffer solution)清洗5次，並移除殘餘液體。接著，加入200μL的四甲基聯苯胺(3,3',5,5'-tetramethylbenzidine，TMB)於室溫下反應15分鐘後，加入50μL的終止液(Stop Solution)並置於搖擺震盪器搖擺數秒，使微量多孔盤中的試劑均勻反應。接下來，利用酵素免疫分析儀於450nm下測定吸光值，依據標準曲線計算出血清中胰島素含量。 A commercially available rat insulin enzyme immunosorbent assay reagent set (Rat Insulin ELISA, Mercodia, Sweden) was used for insulin content determination. Pipette 10 μL of serum and standard solution into a 96-well micro multi-well plate respectively, and then add 100 μL of 1 times the enzyme conjugate solution and place on a rocking shaker at room temperature for 2 hours. Wash with 1 times wash buffer solution (wash buffer solution) 5 times, and remove residual liquid. Next, add 200μL of tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB) and react for 15 minutes at room temperature. Add 50μL of Stop Solution and place in a rocking shaker for a few seconds. , So that the reagents in the micro porous plate react uniformly. Next, use an enzyme immunoassay to measure the absorbance at 450nm, and calculate the serum insulin content based on the standard curve.
表5係在餵食植物醱酵凍乾粉第4週時，大鼠血清中胰島素的含量。實施例1~3的大鼠在餵食植物醱酵凍乾粉後，胰島素含量高於比較例1。因此，植物醱酵凍乾粉可促使胰島素分泌增加。顯示植物醱酵凍乾粉可能有利於修復受損的胰臟β細胞。 Table 5 shows the content of insulin in the serum of rats during the 4th week of feeding the freeze-dried plant fermented powder. After the rats in Examples 1 to 3 were fed the freeze-dried plant fermented powder, the insulin content was higher than that in Comparative Example 1. Therefore, the plant fermented freeze-dried powder can promote the increase of insulin secretion. It shows that the freeze-dried plant fermented powder may be beneficial to repair damaged pancreatic β cells.
表5
(3)植物醱酵凍乾粉於調節口服葡萄糖耐受性試驗(Oral glucose tolerance test，OGTT)之效果 (3) The effect of plant ferment freeze-dried powder on the adjustment of oral glucose tolerance test (OGTT)
依照上述「健康食品之調節血糖功能評估方法」中的葡萄糖耐受性測定(Glucose tolerance test)，測試大鼠進食後對葡萄糖耐受性之情形。在持續餵食大鼠植物醱酵凍乾粉至第4週時，測量葡萄糖耐受性。將大鼠空腹10小時至14小時後，以管餵方式給予大鼠葡萄糖(1g/kg bw)，然後分別在給糖後的0分鐘、30分鐘、60分鐘、90分鐘、120分鐘和180分鐘採血測定血糖值。 According to the Glucose Tolerance Test in the above-mentioned "Methods for the Evaluation of Blood Glucose Regulation Function of Healthy Foods", the glucose tolerance of rats after eating was tested. Glucose tolerance was measured when the rat plant fermented freeze-dried powder was continuously fed to the 4th week. After the rats were fasted for 10 hours to 14 hours, the rats were given glucose (1g/kg bw) by tube feeding, and then at 0 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes and 180 minutes after the sugar administration. Blood was collected to determine blood glucose level.
請同時參閱圖1及圖2，其中圖1係繪示根據本發明一實施例之血漿葡萄糖濃度的折線圖。X軸係時間，單位為分鐘。Y軸是血漿葡萄糖濃度，單位為毫克/分升(mg/dL)。在圖1中，圖號○代表控制組，圖號△代表實施例1，圖號◇代表實施例2，圖號□代表實施例3，且圖號▲代表比較例1。圖2係繪示根據圖1之血漿葡萄糖濃度的折線下之面積的直條圖。X軸由左至右為控制組、實施例1~3與比較例1。Y軸為曲線下面積(area under curve，AUG)，單位為血漿葡萄糖濃度×分鐘數〔(mg/dL)×min〕。 Please refer to FIG. 1 and FIG. 2 at the same time. FIG. 1 is a line graph of plasma glucose concentration according to an embodiment of the present invention. X axis system time, the unit is minutes. The Y-axis is the plasma glucose concentration in milligrams per deciliter (mg/dL). In Figure 1, the figure number ○ represents the control group, the figure number △ represents the embodiment 1, the figure number ◇ represents the embodiment 2, the figure number □ represents the embodiment 3, and the figure number ▲ represents the comparative example 1. Fig. 2 is a bar graph showing the area under the broken line of the plasma glucose concentration according to Fig. 1. The X axis from left to right is the control group, Examples 1 to 3 and Comparative Example 1. The Y-axis is the area under curve (AUG), the unit is plasma glucose concentration × minutes [(mg/dL) × min].
結果顯示，相較於比較例1，實施例1~3之大鼠在餵食植物醱酵凍乾粉後，可顯著性的降低血漿葡萄糖濃度。 The results show that compared with Comparative Example 1, the rats in Examples 1 to 3 can significantly reduce the plasma glucose concentration after being fed the freeze-dried plant fermented powder.
(4)植物醱酵凍乾粉於調節胰島素阻抗測定(Insulin resistance)之效果 (4) The effect of freeze-dried plant fermented powder in regulating insulin resistance
胰島素阻抗(Insulin Resistance，IR)是指細胞(特別是肝臟、肌肉、脂肪細胞)對胰島素的敏感性降低，血液的葡萄糖無法順利進入細胞中，身體為補償此反應，讓胰臟分泌更多的胰島素，導致血液中有高濃度胰島素的現象。 Insulin Resistance (IR) refers to the decreased sensitivity of cells (especially liver, muscle, and fat cells) to insulin. Blood glucose cannot enter the cells smoothly. In order to compensate for this response, the body makes the pancreas secrete more Insulin causes a high concentration of insulin in the blood.
利用胰島素阻抗之恆定模式的評估方法(Homeostasis model assessment method-insulin resistance index，HOMA-IR index)，於持續餵食大鼠植物醱酵凍乾粉至第4週時，測量大鼠空腹胰島素與空腹血糖值，並以下式(III)計算胰島素阻抗程度。倘數值越高，代表胰島素阻抗程度越高。 Use the Homeostasis model assessment method-insulin resistance index (HOMA-IR index) to measure the fasting insulin and fasting blood glucose of rats when the rats were continuously fed the freeze-dried plant fermented powder to the 4th week. Value, and the following formula (III) to calculate the degree of insulin resistance. The higher the value, the higher the degree of insulin resistance.
胰島素阻抗程度=〔空腹胰島素(μU/mL)×空腹血糖值(mmol/L)〕/22.5.....(III) Insulin resistance level = (fasting insulin (μU/mL) × fasting blood glucose value (mmol/L))/22.5...(III)
請參閱圖3，其係繪示根據本發明一實施例之胰島素阻抗程度的直條圖。X軸由左至右為控制組、實施例1~3與比較例1。Y軸為胰島素阻抗程度。在圖3中，數據係使用杜凱多重分析法(Tukey's multiple analysis method)，當樣本為每組10隻大鼠時(n=10)，結果以平均值±標準差(standard deviation，SD)表示。此外，不同上標字母的數值間，代表組間在統計上具顯著性差異(P<0.05)。 Please refer to FIG. 3, which shows a bar graph of insulin resistance according to an embodiment of the present invention. The X axis from left to right is the control group, Examples 1 to 3 and Comparative Example 1. The Y axis is the degree of insulin resistance. In Figure 3, the data uses Tukey's multiple analysis method. When the sample is 10 rats per group (n=10), the results are expressed as mean±standard deviation (SD) . In addition, the values of different superscript letters represent statistically significant differences between groups (P<0.05).
結果顯示，實施例1~3的大鼠，其胰島素阻抗程度均低於比較例1的大鼠。因此，在餵食植物醱酵凍乾粉後，確實可降低胰島素阻抗程度，有可能恢復細胞對胰島 素的敏感性。 The results showed that the insulin resistance of the rats of Examples 1 to 3 was lower than that of the rats of Comparative Example 1. Therefore, after feeding the plant fermented freeze-dried powder, it can indeed reduce the degree of insulin resistance, and it is possible to restore the cell to the islet Susceptibility.
(5)植物醱酵凍乾粉於調節醣化血色素測定(Glycosylated Hb)之效果 (5) The effect of plant fermented freeze-dried powder in regulating Glycosylated Hb
血液中的葡萄糖在進入紅血球後，會與血色素(hemoglobin)互相結合形成醣化血色素，且不易分離。當血液中高濃度葡萄糖狀況持續越久，醣化血色素的百分比就越高。 After the glucose in the blood enters the red blood cells, it will combine with hemoglobin to form glycosylated hemoglobin, and it is not easy to separate. The longer the condition of high concentration of glucose in the blood lasts, the higher the percentage of glycated hemoglobin.
於持續餵食大鼠植物醱酵凍乾粉至第4週時，利用市售的尊爵醣化血色素分析試劑組(Premier Hb9210 Glycated hemoglobin assay，Trinity Biotech，USA)，搭配Premier Hb 9210系統，檢測大鼠的醣化血色素的百分比。 When the rat plant ferment freeze-dried powder was continuously fed to the 4th week, the rats were detected by using the commercially available Premier Hb9210 Glycated hemoglobin assay (Trinity Biotech, USA) with Premier Hb 9210 system The percentage of glycosylated hemoglobin.
結果如表6所示，實施例1~3的大鼠之醣化血色素的百分比低於比較例1。顯示餵食植物醱酵凍乾粉後，醣化血色素降低，代表血液中葡萄糖濃度降低。 The results are shown in Table 6. The percentage of glycated hemoglobin in the rats of Examples 1 to 3 is lower than that of Comparative Example 1. It shows that after feeding plant fermented freeze-dried powder, the glycosylated hemoglobin decreases, which means that the glucose concentration in the blood decreases.
表6
(6)植物醱酵凍乾粉於調節血漿脂肪及脂蛋白之效果 (6) The effect of plant ferment freeze-dried powder in regulating plasma fat and lipoprotein
研究顯示，許多糖尿病患者合併有血漿脂肪異常的症狀。血漿脂肪與特殊蛋白結合成脂蛋白後，可溶於血漿中進入血液循環。前述血漿脂肪包括三酸甘油脂 (Triglyceride，TG)及總膽固醇(Total cholesterol，TC)。前述血漿脂蛋白包括極低密度脂蛋白(Very low density lipoprotein，VLDL)、低密度脂蛋白(Low-density lipoprotein，LDL)和高密度脂蛋白(High-density lipoprotein，HDL)，其中VLDL及LDL易造成動脈硬化，屬於劣質膽固醇。而HDL能清除血管壁上沈積的膽固醇，預防動脈硬化的進行，屬於優質膽固醇。 Studies have shown that many diabetic patients have symptoms of abnormal plasma fat. Plasma fat is combined with special proteins to form lipoproteins, which can be dissolved in plasma and enter the blood circulation. The aforementioned plasma fats include triglycerides (Triglyceride, TG) and total cholesterol (Total cholesterol, TC). The aforementioned plasma lipoproteins include very low density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL), among which VLDL and LDL are easy to Causes arteriosclerosis, which belongs to low-quality cholesterol. HDL can remove cholesterol deposited on blood vessel walls and prevent the progress of arteriosclerosis. It is a high-quality cholesterol.
在本實施例中，持續餵食大鼠植物醱酵凍乾粉至第4週時，委送醫事機構檢測三酸甘油脂、總膽固醇、高密度脂蛋白及低密度脂蛋白，結果如表7所示(單位：mg/dL)。 In this example, when the rat plant fermented freeze-dried powder was continuously fed to the 4th week, the medical institution was commissioned to test triglycerides, total cholesterol, high-density lipoprotein, and low-density lipoprotein. The results are shown in Table 7. Show (unit: mg/dL).
表7顯示，在餵食植物醱酵凍乾粉後，相較於比較例1，實施例3的三酸甘油脂及低密度脂蛋白濃度均有顯著性降低，且實施例1~實施例3的總膽固醇均具有顯著性降低。此外，相較於比較例1，實施例1~實施例3的高密度脂蛋白並無顯著性差異。 Table 7 shows that after feeding the freeze-dried plant fermented powder, compared with Comparative Example 1, the triglyceride and low-density lipoprotein concentrations of Example 3 are significantly reduced, and the results of Examples 1 to 3 There was a significant reduction in total cholesterol. In addition, compared with Comparative Example 1, the high-density lipoproteins of Examples 1 to 3 have no significant difference.
因此，植物醱酵凍乾粉確實可降低三酸甘油脂、總膽固醇及低密度脂蛋白，可有益於調節血漿脂肪及脂蛋白。 Therefore, the freeze-dried plant fermented powder can indeed reduce triglycerides, total cholesterol and low-density lipoprotein, which can be beneficial to regulate plasma fat and lipoprotein.
表7
綜而言之，由上述數個實施例證實，植物醱酵物具有調節血糖之功效，故可應用於製備調節血糖之組成物的用途，可減緩及/或預防高血糖及/或其併發症。 In summary, it is demonstrated by the above several examples that the plant ferment has the effect of regulating blood sugar, so it can be applied to the preparation of blood sugar regulating composition, which can slow down and/or prevent hyperglycemia and/or its complications.
其次，由上述實施例可知，本發明之植物醱酵物及其製造方法、含彼之組成物暨其用途，其優點在於利用多菌種乳桿菌菌液加入植物基質中進行醱酵後，可得到植物醱酵物。藉由投予含有植物醱酵物的組成物，可減緩及/或預防高血糖及/或其併發症。 Secondly, it can be seen from the above-mentioned embodiments that the plant fermented product of the present invention, its manufacturing method, the composition containing it, and its use have the advantage of adding the multi-strain Lactobacillus broth to the plant matrix for fermenting, it can be obtained Plant ferment. By administering a composition containing plant ferment, it can slow down and/or prevent hyperglycemia and/or its complications.
需補充的是，本發明雖以特定的製程、特定的分析方法或特定儀器作為例示，說明本發明之植物醱酵物及其製造方法，惟本發明所屬技術領域中任何具有通常知識者可知，本發明並不限於此，在不脫離本發明之精神和範圍內，本發明之植物醱酵物及其製造方法亦可使用其他製程、其他的分析方法或其他儀器進行。 It should be added that although the present invention uses a specific process, a specific analytical method or a specific instrument as an example to illustrate the plant ferment and its manufacturing method of the present invention, anyone with ordinary knowledge in the technical field of the present invention can know that the present invention The invention is not limited to this. Without departing from the spirit and scope of the invention, the plant ferment and its manufacturing method of the invention can also be carried out using other processes, other analytical methods or other instruments.
雖然本發明已以數個實施例揭露如上，然其並非用以限定本發明，在本發明所屬技術領域中任何具有通常知識者，在不脫離本發明之精神和範圍內，當可作各種之更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。 Although the present invention has been disclosed in several embodiments as above, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs can make various modifications without departing from the spirit and scope of the present invention. Modifications and modifications, therefore, the scope of protection of the present invention shall be subject to those defined by the attached patent application scope.
<110> 陳俊宏 <110> Chen Junhong
<120> 植物醱酵物及其製造方法、含彼之組成物暨其用途 <120> Plant ferment and its manufacturing method, composition containing it and its use
<130> 無 <130> None
<160> 6 <160> 6
<210> 1 <210> 1
<211> 9 <211> 9
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 引子 <223> Introduction
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<211> 10 <211> 10
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 引子 <223> Introduction
<400> 2 <400> 2
<210> 3 <210> 3
<211> 8 <211> 8
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 引子 <223> Introduction
<400> 3 <400> 3
<210> 4 <210> 4
<211> 10 <211> 10
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
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<212> DNA <212> DNA
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<220> <220>
<223> 引子 <223> Introduction
<400> 5 <400> 5
<210> 6 <210> 6
<211> 10 <211> 10
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| CN106858579A (en) * | 2017-04-11 | 2017-06-20 | 姜红成 | Prepare the fermentation composition and preparation method of the plant enzyme for being applied to diabetes conditioning and routine servicing |
| CN107692219A (en) * | 2017-07-31 | 2018-02-16 | 青岛国海生物制药有限公司 | A kind of hypoglycemic fruit zymotic fluid and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106858579A (en) * | 2017-04-11 | 2017-06-20 | 姜红成 | Prepare the fermentation composition and preparation method of the plant enzyme for being applied to diabetes conditioning and routine servicing |
| CN107692219A (en) * | 2017-07-31 | 2018-02-16 | 青岛国海生物制药有限公司 | A kind of hypoglycemic fruit zymotic fluid and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| Yu-Jun Wan , Origin of Hypoglycemic Benefits of Probiotic-Fermented Carrot Pulp, J. Agric. Food Chem. 2019, 67, 895−904. * |
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