TW202325324A - Fermentation complex with anti-aging effect similar to nmn. - Google Patents
Fermentation complex with anti-aging effect similar to nmn. Download PDFInfo
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本發明提供一種發酵組合物,可以有效的延緩老化相關基因表達、提高抗氧化物指標SOD及GSH含量、降低組織器官氧化物濃度,達到抗老化的效果。 The invention provides a fermentation composition, which can effectively delay the expression of aging-related genes, increase the content of antioxidant indexes SOD and GSH, reduce the concentration of oxides in tissues and organs, and achieve the effect of anti-aging.
NMN(煙醯胺單核苷酸)是維生素B3(菸酸)的衍生物之一,也是NAD+生物合成的中間產物,是由磷酸基團和含有核糖(ribose)和煙醯胺(nicotinamide)的核苷反應形成的生物活性核苷酸。 NMN (nicotinamide mononucleotide) is one of the derivatives of vitamin B3 (niacin) and is also an intermediate product of NAD+ biosynthesis. It is composed of a phosphate group and contains ribose and nicotinamide. Biologically active nucleotides formed by the reaction of nucleosides.
NMN目前已被日本核准可使用於食品添加物中,日本是以發酵技術產生出NMM後,以純化方式得到高濃度NMN物質,但至今除日本外,尚無其他國家核准NMN使用於食品中,目前是受保健品訴求之NMN皆為添加維生素B3仿造NMN物質,維生素B3雖為NMN前驅物質,但在發揮作用前仍需要體內的酵素進行轉換,生成NAD+後才具有活性。 NMN has been approved by Japan to be used in food additives. After NMM is produced by fermentation technology in Japan, high-concentration NMN substances are obtained by purification. However, no other country has approved the use of NMN in food except Japan. At present, the NMN that is sought after by health care products is all added vitamin B3 to imitate NMN substances. Although vitamin B3 is the precursor of NMN, it still needs to be converted by enzymes in the body before it can play a role, and it will become active after generating NAD+.
然而,市售NMN訴求的保健品,實際上皆為添加維生素B3,在老化的族群中,其體內的代謝率並無法完整的將維生素B3轉化為NAD+,因此攝取維生素B3很難達到如同NMN的延緩老化作用,另外在一些醫師的研究中,NMN主要能提高細胞的再生能力,但對於癌細胞是否 有相同作用,並無完整研究證實,因此可能存在食用上的風險。 However, the health products marketed by NMN are actually supplemented with vitamin B3. In the aging population, the metabolic rate in the body cannot completely convert vitamin B3 into NAD+, so it is difficult to achieve the same level of vitamin B3 intake as NMN. In addition, in the research of some physicians, NMN can mainly improve the regenerative ability of cells, but whether cancer cells It has the same effect, and there is no complete research to confirm it, so there may be risks in consumption.
有鑑於此,相關領域亟需開發一種類似NMN成分且具有延緩老化的潛力的組合物。 In view of this, there is an urgent need in related fields to develop a composition similar to NMN and having the potential to delay aging.
為解決上述問題,本發明提供一種具有NMN延緩老化功效的發酵組合物之製備方法,包含: In order to solve the above problems, the present invention provides a method for preparing a fermented composition with NMN anti-aging effect, comprising:
(1)蔬果萃取步驟:將不同蔬果分別以物理方式壓榨蔬果至果實或子實體呈碎塊狀,同時保留汁液與果渣或子實體,以取得複數個第一萃取物; (1) Fruit and vegetable extraction step: physically pressing different fruits and vegetables until the fruits or fruiting bodies are in pieces, while retaining the juice and pomace or fruiting bodies to obtain a plurality of first extracts;
(2)負壓破壁工法:添加0.15~1.5%一有機黑糖,以提高該些第一萃取物等滲透壓,並同時以負壓萃取方式在35cmHg~45cmHg近真空環境中,持續萃取3~15天,使蔬果細胞壁破裂,釋放出細胞內營養成分及胞內多醣,以取得複數個第二萃取物; (2) Negative pressure wall breaking method: add 0.15~1.5% organic brown sugar to increase the osmotic pressure of these first extracts, and at the same time use negative pressure extraction method in a near-vacuum environment of 35cmHg~45cmHg, and continuously extract for 3~ After 15 days, the cell walls of fruits and vegetables are broken to release intracellular nutrients and intracellular polysaccharides to obtain multiple second extracts;
(3)低溫發酵階段:以一酵母菌,以比例0.5~1.5%(107CFU/ml)植入該些第二萃取物中,發酵溫度控制在2~12℃,持續發酵3~15天,在此階段菌種處於低活性、讓酵素作用大於菌種作用,減緩糖類的消耗,在發酵過程中防腐,提高纖維聚糖的分解作用,促進細胞壁分解,以取得複數個第一發酵物; (3) Low-temperature fermentation stage: a yeast is implanted into the second extracts at a ratio of 0.5-1.5% (107 CFU/ml), the fermentation temperature is controlled at 2-12°C, and the fermentation is continued for 3-15 days. At this stage, the bacteria are in low activity, so that the action of the enzyme is greater than the action of the bacteria, slowing down the consumption of sugars, antisepsis during the fermentation process, improving the decomposition of celloglycans, and promoting the decomposition of cell walls, so as to obtain multiple first fermented products;
(4)深層發酵階段:以一乳酸菌,以比例0.5~1.5%(107CFU/ml);0.15~0.25%一異麥芽寡糖植入該些第一發酵物中,發酵溫度控制在20~35℃,持續發酵5~25天,在此階段乳酸菌消耗糖類生成乳酸,降低pH值達到防腐作用,且提高酸度降低黏稠度,以取得複數個第二發酵物; (4) Submerged fermentation stage: implant a lactic acid bacterium at a ratio of 0.5-1.5% (107 CFU/ml); 0.15-0.25%-isomaltooligosaccharide into these first fermented products, and control the fermentation temperature at 20-35 ℃, continuous fermentation for 5 to 25 days, during which lactic acid bacteria consume sugars to generate lactic acid, lower the pH value to achieve antiseptic effect, and increase the acidity to reduce the viscosity to obtain multiple second fermented products;
(5)螯合發酵階段:以一醋酸菌,以比例0.5~1.5%(107CFU/ml);2.5~5.5%一D-山梨醇植入該些第二發酵物中,發酵溫度控制在10~25℃,持續發酵5~20
天,在此階段耗氧環境消耗葡萄糖產生醋酸,消耗乙醇且提高酸度降低黏稠度,以取得複數個第三發酵物;以及
(5) Chelating fermentation stage: 0.5-1.5% (107CFU/ml) of acetic acid bacteria; 2.5-5.5%-D-sorbitol is implanted into these second fermentation products, and the fermentation temperature is controlled at 10~ 25 ℃,
(6)乾燥造粒步驟:將通過(1)-(5)步驟發酵完成之該些第三發酵物,進行過濾以取得複數個發酵液並保留該些發酵液,再將該些發酵液以等比例混合後,以噴霧乾燥造粒,取得該發酵組合物。 (6) Drying and granulation step: filter the third fermented product fermented through steps (1)-(5) to obtain a plurality of fermented liquids and retain these fermented liquids, and then use these fermented liquids as After mixing in equal proportions, they are spray-dried and granulated to obtain the fermented composition.
本發明另提供一種具有NMN延緩老化功效的發酵組合物,包含一蔬果原料,該蔬果原料包含松露、菇蕈類子實體、酪梨、番茄或毛豆,並透過一特殊發酵製備方法取得一發酵組合物。 The present invention also provides a fermented composition with NMN anti-aging effect, which includes a vegetable and fruit raw material, the vegetable and fruit raw material includes truffle, mushroom fruiting body, avocado, tomato or edamame, and a fermented composition is obtained through a special fermentation preparation method thing.
在本發明一實施例中,該具有NMN延緩老化功效的發酵組合物之製備方法,其中該蔬果萃取步驟進一步包含將該些蔬果與水混合的步驟。 In one embodiment of the present invention, the preparation method of the fermented composition having NMN anti-aging effect, wherein the step of extracting the fruits and vegetables further includes the step of mixing the fruits and vegetables with water.
在本發明一實施例中,該具有NMN延緩老化功效的發酵組合物之製備方法,其中該蔬果包含松露、菇蕈類子實體、酪梨、番茄或毛豆。 In one embodiment of the present invention, the preparation method of the fermented composition having NMN anti-aging effect, wherein the vegetables and fruits include truffles, fruiting bodies of mushrooms, avocados, tomatoes or edamame.
在本發明一實施例中,該具有NMN延緩老化功效的發酵組合物之製備方法,其中該蔬果萃取步驟之蔬果為松露或菇蕈類子實體時,在以物理方式壓榨前,進一步包含以65~100℃熱水淨泡25~35分鐘軟化之步驟。 In one embodiment of the present invention, the preparation method of the fermented composition with NMN anti-aging effect, wherein when the vegetables and fruits in the vegetable and fruit extraction step are truffles or fruiting bodies of mushrooms, before pressing in a physical way, further comprising 65 Soak in hot water at ~100°C for 25-35 minutes to soften.
在本發明一實施例中,該具有NMN延緩老化功效的發酵組合物之製備方法,其中該些蔬果與水混合的比例分別為松露或菇蕈類子實體比水1:40;酪梨比水1:25;番茄比水1:1;毛豆比水1:30。 In one embodiment of the present invention, the preparation method of the fermented composition with NMN anti-aging effect, wherein the mixing ratio of the vegetables and fruits to water is 1:40 to the fruit body of truffles or mushrooms, and 1:40 to the water ratio of avocado 1:25; tomato to water 1:1; edamame to water 1:30.
在本發明一實施例中,該具有NMN延緩老化功效的發酵組合物之製備方法,其中該些第二萃取物pH值介於5.5~7;該些第一發酵物pH值介於5~6.5;該些第二發酵物pH值介於4.5~5.5;該些第三發酵物pH值介於3~3.5。 In one embodiment of the present invention, the preparation method of the fermented composition with NMN anti-aging effect, wherein the pH value of the second extracts is between 5.5 and 7; the pH value of the first fermented products is between 5 and 6.5 The pH value of the second fermented product is between 4.5 and 5.5; the pH value of the third fermented product is between 3 and 3.5.
在本發明一實施例中,該具有NMN延緩老化功效的發酵組合物之 製備方法,其中該些第二萃取物糖度介於21~28°Brix;該些第一發酵物糖度介於15~34°Brix;該些第二發酵物糖度介於15~38°Brix;該些第三發酵物糖度介於10~50°Brix。 In one embodiment of the present invention, the fermented composition with NMN anti-aging effect The preparation method, wherein the sugar content of the second extracts is between 21-28°Brix; the sugar content of the first fermented products is between 15-34°Brix; the sugar content of the second fermented products is between 15-38°Brix; The sugar content of these third fermented products is between 10~50°Brix.
在本發明一實施例中,具有NMN延緩老化功效的發酵組合物之製備方法,其中該乳酸菌係選自由植物乳桿菌(L.plantarum)、保加利亞乳桿菌(L.delbrueckii)、乳酸乳球菌(L.lactis)、嗜酸乳桿菌(L.acidophilus)和比菲德氏菌(B.bifidum)所組成之群組。 In one embodiment of the present invention, the preparation method of a fermented composition with NMN anti-aging effect, wherein the lactic acid bacteria are selected from Lactobacillus plantarum (L.plantarum), Lactobacillus bulgaricus (L.delbrueckii), Lactococcus lactis (Lactococcus .lactis), acidophilus Lactobacillus (L.acidophilus) and Bifidella (B.bifidum).
在本發明一實施例中,具有NMN延緩老化功效的發酵組合物之製備方法,其中該酵母菌或該醋酸菌係選自由白麴菌(S.fibuligera)、釀酒酵母(S.cerevisiae)、膜畢赤酵母(P.faciens)、粟酒裂殖酵母(S.pombe)、弱氧化促酸菌(A.hansenii)、木質醋酸菌(A.xylinum)和弱氧化醋酸桿菌(A.suboxydans)所組成之群組。 In one embodiment of the present invention, the preparation method of the fermented composition with NMN anti-aging effect, wherein the yeast or the acetic acid bacteria are selected from the group consisting of S. fibuligera, S. cerevisiae, membrane Pichia pastoris (P.faciens), Schizosaccharomyces pombe (S.pombe), weak oxidative acid-promoting bacteria (A.hansenii), xylinum acetic acid bacteria (A.xylinum) and weak oxidative acetic acid bacteria (A.suboxydans) composed of groups.
在本發明一實施例中,具有NMN延緩老化功效的發酵組合物用於製備延緩老化之藥物的用途,其延緩老化係指延緩老化相關基因表達、提高抗氧化物指標SOD及GSH含量、降低組織器官氧化物濃度。 In one embodiment of the present invention, the fermented composition with NMN aging-delaying effect is used to prepare a drug for delaying aging, and the delaying aging refers to delaying the expression of aging-related genes, increasing the content of antioxidant indicators SOD and GSH, reducing tissue Organ oxide concentrations.
本發明之主要目的另提供一具有NMN延緩老化功效的發酵組合物用於製備延緩老化之一藥物的用途,其中該藥物可以用於延緩心臟、腦部、肝臟的老化。 The main purpose of the present invention is to provide a fermented composition with NMN anti-aging effect for preparing a drug for delaying aging, wherein the drug can be used for delaying the aging of the heart, brain and liver.
圖1A-1D顯示溫度梯度發酵技術與傳統發酵技術於糖度、PH值、纖維素、 多醣體的差異。 Figure 1A-1D shows the temperature gradient fermentation technology and traditional fermentation technology in the sugar content, pH value, cellulose, Differences in polysaccharides.
圖2A-2C顯示本發明具有NMN延緩老化功效的發酵組合物對於小鼠血球細胞基因表達量的結果圖。 2A-2C show the results of the fermented composition with NMN anti-aging effect of the present invention on the gene expression of mouse hemocytes.
圖3A-3B為本發明具有NMN延緩老化功效的發酵組合物對於小鼠血液抗氧化物指標濃度的結果圖。 3A-3B are diagrams showing the results of the fermented composition with NMN aging-delaying effect of the present invention on the concentration of antioxidant indicators in mouse blood.
圖4A-4C為本發明具有NMN延緩老化功效的發酵組合物對於小鼠組織器官中氧化物濃度的結果圖。 4A-4C are graphs showing the results of the fermented composition with NMN aging-delaying effect of the present invention on the concentration of oxides in mouse tissues and organs.
圖5為本發明具有NMN延緩老化功效的發酵組合物對於延長小鼠壽命試驗各項結果圖。 Fig. 5 is a graph showing the results of the experiment on prolonging the lifespan of mice with the fermented composition with NMN anti-aging effect of the present invention.
本發明將可由以下的實施例說明而得到充分瞭解,使得熟習本技藝之人士可以據以完成之,然而本案之實施並非可由下列實施例而被限制其實施型態,熟習本技藝之人士仍可依據除既揭露之實施例的精神推演出其他實施例,該等實施例皆當屬於本發明之範圍。 The present invention will be fully understood by the following examples, so that those skilled in the art can complete it. However, the implementation of this case cannot be limited by the following examples. Those who are familiar with the art can still Other embodiments can be deduced according to the spirit of the disclosed embodiments, and these embodiments should all belong to the scope of the present invention.
實施例一、類似NMN活性之發酵組合物的製備方法
以一種或多種菌種之乳酸乳球菌(L.lactis)、嗜酸乳桿菌(L.acidophilus)、比菲德氏菌(B.bifidum)、弱氧化促酸菌(A.hansenii)、木質醋酸菌(A.xylinum)或弱氧化醋酸桿菌(A.suboxydans),分別與松露、酪梨、番茄、毛豆等不同的蔬果種類進行溫度梯度發酵調控,並將發酵完成之發酵液進行過濾後保留發酵液;發酵組合物的溫度梯度發酵調控步驟包含: Lactococcus lactis (L.lactis), Lactobacillus acidophilus (L.acidophilus), Bifidum (B.bifidum), weak oxidation acid-promoting bacteria (A.hansenii), wood acetic acid xylinum or A.suboxydans, and truffles, avocados, tomatoes, edamame and other different types of vegetables and fruits for temperature gradient fermentation control, and the fermented broth is filtered and reserved for fermentation liquid; the temperature gradient fermentation control step of the fermentation composition comprises:
(1)蔬果萃取步驟:將不同蔬果分別以物理方式壓榨蔬果至果實或子實體呈 碎塊狀,同時保留汁液與果渣或子實體以取得複數個第一萃取物; (1) Vegetable and fruit extraction step: press different vegetables and fruits in a physical way until the fruit or fruiting bodies appear Crushed, while retaining juice and pomace or fruiting bodies to obtain multiple first extracts;
(2)負壓破壁工法:添加0.15~1.5%一有機黑糖,以提高該些第一萃取物等滲透壓,並同時以負壓萃取方式在35cmHg~45cmHg近真空環境中,持續萃取3~15天,使蔬果細胞壁破裂,釋放出細胞內營養成分及胞內多醣,以取得複數個第二萃取物; (2) Negative pressure wall breaking method: add 0.15~1.5% organic brown sugar to increase the osmotic pressure of these first extracts, and at the same time use negative pressure extraction method in a near-vacuum environment of 35cmHg~45cmHg, and continuously extract for 3~ After 15 days, the cell walls of fruits and vegetables are broken to release intracellular nutrients and intracellular polysaccharides to obtain multiple second extracts;
(3)低溫發酵階段:以一酵母菌,以比例0.5~1.5%(107CFU/ml)植入該些第二萃取物中,發酵溫度控制在2~12℃,持續發酵3~15天,在此階段菌種處於低活性、讓酵素作用大於菌種作用,減緩糖類的消耗,在發酵過程中防腐,提高纖維聚糖的分解作用,促進細胞壁分解,以取得複數個第一發酵物; (3) Low-temperature fermentation stage: a yeast is implanted into the second extracts at a ratio of 0.5-1.5% (107 CFU/ml), the fermentation temperature is controlled at 2-12°C, and the fermentation is continued for 3-15 days. At this stage, the bacteria are in low activity, so that the action of the enzyme is greater than the action of the bacteria, slowing down the consumption of sugars, antisepsis during the fermentation process, improving the decomposition of celloglycans, and promoting the decomposition of cell walls, so as to obtain multiple first fermented products;
(4)深層發酵階段:以一乳酸菌,以比例0.5~1.5%(107CFU/ml);0.15~0.25%一異麥芽寡糖植入該些第一發酵物中,發酵溫度控制在20~35℃,持續發酵5~25天,在此階段乳酸菌消耗糖類生成乳酸,降低pH值達到防腐作用,且提高酸度降低黏稠度,以取得複數個第二發酵物; (4) Submerged fermentation stage: implant a lactic acid bacterium at a ratio of 0.5-1.5% (107 CFU/ml); 0.15-0.25%-isomaltooligosaccharide into these first fermented products, and control the fermentation temperature at 20-35 ℃, continuous fermentation for 5 to 25 days, during which lactic acid bacteria consume sugars to generate lactic acid, lower the pH value to achieve antiseptic effect, and increase the acidity to reduce the viscosity to obtain multiple second fermented products;
(5)螯合發酵階段:以一醋酸菌,以比例0.5~1.5%(107CFU/ml);2.5~5.5%一D-山梨醇植該些第二發酵物中,發酵溫度控制在10~25℃,持續發酵5~20天,在此階段耗氧環境消耗葡萄糖產生醋酸,消耗乙醇且提高酸度降低黏稠度,以取得複數個第三發酵物;以及 (5) Chelation fermentation stage: with acetic acid bacteria, in proportion 0.5~1.5% (107CFU/ml); ℃, continuous fermentation for 5-20 days, during this stage, the oxygen-consuming environment consumes glucose to produce acetic acid, consumes ethanol and increases acidity to reduce viscosity, so as to obtain multiple third fermented products; and
(6)乾燥造粒步驟:將通過(1)-(5)步驟發酵完成之該些第三發酵物,進行過濾以取得複數個發酵液並保留該些發酵液,再將該些發酵液以等比例混合後,以噴霧乾燥造粒,取得該發酵組合物。 (6) Drying and granulation step: filter the third fermented product fermented through steps (1)-(5) to obtain a plurality of fermented liquids and retain these fermented liquids, and then use these fermented liquids as After mixing in equal proportions, they are spray-dried and granulated to obtain the fermented composition.
在一實施例中,蔬果萃取步驟進一步包含將該些蔬果與水混合的步驟。 In one embodiment, the step of extracting the fruits and vegetables further includes a step of mixing the fruits and vegetables with water.
在一實施例中,該蔬果包含松露、菇蕈類子實體、酪梨、番茄或毛豆。 In one embodiment, the fruits and vegetables include truffles, fruiting bodies of mushrooms, avocados, tomatoes or edamame.
在一實施例中,該蔬果萃取步驟之蔬果為松露或菇蕈類子實體時,在以物理方式壓榨前,進一步包含以65~100℃熱水淨泡25~35分鐘軟化之步驟。 In one embodiment, when the fruits and vegetables in the vegetable and fruit extraction step are truffles or fruit bodies of mushrooms, the step of soaking in hot water at 65-100° C. for 25-35 minutes to soften them is further included before physically pressing.
在一實施例中,該些蔬果與水混合的比例分別為松露或菇蕈類子實體比水1:40;酪梨比水1:25;番茄比水1:1;毛豆比水1:30。 In one embodiment, the mixing ratio of these fruits and vegetables to water is 1:40 to the fruiting bodies of truffles or mushrooms; 1:25 to water from avocado; 1:1 to water from tomatoes; 1:30 to water from soybean .
在一實施例中,該些第二萃取物pH值介於5.5~7;該些第一發酵物pH值介於5~6.5;該些第二發酵物pH值介於4.5~5.5;該些第三發酵物pH值介於3~3.5。 In one embodiment, the pH of the second extracts is between 5.5 and 7; the pH of the first fermented products is between 5 and 6.5; the pH of the second fermented products is between 4.5 and 5.5; The pH value of the third fermented product is between 3 and 3.5.
在一實施例中,該些第二萃取物糖度介於21~28°Brix;該些第一發酵物糖度介於15~34°Brix;該些第二發酵物糖度介於15~38°Brix;該些第三發酵物糖度介於10~50°Brix。 In one embodiment, the sugar content of the second extracts is between 21-28°Brix; the sugar content of the first fermented products is between 15-34°Brix; the sugar content of the second fermented products is between 15-38°Brix ; The sugar content of these third fermentation products is between 10 ~ 50 ° Brix.
實施例二、溫度梯度發酵調控實驗Embodiment two, temperature gradient fermentation control experiment
本發明之溫度梯度發酵調控技術與傳統的發酵技術有著很大的區別,本發明之溫度梯度發酵調控技術可以具有更完整的發酵效果,且能保留更多的纖維素及釋放出更多的胞內多醣。 The temperature gradient fermentation control technology of the present invention is very different from the traditional fermentation technology. The temperature gradient fermentation control technology of the present invention can have a more complete fermentation effect, and can retain more cellulose and release more cells. Endopolysaccharides.
本發明之溫度梯度發酵調控技術對比傳統發酵技術實驗,(T)為溫度梯度發酵技術組;(C)為傳統發酵組(傳統發酵溫度接控制在25~30℃)。 The temperature gradient fermentation control technology of the present invention is compared with the traditional fermentation technology experiment, (T) is the temperature gradient fermentation technology group; (C) is the traditional fermentation group (the traditional fermentation temperature is directly controlled at 25~30°C).
結果如圖1A所示,傳統發酵組糖度變化大,每階段的糖度下降快速,容易造成發酵終點提早達成,且糖度降低無法抑制腐敗菌,可能造成敗槽。溫度梯度發酵將糖度控制在至少15以上,直到螯合發酵階段(醋酸菌)才完整將碳源進行發酵,降低至15°Brix以下。 The results are shown in Figure 1A. The sugar content of the traditional fermentation group changed greatly, and the sugar content decreased rapidly at each stage, which easily caused the fermentation end point to be reached earlier, and the reduction of sugar content could not inhibit spoilage bacteria, which may lead to tank failure. The temperature gradient fermentation controls the sugar content at least above 15, and the carbon source is not completely fermented until the chelating fermentation stage (acetic acid bacteria), and the carbon source is reduced to below 15°Brix.
結果如圖1B所示,傳統發酵組因溫度適合酵母菌株生長,菌種快速生長並消耗碳源,此時pH值快速降低至3.8以下,造成發酵菌株反應快速達到終點,在後續菌株投入後作用無法有效達成生長發酵,以往會在進入下個發酵階段前進行pH值的調整,加入例如碳酸鈣、氫氧化鈉等化合物,對發酵產品來說可能無形增加無機礦物鹽的攝入(如鈉),運用溫度梯度發酵 技術,在酵母發酵階段以低溫處理,使得發酵液在進入後續發酵階段前提供其他菌種良好的生長環境,能確保發酵終點的延長。螯合發酵階段以15~20℃處理,此時醋酸菌在耗氧環境下能大量生成有機酸,使產品pH值達到3左右,提供防腐作用。 The results are shown in Figure 1B. Because the temperature of the traditional fermentation group is suitable for the growth of yeast strains, the strains grow rapidly and consume carbon sources. At this time, the pH value quickly drops below 3.8, causing the fermentation strains to quickly reach the end of the reaction. Growth and fermentation cannot be effectively achieved. In the past, the pH value was adjusted before entering the next fermentation stage. Compounds such as calcium carbonate and sodium hydroxide were added, which may invisibly increase the intake of inorganic mineral salts (such as sodium) for fermented products. , using a temperature gradient to ferment Technology, low temperature treatment in the yeast fermentation stage, so that the fermentation broth can provide a good growth environment for other strains before entering the subsequent fermentation stage, which can ensure the extension of the fermentation end point. The chelating fermentation stage is treated at 15-20°C. At this time, acetic acid bacteria can generate a large amount of organic acids in an oxygen-consuming environment, making the product pH value reach about 3, providing antiseptic effect.
結果如圖1C所示,草本植物細胞壁以纖維聚糖為主成分,在傳統發酵方法中,菌株活性大於酵素活性,容易使pH值、碳源快速降低,發酵終點提前達成,多數草本植物細胞無法完整進行分解,其成分釋放比例有限;溫度梯度發酵技術中,低溫發酵階段控制酵素活性大於菌種活性,使得碳源下降緩慢,對細胞壁的分解能更完全,結果中表明溫度梯度發酵技術纖維素含量皆高於傳統發酵技術,表明細胞壁被分解的比例高。 The results are shown in Figure 1C. The cell wall of herbal plants is mainly composed of celloglycan. In the traditional fermentation method, the activity of the strain is greater than that of the enzyme, which can easily reduce the pH value and carbon source rapidly, and the fermentation end point is reached in advance. Most of the herbal plant cells cannot Decomposition is complete, and the release ratio of its components is limited; in the temperature gradient fermentation technology, the activity of the enzyme in the low-temperature fermentation stage is greater than that of the bacteria, so that the carbon source decreases slowly, and the decomposition of the cell wall can be more complete. The results show that the cellulose content of the temperature gradient fermentation technology All are higher than traditional fermentation technology, indicating that the proportion of cell wall is decomposed is high.
結果如圖1D所示,真菌細胞壁以幾丁聚糖為主成分,必須將細胞壁破壞後才能釋放出胞內多醣,在傳統發酵方法中,菌株活性大於酵素活性,容易使pH值、碳源快速降低,發酵終點提前達成,多數真菌細胞無法完整進行分解,其成分釋放比例有限;溫度梯度發酵技術中,低溫發酵階段控制酵素活性大於菌種活性,使得碳源下降緩慢,對細胞壁的分解能更完全,結果中表明溫度梯度發酵技術多醣體含量皆高於傳統發酵技術,表明釋放的胞內多醣比例能有效提高。 The results are shown in Figure 1D. The fungal cell wall is mainly composed of chitosan, and the intracellular polysaccharide must be released after the cell wall is destroyed. In the traditional fermentation method, the activity of the strain is greater than the activity of the enzyme, and it is easy to rapidly increase the pH value and carbon source. Reduced, the fermentation end point is reached ahead of time, most fungal cells cannot be completely decomposed, and the release ratio of its components is limited; in the temperature gradient fermentation technology, the low-temperature fermentation stage controls the activity of the enzyme to be greater than the activity of the bacteria, so that the carbon source decreases slowly and the decomposition of the cell wall can be more complete , the results show that the polysaccharide content of the temperature gradient fermentation technology is higher than that of the traditional fermentation technology, indicating that the proportion of intracellular polysaccharides released can be effectively increased.
實施例三、實驗動物與實驗方法Embodiment three, experimental animal and experimental method
本發明類NMN發酵組合物具有延緩衰老的效果。依中華民國衛生福利部公告之「健康食品之延緩衰老保健功效評估方法」進行分析,本發明類NMN發酵組合物獲得至少一項指標項目之正反應。本發明類NMN發酵組合物可防止、延緩、抑制一個體的外觀老化、功能上的衰退及行為遲緩。 以下動物實驗以60公斤之成人為基準,劑量換算以人體每日每公斤體重人體建議食用量1g/天換算動物劑量,分成下述5組: The NMN-like fermented composition of the invention has the effect of delaying aging. According to the "Evaluation Method of Anti-aging Health Care Effect of Health Food" announced by the Ministry of Health and Welfare of the Republic of China, the NMN-like fermented composition of the present invention obtained a positive reaction of at least one index item. The NMN-like fermented composition of the present invention can prevent, delay and inhibit an individual's appearance aging, functional decline and behavioral retardation. The following animal experiments are based on a 60 kg adult, and the dose conversion is based on the recommended human consumption per kg body weight per day of 1g/day. The animal dose is divided into the following 5 groups:
(1.)低劑量0.5倍劑組(LD,low dose) (1.) Low dose 0.5 times dose group (LD, low dose)
(2.)中劑量1倍劑量組(MD,medium dose)
(2.)
(3.)高劑量1.5倍劑量為HD(high dose) (3.) High dose 1.5 times the dose is HD (high dose)
(4.)健康組(WT,wild type):未加入任何樣本 (4.) Healthy group (WT, wild type): no samples were added
(5.)老化組(C,control):未加入任何樣本 (5.) Aging group (C, control): no samples added
本發明動物實驗使用的為C57BL/6品種的小鼠,該小鼠週齡為6~12周,並依照(1)健康組、(2)老化組、(3)低劑量組、(4)中劑量組、(5)高劑量組,各組別每組分配10隻雌鼠以及10隻雄鼠。 The animal experiment of the present invention uses the mouse of C57BL/6 breed, and this mouse week age is 6~12 weeks, and according to (1) healthy group, (2) aging group, (3) low dose group, (4) In the middle dose group and (5) high dose group, 10 female mice and 10 male mice were assigned to each group.
將上述小鼠建立D-半乳糖誘發氧化壓力老化動物模型,將小鼠誘導依照(1)健康組、(2)老化組、(3)低劑量組、(4)中劑量組、(5)高劑量組,各組別每組分配10隻雌鼠以及10隻雄鼠,並且對健康組小鼠注射0.1-0.3g/kg bw的生理食鹽水;對對照組、低劑量組、中劑量組、高劑量組注射0.1-0.3g/kg bw的D-半乳糖液,誘導6~10周後取血檢測脂質氧化丙二醛(malondialdehyde,MDA)。
The above-mentioned mice were established as D-galactose-induced oxidative stress aging animal models, and the mice were induced according to (1) healthy group, (2) aging group, (3) low-dose group, (4) medium-dose group, (5) In the high-dose group, 10 female mice and 10 male mice were assigned to each group, and the mice in the healthy group were injected with 0.1-0.3g/kg bw of normal saline; for the control group, low-dose group, and middle-
本發明實驗結果圖中,a表示與WT組別相比,有統計學上顯著的差異(p<0.01);b表示與C組別相比有統計學上顯著的差異(p<0.01)。 In the graph of the experimental results of the present invention, a represents a statistically significant difference (p<0.01) compared with the WT group; b represents a statistically significant difference compared with the C group (p<0.01).
3.1延緩衰老相關基因表達試驗3.1 Anti-aging related gene expression test
以D-半乳糖誘導小鼠老化,再分別以3種不同的劑量(LD、MD、HD)餵食小鼠12周後,取小鼠血液樣品分離血球,並分析血球細胞中相關衰老基因的表達。其中SIRT1掌管同源染色體的重組及斷裂DNA的修復;SIRT6 掌管鹼基對錯誤序列的修復,同時能促進同源染色體的重組;SIRT7可將斷裂DNA末端進行連接,修復DNA損傷。 Induce the aging of mice with D-galactose, and then feed the mice with 3 different doses (LD, MD, HD) for 12 weeks, take blood samples from the mice to separate blood cells, and analyze the expression of related aging genes in blood cells . Among them, SIRT1 is in charge of the recombination of homologous chromosomes and the repair of broken DNA; SIRT6 It is in charge of the repair of base pair error sequences and can promote the recombination of homologous chromosomes; SIRT7 can connect the ends of broken DNA and repair DNA damage.
如圖所示,連續使用12周後,使用不同濃度的組別皆能有效提升SIRT1、SIRT6、SIRT7表達量,相較於老化組,DNA修復能力具有顯著的改善效果。 As shown in the figure, after 12 weeks of continuous use, groups with different concentrations can effectively increase the expression levels of SIRT1, SIRT6, and SIRT7. Compared with the aging group, the DNA repair ability has a significant improvement effect.
3.2血液生化實驗3.2 Blood biochemical experiments
以D-半乳糖誘導小鼠老化,依照3種低、中、高劑量管餵12周後,取小鼠血液樣品,分析抗氧化物指標SOD及GSH含量,因老化被視為是氧化物過多造成細胞的氧化而導致,因此其抗氧化指標越高,延緩老化的能力越強。 D-galactose was used to induce aging in mice. After 12 weeks of tube-feeding according to three low, medium and high doses, blood samples were taken from mice to analyze the content of antioxidant indicators SOD and GSH, because aging is considered to be excessive oxidation It is caused by the oxidation of cells, so the higher the antioxidant index, the stronger the ability to delay aging.
結果如圖所示,連續使用12周後,使用不同濃度的組別皆能有效提升抗氧化物質濃度,相較於老化組,抗氧化能力具有顯著的改善效果。 The results are shown in the figure. After 12 weeks of continuous use, the groups using different concentrations can effectively increase the concentration of antioxidant substances. Compared with the aging group, the antioxidant capacity has a significant improvement effect.
3.3組織、器官老化之生物活性指標測定3.3 Determination of biological activity indicators of tissue and organ aging
以D-半乳糖誘導小鼠老化,依照3種低、中、高劑量管餵12周後,取小鼠器官組織作為樣品,分別針對大腦組織、肝臟組織及心臟組織,分析其組織內氧化物的濃度,8-oxo-dG為人體中經常存在的氧化物,當氧化物濃度越高,組織器官內的細胞老化速度越快。 D-galactose was used to induce aging in mice, and after 12 weeks of tube-feeding according to three low, medium and high doses, mouse organ tissues were taken as samples, and the oxides in the tissues were analyzed for brain tissue, liver tissue and heart tissue respectively. The concentration of 8-oxo-dG is an oxide that often exists in the human body. When the concentration of oxides is higher, the aging speed of cells in tissues and organs is faster.
大腦為老化表現最主要的器官,氧化物質會導致組織內產生8-oxo-dG產物,產物濃度越高表示老化現象越嚴重,而若是能在不同器官中降低氧化物的濃度,例如大腦,能降低阿茲海默症或帕金森氏症的罹患機率;在心臟能降低心血管疾病的風險;在肝臟能減少肝臟代謝負擔,穩定肝指數。 The brain is the most important organ for aging. Oxidative substances will lead to the production of 8-oxo-dG products in the tissue. The higher the concentration of the product, the more serious the aging phenomenon. If the concentration of oxides can be reduced in different organs, such as the brain, it can Reduce the risk of Alzheimer's disease or Parkinson's disease; reduce the risk of cardiovascular disease in the heart; reduce the metabolic burden of the liver and stabilize the liver index in the liver.
結果如圖所示,老化產物與年輕小鼠組別無差異,表示能延緩腦部老化,而肝臟與心臟也是容易老化的器官,在這兩個器官中,也同樣能具有延緩老化產物生成的作用。 The results are shown in the figure, there is no difference between the aging products and the young mouse group, which means that it can delay the aging of the brain, and the liver and the heart are also organs that are prone to aging. In these two organs, the production of aging products can also be delayed effect.
3.4存活期試驗3.4 Survival test
以D-半乳糖誘導小鼠老化,依照3種不同劑量管餵直至小鼠自然死亡,並統計死亡數據,分析平均壽命及最大存活天數,以統計學分析顯著性差異,評估本發明是否具有延長壽命的作用。 Induce the aging of mice with D-galactose, feed them by tube according to 3 different doses until the mice die naturally, and count the death data, analyze the average life span and the maximum survival days, and use statistical analysis to evaluate whether the present invention has prolongation The role of life.
本存活期試驗中,直接進行小鼠的長期觀察,評估對生物體的延長壽命是否有直接的功效,藉由對小鼠施加三種不同劑量(HD、MD、LD)測試,每日餵食直至自然死亡,並統計數據結果。 In this survival test, long-term observation of mice is directly carried out to evaluate whether there is a direct effect on prolonging the life of the organism. By applying three different doses (HD, MD, LD) to the mice, they are fed daily until natural Death, and statistics result.
結果如圖所示,死亡率達50%時,未使用本發明的組別存活118天,而使用本發明且達建議劑量則延長壽命達200%,結果顯示本發明具有良好的延長壽命效果;未使用本發明的組別最長存活127天,使用本發明且達建議劑量組別最長則存活263天,延長207%的壽命。 The results are shown in the figure, when the mortality rate reached 50% , the group that did not use the present invention survived for 118 days, while using the present invention and reaching the recommended dose prolongs life by 200%. The results show that the present invention has a good effect of prolonging life; The longest survival of the group not using the present invention was 127 days, and the longest survival of the group using the present invention and reaching the recommended dose was 263 days, prolonging the life span of 207%.
根據動物試驗,本發明有助於延緩衰好之功效。與降低DNA氧化產物8-oxo-dG的含量,有效減緩腦部脂質過氧化的現象、降低腦部氧化損傷的程度,進而改善腦部氧化壓力上昇、腦部老化等現象。 According to animal experiments, the present invention helps to delay aging. And reduce the content of DNA oxidation product 8-oxo-dG, effectively slow down the phenomenon of lipid peroxidation in the brain, reduce the degree of oxidative damage in the brain, and then improve the phenomenon of increased brain oxidative stress and brain aging.
經上述實驗結果證實,在延緩老化相關基因表達、提高抗氧化物指標SOD及GSH含量、降低組織器官氧化物濃度等老化相關指標上,本發明之發酵組合物具有能有效的延緩老化的效果。 The above experimental results prove that the fermentation composition of the present invention has the effect of effectively delaying aging in terms of delaying the expression of aging-related genes, increasing the content of antioxidant indicators SOD and GSH, and reducing the concentration of oxides in tissues and organs.
上述實驗數據為在特定條件之下所獲得的初步實驗結果,其僅用以易於瞭解或參考本發明之技術內容而已,其尚需進行其他相關實驗。該 實驗數據及其結果並非用以限制本發明之權利範圍。 The above experimental data are preliminary experimental results obtained under specific conditions, which are only used for easy understanding or reference to the technical content of the present invention, and other related experiments are still required. Should Experimental data and results thereof are not intended to limit the scope of the present invention.
前述較佳實施例僅舉例說明本發明及其技術特徵,該實施例之技術仍可適當進行各種實質等效修飾及/或替換方式予以實施;因此,本發明之權利範圍須視後附申請專利範圍所界定之範圍為準。 The above-mentioned preferred embodiments only illustrate the present invention and its technical characteristics, and the technology of this embodiment can still be implemented in various substantially equivalent modifications and/or replacements; therefore, the scope of rights of the present invention depends on the appended patent application The scope defined by the scope shall prevail.
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