WO2024026740A1 - 具有nmn推迟老化功效的发酵组合物 - Google Patents

具有nmn推迟老化功效的发酵组合物 Download PDF

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WO2024026740A1
WO2024026740A1 PCT/CN2022/110018 CN2022110018W WO2024026740A1 WO 2024026740 A1 WO2024026740 A1 WO 2024026740A1 CN 2022110018 W CN2022110018 W CN 2022110018W WO 2024026740 A1 WO2024026740 A1 WO 2024026740A1
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fermentation
preparation
aging
fruits
vegetables
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PCT/CN2022/110018
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English (en)
French (fr)
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黄柽
赖怡君
赖柏儒
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大汉酵素生物科技股份有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/13—Nucleic acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts

Definitions

  • the invention provides a fermentation composition that can effectively delay the expression of aging-related genes, increase the content of antioxidant indicators SOD and GSH, and reduce the oxidation concentration of tissues and organs to achieve anti-aging effects.
  • NMN nicotinamide mononucleotide
  • vitamin B3 nicotinic acid
  • NAD + biosynthesis is composed of a phosphate group and a core containing ribose and nicotinamide.
  • Biologically active nucleotides formed by glycoside reactions.
  • NMN has been approved by Japan for use in food additives. Japan uses fermentation technology to produce NMM and then purifies it to obtain high-concentration NMN substances. However, except for Japan, no other country has approved the use of NMN in food. NMN that is currently required by health care products is an imitation NMN substance added with vitamin B3. Although vitamin B3 is the precursor substance of NMN, it still needs enzymes in the body to convert it before it can take effect, and it can only become active after generating NAD + .
  • NMN can mainly improve the regeneration ability of cells.
  • the present invention provides a preparation method of a fermentation composition with NMN delaying aging effect, including:
  • Fruit and vegetable extraction step physically squeeze different fruits and vegetables until the fruits or fruiting bodies are in pieces, while retaining the juice and pomace or fruiting bodies to obtain multiple first extracts;
  • Negative pressure wall breaking method i.e., negative pressure wall breaking extraction treatment: Add 0.15 to 1.5% brown sugar to increase the isotonic pressure of the first extract, and at the same time use negative pressure extraction at 35 to 45 cmHg In a near-vacuum environment, extraction is continued for 3 to 15 days to rupture the cell walls of fruits and vegetables, releasing intracellular nutrients and intracellular polysaccharides to obtain multiple second extracts;
  • a yeast strain is implanted into the second extract prepared in step 2) at a ratio of 0.5 to 1.5% (10 7 CFU/ml).
  • the fermentation temperature is controlled at 2 to 12°C and the fermentation is continued. 3 to 15 days.
  • the bacteria are in low activity, allowing the enzyme action to be greater than the action of the bacteria, slowing down the consumption of sugars, preservatives during the fermentation process, improving the decomposition of cellulose, and promoting the decomposition of cell walls to obtain multiple first ferment;
  • Submerged fermentation stage A lactic acid bacterium is implanted into the first fermentation material prepared in step 3) at a ratio of 0.5 to 1.5% (10 7 CFU/ml); 0.15 to 0.25% of isomaltooligosaccharide.
  • the fermentation temperature Control at 20-35°C and continue fermentation for 5-25 days.
  • lactic acid bacteria consume sugar to generate lactic acid, lower the pH value to achieve preservative effect, and increase acidity and reduce viscosity to obtain multiple secondary fermentation products;
  • Chelation fermentation stage Use an acetic acid bacteria to implant D-sorbitol into the second fermentation material prepared in step 4) at a ratio of 0.5 to 1.5% (107CFU/ml) and 2.5 to 5.5%, respectively.
  • the fermentation temperature Control the temperature at 10 to 25°C and continue fermentation for 5 to 20 days.
  • the oxygen-consuming environment consumes glucose to produce acetic acid, consumes ethanol, and increases the acidity and reduces the viscosity to obtain multiple third fermentation products; and
  • Drying and granulating step Filter the third fermented product prepared in step 5) respectively to obtain multiple fermentation liquids and retain the fermentation liquid, then mix the fermentation liquids in equal proportions, and spray-dry and granulate to obtain The fermentation composition.
  • the present invention also provides a fermentation composition with NMN delaying aging effect, including vegetable and fruit raw materials, wherein the vegetable and fruit raw materials include one or more of truffles, mushroom fruiting bodies, avocados, tomatoes or edamame, and through A special fermentation preparation method is used to obtain the fermentation composition.
  • the preparation method of the fermentation composition with NMN delaying aging effect wherein the fruit and vegetable extraction step further includes the step of mixing the fruits and vegetables with water.
  • the preparation method of the fermentation composition with NMN delaying aging effect wherein the fruits and vegetables include one or more of truffles, mushroom fruiting bodies, avocados, tomatoes or edamame.
  • the preparation method of the fermentation composition with NMN delaying aging effect wherein when the fruits and vegetables in the fruit and vegetable extraction step are truffles or mushroom fruiting bodies, before being physically pressed, further comprising: Soak in hot water at 65-100°C for 25-35 minutes to soften.
  • the preparation method of the fermentation composition with NMN delaying aging effect wherein the mixing ratio of the fruits and vegetables to water is respectively: the ratio of truffles or mushroom fruiting bodies to water is 1:40; The ratio of avocado to water is 1:25; the ratio of tomatoes to water is 1:1; the ratio of edamame to water is 1:30.
  • the preparation method of the fermentation composition with NMN delaying aging effect wherein the pH value of the second extract is between 5.5 and 7; the pH value of the first fermentation product 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.
  • the preparation method of the fermentation composition with NMN delaying aging effect wherein the second extract sugar content (Degrees Brix, °Bx) is between 21 and 28°Bx; the first fermentation
  • the sugar content of the fermented product ranges from 15 to 34°Bx; the sugar content of the second fermented product ranges from 15 to 38°Bx; and the sugar content of the third fermented product ranges from 10 to 50°Bx.
  • lactic acid bacteria are selected from the group consisting of Lactobacillus plantarum (L.plantarum), Lactobacillus bulgaricus (L.delbrueckii), Lactococcus lactis (L. .lactis), one of L. acidophilus (L. acidophilus) or B. bifidum (B. bifidum).
  • yeast or the acetic acid bacteria are selected from the group consisting of S. fibuligera, S. cerevisiae, membrane In P. faciens, S. pombe, A. hansenii, A. xylinum or A. suboxydans kind of.
  • the fermentation composition with the effect of NMN on delaying aging is used to prepare drugs that delay aging.
  • the delaying of aging refers to delaying the expression of aging-related genes, increasing the content of antioxidant indicators SOD and GSH, and reducing tissue Organ oxygen concentration.
  • the main purpose of the present invention is to provide a fermentation composition with the effect of NMN on delaying aging for use in preparing drugs that delay aging, wherein the drugs can be used to delay the aging of the heart, brain, and liver.
  • Figures 1A-1D show the differences in sugar content, pH value, cellulose, and polysaccharides between temperature gradient fermentation technology and traditional fermentation technology.
  • Figures 2A-2C show the results of the fermentation composition with NMN anti-aging effect of the present invention on the gene expression of mouse blood cells.
  • Figures 3A-3B are graphs showing the results of the fermentation composition with the NMN anti-aging effect of the present invention on the concentration of antioxidant indicators in the blood of mice.
  • Figures 4A-4C are graphs showing the results of the fermentation composition with the NMN aging-delaying effect of the present invention on the concentration of oxidants in mouse tissues and organs.
  • Figure 5 is a graph showing the results of the test on prolonging the life span of mice using the fermentation composition with NMN delaying aging effect of the present invention.
  • Example 1 Preparation method of fermentation composition with similar NMN activity
  • Temperature gradient fermentation is controlled with lactic acid bacteria, yeast and acetic acid bacteria respectively with different types of fruits and vegetables such as truffles, avocados, tomatoes (tomatoes), edamame, etc., and the fermentation liquid after fermentation is filtered to retain the fermentation liquid; the fermentation composition
  • the temperature gradient fermentation control steps include:
  • Fruit and vegetable extraction step physically squeeze different fruits and vegetables until the fruits or fruiting bodies are in the form of broken pieces, while retaining the juice and pomace or fruiting bodies to obtain multiple first extracts;
  • Negative pressure wall-breaking extraction treatment Add 0.15 to 1.5% brown sugar to the first extracts respectively to increase the isotonic pressure of the first extracts, and at the same time use negative pressure extraction to close the range of 35 to 45 cmHg. In a vacuum environment, the extraction is continued for 3 to 15 days to rupture the cell walls of fruits and vegetables, releasing intracellular nutrients and intracellular polysaccharides, and obtaining multiple second extracts respectively;
  • a yeast is implanted into multiple second extracts at a ratio of 0.5 to 1.5% (10 7 CFU/ml), the fermentation temperature is controlled at 2 to 12°C, and the fermentation is continued for 3 to 15 Days, at this stage, the bacteria are in low activity, allowing the enzyme action to be greater than the action of the bacteria, slowing down the consumption of sugars, preventing corrosion during the fermentation process, improving the decomposition of cellulose, promoting the decomposition of cell walls, and obtaining multiple first fermentations.
  • the yeast is selected from one of S. fibuligera, S. cerevisiae, P. faciens or S. pombe;
  • Submerged fermentation stage A lactic acid bacterium is implanted into multiple first fermentation materials at a ratio of 0.5 to 1.5% (10 7 CFU/ml); 0.15 to 0.25% isomaltooligosaccharide, and the fermentation temperature is controlled at 20 to 35°C, continuous fermentation for 5 to 25 days.
  • lactic acid bacteria consume sugar to generate lactic acid, lower the pH value to achieve a preservative effect, and increase acidity and reduce viscosity to obtain multiple second fermentations.
  • the lactic acid bacteria are selected from plants.
  • Lactobacillus (L.plantarum), Lactobacillus bulgaricus (L.delbrueckii), Lactococcus lactis (L.lactis), Lactobacillus acidophilus (L.acidophilus) or B.bifidum ;
  • Chelation fermentation stage Use an acetic acid bacteria to implant into multiple second fermentation materials at a ratio of 0.5 to 1.5% (107CFU/ml); 2.5 to 5.5% D-sorbitol, and the fermentation temperature is controlled at 10 to 25°C, continuous fermentation for 5 to 20 days.
  • the oxygen-consuming environment consumes glucose to produce acetic acid, consumes ethanol, increases acidity and reduces viscosity, and obtains multiple third fermentation products.
  • the acetic acid bacteria are selected from weak oxidizing acid-promoting bacteria. (A. hansenii), one of A. xylinum or A. suboxydans; and
  • Drying and granulating step filter the third fermentation product obtained by chelate fermentation in step 5) respectively to obtain multiple fermentation liquids and retain the fermentation liquids, and then mix the fermentation liquids in equal proportions. , spray drying and granulating to obtain the fermentation composition.
  • Aspergillus fibuligera is used as an example of yeast, and other yeasts such as Saccharomyces cerevisiae (S.cerevisiae), Pichia pastoris (P.faciens) or Schizosaccharomyces pombe (S. pombe) are applicable to the present invention; Lactobacillus plantarum (L. plantarum) is taken as an example for illustration, and other lactic acid bacteria such as: Lactobacillus bulgaricus (L. delbrueckii), Lactococcus lactis (L. lactis), Lactobacillus acidophilus ( L. acidophilus) or B. bifidum (B.
  • the acetic acid bacteria are weakly oxidizing acid-promoting bacteria (A. hansenii) as an example, and other acetic acid bacteria such as xylinum acetic acid bacteria (A. xylinum) or Weakly oxidizing Acetobacter (A. suboxydans) are all suitable for use in the present invention.
  • the fruit and vegetable extraction step further includes the step of mixing the fruits and vegetables with water.
  • the fruits and vegetables include truffles, mushroom fruiting bodies, avocados, tomatoes or edamame.
  • the step further includes a softening step of soaking in hot water at 65 to 100° C. for 25 to 35 minutes before physically pressing.
  • the mass ratios of the fruits and vegetables mixed with water are: the ratio of truffles or mushroom fruiting bodies to water is 1:40; the ratio of avocados to water is 1:25; the ratio of tomatoes to water The ratio of edamame to water is 1:30.
  • the pH value of the second extract is between 5.5 and 7; the pH value of the first fermentation product is between 5 and 6.5; the pH value of the second fermentation product is between 4.5 and 5.5; The pH value of the third fermentation product is between 3 and 3.5.
  • the sugar degree (Degrees Brix, °Bx) of the second extract is between 21 and 28°Bx; the sugar degree of the first fermentation product is between 15 and 34°Bx; and the sugar degree of the second fermentation product is between 15 and 34°Bx.
  • the sugar content of the third fermented product is between 10 and 50°Bx.
  • 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. Endopolysaccharide.
  • 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 (traditional fermentation temperature is controlled at 25-30°C).
  • the results of fermentation sugar content are shown in Figure 1A.
  • the sugar content of the traditional fermentation group changes greatly, and the sugar content of each stage decreases rapidly, which can easily cause the fermentation end point to be reached early, and the lower sugar content cannot inhibit spoilage bacteria, which may cause tank failure.
  • Temperature gradient fermentation controls the sugar content to at least 15 degrees Brix (°Bx). It is not until the chelate fermentation stage (acetic acid bacteria) that the carbon source is completely fermented and reduced to below 15°Bx.
  • the pH value results of the fermentation broth are shown in Figure 1B.
  • the strains grow rapidly and consume carbon sources.
  • the pH value quickly drops below 3.8, causing the fermentation strain reaction to quickly reach the end point.
  • the pH value was adjusted before entering the next fermentation stage, and compounds such as calcium carbonate and sodium hydroxide were added, which may invisibly increase the intake of inorganic mineral salts for fermented products.
  • the temperature gradient fermentation technology is used to treat the yeast at low temperature during the fermentation stage, so that the fermentation liquid provides a good growth environment for other bacterial species before entering the subsequent fermentation stage, which can ensure the extension of the fermentation end point.
  • the chelation fermentation stage is processed at 15 to 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 reach about 3, providing a preservative effect.
  • the results of fermented cellulose content are shown in Figure 1C.
  • the cell wall of herbal plants is mainly composed of cellulose.
  • the strain activity is greater than the enzyme activity, which easily reduces the pH value and carbon source quickly, and the fermentation end point is reached early.
  • Most of the Herbal plant cells cannot be completely decomposed, and the release ratio of their components is limited; in temperature gradient fermentation technology, the activity of controlled enzymes in the low-temperature fermentation stage is greater than the activity of the bacteria, which makes the carbon source decrease slowly and decompose the cell wall more completely.
  • the results show that temperature gradient fermentation The cellulose content of the technology is higher than that of traditional fermentation technology, indicating that the proportion of cell walls being decomposed is high.
  • the results of fermented polysaccharide content are shown in Figure 1D.
  • the fungal cell wall is mainly composed of chitosan.
  • the cell wall must be destroyed to release the intracellular polysaccharide.
  • the strain activity is greater than the enzyme activity, which easily changes the pH value, The carbon source decreases rapidly and the fermentation endpoint is reached early.
  • Most fungal cells cannot be completely decomposed and the release ratio of their components is limited.
  • temperature gradient fermentation technology the activity of controlled enzymes in the low-temperature fermentation stage is greater than the activity of the bacteria, causing the carbon source to decrease slowly and affecting the cell wall.
  • the decomposition 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.
  • (T) is the temperature gradient fermentation technology group
  • (C) is the traditional fermentation group (traditional fermentation temperatures are all controlled at 25-30°C).
  • the NMN-like fermentation composition of the present invention has the effect of delaying aging. According to the "Method for Evaluation of the Health-care Effect of Delaying Aging of Healthy Foods", the NMN-like fermentation composition of the present invention obtained a positive response for at least one indicator item.
  • the NMN-like fermentation composition of the present invention can prevent, delay, and inhibit individual appearance aging, functional decline, and behavioral retardation.
  • the following animal experiments are based on a 60 kg adult.
  • the dose conversion is based on the recommended human daily consumption of 1g/day per kilogram of body weight.
  • the animal dose is divided into the following 5 groups:
  • mice of the C57BL/6 variety The mice are 6 to 12 weeks old. In the medium-dose group and (5) high-dose group, 10 female mice and 10 male mice were randomly assigned to each group.
  • mice were used to establish a D-galactose-induced oxidative stress aging animal model, and the mice were induced according to (1) healthy group, (2) aging group, (3) low-dose group, (4) medium-dose group, (5)
  • the high-dose group 10 female mice and 10 male mice were allocated to each group, and the mice in the healthy group were injected with 0.1-0.3g/kg bw of physiological saline; the control group, low-dose group, medium-dose group, The high-dose group was injected with 0.1-0.3g/kg bw D-galactose solution, and blood was taken to detect lipid oxidation malondialdehyde (MDA) 6 to 10 weeks after induction.
  • MDA lipid oxidation malondialdehyde
  • D-galactose was used to induce aging in mice, and then the mice were fed with three different doses (LD, MD, HD) for 12 weeks. After 12 weeks, mouse blood samples were taken to isolate blood cells, and the expression of related aging genes in the blood cells was analyzed. .
  • SIRT1 is responsible for the recombination of homologous chromosomes and the repair of broken DNA
  • SIRT6 is responsible for the repair of base pair error sequences and can also promote the recombination of homologous chromosomes
  • SIRT7 can connect the ends of broken DNA and repair DNA damage.
  • the groups using the fermentation composition of the present invention at different concentrations can effectively increase the expression levels of SIRT1, SIRT6, and SIRT7. Compared with the aging group, the DNA repair ability has been significantly improved. Effect.
  • D-galactose was used to induce aging in mice. After 12 weeks of tube feeding at three low, medium and high doses, blood samples were taken from the mice to analyze the content of antioxidant indicators SOD and GSH. Aging is considered to be an oxidation peroxide. It is mostly caused by the oxidation of cells, so the higher its antioxidant index, the stronger its ability to delay aging.
  • D-galactose was used to induce aging in mice. After 12 weeks of tube feeding at three low, medium and high doses, the mouse organ tissues were taken as samples. The brain tissue, liver tissue and heart tissue were analyzed to analyze the oxidation in the tissues.
  • the concentration of 8-oxo-dG is an oxide that often exists in the human body. When the concentration of oxides is higher, the cells in tissues and organs age faster.
  • the brain is the most important organ showing signs of aging. Oxidative substances will cause the production of 8-oxo-dG products in tissues. The higher the product concentration, the more serious the aging phenomenon. If the concentration of oxidants can be reduced in different organs, such as the brain, it can Reduces the risk of Alzheimer's disease or Parkinson's disease; reduces the risk of cardiovascular disease in the heart; reduces liver metabolic burden and stabilizes liver index.
  • D-galactose was used to induce aging in mice, and the mice were tube-fed according to three different doses until the mice died naturally.
  • the death data were collected, the average life span and maximum survival days were analyzed, and the significant differences were statistically analyzed to evaluate whether the present invention has the ability to prolong the life of the mice. The role of longevity.
  • mice In this survival period test, long-term observation of mice was directly carried out to evaluate whether it has a direct effect on extending the life span of organisms.
  • the test was conducted by applying three different doses (HD, MD, LD) to mice, and fed them every day until they naturally Death, and statistics of the results.
  • the fermentation composition of the present invention helps delay aging. It reduces the content of DNA oxidation product 8-oxo-dG, effectively slows down the phenomenon of lipid peroxidation in the brain, reduces the degree of oxidative damage in the brain, and thereby improves the rise in oxidative stress in the brain and brain aging.
  • the above experimental results confirm 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 oxidation concentration of tissues and organs.

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Abstract

一种具有NMN推迟老化功效的发酵组合物及其在用于制备推迟老化的药物中的用途。该发酵组合物将蔬果原料通过特殊发酵制备方法（包括蔬果萃取，负压破壁，低温发酵，深层发酵，螯合发酵，干燥造粒等步骤）取得，其中所述蔬果原料包含松露、菇覃类子实体、酪梨、西红柿或毛豆。

Description

具有NMN推迟老化功效的发酵组合物 技术领域
本发明提供一种发酵组合物，可以有效的推迟老化相关基因表达、提高抗氧化物指标SOD及GSH含量、降低组织器官氧化物浓度，达到抗老化的效果。
背景技术
NMN(烟酰胺单核苷酸)是维生素B3(烟酸)的衍生物之一，也是NAD +生物合成的中间产物，是由磷酸基团和含有核糖(ribose)和烟酰胺(nicotinamide)的核苷反应形成的生物活性核苷酸。
NMN目前已被日本核准可使用于食品添加物中，日本是以发酵技术产生出NMM后，以纯化方式得到高浓度NMN物质，但至今除日本外，尚无其他国家核准NMN使用于食品中，目前是受保健品要求的NMN皆为添加维生素B3仿造NMN物质，维生素B3虽为NMN前驱物质，但在发挥作用前仍需要体内的酵素进行转换，生成NAD +后才具有活性。
然而，市售NMN要求的保健品，实际上皆为添加维生素B3，在老化的族群中，其体内的代谢率并无法完整的将维生素B3转化为NAD+，因此摄取维生素B3很难达到如同NMN的推迟老化作用，另外在一些医师的研究中，NMN主要能提高细胞的再生能力，但对于癌细胞是否有相同作用，并无完整研究证实，因此可能存在食用上的风险。
有鉴于此，相关领域亟需开发一种类似NMN成分且具有推迟老化的潜力的组合物。
发明内容
为解决上述问题，本发明提供一种具有NMN推迟老化功效的发酵组合物的制备方法，包含:
(1)蔬果萃取步骤：将不同蔬果分别以物理方式压榨蔬果直至果实或子实体呈碎块状，同时保留汁液与果渣或子实体，以取得多个第一萃取物；
(2)负压破壁工法(即负压破壁萃取处理)：添加0.15～1.5％黑糖，以提高所述第一萃取物的等渗透压，并同时以负压萃取方式在35～45cmHg的近真空环境中，持续萃取3～15天，使蔬果细胞壁破裂，释放出细胞内营养成分及胞内多糖，以取得多个第二萃取物；
(3)低温发酵阶段：以一酵母菌，以比例0.5～1.5％(10 7CFU/ml)分别植入步骤2)制备的第二萃取物中，发酵温度控制在2～12℃，持续发酵3～15天，在此阶段菌种处于低活性、让酵素作用大于菌种作用，减缓糖类的消耗，在发酵过程中防腐，提高纤维聚糖的分解作用，促进细胞壁分解，以取得多个第一发酵物；
(4)深层发酵阶段:以一乳酸菌，以比例0.5～1.5％(10 7CFU/ml)；0.15～0.25％的异麦芽寡糖分别植入步骤3)制备的第一发酵物中，发酵温度控制在20～35℃，持续发酵5～25天，在此阶段乳酸菌消耗糖类生成乳酸，降低pH值达到防腐作用，且提高酸度降低黏稠度，以取得多个第二发酵物；
(5)螯合发酵阶段:以一醋酸菌，以比例0.5～1.5％(107CFU/ml)；2.5～5.5％的D-山梨醇分别植入步骤4)制备的第二发酵物中，发酵温度控制在10～25℃，持续发酵5～20天，在此阶段耗氧环境消耗葡萄糖产生醋酸，消耗乙醇且提高酸度降低黏稠度，以取得多个第三发酵物；以及
(6)干燥造粒步骤:将步骤5)制备的第三发酵物，分别进行过滤以取得多个 发酵液并保留发酵液，再将发酵液以等比例混合后，以喷雾干燥造粒，取得所述的发酵组合物。
本发明另提供一种具有NMN推迟老化功效的发酵组合物，包含蔬果原料，其中所述蔬果原料包含松露、菇蕈类子实体、酪梨、西红柿或毛豆中的一种或多种，并通过特殊发酵制备方法取得发酵组合物。
在本发明的实施例中，该具有NMN推迟老化功效的发酵组合物的制备方法，其中所述蔬果萃取步骤进一步包含将所述蔬果与水混合的步骤。
在本发明的实施例中，该具有NMN推迟老化功效的发酵组合物的制备方法，其中所述蔬果包含松露、菇蕈类子实体、酪梨、西红柿或毛豆中的一种或多种。
在本发明的实施例中，该具有NMN推迟老化功效的发酵组合物的制备方法，其中所述蔬果萃取步骤的蔬果为松露或菇蕈类子实体时，在以物理方式压榨前，进一步包含以65～100℃热水浸泡25～35分钟软化的步骤。
在本发明的实施例中，该具有NMN推迟老化功效的发酵组合物的制备方法，其中所述蔬果与水混合的比例分别为：松露或菇蕈类子实体与水的比例为1:40；酪梨与水的比例为1:25；西红柿与水的比例为1:1；毛豆与水的比例为1:30。
在本发明的实施例中，该具有NMN推迟老化功效的发酵组合物的制备方法，其中所述第二萃取物pH值介于5.5～7；所述第一发酵物pH值介于5～6.5；所述第二发酵物pH值介于4.5～5.5；所述第三发酵物pH值介于3～3.5。
在本发明的实施例中，该具有NMN推迟老化功效的发酵组合物的制备方法，其中所述第二萃取物糖度(Degrees Brix,°Bx)介于21～28°Bx；所述第一发酵物糖度介于15～34°Bx；所述第二发酵物糖度介于15～38°Bx；所述第三发酵物糖度 介于10～50°Bx。
在本发明的实施例中，具有NMN推迟老化功效的发酵组合物的制备方法，其中所述乳酸菌选自植物乳杆菌(L.plantarum)、保加利亚乳杆菌(L.delbrueckii)、乳酸乳球菌(L.lactis)、嗜酸乳杆菌(L.acidophilus)或比菲德氏菌(B.bifidum)中的一种。
在本发明的实施例中，具有NMN推迟老化功效的发酵组合物的制备方法，其中所述酵母菌或所述醋酸菌选自白曲菌(S.fibuligera)、酿酒酵母(S.cerevisiae)、膜毕赤酵母(P.faciens)、粟酒裂殖酵母(S.pombe)、弱氧化促酸菌(A.hansenii)、木质醋酸菌(A.xylinum)或弱氧化醋酸杆菌(A.suboxydans)中的一种。
在本发明的实施例中，具有NMN推迟老化功效的发酵组合物用于制备推迟老化的药物的用途，其推迟老化是指推迟老化相关基因表达、提高抗氧化物指标SOD及GSH含量、降低组织器官氧化物浓度。
本发明的主要目的是另提供具有NMN推迟老化功效的发酵组合物用于制备推迟老化的药物的用途，其中所述药物可以用于推迟心脏、脑部、肝脏的老化。
附图说明
图1A-1D显示温度梯度发酵技术与传统发酵技术对于糖度、pH值、纤维素、多糖体的差异。
图2A-2C显示本发明具有NMN推迟老化功效的发酵组合物对于小鼠血球细胞基因表达量的结果图。
图3A-3B为本发明具有NMN推迟老化功效的发酵组合物对于小鼠血液抗氧化物指标浓度的结果图。
图4A-4C为本发明具有NMN推迟老化功效的发酵组合物对于小鼠组织器官 中氧化物浓度的结果图。
图5为本发明具有NMN推迟老化功效的发酵组合物对于延长小鼠寿命试验各项结果图。
具体实施方式
本发明将可由以下的实施例说明而得到充分了解，使得熟习本技艺的人士可以据以完成之，然而本案的实施并非可由下列实施例而被限制其实施型态，熟习本技艺的人士仍可依据除此揭露的实施例的精神推演出其他实施例，该等实施例皆当属于本发明的范围。
实施例一、类似NMN活性的发酵组合物的制备方法
以乳酸菌、酵母菌及醋酸菌分别与松露、酪梨、西红柿(番茄)、毛豆等不同的蔬果种类进行温度梯度发酵调控，并将发酵完成的发酵液进行过滤后保留发酵液；发酵组合物的温度梯度发酵调控步骤包含:
(1)蔬果萃取步骤:将不同蔬果分别以物理方式压榨蔬果，直至果实或子实体呈碎块状，同时保留汁液与果渣或子实体，获得多个第一萃取物；
(2)负压破壁萃取处理:分别向第一萃取物中添加0.15～1.5％的黑糖，以提高该些第一萃取物等渗透压，并同时以负压萃取方式在35～45cmHg的近真空环境中，持续萃取3～15天，使蔬果细胞壁破裂，释放出细胞内营养成分及胞内多糖，分别获得多个第二萃取物；
(3)低温发酵阶段:以一酵母菌，以比例0.5～1.5％(10 7CFU/ml)分别植入多个第二萃取物中，发酵温度控制在2～12℃，持续发酵3～15天，在此阶段菌种处于低活性、让酵素作用大于菌种作用，减缓糖类的消耗，在发酵过程中防腐，提高 纤维聚糖的分解作用，促进细胞壁分解，分别获得多个第一发酵物，所述酵母菌选自白曲菌(S.fibuligera)、酿酒酵母(S.cerevisiae)、膜毕赤酵母(P.faciens)或粟酒裂殖酵母(S.pombe)中的一种；
(4)深层发酵阶段:以一乳酸菌，以比例0.5～1.5％(10 7CFU/ml)；0.15～0.25％异麦芽寡糖分别植入多个第一发酵物中，发酵温度控制在20～35℃，持续发酵5～25天，在此阶段乳酸菌消耗糖类生成乳酸，降低pH值达到防腐作用，且提高酸度降低黏稠度，分别获得多个第二发酵物，所述乳酸菌系选自植物乳杆菌(L.plantarum)、保加利亚乳杆菌(L.delbrueckii)、乳酸乳球菌(L.lactis)、嗜酸乳杆菌(L.acidophilus)或比菲德氏菌(B.bifidum)中的一种；
(5)螯合发酵阶段:以一醋酸菌，以比例0.5～1.5％(107CFU/ml)；2.5～5.5％D-山梨醇分别植入多个第二发酵物中，发酵温度控制在10～25℃，持续发酵5～20天，在此阶段耗氧环境消耗葡萄糖产生醋酸，消耗乙醇且提高酸度降低黏稠度，获得多个第三发酵物，所述醋酸菌系选自弱氧化促酸菌(A.hansenii)、木质醋酸菌(A.xylinum)或弱氧化醋酸杆菌(A.suboxydans)中的一种；以及
(6)干燥造粒步骤:将步骤5)螯合发酵制得的第三发酵物，分别进行过滤以取得多个发酵液并保留该些发酵液，再将该些发酵液以等比例混合后，以喷雾干燥造粒，取得该发酵组合物。
本具体实施例一中酵母菌以白曲菌(S.fibuligera)为例，其他酵母菌如酿酒酵母(S.cerevisiae)、膜毕赤酵母(P.faciens)或粟酒裂殖酵母(S.pombe)均适用于本发明；乳酸菌以植物乳杆菌(L.plantarum)为例进行说明，其他乳酸菌如：保加利亚乳杆菌(L.delbrueckii)、乳酸乳球菌(L.lactis)、嗜酸乳杆菌(L.acidophilus)或比菲德氏菌(B.bifidum)均适用于本发明；醋酸菌以弱氧化促酸菌(A.hansenii) 为例，其他醋酸菌如木质醋酸菌(A.xylinum)或弱氧化醋酸杆菌(A.suboxydans)均适用于本发明。
在一实施例中，蔬果萃取步骤进一步包含将蔬果与水混合的步骤。
在一实施例中，该蔬果包含松露、菇蕈类子实体、酪梨、西红柿或毛豆。
在一实施例中，该蔬果萃取步骤的蔬果为松露或菇蕈类子实体时，在以物理方式压榨前，进一步包含以65～100℃热水浸泡25～35分钟软化的步骤。
在一实施例中，该些蔬果与水混合的质量比分别为：松露或菇蕈类子实体与水的比例为1:40；酪梨与水的比例为1:25；西红柿与水的比例为1:1；毛豆与水的比例为1:30。
在一实施例中，所述第二萃取物pH值介于5.5～7；所述第一发酵物pH值介于5～6.5；所述第二发酵物pH值介于4.5～5.5；所述第三发酵物pH值介于3～3.5。
在一实施例中，所述第二萃取物糖度(Degrees Brix,°Bx)介于21～28°Bx；所述第一发酵物糖度介于15～34°Bx；所述第二发酵物糖度介于15～38°Bx；所述第三发酵物糖度介于10～50°Bx。
实施例二、温度梯度发酵调控实验
本发明的温度梯度发酵调控技术与传统的发酵技术有着很大的区别，本发明的温度梯度发酵调控技术可以具有更完整的发酵效果，且能保留更多的纤维素及释放出更多的胞内多糖。
本发明的温度梯度发酵调控技术对比传统发酵技术实验，(T)为温度梯度发酵技术组；(C)为传统发酵组(传统发酵温度接控制在25～30℃)。
Figure PCTCN2022110018-appb-000001
Figure PCTCN2022110018-appb-000002
发酵糖度的结果如图1A所示，传统发酵组糖度变化大，每阶段的糖度下降快速，容易造成发酵终点提早达成，且糖度降低无法抑制腐败菌，可能造成败槽。温度梯度发酵将糖度控制在至少15糖度(Degrees Brix,°Bx)以上，直到螯合发酵阶段(醋酸菌)才完整将碳源进行发酵，降低至15°Bx以下。
发酵液pH值结果如图1B所示，传统发酵组因温度适合酵母菌株生长，菌种快速生长并消耗碳源，此时pH值快速降低至3.8以下，造成发酵菌株反应快速达到终点，在后续菌株投入后作用无法有效达成生长发酵，以往会在进入下个发酵阶段前进行pH值的调整，加入例如碳酸钙、氢氧化钠等化合物，对发酵产品来说可能无形增加无机矿物盐的摄入(如钠)，运用温度梯度发酵技术，在酵母发酵阶段以低温处理，使得发酵液在进入后续发酵阶段前提供其他菌种良好的生长环境，能确保发酵终点的延长。螯合发酵阶段以15～20℃处理，此时醋酸菌在耗氧环境下能大量生成有机酸，使产品pH值达到3左右，提供防腐作用。
发酵纤维素含量结果如图1C所示，草本植物细胞壁以纤维聚糖为主成分，在传统发酵方法中，菌株活性大于酵素活性，容易使pH值、碳源快速降低，发酵终点提前达成，多数草本植物细胞无法完整进行分解，其成分释放比例有限；温度梯度发酵技术中，低温发酵阶段控制酵素活性大于菌种活性，使得碳源下降缓慢，对细胞壁的分解能更完全，结果中表明温度梯度发酵技术纤维素含量皆高于传统发酵技术，表明细胞壁被分解的比例高。
发酵多糖含量结果如图1D所示，真菌细胞壁以几丁聚糖为主成分，必须将细胞壁破坏后才能释放出胞内多糖，在传统发酵方法中，菌株活性大于酵素活性，容易使pH值、碳源快速降低，发酵终点提前达成，多数真菌细胞无法完整进行分解，其成分释放比例有限；温度梯度发酵技术中，低温发酵阶段控制酵素活性大于菌种活性，使得碳源下降缓慢，对细胞壁的分解能更完全，结果中表明温度梯度发酵技术多糖体含量皆高于传统发酵技术，表明释放的胞内多糖比例能有效提高。
图1A-1D中：(T)为温度梯度发酵技术组；(C)为传统发酵组(传统发酵温度皆控制在25～30℃)。
实施例三、实验动物与实验方法
本发明类NMN发酵组合物具有推迟衰老的效果。按照“健康食品之推迟衰老保健功效评估方法”进行分析，本发明的类NMN发酵组合物获得至少一项指针项目的正反应。本发明的类NMN发酵组合物可防止、推迟、抑制个体的外观老化、功能上的衰退及行为迟缓。以下动物实验以60公斤的成人为基准，剂量换算以人体每日每公斤体重人体建议食用量1g/天换算动物剂量，分成下述5组:
(1.)健康组(WT,wild type)：未加入任何样本
(2.)老化组(C,control，对照组)：未加入任何样本
(3.)低剂量0.5倍剂组(LD,low dose)
(4.)中剂量1倍剂量组(MD,medium dose)
(5.)高剂量1.5倍剂量为HD(high dose)
本发明动物实验使用的为C57BL/6品种的小鼠，该小鼠周龄为6～12周，并依照(1)健康组、(2)老化组、(3)低剂量组、(4)中剂量组、(5)高剂量组，每组随机分配10只雌鼠以及10只雄鼠。
将上述小鼠建立D-半乳糖诱发氧化压力老化动物模型，将小鼠诱导依照(1)健康组、(2)老化组、(3)低剂量组、(4)中剂量组、(5)高剂量组，各组别每组分配10只雌鼠以及10只雄鼠，并且对健康组小鼠注射0.1-0.3g/kg bw的生理食盐水；对对照组、低剂量组、中剂量组、高剂量组注射0.1-0.3g/kg bw的D-半乳糖液，诱导6～10周后取血检测脂质氧化丙二醛(malondialdehyde，MDA)。
本发明实验结果图2A-2C、3A-3B、4A-4C中，a表示与WT组别相比，有统计学上显著的差异(p<0.01)；b表示与C组别相比有统计学上显著的差异(p<0.01)。
3.1推迟衰老相关基因表达试验
以D-半乳糖诱导小鼠老化，再分别以3种不同的剂量(LD、MD、HD)喂食小鼠12周后，取小鼠血液样品分离血球，并分析血球细胞中相关衰老基因的表达。其中SIRT1掌管同源染色体的重组及断裂DNA的修复；SIRT6掌管碱基对错误序列的修复，同时能促进同源染色体的重组；SIRT7可将断裂DNA末端进行连接，修复DNA损伤。
如图2A-2C所示，连续使用12周后，使用不同浓度本发明发酵组合物的组别皆能有效提升SIRT1、SIRT6、SIRT7表达量，相较于老化组，DNA修复能力具有显著的改善效果。
3.2血液生化实验
以D-半乳糖诱导小鼠老化，依照3种低、中、高剂量管喂12周后，取小鼠血液样品，分析抗氧化物指标SOD及GSH含量，因老化被视为是氧化物过多造成细胞的氧化而导致，因此其抗氧化指标越高，推迟老化的能力越强。
结果如3A-3B图所示，连续使用12周后，使用不同浓度本发明发酵组合物的组别皆能有效提升抗氧化物质浓度，相较于老化组，抗氧化能力具有显著的改善效果。
3.3组织、器官老化的生物活性指标测定
以D-半乳糖诱导小鼠老化，依照3种低、中、高剂量管喂12周后，取小鼠器官组织作为样品，分别针对大脑组织、肝脏组织及心脏组织，分析其组织内氧化物的浓度，8-oxo-dG为人体中经常存在的氧化物，当氧化物浓度越高，组织器官内的细胞老化速度越快。
大脑为老化表现最主要的器官，氧化物质会导致组织内产生8-oxo-dG产物，产物浓度越高表示老化现象越严重，而若是能在不同器官中降低氧化物的浓度，例如大脑，能降低阿兹海默症或帕金森氏症的罹患机率；在心脏能降低心血管疾病的风险；在肝脏能减少肝脏代谢负担，稳定肝指数。
结果如4A-4C图所示，老化产物与年轻小鼠组别无差异，表示能推迟脑部老化，而肝脏与心脏也是容易老化的器官，在这两个器官中，也同样能具有推迟老化产物生成的作用。
3.4存活期试验
以D-半乳糖诱导小鼠老化，依照3种不同剂量管喂直至小鼠自然死亡，并统计死亡数据，分析平均寿命及最大存活天数，以统计学分析显著性差异，评估本发明是否具有延长寿命的作用。
本存活期试验中，直接进行小鼠的长期观察，评估对生物体的延长寿命是否有直接的功效，通过对小鼠施加三种不同剂量(HD、MD、LD)测试，每日喂食直至自然死亡，并统计数据结果。
结果如图5所示，死亡率达50％时，未使用本发明发酵组合物的组别存活118天，而使用本发明发酵组合物且达建议剂量则延长寿命达200％，结果显示本发明的发酵组合物具有良好的延长寿命效果；未使用本发明的组别最长存活127天，使用本发明发酵组合物且达建议剂量组别最长则存活263天，延长207％的寿命。
根据动物试验，本发明发酵组合物有助于推迟衰老的功效。与降低DNA氧化产物8-oxo-dG的含量，有效减缓脑部脂质过氧化的现象、降低脑部氧化损伤的程度，进而改善脑部氧化压力上升、脑部老化等现象。
经上述实验结果证实，在推迟老化相关基因表达、提高抗氧化物指标SOD及GSH含量、降低组织器官氧化物浓度等老化相关指标上，本发明的发酵组合物具有能有效的推迟老化的效果。
上述实验数据为在特定条件下所获得的初步实验结果，其仅用以易于了解或参考本发明的技术内容而已，其尚需进行其他相关实验。该实验数据及其结果并非用以限制本发明的权利范围。
前述较佳实施例仅举例说明本发明及其技术特征，该实施例的技术仍可适当 进行各种实质等效修饰及/或替换方式予以实施；因此，本发明的权利范围须视权利要求书范围所界定的范围为准。

Claims (12)

  1. 一种具有NMN推迟老化功效的发酵组合物的制备方法，包含:
    (1)蔬果萃取步骤:将多种蔬果分别以物理方式进行压榨处理，压制蔬果直至果实或子实体呈碎块状，同时保留汁液与果渣或子实体，获得多个第一萃取物；
    (2)负压破壁工法:向第一萃取物中分别添加0.15～1.5％黑糖，以提高第一萃取物等渗透压，并同时以负压萃取方式在35～45cmHg的近真空环境中，持续萃取3～15天，使蔬果细胞壁破裂，释放出细胞内营养成分及胞内多糖，获得多个第二萃取物；
    (3)低温发酵阶段:以一酵母菌，以比例0.5～1.5％(10 7CFU/ml)分别植入第二萃取物中，发酵温度控制在2～12℃，持续发酵3～15天，在此阶段菌种处于低活性、让酵素作用大于菌种作用，减缓糖类的消耗，在发酵过程中防腐，提高纤维聚糖的分解作用，促进细胞壁分解，获得多个第一发酵物；
    (4)深层发酵阶段:以一乳酸菌，以比例0.5～1.5％(10 7CFU/ml)；以0.15～0.25％异麦芽寡糖分别植入第一发酵物中，发酵温度控制在20～35℃，持续发酵5～25天，在此阶段乳酸菌消耗糖类生成乳酸，降低pH值达到防腐作用，且提高酸度降低黏稠度，获得多个第二发酵物；
    (5)螯合发酵阶段:以一醋酸菌，以比例0.5～1.5％(10 7CFU/ml)；以2.5～5.5％D-山梨醇分别植入第二发酵物中，发酵温度控制在10～25℃，持续发酵5～20天，在此阶段耗氧环境消耗葡萄糖产生醋酸，消耗乙醇且提高酸度降低黏稠度，获得多个第三发酵物；以及
    (6)干燥造粒步骤:将步骤(5)获得的第三发酵物，分别进行过滤，获得多个发酵液并保留发酵液，再将发酵液以等比例混合，然后以喷雾干燥造粒，取得发 酵组合物。
  2. 如权利要求1所述的制备方法，其特征是，所述蔬果萃取步骤进一步包含将所述蔬果与水混合。
  3. 如权利要求1所述的制备方法，其特征是，所述蔬果为松露、菇蕈类子实体、酪梨、西红柿或毛豆中的一种或多种。
  4. 如权利要求3所述的制备方法，其特征是，所述蔬果萃取步骤的蔬果为松露或菇蕈类子实体时，在以物理方式压榨前，进一步包含以65～100℃热水浸泡25～35分钟软化的步骤。
  5. 如权利要求2所述的制备方法，其特征是，所述些蔬果与水混合的质量比分别为：松露或菇蕈类子实体与水的比例为1:40；酪梨与水的比例为1:25；西红柿与水的比例为1:1；毛豆与水的比例为水1:30。
  6. 如权利要求1所述的制备方法，其特征是，所述第二萃取物糖度(Degrees Brix,°Bx)介于21～28°Bx；所述第一发酵物糖度介于15～34°Bx；所述第二发酵物糖度介于15～38°Bx；所述第三发酵物糖度介于10～50°Bx。
  7. 如权利要求1所述的制备方法，其特征是，所述第二萃取物pH值介于5.5～7；所述第一发酵物pH值介于5～6.5；所述第二发酵物pH值介于4.5～5.5；所述第三发酵物pH值介于3～3.5。
  8. 如权利要求1所述的制备方法，其特征是，所述乳酸菌选自植物乳杆菌(L.plantarum)、保加利亚乳杆菌(L.delbrueckii)、乳酸乳球菌(L.lactis)、嗜酸乳杆菌(L.acidophilus)或比菲德氏菌(B.bifidum)中的一种。
  9. 如权利要求1所述的制备方法，其特征是，所述酵母菌或所述醋酸菌选自白曲菌(S.fibuligera)、酿酒酵母(S.cerevisiae)、膜毕赤酵母(P.faciens)、粟酒裂殖酵母(S.pombe)、弱氧化促酸菌(A.hansenii)、木质醋酸菌(A.xylinum)或弱氧化醋酸杆菌(A.suboxydans)中的一种。
  10. 一种如权利要求1所述方法制备的具有NMN推迟老化功效的发酵组合物，包含蔬果原料，所述蔬果原料包含松露、菇蕈类子实体、酪梨、西红柿或毛豆中的一种或多种，通过制备方法取得的发酵组合物。
  11. 一种如权利要求10所述的发酵组合物在用于制备推迟老化的药物中的用途，其特征是，所述推迟老化是指推迟老化相关基因表达、提高抗氧化物指标SOD及GSH含量、降低组织器官氧化物浓度。
  12. 如权利要求11所述的用途，其特征是，所述药物可以用于推迟心脏、脑部及肝脏的老化。
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