Fruit and vegetable enzyme rich in nicotinamide mononucleotide as well as preparation method and application of fruit and vegetable enzyme

Abstract

The invention relates to the technical field of food processing, in particular to a fruit and vegetable enzyme rich in nicotinamide mononucleotide and a preparation method and application thereof.The preparation method comprises the following steps that cucumber powder, broccoli powder, banana powder and apple powder are mixed to obtain a first mixture, water is added into the mixture, and then autoclaved sterilization is conducted to obtain a second mixture; the mass ratio of the cucumber powder to the broccoli powder to the banana powder to the apple powder is (1-2): (1-2): (1-2): (1-2); and mixing rhizopus oryzae, monascus, saccharomycetes and lactic acid bacteria in equal proportion, inoculating the mixture II with the mixture II, fermenting at 20-30 DEG C for 36-60 hours, centrifuging after fermentation, and taking supernate, thereby obtaining the fruit and vegetable enzyme rich in nicotinamide mononucleotide. The functional characteristics of the enzyme are remarkably improved in the fermentation process, and the enzyme is rich in beta-NMN which is an important precursor substance of NAD < + > on the basis of high-activity lipase and superoxide dismutase, so that the enzyme has the functions of resisting oxidation and delaying cell senescence.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and in particular to a fruit and vegetable enzyme rich in nicotinamide mononucleotide, a preparation method and an application thereof. Background Art

[0002] Enzymes, as a complex functional food, pharmaceutical, and cosmetic ingredient, have garnered significant attention in recent years for their natural active ingredients and metabolic regulatory functions. Cucumbers, broccoli, bananas, and apples are rich in active ingredients such as niacin, β-nicotinamide mononucleotide, dietary fiber, polyphenols, and minerals. Cucumber's cucurbitacins and oligosaccharides can synergistically promote gut microbial balance, while broccoli's sulforaphane and isothiocyanates exhibit significant antioxidant and anti-inflammatory activity. Banana starch and natural sugars provide energy substrates for β-NMN synthesis, while apple's pectin and flavonoids enhance the bioavailability of metabolites.

[0003] β-Nicotinamide mononucleotide (β-NMN), a product of nicotinamide phosphoribosyltransferase, plays an important role in the body and serves as a key precursor to NAD+. β-NMN can be converted to NAD+ in a single, highly efficient catalytic reaction, making it considered the most direct and effective functional supplement. β-NMN exhibits significant pharmacological activity in regulating energy metabolism, delaying cellular aging, and improving insulin sensitivity.

[0004] Currently, the industrial production of compound enzymes still relies primarily on crude processing techniques such as physical mixing or low-temperature drying, and the stability and functional targeting of their active ingredients have not been fully developed. In particular, the ability to enhance the functional properties of enzyme products by synthesizing and accumulating specific functional components through the metabolism of specialized bacterial strains during fruit and vegetable fermentation remains a drawback in the current development and production of enzyme products. Summary of the Invention

[0005] To solve the above problems, the present invention provides a fruit and vegetable enzyme rich in nicotinamide mononucleotide, a preparation method and application.

[0006] The present invention is achieved through the following technical solutions: A method for preparing a fruit and vegetable enzyme rich in nicotinamide mononucleotide comprises the following steps: Cucumber powder, broccoli powder, banana powder and apple powder are mixed to obtain a first mixture, water is added to the mixture and then sterilized under high pressure to obtain a second mixture; the mass ratio of the cucumber powder, broccoli powder, banana powder and apple powder is 1-2:1-2:1-2:1-2; and the mass ratio of the first mixture to water is 1:8-12.

[0007] Rhizopus oryzae, Monascus purpureus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into a second mixture, fermented at 20°C to 30°C for 36 hours to 60 hours, and centrifuged after fermentation to obtain a supernatant rich in nicotinamide mononucleotide fruit and vegetable enzyme; the total weight of the Rhizopus oryzae, Monascus purpureus, yeast and lactic acid bacteria accounts for 2% to 4% of the weight of the second mixture.

[0008] Preferably, the mass ratio of the cucumber powder, broccoli powder, banana powder and apple powder is 2:2:1:1.

[0009] Preferably, the fermentation temperature is 25° C. and the fermentation time is 60 h.

[0010] Preferably, the temperature of the high-pressure sterilization is 121° C., the pressure is 100 KPa to 110 KPa, and the time is 15 min to 25 min.

[0011] Preferably, the effective viable count of Aspergillus oryzae is 1×10 8 ~1×10 9 CFU / g; the effective viable count of Monascus is 5×10 8 CFU / g~2×10 9 CFU / g; effective viable yeast count ≥1×10 9 CFU / g; effective viable bacteria count of lactic acid bacteria 1×10 10 ~1×10 11 CFU / g.

[0012] Preferably, the water is deionized water.

[0013] The nicotinamide mononucleotide-rich fruit and vegetable enzyme prepared by the preparation method.

[0014] The application of the nicotinamide mononucleotide-rich fruit and vegetable enzyme in the preparation of an enzyme product with both NAD+ precursor enrichment and multi-target bioactivity.

[0015] The application of the nicotinamide mononucleotide-rich fruit and vegetable enzyme in the preparation of products with antioxidant and cell aging-delaying functions.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing a fruit and vegetable enzyme rich in nicotinamide mononucleotide, comprising the following steps: Mixing cucumber powder, broccoli powder, banana powder and apple powder to obtain mixture one, sterilizing mixture one by adding water to obtain mixture two; the mass ratio of the cucumber powder, the broccoli powder, the banana powder and the apple powder is 1~2:1~2:1~2:1~2; the mass ratio of the mixture one and water is 1:8~12; inoculating rice root mold, monascus, yeast and lactic acid bacteria into the mixture two in a proportion, and fermenting for 36h~60h at 20℃~30℃, centrifuging the fermented mixture to obtain the supernatant to obtain the fruit and vegetable enzyme rich in nicotinamide mononucleotide; the total weight of the rice root mold, the monascus, the yeast and the lactic acid bacteria accounts for 2%~4% of the weight of the mixture two. The application successfully constructs a high-efficiency enrichment system of beta-nicotinamide mononucleotide in fruit and vegetable enzyme through a multi-strain synergistic fermentation strategy. The yield of beta-NMN reaches 808.8ug / L under the conditions of 25℃ and 60h, which is 20.86 times higher than that of the unfermented group. The fermentation process significantly improves the functional characteristics of the enzyme: the total polyphenol content is increased to 0.3425mg / mL, the increase is 140.4%; the superoxide dismutase activity is 50.9917U / mL, which is increased by 3.74 times; the hydroxyl radical scavenging capacity and the total antioxidant capacity (FRAP) are increased by 1.98 and 2.05 times, respectively. Compared with the prior art, the fermentation process of the application significantly improves the functional characteristics of the enzyme, and the enzyme is rich in beta-NMN, an important precursor of NAD+, and has high activity of lipase and superoxide dismutase, thereby having the functions of antioxidant and anti-aging. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0018] Figure 1 The β-NMN content produced by different fermentation strains of the application for fermenting fruit and vegetable powder.

[0019] Figure 2 The β-NMN content produced by different strains of the application for fermenting fruit and vegetable powder.

[0020] Figure 3 The influence of different fermentation temperatures on the β-NMN content and pH of the product.

[0021] Figure 4 The influence of different fermentation times on the pH of the product.

[0022] Figure 5 Effect of different fermentation time on the content of β-NMN in the product of the present application.

[0023] Figure 6 Activity of functional enzymes in the fruit and vegetable enzyme of the present application Figure 7 Change of antioxidant performance before and after fermentation of the fruit and vegetable enzyme of the present application Figure 6 In the figure, A is the total antioxidant capacity detection result before and after fermentation of the fruit and vegetable enzyme; B is the hydroxyl radical inhibition ability detection result. DETAILED DESCRIPTION

[0024] In order to facilitate the understanding of the present application, the present application will be described more fully below, and the preferred embodiments of the present application are given. However, the present application can be realized in many different forms and is not limited to the embodiments described in the present application. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0025] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the present application in the specification of the present application are only for the purpose of describing the specific embodiments and are not intended to limit the present application.

[0026] The cucumber powder, broccoli powder, banana powder and apple powder used in the present application are purchased from Jiangsu Xinghua Lvs Food Co., Ltd.; the yeast is Angel active dry yeast purchased from Angel Yeast Co., Ltd.; Aspergillus oryzae, Rhizopus oryzae, Aspergillus niger, Monascus, lactic acid bacteria are purchased from Shandong Hezhong Kangyuan Biological Technology Co., Ltd.; Mucor is purchased from Jining Xiangyuan Biological Technology Co., Ltd.; Monascus is purchased from Fujian Gutian Hongxin Liquor Co., Ltd.; Lactobacillus plantarum (ATCC8014) is purchased from Shanghai Collection and Biological Technology Center.

[0027] The effective viable count of yeast is ≥1×10 9 CFU / g; the effective viable count of Aspergillus oryzae, Rhizopus oryzae, Aspergillus niger and Mucor is 1×10 8 ~1×10 9 CFU / g (in terms of spore count); the effective viable count of Monascus is 5×10 8 CFU / g~2×10 9 CFU / g; the effective viable count of lactic acid bacteria is 1×10 10 ~1×10 11 CFU / g; the effective viable count of Monascus is 1×10 6 ~1×10 8 CFU / g.

[0028] The beneficial effects of the present invention are described below through specific examples.

[0029] The inventive concept of the present invention is as follows: Currently, the industrial production of compound enzymes still relies primarily on crude processing techniques such as physical mixing or low-temperature drying, and the stability and functional targeting of their active ingredients have not been fully developed. In particular, the ability to enhance the functional properties of enzyme products by synthesizing and accumulating specific functional components through the metabolism of specialized bacterial strains during fruit and vegetable fermentation remains a drawback in the current development and production of enzyme products.

[0030] Based on this, the present invention provides a preparation method of nicotinamide mononucleotide-rich fruit and vegetable enzymes, comprising mixing cucumber powder, broccoli powder, banana powder and apple powder to obtain a first mixture, adding water to the mixture and sterilizing it under high pressure to obtain a second mixture; the mass ratio of the cucumber powder, broccoli powder, banana powder and apple powder is 1-2:1-2:1-2:1-2; the mass ratio of the first mixture to water is 1:8-12; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the second mixture, fermented at 20°C-30°C for 36h-60h, and centrifuged after fermentation to obtain the supernatant to obtain the nicotinamide mononucleotide-rich fruit and vegetable enzymes.

[0031] The present invention uses Rhizopus oryzae, Monascus purpureus, yeast, and lactic acid bacteria, four microorganisms that are highly complementary in their metabolic functions. Yeast (such as Saccharomyces cerevisiae) can efficiently break down carbohydrates to produce pyruvate and ATP, providing precursors (such as niacin and ribose phosphate) for the synthesis of β-NMN and promoting the accumulation of β-NMN through the NAD+ salvage synthesis pathway. Rhizopus oryzae excels at secreting amylase, cellulase, and protease to degrade the cell walls of fruits and vegetables, releasing more fermentable sugars and amino acids, and providing substrate support for other bacterial species. Monascus spp. is known for its unique secondary metabolic capacity and may promote the synthesis of β-NMN by regulating the activity of NAD+-related enzymes (such as NAD+ kinase). Lactic acid bacteria lower the environmental pH through lactic acid fermentation, inhibiting the growth of miscellaneous bacteria, while creating a slightly acidic environment suitable for yeast and mold. Its NAD+-dependent lactate dehydrogenase (LDH) may also participate in the redox balance, indirectly stabilizing the synthesis of β-NMN and preventing the degradation of β-NMN.

[0032] Secondly, the synergistic effect of the four is reflected in the hierarchy of substrate utilization and the cross-activation of metabolic pathways. Rhizopus oryzae and Monascus purpureus first break down macromolecules (such as cellulose and protein), while yeast and lactic acid bacteria further utilize small molecule products (such as monosaccharides and amino acids) for fermentation, forming an efficient metabolic "relay." In addition, the metabolic pathways of different bacterial species may promote each other. For example, yeast and Monascus purpureus may generate nicotinic acid mononucleotide (NaMN) through different means (such as the Preiss-Handler pathway and the de novo synthesis pathway), and then convert it into β-NMN. The acidic environment created by lactic acid bacteria may also activate the stress metabolism of yeast and enhance the activity of the NAD+ salvage synthesis pathway.

[0033] In contrast, single bacterial species produce lower β-NMN production due to their limited functions. For example, when yeast alone is used, although it has strong glycolysis capabilities, it lacks substrate preprocessing capabilities (such as cellulose degradation), resulting in an insufficient supply of precursors. When Monascus or Rhizopus oryzae alone are used, although they can break down large molecules, they lack efficient glycolysis and NAD+ recycling systems. When lactic acid bacteria alone are used, their metabolism is too simple (primarily producing lactic acid) and cannot provide a sufficient β-NMN synthase system. In addition, other mixed bacterial communities (such as yeast + Rhizopus oryzae + Monascus + fruit wine yeast or yeast + Rhizopus oryzae + Monascus + Aspergillus niger) cannot achieve the synergistic effect of the four bacteria due to functional overlap or metabolic conflicts.

[0034] Therefore, the combination of yeast, Rhizopus oryzae, Monascus purpureus, and lactic acid bacteria is irreplaceable. Through the triple mechanism of substrate synergistic degradation, metabolic pathway complementarity, and environmental homeostasis maintenance, they break through the metabolic bottleneck of a single bacterial species, significantly increasing β-NMN production and significantly improving the functional properties of the enzyme during the fermentation process. On the basis of highly active lipase and superoxide dismutase, they are also rich in β-NMN, an important precursor of NAD+, thus having antioxidant and anti-cellular aging functions.

[0035] Example 1 Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 1:1:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:8; the mixture is sterilized under high pressure at 121°C 100Kpa for 15 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 2% by weight, and the mixture is placed in an incubator at a temperature of 30°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the mixture is centrifuged to obtain the fermentation supernatant to prepare the fruit and vegetable enzyme.

[0036] Comparative Example 1 Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 1:1:1:1 to obtain mixture one, deionized water is added to mixture one, and the mass volume ratio of mixture one to deionized water is 1:8; mixture is sterilized under high pressure at 121°C 100Kpa for 15 minutes to obtain mixture two; no bacterial strain is added, and the mixture is placed in an incubator at a temperature of 30°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain fruit and vegetable enzymes.

[0037] Comparative Example 2 Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 1:1:1:1 to obtain mixture one, deionized water is added to mixture one, and the mass volume ratio of mixture one to deionized water is 1:8; high-pressure sterilization is carried out at 121°C 100Kpa for 15 minutes to obtain mixture two; lactic acid bacteria are inoculated into mixture two at an inoculum amount of 2% by weight, and the mixture is placed in an incubator at a temperature of 30°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain fruit and vegetable enzymes.

[0038] Comparative Example 3 Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 1:1:1:1 to obtain mixture one, deionized water is added to mixture one, and the mass volume ratio of mixture one to deionized water is 1:8; high-pressure sterilization is carried out at 121°C 100Kpa for 15 minutes to obtain mixture two; yeast is inoculated into mixture two at an inoculum amount of 2% by weight, and the mixture is placed in an incubator at a temperature of 30°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain fruit and vegetable enzymes.

[0039] Comparative Example 4 Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 1:1:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:8; the mixture is sterilized by high pressure at 121°C 100Kpa for 15 minutes to obtain mixture two; fruit wine yeast is inoculated into the mixture two at an inoculum amount of 2% by weight, and the mixture is placed in an incubator at a temperature of 30°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0040] Comparative Example 5 The cucumber powder, broccoli powder, banana powder and apple powder were mixed in a mass ratio of 1:1:1:1 to obtain a mixture one, deionized water was added into the mixture one, and the mass-volume ratio of the mixture one and the deionized water was 1:8; 121 ℃ 100 Kpa high-pressure sterilization for 15 min to obtain a mixture two; the Rhizopus oryzae was inoculated into the mixture two at an inoculation amount of 2% by weight, and placed in a incubator at a temperature of 30 ℃ and a shaking bed speed of 220 rpm for fermentation for 48 h, and then centrifuged to obtain the fermentation supernatant to prepare the fruit and vegetable enzyme.

[0041] Comparative example 6 The cucumber powder, broccoli powder, banana powder and apple powder were mixed in a mass ratio of 1:1:1:1 to obtain a mixture one, deionized water was added into the mixture one, and the mass-volume ratio of the mixture one and the deionized water was 1:8; 121 ℃ 100 Kpa high-pressure sterilization for 15 min to obtain a mixture two; the Monascus was inoculated into the mixture two at an inoculation amount of 2% by weight, and placed in a incubator at a temperature of 30 ℃ and a shaking bed speed of 220 rpm for fermentation for 48 h, and then centrifuged to obtain the fermentation supernatant to prepare the fruit and vegetable enzyme.

[0042] Comparative example 7 The cucumber powder, broccoli powder, banana powder and apple powder were mixed in a mass ratio of 1:1:1:1 to obtain a mixture one, deionized water was added into the mixture one, and the mass-volume ratio of the mixture one and the deionized water was 1:8; 121 ℃ 100 Kpa high-pressure sterilization for 15 min to obtain a mixture two; the Aspergillus oryzae was inoculated into the mixture two at an inoculation amount of 2% by weight, and placed in a incubator at a temperature of 30 ℃ and a shaking bed speed of 220 rpm for fermentation for 48 h, and then centrifuged to obtain the fermentation supernatant to prepare the fruit and vegetable enzyme.

[0043] Comparative example 8 The cucumber powder, broccoli powder, banana powder and apple powder were mixed in a mass ratio of 1:1:1:1 to obtain a mixture one, deionized water was added into the mixture one, and the mass-volume ratio of the mixture one and the deionized water was 1:8; 121 ℃ 100 Kpa high-pressure sterilization for 15 min to obtain a mixture two; the Mucor was inoculated into the mixture two at an inoculation amount of 2% by weight, and placed in a incubator at a temperature of 30 ℃ and a shaking bed speed of 220 rpm for fermentation for 48 h, and then centrifuged to obtain the fermentation supernatant to prepare the fruit and vegetable enzyme.

[0044] Comparative example 9 Cucumber powder, broccoli powder, banana powder and apple powder were mixed in a mass ratio of 1:1:1:1 to obtain mixture one, deionized water was added to the mixture one, and the mass-volume ratio of the mixture one to the deionized water was 1:8; 121℃ 100Kpa high pressure sterilization for 15min to obtain mixture two; Aspergillus niger was inoculated into the mixture two at an inoculation amount of 2% by weight, and placed in an incubator at a temperature of 30℃ and a shaking bed speed of 220rpm for fermentation for 48h, and after fermentation, centrifugation was performed to obtain the fermentation supernatant to prepare the fruit and vegetable enzyme.

[0045] Comparative example 10 Cucumber powder, broccoli powder, banana powder and apple powder were mixed in a mass ratio of 1:1:1:1 to obtain mixture one, deionized water was added to the mixture one, and the mass-volume ratio of the mixture one to the deionized water was 1:8; 121℃ 100Kpa high pressure sterilization for 15min to obtain mixture two; Yeast, Rhizopus oryzae, Monascus and wine yeast were mixed in equal proportions, and then inoculated into the mixture two at an inoculation amount of 2% by weight, and placed in an incubator at a temperature of 30℃ and a shaking bed speed of 220rpm for fermentation for 48h, and after fermentation, centrifugation was performed to obtain the fermentation supernatant to prepare the fruit and vegetable enzyme.

[0046] Comparative example 11 Cucumber powder, broccoli powder, banana powder and apple powder were mixed in a mass ratio of 1:1:1:1 to obtain mixture one, deionized water was added to the mixture one, and the mass-volume ratio of the mixture one to the deionized water was 1:8; 121℃ 100Kpa high pressure sterilization for 15min to obtain mixture two; Yeast, Rhizopus oryzae, Monascus and wine yeast were mixed in equal proportions, and then inoculated into the mixture two at an inoculation amount of 2% by weight, and placed in an incubator at a temperature of 30℃ and a shaking bed speed of 220rpm for fermentation for 48h, and after fermentation, centrifugation was performed to obtain the fermentation supernatant to prepare the fruit and vegetable enzyme.

[0047] The β-NMN content in the fruit and vegetable enzyme prepared in example 1, comparative examples 1-11 was determined, and the experimental results are shown in Table 1. Figure 1 Figure 2 ​As shown, the β-NMN yield in the fruit and vegetable enzyme obtained by comparative example 1 without adding bacteria was 38.8 μg / L; the β-NMN yield in the fruit and vegetable enzyme obtained by comparative example 2 with only adding lactic acid bacteria was 80.5 μg / L; the β-NMN yield in the fruit and vegetable enzyme obtained by comparative example 3 with only adding yeast was 227.67 μg / L; the β-NMN yield in the fruit and vegetable enzyme obtained by comparative example 4 with only adding wine yeast was 121 μg / L; the β-NMN yield in the fruit and vegetable enzyme obtained by comparative example 5 with only adding Rhizopus oryzae was 175.8 μg / L; In comparative example 6, the yield of β-NMN in the fruit and vegetable enzyme obtained by adding only Monascus was 171.1 μg / L; in comparative example 7, the yield of β-NMN in the fruit and vegetable enzyme obtained by adding only Aspergillus oryzae was 50.8 μg / L; in comparative example 8, the yield of β-NMN in the fruit and vegetable enzyme obtained by adding only Mucor was 75.5 μg / L; in comparative example 9, the yield of β-NMN in the fruit and vegetable enzyme obtained by Aspergillus niger was 85.9 μg / L; in comparative examples 10 and 11, the yield of β-NMN in the fruit and vegetable enzyme obtained by fermentation of mixed strains was less than 500 μg / L. In Example 1, the yield of β-NMN in the fruit and vegetable enzyme obtained by fermentation of yeast, Rhizopus oryzae, Monascus and lactic acid bacteria was as high as 625.32 μg / L, which was much higher than the yield of β-NMN in the fruit and vegetable enzyme obtained by not using strains or using a single strain. The β-NMN production in fruit and vegetable enzymes obtained by mixed fermentation of several other strains is also much lower than that of mixed fermentation of yeast, Rhizopus oryzae, Monascus purpureus and lactic acid bacteria. This shows that only by using the collaborative fermentation of yeast, Rhizopus oryzae, Monascus purpureus and lactic acid bacteria can the β-NMN production in fruit and vegetable enzymes be significantly increased. Therefore, the present invention adopts Rhizopus oryzae, Monascus purpureus, yeast and lactic acid bacteria inoculation in equal proportions to construct a collaborative fermentation system.

[0048] Example 2: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 1:1:2:2 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:10; high-pressure sterilization is performed at 121°C 105Kpa for 20 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 3% by weight, and the mixture is placed in an incubator at a temperature of 30°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0049] Example 3: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:10; high-pressure sterilization is performed at 121°C 105Kpa for 20 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 3% by weight, and the mixture is placed in an incubator at a temperature of 30°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0050] Example 4: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 1:2:2:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:10; high-pressure sterilization is performed at 121°C 105Kpa for 20 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 3% by weight, and the mixture is placed in an incubator at a temperature of 30°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0051] Example 5: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:1:2:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:10; high-pressure sterilization is performed at 121°C 105Kpa for 20 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 3% by weight, and the mixture is placed in an incubator at a temperature of 30°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0052] The β-NMN content in the fruit and vegetable enzymes prepared in Examples 1 to 5 was measured respectively, and the results are shown in Table 1. The fruit and vegetable enzymes prepared in Examples 1 to 5 all contained relatively high levels of β-NMN.

[0053] A comparative analysis of β-NMN production revealed that the higher levels of β-NMN and NMN precursors in cucumber and broccoli powders are key to increasing β-NMN production. Banana and apple powders are rich in fructose, which, at lower ratios, can provide the carbon source required for microbial growth. Therefore, a 2:2:1:1 ratio of broccoli powder: banana powder: apple powder resulted in the highest β-NMN production.

[0054] Table 1 Yield of β-NMN obtained from different raw material ratios Comparative Example 12: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:12; high-pressure sterilization is performed at 121°C 110Kpa for 25 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 4% by weight, and the mixture is placed in an incubator at a temperature of 10°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0055] Comparative Example 13: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:10; high-pressure sterilization is performed at 121°C 110Kpa for 25 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 4% by weight, and the mixture is placed in an incubator at a temperature of 15°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0056] Comparative Example 14: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:12; high-pressure sterilization is performed at 121°C 110Kpa for 25 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 4% by weight, and the mixture is placed in an incubator at a temperature of 40°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0057] Example 6: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:12; high-pressure sterilization is performed at 121°C 110Kpa for 25 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 4% by weight, and the mixture is placed in an incubator at a temperature of 20°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0058] Example 7: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:12; high-pressure sterilization is performed at 121°C 110Kpa for 25 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 4% by weight, and the mixture is placed in an incubator at a temperature of 25°C and a shaker speed of 220rpm for fermentation for 48 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0059] The β-NMN content in the fruit and vegetable enzymes prepared in Comparative Examples 12 to 14, Example 3, and Example 6 to 7 was measured respectively. The results are as follows: Figure 3 As shown in the figure, different fermentation temperatures have a significant effect on the synthesis and pH of β-nicotinamide mononucleotide in the enzyme. When the fermentation temperature rises from 10°C to 25°C, the β-NMN content gradually increases from 233.95μg / L to 678.47μg / L, reaching a peak. At this time, the corresponding pH value of the system after 48 hours is 3.92, showing a mild acidic environment. Figure 4 As shown in Figure 2, the optimal temperature maintains the activity of nicotinamide mononucleotide adenylyltransferase and promotes the biosynthesis of β-NMN. Therefore, according to the experimental results, the β-NMN content is higher when the fermentation temperature is between 20°C and 30°C, and the β-NMN content is the highest when the fermentation temperature is 25°C.

[0060] Comparative Example 15 Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:10; high-pressure sterilization is performed at 121°C 105Kpa for 20 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 3% by weight, and the mixture is placed in an incubator for fermentation at a temperature of 25°C and a shaker speed of 220rpm for 10 hours. After the fermentation is completed, the mixture is centrifuged to obtain the fermentation supernatant to prepare the fruit and vegetable enzyme.

[0061] Comparative Example 16 Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:10; high-pressure sterilization is performed at 121°C 105Kp for 20 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 3% by weight, and the mixture is placed in an incubator at a temperature of 25°C and a shaker speed of rpm for fermentation for 24 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0062] Comparative Example 17 Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:10; high-pressure sterilization is performed at 121°C 105Kp for 20 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 3% by weight, and the mixture is placed in an incubator at a temperature of 25°C and a shaker speed of 220 rpm for fermentation for 72 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0063] Example 8: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:10; high-pressure sterilization is performed at 121°C 105Kp for 20 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 3% by weight, and the mixture is placed in an incubator at a temperature of 25°C and a shaker speed of 220 rpm for fermentation for 36 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0064] Example 9: A method for preparing fruit and vegetable enzymes Cucumber powder, broccoli powder, banana powder and apple powder are mixed in a mass ratio of 2:2:1:1 to obtain mixture one, deionized water is added to the mixture one, and the mass volume ratio of the mixture one to the deionized water is 1:10; high-pressure sterilization is performed at 121°C 105Kp for 20 minutes to obtain mixture two; Rhizopus oryzae, Monascus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into the mixture two at an inoculum amount of 3% by weight, and the mixture is placed in an incubator for fermentation at a temperature of 25°C and a shaker speed of 220rpm for 60 hours. After the fermentation is completed, the fermentation supernatant is centrifuged to obtain the fruit and vegetable enzyme.

[0065] The β-NMN content in the fruit and vegetable enzymes prepared in Comparative Examples 15 to 17, Example 7, and Example 8 to Example 9 was measured respectively. The β-NMN content of the combined bacterial fermentation system at different fermentation times was as follows: Figure 5 As shown in Figure 2, the β-NMN content in the product rose to over 600 μg / L between 36 and 60 hours, reaching a peak of 808.81 μg / L at 60 hours, a 20.86-fold increase from the initial value. When the fermentation time was extended to 72 hours, the β-NMN concentration decreased to 594.91 μg / L.

[0066] The fruit and vegetable enzymes prepared by the preparation methods of the fruit and vegetable enzymes described in Examples 1 to 9 were tested for chemical component content, effective enzyme activity, and antioxidant capacity. The effects produced by Examples 1 to 9 are similar. For the convenience of subsequent discussion and reference, the experimental results of Example 1 shall prevail. The relevant test methods and test results are as follows:

[0067] Experimental Example 1: Determination of chemical components in fruit and vegetable enzymes Fermented liquid was obtained by centrifugation according to the method described in Example 9, and the supernatant was used as a pre-test sample. The total polyphenol, reducing sugar, and protein contents of the fruit and vegetable ferments were determined using the Folin-Ciocalteu colorimetric method, a reducing sugar detection kit, a DNS microplate method, and a BCA protein quantification kit, respectively. The contents of different biological nutrients in the fruit and vegetable ferments varied, with protein content being the highest. The results are shown in Table 2. During the fermentation process, plant polysaccharides generally serve as a carbon source for microbial growth. In the early stages of fermentation, the fermenting microbial community utilizes the sugars of the fruits and vegetables themselves for growth, metabolism, and reproduction. As fermentation proceeds, the reducing sugar content in the late fermentation system decreases by 3.72 times. Phenols, as a substance that is easily oxidized, are degraded into small phenols by lactic acid bacteria during fruit and vegetable fermentation due to the production of phenolic acid esterase, resulting in a 2.4-fold increase in their content. Furthermore, the organic acids produced prevent the degradation of phenols into other substances, thereby improving their antioxidant capacity. The proteins in fruit and vegetable enzymes are mainly enzymes. The results show that the total amount of protein before and after fermentation remains almost unchanged, but the activity of different enzymes has changed significantly. This indicates that during the fermentation process, microorganisms significantly increase the activity and content of specific enzymes, such as SOD, through mechanisms such as zymogen activation and cofactor binding without consuming protein, and promote the production and stabilization of small molecular functional substances, such as antioxidant phenols and organic acids, ultimately enhancing the biological activity of fruit and vegetable enzymes.

[0068] Table 2 Analysis of nutritional components of fruits and vegetables before and after fermentation Experimental Example 2: Determination of the activity of functional enzymes in fruit and vegetable enzymes The activities of amylase, lipase and superoxide dismutase of the fruit and vegetable enzymes prepared in Example 9 were detected using an amylase (AMS) detection kit (iodine-starch microplate method), a lipase (LPS) activity kit and a superoxide dismutase (SOD) activity detection kit (WST-1 method).

[0069] SOD, amylase, and lipase are the main functional enzymes of fruit and vegetable enzymes. SOD can remove excess superoxide anion free radicals produced in the body, delay aging, and improve the body's immunity. Lipase and amylase can promote the degradation of related macromolecules, making it more conducive to their absorption by the human body. Figure 6 As shown in the figure, SOD increased by 3.74 times compared with that before fermentation, indicating that the fermented product has good antioxidant activity. The activity of amylase decreased significantly, which corresponds to the decrease in reducing sugar content. The enzyme activity of lipase did not change significantly before and after fermentation. Therefore, the fruit and vegetable enzymes produced by microbial synergistic fermentation have high SOD enzyme activity, low sugar content and long-lasting lipase activity.

[0070] Experimental Example 3: Determination of Antioxidant Capacity in Fruit and Vegetable Enzymes The total antioxidant capacity (FRAP method) kit and the hydroxyl radical (OH-) assay kit were used to detect the changes in the total antioxidant capacity of the fruit and vegetable enzymes prepared in Example 9 before and after fermentation.

[0071] The total antioxidant capacity and hydroxyl radical scavenging capacity are used to characterize the in vitro antioxidant capacity of fruit and vegetable enzymes. Hydroxyl free radicals are a very aggressive reactive oxygen species that are closely related to the occurrence of many diseases in the body, but can be scavenged by SOD. The total antioxidant capacity is the most direct indicator of the antioxidant capacity of fruit and vegetable enzymes. The experimental results are as follows Figure 7 As shown in the results, the total antioxidant capacity of the fermented fruit and vegetable enzymes was 0.5483 μmol Trolox / mL, which was 2.05 times higher than that of the unfermented group, and the ability to inhibit hydroxyl radicals was 1.98 times higher. Microbial synergistic fermentation improved the antioxidant capacity of fruit and vegetable enzymes.

[0072] This study successfully constructed a highly efficient enrichment system for β-nicotinamide mononucleotide (β-NMN) in fruit and vegetable enzymes using a multi-strain synergistic fermentation strategy. Using cucumber powder, broccoli powder, banana powder, and apple powder as a composite matrix, and employing a combined fermentation system of Rhizopus oryzae, Monascus purpureus, yeast, and lactic acid bacteria, the fermentation yield reached 808.8 μg / L at 25°C for 60 hours, a 20.86-fold increase compared to the unfermented control. The fermentation process significantly improved the functional properties of the fruit and vegetable enzymes: total polyphenol content increased to 0.3425 mg / mL, a 140.4% increase; superoxide dismutase activity reached 50.9917 U / mL, a 3.74-fold increase; and hydroxyl radical scavenging capacity and total antioxidant capacity (FRAP) increased by 1.98 and 2.05 times, respectively. The present invention demonstrates the metabolic characteristics of multi-bacteria synergistic fermentation regulating β-NMN synthesis in fruit and vegetable matrices, provides an innovative process for the development of enzyme products that have both NAD+ precursor enrichment and multi-target bioactivity, and provides new ideas and references for the development of enzyme powder rich in β-NMN in the fields of food, medicine, cosmetics, etc.

[0073] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0074] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. A person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims.

Claims

1. A method for preparing fruit and vegetable enzymes rich in nicotinamide mononucleotide, characterized in that: The following steps are involved: Mixing cucumber powder, broccoli powder, banana powder and apple powder to obtain a first mixture, adding water to the mixture and sterilizing it under high pressure to obtain a second mixture; the mass ratio of the cucumber powder, broccoli powder, banana powder and apple powder is 1-2:1-2:1-2:1-2; the mass ratio of the first mixture to water is 1:8-12; Rhizopus oryzae, Monascus purpureus, yeast and lactic acid bacteria are mixed in equal proportions and inoculated into a second mixture, fermented at 20°C to 30°C for 36 hours to 60 hours, and centrifuged after fermentation to obtain a supernatant rich in nicotinamide mononucleotide fruit and vegetable enzyme; the total weight of the Rhizopus oryzae, Monascus purpureus, yeast and lactic acid bacteria accounts for 2% to 4% of the weight of the second mixture.

2. The preparation method according to claim 1, wherein The mass ratio of the cucumber powder, broccoli powder, banana powder and apple powder is 2:2:1:

1.

3. The preparation method according to claim 1, wherein The fermentation temperature is 25° C. and the fermentation time is 60 h.

4. The preparation method according to claim 1, wherein The high-pressure sterilization temperature is 121° C., the pressure is 100 KPa to 110 KPa, and the time is 15 min to 25 min.

5. The preparation method according to claim 1, wherein The effective viable bacteria count of Aspergillus oryzae is 1×10 8 ~1×10 9 CFU / g; the effective viable count of Monascus is 5×10 8 CFU / g ~2×10 9 CFU / g; effective viable yeast count ≥1×10 9 CFU / g; effective viable bacteria count of lactic acid bacteria 1×10 10 ~1×10 11 CFU / g.

6. The preparation method according to claim 1, wherein The water is deionized water.

7. The nicotinamide mononucleotide-rich fruit and vegetable enzyme prepared by the preparation method according to claim 1.

8. Use of the nicotinamide mononucleotide-rich fruit and vegetable enzyme according to claim 7 in the preparation of an enzyme product with both NAD+ precursor enrichment and multi-target bioactivity.

9. Use of the nicotinamide mononucleotide-rich fruit and vegetable enzyme according to claim 7 in preparing a product having antioxidant and cell aging-delaying functions.

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