Preparation method of fermented beverage
By using fermentation processes with Saccharomyces cerevisiae and Bacillus rennet, the problem of the indigestibility of NMN from natural yeast sources has been solved, resulting in an absorbable fermented beverage containing NMN that has antioxidant functions and health benefits.
Patent Information
- Application Number
- CN202410555651.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-07
- Publication Date
- 2025-11-07
AI Technical Summary
In existing technologies, β-nicotinamide mononucleotide (NMN) raw materials are mainly derived from natural yeast, which makes them difficult for the human body to digest, causing high uric acid and health problems. Therefore, they need to be consumed in limited quantities or after hydrolysis.
A fermented beverage containing β-nicotinamide mononucleotide (NMN) was prepared by fermenting a sugar-containing substrate with Saccharomyces cerevisiae BM2, inducing yeast autolysis through protease, and then combining it with secondary fermentation by Bacillus coagulans.
A fermented beverage containing β-nicotinamide mononucleotide (NMN) that can be effectively absorbed was prepared. It has a natural tea and fruit flavor, is alcohol-free, and the NMN content is proportional to the amount of yeast inoculated, providing antioxidant function.
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Figure CN120898930A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a preparation method of a fermented beverage, in particular, a technical improvement of a preparation method of a fermented beverage containing nicotinamide mononucleotide (NMN). BACKGROUND
[0002] Kombucha Tea is a fermented beverage made from acetic acid bacteria, lactic acid bacteria and yeast fermentation of plant materials (such as tea, fruit juice, herbal extract), which is a high-bioactive compound-containing and alcohol-free beverage. It attracts more and more consumers to drink because of its health-promoting properties.
[0003] Nicotinamide mononucleotide (NMN) exists in various organisms, and as a key intermediate for the synthesis of coenzyme NAD+ (nicotinamide adenine dinucleotide), recent studies have shown that taking nicotinamide mononucleotide (NMN) can increase the concentration of coenzyme NAD+ (nicotinamide adenine dinucleotide) in the human body, providing anti-aging functions and extending life functions.
[0004] Currently, the common raw materials of nicotinamide mononucleotide (NMN) in Taiwan are mostly from natural yeast sources. However, the cell wall of yeast and high RNA / DNA ratio nucleic acid are difficult to be digested by the human body, and direct consumption will cause high uric acid in the human body, leading to gout and urinary stones, and therefore requires limited consumption or consumption after hydrolysis.
[0005] Therefore, it is necessary to provide a novel and progressive preparation method of a fermented beverage to solve the above problems. SUMMARY
[0006] The main purpose of the present application is to provide a preparation method of a fermented beverage, which can prepare a fermented beverage containing nicotinamide mononucleotide (NMN) active probiotics.
[0007] To achieve the above object, the present application provides a preparation method of a fermented beverage, comprising the following steps: preparing a sugar-containing fermentation substrate, the sugar content of the sugar-containing fermentation substrate ranges from 0.50% (w / v, weight / volume) to 20.00% (w / v, weight / volume); fermentation, inoculating Saccharomyces cerevisiae BM2 for preparing nicotinamide mononucleotide (NMN) into the sugar-containing fermentation substrate, and then culturing and fermenting the sugar-containing fermentation substrate containing Saccharomyces cerevisiae BM2 until a fermentation broth with residual sugar content <0.5% (w / v, weight / volume) is formed; inducing autolysis of Saccharomyces cerevisiae, adding protease to the fermentation broth to induce autolysis of Saccharomyces cerevisiae BM2 to produce an autolysed Saccharomyces cerevisiae fermentation broth; secondary fermentation, inoculating Bacillus coagulans into the autolysed Saccharomyces cerevisiae fermentation broth for secondary aerobic fermentation to produce a secondary fermentation broth, and the secondary fermentation broth contains nicotinamide mononucleotide (NMN).
[0008] As described above, the sugar-containing fermentation substrate is a substrate of 0.50% (w / v, weight / volume) to 5.00% (w / v, weight / volume), and sugar is added to the substrate to adjust the sugar content to a range of 0.50% (w / v, weight / volume) to 20.00% (w / v, weight / volume); wherein the substrate includes at least one of tea soup, plant extract, and vegetable and fruit juice.
[0009] As described above, the protease is derived from pineapple, papaya, fig, kiwi, or any combination thereof.
[0010] As described above, in the fermentation step, the inoculation amount of Saccharomyces cerevisiae BM2 ranges from 0.50% (v / v, volume / volume) to 20.00% (v / v, volume / volume).
[0011] As described above, in the step of inducing autolysis of Saccharomyces cerevisiae, the amount of protease added ranges from 1.00 U / mL (Units per milliliter) to 5.00 U / mL (Units per milliliter).
[0012] As described above, in the step of inducing autolysis of the Saccharomyces cerevisiae, after adding the protease to the fermentation broth, the fermentation broth to which the protease has been added is incubated at a temperature of 37°C to 55°C for 4 hours to 36 hours to induce autolysis of the Saccharomyces cerevisiae (Saccharomyces cerevisiae BM2).
[0013] As described above, before the fermentation step, the sugar-containing fermentation substrate is subjected to pasteurization in a closed tank.
[0014] As described above, in the fermentation step, after inoculating the Saccharomyces cerevisiae (Saccharomyces cerevisiae BM2) into the sugar-containing fermentation substrate, the fermentation broth is incubated at 18°C to 30°C for 8 hours to 48 hours until the residual sugar content of the fermentation broth is <0.5% (w / v, weight / volume).
[0015] As described above, in the secondary fermentation step, the non-fermented sugar-adjusted sweetening is added to the secondary fermentation broth.
[0016] The sugar-containing fermentation substrate is 2.50% (w / v, weight / volume) to 5.00% (w / v, weight / volume) of the substrate, and sugar is added to the substrate to adjust the sugar content to a sugar content range of 2.00% (w / v, weight / volume) to 5.00% (w / v, weight / volume); wherein the sugar is sucrose; the substrate includes at least one of tea soup, plant extract, and fruit and vegetable juice; in the fermentation step, the inoculation amount of the Saccharomyces cerevisiae BM2 is in the range of 2.00% (v / v, volume / volume) to 5.00% (v / v, volume / volume); the protease is derived from pineapple, papaya, fig, kiwi, or any combination thereof; in the step of inducing autolysis of the Saccharomyces cerevisiae, after adding the protease to the fermentation broth, the fermentation broth with the added protease is incubated at a temperature of 50°C to 55°C for 4 hours to 12 hours to induce autolysis of the Saccharomyces cerevisiae BM2; before the fermentation step, the sugar-containing fermentation substrate is subjected to pasteurization in a sealed tank at a temperature of 85°C for 30 minutes; in the fermentation step, after inoculating the Saccharomyces cerevisiae BM2 into the sugar-containing fermentation substrate, the fermentation broth is incubated at 18°C to 30°C for 8 hours to 48 hours until the residual sugar content of the fermentation broth is <0.5% (w / v, weight / volume); in the secondary fermentation step, the autolysed Saccharomyces cerevisiae fermentation broth is subjected to secondary aerobic fermentation at a temperature of 30°C to 35°C with a stirring speed of 200 rpm to 400 rpm until the alcohol concentration in the autolysed Saccharomyces cerevisiae fermentation broth during the fermentation process is <0.5% (w / v, weight / volume); in the secondary fermentation step, non-fermented sugar is added to the secondary fermentation broth to adjust the sweetness.
[0017] The present application has the following advantages:
[0018] The present application provides a preparation method of a fermented beverage, which can produce a fermented beverage containing β-nicotinamide mononucleotide (NMN) active probiotics. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 A block diagram of the preparation process of a preferred embodiment of the present application. DETAILED DESCRIPTION
[0020] The following only illustrates possible implementation manners of the present application by way of examples, but is not used to limit the scope of the present application.
[0021] Reference is made to Figure 1 , which shows a preferred embodiment of the present application, a method for preparing a fermented beverage of the present application, comprising the following steps: step S1 : preparing a sugar-containing fermentation substrate, the sugar content of the sugar-containing fermentation substrate ranges from 0.50% (w / v, weight / volume) to 20.00% (w / v, weight / volume); step S2: fermentation, inoculating Saccharomyces cerevisiae BM2 for preparing nicotinamide mononucleotide (NMN) into the sugar-containing fermentation substrate, and then incubating and fermenting the sugar-containing fermentation substrate containing Saccharomyces cerevisiae BM2 until the residual sugar content of the fermentation broth is less than 0.5% (w / v, weight / volume); step S3: inducing autolysis of Saccharomyces cerevisiae, adding protease to the fermentation broth to induce autolysis of Saccharomyces cerevisiae BM2 to produce autolysed Saccharomyces cerevisiae fermentation broth; step S4: secondary fermentation, inoculating Bacillus coagulans into the autolysed Saccharomyces cerevisiae fermentation broth for secondary aerobic fermentation to produce secondary fermentation broth, the secondary fermentation broth containing nicotinamide mononucleotide (NMN).
[0022] <0.5% (w / v, weight / volume) of the fermentation broth; step S3: inducing autolysis of Saccharomyces cerevisiae, adding protease to the fermentation broth to induce autolysis of Saccharomyces cerevisiae BM2 to produce autolysed Saccharomyces cerevisiae fermentation broth; step S4: secondary fermentation, inoculating Bacillus coagulans into the autolysed Saccharomyces cerevisiae fermentation broth for secondary aerobic fermentation to produce secondary fermentation broth, the secondary fermentation broth containing nicotinamide mononucleotide (NMN).
[0023] In this embodiment, the Saccharomyces cerevisiae BM2 is Saccharomyces cerevisiae BM2 (Genebank accession number: PP338187).
[0024] After the Saccharomyces cerevisiae BM2 is induced to autolyze, macromolecules in the cell are degraded into various biological small molecules and overflow the cell, including nicotinamide mononucleotide (NMN).
[0025] The sugar-containing fermentation substrate is a substrate of 0.50% (w / v, weight / volume) to 5.00% (w / v, weight / volume) to which sugar is added to adjust the sugar content to a sugar content range of 0.50% (w / v, weight / volume) to 20.00% (w / v, weight / volume); wherein the substrate comprises at least one of tea soup, plant extract, and fruit and vegetable juice. Preferably, the sugar-containing fermentation substrate is a substrate of 2.50% (w / v, weight / volume) to 5.00% (w / v, weight / volume) to which sugar is added to adjust the sugar content to a sugar content range of 2.00% (w / v, weight / volume) to 5.00% (w / v, weight / volume); wherein the sugar is sucrose. In other embodiments, the sugar can also be glucose, fructose, or maltose, but is not limited thereto.
[0026] Further, the tea soup can be green tea, black tea, oolong tea, coffee leaf tea, Pu'er tea, glutinous rice fragrant leaf tea, variegated leaf tea, or chrysanthemum tea, but is not limited thereto. The plant extract can be a wolfberry extract or a red date extract, but is not limited thereto.
[0027] The tea soup or plant extract is weighed with tea leaves or plant material, and water is added and heated at a temperature of 70°C to 100°C (preferably 85°C) for 5 minutes to 45 minutes (preferably 30 minutes of extraction). After extraction, the tea leaves or plant material are filtered out, and only the filtrate is left.
[0028] The protease is derived from pineapple, papaya, fig, kiwi, or any combination thereof. Preferably, the protease is derived from pineapple or is a protease mixture derived from multiple fruits.
[0029] Before the fermentation step, the sugar-containing fermentation substrate is subjected to pasteurization in a sealed tank. Specifically, pasteurization is performed at a temperature of 85°C for 30 minutes. In this way, many miscellaneous bacteria can be avoided from growing in the sugar-containing fermentation substrate at the initial stage of fermentation.
[0030] In the fermentation step, the inoculation amount of the Saccharomyces cerevisiae BM2 is in the range of 0.50% (v / v, volume / volume) to 20.00% (v / v, volume / volume). Preferably, the inoculation amount of the Saccharomyces cerevisiae BM2 is in the range of 2.00% (v / v, volume / volume) to 5.00% (v / v, volume / volume).
[0031] Specifically, in the fermentation step, after inoculating Saccharomyces cerevisiae BM2 into the sugar-containing fermentation substrate, the fermentation liquid is incubated at 18-30°C for 8-48 hours until the residual sugar content of the fermentation liquid is <0.5% (w / v, weight / volume).
[0032] In the step of inducing autolysis of Saccharomyces cerevisiae BM2, the amount of protease added ranges from 1.00 U / mL (Units per milliliter) to 5.00 U / mL (Units per milliliter). In the step of inducing autolysis of Saccharomyces cerevisiae BM2, after adding protease to the fermentation liquid, the fermentation liquid with added protease is incubated at a temperature of 37-55°C (preferably, a temperature of 50-55°C) for 4-36 hours (preferably, 4-12 hours) to induce autolysis of Saccharomyces cerevisiae BM2.
[0033] In the secondary fermentation step, the Saccharomyces cerevisiae BM2 fermentation liquid that has been induced to autolyze is subjected to secondary aerobic fermentation at a temperature of 30-35°C with stirring at a stirring speed of 200-400 rpm until the alcohol concentration in the Saccharomyces cerevisiae BM2 fermentation liquid that has been induced to autolyze is <0.5% (w / v, weight / volume) (end of fermentation). The residual sugar and alcohol serve as carbon sources for fermentation of Bacillus coagulans, and the water-soluble proteins (yeast extract) released after autolysis of Saccharomyces cerevisiae BM2 serve as nitrogen sources.
[0034] In the secondary fermentation step, non-fermentable sugar is added to the secondary fermentation liquid to adjust the sweetness. The non-fermentable sugar is inulin, mannitol, or sorbitol, i.e., a fermentation beverage containing nicotinamide mononucleotide (NMN) is completed; further, the fermentation beverage containing nicotinamide mononucleotide (NMN) has a natural tea aroma or a vegetable and fruit juice aroma, contains no alcohol, and has a content of nicotinamide mononucleotide (NMN) that is directly proportional to the inoculation amount of Saccharomyces cerevisiae BM2, has a sour taste of a carboxylic acid, and contains live Bacillus coagulans probiotics.
[0035] Therefore, the sugar-containing fermentation substrate is suitable for the growth of the Saccharomyces cerevisiae BM2, and causes the Saccharomyces cerevisiae BM2 to autolyze in the sugar-containing fermentation substrate to produce β-nicotinamide mononucleotide (NMN), so as to produce a kombucha-like flavored and β-nicotinamide mononucleotide (NMN) active probiotic.
[0036] (Example One)
[0037] 1. 10 grams of black tea was weighed, 450 mL of hot water at 95°C was added to brew for 5 minutes, and then about 400 mL of tea soup was filtered out after the tea residue was removed. 8 grams of 2% (w / v, weight / volume) sucrose was added, and sterilized in a 85°C water bath for 30 minutes.
[0038] 2. After the tea soup was cooled to room temperature, 2% (v / v, volume / volume) of the Saccharomyces cerevisiae BM2 (cultured in potato dextrose broth) was inoculated, and incubated at 26°C for 12 hours. Before inducing the autolysis of the Saccharomyces cerevisiae BM2, the content of β-nicotinamide mononucleotide (NMN) was analyzed, and no detection was found. 0.4 grams of salt-free tenderizer (containing papain and bromelain) was added to the 50°C water bath to induce the autolysis of the Saccharomyces cerevisiae BM2 for 4 hours.
[0039] 3. 2% (v / v, volume / volume) of Bacillus coagulans (cultured in MRS broth) was inoculated into the Saccharomyces cerevisiae fermentation broth that had been induced to autolyze, and incubated at 30°C with shaking at 200 rpm for 24 hours. The content of β-nicotinamide mononucleotide (NMN) was analyzed to be 54.5 ± 9.1 ppm (parts per million).
[0040] 4. A (black tea) fermented beverage containing β-nicotinamide mononucleotide (NMN) was prepared, which scavenged 50% DPPH (1,1-diphenyl-2-trinitrophenylhydrazyl radical) at IC50 (half maximal inhibitory concentration) = 1.66 mg / mL.
[0041] (Example Two)
[0042] 1. Pineapple was juiced and the juice was filtered to remove the pulp. The sugar content was adjusted to 6°Bx (Brix) using water and fructose. The juice was sterilized by heating at 85°C for 30 minutes.
[0043] 2. After cooling to room temperature, 20.00% (v / v, volume / volume) of Saccharomyces cerevisiae BM2 (cultured in 2% sucrose water) was inoculated and incubated at 23°C for 8 hours. Before inducing autolysis, the β-nicotinamide mononucleotide (NMN) content was analyzed and no NMN was detected. One-thousandth of bromelain (85K CDU / mg (Casein Digestion Unit)) was added to induce autolysis of the Saccharomyces cerevisiae BM2 at 55°C for 8 hours.
[0044] 3. 1% (v / v, volume / volume) of Bacillus coagulans (cultured in MRS Broth) was inoculated and incubated at 30°C with shaking at 200 rpm for 24 hours. The β-nicotinamide mononucleotide (NMN) content was analyzed and found to be 92.9 ± 3.5 ppm.
[0045] (Example Three)
[0046] 1. 20 grams of oolong tea was weighed and infused in 450 mL of hot water at 98°C for 5 minutes. The tea soup was filtered to remove the tea residue, and about 400 mL of tea soup was obtained. 8 grams (2% (w / v, weight / volume)) of fructose was added, and the tea soup was sterilized by heating at 85°C for 30 minutes.
[0047] 2. After the tea soup is cooled to room temperature, inoculate 1% (v / v, volume / volume) of the Saccharomyces cerevisiae BM2 (cultured in potato dextrose broth) and incubate at 30°C for 48 hours. Before inducing autolysis of the Saccharomyces cerevisiae BM2, analyze the β-nicotinamide mononucleotide (NMN) in the fermentation broth. No results are detected. Add one-thousandth of the bromelain (85K CDU / mg (Casein Digestion Unit)) to the 5°C water bath to induce autolysis of the Saccharomyces cerevisiae BM2 for 8 hours.
[0048] 3. Inoculate 0.5% (v / v, volume / volume) of Bacillus coagulans (cultured in MRS broth) and incubate at 30°C with shaking at 200 rpm for 24 hours. Analyze the β-nicotinamide mononucleotide (NMN) content, which is 41.1 ± 9.8 ppm.
[0049] 4. Prepare a fermented beverage containing β-nicotinamide mononucleotide (NMN) (oolong tea), which has a DPPH (1,1-diphenyl-2-picrylhydrazyl radical) scavenging concentration of IC50 (half maximal inhibitory concentration) = 26 μg / mL (micrograms per milliliter).
[0050] By the above method, the present application can prepare a fermented beverage containing β-nicotinamide mononucleotide (NMN) active probiotics, which is beneficial for the effective absorption and utilization of β-nicotinamide mononucleotide (NMN) by the human body.
Claims
1. A method of preparing a fermented beverage, characterized in that, comprising the steps of: providing a sugar-containing fermentation medium having a sugar content ranging from 0.50 w / v% to 20.00 w / v%; fermenting the sugar-containing fermentation medium inoculated with Saccharomyces cerevisiae BM2 for producing β-nicotinamide mononucleotide, followed by incubating the Saccharomyces cerevisiae -containing fermentation medium until a residual sugar content <0.5 w / v% is formed; inducing autolysis of the Saccharomyces cerevisiae by adding protease to the fermentation medium to induce autolysis of the Saccharomyces cerevisiae BM2 to produce an autolysed Saccharomyces cerevisiae fermentation medium; secondary fermentation by inoculating Bacillus coagulans to the autolysed Saccharomyces cerevisiae fermentation medium for secondary aerobic fermentation to produce a secondary fermentation medium having β-nicotinamide mononucleotide.
2. The method of preparing a fermented beverage according to claim 1, characterized in that, the sugar-containing fermentation medium is a 0.50 w / v% to 5.00 w / v% medium to which sugar is added to adjust the sugar content to a sugar content ranging from 0.50 w / v% to 20.00 w / v%; wherein the medium comprises at least one of tea infusion, plant extract, and fruit and vegetable juice.
3. The method of preparing a fermented beverage according to claim 1, characterized in that, the protease is derived from pineapple, papaya, fig, kiwifruit, or any combination thereof.
4. The method of preparing a fermented beverage according to claim 1, characterized in that, in the fermentation step, the inoculation amount of the Saccharomyces cerevisiae BM2 ranges from 0.50 v / v% to 20.00 v / v%.
5. The method of preparing a fermented beverage according to claim 1, wherein in the step of inducing autolysis of the Saccharomyces cerevisiae, the amount of protease added ranges from 1.00 U / mL to 5.00 U / mL.
6. The method of preparing a fermented beverage according to claim 1, wherein in the step of inducing autolysis of the Saccharomyces cerevisiae, after adding protease to the fermentation medium, the protease-added fermentation medium is incubated at a temperature ranging from 37°C to 55°C for 4 hours to 36 hours to induce autolysis of the Saccharomyces cerevisiae BM2.
7. The method of preparing a fermented beverage according to claim 1, wherein before the fermentation step, the sugar-containing fermentation medium is subjected to pasteurization in a closed tank.
8. The method of preparing a fermented beverage according to claim 1, characterized in that, in the fermentation step, after inoculating the Saccharomyces cerevisiae BM2 into the sugar-containing fermentation medium, the fermentation medium is incubated at 18°C to 30°C for 8 hours to 48 hours until a residual sugar content <0.5 w / v% is formed.
9. The method of preparing a fermented beverage according to claim 1, wherein in the secondary fermentation step, the secondary fermentation medium is added with non-fermenting sugar to adjust sweetness.
10. The method of preparing a fermented beverage according to claim 5, wherein The sugar-containing fermentation substrate is 2.50 w / v% to 5.00 w / v% substrate, and sugar is added to the substrate to adjust the sugar content to a sugar content range of 2.00 w / v% to 5.00 w / v%; wherein the sugar is sucrose; the substrate includes at least one of tea soup, plant extract, fruit and vegetable juice; in the fermentation step, the inoculation amount of Saccharomyces cerevisiae BM2 is in the range of 2.00 v / v% to 5.00 v / v%; the protease is derived from pineapple, papaya, fig, kiwi or any combination thereof; in the step of inducing autolysis of Saccharomyces cerevisiae, after adding protease to the fermentation broth, the fermentation broth with added protease is incubated at a temperature of 50°C to 55°C for 4 hours to 12 hours to induce autolysis of Saccharomyces cerevisiae BM2; before the fermentation step, the sugar-containing fermentation substrate is subjected to pasteurization in a sealed tank at a temperature of 85°C for 30 minutes; in the fermentation step, after inoculating Saccharomyces cerevisiae BM2 into the sugar-containing fermentation substrate, it is incubated at 18°C to 30°C for 8 hours to 48 hours until the residual sugar content of the fermentation broth is <0.5 w / v%; in the secondary fermentation step, the Saccharomyces cerevisiae fermentation broth after induction of autolysis is subjected to secondary aerobic fermentation at a temperature of 30°C to 35°C with stirring at a stirring speed of 200 rpm to 400 rpm until the alcohol concentration in the Saccharomyces cerevisiae fermentation broth after induction of autolysis is <0.5 w / v% during fermentation; in the secondary fermentation step, non-fermented sugar is added to the secondary fermentation broth to adjust the sweetness.