CN112869164B - Preparation method of broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) - Google Patents
Preparation method of broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) Download PDFInfo
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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
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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
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/09—Mashed or comminuted products, e.g. pulp, purée, sauce, or products made therefrom, e.g. snacks
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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
- A23L33/13—Nucleic acids or derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses a preparation method of a broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN), which comprises the following steps: the method comprises the following steps: pre-treating, namely, respectively selecting, cleaning, carrying out ozone sterilization, leaching, draining and pulping on the broccoli, pumping the broccoli into a sterile fermentation tank through a thick pulp pump, adding a red date extraction concentrated solution (containing adenosine cyclophosphate), a ginseng extraction concentrated solution (containing ATP) and water, stirring and mixing uniformly, and carrying out the second step: performing enzymolysis before fermentation, namely adding pectinase and cellulase into the mixed pulp obtained by the pretreatment in the step one to perform enzymolysis, and performing the step three: and (3) fermentation, namely adding glucose, nicotinamide, magnesium sulfate and dipotassium hydrogen phosphate into the mixed liquor obtained by the second enzymolysis, stirring for dissolving, heating, inactivating enzymes and sterilizing, and performing the fourth step: and (3) post-treatment, namely, compared with the prior art (CN 110559224A, the broccoli extract contains about 1.4% w/w of NMN) and the environmental pollution of the fermentation liquor obtained by the fermentation in the step three is greatly reduced compared with the traditional method.
Description
Technical Field
The invention relates to the technical field of functional food preparation by a fermentation method, in particular to a preparation method of a broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN).
Background
Nicotinamide Mononucleotide (NMN) is an endogenous substance in the human body and is produced by nicotinamide under the catalysis of nicotinamide phosphoribosyltransferase, and then NMN produces NAD + (nicotinamide adenine dinucleotide) under the catalysis of nicotinamide mononucleotide adenylyltransferase. The physiological effects of NMN in humans are all accomplished by conversion to NAD +. The existing scientific research shows that NMN can improve ischemic brain injury and Alzheimer's disease, has therapeutic effect on Parkinson's disease, degenerative disease ] and obesity, and can also relieve myocardial ischemia reperfusion injury, cerebral hemorrhage and cerebral infarction hemorrhage injury, etc.; the most popular recent research is the development and application of NMN in the aspects of anti-aging, medicine and health care products. The American scientist David Sinclair has proved that NMN has the effect of reversing physiological aging in mice, and Japan takes the lead to push out the main beating product in 2016, but the content of beta-nicotinamide mononucleotide is only 3000mg (only 10 days are eaten), but the price is nearly 7000 yuan. The poor cost performance of the japanese beta-nicotinamide mononucleotide product limits the popularization of the revolutionary anti-aging substance, and almost falls into rich privilege. In 2018, a dietary supplement product Reinvigoror (China: revitto) based on research results of Harvard medical college was issued by Herbalmax (American biotechnology company), the main component of the product is NMN, and the price of the drug-grade NMN is successfully reduced by more than 90% by Herbalmax through a biological enzyme catalysis technology (the price of basic editions such as Jingdong, tecatus, suning and the like is not more than 2000 yuan), so that the administration cost of the NMN enters the bearing range of general high-income groups. In 2018, NMN proved by experiment of Greenwich aging biology center of Harvard university can prolong the life of mammals by more than 30%. For this reason, the main discoverer of NMN anti-aging efficacy-professor of harvard medical college, paul, F, galen bioscience aging biology, leading to David Sinclair, was also commented by the times journal as "100 most influential people worldwide". 11.2.2020, professor David Sinclair at the medical college of harvard university and professor Lindsay Wu at university of new south wals brought a new result to the research team: NMN can reverse oocyte senescence, thereby restoring fertility to women during reproductive senescence. In addition to pharmaceuticals and nutraceuticals containing NMN as an active ingredient, NMN is also found in emulsions of natural foods such as vegetables, fruits, meats, and mammals. According to the FDA equivalent principle, one adult of 70kg should supplement 600mg of NMN every day, and one adult should supplement the same amount of NMN, then need eat 54-240 kg of broccoli, therefore, it is lower to see, supplement the efficiency through eating the broccoli source directly, and prepare a broccoli extract (containing more than 5.0% w/w of NMN) of high containing NMN through the fermentation method can reach this effect relatively fast.
A broccoli extract, a preparation method and application thereof are disclosed in the prior patent CN 110559224A. Adding an extraction solvent into broccoli powder, performing ultrasonic-assisted extraction for 60-90 min, collecting an extracting solution, cooling, centrifuging, performing reduced-pressure filtration and filter membrane ultrafiltration, and collecting filtrate; c) And (4) carrying out vacuum freeze drying on the collected filtrate to obtain the broccoli extract. The content of nicotinamide mononucleotide in the broccoli extract prepared by the method is only 13.724. Mu.g/mg (about equivalent to 1.4% w/w).
At present, other production processes reported in China adopt a chemical synthesis method and an enzymatic method, wherein the chemical synthesis method uses ribose nicotinate as a raw material and uses phosphorus oxychloride for phosphorylation to obtain NMN (reference: CHEM. COMMON.1999, 729-730), the yield is low, the product purity is poor, a large amount of organic solvent is used, the environmental damage is serious, and the phosphorus oxychloride is a relatively dangerous reagent. A holoenzyme process (CN 201611245619) uses nicotinamide ribose as a substrate, and reacts in the presence of phosphate donors such as ATP and under the catalysis of nicotinamide ribokinase or recombinant cells containing nicotinamide ribokinase to generate beta-nicotinamide mononucleotide. The nicotinamide ribokinase used is from Saccharomyces cerevisiae.
However, in the two existing methods, a large amount of organic solvents and acid and alkali are used in the actual production process, so that the environment is greatly polluted.
Disclosure of Invention
1. Technical problem to be solved
The invention aims to solve the problem that a large amount of organic solvents and acid and alkali are used in the actual production process in the prior art, which causes great pollution to the environment, and provides a preparation method of a broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN).
2. Technical scheme
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) comprises the following steps:
the method comprises the following steps: pre-treating, selecting, cleaning, sterilizing with ozone, leaching, pulping, adding into sterile fermentation tank with thick pulp pump, adding concentrated extractive solution of fructus Jujubae (containing adenosine cyclophosphate), concentrated extractive solution of Ginseng radix (containing ATP), and water, stirring, and mixing.
Step two: and (3) performing enzymolysis before fermentation, namely adding pectinase and cellulase into the mixed pulp obtained by the pretreatment in the step one to perform enzymolysis, wherein the enzymolysis temperature is 40-55 ℃, the enzymolysis pH is 3.4-6.5, and the enzymolysis time is 2-8 h.
Step three: and (2) fermenting, namely adding glucose, nicotinamide, magnesium sulfate and dipotassium hydrogen phosphate into the mixed liquor obtained by the enzymolysis in the second step respectively, stirring for dissolving, heating, carrying out enzyme deactivation and sterilization, cooling the mixed liquor to 20-32 ℃, inoculating a starter saccharomyces cerevisiae with the inoculation amount of 0.05-0.1%, maintaining the temperature at 20-32 ℃, fermenting for 3-7 days, intermittently stirring in the fermentation process, and stirring for 20-30 minutes every 12 hours, wherein the stirring mode can be mechanical stirring or stirring by introducing sterile compressed air.
Step four: performing post-treatment, namely sterilizing, cooling, performing centrifugal separation on the fermentation liquor obtained in the third step to collect filtrate, concentrating the filtrate at low temperature under reduced pressure until the relative density of the extract is 1.01-1.05, and performing vacuum freeze drying to obtain the high-NMN-content broccoli extract, wherein the NMN content can reach more than 5.0% (w/w), and the extract can be used for anti-aging functional food; wherein the sterilization temperature is controlled at 60-90 deg.C, preferably 75-85 deg.C, and the sterilization time is 10-40 min, preferably 20-30 min; wherein the concentration temperature is 30-50 deg.C, preferably 35-45 deg.C, and vacuum degree is below 80 pa.
Preferably, 300-600 parts of broccoli, 100-300 parts of red date extract concentrated solution (containing adenosine cyclophosphate), 100-300 parts of ginseng extract concentrated solution (containing ATP) and 200-500 parts of water in the first step.
Preferably, the enzymolysis temperature in the second step is most preferably 48-52 ℃.
Preferably, the enzymolysis in the second step is most preferably pH4.0-5.0.
Preferably, the enzymolysis time in the second step is preferably 4-5h.
Preferably, the addition amount of the pectinase in the second step is 0.01-0.07%; wherein the addition amount of cellulase (1 ten thousand u/g) is 0.05-0.15%.
Preferably, the addition amount of the pectinase in the second step is preferably 0.03-0.05%.
Preferably, the addition amount of the cellulase (1 ten thousand u/g) in the second step is preferably 0.1-0.12%.
Preferably, the fermentation strain obtained in the third step is obtained by separating and screening fresh broccoli, is used as an original strain, and is subjected to activation (slant culture), expanding culture (liquid culture) and Karschner's jar amplification culture, fermentation broth is subjected to centrifugal separation after fermentation is finished to obtain saccharomyces cerevisiae bacterial sludge, and the saccharomyces cerevisiae bacterial sludge is inoculated to the broccoli enzymolysis mixed liquor for fermentation; or directly adding the yeast liquid in the Kaschin tank into the blue flower enzymolysis mixed liquid for fermentation without centrifugal separation.
Preferably, the three steps comprise 1-8% of glucose, 0.1-1.0% of nicotinamide, 0.01-0.03% of magnesium sulfate and 0.2-0.3% of dipotassium phosphate, wherein the enzyme inactivation and sterilization temperature is controlled at 60-90 ℃, preferably 75-85 ℃, and the enzyme inactivation and sterilization time is 10-40 minutes, preferably 20-30 minutes.
3. Advantageous effects
Compared with the prior art, the invention has the advantages that:
in the invention, the product with high NMN content (containing more than 5.0% of NMN w/w) is produced at lower cost, compared with the prior art (CN 110559224A, the broccoli extract contains about 1.4% of NMN w/w), and the environmental pollution is greatly reduced compared with the traditional method.
Drawings
FIG. 1 is a sample map of a preparation method of a broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) according to example 3 of the present invention;
FIG. 2 is a map of a control sample in example 3 of a method for preparing a broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1:
a method for preparing broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) comprises the following steps:
the method comprises the following steps: pre-treating, selecting, cleaning, sterilizing with ozone, leaching, draining, pulping, pumping into sterile fermentation tank with thick pulp pump, adding fructus Jujubae extract concentrate (containing adenosine cyclophosphate), ginseng radix extract concentrate (containing ATP), and water, stirring, and mixing.
Step two: and (3) performing enzymolysis before fermentation, namely adding pectinase and cellulase into the mixed pulp obtained by the pretreatment in the step one to perform enzymolysis, wherein the enzymolysis temperature is 40-55 ℃, the enzymolysis pH is 3.4-6.5, and the enzymolysis time is 2-8 h.
Step three: and (2) fermenting, namely adding glucose, nicotinamide, magnesium sulfate and dipotassium hydrogen phosphate into the mixed liquor obtained by the enzymolysis in the second step respectively, stirring for dissolving, heating, carrying out enzyme deactivation and sterilization, cooling the mixed liquor to 20-32 ℃, inoculating a starter saccharomyces cerevisiae with the inoculation amount of 0.05-0.1%, maintaining the temperature at 20-32 ℃, fermenting for 3-7 days, intermittently stirring in the fermentation process, and stirring for 20-30 minutes every 12 hours, wherein the stirring mode can be mechanical stirring or stirring by introducing sterile compressed air.
Step four: performing post-treatment, namely sterilizing, cooling, performing centrifugal separation on the fermentation liquor obtained in the third step to collect filtrate, concentrating the filtrate at low temperature under reduced pressure until the relative density of the extract is 1.01-1.05, and performing vacuum freeze drying to obtain the broccoli extract with high NMN content, wherein the NMN content can reach more than 5.0% (w/w), and the broccoli extract can be used for anti-aging functional foods; wherein the sterilization temperature is controlled at 60-90 deg.C, preferably 75-85 deg.C, and the sterilization time is 10-40 min, preferably 20-30 min; wherein the concentration temperature is 30-50 deg.C, preferably 35-45 deg.C, and vacuum degree is below 80 pa.
Example 2:
a method for preparing broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) comprises the following steps:
the method comprises the following steps: pre-treatment, namely, respectively selecting, cleaning, carrying out ozone sterilization, leaching and draining, pulping the broccoli, pumping the broccoli into a sterile fermentation tank through a thick slurry pump, adding red date extract concentrated solution (containing adenosine cyclophosphate), ginseng extract concentrated solution (containing ATP) and water, and uniformly stirring and mixing, wherein in the step one, 300-600 parts of broccoli, 100-300 parts of red date extract concentrated solution (containing adenosine cyclophosphate), 100-300 parts of ginseng extract concentrated solution (containing ATP) and 200-500 parts of water are added.
Step two: performing enzymolysis before fermentation, namely adding pectinase and cellulase into the mixed pulp obtained by the pretreatment in the first step for enzymolysis, wherein the enzymolysis temperature is 40-55 ℃, the enzymolysis pH is 3.4-6.5, and the enzymolysis time is 2-8 h, the enzymolysis temperature in the second step is most preferably 48-52 ℃, the enzymolysis pH in the second step is most preferably 4.0-5.0, the enzymolysis time in the second step is preferably 4-5h, and the addition amount of the pectinase in the second step is 0.01-0.07%; wherein the addition amount of cellulase (1 ten thousand u/g) is 0.05-0.15%, the addition amount of pectinase in the second step is preferably 0.03-0.05%, and the addition amount of cellulase (1 ten thousand u/g) in the second step is preferably 0.1-0.12%.
Step three: fermenting, namely adding glucose, nicotinamide, magnesium sulfate and dipotassium hydrogen phosphate into the mixed liquor obtained by the enzymolysis in the second step respectively, stirring for dissolving, heating, carrying out enzyme deactivation and sterilization, cooling the mixed liquor to 20-32 ℃, inoculating a starter saccharomyces cerevisiae with the inoculum size of 0.05-0.1%, maintaining the temperature at 20-32 ℃, fermenting for 3-7 days, intermittently stirring in the fermentation process, stirring for 20-30 minutes every 12 hours, stirring mechanically or introducing sterile compressed air for stirring, separating and screening the fermentation strain obtained in the third step from fresh broccoli, taking the fermentation strain as an original strain, carrying out activation (slant culture), expanding culture (liquid culture) and Karschner's jar amplification culture, carrying out centrifugal separation on the fermentation liquor after the fermentation is finished to obtain saccharomyces cerevisiae bacterial sludge, and inoculating the saccharomyces cerevisiae bacterial sludge into the broccoli enzymolysis mixed liquor for fermentation; or directly adding yeast liquid in a Kaschin tank into the blue flower enzymolysis mixed liquid for fermentation without centrifugal separation, wherein in the step three, the glucose is 1-8%, the nicotinamide is 0.1-1.0%, the magnesium sulfate is 0.01-0.03%, and the dipotassium hydrogen phosphate is 0.2-0.3%, wherein the enzyme inactivation and sterilization temperature is controlled at 60-90 ℃, preferably 75-85 ℃, and the enzyme inactivation and sterilization time is 10-40 minutes, preferably 20-30 minutes.
Step four: performing post-treatment, namely sterilizing, cooling, performing centrifugal separation on the fermentation liquor obtained in the third step to collect filtrate, concentrating the filtrate at low temperature under reduced pressure until the relative density of the extract is 1.01-1.05, and performing vacuum freeze drying to obtain the high-NMN-content broccoli extract, wherein the NMN content can reach more than 5.0% (w/w), and the extract can be used for anti-aging functional food; wherein the sterilization temperature is controlled at 60-90 deg.C, preferably 75-85 deg.C, and the sterilization time is 10-40 min, preferably 20-30 min; wherein the concentration temperature is 30-50 deg.C, preferably 35-45 deg.C, and vacuum degree is below 80 pa.
Example 3:
referring to fig. 1-2, a method for preparing a broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) comprises the following steps:
the method comprises the following steps: pre-treatment, namely, respectively selecting, cleaning, carrying out ozone sterilization, leaching and draining, pulping the broccoli, pumping the broccoli into a sterile fermentation tank through a thick slurry pump, adding red date extract concentrated solution (containing adenosine cyclophosphate), ginseng extract concentrated solution (containing ATP) and water, and uniformly stirring and mixing, wherein in the step one, 300-600 parts of broccoli, 100-300 parts of red date extract concentrated solution (containing adenosine cyclophosphate), 100-300 parts of ginseng extract concentrated solution (containing ATP) and 200-500 parts of water are added.
Step two: performing enzymolysis before fermentation, namely adding pectinase and cellulase into the mixed pulp obtained by the pretreatment in the first step for enzymolysis, wherein the enzymolysis temperature is 40-55 ℃, the enzymolysis pH is 3.4-6.5, and the enzymolysis time is 2-8 h, the enzymolysis temperature in the second step is most preferably 48-52 ℃, the enzymolysis pH in the second step is most preferably 4.0-5.0, the enzymolysis time in the second step is preferably 4-5h, and the addition amount of the pectinase in the second step is 0.01-0.07%; wherein the addition amount of cellulase (1 wu/g) is 0.05-0.15%, the addition amount of pectinase in the second step is preferably 0.03-0.05%, and the addition amount of cellulase (1 wu/g) in the second step is preferably 0.1-0.12%.
Step three: fermenting, namely adding glucose, nicotinamide, magnesium sulfate and dipotassium hydrogen phosphate into the mixed liquor obtained by the enzymolysis in the second step respectively, stirring for dissolving, heating, carrying out enzyme deactivation and sterilization, cooling the mixed liquor to 20-32 ℃, inoculating a starter saccharomyces cerevisiae with the inoculum size of 0.05-0.1%, maintaining the temperature at 20-32 ℃, fermenting for 3-7 days, intermittently stirring in the fermentation process, stirring for 20-30 minutes every 12 hours, stirring mechanically or introducing sterile compressed air for stirring, separating and screening the fermentation strain obtained in the third step from fresh broccoli, taking the fermentation strain as an original strain, carrying out activation (slant culture), expanding culture (liquid culture) and Karschner's jar amplification culture, carrying out centrifugal separation on the fermentation liquor after the fermentation is finished to obtain saccharomyces cerevisiae bacterial sludge, and inoculating the saccharomyces cerevisiae bacterial sludge into the broccoli enzymolysis mixed liquor for fermentation; or directly adding yeast liquid in a Kaschin tank into the blue flower enzymolysis mixed liquid for fermentation without centrifugal separation, wherein in the step three, the glucose is 1-8%, the nicotinamide is 0.1-1.0%, the magnesium sulfate is 0.01-0.03%, and the dipotassium hydrogen phosphate is 0.2-0.3%, wherein the enzyme inactivation and sterilization temperature is controlled at 60-90 ℃, preferably 75-85 ℃, and the enzyme inactivation and sterilization time is 10-40 minutes, preferably 20-30 minutes.
Step four: performing post-treatment, namely sterilizing, cooling, performing centrifugal separation on the fermentation liquor obtained in the third step to collect filtrate, concentrating the filtrate at low temperature under reduced pressure until the relative density of the extract is 1.01-1.05, and performing vacuum freeze drying to obtain the broccoli extract with high NMN content, wherein the NMN content can reach more than 5.0% (w/w), and the broccoli extract can be used for anti-aging functional foods; wherein the sterilization temperature is controlled at 60-90 deg.C, preferably 75-85 deg.C, and the sterilization time is 10-40 min, preferably 20-30 min; wherein the concentration temperature is 30-50 deg.C, preferably 35-45 deg.C, and vacuum degree is below 80 pa.
In the invention, the fermentation strain saccharomyces cerevisiae is obtained by separating and screening fresh broccoli and is used as an original strain to be prepared into a glycerol freezing pipe for preservation at (-80 ℃). And performing slant culture activation (culture medium: 2% of glucose, 1% of peptone, 1% of yeast extract powder, 1.5% of agar powder, 28 +/-2 ℃ and 42 hours) and liquid culture expansion culture (culture medium: 2% of glucose, 1% of peptone, 1% of yeast extract powder, 1.5% of agar powder, 250ml shaking table vibration, 28 +/-2 ℃ and 24 hours), inoculating the culture solution into a 10-liter Karschner jar filled with 5kg of wort (12 ℃) for amplification culture (25 ℃ and 72 hours), and after the fermentation is finished, centrifuging the fermentation liquor by a refrigerated centrifuge (4000 rpm and 10 minutes) to separate 120g of saccharomyces cerevisiae bacterial sludge (water content: 80%).
In the invention, the content determination method of the broccoli extract NMN comprises the following steps of: weighing the obtained stigma croci Sativi extract about 0.1g, precisely weighing, adding into 100ml volumetric flask, adding water 50ml for dissolving, ultrasonic treating for 10min, adding water for diluting to 100ml, and mixing. Putting part of the solution into a centrifuge tube, centrifuging at 4500r/min for 20min, collecting supernatant, and filtering with 0.45 μm filter membrane. And precisely weighing an appropriate amount of NMN reference substance to prepare a reference substance solution containing about 0.1mg/ml of NMN for later use.
In the invention, the chromatographic conditions of the high performance liquid chromatography are as follows: shim-packVP-ODS (150 mm. Times.4.6 mm,5 μm) column Shijin, column temperature: 40 ℃; mobile phase: a is 0.1% acetic acid water solution, B is methanol solution containing 2mmol/L ammonium acetate; flow rate 1.0mL/min, detection wavelength: 244nm, and the sample size is 10 μ L.
In the present invention, the gradient elution conditions are as follows:
| time (minutes) | Mobile phase A | |
| 0 | 95 | 5 |
| 3 | 65 | 35 |
| 5 | 0 | 100 |
| 8.1 | 95 | 5 |
| 12 | 95 | 5 |
。
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (7)
1. A preparation method of broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) is characterized by comprising the following steps:
the method comprises the following steps: pre-treating, namely respectively selecting, cleaning, carrying out ozone sterilization, leaching, draining and pulping broccoli, pumping into a sterile fermentation tank through a thick slurry pump, adding red date extract concentrated solution containing adenosine cyclophosphate, ginseng extract concentrated solution containing ATP and water, and uniformly stirring and mixing;
step two: performing enzymolysis before fermentation, namely adding pectinase and cellulase into the mixed pulp obtained by the pretreatment in the step one for enzymolysis, wherein the enzymolysis temperature is 40-55 ℃, the enzymolysis pH is 3.4-6.5, and the enzymolysis time is 2-8 h;
step three: fermenting, namely adding glucose, nicotinamide, magnesium sulfate and dipotassium hydrogen phosphate into the mixed liquor obtained by the enzymolysis in the second step respectively, stirring for dissolving, heating, carrying out enzyme deactivation and sterilization, cooling the mixed liquor to 20-32 ℃, inoculating a starter saccharomyces cerevisiae with the inoculation amount of 0.05-0.1%, maintaining the temperature at 20-32 ℃, fermenting for 3-7 days, intermittently stirring in the fermentation process, stirring for 20-30 minutes every 12 hours, wherein the stirring mode is mechanical stirring or stirring by introducing sterile compressed air;
step four: post-treatment, namely sterilizing, cooling, centrifugally separating the fermentation liquor obtained in the third step, collecting filtrate, concentrating the filtrate at low temperature under reduced pressure until the relative density of the extract is 1.01-1.05, and freeze-drying the extract in vacuum to obtain the broccoli extract with high NMN content, wherein the NMN content is more than 5.0% (w/w), and the broccoli extract is used for anti-aging functional food; wherein the sterilization temperature is controlled at 60-90 ℃, and the sterilization time is 10-40 minutes; wherein the concentration temperature is 30-50 deg.C and vacuum degree is below 80 Pa;
300-600 parts of broccoli, 100-300 parts of red date extract concentrated solution containing adenosine cyclophosphate, 100-300 parts of ginseng extract concentrated solution containing ATP and 200-500 parts of water in the first step; in the second step, the addition amount of the pectinase is 0.01-0.07%; wherein the addition amount of 1 ten thousand U/g of cellulase is 0.05-0.15%; the fermentation strain obtained in the third step is obtained by separating and screening fresh broccoli, is used as an original strain, is subjected to slant activation culture, liquid expansion culture and Ka's tank amplification culture, fermentation broth is subjected to centrifugal separation after fermentation is finished to obtain saccharomyces cerevisiae bacterial sludge, and is inoculated to the broccoli enzymolysis mixed liquor for fermentation; or directly adding the yeast liquid in the Kaschin tank into the broccoli enzymolysis mixed liquid for fermentation without centrifugal separation.
2. The method for preparing broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) according to claim 1, wherein the enzymolysis temperature in the second step is 48-52 ℃.
3. The method for preparing broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) according to claim 1, wherein the pH value of enzymolysis in the second step is 4.0-5.0.
4. The method for preparing broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) according to claim 1, wherein the enzymolysis time in the second step is 4-5h.
5. The method for preparing broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) according to claim 1, wherein the amount of pectinase added in the second step is 0.03% -0.05%.
6. The method for preparing broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) according to claim 1, wherein the cellulase is added in an amount of 0.1-0.12% per 1 million U/g in the second step.
7. The method for preparing broccoli extract with high content of beta-Nicotinamide Mononucleotide (NMN) according to claim 1, wherein glucose is 1-8%, nicotinamide is 0.1-1.0%, magnesium sulfate is 0.01-0.03%, and dipotassium phosphate is 0.2-0.3% in the step three.
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