Polygonatum sibiricum fermentation product as well as preparation method and application thereof

Abstract

The invention provides a preparation method of a polygonatum sibiricum fermentation product, which comprises the following steps: A) pretreating a polygonatum sibiricum raw material, and mixing with water to obtain polygonatum sibiricum slurry; b) heating the polygonatum sibiricum slurry, mixing the heated polygonatum sibiricum slurry with a nitrogen source and a phosphorus source, and performing primary sterilization to obtain sterilized polygonatum sibiricum slurry; c) inoculating the sterilized rhizoma polygonati slurry with saccharomycetes, and fermenting to obtain fermentation liquor; d) adding a carbon source into the fermentation liquor, performing secondary sterilization, inoculating lactic acid bacteria, performing secondary fermentation, and performing third sterilization to obtain secondary fermentation liquor; and E) adding a compound enzyme into the secondary fermentation liquid for enzymolysis, then sterilizing for four times, and centrifuging to obtain the polygonatum sibiricum fermentation product. 3000 Da), a small molecule polypeptide (lt; the polygonatum kingianum fermentation product prepared by the method disclosed by the invention has the advantages that the conversion of polygonatum kingianum saponin can be promoted, the contents of diosgenin and trilliin are increased and calibrated, and the polygonatum kingianum fermentation product has the effects of improving the reaction capability and the exercise capability.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation, and in particular to a polygonatum fermentation product and a preparation method and application thereof. Background Art

[0002] Polygonatum sibiricum is the dried rhizome of Polygonatum kingianum Coll.et Hemsl., Polygonatum sibiricum Red. or Polygonatum cyrtonema Hua, which belongs to the Liliaceae family. It has the effects of nourishing qi and yin, strengthening the spleen, moistening the lungs and benefiting the kidneys. It is the first batch of food ingredients to be included in the catalog of medicine and food homology, rich in important active substances such as polysaccharides, flavonoids, polyphenols, anthraquinones, steroidal saponins, alkaloids and essential amino acids.

[0003] Raw Polygonatum has a numb tongue feeling and irritates the throat mucosa. It is not suitable for direct consumption. The skin that comes into contact with raw Polygonatum or juice will feel itchy. Therefore, it is necessary to use steaming, wine making, auxiliary material making (turmeric, black bean juice, honey, etc.) and other processing methods to improve the taste and efficacy. Microbial fermentation technology has a place in the processing of Polygonatum due to its mild reaction conditions, stable process and easy quality control. However, the existing Polygonatum fermentation processing technology (enzymatic hydrolysis followed by fermentation or just fermentation) makes it difficult for small molecular active substances to be converted into intracellular substances or macromolecular substances by microbial metabolism, resulting in low contents of small molecular polysaccharides (<3000Da), small molecular polypeptides (<1000Da), diosgenin and trillium glycosides in Polygonatum fermentation products (Polygonatum saponins are metabolized into diosgenin and trillium glycosides).

[0004] Responsiveness and athleticism are two different physiological traits of the human body that play an important role in daily life, work, and sports. Responsiveness generally refers to an individual's ability to respond quickly and appropriately to external stimuli. This includes the perception of changes in the outside world, the speed of processing information, and the ability to react based on this information. The quality of responsiveness can affect an individual's performance in emergency situations, such as encountering an emergency while driving a car, or responding quickly to an opponent's actions in a sports game. Athleticism refers to an individual's ability to perform various physical activities, including strength, speed, endurance, coordination, agility, etc. These abilities directly affect an individual's performance in sports games and their ability to complete various tasks in daily life.

[0005] Therefore, it is very necessary to provide a product that can improve reaction ability and movement ability. Summary of the invention

[0006] In view of this, the present invention provides a method for preparing a polygonatum fermentation product. The polygonatum fermentation product prepared by the present invention can better improve reaction ability and motor ability.

[0007] The present invention provides a method for preparing a fermented product of polygonatum, comprising the following steps:

[0008] A) the raw material of polygonatum sibiricum is mixed with water after pretreatment to obtain polygonatum sibiricum slurry;

[0009] B) the polygonatum slurry is heated and mixed with a nitrogen source and a phosphorus source, and then sterilized once to obtain a sterilized polygonatum slurry;

[0010] C) inoculating the sterilized polygonatum slurry with yeast, fermenting it, and obtaining a fermentation liquid;

[0011] D) adding a carbon source to the fermentation broth, sterilizing it for a second time, then inoculating lactic acid bacteria, fermenting it for a second time, and sterilizing it for a third time to obtain a second fermentation broth;

[0012] E) Add compound enzyme to the secondary fermentation broth for enzymatic hydrolysis, and then sterilize and centrifuge for four times to obtain the broth.

[0013] Preferably, the raw material of polygonatum sibiricum in step A) is dried polygonatum sibiricum, fresh polygonatum sibiricum or polygonatum sibiricum decoction pieces; the raw material of polygonatum sibiricum is pretreated by one of low temperature pulverization, colloid mill wet pulverization or steam explosion;

[0014] The mass ratio of the polygonatum raw material to water is 1: (5-50).

[0015] Preferably, the heating in step B) is heating in a water bath at 90-100°C for 2-6 hours;

[0016] The nitrogen source is added in an amount of 0.5wt% to 5.0wt% of the polygonatum slurry; the phosphorus source is added in an amount of 0.05wt% to 0.1wt% of the polygonatum slurry;

[0017] The nitrogen source is selected from one of yeast extract powder, ammonium sulfate, ammonium chloride, casein, soy protein isolate and peptone; the phosphorus source is selected from one of potassium dihydrogen phosphate, ammonium dihydrogen phosphate and sodium dihydrogen phosphate;

[0018] The primary sterilization temperature is 60-121°C and the time is 15-240 min.

[0019] Preferably, the yeast in step C) includes one or two of Saccharomyces cerevisiae, Candida, Kluyveromyces marxianus and Hansenula anomala, and any two of the above four strains are mixed for fermentation at a mass ratio of 1:1;

[0020] The inoculation amount of the yeast is 1g:100 ml~1g:2000 ml;

[0021] The fermentation parameters include: ventilation volume 0~100 L / min, temperature 25~45 ℃, time 18~26 h, rotation speed 10~100 r / min, and tank pressure 0~0.08 Mpa.

[0022] Preferably, the amount of the carbon source added in step D) is 1.0wt%~5.0wt% of the fermentation broth; the carbon source includes one or more of glucose, oligofructose, sucrose and maltose; and the parameters of the secondary sterilization include sterilization at 60~90℃ for 30~90min.

[0023] Preferably, in step D), the lactic acid bacteria include one or two of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus and Lactobacillus rhamnosus, and any two of the above four strains are mixed and fermented in a mass ratio of 1:1; the inoculation amount of the lactic acid bacteria is 1g:100 ml~1g:2000 ml;

[0024] The parameters of secondary fermentation include: temperature 25~45 ℃, time 45~60 h, speed 10~100 r / min, tank pressure 0~0.08 Mpa;

[0025] The parameters of the tertiary sterilization include sterilization at 60~100℃ for 15~60 min.

[0026] Preferably, in step E), the amount of the compound enzyme added is 0.05-0.50wt%; the compound enzyme includes glycoside hydrolase and protease; the mass ratio of the glycoside hydrolase to the protease is 9:5-5:9; the glycoside hydrolase is selected from cellulase, mannanase, hemicellulase or β-D-glucosidase; the protease is selected from papain, acid protease, neutral protease, flavor protease or pepsin; the enzymolysis parameters include enzymolysis at pH 3.0-7.0 and temperature 30-60°C for 30-180min;

[0027] The four sterilization parameters include sterilization at 60-100°C for 15-60 min.

[0028] The present invention provides a polygonatum fermentation product, which is prepared by the preparation method described in any one of the above technical solutions.

[0029] The present invention provides the use of a fermented product of polygonatum sibiricum prepared by the preparation method described in the above technical scheme in the preparation of a product for improving reaction ability and motor ability.

[0030] The present invention provides a polygonatum product, comprising the polygonatum fermentation product described in the above technical solution.

[0031] Compared with the prior art, the present invention provides a method for preparing a fermented product of polygonatum, comprising the following steps: A) the polygonatum raw material is pretreated and mixed with water to obtain a polygonatum slurry; B) the polygonatum slurry is heated and mixed with a nitrogen source and a phosphorus source, and then sterilized once to obtain a sterilized polygonatum slurry; C) the sterilized polygonatum slurry is inoculated with yeast and fermented to obtain a fermentation liquid; D) a carbon source is added to the fermentation liquid and then sterilized twice, and then lactic acid bacteria are inoculated, fermented twice, and sterilized three times to obtain a secondary fermentation liquid; E) a composite enzyme is added to the secondary fermentation liquid for enzymolysis, and then sterilized four times and centrifuged to obtain. The method of the present invention can increase the content of small molecular polysaccharides (<3000Da) and small molecular polypeptides (<1000Da) in the polygonatum fermentation product; it can promote the conversion of polygonatum saponin, increase the content of diosgenin and trillium glycosides and calibrate them; the product fermented by the polygonatum of the present invention has the effect of improving reaction ability and motor ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Effects of the test substances on the movement frequency and activity level of zebrafish. DETAILED DESCRIPTION

[0033] The present invention provides a fermented product of Polygonatum sibiricum and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and they all fall within the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0034] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0035] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0036] The content of small molecule active substances in existing Polygonatum sibiricum fermented products is low, and there are few reports on the improvement of reaction ability and motor ability. The present invention uses physical pretreatment, microbial fermentation and bioenzyme hydrolysis to avoid the absorption of small molecules by microorganisms, and the fermentation strain can also be hydrolyzed and utilized. The obtained Polygonatum sibiricum fermented product has a high content of small molecule active substances and has been proven to have good auxiliary improvement of reaction ability and motor ability (assisting improvement of memory function).

[0037] The polygonatum fermentation product prepared by the present invention has a small molecule polysaccharide (<3000Da) content of 15-20%, a small molecule polypeptide (<1000Da) content of 5-10%, a diosgenin content of 1.5-2%, and a trillium glycoside content of 0.5-1%, and can better improve reaction ability and motor ability (assisting in improving memory function).

[0038] The present invention provides a method for preparing a fermented product of polygonatum, comprising the following steps:

[0039] A) the raw material of polygonatum sibiricum is mixed with water after pretreatment to obtain polygonatum sibiricum slurry;

[0040] B) the polygonatum slurry is heated and mixed with a nitrogen source and a phosphorus source, and then sterilized once to obtain a sterilized polygonatum slurry;

[0041] C) inoculating the sterilized polygonatum slurry with yeast, fermenting it, and obtaining a fermentation liquid;

[0042] D) adding a carbon source to the fermentation broth, sterilizing it for a second time, then inoculating lactic acid bacteria, fermenting it for a second time, and sterilizing it for a third time to obtain a second fermentation broth;

[0043] E) Add compound enzyme to the secondary fermentation broth for enzymatic hydrolysis, and then sterilize and centrifuge for four times to obtain the broth.

[0044] The method for preparing the polygonatum sibiricum fermentation product provided by the invention firstly pretreats the polygonatum sibiricum raw material.

[0045] The raw material of polygonatum sibiricum described in the present invention is dried polygonatum sibiricum, fresh polygonatum sibiricum or polygonatum sibiricum decoction pieces; preferably polygonatum sibiricum decoction pieces.

[0046] Specifically, the pretreatment of the polygonatum raw material includes: low temperature crushing, colloid mill wet crushing or steam explosion;

[0047] The crushed polygonatum sibiricum is mixed with water to obtain polygonatum sibiricum slurry.

[0048] The mass ratio of the polygonatum raw material to water is 1:(5-50), and can be 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, or any value between the above two.

[0049] The polygonatum slurry is heated; the heating is preferably water bath heating; the heating is 90-100°C water bath heating for 2-6h; it can be 90-100°C water bath heating for 2h, 3h, 4h, 4h, 5h or 6h.

[0050] A nitrogen source and a phosphorus source are added to the above-mentioned hot water-treated polygonatum slurry.

[0051] The nitrogen source of the invention is selected from one of yeast extract powder, ammonium sulfate, ammonium chloride, casein, soybean protein isolate and peptone; the phosphorus source is selected from one of potassium dihydrogen phosphate, ammonium dihydrogen phosphate and sodium dihydrogen phosphate.

[0052] According to the present invention, the added amount of the nitrogen source is 0.5wt%~5.0wt% of the polygonatum slurry; specifically, it can be 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt% or 5wt%; or any point value between any two of the above.

[0053] The phosphorus source is added in an amount of 0.05wt% to 0.1wt% of the polygonatum slurry, and can be specifically 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt% or 0.1wt% or any value between the above two.

[0054] After mixing, sterilize once to obtain sterilized polygonatum slurry; the temperature of the primary sterilization is 60-121°C, and the time is 15-240 min; preferably, the temperature of the primary sterilization is 60-100°C, and the time is 15-100 min; more preferably, the temperature of the primary sterilization is 60-90°C, and the time is 15-90 min.

[0055] The sterilized polygonatum slurry is inoculated with yeast for fermentation to obtain a fermentation liquid. The yeast includes but is not limited to one or a combination of Saccharomyces cerevisiae, Candida, Kluyveromyces marxianus, and Hansenula anomala.

[0056] The inoculation volume is 1g:100 ml~1g:2000 ml, specifically 1g:100 ml, 1g:200 ml, 1g:300 ml, 1g:400 ml, 1g:500 ml, 1g:600 ml, 1g:800 ml, 1g:1500 ml or 1g:2000 ml.

[0057] The mass ratio of the two mixed strains is 1:1.

[0058] Fermentation parameters include: aeration rate of 0 to 100 L / min, preferably 10 to 90 L / min, more preferably 15 to 80 L / min, and particularly preferably 20 to 70 L / min.

[0059] In some embodiments, the fermentation temperature is 25-45° C., the fermentation time is 18-26 h, the rotation speed is 10-100 r / min, and the tank pressure is 0-0.08 Mpa.

[0060] In some embodiments, the fermentation temperature is 27-42°C, the fermentation time is 20-25 h, the rotation speed is 20-90 r / min, and the tank pressure is 0.02-0.08 Mpa;

[0061] In some embodiments, the fermentation temperature is 30-40° C., the fermentation time is 22-24 h, the rotation speed is 40-70 r / min, and the tank pressure is 0.04-0.06 Mpa.

[0062] A carbon source is added to the fermentation liquid; the amount of the carbon source added is 1.0wt% to 5.0wt% of the fermentation liquid; specifically, it can be 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt% or 5wt%; or any point value between any two of the above. The carbon source includes one or more of glucose, oligofructose, sucrose and maltose.

[0063] After adding the carbon source, secondary sterilization is performed, and the parameters of the secondary sterilization include sterilization at 60-90°C for 30-90 min; preferably, sterilization at 70-90°C for 30-80 min; more preferably, sterilization at 80-90°C for 30-60 min.

[0064] After secondary sterilization, lactic acid bacteria are inoculated for secondary fermentation. The lactic acid bacteria include one or two of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, and Lactobacillus rhamnosus, and the mass ratio of the two bacteria for mixed fermentation is 1:1; the inoculation amount of the lactic acid bacteria is 1g:100 ml~1g:2000 ml;

[0065] The inoculation volume is 1g:100 ml~1g:2000 ml, specifically 1g:100 ml, 1g:200 ml, 1g:300 ml, 1g:400 ml, 1g:500 ml, 1g:600 ml, 1g:800 ml, 1g:1500 ml or 1g:2000 ml.

[0066] The mass ratio of the two mixed strains is 1:1.

[0067] The parameters of the secondary fermentation of the present invention include: temperature 25-45°C, time 45-60 h, rotation speed 10-200 r / min, tank pressure 0-0.08 Mpa;

[0068] In some embodiments, the secondary fermentation temperature is 27-42°C, the time is 46-58 h, the rotation speed is 20-90 r / min, and the tank pressure is 0.02-0.08 Mpa;

[0069] In some embodiments, the secondary fermentation temperature is 30-40° C., the time is 48-55 h, the rotation speed is 40-70 r / min, and the tank pressure is 0.04-0.06 Mpa.

[0070] After the secondary fermentation, the secondary fermentation liquid is obtained by three sterilizations. The parameters of the three sterilizations of the present invention include sterilization at 60-100°C for 15-60 min; preferably, sterilization at 70-90°C for 20-50 min; more preferably, sterilization at 80-90°C for 30-40 min.

[0071] The composite yeast fermentation and composite lactic acid bacteria fermentation of the present invention have a synergistic effect. The combination of the specific fermentation strains can metabolize and produce more abundant biological enzymes, forming a synergistic effect, thereby improving the conversion of active ingredients and ultimately improving the reaction ability and motor ability.

[0072] After cooling the fermented polygonatum sibiricum liquid, a composite enzyme is added for enzymolysis. The composite enzyme of the present invention is added in an amount of 0.05-0.50wt%; 0.05wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, or 0.5wt%; or any value between any two of the above.

[0073] The composite enzyme of the present invention comprises glycoside hydrolase and protease; the mass ratio of the glycoside hydrolase to the protease is 9:5 to 5:9; specifically, it can be 9:5, 8:6, 7:7, 6:8, 5:9,

[0074] According to the present invention, the glycoside hydrolase is selected from cellulase, mannanase, hemicellulase or β-D-glucosidase; the protease is selected from papain, acid protease, neutral protease, flavor protease or pepsin. The enzymolysis parameters include enzymolysis at pH 3.0-7.0 and temperature 30-60°C for 30-180 min.

[0075] In some embodiments, the enzymatic hydrolysis parameters include enzymatic hydrolysis at pH 4.0-6.0 and temperature 40-58° C. for 50-160 min.

[0076] In some embodiments, the enzymatic hydrolysis parameters include enzymatic hydrolysis at pH 4.5-5.8 and temperature 45-57° C. for 90-140 min.

[0077] In some embodiments, the enzymatic hydrolysis parameters include treatment at pH 5.5 and temperature 55° C. for 120 min.

[0078] The four sterilization parameters of the present invention include sterilization at 60-100°C for 15-60 min. Preferably, sterilization at 70-90°C for 20-50 min; more preferably, sterilization at 80-90°C for 30-40 min.

[0079] The sterilized fermented Polygonatum sibiricum hydrolysate is centrifuged and filtered until it is clarified, and the obtained supernatant is used for later use. The present invention does not limit the specific operation of the centrifugal filtration, which is well known to those skilled in the art.

[0080] The present invention uses physical pretreatment, microbial fermentation and the synergistic effect of the above-mentioned specific composite bio-enzyme hydrolysis to avoid small-molecule substances being absorbed by microorganisms, and can also hydrolyze and utilize the fermentation strains, so that the obtained polygonatum fermentation product has a high content of small-molecule active substances.

[0081] The supernatant obtained above is used to prepare the desired extract or powder. The present invention does not limit the specific operation of preparing the extract or powder, which is well known to those skilled in the art.

[0082] The present invention provides a polygonatum fermentation product, which is prepared by the preparation method described in any one of the above technical solutions.

[0083] The preparation method of the present invention has been clearly described above and will not be repeated here.

[0084] The present invention provides the use of a fermented product of polygonatum sibiricum prepared by the preparation method described in the above technical scheme in the preparation of a product for improving reaction ability and motor ability.

[0085] The polygonatum sibiricum fermentation product prepared by the process of the invention has a small molecule polysaccharide (<3000Da) content of 15-24%, a small molecule polypeptide (<1000Da) content of 5-10%, a diosgenin content of 1.5-2%, and a trillium glycoside content of 0.5-1%.

[0086] The inventors found that the Polygonatum sibiricum fermentation product prepared by the present invention significantly improved the movement frequency and activity of zebrafish, which was significantly higher than the control group (P<0.05). This is largely related to the significant increase in the molecular polysaccharides (<3000Da), small molecular polypeptides (<1000Da), diosgenin and trillium saponin in the Polygonatum sibiricum fermentation product prepared in the example.

[0087] The present invention provides a polygonatum product, comprising the polygonatum fermentation product described in the above technical solution.

[0088] The invention provides a preparation method of a polygonatum fermentation product, comprising the following steps: A) pre-treating a polygonatum raw material and mixing it with water to obtain a polygonatum slurry; B) heating the polygonatum slurry and mixing it with a nitrogen source and a phosphorus source, and then sterilizing it once to obtain a sterilized polygonatum slurry; C) inoculating yeast into the sterilized polygonatum slurry and fermenting it to obtain a fermentation liquid; D) adding a carbon source to the fermentation liquid and then sterilizing it twice, and then inoculating lactic acid bacteria, fermenting it twice, and sterilizing it three times to obtain a secondary fermentation liquid; E) adding a composite enzyme to the secondary fermentation liquid for enzymolysis, and then sterilizing it four times and centrifuging it to obtain the secondary fermentation liquid.

[0089] It should be understood that in various embodiments of the present application, the size of the sequence number of each process does not mean the order of execution, some or all of the steps can be executed in parallel or successively, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiment of the present application. The numerical ranges and parameters involved in the present invention have presented the relevant numerical values ​​in the specific embodiments as accurately as possible. However, any numerical value inevitably contains the standard deviation due to individual test methods in nature. Therefore, unless otherwise clearly stated, it should be understood that all numerical ranges or specific data used in the present disclosure can have certain reasonable deviations within a certain range, for example: within 1% or 0.5%.

[0090] Some cases are recorded in the embodiments and comparative examples of the present invention, wherein the embodiments show certain implementation modes of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0091] In order to further illustrate the present invention, a fermented product of Polygonatum sibiricum provided by the present invention and a preparation method and application thereof are described in detail below in conjunction with embodiments.

[0092] Example 1

[0093] A fermented product of polygonatum sibiricum that helps improve reaction ability and exercise ability comprises the following steps:

[0094] (1) grinding the raw material of Polygonatum sibiricum by a colloid mill;

[0095] (2) mixing the crushed polygonatum sibiricum with water in a ratio of 1:10 to obtain a polygonatum slurry;

[0096] (3) treating the polygonatum slurry obtained above in hot water at 95°C for 2 h;

[0097] (4) Add 0.5% yeast extract powder and 0.05% potassium dihydrogen phosphate (5) to the hot water-treated polygonatum slurry, mix well, and sterilize at 121°C for 15 min;

[0098] (5) The sterilized Polygonatum slurry was inoculated with Saccharomyces cerevisiae at an inoculation volume of 1 g:100 ml, aeration volume of 20 L / min, temperature of 30 °C, time of 22 h, rotation speed of 50 r / min, and tank pressure of 0.05 MPa;

[0099] (6) After fermentation, add 1.0% sucrose to the fermentation liquid, mix well, and sterilize at 90°C for 30 minutes;

[0100] (7) The fermentation liquid was inoculated with Lactobacillus plantarum at an inoculation amount of 1 g:100 ml. The fermentation conditions were as follows: temperature 37 °C, time 48 h, rotation speed 150 r / min, tank pressure 0.05 MPa. After the fermentation was completed, the liquid was sterilized at 90 °C for 30 min to obtain the Polygonatum sibiricum fermentation liquid.

[0101] (8) After cooling the above-mentioned Polygonatum sibiricum fermentation liquid, 0.05% of a composite enzyme, β-D-glucosidase and papain in a ratio of 9:5, was added, and the mixture was treated at pH 5.5 and temperature 55°C for 120 min to obtain a fermented Polygonatum sibiricum enzymatic hydrolyzate.

[0102] (9) The fermented Polygonatum sibiricum hydrolysate was sterilized at 90°C for 30 min, centrifuged and filtered until clear, and the obtained supernatant was freeze-dried in vacuum.

[0103] Example 2

[0104] A fermented product of polygonatum sibiricum which is helpful to improve reaction ability and exercise ability comprises the following steps:

[0105] The difference from Example 1 is that yeast fermentation uses Saccharomyces cerevisiae and Kluyveromyces marxianus in a mass ratio of 1:1, and the other steps are consistent with Example 1.

[0106] Example 3

[0107] A fermented product of polygonatum sibiricum which is helpful to improve reaction ability and exercise ability comprises the following steps:

[0108] The difference from Example 1 is that the lactic acid bacteria fermentation uses a composite fermentation of Lactobacillus plantarum and Lactobacillus acidophilus in a mass ratio of 1:1, and the other steps are consistent with Example 1.

[0109] Example 4

[0110] A fermented product of polygonatum sibiricum which is helpful to improve reaction ability and exercise ability comprises the following steps:

[0111] The difference from Example 1 is that the yeast fermentation uses Saccharomyces cerevisiae and Kluyveromyces marxianus in a mass ratio of 1:1, and the lactic acid bacteria fermentation uses Lactobacillus plantarum and Lactobacillus acidophilus in a mass ratio of 1:1. The other steps are consistent with Example 1.

[0112] Example 5

[0113] A fermented product of polygonatum sibiricum which is helpful to improve reaction ability and exercise ability comprises the following steps:

[0114] The difference from Example 1 is that the enzymatic hydrolysis uses a combined enzymatic hydrolysis of hemicellulase and neutral protease in a mass ratio of 1:1, and the other steps are consistent with Example 1.

[0115] Comparative Example 1

[0116] A fermented product of polygonatum sibiricum which is helpful to improve reaction ability and exercise ability comprises the following steps:

[0117] The difference from Example 1 is that the processed raw materials are polygonatum decoction pieces, and the other steps are consistent with Example 1.

[0118] Comparative Example 2

[0119] A fermented product of polygonatum sibiricum which is helpful to improve reaction ability and exercise ability comprises the following steps:

[0120] The difference from Example 1 is that the polygonatum slurry is only subjected to microbial fermentation processing, and the other steps are consistent with Example 1.

[0121] Comparative Example 3

[0122] A fermented product of polygonatum sibiricum which is helpful to improve reaction ability and exercise ability comprises the following steps:

[0123] The difference from Example 1 is that the polygonatum slurry is only subjected to compound enzyme hydrolysis, and the other steps are consistent with Example 1.

[0124] Comparative Example 4

[0125] A fermented product of polygonatum sibiricum which is helpful to improve reaction ability and exercise ability comprises the following steps:

[0126] The difference from Example 1 is that the Polygonatum sibiricum slurry is firstly subjected to enzymatic hydrolysis and then fermented, and the other steps are consistent with Example 1.

[0127] Test Example 1

[0128] The fermented products of Polygonatum cyrtonema prepared in the above examples and comparative examples were tested, and the results are shown in Table 1

[0129] Table 1 Test results of indicators in the examples and comparative examples

[0130] Sample name Small molecule polysaccharides (<3000Da)% Small molecule peptides (<1000Da)% Diosgenin% Trillium glycosides% Example 1 20.23±0.54** 4.92±0.33** 1.44±0.11** 0.65±0.11** Example 2 23.98±0.57**# 5.31±0.23**# 1.50±0.09**# 0.73±0.09**# Example 3 23.34±0.54**# 6.61±0.46**## 1.55±0.23**# 0.77±0.08**## Example 4 27.44±0.47**## 7.21±0.53**## 1.79±0.17**## 0.83±0.12**## Example 5 21.84±0.56** 5.31±0.45** 1.48±0.08** 0.71±0.14**# Comparative Example 1 5.97±0.34 1.88±0.21 0.23±0.02 0.15±0.04 Comparative Example 2 2.47±0.41 1.44±0.13 0.27±0.03 0.19±0.07 Comparative Example 3 4.50±0.33 1.03±0.09 0.39±0.02 0.18±0.03 Comparative Example 4 7.33±0.29 0.17±0.03 0.24±0.03 0.12±0.01

[0131] Note: ** indicates that each group of the examples is compared with the highest value of the comparative example, P < 0.01; # and ## respectively indicate that Examples 2-5 are compared with Example 1, P < 0.05 and P < 0.01. In the following figure, ** has the same meaning as # and ##.

[0132] It can be concluded from Table 1 that the content of small molecule polysaccharides (<3000Da), small molecule polypeptides (<1000Da), diosgenin and trillium saponin in the embodiment is significantly higher than that in the comparative example (P<0.01), which prevents small molecule substances from being used by microorganisms to be converted into intracellular substances. At the same time, enzymatic hydrolysis after fermentation can further hydrolyze and convert microbial cells and macromolecule substances. Compared with Example 1, the content of small molecule polysaccharides (<3000Da), small molecule polypeptides (<1000Da), diosgenin and trillium saponin in the fermented products of Polygonatum cyrtonema prepared by using composite bacterial agents in Examples 2-5 is relatively high. In Example 4, the composite bacterial agent of Saccharomyces cerevisiae and Kluyveromyces marxianus is used for yeast fermentation, and the active ingredients are significantly increased when the composite bacterial agent of Lactobacillus plantarum and Lactobacillus acidophilus is used for lactic acid bacteria fermentation (P<0.01). The main reason is that the increase in fermentation strains can metabolize and produce more abundant biological enzymes, forming a synergistic effect, thereby improving the conversion of active ingredients. The fermentation product of Polygonatum sibiricum prepared by the above process has a small molecule polysaccharide (<3000Da) content of 15-24%, a small molecule polypeptide (<1000Da) content of 5-10%, a diosgenin content of 1.5-2%, and a trillium glycoside content of 0.5-1%.

[0133] Test Example 2

[0134] Evaluation of the effect of Polygonatum sibiricum fermentation products in improving reaction ability and motor ability (memory)

[0135] 1 Experimental steps

[0136] Select healthy zebrafish that are 5 days old after fertilization. Test process: zebrafish spawning → collect zebrafish eggs → select zebrafish eggs → normal zebrafish eggs → 5-day-old zebrafish eggs (test preparation←sample) → blank control / model control / test → culture at 28±1℃ for 48±1h → measure exercise volume and calculate exercise promotion rate.

[0137] 2 Test Grouping

[0138] The test requires setting up a blank control group (fish embryo culture medium, pH adjusted to 5.8), a model control group (aluminum chloride) and a test substance group.

[0139] (Aluminum chloride and the test substance). If an organic solvent is used as a solubilizing agent, the solvent concentration in each group of solutions must be the same.

[0140] Blank control group setting: 24 fish embryos were randomly selected and placed in a 96-well plate, with each well containing one fish embryo and 0.2 mL of fish embryo culture medium.

[0141] Model control group setting: 24 fish embryos were randomly selected and placed in a 96-well plate, with each well containing one fish embryo and 0.2 mL of aluminum chloride solution (50 µM).

[0142] Test group setting: 24 fish embryos were randomly selected and placed in a 96-well plate, with each well containing one fish embryo and 0.2 mL of fish embryo culture medium containing aluminum chloride (50 µM) and the test substance (0.43 g / L).

[0143] Then place them in a constant temperature box at 28℃±1℃ for 48h±1h, and calculate the survival rate of fish embryos.

[0144] 3. Record your activity level

[0145] The 96-well plate was placed in a zebrafish movement recorder. After adapting to the white light environment for 15 minutes, 5 minutes of darkness was started, followed by 5 minutes of white light and 5 minutes of darkness. The activity of each zebrafish in the dark environment was recorded using infrared tracking software.

[0146] 4 Data and result calculation

[0147] The movement trajectory of zebrafish embryos can be recorded by infrared and the trajectory can be measured and analyzed.

[0148] Measure the activity of each fish and calculate the activity promotion rate:

[0149] Where: S—mean value of zebrafish activity in the test substance treatment group;

[0150] M—mean value of zebrafish activity in the model control group;

[0151] B—The average activity level of zebrafish in the blank control group.

[0152] The mean and standard error of each test group were calculated, and the statistical results were expressed as mean ± standard error. The data were analyzed by variance analysis using statistical software, and the activity between the test group and the model control group was tested by two-tailed T test to obtain the p value. p<0.05 indicated a significant difference.

[0153] Figure 1 Effects of the test substance on the movement frequency and activity of zebrafish. Figure 1It can be seen that the fermented product of Polygonatum cyrtonema prepared in the example has a significant improvement on the movement frequency and activity of zebrafish, which is significantly higher than the control example (P<0.05). This is closely related to the significant increase in the molecular polysaccharide (<3000Da), small molecular polypeptide (<1000Da), diosgenin and trillium saponin in the fermented product of Polygonatum cyrtonema prepared in the example. In particular, in Examples 2, 3, and 4, the main components of the fermented product of Polygonatum cyrtonema increased due to the more abundant enzyme system of the synergistic fermentation metabolism of the mixed bacteria, so that the movement frequency and activity of the zebrafish in the corresponding experimental group increased significantly (P<0.05).

[0154] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a fermented product of Polygonatum sibiricum, characterized in that: The steps include: A) the raw material of polygonatum sibiricum is mixed with water after pretreatment to obtain polygonatum sibiricum slurry; B) the polygonatum slurry is heated and mixed with a nitrogen source and a phosphorus source, and then sterilized once to obtain a sterilized polygonatum slurry; C) inoculating the sterilized polygonatum slurry with yeast, fermenting it, and obtaining a fermentation liquid; D) adding a carbon source to the fermentation broth, sterilizing it for a second time, then inoculating lactic acid bacteria, fermenting it for a second time, and sterilizing it for a third time to obtain a second fermentation broth; E) Add compound enzyme to the secondary fermentation broth for enzymatic hydrolysis, and then sterilize and centrifuge for four times to obtain the broth.

2. The preparation method according to claim 1, characterized in that: In step A), the raw material of polygonatum sibiricum is dried polygonatum sibiricum, fresh polygonatum sibiricum or polygonatum sibiricum decoction pieces; the raw material of polygonatum sibiricum is pretreated by one of low temperature crushing, colloid mill wet crushing or steam explosion; The mass ratio of the polygonatum raw material to water is 1: (5-50).

3. The preparation method according to claim 1, characterized in that: The heating in step B) is heating in a water bath at 90-100°C for 2-6 hours; The nitrogen source is added in an amount of 0.5wt% to 5.0wt% of the polygonatum slurry; the phosphorus source is added in an amount of 0.05wt% to 0.1wt% of the polygonatum slurry; The nitrogen source is selected from one of yeast extract powder, ammonium sulfate, ammonium chloride, casein, soy protein isolate and peptone; the phosphorus source is selected from one of potassium dihydrogen phosphate, ammonium dihydrogen phosphate and sodium dihydrogen phosphate; The primary sterilization temperature is 60-121°C and the time is 15-240 min.

4. The preparation method according to claim 1, characterized in that: In step C), the yeast comprises one or two of Saccharomyces cerevisiae, Candida, Kluyveromyces marxianus and Hansenula anomala, any two of the above four strains are mixed for fermentation at a mass ratio of 1:1, and the inoculation amount of the yeast is 1g:100 ml~1g:2000 ml; The fermentation parameters include: ventilation volume 0~100 L / min, temperature 25~45 ℃, time 18~26 h, rotation speed 10~100 r / min, and tank pressure 0~0.08 Mpa.

5. The preparation method according to claim 1, characterized in that: In step D), the amount of the carbon source added is 1.0 wt% to 5.0 wt% of the fermentation broth; the carbon source includes one or more of glucose, oligofructose, sucrose and maltose; and the parameters of the secondary sterilization include sterilization at 60 to 90 °C for 30 to 90 min.

6. The preparation method according to claim 1, characterized in that: Step D) the lactic acid bacteria include one or two of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus and Lactobacillus rhamnosus, and any two of the above four strains are mixed and fermented in a mass ratio of 1:1; the inoculation amount of the lactic acid bacteria is 1g:100 ml~1g:2000 ml; The parameters of secondary fermentation include: temperature 25~45 ℃, time 45~60 h, speed 10~200 r / min, tank pressure 0~0.08Mpa; The parameters of the tertiary sterilization include sterilization at 60~100℃ for 15~60 min.

7. The preparation method according to claim 1, characterized in that: Step E) the compound enzyme is added in an amount of 0.05-0.50 wt %; the compound enzyme comprises glycoside hydrolase and protease; the mass ratio of the glycoside hydrolase to the protease is 9:5-5:9; the glycoside hydrolase is selected from cellulase, mannanase, hemicellulase or β-D-glucosidase; the protease is selected from papain, acid protease, neutral protease, flavor protease or pepsin; the enzymolysis parameters include enzymolysis at pH 3.0-7.0 and temperature 30-60°C for 30-180 min; The four sterilization parameters include sterilization at 60-100°C for 15-60 min.

8. A fermented product of Polygonatum cyrtonema, characterized in that: The method is prepared according to any one of claims 1 to 7.

9. Use of the polygonatum fermentation product prepared by the preparation method according to any one of claims 1 to 7 in the preparation of products for improving reaction ability and motor ability.

10. A polygonatum product, characterized in that: The invention comprises the polygonatum fermentation product as claimed in claim 8.

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