Rhizoma polygonati fermented beverage and preparation method thereof

Abstract

The invention provides a preparation method of a rhizoma polygonati fermented beverage, and belongs to the technical field of functional beverage development. The preparation method disclosed by the invention comprises the following steps: (1) adding papain and cellulase into polygonatum sibiricum pulp for primary enzymolysis, deactivating the enzyme after the enzymolysis is finished, and separating residues and supernate; (2) preparing the residues into a suspension, adding amylase for saccharifying enzymolysis, deactivating the enzyme after the enzymolysis is finished, and mixing with the primary enzymolysis supernatant to obtain a mixed enzymolysis solution; (3) inoculating the mixed enzymatic hydrolysate with saccharomyces cerevisiae and lactobacillus acidophilus complex microbial inoculants, and performing sealed fermentation to obtain rhizoma polygonati fermentation liquor; and (4) clarifying and filtering the polygonatum sibiricum fermentation liquor to obtain the polygonatum sibiricum fermentation beverage. The novel rhizoma polygonati fermented beverage is developed through secondary enzymolysis and strain combined fermentation, and the fermented beverage is high in effective component content, excellent in physiological activity, good in sensory quality and easy to accept by consumers.

Description

Technical Field

[0001] The invention belongs to the technical field of functional beverage development, and in particular relates to a polygonatum fermented beverage and a preparation method thereof. Background Art

[0002] With the acceleration of modern life and the continuous improvement of people's health awareness, the demand for beverages with health care functions is growing. Although traditional carbonated drinks, fruit juice drinks, etc. can satisfy the taste preferences of some consumers, they often lack deep health care effects.

[0003] As a traditional Chinese medicinal material, Polygonatum sibiricum has long been recorded in traditional Chinese medical books. It has many functions such as nourishing yin and moistening the lungs, tonifying the spleen and replenishing qi, benefiting the kidneys and replenishing essence. It is rich in Polygonatum sibiricum polysaccharides, steroidal saponins, flavonoids and other active ingredients.

[0004] However, products made from the original form of Polygonatum or simple processing methods have many limitations. As far as direct consumption is concerned, it tastes bitter and difficult to swallow, which seriously affects consumer acceptance. More importantly, the absorption and utilization rate of the effective ingredients in Polygonatum by the human body is relatively low. From the perspective of its structural characteristics, the cell wall structure of Polygonatum is extremely complex, mainly composed of cellulose, lignin and other components. These substances act like a solid barrier, strictly hindering the release and absorption of effective ingredients in the cell, such as polysaccharides and saponins. At the same time, the effective ingredients contained in Polygonatum that have not been specially treated are mostly in the form of macromolecules. This macromolecular structure makes it difficult for them to penetrate the human intestinal barrier, and thus cannot fully exert their due health benefits.

[0005] Therefore, it is necessary to provide consumers with a novel polygonatum functional beverage that is delicious, rich in nutritional and health benefits, and suitable for daily drinking. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a method for preparing a fermented polygonatum beverage, wherein the prepared beverage has a high content of polygonatum effective ingredients and excellent physiological activity.

[0007] The preparation method of the present invention comprises the following steps:

[0008] (1) adding papain and cellulase to polygonatum slurry for preliminary enzymolysis, inactivating the enzymes after the enzymolysis, and separating the residue and the supernatant; (2) making the residue into a suspension, adding amylase for saccharification enzymolysis, inactivating the enzymes after the enzymolysis, and mixing with the preliminary enzymolysis supernatant to obtain a mixed enzymolysis liquid; (3) inoculating the mixed enzymolysis liquid with a composite bacterial agent of saccharomyces cerevisiae and Lactobacillus acidophilus for sealed fermentation to obtain a polygonatum fermentation liquid; the number of live saccharomyces cerevisiae in the composite bacterial agent is 2×10 8 -2×10 9CFU / mL, the viable count of Lactobacillus acidophilus is 1×10 8 -1×10 9 CFU / mL; (4) the polygonatum fermented liquid is clarified and filtered to obtain a polygonatum fermented beverage.

[0009] Preferably, in the step (1), during the initial enzymatic hydrolysis, the enzymatic activity of papain is 1000-2000 U / mL, and the enzymatic activity of cellulase is 2000-4000 U / mL.

[0010] Preferably, in step (1), during the initial enzymatic hydrolysis, the temperature is 45-50°C and the time is 2-3 hours.

[0011] Preferably, in step (2), during saccharification and enzymatic hydrolysis, the enzymatic activity of amylase is 1500-2500 U / mL.

[0012] Preferably, in step (2), during the saccharification and enzymatic hydrolysis, the temperature is 55-60° C. and the time is 1.5-2.5 h.

[0013] Preferably, in steps (1) and (2), the temperature for inactivating the enzyme is 90-100° C. and the time is 8-10 min.

[0014] Preferably, in step (3), the inoculation amount of the composite bacterial agent is 1%-3% of the volume of the mixed enzymatic hydrolysate.

[0015] Preferably, in step (3), the temperature of the sealed fermentation is 32-37°C and the time is 48-72 hours.

[0016] The present invention also provides a fermented polygonatum beverage prepared by the preparation method; preferably, it further comprises one or more of fruit and vegetable juice, flavoring agent and stabilizer.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention uses papain and cellulase to perform preliminary enzymolysis on polygonatum sibiricum pulp, which can decompose proteins into small molecule peptides and amino acids, decompose cell wall cellulose to release effective components in cells, and then further release sugar substances through amylase saccharification and enzymolysis, thereby improving the utilization rate of effective components of polygonatum sibiricum. According to detection, the release amount of nutrients such as polysaccharides, steroidal saponins and flavonoids after enzymolysis is significantly increased. Subsequent fermentation with a composite bacterial agent of saccharomyces cerevisiae and lactobacillus acidophilus hydrolyzes steroidal saponins into secondary saponins or aglycones with higher activity and solubility, and converts flavonoids into glycoside forms, thereby improving their stability and absorption utilization rate, and the polygonatum sibiricum polysaccharide is fermented and degraded into oligosaccharides and monosaccharides, which are more easily absorbed by the human body to exert physiological activity.

[0019] During the fermentation process of the composite bacterial agent of the present invention, flavor substances such as organic acids, alcohols, and esters are produced, giving the beverage a unique flavor. At the same time, the early enzymatic hydrolysis improves the bitter taste of polygonatum, making the final beverage taste better and more acceptable to consumers. According to sensory evaluation, the beverage fermented with the composite bacterial agent scored high in terms of aroma, taste, etc. In addition, the bioactive substances produced by fermentation work synergistically with the original effective ingredients of polygonatum, enhancing the health functions of the beverage, such as significantly improving the antioxidant capacity. According to tests, the antioxidant capacity of the fermented beverage is greatly improved compared with the mixed enzymatic hydrolyzate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The polygonatum fermented beverage prepared by the present invention;

[0021] Figure 2 The effects of different enzyme activities and enzymatic hydrolysis times on saccharification enzymatic hydrolysis efficiency. DETAILED DESCRIPTION

[0022] The present invention provides a method for preparing a fermented polygonatum beverage, comprising the following steps:

[0023] (1) Papain and cellulase are added to the polygonatum slurry for preliminary enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzymes are inactivated and the residue and supernatant are separated.

[0024] In the present invention, the method for preparing the polygonatum slurry is preferably as follows: fresh, pest-free polygonatum rhizomes are selected, cleaned, steamed and dried nine times, cut into thin slices or small pieces, and distilled water is added at a mass ratio of 1:5-10, more preferably 1:8, and a high-speed blender is used to make a uniform and fine polygonatum slurry; more preferably, the pH value of the polygonatum slurry is adjusted to 5-6. The polygonatum rhizomes of the present invention are steamed and dried nine times, which can remove the irritation and toxic components of polygonatum, enhance the medicinal effect of polygonatum, and improve the taste of polygonatum.

[0025] In the present invention, the enzymatic activity of papain is preferably 1000-2000U / mL, more preferably 1200-1800U / mL, and more preferably 1500U / mL; the enzymatic activity of cellulase is preferably 2000-4000U / mL, more preferably 2400-3600U / mL, and more preferably 3000U / mL. The polygonatum slurry is subjected to preliminary enzymolysis by papain and cellulase. Papain can decompose the protein in polygonatum and hydrolyze it into small molecule peptides and amino acids, so that the nutrients in polygonatum are more easily absorbed by the human body, and also help to improve the taste and flavor of the beverage; cellulase can decompose the cellulose in the cell wall of polygonatum and rupture the cell wall, thereby releasing the effective components such as polysaccharides, saponins, flavonoids, etc. in the cell, and improving the availability of the effective components in polygonatum.

[0026] In the present invention, the preferred temperature for the initial enzymolysis is 45-50°C, more preferably 48°C; the preferred time for the initial enzymolysis is 2-3h, more preferably 2.5h; more preferably, slow stirring is performed during the initial enzymolysis to ensure sufficient contact between the enzyme and the polygonatum.

[0027] In the present invention, the residue and the supernatant are preferably separated by centrifugation. The centrifugal speed is further preferably 3000-4000 r / min, more preferably 3500 r / min. The centrifugal time is further preferably 8-12 min, more preferably 10 min.

[0028] (2) The initial enzymatic hydrolysis residue separated in step (1) is made into a suspension, and amylase is added to perform saccharification and enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated and the suspension is mixed with the initial enzymatic hydrolysis supernatant to obtain a mixed enzymatic hydrolysis solution.

[0029] In the present invention, the residue is preferably added to distilled water at a mass ratio of 1:3-5, and stirred again to form a uniform suspension, and the mass ratio is further preferably 1:4; and the pH value of the suspension is more preferably adjusted to 4.5-5.5.

[0030] In the present invention, the enzymatic activity of the amylase is preferably 1500-2500U / mL, more preferably 1700-2200U / mL, and more preferably 2000U / mL. Amylase is added to the residue after the preliminary enzymatic hydrolysis for saccharification and enzymatic hydrolysis, and the amylase can decompose the starch in the residue into glucose and other sugar substances. On the one hand, this can further improve the utilization rate of the effective ingredients in Polygonatum sibiricum, so that more sugar substances are released from the residue into the solution; on the other hand, the generated glucose and other sugar substances can provide sufficient carbon source for subsequent fermentation, which is beneficial to the growth and fermentation of microorganisms.

[0031] In the present invention, the saccharification and enzymatic hydrolysis temperature is preferably 55-60°C, more preferably 58°C; the saccharification and enzymatic hydrolysis time is preferably 1.5-2.5h, more preferably 2h; more preferably, slow stirring is performed during the saccharification and enzymatic hydrolysis to ensure sufficient contact between the enzyme and the residue.

[0032] In the present invention, the preferred enzyme inactivation temperature after the preliminary enzymatic hydrolysis and saccharification enzymatic hydrolysis is 90-100°C for 8-10 minutes. After the enzymatic hydrolysis is completed, the enzyme inactivation treatment is carried out in a timely manner to inactivate papain, cellulase and amylase, stop the enzymatic hydrolysis reaction, prevent the destruction of effective ingredients or the generation of bad flavors due to excessive enzymatic hydrolysis, and also provide a stable system for subsequent separation operations.

[0033] (3) inoculating a composite bacterial agent of saccharomyces cerevisiae and Lactobacillus acidophilus into the mixed enzymatic hydrolysate obtained in step (2) for sealed fermentation to obtain a polygonatum fermented liquid;

[0034] In the present invention, the number of live yeast cells in the composite bacterial agent is 2×108 -2×10 9 CFU / mL, the number of viable Lactobacillus acidophilus is 1×10 8 -1×10 9 CFU / mL, and the ratio of the number of live bacteria of Saccharomyces cerevisiae and Lactobacillus acidophilus in the composite bacterial agent is further preferably 2:1. The composite bacterial agent is inoculated in the mixed enzymatic hydrolysate for fermentation, and the composite bacterial agent can use the nutrients such as sugars and amino acids in the mixed enzymatic hydrolysate for metabolic activities. In this process, microorganisms will produce a variety of enzymes and metabolites to further decompose and transform the components in Polygonatum sibiricum, making the effective ingredients in Polygonatum sibiricum more stable and easier to absorb. Specifically:

[0035] The enzymes produced by Saccharomyces cerevisiae and Lactobacillus acidophilus can cause steroidal saponins to undergo hydrolysis reactions, gradually hydrolyzing their glycosyl parts to generate secondary saponins or aglycones, thus simplifying the structure of saponins. The hydrolyzed products have better biological activity and solubility, thereby improving their efficacy and stability in beverages. Saccharomyces cerevisiae and Lactobacillus acidophilus can also convert flavonoids in Polygonatum sibiricum into their glycoside forms, and increase their water solubility and stability by connecting glycosyl groups to the hydroxyl groups of flavonoids. During the fermentation process, the glycoside transferases produced by Saccharomyces cerevisiae and Lactobacillus acidophilus can catalyze the combination of flavonoids with sugars such as glucose and galactose to form flavonoid glycosides, which have better solubility in beverages and are easier to be absorbed and utilized by the human body. Polygonatum sibiricum polysaccharides have a large molecular weight, and the efficiency of their direct absorption and utilization by the human digestive system is relatively low. However, the oligosaccharides and monosaccharides formed after fermentation and degradation have simpler molecular structures and are more easily absorbed by the human gastrointestinal tract into the blood circulation, thereby being able to exert their physiological activities more quickly and effectively. At the same time, after fermentation treatment, some unique flavor substances and bioactive substances, such as organic acids, alcohols, esters, etc., can be produced, which give the fermented Polygonatum sibiricum beverage a unique flavor and taste and increase its health function.

[0036] In the present invention, the preferred inoculation amount of the composite bacterial agent is 1%-3% of the volume of the mixed enzymatic solution, and more preferably 2%. The preferred sealed fermentation temperature is 32-37°C, and more preferably 35°C; the preferred sealed fermentation time is 48-72h, and more preferably 60h.

[0037] (4) clarifying and filtering the polygonatum fermented liquid obtained by fermentation in step (3) to obtain a polygonatum fermented beverage.

[0038] In the present invention, preferably, a 0.22-0.45 μm filter membrane is used for filtration, and more preferably, a 0.3 μm filter membrane is used to remove the precipitate and impurities in the fermentation liquid to obtain a clear and transparent Polygonatum fermented beverage.

[0039] In the present invention, it is preferred that a stabilizer is added to the fermented beverage of polygonatum obtained in step (4) to prevent the beverage from precipitation, stratification and the like during storage. It is further preferred that the stabilizer is selected from one or more of xanthan gum and carboxymethyl cellulose, and the added amount is 0.1wt%-0.2wt%. It is preferred that a flavoring agent is also added to adjust the sweetness or acidity of the fermented beverage. It is further preferred that the flavoring agent is selected from one or more of sucrose, honey, and citric acid, and the added amount is 0.1wt%-5wt%. It is preferred that fruit and vegetable juice is also added to enrich the taste and nutritional components of the fermented beverage and give it a natural fruity aroma and color. It is further preferred that the fruit and vegetable juice is selected from apple juice, orange juice, pear juice, peach juice, carrot juice, cucumber juice, and spinach juice, and the added amount is 0.1wt%-30wt%.

[0040] The present invention also provides a fermented polygonatum beverage prepared by the above preparation method. As an implementation method, the fermented beverage is aseptically packaged in a sterile glass bottle or a sterile Tetra Pak package, and is stored in a cool and dry place after packaging.

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] A fermented polygonatum beverage, the preparation steps of which are as follows:

[0044] (1) Preparation of polygonatum slurry: fresh, pest-free rhizomes of polygonatum sibiricum are selected, cleaned, steamed and dried for nine times, cut into thin slices or small pieces, added with distilled water at a mass ratio of 1:8, and blended into a uniform and fine polygonatum slurry using a high-speed blender;

[0045] (2) Preliminary enzymatic hydrolysis: add papain and cellulase to the polygonatum slurry prepared in step (1), wherein the enzymatic activity of papain is 1500 U / mL and the enzymatic activity of cellulase is 3000 U / mL, perform enzymatic hydrolysis at 48°C for 2.5 h, inactivate the enzymes at 100°C for 10 min after the enzymatic hydrolysis, centrifuge the enzymatic hydrolyzate at 3500 r / min for 10 min, and separate the residue and the supernatant;

[0046] (3) Saccharification and enzymatic hydrolysis: distilled water was added to the residue in step (2) at a mass ratio of 1:4, and the mixture was stirred into a uniform suspension. Amylase with an activity of 2000 U / mL was added to the suspension. The suspension was saccharified and enzymatically hydrolyzed at 58° C. for 2 h. After the enzymatic hydrolysis, the enzyme was inactivated at 100° C. for 10 min.

[0047] (4) mixing the enzymatic hydrolysates of step (2) and step (3) to obtain a mixed enzymatic hydrolysate;

[0048] (5) Sealed fermentation: Inoculate the mixed enzymatic hydrolysate of step (4) with a composite bacterial agent of saccharomyces cerevisiae and Lactobacillus acidophilus, wherein the number of live saccharomyces cerevisiae in the composite bacterial agent is 2×10 9 CFU / mL, the number of viable Lactobacillus acidophilus is 1×10 9 CFU / mL, the inoculation amount was 2% of the volume of the mixed enzymatic hydrolysate, and the fermentation was sealed at 35°C for 60 h;

[0049] (6) Clarification and filtration: Filter the Polygonatum cyrtonema fermented broth from step (5) using a 0.3 μm filter membrane to remove precipitates and impurities in the fermented broth to obtain a clear and transparent Polygonatum cyrtonema fermented beverage.

[0050] Example 2

[0051] A fermented polygonatum beverage, the preparation steps of which are as follows:

[0052] (1) Preparation of Polygonatum sibiricum slurry: Select fresh, disease-free and insect-free Polygonatum sibiricum rhizomes, clean them, steam and dry them nine times, cut them into thin slices or small pieces, add distilled water at a mass ratio of 1:5, and use a high-speed blender to make a uniform and fine Polygonatum slurry.

[0053] (2) Preliminary enzymatic hydrolysis: papain and cellulase were added to the polygonatum slurry. The enzymatic activity of papain was 1000 U / mL and that of cellulase was 2000 U / mL. The enzymatic hydrolysis was carried out at 45°C for 3 h. After the enzymatic hydrolysis, the enzymes were inactivated at 90°C for 10 min. The enzymatic hydrolyzate was centrifuged at 3000 r / min for 12 min to separate the residue and the supernatant.

[0054] (3) Saccharification and enzymatic hydrolysis: distilled water was added to the residue at a mass ratio of 1:3 and stirred into a uniform suspension. Amylase with an activity of 1500 U / mL was added to the suspension. Saccharification and enzymatic hydrolysis was carried out at 55°C for 2.5 h. After the enzymatic hydrolysis, the enzyme was inactivated at 90°C for 8 min.

[0055] (4) Mixed enzymatic hydrolysate: The enzymatic hydrolysate of the preliminary enzymatic hydrolysis and the saccharification enzymatic hydrolysis are mixed to obtain a mixed enzymatic hydrolysate.

[0056] (5) Sealed fermentation: The mixed enzymatic hydrolysate was inoculated with a composite bacterial agent of saccharomyces cerevisiae and Lactobacillus acidophilus, and the number of live saccharomyces cerevisiae in the composite bacterial agent was 2×10 8 CFU / mL, the number of viable Lactobacillus acidophilus is 1×10 8 CFU / mL, the inoculation amount was 1% of the volume of the mixed enzymatic hydrolysate, and the fermentation was carried out in a sealed state at a fermentation temperature of 32°C for 72 h.

[0057] (6) Clarification and filtration: The fermented polygonatum sibiricum broth was filtered using a 0.22 μm filter membrane to remove the precipitate and impurities in the fermented broth to obtain a clear and transparent fermented polygonatum sibiricum beverage, which was then blended with orange juice at a rate of 1 wt %.

[0058] Example 3

[0059] A fermented polygonatum beverage, the preparation steps of which are as follows:

[0060] (1) Preparation of Polygonatum sibiricum slurry: Select fresh, disease-free and insect-free Polygonatum sibiricum rhizomes, clean them, steam and dry them nine times, cut them into thin slices or small pieces, add distilled water at a mass ratio of 1:10, and use a high-speed blender to make a uniform and fine Polygonatum slurry.

[0061] (2) Preliminary enzymatic hydrolysis: papain and cellulase were added to the polygonatum slurry. The enzymatic activity of papain was 2000 U / mL and that of cellulase was 4000 U / mL. The enzymatic hydrolysis was carried out at 50°C for 2 h. After the enzymatic hydrolysis, the enzymes were inactivated at 100°C for 8 min. The enzymatic hydrolyzate was centrifuged at 4000 r / min for 8 min to separate the residue and the supernatant.

[0062] (3) Saccharification and enzymatic hydrolysis: distilled water was added to the residue at a mass ratio of 1:5 and stirred into a uniform suspension. Amylase with an activity of 2000 U / mL was added to the suspension. Saccharification and enzymatic hydrolysis was carried out at 60°C for 1.5 h. After the enzymatic hydrolysis, the enzyme was inactivated at 100°C for 9 min.

[0063] (4) Mixed enzymatic hydrolysate: The enzymatic hydrolysate of the preliminary enzymatic hydrolysis and the saccharification enzymatic hydrolysis are mixed to obtain a mixed enzymatic hydrolysate.

[0064] (5) Sealed fermentation: The mixed enzymatic hydrolysate was inoculated with a composite bacterial agent of saccharomyces cerevisiae and Lactobacillus acidophilus, and the number of live saccharomyces cerevisiae in the composite bacterial agent was 2×10 9 CFU / mL, the number of viable Lactobacillus acidophilus is 1×10 9 CFU / mL, the inoculation amount was 3% of the volume of the mixed enzymatic hydrolysate, and the fermentation was carried out in a sealed state at a fermentation temperature of 37°C for 48 h.

[0065] (6) Clarification and filtration: The fermented polygonatum sibiricum broth was filtered using a 0.45 μm filter membrane to remove the precipitate and impurities in the fermented broth to obtain a clear and transparent fermented polygonatum sibiricum beverage, and 0.1 wt % sodium carboxymethyl cellulose, 3 wt % honey and 0.1 wt % citric acid were added.

[0066] Example 4

[0067] A fermented polygonatum beverage, the preparation steps of which are as follows:

[0068] (1) Preparation of Polygonatum sibiricum slurry: Select fresh, disease-free and insect-free Polygonatum sibiricum rhizomes, clean them, steam and dry them nine times, cut them into thin slices or small pieces, add distilled water at a mass ratio of 1:7, and use a high-speed blender to make a uniform and fine Polygonatum slurry.

[0069] (2) Preliminary enzymatic hydrolysis: papain and cellulase were added to the polygonatum slurry. The enzymatic activity of papain was 1800 U / mL and that of cellulase was 3600 U / mL. The enzymatic hydrolysis was carried out at 48°C for 2.5 h. After the enzymatic hydrolysis, the enzymes were inactivated at 95°C for 9 min. The enzymatic hydrolyzate was centrifuged at 3500 r / min for 10 min to separate the residue and the supernatant.

[0070] (3) Saccharification and enzymatic hydrolysis: distilled water was added to the residue at a mass ratio of 1:4 and stirred into a uniform suspension. Amylase with an activity of 2500 U / mL was added to the suspension. Saccharification and enzymatic hydrolysis was carried out at 58°C for 1.5 h. After the enzymatic hydrolysis, the enzyme was inactivated at 95°C for 9 min.

[0071] (4) Mixed enzymatic hydrolysate: The enzymatic hydrolysate of the preliminary enzymatic hydrolysis and the saccharification enzymatic hydrolysis are mixed to obtain a mixed enzymatic hydrolysate.

[0072] (5) Sealed fermentation: The mixed enzymatic hydrolysate was inoculated with a composite bacterial agent of saccharomyces cerevisiae and Lactobacillus acidophilus, and the number of live saccharomyces cerevisiae in the composite bacterial agent was 1.5×10 9 CFU / mL, the number of viable Lactobacillus acidophilus was 7.5×10 8 CFU / mL, the inoculation amount was 2% of the volume of the mixed enzymatic hydrolysate, and the fermentation was sealed at 35°C for 60 h.

[0073] (6) Clarification and filtration: The fermented polygonatum sibiricum broth was filtered using a 0.35 μm filter membrane to remove the precipitate and impurities in the fermented broth to obtain a clear and transparent fermented polygonatum sibiricum beverage, which was then blended with carrot juice at 5 wt % and 1 wt % sucrose was added.

[0074] Example 5

[0075] Effects of papain and cellulase on nutrient release from polygonatum

[0076] Experimental Preparation

[0077] 1. Material pretreatment: Select fresh, pest-free and uniform quality Polygonatum rhizomes, carefully wash them to remove surface impurities and dirt, steam and dry them nine times, then cut them into thin slices or small pieces, place them in a cool and ventilated place to dry for later use.

[0078] 2. Reagent Preparation

[0079] Glucose standard solution, rutin standard solution, anthrone reagent and vanillin-glacial acetic acid reagent.

[0080] Instrument debugging and calibration: debug and calibrate experimental instruments such as high-speed grinders, constant temperature water baths, centrifuges, spectrophotometers, and high-performance liquid chromatographs (HPLC) to ensure that the instruments work properly and the measurement data is accurate and reliable.

[0081] 3. Enzymatic hydrolysis experiment operation

[0082] 3.1. Preparation of Polygonatum slurry: Accurately weigh 100 g of pretreated Polygonatum sibiricum pieces, add distilled water that has been pre-boiled and cooled to room temperature at a mass ratio of 1:5, then transfer it to a high-speed grinder, stir and grind it at 10,000 r / min for 5 minutes to form a uniform and fine Polygonatum slurry.

[0083] 3.2. Enzyme solution addition and mixing: According to the enzyme activity combination designed in the experiment, different volumes of papain and cellulase preparations were accurately measured and added to the polygonatum slurry, as shown in Table 1. Use a pipette to fully mix the enzyme solution and polygonatum slurry to ensure that the enzyme is evenly distributed in the reaction system, and pay attention to avoid generating too many bubbles.

[0084] Table 1 Enzyme activities of papain and cellulase in different treatments

[0085] deal with Papain enzyme activity (U / mL Polygonatum sibiricum serum) Cellulase activity (U / mL Polygonatum sibiricum serum) 1 500 1000 2 1000 2000 3 1500 3000 4 2000 4000

[0086] 3.3. Enzyme hydrolysis reaction: The container containing the mixed enzyme solution was quickly transferred to a constant temperature water bath and the enzymolysis reaction was carried out at 45°C. During the enzymolysis process, a magnetic stirrer was used to stir slowly and continuously to ensure that the enzyme was in full contact with the Polygonatum slurry without causing excessive shearing or damage to the enzyme molecules. The enzymolysis reaction lasted for 2.5 hours, during which the temperature changes of the reaction system were closely observed to ensure that the temperature fluctuation was controlled within ±0.5°C.

[0087] 3.4. Enzyme inactivation: After the enzymatic reaction is completed, the reaction vessel is immediately taken out of the constant temperature water bath and placed in a water bath preheated to 95°C for enzyme inactivation. The enzyme inactivation time is strictly controlled within 10 minutes to ensure that papain and cellulase are completely inactivated and the enzymatic reaction is stopped. During the enzyme inactivation process, continue to stir to ensure that the reaction system is heated evenly.

[0088] 3.5. Centrifugal separation: After the enzyme is inactivated, the reaction solution is quickly transferred to a centrifuge tube and centrifuged at 3500r / min for 15 minutes using a high-speed centrifuge. After the centrifugation is completed, carefully remove the centrifuge tube, transfer the supernatant to a clean container, mark the group and repeat number, and use it for the subsequent determination of nutrient content.

[0089] 4. Nutrient content determination

[0090] 4.1. Determination of polysaccharide content (anthrone-sulfuric acid method)

[0091] Standard curve drawing: Take a series of glucose standard solutions of different concentrations (0, 0.02, 0.04, 0.06, 0.08, 0.10 mg / mL), accurately pipette 1.0 mL into stoppered test tubes, add 5.0 mL of anthrone reagent to each, quickly place the test tubes in a boiling water bath and heat for 10 minutes, then immediately take them out and cool them to room temperature in cold water. Use a spectrophotometer at a wavelength of 620 nm, with a blank solution (distilled water) as a reference, to measure the absorbance of each standard solution. Use glucose concentration as the horizontal axis and absorbance as the vertical axis to draw a standard curve and obtain the regression equation.

[0092] Sample determination: Accurately pipette 1.0 mL of the enzymatic supernatant into a stoppered test tube, add anthrone reagent and perform color reaction and absorbance determination according to the method of drawing a standard curve. Calculate the polysaccharide content in the sample according to the standard curve regression equation, expressed as glucose equivalent (mg / g Polygonatum odoratum).

[0093] 4.2. Determination of steroidal saponin content (vanillin-glacial acetic acid colorimetric method)

[0094] Standard curve drawing: Prepare a series of diosgenin standard solutions of different concentrations (0, 0.05, 0.10, 0.15, 0.20, 0.25 mg / mL), take 1.0 mL of each solution and place it in a stoppered test tube, add 0.2 mL of vanillin-glacial acetic acid reagent and 0.8 mL of perchloric acid, shake well and heat in a 60°C water bath for 15 minutes, then take out and cool to room temperature, and add 5.0 mL of glacial acetic acid to make up the volume. Use a spectrophotometer at a wavelength of 540 nm, with a blank solution (reagent mixture without diosgenin) as a reference, to measure the absorbance of each standard solution. Use diosgenin concentration as the horizontal axis and absorbance as the vertical axis to draw a standard curve and obtain a regression equation.

[0095] Sample determination: Accurately pipette 1.0 mL of the enzymatic supernatant into a stoppered test tube, add the corresponding reagents for color development and absorbance determination according to the steps of drawing the standard curve above. Calculate the content of steroidal saponins in the sample according to the regression equation of the standard curve, expressed as diosgenin equivalent (mg / g Polygonatum odoratum).

[0096] 4.3. Determination of flavonoid content (spectrophotometer method)

[0097] Standard curve drawing: prepare a series of rutin standard solutions of different concentrations (0, 0.02, 0.04, 0.06, 0.08, 0.10 mg / mL), draw 2.0 mL respectively and place in a 25 mL volumetric flask, add 1.0 mL of 5% sodium nitrite solution, shake well, add 1.0 mL of 10% aluminum nitrate solution after standing for 6 min, shake well, stand for 6 min, then add 10.0 mL of 4% sodium hydroxide solution, make up to the scale with distilled water, shake well. After 15 min, use a spectrophotometer at a wavelength of 510 nm, with a blank solution (agent mixture without rutin) as a reference, measure the absorbance of each standard solution. With rutin concentration as the horizontal coordinate and absorbance as the vertical coordinate, draw a standard curve to obtain a regression equation.

[0098] Sample determination: Accurately pipette an appropriate amount of 2.0 mL of the enzymatic supernatant into a 25 mL volumetric flask, add the corresponding reagents for color reaction and absorbance determination according to the method of drawing a standard curve. Calculate the content of flavonoids in the sample according to the regression equation of the standard curve, expressed as rutin equivalent (mg / g Polygonatum odoratum).

[0099] 5. Data Recording and Analysis

[0100] After the experiment, the recorded data were sorted and analyzed. First, the mean and standard deviation of each group of data were calculated to evaluate the discreteness and reliability of the data. Statistical analysis software (Excel) was used to perform variance analysis (ANOVA) to compare the significance of the differences in the content of polysaccharides, steroidal saponins and flavonoids between the different enzyme activity combination treatment groups and the control group.

[0101] 6. Test results

[0102] As shown in Table 2, papain and cellulase have a significant effect on the release of nutrients such as polysaccharides, steroidal saponins and flavonoids in Polygonatum. As the enzyme activity increases, the nutrient release gradually increases, among which the enzyme activity combination of treatment 4 (2000U / mL papain + 4000U / mL cellulase) has the most significant effect in promoting nutrient release.

[0103] Table 2 Content of effective components of Polygonatum sibiricum in enzymatic hydrolysate under different enzyme activities

[0104] deal with Polysaccharide content (mg / g) Steroidal saponins (mg / g) Flavonoids (mg / g) 1 25.53±0.62 12.10±0.30 10.50±0.30 2 32.43±0.65 15.20±0.32 13.12±0.30 3 38.27±0.70 18.53±0.55 15.62±0.41 4 42.53±0.68 20.83±0.65 17.77±0.45

[0105] Example 6

[0106] Effect of amylase on saccharification and enzymatic hydrolysis of Polygonatum cyrtonema residue

[0107] 1. Experimental Materials and Instruments

[0108] Materials: Polygonatum sibiricum residue after enzymatic hydrolysis (2000 U / mL papain + 4000 U / mL cellulase, 2.5 h) and separation in Example 5, amylase preparation, distilled water, DNS reagent.

[0109] Instruments: constant temperature water bath, saccharimeter, spectrophotometer, electronic balance, pH meter, magnetic stirrer, centrifuge, etc.

[0110] 2. Experimental Design

[0111] Enzyme activity setting: set up multiple groups of experiments, take 100g of Polygonatum sibiricum residue in each group, add distilled water at a mass ratio of 1:4 to make a suspension. Add amylase with different enzyme activities respectively, which are:

[0112] Group 1: amylase activity 1000U / mL suspension;

[0113] Group 2: amylase activity 1500U / mL suspension;

[0114] Group 3: amylase activity 2000U / mL suspension;

[0115] Group 4: amylase activity 2500U / mL suspension;

[0116] ck: no amylase added.

[0117] Set up 3 repetitions per set.

[0118] Enzymatic hydrolysis condition control: The suspension was placed in a 55°C constant temperature water bath and the enzymatic hydrolysis reaction was carried out under continuous stirring (stirring speed was controlled at 150r / min). The enzymatic hydrolysis time was set to 1.0h, 1.5h, 2h, 2.5h and 3.0h respectively to study the effect of enzymatic hydrolysis time on saccharification effect. After the enzymatic hydrolysis was completed, the reaction vessel was immediately placed in a 95°C water bath to inactivate the enzyme for 10min, and then cooled to room temperature.

[0119] 3. Detection indicators and methods

[0120] Determination of reducing sugar content (DNS method)

[0121] Standard curve drawing: accurately prepare a series of glucose standard solutions of different concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 mg / mL). Pipette 1.0 mL of standard solution into test tubes respectively, add 3.0 mL of DNS reagent, mix well and heat in a boiling water bath for 5 minutes, then quickly cool to room temperature and dilute to 25 mL with distilled water. Use a spectrophotometer at a wavelength of 540 nm, with a blank solution (distilled water instead of glucose standard solution) as a reference, to measure the absorbance of each standard solution. Use glucose concentration as the horizontal axis and absorbance as the vertical axis to draw a standard curve and obtain the regression equation.

[0122] Sample determination: Accurately pipette 1.0 mL of the enzymatic supernatant into a test tube, add DNS reagent for color reaction and absorbance determination according to the method of drawing a standard curve. Calculate the reducing sugar content in the sample according to the standard curve regression equation, expressed as glucose equivalent (mg / g Polygonatum residue).

[0123] 4. Test results

[0124] like Figure 2 As shown in the figure, amylase plays a key role in promoting the production of reducing sugars. Only under the action of amylase can the starch in the residue of Polygonatum odoratum be effectively hydrolyzed into reducing sugars. During the whole enzymatic hydrolysis process, the performance of different enzyme activity groups at various time stages fully demonstrated the complex interaction between enzyme activity and enzymatic hydrolysis time. In the early stage of enzymatic hydrolysis, enzyme activity is the key factor determining the rate of reducing sugar production and the increase in content. The high enzyme activity group can quickly accumulate reducing sugars, but as time goes by, substrate concentration, product inhibition, enzyme stability, etc. gradually participate in and affect the reaction process, so that the effect of enzyme activity on reducing sugar content presents different characteristics at different stages. Although the differences between the experimental groups narrowed in the later stage of the reaction, Groups 3 and 4 always maintained relatively high levels of reducing sugars, indicating that their higher enzyme activity played an important role in the whole enzymatic hydrolysis process and maintained a certain catalytic ability even when the reaction was close to equilibrium. Therefore, in order to quickly obtain more reducing sugars, amylases with higher enzyme activity can be preferentially selected and the enzymatic hydrolysis time can be controlled at 1.5-2.5h.

[0125] Example 7

[0126] Experimental study on the effect of fermentation agent on the quality of Polygonatum cyrtonema fermented beverage

[0127] 1. Experimental Design

[0128] This experiment set up 3 treatment groups and 1 control group, and each treatment group was set up with 3 replications.

[0129] Treatment 1: Saccharomyces cerevisiae was inoculated alone at a volume of 2% of the mixed enzymatic solution (viable count was 2×10 9 CFU / mL).

[0130] Treatment 2: Inoculate Lactobacillus acidophilus alone, with an inoculation volume of 2% of the volume of the mixed enzymatic hydrolysate (viable count of 1×10 9 CFU / mL).

[0131] Treatment 3: Inoculation of a composite bacterial agent of Saccharomyces cerevisiae and Lactobacillus acidophilus (the number of live bacteria of Saccharomyces cerevisiae was 2×10 9 CFU / mL, the number of viable Lactobacillus acidophilus is 1×10 9 CFU / mL), and the inoculation volume was 2% of the volume of the mixed enzymatic hydrolysate.

[0132] Treatment 4: Inoculation of a composite bacterial agent of Saccharomyces cerevisiae and Lactobacillus acidophilus (the number of live bacteria of Saccharomyces cerevisiae was 1×10 9 CFU / mL, the number of viable Lactobacillus acidophilus is 2×10 9 CFU / mL), and the inoculation volume was 2% of the volume of the mixed enzymatic hydrolysate.

[0133] Control group: No bacterial agent was inoculated, and only the mixed enzymatic hydrolysate was placed in the same environment.

[0134] Fermentation conditions: Transfer the inoculated mixed enzymatic hydrolysate to a fermentation tank, seal it, place it in a constant temperature incubator, and ferment it at 35°C for 48 hours.

[0135] 2. Detection indicators

[0136] The polysaccharide content, steroidal saponin content and flavonoid content were the same as in Example 5. The organic acid content (HPLC), alcohol content (gas chromatography-mass spectrometry), pH value, antioxidant capacity (DPPH free radical scavenging rate method) and sensory quality evaluation were evaluated. Detailed sensory evaluation indicators and standards were formulated, including appearance (color, transparency, clarity, full score 20 points), aroma (richness, coordination, characteristic aroma, full score 30 points), taste (sweetness, acidity, bitterness, smoothness, mellowness, full score 40 points) and overall preference (full score 10 points).

[0137] 3. Test results

[0138] As shown in Table 3, after fermentation, the content of polysaccharides, steroidal saponins and flavonoids in each treatment group was reduced compared with the mixed enzymatic hydrolysate, but the retention of effective ingredients in the fermentation broth of treatment 3 (compound bacterial agent 2:1) was relatively good. This is because the synergistic effect between Saccharomyces cerevisiae and Lactobacillus acidophilus is more effective under the ratio of compound bacterial agent. While promoting the production and metabolism of flavor substances, it plays a certain role in protecting and transforming the effective ingredients in Polygonatum sibiricum, making its loss during the fermentation process relatively small. When the strain is fermented alone, the decline in effective ingredients is relatively large. This is because the metabolic pathway of a single bacterial agent is relatively simple, and the decomposition or transformation of the effective ingredients is more direct and intense. The content of effective ingredients in treatment 4 (compound bacterial agent 1:2) is also retained to a certain extent, but the overall effect is slightly inferior to treatment 3.

[0139] After inoculation, the content of organic acids and alcohols in each treatment group increased significantly. Treatment 2, inoculated with Lactobacillus acidophilus alone, produced the most lactic acid, which is closely related to the metabolic characteristics of Lactobacillus acidophilus, which tends to produce a large amount of lactic acid during the fermentation process. Treatment 1, inoculated with Saccharomyces cerevisiae alone, produced a relatively prominent amount of ethanol, which is consistent with the fermentation characteristics of Saccharomyces cerevisiae. In the composite bacterial agent treatment groups of Treatments 3 and 4, the content of various substances was relatively more balanced, and the total amount was also higher. This shows that the composite bacterial agent can promote the synergistic metabolism between the two bacteria during the fermentation process, allowing a variety of flavor substances to be produced, enriching the flavor level of the beverage, and helping to improve the taste quality of the beverage, making it more coordinated and mellow.

[0140] The pH value of the fermentation liquid decreased significantly after inoculation of the bacterial agent. The fermentation liquid in treatment 2 inoculated with Lactobacillus acidophilus alone had the lowest pH value and the strongest acidity, which was mainly due to the large amount of organic acids produced by it. The pH value of the composite bacterial agent treatment group of treatment 3 and treatment 4 was in a relatively moderate range, which not only maintained a certain acidity but also did not make the beverage too sour and astringent, which was more in line with consumers' taste preferences. At the same time, the antioxidant capacity of the beverages after fermentation was significantly improved, and the antioxidant capacity of treatment 3 was the strongest. This is because the composite bacterial agent of treatment 3 can more effectively stimulate the active transformation of antioxidant components in Polygonatum odoratum during the fermentation process, so that it releases more metabolites with antioxidant functions, such as certain phenolic and flavonoid metabolic derivatives. These substances not only enhance the antioxidant capacity of the beverage, but also interact with the organic acids, alcohols, esters, etc. produced by fermentation, further optimizing the flavor and taste of the beverage.

[0141] Table 3 Effects of different fermentation agents on the quality of Polygonatum cyrtonema fermented beverages

[0142]

[0143]

[0144] 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 beverage of polygonatum, characterized in that: The following steps are involved: (1) adding papain and cellulase to polygonatum slurry for preliminary enzymolysis, killing the enzymes after the enzymolysis, and separating the residue and the supernatant; (2) the residue is made into a suspension, amylase is added to perform saccharification and enzymolysis, the enzyme is inactivated after the enzymolysis is completed, and the suspension is mixed with the initial enzymolysis supernatant to obtain a mixed enzymolysis solution; (3) The mixed enzymatic hydrolysate is inoculated with a composite bacterial agent of saccharomyces cerevisiae and Lactobacillus acidophilus for sealed fermentation to obtain a Polygonatum sibiricum fermentation liquid; the number of live bacteria of saccharomyces cerevisiae in the composite bacterial agent is 2×10 8 -2×10 9 CFU / mL, the viable count of Lactobacillus acidophilus is 1×10 8 -1×10 9 CFU / mL; (4) The polygonatum fermented liquid is clarified and filtered to obtain a polygonatum fermented beverage.

2. The preparation method according to claim 1, characterized in that: In the step (1), during the initial enzymatic hydrolysis, the enzymatic activity of papain is 1000-2000 U / mL, and the enzymatic activity of cellulase is 2000-4000 U / mL.

3. The preparation method according to claim 1, characterized in that: In the step (1), during the initial enzymatic hydrolysis, the temperature is 45-50° C. and the time is 2-3 hours.

4. The preparation method according to claim 1, characterized in that: In the step (2), during the saccharification and enzymatic hydrolysis, the enzymatic activity of the amylase is 1500-2500 U / mL.

5. The preparation method according to claim 1, characterized in that: In the step (2), during the saccharification and enzymatic hydrolysis, the temperature is 55-60° C. and the time is 1.5-2.5 h.

6. The preparation method according to claim 1, characterized in that: In the steps (1) and (2), the temperature for inactivating the enzyme is 90-100° C. and the time is 8-10 minutes.

7. The preparation method according to claim 1, characterized in that: In the step (3), the inoculation amount of the composite bacterial agent is 1%-3% of the volume of the mixed enzymatic hydrolysate.

8. The preparation method according to claim 1, characterized in that: In the step (3), the temperature of the sealed fermentation is 32-37°C and the time is 48-72 hours.

9. A fermented polygonatum beverage prepared by the preparation method according to any one of claims 1 to 8.

10. The fermented polygonatum beverage according to claim 9, characterized in that: It also includes one or more of fruit and vegetable juice, flavoring agent and stabilizer.

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