Polygonatum sibiricum fermentation product preparation method capable of increasing polysaccharide content, product and application

Abstract

The invention relates to a polygonatum kingianum fermented product capable of increasing the polysaccharide content and preparation and application thereof. Rhizopus arrhizus, aspergillus oryzae and Ferbienberg's yeast in a mass ratio of 3: 2: 1 are adopted to form a composite strain for single-time variable-temperature fermentation; the prepared rhizoma polygonati fermentation product is golden yellow, has the due raw material fragrance and fermentation flower fragrance of rhizoma polygonati, is free of numb and astringent taste, pure in taste and rich in polysaccharide. According to the rhizoma polygonati fermented product, aldehydes, alcohols and acid substances with green grass taste are reduced, the content of ester substances and benzene ring aromatic substances is increased, and the flavor characteristics of the rhizoma polygonati fermented product and the rhizoma polygonati fermented beverage are improved. A simple and efficient solution is adopted, the utilization rate of a rhizoma polygonati raw material can be remarkably increased, the content of polysaccharide and polypeptide of a rhizoma polygonati fermentation product is increased, the oxidation resistance and the xanthine oxidase inhibiting capacity of the rhizoma polygonati fermentation product are remarkably improved, and the rhizoma polygonati fermentation product has good uric acid reducing potential; and a new path is opened up for developing highly-processed polygonatum sibiricum products with unique effects and high additional values.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of processing of medicinal and edible Chinese herbal materials, and more particularly relates to a fermented product of Polygonatum sibiricum with improved polysaccharide content, a preparation method thereof, the product, and an application of the product. BACKGROUND

[0002] Fujian is an important production area of Polygonatum sibiricum with rich resources, as it is endowed with superior ecological conditions. As an important representative of the characteristic edible and medicinal dual-purpose agricultural product and characteristic local medicinal material "Fujian Nine Ingredients" widely planted in Fujian Province, the planting area of Polygonatum sibiricum is more than 200,000 mu, and the resources are rich. It enjoys a good reputation across the country for its unique geographical indications and dual quality of medicine and food. Although it has a long history of consumption, raw Polygonatum sibiricum has problems of poor taste (astringent, bitter, astringent, and fishy) and irritation.

[0003] Currently, the overall processing of Polygonatum sibiricum is still dominated by rough processing, and the main products are nine-processed Polygonatum sibiricum, Polygonatum sibiricum tea, and refined aconite, etc. There is serious homogeneity, low comprehensive utilization rate, and a lack of deep processing products. Nine-processed Polygonatum sibiricum is a kind of Polygonatum sibiricum processed by nine times of steaming and nine times of drying, which has high medicinal value and health care functions, and is widely used in traditional Chinese medicine treatment and health care. At present, the main product is nine-processed Polygonatum sibiricum tea, but the production process has the problems of long time consumption and nearly 50% loss of polysaccharides. In addition, many known reports on fermented Polygonatum sibiricum beverages use lactic acid bacteria for fermentation, which have the defects of single product flavor, low raw material utilization rate, and low polysaccharide content.

[0004] Chinese invention patent application CN115197976A provides a method for preparing polyflora polygonatum prebiotics by microbial fermentation. The invention uses Aspergillus oryzae to ferment polyflora polygonatum, then water extracts, and uses yeast to ferment the water extract again to prepare polyflora polygonatum fermentation broth. The content of active ingredients such as sugar, polyphenol and β-glucan in the obtained polyflora polygonatum fermentation broth is significantly increased, which has good antioxidant activity and promotes the growth of probiotics, and can be used as prebiotics to promote the growth of probiotics. Chinese invention patent application CN107753741A provides a deep processing method of polyflora polygonatum and its product. The invention uses the power of microorganisms to ferment polyflora polygonatum. First, the powerful starch sugar and protein decomposition ability of Aspergillus niger and Aspergillus oryzae is used, then the macromolecular substances are enzymatically hydrolyzed into small molecular substances, and then Cordyceps militaris is used for secondary fermentation. The active ingredients produced by Cordyceps militaris itself, such as cordycepin and polysaccharide, are used to give the fermented polyflora polygonatum better treatment and health care effect. The above methods mainly rely on single strain fermentation or use multiple fermentation and multiple extraction processes, which are complicated and inefficient. The active ingredients mainly focus on increasing the content of polysaccharide, polyphenol and antioxidant activity. Therefore, there is still a lack of simple and effective fermentation technology in the prior art to effectively increase the content of polysaccharide and polypeptide substances in the fermented polyflora polygonatum product. There is also no fermented polyflora polygonatum product with high polysaccharide and polypeptide content, antioxidant activity and significant xanthine oxidase XOD inhibition capacity to reduce uric acid. SUMMARY

[0005] In view of the above technical problems, the present application provides a preparation method, product and application of a polyflora polygonatum fermentation product with increased polysaccharide content, in order to effectively utilize starch, protein and sugar substances in polyflora polygonatum, increase the utilization rate of polyflora polygonatum raw materials, and increase the content of small molecular polysaccharide and polypeptide in polyflora polygonatum product, improve its structural characteristics, so that the polyflora polygonatum product is not only beneficial to the digestion and absorption of the body, but also exposes more active groups, improves the antioxidant capacity, XOD inhibition capacity and other biological activities, so as to open up a broader application prospect in the field of uric acid-lowering health food, beverage, condiment, auxiliary drug, etc.

[0006] To achieve the above-mentioned purpose, in a first aspect, the present application provides a preparation method of a polyflora polygonatum fermentation product with increased polysaccharide content, comprising the following steps:

[0007] Mixing polyflora polygonatum powder with water at a mass ratio of 1:5-8, inoculating with a composite strain for single temperature variation fermentation, to obtain the polyflora polygonatum fermentation product;

[0008] The complex strain is composed of Rhizopus arrhizus, Aspergillus oryzae and Cyberlindnera fabianii, and the mass ratio of the three is 3:2:1;

[0009] The inoculation amount of the complex strain is 0.5% to 1% of the mass of the polygonatum sibiricum powder;

[0010] The single temperature variation fermentation condition is: 40±1℃ fermentation for 2h, 45±1℃ fermentation for 1h, and 58±1℃ fermentation for 24 to 48h.

[0011] Compared with the traditional single strain or complex multiple fermentation and multiple extraction process, the above technical scheme adopts Rhizopus arrhizus, Aspergillus oryzae and Cyberlindnera fabianii to form a complex strain with a mass ratio of 3:2:1 for single temperature variation fermentation, which provides a simpler and more efficient solution. Not only can the utilization rate of polygonatum sibiricum raw materials be significantly improved, but also the polysaccharides wrapped in polygonatum sibiricum starch and protein can be fully released. Large molecular polysaccharides are cut into small molecular polysaccharides, and proteins are fully hydrolyzed into small molecular peptides. The polysaccharide and polypeptide content of polygonatum sibiricum fermented beverage is improved. More active groups are fully exposed, the antioxidant capacity of polygonatum sibiricum fermented product is improved, it has xanthine oxidase XOD inhibition capacity and uric acid reduction potential, and it opens up a new path for the development of high value-added polygonatum sibiricum deep processing products with unique efficacy.

[0012] In some embodiments, the Rhizopus arrhizus strain is CMR21 with high amylase and glucoamylase production, with the accession number CCTCC NO: M20242064 and the preservation date of September 25, 2024. The Rhizopus arrhizus CMR21 has been preserved in the China Center for Type Culture Collection in Wuhan. The Aspergillus oryzae strain is MJY2-5 with high protease activity, with the accession number CCTCC NO: M 20221400 and the preservation date of September 9, 2022. The Aspergillus oryzae MJY2-5 has been preserved in the China Center for Type Culture Collection in Wuhan. The Cyberlindnera fabianii strain is JGM9-1 with aroma production, with the accession number CCTCC NO: M 20221401 and the preservation date of September 9, 2022. The Cyberlindnera fabianii JGM9-1 has been preserved in the China Center for Type Culture Collection in Wuhan. In these embodiments, the starch in the polygonatum sibiricum is hydrolyzed into sugar substances by fermentation of the Rhizopus arrhizus CMR21 with high amylase and glucoamylase production; the protein in the polygonatum sibiricum is decomposed into small molecular peptides and amino acid substances by fermentation of the Aspergillus oryzae MJY2-5 with high protein utilization rate; and the sugar in the polygonatum sibiricum produces aroma substances to enhance the flavor characteristics of the polygonatum sibiricum by fermentation of the Cyberlindnera fabianii JGM9-1 with aroma production. The combined bacteria such as the Rhizopus arrhizus CMR21, the Aspergillus oryzae MJY2-5 and the Cyberlindnera fabianii JGM9-1 are used for temperature-variable fermentation, which can significantly improve the utilization rate of the polygonatum sibiricum raw materials. Since the polysaccharides wrapped in the starch and protein are fully released, the large molecular polysaccharides are cut into small molecular polysaccharides, the polysaccharide content of the polygonatum sibiricum fermentation product is improved, the polygonatum sibiricum protein is fully hydrolyzed into peptides, the polypeptide content is greatly improved, and thus the antioxidant capacity and the xanthine oxidase XOD inhibition capacity of the polygonatum sibiricum product are improved.

[0013] In some embodiments of the present application, the polygonatum sibiricum powder is prepared by a preparation method comprising the following steps: subjecting polygonatum sibiricum rhizomes to steaming, drying and low-temperature crushing treatment to obtain the polygonatum sibiricum powder. In such embodiments, the polygonatum sibiricum from the desired production area can be strictly selected to achieve the purpose of controllable quality of the polygonatum sibiricum powder and optimization of the polygonatum sibiricum fermentation product.

[0014] In a more preferred embodiment, the steaming treatment comprises: drying the selected healthy polyfloral polygonatum tubers or rhizomes to a water content of ≤30%, washing, removing the rhizome skin and root hairs, steaming for 100-140 min, and drying at 50-60℃ after drying. The low-temperature crushing comprises: freezing the dried polygonatum at -40℃, and then crushing to below 60 mesh in an environment of -30℃ to -40℃. In this way, the cumbersome steps of traditional Chinese medicine processing "nine steaming and nine drying" can be simplified, and enzymes can be inactivated to preserve glycosides, promote conversion, and improve efficacy. The low-temperature crushing of -40℃ freezing and -30℃ to -40℃ crushing can prevent heat-sensitive loss and low-temperature oxidation, and the crushing in an extremely low-temperature environment can maximize the avoidance of the degradation, destruction or volatilization of heat-sensitive active ingredients caused by friction heat generated during conventional crushing. Through steaming and moderate drying (drying + 50-60℃ drying), the polygonatum tuber structure is loose, the cell wall is partially broken, the starch is gelatinized, and the polysaccharide and other macromolecular substances are partially degraded or structurally changed. This greatly improves the accessibility and utilization rate of the substrate by the complex strains in the subsequent fermentation. Raw polygonatum is rich in mucilage, and conventional crushing is extremely easy to stick and form a ball, making it difficult to obtain fine powder, and the crushing process generates a lot of heat. Low-temperature crushing (-30℃ to -40℃) is an effective method to solve this problem. Low temperature makes the mucilage brittle, greatly reducing the stickiness of the material, making it possible to crush to below 60 mesh, and ensuring the smooth progress of the subsequent process. The low-temperature crushing obtains fine powder (≥60 mesh), increases the contact surface area of the material and the complex strains, and enables the enzymes secreted by the complex strains to act more efficiently on the substrate, promoting conversion, improving fermentation efficiency and product yield.

[0015] In a second aspect of the present application, a polygonatum fermentation product with improved polysaccharide content prepared by the preparation method of the first aspect of the present application is provided. Due to the variable-temperature fermentation in the preparation method of the first aspect of the present application, the low temperature in the early stage is suitable for strain growth, the middle stage promotes enzymatic hydrolysis, and the high temperature in the later stage can inactivate miscellaneous bacteria and promote the production of flavor substances by Maillard reaction. The synergistic effect of the protease of Aspergillus oryzae MJY2-5 and the amylase of Rhizopus arrhizus CMR21 effectively degrades the polysaccharide of polygonatum, improves the utilization rate of raw materials, and makes the polygonatum fermentation product have both the inherent raw material aroma of polygonatum and the fermentation floral aroma. The ester aroma substances can still be produced by the JGM9-1 Saccharomyces boulardii at 58℃, and in addition, the high-temperature stage also has the effect of pasteurization, making the polygonatum fermentation product very safe. The polygonatum fermentation product provided by the present application has the advantages of high polysaccharide content, high polypeptide content, strong antioxidant capacity and XOD inhibition capacity.

[0016] Further preferably, the polysaccharide content in the above-mentioned fermented product of Rhizoma Polygonati is ≥ 0.74 g / 100 mL, and the polypeptide content is ≥ 3.12 g / 100 mL. The high yield of polysaccharide and the breakthrough of polypeptide content depend on the complex strain, the ratio and the variable-temperature fermentation process, which are beneficial to the popularization and application of the fermented product of Rhizoma Polygonati provided by the present application in the development of subsequent functional foods, medicines and health products with the function of reducing uric acid, and help to provide technical indicators for the production and quality control management of the product.

[0017] In a third aspect of the present application, a fermented beverage of Rhizoma Polygonati is provided, which is prepared by a preparation method comprising the following steps:

[0018] The fermented product of Rhizoma Polygonati with increased polysaccharide content prepared by the preparation method of the first aspect of the present application is filtered, blended and sterilized to obtain the fermented beverage of Rhizoma Polygonati. Specifically, the fermented product of Rhizoma Polygonati is filtered through a 100-mesh or larger filtering device to obtain filter residue (Rhizoma Polygonati), and then the filter residue and water are added to warm water at about 50℃ in a ratio of 1:3-5 for 0.5-1 h, and then filtered. The two filtrates are combined, boiled, and then centrifuged or plate-frame filtered to obtain a fermentation liquor. The fermentation liquor is adjusted to have a soluble solid content of 10-14% by white granulated sugar, and a pH value of 4.1-4.2 by citric acid, and then hot canned and sterilized at 100℃ for 15 min.

[0019] The preparation method of the present application has stable process and high raw material utilization rate, and the fermented beverage of Rhizoma Polygonati prepared thereby has an ethanol content of less than 0.1%, a golden yellow color, inherent raw material aroma and fermentation floral aroma of Rhizoma Polygonati, no astringent taste, pure taste, rich in polysaccharide and polypeptide, and pure natural characteristics, nutritional and probiotic effects.

[0020] In a more preferred fermented beverage of Rhizoma Polygonati, the polysaccharide content is ≥ 0.45 g / 100 mL, and the polypeptide content is ≥ 1.8 g / 100 mL.

[0021] Further preferably, in the fermented beverage of Rhizoma Polygonati, the polysaccharide has a molecular weight of 4000-4500 Da, and the ethanol content is ≤ 0.1%. The fermented beverage of Rhizoma Polygonati provided by the present application has high raw material utilization rate and rich nutrition, and has good antioxidant capacity and XOD inhibition capacity due to the polysaccharide content of ≥ 0.45 g / 100 mL and the polypeptide content of ≥ 1.8 g / 100 mL, which breaks through the bottleneck of traditional fermented Rhizoma Polygonati only having polysaccharide, polyphenol and other nutritional ingredients and antioxidant capacity.

[0022] In a fourth aspect of the present application, the polysaccharide fermented product of Rhizoma Polygonati prepared by the preparation method of the first aspect of the present application is used in the preparation of functional foods or health products with the function of reducing uric acid.

[0023] The above summary related to the invention is only a summary of the technical solutions of the present application. In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, and then implement the content recorded in the specification and drawings, and in order to enable the above and other purposes, characteristics and advantages of the present application to be more easily understood, the following will be described in combination with the specific embodiments of the present application and the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0024] The accompanying drawings are only used to show the principles, implementation manners, applications, characteristics and effects of the specific embodiments of the present application and other related contents, and cannot be considered as limitations to the present application.

[0025] In the drawings:

[0026] Figure 1 The relative molecular mass distribution graph of sample A described in specific embodiment eleven;

[0027] Figure 2 The relative molecular mass distribution graph of sample B described in specific embodiment one. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, and then implement the content recorded in the specification and drawings, and in order to enable the above and other purposes, characteristics and advantages of the present application to be more easily understood, the following will be described in combination with the specific embodiments of the present application and the accompanying drawings. The embodiments recorded in the present text are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0029] In the present text, the term "embodiment" means that the specific features, structures or characteristics described in combination with the embodiments can be contained in at least one embodiment of the present application. The term "embodiment" appearing at various positions in the specification does not necessarily refer to the same embodiment, and does not particularly limit the independence or association between other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment can be combined in any manner to form a corresponding implementable technical solution.

[0030] Unless otherwise defined, the meanings of the technical terms used in the present text are the same as those generally understood by those skilled in the art to which the present application belongs; the use of related terms in the present text is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0031] In the description of the present application, the term "and / or" is a description of the logical relationship between the objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in the present text generally represents that the associated objects before and after are a "or" logical relationship.

[0032] In the present application, the terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual number, primary or secondary, or order relationship between the entities or operations.

[0033] In the present application, the "includes", "contains", "has", or other similar expressions used in the statements are intended to cover non-exclusive inclusion, and the expressions do not exclude the presence of additional elements in the process, method or product comprising the elements, so that the process, method or product comprising a series of elements can not only include those defined elements, but also include other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0034] As the same understanding as in the "Guidelines for Examination", in the present application, the expressions such as "greater than", "less than", "exceed" are understood as not including the number; the expressions such as "above", "below", "within" are understood as including the number. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times", etc., unless otherwise explicitly limited.

[0035] In the description of the embodiments of the present application, the spatial-related expressions used, such as "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "perpendicular", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. The indicated orientation or position relationship is based on the orientation or position relationship shown in the specific embodiment or the drawing, and is only for the convenience of describing the specific embodiments of the present application or for the reader to understand, and does not indicate or imply that the indicated device or component must have a specific position, a specific orientation, or be constructed or operated in a specific orientation, and therefore cannot be understood as a limitation on the embodiments of the present application.

[0036] Unless otherwise explicitly specified or limited, in the description of the embodiments of the present application, the terms "mount", "connect", "connect", "fix", "set", etc. should be understood in a broad sense. For example, the "connection" can be a fixed connection, or a detachable connection, or an integral setting; it can be a mechanical connection, or an electrical connection, or a communication connection; it can be directly connected, or indirectly connected through an intermediate medium; it can be the internal communication or interaction relationship of two elements. For those skilled in the art to which the present application belongs, the specific meaning of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0037] Although Polygonatum cyrtonema has a history of 5,000 years as a food and medicine, it is difficult to be directly consumed due to the presence of bitter and astringent components such as saponins and alkaloids in the raw product. The traditional processing method (such as nine steaming and nine processing of Polygonatum) has defects such as a long processing time of up to 30 days and a loss of nearly 50% of polysaccharides. Modern fermentation technology also faces multiple bottlenecks, such as single flavor of lactic acid bacteria fermentation products and low polysaccharide content. The operation is complicated due to the use of multiple fermentation and extraction processes, and the step-by-step fermentation method is prone to environmental pollution risk and fluctuation of the components of the fermented Polygonatum product. The fine processing of Polygonatum urgently needs an innovative solution that can simultaneously solve the problems of taste improvement, active ingredient preservation, and process simplification.

[0038] The present application breaks through three technical barriers at one time by using the compound strain of Rhizopus arrhizus CMR21+ Aspergillus oryzae MJY2-5+ Fibrobacter succinogenes JGM9-1 (mass ratio 3:2:1) for single temperature variation fermentation: (1) Aspergillus oryzae directionally degrades astringent substances to achieve effective detoxification and taste improvement, and simultaneously sterilizes and produces aroma at a high temperature of 58℃; (2) The polysaccharide yield is increased to ≥0.74g / 100mL, which is significantly higher than that of traditional processes, and the polypeptide content reaches ≥3.12g / 100mL, breaking the upper limit of existing technology; (3) For the first time, it is found that specific polypeptides in the fermentation product have strong xanthine oxidase inhibition capacity (XOD inhibition rate ≥52%), providing a new material basis for uric acid-lowering functional foods and a reliable technical path for promoting the upgrading of the Polygonatum industry from low-value medicinal slices to high-value functional foods.

[0039] The biological materials used in the embodiments of the present application, Rhizopus arrhizus CMR21, Aspergillus oryzae MJY2-5 and Fibrobacter succinogenes JGM9-1, are strains independently selected by the applicant, and their preservation numbers and dates are CCTCC NO: M20242064, September 25, 2024; CCTCC NO: M 20221400, September 09, 2022; and CCTCC NO: M20221401, September 09, 2022, respectively.

[0040] In the present application, all chemical reagents are of analytical purity grade and come from conventional commercially available products unless otherwise specified; and all experimental instruments are conventional commercially available equipment.

[0041] In the present application, the Polygonatum powder refers to the healthy Polygonatum cyrtonema tubers without impurities, diseases, insect damage, spots, and mold changes, which are dried to a water content of ≤30%, washed, and then the epidermis and root hairs are removed. The Polygonatum cyrtonema is steamed for 100-140min, dried at 50-60℃ after drying, then low-temperature crushed: the dried Polygonatum cyrtonema is frozen at-40℃, then crushed to 60 mesh or less under the condition of-30℃ to-40℃, and the water content of the Polygonatum powder is ≤8%.

[0042] The "enzyme activity unit" referred to in the present application is defined as: the amount of enzyme required to hydrolyze the substrate to produce 1 μmol of glucose per minute at 40℃ and pH 5.0, which is recorded as "1 U".

[0043] Unless otherwise defined, the terms used in the present application have the meanings commonly understood by those skilled in the art. If there is a conflict, the definition in the present specification shall prevail. Unless otherwise specified, the test methods used in the present application can use the methods commonly used by those skilled in the art.

[0044] When the numerical range "a to b" is described in the examples, the end point values a and b are included, as well as all sub-ranges therebetween (such as a to 0.8b).

[0045] In the present application, the determination of total acid refers to the potentiometric titration method in GB 12456-2021 "Determination of Total Acid in Food". The specific steps are: take 10 mL of Huangjing fermentation product, add 60 mL of distilled water in a 250 mL beaker, insert the electrode, put in a rotor, place on an electromagnetic stirrer, start stirring, and titrate with 0.1 mol / L sodium hydroxide standard titration solution. Record the volume of sodium hydroxide standard titration solution consumed at the end point pH=8.2. At the same time, do a blank test.

[0046] In the present application, the determination of soluble solids refers to GB / T 12143-2008 "General Analysis Method for Beverages" using a handheld sugar meter to directly read out the data. The determination of pH value refers to GB 5009.237-2016 "Determination of pH Value of Food" using a pH meter to directly read out the data.

[0047] In the present application, the determination of total sugar refers to GB 5009.7-2016 "Determination of Reducing Sugar in Food". The specific steps are: take 1.0 g (accurate to the third decimal place), place in a 250 mL volumetric flask, add 50 mL of water, slowly add 5 mL of zinc acetate solution and 5 mL of potassium ferrocyanide solution, add water to the mark, mix well, stand for 30 min, filter with dry filter paper, discard the initial filtrate, take 10 mL of the subsequent filtrate in a 100 mL volumetric flask, add 5 mL of hydrochloric acid and 10 mL of water, and heat in a 68℃ water bath for 15 min. Cool to room temperature, titrate with 200 g / L sodium hydroxide to neutralize and dilute to volume. Take 5 mL of each of the Filing reagent methanol and ethyl alcohol, and 10 mL of the sample solution under heating conditions with 1 g / L glucose standard solution.

[0048] The determination of polysaccharide in the application refers to the determination results of the two samples of Huangjing fermentation products A and B obtained according to SN / T 4260-2015 “Determination of Crude Polysaccharide in Plant-derived Food for Export Phenol-sulfuric Acid Method”. Sample treatment: weigh 10 mL of the sample into a 50 mL centrifuge tube with a stopper, slowly add 40 mL of anhydrous ethanol, and simultaneously use a vortex shaker to shake, so as to mix uniformly, and place in an ultrasonic extractor for ultrasonic extraction for 30 min. After the extraction is completed, centrifuge at 4000 r / min for 10 min, and discard the supernatant. The insoluble substance is washed with 20 mL of 80% ethanol solution and centrifuged.

[0049] The determination of acid-soluble protein content (polypeptide content) in the application refers to the method disclosed in Zhang Guozhi et al. “Preparation of Angiotensin Converting Enzyme (ACE) Inhibitory Peptide by Hydrolyzing Alcohol-washed Peanut Protein with 2709 Alkaline Protease”. The determination results of the two samples of Huangjing fermentation products A and B are obtained. The determination method is as follows: weigh 5.00 mL of the sample, add 25 mL of 15% trichloroacetic acid solution, stir uniformly, stand for 5 min, quantitatively transfer the solution, centrifuge at 4000 r / min for 10 min, and take the supernatant to detect the protein content. The protein content detection refers to GB 5009.5-2016 “Determination of Protein in Food”, and the Kjeldahl method is adopted.

[0050] DPPH free radical scavenging capacity, ABTS ＋ The free radical scavenging capacity refers to GB / T 39100-2020 “Determination Method of Polypeptide Antioxidation”.

[0051] The test method of XOD inhibition rate is as follows: first add 50 μL of xanthine oxidase (0.1 U·mL -1 ), 50 μL of enzyme hydrolysate, 150 μL of phosphate buffer solution, add 50 μL of xanthine oxidase (0.1 U·mL -1 ) and 50 μL of enzyme hydrolysate in the double wells, incubate at 37°C for 10 min, then add 150 μL of xanthine solution (0.3 mmol·L -1 ) in the double wells, incubate at 37°C for 15 min, and then measure the optical density at 292 nm. The optical density of the double wells minus the optical density of the single well is the optical density of the test group D A ; after the enzyme hydrolysate in the above is replaced by the phosphate buffer solution, the optical density of the blank group is calculated as D0.

[0052] The XOD inhibition rate (I) is calculated according to formula (1):

[0053] I (%) = [(D0-D A ) / D0] × 100% (1)

[0054] In the formula, I represents the XOD inhibition rate, D0 represents the optical density of the blank group, and D AThe optical density of the test group is represented.

[0055] Example One

[0056] A preparation method of a fermented product of Polygonatum sibiricum

[0057] Take Polygonatum cyathopetalum tubers, dry them to a moisture content of 28%, wash and remove the skin and hairs, steam them for 120 min, dry them at 55°C, freeze the dried Polygonatum sibiricum at -40°C for 24 h, and grind them to 80 mesh (a screening rate ≥ 95%) under a -35°C environment to obtain Polygonatum sibiricum powder. Then mix the Polygonatum sibiricum powder with water at a mass ratio of 1:6, and inoculate a composite strain (a total amount of 0.8% of the mass of the Polygonatum sibiricum powder). The composite strain is obtained by mixing Rhizopus arrhizus CMR21 (a preservation number of CCTCC NO: M20242064), Aspergillus oryzae MJY2-5 (a preservation number of CCTCC NO: M 20221400), and Scheffersomyces fibuliger JGM9-1 (a preservation number of CCTCC NO: M 20221401) at a mass ratio of 3:2:1.

[0058] Perform temperature-variable fermentation: 40°C±0.5°C for 2 h, 45°C±0.5°C for 1 h, 58°C±0.5°C for 36 h, and terminate the fermentation to obtain a slurry-like polysaccharide Polygonatum sibiricum fermentation product.

[0059] According to the above polysaccharide determination method, the polysaccharide content of the polysaccharide Polygonatum sibiricum fermentation product in this example is 0.74 g / 100 mL.

[0060] Example Two

[0061] A preparation method of a fermented product of Polygonatum sibiricum

[0062] The difference from Example One is that Polygonatum cyathopetalum tubers are taken, dried to a moisture content of 28%, washed and peeled, steamed for 120 min, dried at 55°C, frozen at -40°C for 24 h, and ground to 60 mesh under a -35°C environment to obtain Polygonatum sibiricum powder. Then mix the Polygonatum sibiricum powder with water at a mass ratio of 1:5, inoculate a composite strain at a total amount of 0.5% of the mass of the Polygonatum sibiricum powder. Perform temperature-variable fermentation: 40°C±0.5°C for 2 h, 45°C±0.5°C for 1 h, 58°C±0.5°C for 24 h, and terminate the fermentation to obtain a slurry-like polysaccharide Polygonatum sibiricum fermentation product.

[0063] Example Three

[0064] A preparation method of a fermented product of Polygonatum sibiricum

[0065] The difference from Example One is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (CCTCC NO: M20242064), Aspergillus oryzae MJY2-5 (CCTCC NO: M20221400) and Wickerhamiella sechophila JGM9-1 (CCTCC NO: M20221401) in a mass ratio of 3:2:2. Other steps are the same as those in Example One.

[0066] Example Four

[0067] A preparation method of a fermented product of Polygonatum sibiricum

[0068] The difference from Example One is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (CCTCC NO: M20242064), Aspergillus oryzae MJY2-5 (CCTCC NO: M20221400) and Wickerhamiella sechophila JGM9-1 (CCTCC NO: M20221401) in a mass ratio of 0:0:2. Other steps are the same as those in Example One.

[0069] Example Five

[0070] A preparation method of a fermented product of Polygonatum sibiricum

[0071] The difference from Example One is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (CCTCC NO: M20242064), Aspergillus oryzae MJY2-5 (CCTCC NO: M20221400) and Wickerhamiella sechophila JGM9-1 (CCTCC NO: M20221401) in a mass ratio of 3:3:1. Other steps are the same as those in Example One.

[0072] Example Six

[0073] A preparation method of a fermented product of Polygonatum sibiricum

[0074] The difference from Example One is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (CCTCC NO: M20242064), Aspergillus oryzae MJY2-5 (CCTCC NO: M20221400) and Wickerhamiella sechophila JGM9-1 (CCTCC NO: M20221401) in a mass ratio of 4:2:1. Other steps are the same as those in Example One.

[0075] Example Seven

[0076] Preparation method of a fermented product of polygonatum sibiricum

[0077] The difference from example one is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (preserved number CCTCC NO: M20242064): Aspergillus oryzae MJY2-5 (preserved number CCTCC NO: M20221400): Wickerhamiella sechophila JGM9-1 (preserved number CCTCC NO: M20221401) in a mass ratio of 0:2:1. Other steps are the same as example one.

[0078] Example eight

[0079] Preparation method of a fermented product of polygonatum sibiricum

[0080] The difference from example one is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (preserved number CCTCC NO: M20242064): Aspergillus oryzae MJY2-5 (preserved number CCTCC NO: M20221400): Wickerhamiella sechophila JGM9-1 (preserved number CCTCC NO: M20221401) in a mass ratio of 2:2:1. Other steps are the same as example one.

[0081] Example nine

[0082] Preparation method of a fermented product of polygonatum sibiricum

[0083] The difference from example one is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (preserved number CCTCC NO: M20242064): Aspergillus oryzae MJY2-5 (preserved number CCTCC NO: M20221400): Wickerhamiella sechophila JGM9-1 (preserved number CCTCC NO: M20221401) in a mass ratio of 3:1:1. Other steps are the same as example one.

[0084] Example ten

[0085] Preparation method of a fermented product of polygonatum sibiricum

[0086] The difference from example one is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (preserved number CCTCC NO: M20242064): Aspergillus oryzae MJY2-5 (preserved number CCTCC NO: M20221400): Wickerhamiella sechophila JGM9-1 (preserved number CCTCC NO: M20221401) in a mass ratio of 3:0:1. Other steps are the same as example one.

[0087] Example Eleven

[0088] A preparation method of a fermented product of Rhizoma Polygonati

[0089] The difference from Example One is that the Rhizoma Polygonati powder and water are not inoculated with the composite strain after mixing, and the operation of the temperature variation fermentation stage is still operated according to the steps of Example One.

[0090] Example Twelve

[0091] A preparation method of a fermented product of Rhizoma Polygonati

[0092] The difference from Example One is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (CCTCC NO: M20242064), Aspergillus oryzae MJY2-5 (CCTCC NO: M20221400), and Wickerhamiella seylanensis JGM9-1 (CCTCC NO: M20221401) at a mass ratio of 3:3:0. The other steps are the same as those of Example One.

[0093] Example Thirteen

[0094] A preparation method of a fermented product of Rhizoma Polygonati

[0095] The difference from Example One is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (CCTCC NO: M20242064), Aspergillus oryzae MJY2-5 (CCTCC NO: M20221400), and Wickerhamiella seylanensis JGM9-1 (CCTCC NO: M20221401) at a mass ratio of 4:0:0. The other steps are the same as those of Example One.

[0096] Example Fourteen

[0097] A preparation method of a fermented product of Rhizoma Polygonati

[0098] The difference from Example One is that the composite strain is obtained by mixing Rhizopus arrhizus CMR21 (CCTCC NO: M20242064), Aspergillus oryzae MJY2-5 (CCTCC NO: M20221400), and Wickerhamiella seylanensis JGM9-1 (CCTCC NO: M20221401) at a mass ratio of 0:3:0. The other steps are the same as those of Example One.

[0099] The fermented products of Rhizoma Polygonati obtained in Example One, Example Three to Example Fourteen are tested for sensory evaluation, flavor, and polysaccharide content, and the results are shown in Table One.

[0100] Table 1. Results of sensory, flavor and polysaccharide content tests of the fermented product of Rhizoma Polygonati

[0101] The fermented product of Rhizoma Polygonati prepared in Example 11 was named Sample A, and the fermented product of Rhizoma Polygonati prepared in Example 1 was named Sample B. The total acid, soluble solids, pH value, total sugar, polysaccharide, relative molecular weight and polypeptide of Sample A and Sample B were determined according to the above-mentioned methods, respectively. The determination results are shown in Table 2.

[0102] Table 2. Results of total acid, soluble solids, pH value, total sugar, polysaccharide, relative molecular weight and polypeptide of Sample A and Sample B

[0103] Meanwhile, please refer to the relative molecular weight distribution diagram of Sample A shown in FIG. 1, and the relative molecular weight distribution diagram of Sample B shown in FIG. 2. Figure 1 Figure 2

[0104] From the above results, it can be seen that, by using the fermentation of Rhizopus CMR21 with high amylase and glucoamylase production, the starch substances in Rhizoma Polygonati can be hydrolyzed into sugar substances; by using the fermentation of Aspergillus oryzae MJY2-5 with high protein utilization rate, the protein substances in Rhizoma Polygonati can be decomposed into small molecular peptides and amino acid substances; by using the fermentation of Starmerella fibulifera JGM9-1 producing aroma substances, the sugar in Rhizoma Polygonati can be used to produce aroma substances, and the flavor of Rhizoma Polygonati can be improved. By using the combined bacteria temperature-variable fermentation of Rhizopus CMR21, Aspergillus oryzae MJY2-5 and Starmerella fibulifera JGM9-1, the starch in Rhizoma Polygonati raw materials can be hydrolyzed into reducing sugar, the growth of microorganisms can be more effectively promoted, the protein can be hydrolyzed into small molecular peptides and free amino acids, the nutrition of the fermentation liquor can be improved, the polysaccharide wrapped in starch and protein can be released, the large molecular polysaccharide can be cut into small molecular polysaccharide, thereby the utilization rate of Rhizoma Polygonati raw materials can be improved, the polysaccharide and polypeptide contents of the fermented Rhizoma Polygonati beverage can be improved, and the antioxidant capacity and XOD inhibition capacity of the fermentation liquor can be improved.

[0105] The Sample A and Sample B were filtered (through a 200-mesh sieve), and the obtained filtrates were respectively mixed with purified water at a volume ratio of 1:3, then pasteurized at 72°C for 15s, and then canned to obtain the fermented Rhizoma Polygonati beverage A and the fermented Rhizoma Polygonati beverage B.

[0106] The fermented Rhizoma Polygonati beverage A and the fermented Rhizoma Polygonati beverage B were subjected to flavor substance determination, and the results are shown in Table 3.

[0107] Table 3. Results of flavor substance determination of the fermented Rhizoma Polygonati beverage A and the fermented Rhizoma Polygonati beverage B ​​

[0108]

[0109] From the results of Table III, it can be seen that the use of Rhizopus arrhizus CMR21, Aspergillus oryzae MJY2-5, and Phaffia JGM9-1 complex fermentation can reduce the content of aldehydes such as hexanal, pentanal, decanal, and 3-methylbutanal, alcohols such as 1-hexanol, 1-heptanol, and 3-methyl-1-butanol, and acids such as acetic acid, octanoic acid, and n-decanoic acid in the fermented liquid of Polygonatum, and can increase the content of esters such as 1,2-benzenedicarboxylic acid bis(2-methylpropyl) ester, dibutyl phthalate, and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, and benzene ring aromatic substances, thereby improving the aroma of the fermented liquid, and enhancing the flavor characteristics of the fermented product of Polygonatum and the fermented beverage of Polygonatum.

[0110] The sample A and sample B were subjected to DPPH free radical scavenging capacity, ABTS ＋ radical scavenging capacity, and XOD inhibition rate determination, and the determination results are shown in Table IV.

[0111] Table IV Determination results of antioxidant activity and XOD inhibition capacity

[0112] Among them, EC50 (Half Maximal Effective Concentration) refers to the sample concentration required to achieve 50% DPPH free radical scavenging capacity or ABTS + radical scavenging rate. The lower the EC50 value, the stronger the antioxidant capacity of the sample.

[0113] XOD enzyme (xanthine oxidase) is a kind of oxidoreductase widely existing in organisms, mainly involved in purine metabolism, catalyzing the conversion of hypoxanthine into xanthine, and further generating uric acid and active oxygen. Therefore, inhibiting the activity of XOD enzyme is an effective way to inhibit the generation of uric acid. XOD is a metalloflavoprotein with very high molecular weight, and the key site catalyzed by it is the molybdenum center of molybdopterin (Mo-pt). Glu802, Arg880 and Glu1261 are key residues for catalytic reaction. The applicant's previous studies have shown that small molecule peptides with XOD inhibition ability form different forces with Glu802, Arg880 and Glu1261 in XOD through hydrophobic force, hydrogen bond, π-π stacking and salt bridge interaction, enter the active center of XOD, and interact with the amino acid residues distributed around molybdopterin, thereby occupying the catalytic center of XOD, hindering the entry of substrate xanthine, and reducing the catalytic activity of XOD. From the results in Table Four, it can be seen that the DPPH free radical scavenging capacity, ABTS ＋ free radical scavenging capacity were significantly improved, and the XOD inhibition rate was increased by 5.79%.

[0114] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of the present application, this does not limit the patent protection scope of the present application. Any equivalent structure or equivalent process substitution or modification based on the essential concept of the present application, using the content described in the specification and drawings, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, etc., are all included in the patent protection scope of the present application.

Claims

1. A method for preparing a fermented product of polygonatum with increased polysaccharide content, characterized in that: The following steps are involved: The polygonatum powder and water are mixed in a mass ratio of 1:5 to 8, and the composite bacteria are inoculated to perform a single temperature-variable fermentation to obtain the polygonatum fermentation product; The composite bacterial strain is composed of Rhizopus arrhizus, Aspergillus oryzae and Lindnerella fabienne, with the mass ratio of the three being 3:2:1; The inoculation amount of the composite bacteria is 0.5% to 1% of the mass of the polygonatum powder; The single temperature-variable fermentation conditions are: fermentation at 40±1°C for 2 hours, fermentation at 45±1°C for 1 hour, and fermentation at 58±1°C for 24 to 48 hours.

2. The preparation method according to claim 1, characterized in that The Rhizopus arrhizus strain is CMR21, which produces high levels of amylase and saccharifying enzymes and is deposited in the China Center for Type Culture Collection in Wuhan with a deposit number of CCTCC NO: M20242064 and a deposit date of September 25, 2024. The Aspergillus oryzae strain is MJY2-5 with high protease activity, which is deposited in the China Center for Type Culture Collection in Wuhan with a deposit number of CCTCC NO: M 20221400 and a deposit date of September 9, 2022; The Fabianseber Lindner yeast strain is the aroma-producing type JGM9-1, which is deposited in the China Center for Type Culture Collection in Wuhan, with a deposit number of CCTCC NO: M 20221401 and a deposit date of September 9, 2022.

3. The preparation method according to claim 1, characterized in that The polygonatum powder is prepared by a preparation method comprising the following steps: steaming, drying and low-temperature pulverizing polygonatum tubers to obtain the polygonatum powder.

4. The preparation method according to claim 3, characterized in that The steaming and drying treatment comprises: airing the Polygonatum cyrtonema tuber until the moisture content is ≤30%, washing and removing the epidermis and roots, steaming for 100-140 minutes, drying in the sun and then drying at 50-60° C.; The low-temperature crushing comprises: freezing the dried polygonatum at -40°C, and then crushing it to less than 60 meshes in an environment of -30°C to -40°C.

5. A fermented product of Polygonatum sibiricum prepared by the preparation method according to any one of claims 1 to 4.

6. The fermented product of Polygonatum sibiricum according to claim 5, characterized in that The polysaccharide content is ≥0.74g / 100mL, and the polypeptide content is ≥3.12g / 100mL.

7. A fermented beverage of Polygonatum sibiricum, characterized in that: Prepared by a preparation method comprising the following steps: The polygonatum fermented product prepared by the preparation method according to any one of claims 1 to 4 is filtered, blended and sterilized to obtain the polygonatum fermented beverage.

8. The fermented polygonatum beverage according to claim 7, characterized in that The polysaccharide content is ≥0.45g / 100mL, and the polypeptide content is ≥1.8g / 100mL.

9. The fermented polygonatum beverage according to claim 8, characterized in that The polysaccharide has a molecular weight of 4000-4500 Da and an ethanol content of ≤0.1%.

10. Use of a fermented product of Polygonatum sibiricum prepared by the preparation method according to any one of claims 1 to 4 in the preparation of food or health products having uric acid-lowering efficacy.

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