Method for increasing content of total saponins in polygonatum kingianum by utilizing solid-state fermentation of aspergillus niger

Abstract

The invention discloses a method for increasing the content of total saponins in polygonatum kingianum through solid-state fermentation of aspergillus niger. According to the method, the polygonatum kingianum is subjected to solid-state fermentation by utilizing the aspergillus niger, the content of total saponins in the polygonatum kingianum after solid-state fermentation is 2-3 times that of the total saponins in unfermented polygonatum kingianum, and high-quality raw materials and technical supports are provided for increasing the additional value of polygonatum kingianum resources and developing functional products. According to the method disclosed by the invention, the polygonatum kingianum original product is fermented, so that the content of active ingredients of polygonatum kingianum is comprehensively increased, redundant wastes are not generated, the waste of resources is not caused, and an idea is broadened for developing high-added-value healthy foods and other medicinal products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to a method for improving the content of total saponins in Polygonatum sibiricum by using Aspergillus niger solid-state fermentation BACKGROUND

[0002] Polygonatum sibiricum, a perennial herb of the family Asparagaceae, has rich nutritional and medicinal values. Three commonly used medicinal Polygonatum sibiricum recorded in Chinese Pharmacopoeia are P. sibiricum, P. sibiricum and P. sibiricum. A large number of scientific studies have proved that P. sibiricum contains various active substances, including but not limited to steroidal saponins, polysaccharides, oligosaccharides, triterpene saponins, flavonoids and amino acids, which have the effects of lowering blood sugar, lowering blood lipids, antioxidant, anti-inflammatory, anti-tumor, anti-atherosclerosis, etc. At present, more researches are focused on the extraction and development of P. sibiricum polysaccharides, while P. sibiricum saponins, as the second largest component in P. sibiricum, also have various pharmacological effects such as antibacterial, hypoglycemic, anti-tumor, etc.

[0003] Aspergillus niger is a common and widely used fungus in industrial fermentation production. The colony is black-brown, with a spherical apical cyst at the top, which is covered with a layer of stipe base and a layer of small stems. The small stems are covered with a string of brown-black spheres. It is widely distributed in air, grain, soil, etc. and is an important fermentation strain in industry. Aspergillus niger is also a widely used edible fungus in the world, which has been used for the biotransformation of various compounds, such as isoflavones, saponins and steroids.

[0004] P. sibiricum is a widely used traditional Chinese medicine, but the clinical or edible raw P. sibiricum may cause numbness of the mouth and throat irritation, and contact with fresh raw P. sibiricum may cause skin itching, etc. Therefore, P. sibiricum is often processed by a certain method to reduce or eliminate these irritating feelings. The current processing method of P. sibiricum on the market is usually "nine steaming and nine drying", which has a complex processing procedure and is time-consuming and laborious. Patent CN202310742863.5 discloses a processing method of wine P. sibiricum, which is processed through multiple steps such as washing, drying, first steaming, slicing, second steaming, drying, etc. to obtain P. sibiricum processed products that retain more P. sibiricum polysaccharide active ingredients and improve the content of extract. This method reduces the irritating components of raw P. sibiricum to a certain extent, but the operation procedure is complex, time-consuming and laborious, and the content of active ingredients in P. sibiricum is not improved, which has certain defects. Fermentation is a common method for processing traditional Chinese medicine to reduce the toxicity of traditional Chinese medicine, enhance its characteristics or produce new effects. Compared with traditional methods, it has the advantages of mild conditions, high conversion rate and simple post-processing. P. sibiricum fermentation is mainly liquid fermentation, and there are few studies on solid fermentation. Compared with liquid fermentation, solid-state fermentation is simpler in production process, and there is no waste water and waste gas produced in the whole production process, which is a low-investment, low-energy-consumption and high-yield fermentation method. SUMMARY

[0005] Therefore, the application provides a method for improving the content of total saponins in Yunnan polygonatum by using Aspergillus niger solid-state fermentation.

[0006] To achieve the above technical purposes, the technical solutions implemented by the application are as follows:

[0007] A method for improving the content of total saponins in Yunnan polygonatum by using Aspergillus niger solid-state fermentation, comprising the following steps:

[0008] A. Fresh Yunnan polygonatum is washed, sliced, dried to constant weight, crushed and sieved to obtain Yunnan polygonatum powder for standby;

[0009] B. A potato glucose agar culture medium plate is prepared, Aspergillus niger seed liquid stored in a glycerol tube is inoculated on the plate for activation, and after activation, an inoculation ring is used to inoculate in a potato glucose liquid culture medium for expansion culture, and then Aspergillus niger spore suspension is obtained for standby;

[0010] C. A Yunnan polygonatum powder fermentation substrate is prepared and high-pressure steam sterilized, Aspergillus niger spore suspension is inoculated, and solid-state fermentation is carried out in a constant-temperature incubator to obtain a fermentation product;

[0011] D. After solid-state fermentation is completed, the content of total saponins in the fermentation product is measured;

[0012] E. Design Expert 13.0 software is applied, single-factor test and response surface design analysis test are carried out, a mathematical regression model is established, and the process of Aspergillus niger solid-state fermentation of Yunnan polygonatum is optimized.

[0013] Further, the temperature in the vacuum drying box in step A is 60 DEG C, and the sieving mesh size is 80 mesh.

[0014] Further, the Aspergillus niger strain used in step B has a preservation number of CGMCC 3.5487 (China General Microbiological Culture Collection Center).

[0015] Further, the culture media used in step B all need to be sterilized in a high-pressure steam sterilization pot at 121 DEG C for 20 minutes, and then the experimental instruments all need to be ultraviolet sterilized in a biological safety cabinet for 15-30 minutes before use, and the experimental operations all need to be carried out in sterile conditions.

[0016] Further, the activation temperature of Aspergillus niger in step B is 30 DEG C, and the incubation in the constant-temperature incubator is for 5 days; the temperature of Aspergillus niger expansion culture is 30 DEG C, and the incubation in the constant-temperature shaker with a rotation speed of 200 r / min is for 3 days.

[0017] Further, the fermentation substrate in step C is Yunnan rhizoma polygonati powder with a water content of 35-45%, the inoculation amount of Aspergillus niger is 8-16%, and the fermentation time is 6-7 days, and the pH of the fermentation substrate is 4.

[0018] Further, the fermentation temperature during the solid-state fermentation in step C is 32°C, and the environmental humidity is 80%-100%.

[0019] Further, the single factors in step E include the water content of the fermentation substrate, the fermentation time, and the pH of the fermentation substrate.

[0020] The beneficial effects of the present application are as follows:

[0021] 1) The present application uses Aspergillus niger to solid-state ferment Yunnan rhizoma polygonati, which can effectively improve the total saponin content in Yunnan rhizoma polygonati. Through the metabolic action of the strain, the total saponin content of the fermented Yunnan rhizoma polygonati is improved, providing high-quality raw materials for the development of high-value health foods and medicinal products from Yunnan rhizoma polygonati.

[0022] 2) The complex enzyme system (such as cellulase, pectinase, and β-glucosidase) produced by Aspergillus niger can efficiently degrade structural polysaccharides (such as cellulose and hemicellulose) in the cell wall of Yunnan rhizoma polygonati, break down the packaging barrier of saponin components, promote the release and transformation of saponins, and simultaneously improve the texture and palatability of Yunnan rhizoma polygonati, solving the problem of complex process flow in traditional Yunnan rhizoma polygonati processing technology, and increasing the content of bioactive components in Yunnan rhizoma polygonati.

[0023] 3) Through the solid-state fermentation process, the total saponin content in Yunnan rhizoma polygonati is increased by 2-3 times compared to the total saponin content in unfermented Yunnan rhizoma polygonati, providing an excellent fermentation process for the subsequent development of Yunnan rhizoma polygonati products.

[0024] 4) The fermentation process uses the whole tissue of Yunnan rhizoma polygonati as the substrate without peeling or other treatments, and the raw material utilization rate reaches 100%. The product after fermentation can be applied to food or drug development without overly complex separation, which conforms to the concept of green and low-carbon production.

[0025] 5) The present application optimizes the fermentation parameters (water content of Yunnan rhizoma polygonati, inoculation amount, fermentation time, and solution pH) based on the response surface method, establishes a stable process model, and provides precise control basis for large-scale production.

[0026] 6) The fermentation process of the present application is simple to operate, and only requires simple mixing of raw materials and strains without complex process flow or expensive equipment. The entire fermentation process only needs to maintain a suitable environmental temperature without additional operations or manual intervention, greatly reducing the production cost and having high practicality and broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1The graph shows the effect of water content in Polygonatum sibiricum on the total saponin content of Polygonatum yunnanense.

[0028] Figure 2 Figure showing the effect of Aspergillus niger inoculum amount on the total saponin content of Polygonatum yunnanense;

[0029] Figure 3 Figure showing the effect of fermentation time on the total saponin content of Polygonatum yunnanense;

[0030] Figure 4 The graph shows the effect of solution pH on the total saponin content of Polygonatum odoratum.

[0031] Figure 5 The surface plot and contour plot show the response of the moisture content (A) and fermentation time (B) of Polygonatum odoratum to the total saponin content of Polygonatum odoratum.

[0032] Figure 6 The surface plot and contour plot show the response of water content (A) and solution pH (C) of Polygonatum odoratum to the total saponin content of Polygonatum odoratum.

[0033] Figure 7 The surface plot and contour plot show the response of fermentation time (B) and solution pH (C) to the total saponin content of Polygonatum yunnanense;

[0034] Figure 8 This is a flowchart of the experimental technique. Detailed Implementation

[0035] To further illustrate the present invention, the following describes in detail a method for increasing the total saponin content in Polygonatum yunnanensis using solid-state fermentation with Aspergillus niger, in conjunction with embodiments and accompanying drawings.

[0036] Example 1

[0037] like Figure 8 As shown, a method for increasing the total saponin content in Polygonatum yunnanense using solid-state fermentation with Aspergillus niger includes the following specific steps:

[0038] (I) Raw material pretreatment

[0039] 1. Cleaning: Wash the fresh Polygonatum yunnanense purchased from Qianxinan Prefecture, Guizhou Province, with clean water several times to remove surface mud and impurities, and finally wash it three more times with deionized water.

[0040] 2. Drying: Place the washed fresh Polygonatum yunnanense slices in a vacuum drying oven at 60℃ and dry until constant weight;

[0041] 3. Crushing: Use a pulverizer to crush the dried Polygonatum odoratum slices and pass them through an 80-mesh sieve to obtain Polygonatum odoratum powder, which is then set aside for later use.

[0042] (II) Preparation of Seed Liquid

[0043] The strain activation and expansion culture operation were carried out under sterile conditions, and all consumables required in the biological safety cabinet were sterilized in the high-pressure sterilization pot and irradiated under the ultraviolet lamp for 30 minutes before use.

[0044] 1. Preparation of PDA medium: precisely weigh 2.6 g of potato glucose powder on an electronic balance, add 100 mL of pure water to a clean conical flask, and then add 1.7 g of agar to obtain PDA solid medium, or do not add agar to obtain PDA liquid medium;

[0045] 2. Activation of Aspergillus niger: place the prepared PDA solid medium and the washed and packaged flat plate together in the high-pressure steam sterilization pot and sterilize at 121°C for 20 minutes. After high-pressure sterilization, place the inoculation ring and sealing film together in the biological safety cabinet and ultraviolet sterilize for 20 minutes. Immediately pour the PDA solid medium into the flat plate when it has not completely solidified (an intact smooth layer is formed on the flat plate). Use the inoculation ring to streak the glycerol frozen seed liquid of the Aspergillus niger strain on the flat plate. After inoculation, wrap and seal the flat plate with sealing film, and place it in a 30°C incubator for culture. The culture time is 5 days, and the incubator is kept sterile to avoid affecting the culture of Aspergillus niger.

[0046] 3. Preparation of Aspergillus niger spore suspension: after 5 days of activation, use a clean, ultraviolet-sterilized inoculation ring to scrape the well-grown and pure Aspergillus niger spores and transfer them to the PDA liquid medium (sterilized after high-pressure sterilization; all operations are sterile in the biological safety cabinet). Place the inoculated conical flask in a constant-temperature shaker for culture at a temperature of 30°C and a speed of 200 r / min for 3 days, and then store it for later use.

[0047] (III) Preparation of related reagents

[0048] 1. 1% hydrochloric acid solution: precisely weigh 1 g of HCl powder and add 99 g of pure water to a beaker to prepare a 1% hydrochloric acid solution for later use.

[0049] 2. 1% sodium hydroxide solution: precisely weigh 1 g of NaOH powder and add 99 g of pure water to a beaker to prepare a 1% sodium hydroxide solution for later use.

[0050] 3. Preparation of solutions with different pH values: based on pure water (pH 6.5), add 1% hydrochloric acid solution to adjust the solution to acidic to obtain solutions with pH values of 3, 4, and 5; add 1% sodium hydroxide solution to adjust the solution to alkaline to obtain solutions with pH values of 8 and 9.

[0051] 4. Preparation of water-saturated n-butanol solution: Mix n-butanol and distilled water according to the volume ratio of 1:1, shake well, and then mix evenly. Ultrasonic assisted treatment for 10 minutes can accelerate the layering. Or after standing overnight, the upper layer is the water-saturated n-butanol solution, which is ready for use.

[0052] 5. 5% vanillin-glacial acetic acid solution: Accurately weigh 0.5 g of vanillin solid powder into a 10 mL glacial acetic acid solution beaker to obtain a 5% vanillin-glacial acetic acid solution for use.

[0053] (IV) The first single factor: the water content of Huangjing is investigated by Aspergillus niger solid-state fermentation

[0054] First, accurately weigh the sieved Yunnan Huangjing powder and mix it with distilled water at pH 6.5 according to the preset ratio to form the fermentation medium. Using single factor experimental design, set the water content of Huangjing as the key variable, and divide it into five gradients: 30%, 35%, 40%, 45%, and 50%, with corresponding ratios of 14 g powder + 6 mL water, 13 g powder + 7 mL water, 12 g powder + 8 mL water, 11 g powder + 9 mL water, and 10 g powder + 10 mL water. After sterilization in a 121℃ high-pressure steam sterilization pot for 20 minutes, 10% of the Aspergillus niger spore suspension is inoculated for solid-state fermentation. The fermentation process is strictly controlled at a temperature of 32℃ and an environmental humidity of 80% under constant temperature and humidity conditions, and the fermentation time is 6 days.

[0055] (V) Determination of total saponin content in Aspergillus niger fermented Yunnan Huangjing fermentation product

[0056] 1. Preparation of diosgenin standard solution: Accurately weigh 2.50 mg of diosgenin standard product dried to constant weight at 60℃ into a 10 mL volumetric flask. Dilute to the mark with methanol and shake well to obtain a control solution containing 0.25 mg of diosgenin per 1 mL. Store at 4℃ for future use.

[0057] 2. Preparation of standard curve

[0058] a. Accurately take 0.02 mL, 0.05 mL, 0.10 mL, 0.20 mL, 0.30 mL, 0.40 mL, 0.50 mL, and 0.60 mL of the control solution and place them in 10 mL stoppered test tubes, respectively. Dry the methanol in a 60℃ water bath;

[0059] b. Add 1.0 mL of freshly prepared 5% (w / v) vanillin-glacial acetic acid solution: perchloric acid (2:8) mixture to each, shake well, and place in a 60℃ water bath for 25 minutes,

[0060] c. After the water bath, quickly remove and cool in cold water, then add 5 mL of glacial acetic acid and shake well.

[0061] d. With reagent blank as control, the absorbance value A was determined at 560 nm. The standard curve was drawn with A value as ordinate and the content of diosgenin in the test tube with plug as abscissa. The standard curve obtained was Y = 17.627X + 0.0018 (R 2 = 0.9996)

[0062] 3. Extraction of total saponins of Yunnan Polygonatum

[0063] a. Sample pretreatment: the solid-state fermentation product of Yunnan Polygonatum was placed in a vacuum drying oven at 60°C and dried to constant weight, and then 1.0 g of the sample was accurately weighed and reserved;

[0064] b. Ultrasonic-assisted extraction: 15 mL of 80% ethanol solution was added to the sample, and ultrasonic treatment was performed at 50°C for 40 min;

[0065] c. Solid-liquid separation: the mixture was filtered through filter paper, and the filter residue and filter paper were washed with 80% ethanol solution for multiple times;

[0066] d. Filtrate concentration: the filtrate and washing liquid were combined and evaporated to dryness to obtain a dry residue;

[0067] e. Alkali dissolution: 20 mL of 1% NaOH solution was added to the residue, and the mixture was stirred thoroughly until complete dissolution;

[0068] f. Solvent extraction: the above solution was extracted with water-saturated n-butanol for 3 times (30 mL, 10 mL, 10 mL), and the n-butanol extraction phases were combined;

[0069] g. Solvent removal: the n-butanol extraction liquid was evaporated to dryness, and the residue was collected;

[0070] h. Volumetric treatment: the residue was dissolved with methanol and made up to 5 mL, and after thorough mixing, the test sample solution was labeled.

[0071] 4. Determination of the content of total saponins of Yunnan Polygonatum

[0072] 0.1 mL of the test sample solution was accurately taken in a test tube with plug, methanol was evaporated in a water bath at 60°C, and coloration was performed according to the above method. With reagent blank as control, the A value was determined at 560 nm, and the content was calculated according to the standard curve, and the average value of three times was taken as the measurement result. The total saponin content in the Yunnan Polygonatum powder without fermentation was 0.080 mg / g.

[0073] First single factor: experimental results of water content of Polygonatum

[0074] The effect of this single factor on the content of total saponins of Yunnan Polygonatum is shown in Table 1. Figure 1It is verified by experiments that the more the amount of distilled water added in the solid culture medium of Huangjing, the more the total saponin content in the fermentation product increases first and then decreases, and when the ratio of Huangjing powder to distilled water is 12g:8mL (i.e. the water content of Huangjing is 40%) in the solid state fermentation of Yunnan Huangjing by Aspergillus niger, the total saponin content in the fermentation product reaches the maximum value. It is presumed that in the early stage of fermentation, appropriate amount of water is conducive to the growth and metabolic activity of microorganisms, and sufficient water can make microorganisms more easily absorb nutrients and promote enzyme activity, thereby increasing the yield of total saponin; however, when the water content is too large, the culture medium may become too wet and viscous or even liquefied, and Aspergillus niger is an aerobic microorganism, which makes the oxygen supply in the culture medium insufficient, thereby affecting the normal growth and metabolism of aerobic microorganisms and reducing the content of total saponin.

[0075] Example 2

[0076] A method for improving the content of total saponin in Yunnan Huangjing by solid state fermentation of Aspergillus niger, the second single factor: the inoculation amount of Aspergillus niger seed liquid, the operation steps are as follows:

[0077] The difference from Example 1 is that the inoculation amount of Aspergillus niger seed liquid is set as the key variable, which is divided into 5 gradients, 8% (v / v), 10% (v / v), 12% (v / v), 14% (v / v), and 16% (v / v), and the ratio of Huangjing powder to distilled water in the fermentation substrate is 12g:8mL (i.e. the water content of Huangjing is 40%), and the fermentation process is still carried out under the constant temperature and humidity conditions of temperature 32℃ and environmental humidity 80%-100%, and the fermentation time is 6 days. The remaining steps remain the same as those in Example 1.

[0078] The experimental results of the second single factor: the inoculation amount of Aspergillus niger seed liquid

[0079] The influence of this single factor on the total saponin content of Yunnan Huangjing is shown in Figure 2 It is verified by experiments that with the increase of the inoculation amount of Aspergillus niger, the total saponin content in the fermentation product generally shows a relatively regular trend, and when the inoculation amount of Aspergillus niger seed liquid is 14%, the total saponin content in the fermentation product reaches the maximum value. It is presumed that before the inoculation amount is 14%, the inoculation amount is relatively high but not excessive, and Aspergillus niger can fully utilize the nutrients in the culture medium to promote saponin synthesis, resulting in the increase of total saponin content; when the inoculation amount is too large, with the passage of time and the increase of the number of bacteria, the nutrients in the culture medium are gradually depleted, which inhibits the growth of the bacteria and is not conducive to the metabolic activity of the bacteria, thereby reducing the total saponin content.

[0080] Example 3

[0081] A method for improving the content of total saponin in Yunnan Huangjing by solid state fermentation of Aspergillus niger, the third single factor: fermentation time, the operation steps are as follows:

[0082] The difference from Example 1 is that the fermentation time is set as the key variable, which is divided into 5 gradients, 4 days, 5 days, 6 days, 7 days and 8 days. The ratio of polygonatum kingianum powder to distilled water in the fermentation substrate is 12g:8mL (i.e. the water content of polygonatum kingianum is 40%), the inoculation amount of aspergillus niger seed liquid is 14%, and the fermentation process is still carried out under the constant temperature and humidity conditions of temperature 32℃ and environmental humidity 80%. The remaining steps remain the same as Example 1.

[0083] Third single factor: fermentation time experimental results

[0084] The effect of this single factor on the total saponin content of polygonatum kingianum is shown in Figure 3 Through experimental verification, with the increase of fermentation time, the total saponin content in the fermentation product first increases and then decreases, and reaches the peak on the 6th day of fermentation. The possible reason is that during the fermentation period of 4-6 days, the nutrients in the culture medium are sufficient, the growth and metabolic activity of aspergillus niger is high, and the enzymes secreted can effectively decompose the components in polygonatum kingianum, so the total saponin content gradually increases until it reaches the highest point; during the fermentation period of 7-8 days, the nutrients in the culture medium are gradually consumed, and the growth and metabolic activity of aspergillus niger is limited, resulting in a decrease in saponin conversion rate.

[0085] Example 4

[0086] A method for improving the total saponin content in polygonatum kingianum by using aspergillus niger solid-state fermentation, the fourth single factor: solution pH, the operation steps are as follows:

[0087] The difference from Example 1 is that the solution pH is set as the key variable, which is divided into 5 gradients, 3, 4, 5, 6.5 and 8. The ratio of polygonatum kingianum powder to distilled water of different pH in the fermentation substrate is 12g:8mL (i.e. the water content of polygonatum kingianum is 40%), the inoculation amount of aspergillus niger seed liquid is 14%, and the fermentation time is 6 days. The fermentation process is still carried out under the constant temperature and humidity conditions of temperature 32℃ and environmental humidity 80%. The remaining steps remain the same as Example 1.

[0088] Fourth single factor: solution pH experimental results

[0089] The effect of this single factor on the total saponin content of polygonatum kingianum is shown in Figure 4 Through experimental verification, when aspergillus niger is used for solid-state fermentation of polygonatum kingianum, the total saponin content in the fermentation product reaches the maximum value when the pH of the added solution is 4.

[0090] Optimization of fermentation process by response surface method

[0091] On the basis of the results of the previous single-factor experiment, it was found that the total saponin content in the fermentation product of Polygonatum sibiricum increased first and then decreased with the increase of the moisture content of Polygonatum sibiricum, fermentation time and solution pH, and the effect of the three single factors on the total saponin content in the fermentation product of Aspergillus niger was more obvious. When the inoculum size of Aspergillus niger was studied, it was found that the total saponin content in the fermentation product did not change significantly by changing the gradient. Therefore, when the process of Aspergillus niger fermentation of Polygonatum sibiricum was optimized by response surface method, the total saponin content in the fermentation product was taken as the response value, the moisture content of Polygonatum sibiricum (A), fermentation time (B) and solution pH (C) were taken as independent variables, three levels (0, +1, -1) were selected respectively, BBD (Box-Behnken Design) response surface method was used to design and analyze the experiment of three factors and three levels by using Design Expert 13.0 software, the experiment was carried out according to the experimental conditions given by the software, and the experimental results were recorded. The experimental design factors and levels are shown in Table 1, and the experimental design conditions and results are shown in Table 2.

[0092] Table 1 Experimental design factors and levels

[0093] Factor -1 0 1 Water content of Huangjiin (%) 35 40 45 Fermentation time (day) 5 6 7 Solution pH 3 4 5

[0094] Table 2 Experimental design conditions and results

[0095]

[0096] Response surface experiment results

[0097] In this study, the response surface BBD experimental design and the response value of total saponin content in Table 2 were analyzed by regression fitting using Design-Expert 13.0 software, a second-order response surface regression model was used, and the regression equation of total saponin content was obtained as follows: Y (total saponin content) = 0.2674-0.005875×A+0.006875×B-0.006×C+0.0105×AB+0.00225×

[0098] AC+0.01525×BC-0.03845×A^2-0.01845×B^2-0.0387×C^2

[0099] The variance analysis of the fitted model of the response value of total saponin content was carried out, and the significance of each factor and the results of variance analysis of the multiple regression model are shown in Table 3. The variance analysis table of the response surface model is shown in Table 4. Figure 5-7 The response surface plots and contour plots of the total saponin content of Polygonatum sibiricum from three groups of the moisture content of Polygonatum sibiricum (A) and fermentation time (B), the moisture content of Polygonatum sibiricum (A) and solution pH (C), and fermentation time (B) and solution pH (C) are shown in Figures 1-3 respectively.

[0100] Analysis Figure 5From the three-dimensional response surface graph in Fig. 4, it can be seen that the moisture content of Huangjing and the fermentation time have significant interaction on the total saponin content. When the moisture content of Huangjing is 39%~40% and the fermentation time is about 6 days, the total saponin content reaches the maximum value (about 0.26 mg / g), indicating that at this time, Huangjing provides Aspergillus niger with a suitable moisture and nutrient environment, which is conducive to the accumulation and transformation of saponins. This trend is also shown in the contour plot. The contour plot is in the shape of an approximate ellipse, indicating that the process parameters are more sensitive to the change of saponin content in this region. Too high or too low moisture content and too long or too short fermentation time will all lead to a decrease in saponin production efficiency.

[0101] Analysis Figure 6 From the three-dimensional response surface graph in Fig. 4, it can be seen that the moisture content of Huangjing and the fermentation time have significant interaction on the total saponin content. When the moisture content of Huangjing is 39%~40% and the fermentation time is about 6 days, the total saponin content reaches the maximum value (about 0.26 mg / g), indicating that at this time, Huangjing provides Aspergillus niger with a suitable moisture and nutrient environment, which is conducive to the accumulation and transformation of saponins. This trend is also shown in the contour plot. The contour plot is in the shape of an approximate ellipse, indicating that the process parameters are more sensitive to the change of saponin content in this region. Too high or too low moisture content and too long or too short fermentation time will all lead to a decrease in saponin production efficiency.

[0102] Analysis Figure 7 From the three-dimensional response surface graph in Fig. 4, it can be seen that the moisture content of Huangjing and the fermentation time have significant interaction on the total saponin content. When the moisture content of Huangjing is 39%~40% and the fermentation time is about 6 days, the total saponin content reaches the maximum value (about 0.26 mg / g), indicating that at this time, Huangjing provides Aspergillus niger with a suitable moisture and nutrient environment, which is conducive to the accumulation and transformation of saponins. This trend is also shown in the contour plot. The contour plot is in the shape of an approximate ellipse, indicating that the process parameters are more sensitive to the change of saponin content in this region. Too high or too low moisture content and too long or too short fermentation time will all lead to a decrease in saponin production efficiency.

[0103] Table 3 Analysis of variance of regression model

[0104]

[0105]

[0106] Note: P≤0.01 indicates that the effect of the factor on the response value is extremely significant (**); P≤0.05 indicates that the effect of the factor on the response value is significant (*).

[0107] From Table 3, it can be seen that the P of the model is less than 0.0001, indicating that the fitting equation has good regression effect and strong significance. The interaction terms AB, BC and the quadratic terms A 2 , B 2 , C 2The influence degree of the results total saponins content reached extremely significant level (P<0.01), the first term A, B and C on the results influence degree was significant level (P<0.05). According to the F test results showed that each factor on the response value total saponins content influence order was B>C>A, namely fermentation time> solution pH> Huangjing water content. And the P=0.1144>0.05 of the misfit term, the model difference was not significant, indicating that the non-test factors had little effect on the total saponins content, the model had good test stability, indicating that the equation was reliable.

[0108] Table 4 response surface model analysis of variance table

[0109] Response value index Mean Standard deviation [R 2 ]]> [R adj 2 ]]> [R pre 2 ]] C.V. % Adeq Precision Total saponin content 2.65 0.0059 0.9864 0.9690 0.8338 2.65 19.1654

[0110] From table 4, the regression coefficient R 2 =0.9864, indicating that the test model was well fitted with the actual test, therefore the regression equation could be used instead of the actual test factor value to analyze the corresponding relationship between each dependent variable and response value, the correction coefficient R adj2 =0.9690, the prediction R pre2 =0.8338, the coefficient of variation C.V.=2.65%<10%, indicating that the model regression equation had high reliability, and could well reflect the true value. The signal-to-noise ratio Adeq Precision=19.1654>4, generally considered that when the signal-to-noise ratio was greater than 4.0, the model was well fitted with the test value.

[0111] In summary, the fitting degree of the response surface quadratic regression equation was good, and the model could be used for the condition prediction and analysis of Aspergillus niger solid-state fermentation of Yunnan Rhizoma Polygonati.

[0112] Through the analysis of the response surface model, in order to make the response value total saponins content result reach the maximum, the effective regression model was used for condition optimization, and the parameter module of the response surface software was used for prediction. The parameter values of each factor after optimization were as follows: Huangjing water content (A): 39.710%, fermentation time (B): 6.149d, solution pH (C): 3.950, and the model predicted that the total saponins content was 0.268mg / g. Considering the test conditions, operability and rationality, the factors were set as follows: Huangjing water content (A): 39.7%, fermentation time (B): 6d, solution pH (C): 4, and three repeated tests were carried out to obtain the verification results of total saponins content. The average value of total saponins content was 0.264mg / g, the relative error between the measured value and the predicted value was 1.5%, the relative error between the predicted value and the measured value of total saponins content was less than 5%, and the specific test results were shown in table 5. The response predicted value was basically consistent with the actual test value, which verified the accuracy and reliability of the model.

[0113] Table 5 verification test results table

[0114]

[0115] The application has been described in detail by the embodiments, and other embodiments can be obtained without creativity according to the embodiments, and these embodiments all belong to the protection scope of the application.

Claims

1. A method for improving the content of total saponins in Panax englerianus by using Aspergillus niger solid-state fermentation, characterized in that, It comprises the following steps: A. Fresh Yunnan rhizoma polygonati is washed, sliced, dried to constant weight, ground and sieved to obtain Yunnan rhizoma polygonati powder for use; B. A potato glucose agar culture medium plate is prepared, and an Aspergillus niger seed liquid stored in a glycerol tube is inoculated on the plate for activation. After activation, the activated culture is inoculated in a potato glucose liquid culture medium with a loop for expansion culture, and then an Aspergillus niger spore suspension is obtained for use; C. A Yunnan rhizoma polygonati powder fermentation substrate is prepared and autoclaved, inoculated with the Aspergillus niger spore suspension, and placed in a constant temperature incubator for solid-state fermentation to obtain a fermentation product; D. After solid-state fermentation, the total saponin content in the fermentation product is measured; E. Design Expert 13.0 software is used to analyze the test through single factor test and response surface design, a mathematical regression model is established, and the process of Aspergillus niger solid-state fermentation of Yunnan rhizoma polygonati is optimized.

2. The method of claim 1, wherein, The temperature in the vacuum drying oven in step A is 60℃, and the sieving mesh size is 80 mesh.

3. The method of claim 1, wherein, The Aspergillus niger strain used in step B has a preservation number of CGMCC 3.5487 (China General Microbiological Culture Collection Center).

4. The method of claim 1, wherein, The culture media used in step B need to be sterilized in a high-pressure steam sterilization pot at 121℃ for 20 minutes. Then, the experimental instruments need to be sterilized with ultraviolet light in a biological safety cabinet for 15-30 minutes before use, and the experimental operation needs to be carried out under sterile conditions.

5. The method of claim 1, wherein, The activation temperature of Aspergillus niger in step B is 30℃, and the culture is incubated in a constant temperature incubator for 5 days. The temperature for expansion culture of Aspergillus niger is 30℃, and the culture is incubated in a constant temperature shaker at a speed of 200 r / min for 3 days.

6. The method of claim 1, wherein, The fermentation substrate in step C is Yunnan rhizoma polygonati powder with a water content of 35-45%, the inoculation amount of Aspergillus niger is 8-16%, the fermentation time is 6-7 days, and the pH of the fermentation substrate is 4.

7. The method of claim 1, wherein, The fermentation temperature during solid-state fermentation in step C is 32℃, and the environmental humidity is 80%-100%.

8. The method of claim 1, wherein, The single factors in step E include the water content of the fermentation substrate, the fermentation time, and the pH of the fermentation substrate.

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