Method and system for optimizing solid-state fermentation process of diaphragma juglandis

Abstract

The invention belongs to but not limited to the technical field of solid-state fermentation of diaphragma juglandis, and discloses a solid-state fermentation process optimization method and system for diaphragma juglandis. Activating an aspergillus niger strain and preparing a bacterial suspension; optimizing solid state fermentation process conditions by utilizing a single factor and response surface test; aspergillus niger solid state fermentation of diaphragma juglandis. According to the method, the diaphragma juglandis is taken as a raw material, solid-state fermentation process conditions are optimized by utilizing a single factor and response surface test, and the optimal fermentation conditions for solid-state fermentation of the diaphragma juglandis by the aspergillus niger are determined as follows: the fermentation time is 4 days, the fermentation temperature is 28 DEG C, the moisture content is 65%, the inoculum size of a bacterial solution is 1 * 10 < 7 > CFU / g, and the polyphenol content is 95.32 mg GAE / g DW at the moment. The method is a feasible technology for increasing the polyphenol content in the diaphragma juglandis through solid state fermentation of the diaphragma juglandis by using the aspergillus niger.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the technical field of solid-state fermentation of walnut pith wood, and in particular relates to a method and system for optimizing the solid-state fermentation process of walnut pith wood. Background Art

[0002] Walnut heartwood is a dry woody membrane located between walnut kernels. During walnut processing, heartwood is usually used as fuel or discarded as garbage, which not only causes a waste of resources but also causes environmental pollution. Therefore, the development and utilization of walnut heartwood is of great significance. Heartwood is rich in various bioactive ingredients such as phenols, flavonoids, saponins, polysaccharides and alkaloids. In the field of traditional Chinese medicine, it is mainly used to treat various diseases such as kidney deficiency, diarrhea, and reproductive diseases. At present, many researchers have conducted qualitative and quantitative studies on the bioactivity of polyphenol compounds in heartwood. Liu et al. successfully identified 200 phenolic compounds in heartwood using UHPLC-Q-Orbitrap HRMS and UHPLC-MS / MS techniques. Wang and Liu et al. found that heartwood polyphenols have antioxidant, anti-inflammatory and anti-diabetic functional activities. However, bioactive ingredients such as polyphenols in walnut heartwood can covalently bind to macromolecules such as cellulose to form insoluble bonded polyphenols, which are difficult to extract and release, reducing the bioavailability of polyphenols and their preventive effects on diseases.

[0003] Fermentation is divided into two types: solid-state fermentation and liquid-state fermentation. Solid-state fermentation refers to the fermentation process in which one or more microorganisms grow on a solid matrix in the absence of free water. Compared with liquid-state fermentation, solid-state fermentation can not only better simulate the natural habitat of microorganisms, but also has the advantages of low production cost, simple operation and environmental friendliness.

[0004] The factors affecting solid-state fermentation include microbial species, fermentation temperature, fermentation time and fermentation substrate. In the process of solid-state fermentation, commonly used microorganisms include filamentous fungi, yeast and bacteria. Among them, filamentous fungi are the most commonly used. Since filamentous fungi can secrete a variety of hydrolases and have a significant ability to degrade lignocellulose cell walls, they are often used in the treatment of industrial and agricultural waste, the production of enzyme preparations and the extraction and release of bioactive components.

[0005] Walnut is an important economic and ecological crop in Yunnan Province. Walnut kernel has rich nutritional value. Walnut pith is discarded after walnut processing, causing serious environmental pollution and waste of resources. Therefore, the development and utilization of walnut pith is of great significance. Therefore, a method for optimizing the solid-state fermentation process of walnut pith is needed to promote the release of insoluble bonded polyphenols during the solid-state fermentation of pith by Aspergillus niger, increase the content of soluble polyphenols, and improve the bioavailability of polyphenols.

[0006] In summary, the problems existing in the prior art are: the bioactive ingredients such as polyphenols in walnut pith can be covalently bonded with macromolecules such as cellulose to form insoluble bonded polyphenols, which are difficult to extract and release, thereby reducing the bioavailability of polyphenols and their preventive effect on diseases; the prior art cannot effectively extract and release the bioactive ingredients such as insoluble bonded polyphenols in walnut pith, thereby hindering the development and utilization of walnut pith.

[0007] The difficulty of solving the above technical problems: Most of the walnut pith polyphenols exist in the form of insoluble bonds, which are difficult to extract and release, and require complex extraction steps, which not only increases the production cost, but may also affect the biological activity of the polyphenols; in addition, polyphenols have strong antioxidant activity, but are easily affected by oxidation during the extraction process and lose their activity. In order to maintain the antioxidant activity of polyphenols, appropriate antioxidant measures need to be taken during the extraction process, such as avoiding light and keeping low temperature; secondly, there may be problems such as environmental pollution during the extraction of insoluble bonded polyphenols.

[0008] The significance of solving the above technical problems: solid-state fermentation has the advantages of low production cost, simple operation and environmental friendliness. Aspergillus niger, as a safe filamentous fungus, can secrete a variety of hydrolases, has a significant ability to degrade lignocellulose cell walls, can effectively release insoluble bonded polyphenols, increase the content of soluble polyphenols, improve the body's absorption and bioutilization of walnut pith polyphenols, promote human health, and provide a scientific basis for the further development and utilization of walnut pith wood. At the same time, it can solve the environmental pollution problems caused by walnut processing, realize the comprehensive utilization of walnut processing waste, and promote the development of the walnut industry in Yunnan Province. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention provides a method and system for optimizing the solid-state fermentation process of walnut pith wood, and uses Aspergillus niger as a fermentation strain to optimize the solid-state fermentation process of walnut pith wood.

[0010] The present invention is achieved by a method for optimizing the solid-state fermentation process of walnut pith wood, comprising the following steps:

[0011] Step 1, pre-treating the walnut core wood;

[0012] Step 2, activating the Aspergillus niger strain and preparing a bacterial suspension;

[0013] Step 3, using single factor and response surface experiments to optimize the solid-state fermentation process conditions;

[0014] Step 4: Solid-state fermentation of walnut pith wood by Aspergillus niger.

[0015] Furthermore, in step 1, the dried walnut pith wood is ground into powder, passed through a 50-mesh sieve, and stored in a -20°C refrigerator away from light; and the moisture content of the pith wood is determined before solid-state fermentation.

[0016] Further, in step 2, the Aspergillus niger strain is taken out from the -20°C refrigerator, revived in a 37°C water bath, and the revived Aspergillus niger is cultured on potato dextrose agar medium, cultured in a constant temperature incubator at 28°C for 10 days, and the bacterial suspension is collected using a sterile physiological saline solution (0.9%), the concentration of the bacterial suspension is calculated using a McFarland turbidimeter, and used for the preparation of inoculum as needed.

[0017] Furthermore, in the single factor experiment in step 3, polyphenol content was used as the evaluation index, and fermentation time (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h), fermentation temperature (24 ° C, 26 ° C, 28 ° C, 30 ° C, 32 ° C), strain inoculation concentration (1 × 10 5 CFU / g, 1×10 6 CFU / g, 1×10 7 CFU / g, 1×10 8 CFU / g, 1×10 9 CFU / g) and moisture content (55%, 60%, 65%, 70%, 75%) were used as factors, and single factor experiments were carried out to determine the effects of each factor on the solid-state fermentation of wood.

[0018] Furthermore, in step 3, the response surface optimization experiment was conducted: based on the single factor experiment, polyphenol content was used as the evaluation index, and fermentation time, fermentation temperature, strain inoculation concentration, and moisture content were selected as independent variables. The Design-Expert software was used to conduct a 4-factor 5-level Central Composite Design (CCD) experimental design to determine the optimal process conditions for solid-state fermentation of Aspergillus niger with split wood.

[0019] Furthermore, the optimal fermentation conditions for solid-state fermentation of Aspergillus niger were as follows: fermentation time of 4 days, fermentation temperature of 28 °C, moisture content of 65%, inoculation volume of 1 × 10 7 CFU / g, and the polyphenol content was 95.32 mg GAE / g DW.

[0020] Further, in step 4, 10 g of walnut wood powder is weighed into a 250 mL conical flask, an appropriate amount of sterile distilled water is added, the mixture is fully mixed, and the mixture is soaked for 1 hour, sterilized at 121° C. for 30 minutes, and after sterilization, the mixture is placed under a sterile operating table for ultraviolet sterilization for 60 minutes, and cooled for use; different concentrations of Aspergillus niger solution are inoculated and placed in a constant temperature incubator at different temperatures for culture.

[0021] Another object of the present invention is to provide a walnut core wood solid-state fermentation process optimization method and a walnut core wood solid-state fermentation process optimization system, comprising:

[0022] A pretreatment module, used for pretreatment of walnut core wood;

[0023] The strain activation and bacterial suspension preparation module is used for activating Aspergillus niger strains and preparing bacterial suspensions;

[0024] Process optimization module, used to optimize solid-state fermentation process conditions using single factor and response surface experiments;

[0025] Solid-state fermentation module, used for solid-state fermentation of walnut pith wood by Aspergillus niger.

[0026] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0027] First, the present invention uses walnut split wood as raw material and optimizes the solid-state fermentation process conditions by using single factor and response surface test. Through single factor and response surface optimization test, the optimal fermentation conditions of Aspergillus niger solid-state fermentation split wood are determined as follows: fermentation time is 4 days, fermentation temperature is 28°C, moisture content is 65%, bacterial liquid inoculation amount is 1×10 7 CFU / g, and the polyphenol content was 95.32 mg GAE / g DW. The present invention is a feasible technology for solid-state fermentation of Aspergillus niger to promote the release of insoluble bonded polyphenols in the split wood and increase the content of soluble polyphenols. Therefore, the present invention lays a foundation for further studying the application of fermented split wood as a potential antioxidant ingredient in functional foods.

[0028] Second, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0029] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are as follows: Walnut pith contains a large amount of polyphenol compounds, and solid-state fermentation can promote the release of insoluble bonded polyphenols, increase the content and biological activity of soluble polyphenols in pith, improve the body's absorption and biological utilization of pith polyphenols, and promote human health. The present invention can cooperate with walnut processing-related enterprises, solve the environmental pollution problems generated during walnut processing, realize the comprehensive utilization of walnut processing waste, improve walnut deep processing, and promote the development of the walnut industry in Yunnan Province.

[0030] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad: there are relatively few research reports on walnut walnut wood at home and abroad, and there is no report on the invention of using walnut walnut wood to promote the release of polyphenols in walnut wood through solid-state fermentation process. Therefore, the present invention uses Aspergillus niger as the fermentation strain, optimizes the solid-state fermentation process of walnut wood on the basis of single factor experiment, promotes the release of insoluble bonded polyphenols, increases the content of soluble polyphenols, and improves the bioavailability of walnut wood polyphenols.

[0031] (3) The technical solution of the present invention solves a technical problem that people have been eager to solve but have never been successful: the content of insoluble bonded polyphenols in walnut pith wood is relatively high, and there are problems such as low extraction efficiency and great difficulty in purification, which limit the bioavailability of polyphenols in the pith wood. Based on the fact that fermentation can promote the release of insoluble bonds and polyphenols in pith wood, the present invention uses safe and efficient Aspergillus niger as a fermentation strain, optimizes the solid-state fermentation process of walnut pith wood, can effectively release insoluble bonded polyphenols, increase the content of soluble polyphenols, improve the body's absorption and bioutilization of pith wood polyphenols, and promote human health. At the same time, it can solve the environmental pollution problem caused by the walnut processing process, realize the comprehensive utilization of walnut processing waste, improve the deep processing of walnuts, and promote the development of the walnut industry in Yunnan Province.

[0032] Third, the present invention provides a solid-state fermentation process optimization method for walnut pith wood, which solves the problems of low fermentation efficiency and difficulty in extracting functional components in the prior art through systematic process design and optimization steps. Walnut pith wood is rich in active substances such as polyphenols, but its traditional application method is difficult to effectively release these functional components. The present invention significantly increases the polyphenol content in walnut pith wood and enhances its antioxidant activity through a solid-state fermentation process, especially a fermentation process using a black Aspergillus strain.

[0033] In the present invention, the walnut core wood is subjected to meticulous pretreatment, including grinding, sieving and storage in a refrigerator at low temperature and away from light, to ensure that the physical and chemical properties of the raw material do not change and retain its nutritional components to the greatest extent. This treatment method improves the stability of subsequent fermentation and ensures the uniformity and fermentation effect of the raw material in solid-state fermentation. By measuring the moisture content, the necessary basic data is further provided for process optimization.

[0034] The present invention designs the resuscitation and cultivation process in detail for the activation of the Aspergillus niger strain and the preparation of the bacterial suspension, ensuring that the strain can exert its maximum efficiency during the fermentation process. By using potato dextrose agar medium and combining constant temperature culture, a high-quality bacterial suspension is cultivated, the concentration of the bacterial solution is controlled, and the microbial activity during the fermentation process and the stability of the fermentation quality are ensured.

[0035] The present invention systematically optimized the fermentation conditions through single factor experiment and response surface optimization experiment, and finally determined the optimal fermentation conditions, namely, fermentation time 4 days, fermentation temperature 28 ° C, moisture content 65%, bacterial inoculation volume 1×10 7 This optimized process greatly increased the polyphenol content in walnut core wood. The test results showed that the polyphenol content could reach 95.32 mg GAE / g DW, which significantly improved the nutritional value and functional properties of walnut core wood and had good industrial application prospects.

[0036] Fourth, the main parameters of the present invention include key process conditions for solid-state fermentation of walnut pith wood, such as fermentation time, fermentation temperature, strain inoculation concentration and moisture content. Through single factor experiment and response surface optimization, the optimal fermentation conditions finally determined are: fermentation time of 4 days, fermentation temperature of 28°C, moisture content of 65%, Aspergillus niger inoculation amount of 1×10 7 Under these conditions, the polyphenol content of walnut wood reached 95.32 mg GAE / g DW, significantly improving the antioxidant activity and nutritional value of the product.

[0037] The present invention solves the problem that the active ingredients in walnut core wood, such as polyphenols, are difficult to effectively release and utilize in the prior art. The traditional processing method of walnut core wood cannot fully exert the role of its functional ingredients, while the present invention adopts Aspergillus niger solid-state fermentation technology and utilizes microbial metabolic activities to promote the synthesis and accumulation of active substances such as polyphenols, thereby achieving efficient conversion of the functional ingredients of walnut core wood.

[0038] By optimizing the fermentation process, the present invention has achieved significant technical progress. First, the polyphenol content is significantly improved during the fermentation process, and the fermented walnut pith has higher antioxidant capacity and biological activity. Secondly, the present invention improves the taste and palatability of walnut pith through the fermentation process, making it easier to apply to functional foods, health products and other fields. At the same time, after the process is optimized, the fermentation efficiency is higher, and high-quality fermented products can be obtained in a shorter time.

[0039] In addition, the technical progress of the present invention is also reflected in the precise control of process conditions. Through the response surface optimization method, the present invention can accurately regulate the relationship between various parameters in the fermentation process to ensure the quality stability of each batch of fermented products. This refined fermentation process greatly improves the industrial application potential of solid-state fermentation of walnut pith wood, and has good market prospects and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a flow chart of a solid-state fermentation process optimization method for walnut pith wood provided by an embodiment of the present invention;

[0041] Figure 2 This is a structural diagram of a walnut corewood solid-state fermentation process optimization system provided by an embodiment of the present invention;

[0042] Figure 3 1. It is a graph showing the effects of (A) fermentation time, (B) fermentation temperature, (C) bacterial liquid concentration, and (D) water content on the soluble polyphenol content of Pyrrolidone provided in an embodiment of the present invention;

[0043] Figure 4 Graph showing the influence of the interaction of factors on the polyphenol content provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0045] The present invention relates to a solid-state fermentation process optimization method for walnut pith wood, which improves the nutritional value and functionality of walnut pith wood fermentation products through pretreatment, strain activation, process optimization and systematic design of fermentation steps. Walnut pith wood is a natural material rich in bioactive substances, and its antioxidant activity and polyphenol content can be further enhanced through solid-state fermentation, thereby improving its application potential in food and health products.

[0046] First, the pre-treated walnut wood in step 1 is the basis of the entire fermentation process. The walnut wood needs to be properly cleaned and dried before use to remove impurities and reach the appropriate humidity level. The pre-treated walnut wood has a uniform texture, which helps uniform fermentation and nutrient release during the fermentation process. This step ensures the cleanliness of the raw materials and the good physical state during the fermentation process, providing a suitable environment for the subsequent growth and metabolic activities of the strain.

[0047] In step 2, the activation of the Aspergillus niger strain and the preparation of the bacterial suspension are the key to ensure that the fermentation proceeds smoothly. Aspergillus niger is a microorganism commonly used in solid-state fermentation. Under suitable conditions, it can produce abundant enzymes to promote the decomposition and conversion of raw materials. The Aspergillus niger strain is activated by the culture medium to grow rapidly and prepare a high-concentration bacterial suspension. In this step, it is very important to control the activity state of the strain, because the quality of the active strain directly affects the efficiency and product quality of the subsequent fermentation.

[0048] Step 3: Single factor and response surface experiments were used to optimize the fermentation process conditions. Single factor experiments were used to explore the effects of individual factors such as temperature, humidity, inoculation amount, and fermentation time on the fermentation effect. Then, response surface design was used to further optimize the process conditions of multi-factor combination to find the best combination of fermentation parameters. This optimization method can improve the fermentation efficiency and ensure that the fermented walnut pith wood reaches the optimal state in terms of nutritional content and functionality.

[0049] Step 4 is the core step of solid-state fermentation of walnut pith wood by Aspergillus niger. Under optimal fermentation conditions, the activated Aspergillus niger suspension is inoculated onto the pretreated walnut pith wood for solid-state fermentation. During the entire fermentation process, the fermentation temperature, humidity and ventilation conditions are strictly controlled to ensure the normal growth and metabolic activity of Aspergillus niger. The microbial metabolism during the fermentation process can decompose the macromolecules in the walnut pith wood and produce more beneficial small molecule active substances, such as polyphenols and flavonoids.

[0050] Through solid-state fermentation, the nutritional and functional components of walnut wood have been significantly improved. The enzymes produced during the fermentation process can not only decompose the complex organic matter in the raw materials, but also promote the synthesis and accumulation of antioxidant active substances. The optimized fermentation process has greatly increased the antioxidant capacity, total polyphenols and total flavonoids content of walnut wood, thereby improving its application value as a functional food raw material.

[0051] The process optimization method has broad application prospects in industrial production. Through reasonable process design and optimization, large-scale fermentation production of walnut heartwood can be achieved to obtain functional fermentation products with high added value. At the same time, the process provided by the invention can effectively utilize the natural resource of walnut heartwood, reduce waste, and conform to the concept of sustainable development.

[0052] like Figure 1 As shown, a method for optimizing the solid-state fermentation process of walnut pith wood provided by an embodiment of the present invention comprises the following steps:

[0053] (1) Pretreatment of split wood

[0054] The dried walnut pith wood was ground into powder and passed through a 50-mesh sieve, and then stored in a -20°C refrigerator away from light. The moisture content of the pith wood was determined before solid-state fermentation.

[0055] (2) Activation of Aspergillus niger strain and preparation of bacterial suspension

[0056] The Aspergillus niger strain was taken out from a -20°C refrigerator and revived in a 37°C water bath. The revived Aspergillus niger was cultured on potato dextrose agar medium and cultured in a constant temperature incubator at 28°C for 10 days. The bacterial suspension was collected using a sterile saline solution (0.9%), the concentration of the bacterial suspension was calculated using a McFarland turbidimeter, and used for the preparation of inoculum as needed.

[0057] (3) Optimization of solid-state fermentation process conditions of Aspergillus niger

[0058] ① Single factor test

[0059] The polyphenol content was used as the evaluation index, and the fermentation time (0h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h), fermentation temperature (24℃, 26℃, 28℃, 30℃, 32℃), strain inoculation concentration (1×10 5 CFU / g, 1×10 6 CFU / g, 1×10 7 CFU / g, 1×10 8 CFU / g, 1×10 9 CFU / g) and moisture content (55%, 60%, 65%, 70%, 75%) were used as factors, and single factor experiments were carried out to determine the effects of each factor on the solid-state fermentation of wood.

[0060] ②Response surface optimization experiment

[0061] On the basis of single factor experiment, polyphenol content was used as the evaluation index, and fermentation time, fermentation temperature, strain inoculation concentration, and moisture content were selected as independent variables. Design-Expert software was used to conduct a 4-factor 5-level Central Composite Design (CCD) experimental design to determine the optimal process conditions for solid-state fermentation of Aspergillus niger with split wood. The factor level design table is shown in Table 1.

[0062] Table 1 Central Composite Design design factor level table

[0063]

[0064] (4) Solid-state fermentation

[0065] Weigh 10g of walnut wood powder into a 250mL conical flask, add an appropriate amount of sterile distilled water, mix thoroughly, soak for 1h, sterilize at 121℃ for 30min, place under a sterile operating table for UV sterilization for 60min, cool for use. Inoculate different concentrations of Aspergillus niger liquid and culture in a constant temperature incubator at different temperatures.

[0066] (5) Extraction of soluble polyphenols

[0067] After the fermentation, 1 g of the fermented sample of the wood was weighed. The extraction of soluble polyphenols was based on the method reported by Wang et al. with slight modifications. Weigh 1 g of the fermented wood sample, add the extract (methanol: formic acid: water, 80:0.1:19.9, v / v / v) at a solid-liquid ratio of 1:10 (g / mL), and extract by ultrasonic at low temperature for 1 h, centrifuge at 8000 rpm for 15 min at 4 ° C, repeat the above steps twice, and combine the supernatant to obtain the soluble polyphenol extract.

[0068] (6) Determination of polyphenol content

[0069] The polyphenol content was determined as described by Wu et al. with slight modifications. A standard curve was drawn using gallic acid, and the standard curve equation was: y = 0.4419x + 0.1054 (R2 = 0.9992). The results were expressed as the polyphenol content per gram of dried wood sample as gallic acid equivalents (GAE), i.e. mg GAE / g DW.

[0070] (7) Statistical analysis

[0071] All experiments were repeated three times, and the results were expressed as mean ± standard deviation. SPSS 22.0 was used to perform one-way ANOVA and Tukey's multiple test to analyze the significant differences of the experimental data, and Design-Expert 8.0.6 was used for data analysis and drawing. P < 0.05 indicated significant statistical significance.

[0072] like Figure 2 As shown, a walnut core wood solid-state fermentation process optimization method and a walnut core wood solid-state fermentation process optimization system provided by an embodiment of the present invention include:

[0073] A pretreatment module, used for pretreatment of walnut core wood;

[0074] The strain activation and bacterial suspension preparation module is used for activating Aspergillus niger strains and preparing bacterial suspensions;

[0075] Process optimization module, used to optimize solid-state fermentation process conditions using single factor and response surface experiments;

[0076] Solid-state fermentation module, used for solid-state fermentation of walnut pith wood by Aspergillus niger.

[0077] The present invention uses Aspergillus niger as a fermentation strain, optimizes the solid-state fermentation process of the wood stalk on the basis of a single factor test, can promote the release of insoluble bonded polyphenols, increase the content of soluble polyphenols, improve the bioavailability of wood stalk polyphenols, and promote human health. The present invention can be applied in walnut processing related fields, can solve the environmental pollution problem generated in the walnut processing process, realize the comprehensive utilization of walnut processing waste, improve the walnut deep processing, and promote the development of the walnut industry in Yunnan Province.

[0078] Relevant evidence of the technical effects achieved by the embodiments of the present invention.

[0079] 1. Optimization of solid-state fermentation process of Aspergillus niger on Pyrrolidone

[0080] (1) Results of single-factor solid-state fermentation experiments

[0081] The effects of fermentation time, fermentation temperature, bacterial liquid concentration and water content on the polyphenol content of the woody plant are shown in Figure 3 .

[0082] Depend on Figure 3 A shows that as the fermentation time increases, the polyphenol content increases first and then decreases. At the 96th hour of fermentation, the polyphenol content reaches the maximum value of 89.371.40 mg GAE / g DW. Figure 3 B shows that with the increase of fermentation temperature, the polyphenol content increases first and then decreases. When the fermentation temperature is 28℃, the polyphenol content is the highest at 91.480.22mgGAE / g DW. Figure 3 C shows that when the bacterial solution concentration is between 1×105 CFU / g and 1×10 7 CFU / g. With the increase of bacterial concentration, the polyphenol content gradually increased from 62.550.83mg GAE / g DW to 91.181.47mg GAE / g DW. When the bacterial concentration was greater than 1×10 7 CFU / g, the polyphenol content gradually decreased. Figure 3 It can be seen from D that when the moisture content is less than 65%, the polyphenol content gradually increases with the increase of moisture content and reaches a maximum value of 93.300.85 mg GAE / g DW when the moisture content is 65%. Then, as the moisture content increases, the polyphenol content gradually decreases. Therefore, the parameters for the solid-state fermentation of Aspergillus niger are selected as follows: fermentation time of 96 h, fermentation temperature of 28 °C, bacterial liquid concentration of 1×10 7 CFU / g, and the moisture content was 65% for optimization experiments.

[0083] (2) Solid-state fermentation response surface optimization experiment

[0084] The polyphenol content was recorded as Y value, and the fermentation time, fermentation temperature, bacterial liquid concentration and water content were recorded as X respectively.1 , X 2 , X 3 and X 4 The results of response surface experimental design are shown in Table 2, and the results of variance analysis are shown in Table 3.

[0085] Table 2 Response surface experimental design and results

[0086]

[0087]

[0088] Table 3 Analysis of variance table

[0089]

[0090] Note: ** indicates extremely significant (P < 0.01); * indicates significant (P < 0.05); P > 0.05 indicates not significant.

[0091] The binary multiple regression equation obtained by software optimization is: Y = 95.69-0.76X 1 +0.12X 2 -0.75X 3 +0.62X 4 -1.80X 1 X 2 -0.43X 1 X 3 +0.70X 1 X 4 -0.71X 2 X 3 -0.067X 2 X 4 -0.14X 3 X 4 -9.84X 12 -9.19X 22 -7.71X 32 -8.59X 42 .

[0092] As shown in Table 3, the model P < 0.01 is extremely significant, the lack of fit term P = 0.2829 > 0.05 is not significant, and R 2 =0.9996, R 2 adj =0.9986, indicating that the model can accurately predict the polyphenol content with a high degree of fit. 1 , X 3 The effect on polyphenol content was significant (P<0.05), among which the first-order term X 4 and the quadratic term X 12 , X 22 , X32 , X 42 The effects on polyphenol content were extremely significant (P<0.01), and the interaction term X 1 X 2 The influence on polyphenol content is extremely significant (P<0.01). According to the size of the F value, the order of the influence of each factor on polyphenol content is water content (X 4 )>Fermentation time (X 1 )>Bacterial concentration (X 3 )>Fermentation temperature (X 2 ).

[0093] (3) Response surface analysis

[0094] The effects of the interaction of various factors on polyphenol content are shown in Figure 4 .

[0095] Depend on Figure 4 It can be seen that the response surface diagrams are all convex surfaces, indicating that the polyphenol content increases with the independent variable (fermentation time (X 1 ), fermentation temperature (X 2 ), bacterial inoculation concentration (X 3 ) and moisture content (X 4 ) showed a trend of increasing first and then decreasing. 1 , X 2 The contour lines are elliptical and the slope of the response surface is steep, indicating that the pairwise interaction between fermentation time and fermentation temperature has a significant effect on the polyphenol content. 1 and X 3 , X 1 and X 4 , X 2 and X 3 The slope is relatively gentle, indicating that its effect on polyphenol content is weaker.

[0096] The response surface analysis showed that the optimal process conditions were fermentation time 96.08 h, fermentation temperature 28.02 °C, bacterial concentration 1 × 106.95 CFU / g and water content 65.2%. Under these conditions, the polyphenol content of the wood was predicted to be 95.74 mg GAE / g DW. In order to facilitate actual operation, the fermentation conditions were adjusted to fermentation time 96 h, fermentation temperature 28 °C, bacterial concentration 1 × 10 7 CFU / g and moisture content were 65%, and the experiment was repeated 3 times under this condition, at which time the polyphenol content was 95.32 mg GAE / gDW. The actual value was consistent with the predicted value, indicating that the fermentation process was feasible.

[0097] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for optimizing the solid-state fermentation process of walnut pith wood, characterized in that: The following steps are involved: Step 1: pre-treating the walnut pith wood by cleaning, drying and controlling the moisture content to a level suitable for fermentation; Step 2: activating the Aspergillus niger strain by using a suitable culture medium to activate the Aspergillus niger and prepare a high-concentration bacterial suspension; Step 3: Optimize the fermentation process conditions using single-factor experiments and response surface design to investigate the effects of temperature, humidity, inoculum size, and fermentation time on the fermentation effect, and determine the optimal fermentation parameter combination; Step 4: inoculate the activated Aspergillus niger suspension onto the pretreated walnut pith wood for solid-state fermentation. Ferment for a certain period of time under optimized fermentation conditions. After the fermentation is completed, collect and test the product.

2. The walnut corewood solid-state fermentation process optimization method according to claim 1, wherein: In step 1, the dried walnut pith wood is ground into powder, passed through a 50-mesh sieve, and stored in a -20°C refrigerator away from light; and the moisture content of the pith wood is determined before solid-state fermentation.

3. The method for optimizing the solid-state fermentation process of walnut corewood according to claim 1, wherein: In step 2, the Aspergillus niger strain was taken out from the -20°C refrigerator and revived in a 37°C water bath. The revived Aspergillus niger was cultured on potato dextrose agar medium and cultured in a constant temperature incubator at 28°C for 10 days. The bacterial suspension was collected using a 0.9% sterile saline solution, and the concentration of the bacterial suspension was calculated using a McFadden turbidimeter and used for the preparation of the inoculum as needed.

4. The walnut corewood solid-state fermentation process optimization method according to claim 1, wherein: In step 3, single factor experiment: polyphenol content was used as the evaluation index, and the fermentation time was 0h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, and 192h, the fermentation temperature was 24℃, 26℃, 28℃, 30℃, and 32℃, and the fermentation temperature was 1×10 5 CFU / g, 1×10 6 CFU / g, 1×10 7 CFU / g, 1×10 8 CFU / g, 1×10 9 The single factor experiment was carried out with the strain inoculation concentration of 1000 CFU / g and the moisture content of 55%, 60%, 65%, 70% and 75% as factors to determine the effect of each factor on the solid-state fermentation of the wood.

5. The method for optimizing the solid-state fermentation process of walnut corewood according to claim 1, wherein: In step three, response surface optimization experiment: Based on the single-factor experiment, polyphenol content was used as the evaluation index, and fermentation time, fermentation temperature, strain inoculation concentration, and moisture content were selected as independent variables; Design-Expert software was used to perform a 4-factor 5-level Central Composite Design experiment to determine the optimal process conditions for solid-state fermentation of Aspergillus niger with split wood.

6. The method for optimizing the solid-state fermentation process of walnut corewood according to claim 1, wherein: The optimal fermentation conditions for Aspergillus niger solid-state fermentation of wood are as follows: fermentation time 4 days, fermentation temperature 28 °C, moisture content 65%, inoculum size 1×10 7 CFU / g, and the polyphenol content was 95.32mg GAE / gDW.

7. The method for optimizing the solid-state fermentation process of walnut corewood according to claim 1, wherein: In step 4, 10 g of walnut wood powder was weighed into a 250 mL conical flask, an appropriate amount of sterile distilled water was added, the mixture was thoroughly mixed, and the mixture was soaked for 1 hour. The mixture was sterilized at 121° C. for 30 minutes. After sterilization, the mixture was placed under a sterile operating table for ultraviolet sterilization for 60 minutes and cooled for later use. Different concentrations of Aspergillus niger solution were inoculated and placed in a constant temperature incubator at different temperatures for culture.

8. A walnut core wood solid-state fermentation process optimization system for the walnut core wood solid-state fermentation process optimization method according to any one of claims 1 to 7, characterized in that: include: Pretreatment module, used for pretreatment of walnut core wood; Strain activation and bacterial suspension preparation module, used to activate Aspergillus niger strains and prepare bacterial suspensions; Process optimization module, used to optimize solid-state fermentation process conditions using single factor and response surface experiments; Solid-state fermentation module, used for solid-state fermentation of walnut pith wood by Aspergillus niger.

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