Method for preparing soluble dietary fiber by pretreating bean dregs through aspergillus oryzae two-stage method

Abstract

The invention relates to a method for preparing soluble dietary fibers (SDF) by pretreating bean dregs through an aspergillus oryzae two-stage method, and belongs to the field of food processing. The method comprises the following steps: by taking bean dregs as a raw material and optimizing a process, obtaining a section of pretreatment conditions: 2% of glucose, microwave treatment time (700W) of 70s, material-liquid ratio of 1: 4, aspergillus oryzae inoculum size of 7%, initial fermentation pH of 6.8, fermentation temperature of 29 DEG C and fermentation time of 60h; the second-stage pretreatment condition is that the temperature is kept for 36 hours at 50 DEG C The extraction rate of the soluble dietary fibers of the bean dregs reaches 15.13%, compared with untreated bean dregs, the bean dregs are looser and more porous in structure, lower in crystallinity and higher in water and oil holding capacity and expansibility, and the glucose adsorption capacity, the alpha-amylase inhibitory activity and the glucose dialysis delay index are all remarkably increased. According to the method, the SDF extraction rate of the bean dregs is effectively increased, the physicochemical and functional characteristics of the SDF are improved, bean dreg resources are effectively utilized, and the additional value of the bean dregs is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and in particular relates to a method for preparing soluble dietary fiber by pretreating bean dregs through a two-stage Aspergillus oryzae process. Background Art

[0002] Okara is a byproduct of soybean processing. It is rich in nutrients and has high nutritional value. Dietary fiber (DF) accounts for the largest proportion of about 50%-60%, followed by protein, carbohydrates, fat, vitamins, minerals and other nutrients. As the "seventh nutrient", dietary fiber has attracted much attention in promoting gastrointestinal motility, lowering blood sugar and lipids, and preventing cardiovascular and cerebrovascular diseases. Dietary fiber is divided into insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) according to whether it is soluble in water. SDF has a smaller molecular weight, a looser structure, and more active factors. Therefore, it has better adsorption capacity, ion exchange capacity and antioxidant properties than IDF, and has greater development prospects in the fields of food and medicine. The content of SDF in okara is low, about 6%, which affects the utilization rate of okara. Therefore, it can be considered to use chemical, physical or microbiological methods to change the structure and properties of okara DF to achieve the effect of increasing SDF production.

[0003] Chemical modification will pollute the environment and endanger human health, while physical modification consumes a lot of energy. Microbial fermentation is safe, effective, low-cost and easier to achieve industrial production. In the process of microbial fermentation of soybean dregs, a large amount of metabolites such as organic acids, cellulases, and hemicellulases will be produced, which will break the glycosidic bonds between macromolecules in soybean dregs DF, produce new reducing ends, and continuously reduce the degree of polymerization of macromolecules, so that part of IDF is converted into SDF, thereby increasing its yield. The commonly used microorganisms for fermenting soybean dregs are generally molds, such as Aspergillus oryzae, Aspergillus niger, and Trichoderma viride. Among them, Aspergillus oryzae is often used to produce amylase, protease, pectinase, cellulase, etc. It is widely used in soy sauce brewing, fermented black beans, etc., and is recognized as a safe food production strain.

[0004] At present, according to statistics, every 100 kg of soybeans can produce 120 kg of fresh dregs. my country's consumption of soybeans is relatively high, accounting for a large proportion of the world's dregs production, and about 20 million tons of wet dregs are produced a year. In addition to being used as feed and fertilizer, most of the dregs are treated as waste, which has caused serious environmental pollution and waste of resources.

[0005] In recent years, there have been patents related to the extraction of SDF using bean dregs as raw materials. For example, in the patent with application number 202410088555.X, the bean dregs are subjected to strong UV modification, combined fermentation modification by brewer's yeast and plant lactobacillus, and then further processed into jelly products. The SDF extraction rate of the modified bean dregs after hydrolysis by α-amylase, protease, and saccharification enzyme reaches 21.81%. A variety of enzyme preparations are added to this patent, which is costly, and the use of UV modification consumes a lot of energy. In the patent with application number 202310466412.3, the bean dregs are ball-milled, cavitated, and hydrolyzed by cellulase to obtain modified bean dregs. The modified bean dregs are used to prepare a soft candy rich in SDF suitable for diabetics, in which the SDF extraction rate is 6.97%. The patent uses physical modification, which consumes a lot of energy, and the cost of adding enzyme preparations is high. The SDF extraction rate is low after the bean dregs are modified.

[0006] In view of the low utilization rate of okara, an effective preparation method is urgently needed to increase the yield of okara SDF, reduce the preparation cost, and provide the possibility for the development of high SDF products. Summary of the invention

[0007] In view of the current problems of low utilization rate of bean dregs, waste of resources and low yield of extracting bean dregs SDF, the present invention provides a method for preparing bean dregs SDF by pretreating bean dregs in two stages. The present invention aims to improve the extraction rate of bean dregs SDF, reduce the raw material cost of preparing SDF, and improve the physicochemical functional properties of bean dregs SDF. The present invention uses bean dregs as raw materials, adds glucose to supplement the carbon source, and after microwave pretreatment, adds Aspergillus oryzae for solid-state fermentation. The extraction rate of bean dregs SDF is used as an evaluation index, and the parameters such as time, temperature, initial pH, material-liquid ratio, and strain addition of the first fermentation are optimized to obtain the optimal conditions for the first fermentation of Aspergillus oryzae; and then the fermentation system's own endogenous enzymes are used for enzymolysis, and the temperature and time of the second enzymolysis are optimized to obtain the optimal conditions for the second enzymolysis. The preparation by the two-stage process of the present invention not only improves the extraction rate of SDF in bean dregs, enhances its physicochemical functional properties, and improves the quality of SDF; at the same time, the process does not require the addition of additional enzyme preparations, which reduces the procurement cost of enzyme preparations.

[0008] To achieve the above object, the present invention comprises the following steps:

[0009] A method for preparing soluble dietary fiber by pretreating bean dregs using Aspergillus oryzae in two stages, comprising the following process steps:

[0010] (1) grinding and crushing the dried bean dregs and passing through a 100-mesh sieve to obtain bean dregs powder;

[0011] (2) adding glucose to the okara powder, mixing evenly, adding drinking water with a pH of 6.8 at a solid-liquid ratio of 1:4, stirring evenly, and setting aside;

[0012] (3) treating the okara powder with microwaves, adding drinking water with a pH of 6.8 to a solid-liquid ratio of 1:4 after cooling, and stirring evenly to obtain an okara fermentation medium;

[0013] (4) sterilizing the okara fermentation medium at 121° C. for 15-20 min, cooling to room temperature, and setting aside;

[0014] (5) adding 7% (W / W) Aspergillus oryzae mycelium to the cooled okara fermentation medium under sterile conditions, culturing at 29° C. for 60 h, and performing a fermentation step to obtain fermented okara;

[0015] (6) subjecting the fermented okara to a second-stage enzymatic hydrolysis at 50° C. to obtain enzymatically hydrolyzed okara;

[0016] (7) The bean dregs after enzymatic hydrolysis are taken out, and an appropriate amount of clean water is added, and the mixture is extracted in a water bath for 2 h. After centrifugation, alcohol precipitation, elution, and drying, the soluble dietary fiber of the two-stage fermented bean dregs is obtained.

[0017] Preferably, the amount of glucose added in step (2) is 2% of the amount of okara powder added.

[0018] Preferably, in step (2), the pH of the drinking water is adjusted to 6.8 using 1 mol / L sodium hydroxide and 1 mol / L hydrochloric acid.

[0019] Preferably, the power of the microwave in step (3) is 700 W and the processing time is 70 s.

[0020] Preferably, in step (5), the Aspergillus oryzae preserved on the slant is inoculated into a sterilized PDA liquid culture medium, cultured in a shaking incubator at 30° C. and 170 r / min for 48 h for activation, centrifuged at 5000 r / min for 1 min, and the supernatant is discarded to obtain Aspergillus oryzae mycelium.

[0021] Preferably, the time for the second stage enzymatic hydrolysis in step (6) is 36 hours.

[0022] Preferably, the temperature of the water bath extraction in step (7) is 80°C.

[0023] The present invention provides okara soluble dietary fiber obtained by any one of the aforementioned preparation processes.

[0024] With respect to the existing technology, the advantages of the present invention are:

[0025] (1) The present invention utilizes Aspergillus oryzae two-stage method to pretreat bean dregs, so that the extraction rate of SDF from bean dregs is significantly improved. The fermentation preparation process is safe, simple, easy to operate, and can be industrialized for production, which is beneficial to improving the utilization rate of bean dregs, alleviating the waste of bean dregs resources and environmental pollution, and providing a technical reference for the future production of green and efficient soluble dietary fiber products.

[0026] (2) Compared with the conventional SDF, the SDF prepared by the two-stage pretreatment of Aspergillus oryzae has a looser and more porous structure, lower crystallinity, greater water-holding capacity, greater oil-holding capacity and swelling capacity, and improved glucose adsorption capacity, α-amylase inhibitory activity and glucose dialysis delay index, which makes the product have better glucose-lowering potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Effect of different glucose addition amounts on SDF extraction rate

[0028] Figure 2 Effect of different microwave treatment times on SDF extraction rate

[0029] Figure 3 Effect of different fermentation times on SDF extraction rate

[0030] Figure 4 Effects of different fermentation temperatures on SDF extraction rate

[0031] Figure 5 Effect of different solid-liquid ratios on SDF extraction rate

[0032] Figure 6 Effect of different initial pH on SDF extraction rate

[0033] Figure 7 Effect of different inoculation amounts on SDF extraction rate

[0034] Figure 8 Effects of different fermentation times on the enzyme activity of filter paper (under the optimal culture conditions of Aspergillus oryzae)

[0035] Fig. 9 Effects of different enzymatic hydrolysis temperatures on SDF extraction efficiency

[0036] Fig.10 Scanning electron micrographs of three types of SDF

[0037] Fig.11 Fourier transform infrared spectra of three SDFs

[0038] Fig.12 X-ray diffraction patterns of three SDFs

[0039] Fig.13 Thermogravimetric analysis curves of three SDFs

[0040] Fig.14 Monosaccharide HPLC chromatograms of three SDFs and standards

[0041] Fig.15 Determination of glucose adsorption capacity of three SDFs

[0042] Fig.16Determination of α-amylase inhibitory activity of three SDFs

[0043] Fig.17 Determination of Glucose Dialysis Delay Index of Three SDFs DETAILED DESCRIPTION

[0044] Example 1: Strain activation

[0045] The Aspergillus oryzae preserved on the slant was inoculated into the sterilized PDA liquid culture medium, cultured in a shaking incubator at 30°C and 170 rpm for 48 h for activation, and then transferred for two generations for standby use.

[0046] Example 2: Selection of the amount of glucose added

[0047] Take 10g of ground soybean dregs powder through a 100-mesh sieve and put it in a 500ml conical flask. Add 1, 2, 3, 4, and 5% glucose respectively, mix well, adjust the pH of drinking water to 6.8 with 1mol / L sodium hydroxide and 1mol / L hydrochloric acid, add 40ml of drinking water and stir evenly, microwave pretreatment for 70s, add drinking water with a pH of 6.8 to a solid-liquid ratio of 1:4 after cooling, sterilize at 121℃ for 15-20min, inoculate 7% Aspergillus oryzae mycelium after cooling to room temperature, and culture at 30℃ for 4d. Determine the extraction rate of SDF in soybean dregs after fermentation. Figure 1 It can be seen that with the increase of glucose addition, the extraction of SDF first increased and then decreased. When the addition amount was 2%, the extraction rate reached the maximum value of 11.78%. Therefore, 2% was selected as the optimal glucose addition amount.

[0048] Example 3: Selection of microwave treatment time

[0049] Take 10g of ground soybean dregs powder through a 100-mesh sieve and put it in a 500ml conical flask, add 2% glucose, mix well, adjust the pH of drinking water to 6.8 with 1mol / L sodium hydroxide and 1mol / L hydrochloric acid, add 40ml of drinking water and stir well, microwave pretreatment for 40, 50, 60, 70, 80s, add drinking water with a pH of 6.8 to a solid-liquid ratio of 1:4 after cooling, sterilize at 121℃ for 15-20min, inoculate 7% Aspergillus oryzae mycelium after cooling to room temperature, and culture at 30℃ for 4d, and determine the extraction rate of SDF in soybean dregs after fermentation. Figure 2 It can be seen that with the extension of microwave treatment time, the extraction of SDF first increased and then decreased. When the microwave treatment time was 70s, the extraction rate reached the maximum value of 12.82%. Therefore, 70s was selected as the optimal microwave treatment time.

[0050] Example 4: Single factor test of Aspergillus oryzae fermentation culture conditions

[0051] (1) Selection of fermentation time

[0052] Take 10g of ground soybean dregs powder after passing through a 100-mesh sieve and put it in a 500ml conical flask, add 2% glucose, adjust the pH of drinking water to 6.8 with 1mol / L sodium hydroxide and 1mol / L hydrochloric acid, add 40ml of drinking water and mix evenly, microwave pretreatment for 70s, add drinking water with a pH of 6.8 to a solid-liquid ratio of 1:4 after cooling, sterilize at 121℃ for 15-20min, inoculate 7% Aspergillus oryzae mycelium after cooling to room temperature, and culture at 30℃ for 1, 2, 3, 4, and 5d, respectively, to determine the extraction rate of SDF in soybean dregs after fermentation. Figure 3 It can be seen that as the fermentation time increases, the extraction of SDF first increases and then decreases, and the extraction rate reaches a maximum of 10.63% on the 4th day of fermentation. Therefore, the 4th day was selected as the optimal fermentation time.

[0053] (2) Selection of fermentation temperature

[0054] Take 10g of ground soybean dregs powder through a 100-mesh sieve and put it in a 500ml conical flask, add 2% glucose, adjust the pH of drinking water to 6.8 with 1mol / L sodium hydroxide and 1mol / L hydrochloric acid, add 40ml of drinking water and mix evenly, microwave pretreatment for 70s, add drinking water with a pH of 6.8 to a solid-liquid ratio of 1:4 after cooling, sterilize at 121℃ for 15-20min, inoculate 7% Aspergillus oryzae mycelium after cooling to room temperature, and culture at 20, 25, 30, 35, and 40℃ respectively. The culture time is based on the optimal time of the above test, and the extraction rate of SDF in the fermented soybean dregs is determined. Figure 4 It can be seen that with the increase of temperature, the extraction of SDF first increased and then decreased. When the fermentation temperature was 30°C, the extraction rate reached the maximum value of 10.53%. Therefore, 30°C was selected as the optimal fermentation temperature.

[0055] (3) Selection of material-liquid ratio

[0056] Take 10g of ground soybean dregs powder through a 100-mesh sieve and put it in a 500ml conical flask, add 2% glucose, adjust the pH of drinking water to 6.8 with 1mol / L sodium hydroxide and 1mol / L hydrochloric acid, add 20, 30, 40, 50, and 60ml of drinking water respectively, mix evenly, microwave pretreatment for 70s, add drinking water with a pH of 6.8 to the corresponding solid-liquid ratio after cooling, sterilize at 121℃ for 15-20min, inoculate 7% Aspergillus oryzae mycelium after cooling to room temperature, and culture the fermentation time and temperature according to the optimal parameters of the above test, and determine the extraction rate of SDF in soybean dregs after fermentation. Figure 5 It can be seen that with the increase of the material-liquid ratio, the extraction of SDF first increased and then decreased. When the ratio was 1:4, the extraction rate reached the maximum value of 10.32%. Therefore, 1:4 was selected as the optimal fermentation material-liquid ratio.

[0057] (4) Selection of initial pH of fermentation

[0058] Take 10g of ground soybean dregs powder through a 100-mesh sieve and put it in a 500ml conical flask, add 2% glucose, adjust the pH of drinking water to 5.9, 6.2, 6.5, 6.8, and 7.1 with 1mol / L sodium hydroxide and 1mol / L hydrochloric acid, add drinking water according to the optimal solid-liquid ratio of the previous test and mix evenly, microwave pretreatment for 70s, add drinking water of corresponding pH to the optimal solid-liquid ratio of the previous test after cooling, sterilize at 121℃ for 15-20min, inoculate 7% Aspergillus oryzae mycelium after cooling to room temperature, and culture according to the optimal parameters of fermentation time and temperature, and determine the extraction rate of SDF in soybean dregs after fermentation. Figure 6 It can be seen that with the increase of pH value, the extraction of SDF first increases and then decreases. When the pH is 6.8, the extraction rate reaches the maximum value of 10.56%. Therefore, 6.8 is selected as the optimal fermentation pH.

[0059] (5) Selection of inoculation amount

[0060] Take 10g of ground soybean dregs powder through a 100-mesh sieve and put it in a 500ml conical flask, add 2% glucose, adjust drinking water with 1mol / L sodium hydroxide and 1mol / L hydrochloric acid according to the optimal pH of the previous test, add drinking water according to the optimal solid-liquid ratio of the previous test and mix evenly, microwave pretreatment for 70s, add drinking water with the optimal pH of the previous test to the optimal solid-liquid ratio of the previous test after cooling, sterilize at 121°C for 15-20min, cool to room temperature and inoculate 3%, 5%, 7%, 9%, 11% Aspergillus oryzae mycelium respectively, and culture according to the optimal parameters of fermentation time and temperature, and determine the extraction rate of SDF in soybean dregs after fermentation. Figure 7 The results show that with the increase of inoculation amount, the extraction of SDF first increases and then decreases. When the inoculation amount is 7% and 9%, the extraction rate is 10.71% and 10.81% respectively, and the difference is not significant. Therefore, 7% is selected as the optimal strain inoculation amount.

[0061] Example 5: Response surface optimization of Aspergillus oryzae fermentation culture conditions

[0062] (1) Plackett-Burman experimental design

[0063] In order to further investigate the main influencing factors of fermented soybean dregs by Aspergillus oryzae, the SDF extraction rate was used as the response value to carry out PB experimental design. The experimental design and results are shown in Tables 1 to 3 below.

[0064] Table 1: Plackett-Burman Experimental Design Factors and Levels

[0065] Table 2: Plackett-Burman test design and results

[0066] Table 3: Analysis of variance of Plackett-Burman test results

[0067]

[0068] From the design and results of the PB experiment of Aspergillus oryzae fermentation in Tables 1 to 3, it can be seen that the factors that significantly affect the SDF extraction rate are E solid-liquid ratio (P = 0.0003), A temperature (P = 0.0004), B time (P = 0.0039), while C inoculation amount (P = 0.1628) and D pH (P = 0.3768) have no significant effects. Therefore, a ramp test was conducted on the three significant influencing factors selected. After regression analysis of the data obtained from the PB experiment, the multivariate linear equation was obtained: SDF extraction rate = 11.31-0.3686A-0.2383B-0.0833C-0.0500D-0.3917E, R 2 =0.9557, where the coefficients of A, B, C, D, and E are all negative values, so the five influencing factors are negatively correlated with the SDF extraction rate.

[0069] (2) Steepest climbing test

[0070] From the coefficients of the multivariate linear regression equation obtained from the PB design test results, it can be seen that temperature, time and solid-liquid ratio are all negatively correlated with the SDF extraction rate, and their values ​​need to be reduced in the steepest climbing test. The steepest climbing test design and results are shown in Table 4 below.

[0071] Table 4: Steepest climbing test design and results

[0072]

[0073] It can be seen from the steepest climbing test design and results in Table 4 that the SDF extraction rate of Group 3 is the highest, proving that the optimal values ​​of the key variables are near Group 3, namely A (temperature) 31 °C, B (time) 104 h, and E (solid-liquid ratio) 4 ml / g. Therefore, the conditions of Group 3 are taken as the center point of the response surface experiment.

[0074] (3) Response surface methodology experiment

[0075] According to the results of the ramp test, the temperature of 31°C, the time of 104h and the solid-liquid ratio of 4ml / g were used as the center points of the response surface experiment. The response surface experiment design and results are shown in Table 5-6 below.

[0076] Table 5: Response surface analysis experimental design and results

[0077] Table 6: ANOVA of response surface analysis test results

[0078]

[0079] The variance analysis was performed using Design-Expert 13.0 software, and the equation was obtained: extraction rate =13.41-0.5825A-0.1750B+0.0825C-0.1025AB+0.0075AC+0.0225BC-0.3593A 2 -0.2943B 2 -0.6342C 2 ,R 2 =0.9913, the P value of the model is <0.0001, the model is extremely significant, and the lack of fit term is not significant (P>0.05), indicating that the quadratic polynomial regression equation can better describe the true relationship between each factor and the response value. From the F-value values ​​in Table 6, it can be seen that the influence of each factor on the extraction rate is ranked from large to small: A>C 2 >A 2 >B 2 >B>C>AB>BC>AC. The variance analysis showed that the first-order term, the second-order term and the interaction term AB had significant effects on the SDF extraction rate regression model (P<0.05), and the interaction terms BC and AC had no significant effects on the model (P>0.05). The best conditions obtained according to the response optimization results were: fermentation temperature 29.201℃, fermentation time 98.681h, solid-liquid ratio 4.088ml / g, and the estimated extraction rate was 13.555%. In order to facilitate practical application, the best fermentation conditions for the first stage were: fermentation temperature 29℃, fermentation time 99h, solid-liquid ratio 4ml / g, inoculation size 7%, and pH 6.8.

[0080] Under the above optimal conditions, a confirmatory test was carried out, and finally the average extraction rate of soluble dietary fiber was obtained to be 13.46%.

[0081] Example 6: Filter paper enzyme activity assay

[0082] Under the above-mentioned optimal fermentation conditions, the fermented bean dregs at five time points of 36h, 48h, 60h, 72h and 84h were taken to determine the enzyme activity on the filter paper. The fermented bean dregs at each time point were dissolved with an appropriate amount of clean water, centrifuged at 8000r / min for 15min, the supernatant was taken, diluted appropriately, 4 20mL graduated test tubes were taken, 0.5mL enzyme solution and 1.5mL 0.05mol / L, pH 4.5 citric acid buffer solution were added to each, 1.5mL 3,5-dinitrosalicylic acid reagent was added to test tube No. 1 to inactivate the enzyme activity, as a blank control. The 4 test tubes were simultaneously preheated in a 50℃ water bath for 5-10min, and then 50mg filter paper strips (1cm×6cm) were added to each, and kept warm for 1h. After taking out, 1.5mL 3,5-dinitrosalicylic acid reagent was added to test tubes No. 2, 3 and 4 (the action should be quick) to terminate the enzyme reaction. After shaking the four test tubes thoroughly, heat them in a boiling water bath for 5 minutes, take them out, cool them, and then dilute them to the scale with distilled water. Take the solution in test tube No. 1 as the blank control, measure the absorbance at a wavelength of 540nm and record the results. According to the average value of the above three absorbances, find the corresponding glucose content on the standard curve, and calculate the filter paper enzyme activity according to the following formula. The amount of enzyme required to produce 1μmol of glucose from 1ml of crude enzyme solution per hour is one enzyme activity unit (U). The calculation formula for filter paper enzyme activity (U / ml) is as follows:

[0083]

[0084] Where: 5.56 is the conversion coefficient.

[0085] Depend on Figure 8 The results show that as time goes by, the enzyme activity of the filter paper increases first and then decreases, reaching the maximum value at 60h, indicating that the total enzyme activity of cellulose is the highest at this time. Cellulase can degrade cellulose, promote the conversion of IDF to SDF, and improve the extraction rate of okara SDF. Therefore, the fermentation time of the first stage of okara pretreatment is adjusted to 60h.

[0086] Example 7: Determination of the conditions for the second-stage enzymatic hydrolysis

[0087] After the first fermentation of okara, the second enzymatic hydrolysis was carried out, and the enzymatic hydrolysis temperature was set to 45℃, 50℃, and 55℃, and the enzymatic hydrolysis time was set to 24h, 36h, 48h, 60h, and 72h, respectively. After the enzymatic hydrolysis, the extraction rate of fermented okara SDF was measured. Fig. 9As shown in A, at 50°C, the SDF extraction rate first increased and then decreased with the extension of enzymatic hydrolysis time, and the highest extraction rate was 15.13% at 36h; B was at 45°C, the SDF extraction rate first increased and then decreased with the increase of time, and reached a maximum value of 14.25% at 60h; C was at 55°C, and there was no significant difference in the SDF extraction rate within the time period of 24-72h, so 50°C enzymatic hydrolysis for 36h was determined to be the optimal enzymatic hydrolysis condition.

[0088] Through single factor experiment, response surface optimization experiment, enzyme activity determination and enzymatic hydrolysis conditions, the optimal process conditions for preparing soybean dregs SDF by two-stage method of Aspergillus oryzae are as follows: glucose addition 2%, microwave pretreatment (700w) 70s, solid-liquid ratio 1:4, initial pH 6.8, inoculation amount 7%, first fermentation time 60h, first fermentation temperature 29℃, second enzymatic hydrolysis time 36h, second enzymatic hydrolysis temperature 50℃. Under these process conditions, the extraction rate of soybean dregs SDF is 15.13%.

[0089] Example 6: Scanning electron microscopy (SEM)

[0090] The unfermented, first-stage fermented, and second-stage enzymatically extracted SDFs were named U-SDF, F-SDF, and FM-SDF, respectively. The SDF samples were attached to a fixed table with conductive adhesive. After gold spraying, the surface structure of the SDF samples was observed using a field emission scanning electron microscope. The images of different SDFs at 500× and 2000× magnifications are shown in Figure 2. Fig.10 As shown in the figure, the particle surface of U-SDF is flat, with a tight and smooth structure; the particle surface of F-SDF is rough, with a loose structure and a honeycomb structure; the particles of FM-SDF are smaller, fluffy, with a rougher surface, more irregular protrusions and loose holes.

[0091] Example 8: Fourier Transform Infrared Spectroscopy (FTIR)

[0092] The SDF sample was mixed with potassium bromide at a ratio of 1:100. The scanning conditions were from 400 to 4000 cm -1 Record 16 scans with a resolution of 4cm -1 , with potassium bromide as blank control. The spectra of okara SDF extracted under different treatment conditions are shown in Fig.11 shown.

[0093] All SDF at 3400cm -1Broad and strong absorption bands were observed near 2820 cm, which can be attributed to the vibration of hydrogen bonds in the hydroxyl groups of cellulose, hemicellulose and pectin. The absorption peak intensity of SDF increased after fermentation, especially the absorption peak intensity of FM-SDF was the largest, indicating that the structures of cellulose and hemicellulose were destroyed and new hydrogen bonds were generated. -1 The absorption peak at 1750cm -1 A new absorption peak appears at 1600 cm-1, which may be related to the C=O stretching vibration of the -COOH group in uronic acid; -1 The characteristic absorption diffraction peak of CC is at 1381cm -1 The absorption peak at 1200-950cm is caused by the bending vibration of CH; -1 The bands in the range are characteristic areas of carbohydrates (“fingerprint areas”), which mainly come from the vibration of COC bonds and CO bonds in polysaccharides. The difference in the peak strength of the “fingerprint areas” of the three SDFs indicates that during the fermentation of soybean dregs by Aspergillus oryzae, cellulose, hemicellulose, lignin, etc. are enzymatically hydrolyzed, and the glycosidic bonds, ether bonds, etc. are broken to release more COC bonds and CO bonds, making the “fingerprint area” absorption peak of FM-SDF stronger; at 775cm -1 The absorption peak near 611cm is caused by the skeleton vibration of the pyranose ring; -1 The absorption peak is due to the β-CH bending vibration.

[0094] The peak positions of SDF extracted by the three treatment methods were similar, and there was no obvious change in the functional groups and structures, but the absorption peak intensities were different, indicating that the three SDF structures were similar but the component contents were different, and the two-stage fermentation treatment method had the greatest impact on the SDF structure and components. After the structure was destroyed, it had more newly generated hydrogen bonds and aldehydes.

[0095] Example 9: X-ray diffraction analysis (XRD)

[0096] The X-ray diffraction spectrum of SDF was tested using a diffractometer. The diffraction intensity was scanned in the range of 5 to 50° (2θ angle range). The working voltage was 40 kV and the working current was 40 mA. The XRD patterns of different SDFs are shown in Figure 2. Fig.12 As shown. The three SDFs have similar morphologies in the diffraction angle range of 2θ from 5 to 50°, among which 2θ at 22.26° shows a strong diffraction peak, which is consistent with the type I crystal structure of cellulose. The crystallinity of U-SDF, F-SDF and FM-SDF are 13.54%, 11.72% and 10.46%, respectively. The crystallinity of the two fermented SDFs is lower than that of the unfermented ones, and the crystallinity of FM-SDF prepared by the two-stage method is the lowest, and the content of amorphous components increases.

[0097] Example 10: Thermogravimetric Analysis (TGA)

[0098] The SDF sample was placed in an aluminum crucible and heated from 30°C to 650°C at a heating rate of 10°C min-1. The TGA curves of different SDFs are shown in Figure 2. Fig.13 As shown. The curves of the three SDFs are similar and can be divided into three thermal degradation stages. In the first stage (30-210℃), the evaporation temperature and mass loss of U-SDF, F-SDF and FM-SDF are 68.63℃, 42.25℃, 72.16℃ and 8.43%, 11.78%, 7.41%, respectively; in the second stage (210-400℃), U-SDF, F-SDF and FM-SDF all have obvious mass losses, which are 47.92%, 60.23%, 53.99%, respectively. In the third stage (400-650℃), the mass loss of the three SDFs slows down, with the residual mass of U-SDF being the highest at 33.05%, followed by FM-SDF at 18.10%, and F-SDF at the lowest at 11.41%, indicating that the thermal stability of SDF decreases after fermentation, but the thermal stability of FM-SDF prepared by the two-stage method decreases less.

[0099] Example 11: Monosaccharide composition analysis

[0100] Sample hydrolysis: Accurately weigh 10 mg of the sample to be tested into a threaded headspace bottle, add 2 mL of 2 mol / L trifluoroacetic acid, and hydrolyze in an oven at 100°C for 6 hours. Take it out and cool it at room temperature for about 10 minutes, then transfer it to a rotating bottle. After rotary evaporation to dryness, add 1 mL of methanol and rotary evaporation to dryness, repeat three times, then add distilled water to make up to 1 mL, centrifuge and set aside.

[0101] PMP derivatization treatment: prepare 2mg / ml standard solutions of rhamnose, glucose, xylose, arabinose, galacturonic acid, and fucose monosaccharide, and take 5.0mg of each of the six monosaccharides and mix them to prepare a mixed standard solution. Take 300μL of sample solution / standard solution in a centrifuge tube, add 200μL of 0.5mol / LPMP methanol solution, mix well, add 300μL of 0.3mol / LNaOH solution, mix well, cool in a 70℃ water bath for 1h, take out after 10min, add 300μL of 0.3mol / LHCL solution to neutralize, then add 1mL of chloroform and vortex oscillation extraction, centrifuge at 8500r / min for 10min, retain the supernatant, and repeat the extraction three times. After filtering the supernatant with a 0.22μm filter membrane, inject it into the chromatographic injection bottle and wait for the machine.

[0102] Chromatographic conditions: The instrument model is Agilent 1260ⅡPrime; the chromatographic column is EC-C18, 250×4.6mm; the detector is a UV absorption detector.

[0103] Injection conditions: mobile phase was PBS phosphate buffer solution (10 mmol / l): acetonitrile = 83:17 (v / v); flow rate was 0.5 mL / min; injection volume was 10 μL; column temperature was 30°C; wavelength was 250 nm.

[0104] The monosaccharide compositions of the three SDFs are shown in Table 7 below.

[0105] Table 7: Monosaccharide composition

[0106]

[0107] from Fig.14 From the results in Table 7, it can be seen that the main monosaccharides of U-SDF are xylose, arabinose, galacturonic acid and rhamnose; the main monosaccharides of F-SDF and FM-SDF are galacturonic acid, xylose, rhamnose and fucose; after fermentation, the fucose, galacturonic acid, rhamnose and glucose in the two SDFs increased, and arabinose and xylose decreased; the glucose and galacturonic acid in F-SDF increased more, while the fucose and rhamnose in FM-SDF increased more.

[0108] Example 12: Analysis of SDF functional properties

[0109] (1) Water and oil holding capacity and expansion capacity

[0110] To determine the water holding capacity (WHC) of SDF, the SDF sample (0.5 g) was mixed with distilled water (10 mL) at 37°C, allowed to stand for 24 hours, and then centrifuged at 5000 r / min for 15 minutes to separate the supernatant, and the residue was collected and weighed. The calculation formula of WHC is as follows:

[0111] WHC(g / g)=W1 / W2

[0112] Where W1 is the weight of adsorbed water; W2 is the weight of SDF sample.

[0113] To determine the oil holding capacity (OHC) of SDF, SDF sample (0.2 g) was mixed with soybean oil (10 mL) at 37°C, allowed to stand for 1 hour, and then centrifuged at 5000 r / min for 15 minutes to discard the upper layer of oil, collect the residue and weigh it. The calculation formula of OHC is as follows:

[0114] OHC(g / g)=W1 / W2

[0115] Where W1 is the weight of adsorbed oil; W2 is the weight of SDF sample.

[0116] To measure the swelling capacity (SC) of SDF, place the SDF sample (0.5 g) in a 5 ml measuring cylinder, add distilled water to the 5 ml mark, shake well, seal and let stand at room temperature for 24 hours. Read the volume of the SDF sample before and after swelling. The calculation formula of SC is as follows: SC (ml / g) = (V1-V2) / W1

[0117] Where V1 is the volume of SDF after expansion; V2 is the volume of SDF before expansion; W1 is the weight of SDF sample.

[0118] The water holding capacity, oil holding capacity and swelling capacity results of the three SDFs are shown in Table 8 below.

[0119] Table 8: Water and oil holding capacity and swelling capacity of three SDFs

[0120]

[0121] It can be seen from the results in Table 8 that the water holding, oil holding and swelling capacity of SDF after fermentation are better than those of unfermented SDF. The water holding, oil holding and swelling capacity of F-SDF increased by 1.96, 1.01 and 2.02 times, respectively, while that of FM-SDF increased by 2.07, 1.07 and 2.31 times, respectively. Among the three groups of SDF, FM-SDF had the best water holding, oil holding and swelling capacity.

[0122] (2) Glucose adsorption capacity (GAC)

[0123] 1g SDF sample was mixed with 100mL glucose solution (1, 2.5, 5, 10, 15, 30mg / ml), shaken in a constant temperature shaking water bath at 37℃ for 6h, centrifuged at 5000r / min for 15min, and the glucose content in the supernatant was determined by DNS method. Similarly, distilled water was used instead of glucose solution as the blank group, and glucose solution without sample was used as the control group. The calculation formula of GAC is as follows:

[0124]

[0125] Wherein C1 is the glucose content of the sample (mg); C2 is the glucose content of the control group (mg); C0 is the glucose content of the blank group (mg); and M is the weight of the sample (g).

[0126] from Fig.15It can be seen that when the glucose concentration is less than 1 mg / ml, there is no significant difference in the adsorption capacity of the three SDFs. As the glucose concentration increases, the adsorption capacity of the three SDFs increases. After the glucose concentration reaches 15 mg / ml, the adsorption capacity of the three SDFs tends to be flat. Among them, the adsorption capacity of the fermented SDF is always higher than that of the unfermented group, and the adsorption capacity of FM-SDF is the highest. When the glucose concentration is 30 mg / ml, the glucose adsorption capacity of FM-SDF is 106.28 mg / g, which is 1.27 times that of U-SDF.

[0127] (3) α-Amylase activity inhibition ration (α-AAIR)

[0128] 1g SDF sample was mixed with 40mL potato starch solution (4%, w / v, pH 6.5) and 0.04g α-amylase, shaken in a constant temperature shaking water bath at 37°C for 1h, centrifuged at 5000r / min for 10min, and the glucose content of the supernatant was determined. 40mL potato starch solution without sample was used as the control group, and distilled water was used instead of potato starch to remove sugar from the sample as the blank group. The calculation formula of α-AAIR is as follows:

[0129]

[0130] Where Ac is the glucose content in the control group (mg); As is the glucose content in the sample (mg); Ao is the glucose content in the blank group (mg).

[0131] from Fig.16 It can be seen that the α-amylase inhibitory activities of U-SDF, F-SDF and FM-SDF are 7.52%, 11.54% and 12.02%, respectively. Among them, the inhibitory activity of FM-SDF is the highest, which is 1.60 times that of U-SDF, followed by F-SDF, which is 1.53 times that of U-SDF.

[0132] (4) Glucose dialysis retardation index (GDRI)

[0133] 0.2g SDF sample and 10mL (0.2g / 100mL) glucose solution were stirred in a magnetic stirrer for 1h, and then the mixture was transferred to a 15cm (8000-14000MWCO) dialysis bag. Each bag was placed in a beaker containing 200ml distilled water, and oscillated at 160r / min and 37℃ for 1 hour. 1mL of glucose dialysate was drawn every 15min, and the glucose content was determined by phenol-sulfuric acid visible spectrophotometry. At the same time, a control group (with glucose, no sample) and a blank group (with sample, no glucose) were prepared. The glucose dialysis retardation index was calculated as follows:

[0134]

[0135] Wherein A1 is the glucose concentration of the SDF sample solution (mg / ml); A2 is the glucose concentration of the blank group (mg / ml); A3 is the glucose concentration of the control group (mg / ml).

[0136] from Fig.17 It can be seen that as time goes on, the glucose dialysis delay index of the three SDFs first increases and then decreases, and all reach the maximum value at 30 minutes. Among them, the delay index of FM-SDF at 30 minutes is the highest, which is 62.21%, which is 1.63 times that of U-SDF in the same period. Within 60 minutes, the glucose dialysis delay index of FM-SDF is always the highest, followed by F-SDF.

[0137] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing soluble dietary fiber by pretreating bean dregs using a two-stage Aspergillus oryzae method, characterized in that: The following steps are involved: (1) grinding and crushing the dried bean dregs and passing through a 100-mesh sieve to obtain bean dregs powder; (2) adding glucose to the okara powder, adding drinking water with a pH of 6.8 at a solid-liquid ratio of 1:4, and stirring evenly; (3) subjecting the okara powder to microwave treatment, adding drinking water with a pH of 6.8 to a solid-liquid ratio of 1:4 after cooling, and stirring to obtain an okara fermentation medium; (4) sterilizing the okara fermentation medium at 121° C. for 15-20 min, cooling to room temperature, and setting aside; (5) adding 7% (W / W) Aspergillus oryzae mycelium to the cooled okara fermentation medium under sterile conditions, culturing at 29° C. for 60 h, and performing a fermentation step to obtain fermented okara; (6) continuing to culture the fermented okara at 50° C. and performing a second-stage enzymatic hydrolysis to obtain enzymatically hydrolyzed okara; (7) The okara after enzymatic hydrolysis is taken out, an appropriate amount of clean water is added, and the okara is extracted in a water bath for 2 h. After centrifugation, alcohol precipitation, elution, and drying, the okara soluble dietary fiber is obtained.

2. The method for preparing soluble dietary fiber by pretreating bean dregs by two-stage Aspergillus oryzae method according to claim 1, characterized in that: The amount of glucose added in step (2) is 2% of the amount of dregs powder added.

3. The method for preparing soluble dietary fiber by pretreating bean dregs by two-stage Aspergillus oryzae method according to claim 1, characterized in that: In the step (2), 1 mol / L sodium hydroxide and 1 mol / L hydrochloric acid are used to adjust the pH of the drinking water to 6.

8.

4. The method for preparing soluble dietary fiber by pretreating bean dregs by two-stage Aspergillus oryzae method according to claim 1, characterized in that: In the step (3), the power of the microwave is 700 W and the processing time is 70 s.

5. The method for preparing soluble dietary fiber by pretreating bean dregs by two-stage Aspergillus oryzae method according to claim 1, characterized in that: In the step (5), the Aspergillus oryzae stored on the slant is inoculated into a sterilized PDA liquid culture medium, cultured in a shaking incubator at 30° C. and 170 r / min for 48 hours for activation, centrifuged at 5000 r / min for 1 minute, and the supernatant is discarded to obtain the Aspergillus oryzae mycelium.

6. The method for preparing soluble dietary fiber by pretreating bean dregs by two-stage Aspergillus oryzae method according to claim 1, characterized in that: The time of the second stage enzymatic hydrolysis in step (6) is 36 hours.

7. The method for preparing soluble dietary fiber by pretreating bean dregs by two-stage Aspergillus oryzae method according to claim 1, characterized in that: The temperature of the water bath extraction in step (7) is 80°C.

8. Okara soluble dietary fiber prepared by the method according to any one of claims 1 to 7.

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