Fermented rice bran as well as preparation method and application thereof

Abstract

The invention discloses fermented rice bran as well as a preparation method and application thereof, and belongs to the technical field of food preparation. The preparation method of the fermented rice bran comprises the following steps: adding water into rice bran, cooking, cooling, inoculating rhizopus oryzae and lactobacillus fermentum, and fermenting at constant temperature to obtain the fermented rice bran. The prepared fermented rice bran can be used for preparing high-fiber rice bran biscuits, and the biscuits prepared from the fermented rice bran are high in sensory score, good in water-holding capacity and oil-holding capacity, high in content of total phenols and total flavonoids and excellent in oxidation resistance.

Description

Technical Field

[0001] The invention relates to the technical field of food preparation, in particular to fermented rice bran and a preparation method and application thereof. Background Art

[0002] Rice bran is a high value-added byproduct of rice processing, accounting for about 8% of the total weight of rice. Rice bran is rich in protein, fat, carbohydrates, dietary fiber and minerals, as well as unsaturated fatty acids, tocopherols, tocotrienols, oryzanol, phytic acid and other bioactive substances, but at present it is mostly used as feed or abandoned, with extremely low effective utilization rate and product added value.

[0003] Biscuits are snack foods made from wheat flour as the main raw material through baking. With the deepening of health concepts, biscuits are gradually changing from traditional grain and oil foods to functional nutritional foods. Applying rice bran, which is rich in nutrients such as dietary fiber and protein, to biscuits can effectively improve the nutritional value and health benefits of biscuits, but the addition of rice bran will inevitably affect the sensory quality of biscuits, resulting in problems such as increased hardness, reduced specific volume, and poor crispness of the taste. Summary of the invention

[0004] The purpose of the present invention is to provide a fermented rice bran and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention: a method for preparing fermented rice bran, comprising the following steps:

[0007] The rice bran is boiled with water, inoculated with Rhizopus oryzae and Lactobacillus fermentans after cooling, and fermented at a constant temperature to obtain the fermented rice bran.

[0008] Furthermore, the mass ratio of the rice bran to water is 1:2.25; and the cooking time is 60 minutes.

[0009] Further, the fermented Lactobacillus is inoculated in the form of fermentation liquid;

[0010] The ratio of the fermentation liquid to rice bran is 10-12 mL: 100 g; the ratio of the fermentation liquid to Rhizopus oryzae is 11 mL: 3-11 g;

[0011] The method for preparing the fermentation liquid comprises the following steps: activating and culturing the fermented lactobacillus and then fermenting and culturing it to obtain the fermentation liquid.

[0012] Furthermore, the method for preparing the fermentation liquid comprises the following steps: inoculating the fermented Lactobacillus preserved liquid into a liquid culture medium for a first activation culture to obtain a first activated culture liquid; inoculating the first activated culture liquid into a liquid culture medium for a second activation culture to obtain a second activated culture liquid; inoculating the second activated culture liquid into a liquid culture medium for fermentation culture to obtain the fermentation liquid.

[0013] Furthermore, during the first activation culture, the volume ratio of the fermented Lactobacillus preservation solution to the liquid culture medium is 1:150; the temperature of the first activation culture is 33° C., and the time is 24 hours;

[0014] When performing the second activation culture, the volume ratio of the first activation culture solution to the liquid culture medium is 3:100; the temperature of the second activation culture is 33° C., and the time is 24 hours;

[0015] During fermentation, the volume ratio of the second activation culture solution to the liquid culture medium is 3:10; the temperature of the fermentation is 33°C and the time is 24 hours;

[0016] The liquid culture medium consists of apple juice with a volume ratio of 85:15 and a whey powder solution with a concentration of 8 wt.%.

[0017] Furthermore, the temperature of the constant temperature fermentation is 35-39° C., and the time is 36-48 hours.

[0018] The second technical solution of the present invention: fermented rice bran prepared by the above preparation method.

[0019] The third technical solution of the present invention: an application of the above fermented rice bran in the preparation of biscuits.

[0020] Technical solution 4 of the present invention: A high-fiber rice bran biscuit, comprising the following raw materials: a reference raw material and an additive;

[0021] The reference raw materials, in terms of mass percentage, include the following raw materials: 6-14% of the fermented rice bran and 86-94% of low-gluten flour;

[0022] The additives include salt, baking soda, baking powder, whole milk powder, water, powdered sugar, butter and whole egg liquid, and the amounts used are 1%, 0.5%, 1.5%, 10%, 20%, 20-40%, 10-30% and 10-30% of the mass of the reference raw materials respectively.

[0023] Technical solution 5 of the present invention: A method for preparing the above-mentioned high-fiber rice bran biscuits, comprising the following steps:

[0024] The raw materials are premixed and then prepared into dough, which is then left to stand, sheeted, punched and baked to obtain the high-fiber rice bran biscuits.

[0025] The present invention discloses the following technical effects:

[0026] The fermented rice bran prepared by the invention can be used to prepare high-fiber rice bran biscuits. The biscuits prepared by using the fermented rice bran have high sensory scores, good water holding capacity and oil holding capacity, high total phenol and total flavonoid contents, and excellent antioxidant capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The results of the effects of different inoculation amounts of GBJ fermented liquor with Lactobacillus fermentans on the total acid content, reducing sugar content, and sensory organs;

[0029] Figure 2 The results of the effects of different inoculation ratios of Lactobacillus GBJ fermentation broth and Rhizopus oryzae on total acid content, reducing sugar content, and sensory organs;

[0030] Figure 3 The results of the effects of different fermentation temperatures on total acid content, reducing sugar content, and sensory perception;

[0031] Figure 4 The results of the effects of different fermentation times on total acid content, reducing sugar content, and sensory perception;

[0032] Figure 5 The effect of rice bran extract before and after fermentation on DPPH clearance;

[0033] Figure 6 ABTS + Effect of clearance rate;

[0034] Figure 7 are SEM images of F-SDF and B-SDF, wherein A is the SEM image of B-SDF at a magnification of 5000 times, B is the SEM image of B-SDF at a magnification of 10000 times, C is the SEM image of F-SDF at a magnification of 5000 times, and D is the SEM image of F-SDF at a magnification of 10000 times;

[0035] Figure 8 is the gel chromatogram of F-SDF;

[0036] Fig. 9is the ion chromatogram of F-SDF, where A is the monosaccharide standard and B is the monosaccharide composition of F-SDF;

[0037] Fig.10 The effect of fermentation on the antioxidant capacity of SDF, where (A) is the effect of SDF before and after fermentation on the DPPH clearance rate, and (B) is the effect of SDF before and after fermentation on ABTS + The effect of SDF on the scavenging rate, (C) the effect of SDF before and after fermentation on the FRAP reducing ability, (D) the effect of SDF on the scavenging rate of hydroxyl radicals;

[0038] Fig.11 The effect of SDF on the viability of RAW264.7 cells;

[0039] Fig.12 The effect of SDF on the phagocytosis rate of neutral red in RAW264.7 cells;

[0040] Fig.13 The effect of SDF on the release of nitric oxide (NO) in RAW264.7 cells;

[0041] Fig.14 The effect of SDF on the secretion of cytokines (TNF-α, IL-1β, IL-6, IL-10) in RAW264.7 cells, where (A) is IL-1β, (B) is IL-6, (C) is IL-10, and (D) is TNF-α;

[0042] Fig.15 To study the effect of the amount of fermented rice bran added on the quality of biscuits;

[0043] Fig.16 The effect of the amount of powdered sugar added on the quality of cookies;

[0044] Fig.17 The effect of butter addition on biscuit quality;

[0045] Fig.18 The effect of whole egg liquid addition on biscuit quality;

[0046] Fig.19 The biscuits without adding rice bran prepared in Comparative Example 1 and the biscuits with adding fermented rice bran prepared in Example 12 are physical pictures, wherein (A) is the biscuits without adding rice bran prepared in Comparative Example 1, and (B) is the biscuits with adding fermented rice bran prepared in Example 12;

[0047] Fig. 20 Comparison of the antioxidant capacity of different biscuits, where (A) shows the effect of different biscuits on DPPH scavenging rate, and (B) shows the effect of SDF of different biscuits on ABTS +(C) is the effect of different biscuits on the FRAP reducing ability, and (D) is the effect of different biscuits on the hydroxyl radical scavenging ability. DETAILED DESCRIPTION

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] When preparing fermented rice bran, the method for determining the ratio of rice bran to water is as follows: weigh 100.0g of rice bran and pour it into a beaker, add an appropriate amount of distilled water and stir evenly. Observe the water absorption state of the rice bran, and determine the optimal ratio when all the rice bran is moistened and there is no excess free water.

[0050] When preparing fermented rice bran, the method for determining the cooking time of the rice bran is: weigh 200g of rice bran, add 450mL of cold boiled water and cook, and check every 15 minutes after boiling. The cooking time is selected based on the rice bran being cooked through, the hardness is reduced, and there is a certain grain aroma.

[0051] The Rhizopus oryzae and Lactobacillus fermentum GBJ (Lactobacillus fermentum GBJ preserved solution) used in the present invention were purchased from commercial sources. 10 cfu / g; the effective viable count of Lactobacillus fermentum GBJ was 2.4×10 10 cfu / mL.

[0052] The apple juice used in the present invention is Huiyuan apple juice.

[0053] Example 1

[0054] A preparation method of fermented rice bran:

[0055] (1) Culture medium preparation

[0056] Apple juice and a whey powder solution with a concentration of 8 wt.% were mixed in a volume ratio of 85:15, and after packaging, they were sterilized at 108° C. for 15 min and cooled to room temperature to obtain a liquid culture medium.

[0057] (2) Preparation of Lactobacillus fermentum GBJ fermentation broth

[0058] 1 mL of fermented Lactobacillus GBJ was inoculated into 150 mL of liquid culture medium for the first activation culture (the activation culture temperature was 33° C. and the time was 24 h) to obtain the first activation culture solution;

[0059] The first activated culture solution was inoculated into a liquid culture medium (the volume ratio of the first activated culture solution to the liquid culture medium was 3:100, i.e., the inoculation amount was 3%) for a second activation culture (the culture temperature was 33° C., and the culture time was 24 h) to obtain a second activated culture solution;

[0060] The second activated culture solution was inoculated into a liquid culture medium (the volume ratio of the second activated culture solution to the liquid culture medium was 3:10, i.e. the inoculation amount was 3%) for fermentation (the culture temperature was 33°C and the time was 24h) to obtain a fermented Lactobacillus GBJ fermentation solution.

[0061] (3) Weigh 200 g of rice bran, wash it, add 450 mL of boiled water (the material-water mass ratio is 1:2.25 (rice bran: boiled water)), stir it evenly, boil it in boiling water for 60 min, and after the rice bran is cooled, inoculate Rhizopus oryzae and fermentation broth of Lactobacillus fermentum GBJ respectively, mix them evenly, and place them in an incubator for constant temperature fermentation to obtain fermented rice bran.

[0062] Wherein, the usage ratio of fermented lactobacillus GBJ fermented liquid and rice bran is 8mL: 100g (inoculum size 8%, mL / g), 9mL: 100g (inoculum size 9%, mL / g), 10mL: 100g (inoculum size 10%, mL / g), 11mL: 100g (inoculum size 11%, mL / g), 12mL: 100g (inoculum size 12%, mL / g);

[0063] The dosage ratio of Lactobacillus fermentation GBJ fermentation liquid and Rhizopus oryzae is 11 mL: 5 g.

[0064] The temperature of constant temperature fermentation is 33℃ and the time is 36h.

[0065] Example 2

[0066] Same as Example 1, except that the ratio of Lactobacillus fermentum GBJ fermentation liquid to rice bran is 11 mL: 100 g;

[0067] The dosage ratios of Lactobacillus GBJ fermentation broth and Rhizopus oryzae were 11 mL: 3 g, 11 mL: 5 g, 11 mL: 7 g, 11 mL: 9 g, and 11 mL: 11 g, respectively.

[0068] The temperature of constant temperature fermentation is 33℃ and the time is 36h.

[0069] Example 3

[0070] Same as Example 1, except that the ratio of Lactobacillus fermentum GBJ fermentation liquid to rice bran is 11 mL: 100 g;

[0071] The dosage ratio of Lactobacillus fermentum GBJ fermentation liquid and Rhizopus oryzae was 11 mL: 9 g;

[0072] The temperatures of constant temperature fermentation were 31°C, 33°C, 35°C, 37°C and 39°C, and the time was 36h.

[0073] Example 4

[0074] Same as Example 1, except that the ratio of Lactobacillus fermentum GBJ fermentation liquid to rice bran is 11 mL: 100 g;

[0075] The dosage ratio of Lactobacillus fermentum GBJ fermentation liquid and Rhizopus oryzae was 11 mL: 9 g;

[0076] The temperature of constant temperature fermentation was 37°C, and the time was 24h, 30h, 36h, 42h and 48h respectively.

[0077] Effect Example 1

[0078] (I) Determine the total acid content and reducing sugar content in the fermented rice bran prepared in Examples 1 to 4, the determination standard or method is as follows:

[0079] Determination of total acid content: Determination is carried out in accordance with GB5009.239-2016 "National Food Safety Standard Determination of Food Acidity".

[0080] Determination of reducing sugar content: accurately weigh 1g of fermented rice bran, add 50mL of distilled water, grind to homogenate, centrifuge at 3800r / min for 15min, take the supernatant and make it up to 250mL to obtain the sample solution. Take 1mL of the sample solution and determine the reducing sugar content using the 3,5-dinitrosalicylic acid colorimetric method.

[0081] (ii) sensory evaluation of the fermented rice bran prepared in Examples 1 to 4 was performed, and the evaluation method was as follows:

[0082] Select more than 10 teachers or students to conduct sensory evaluation of the color, shape, smell, taste, etc. of rice bran by visual inspection, nose smelling, and mouth tasting. The full score is 10, and the sensory evaluation criteria are shown in Table 1.

[0083] Table 1 Sensory evaluation table

[0084]

[0085] The results of the effects of different inoculation amounts of Lactobacillus fermentum GBJ fermentation broth on total acid content, reducing sugar content, and sensory organs in Example 1 are shown in Table 1. Figure 1 .

[0086] from Figure 1It can be seen that the total acid content of fermented rice bran increases with the increase of inoculation amount, reaching a maximum value of 1.02%±0.01% at an inoculation amount of 12%. This is because Rhizopus oryzae and fermented Lactobacillus GBJ separate and utilize carbohydrates to produce organic acids such as lactic acid during the entire fermentation process, resulting in an increase in the total acid content. The reducing sugar content increases with the increase of inoculation amount, reaching a maximum value of (8.76±0.1) g / 100g at an inoculation amount of 12%, because the increase in the inoculation amount of Rhizopus oryzae increases the output of starch saccharifying enzymes. The sensory score increases first and then decreases with the increase of inoculation amount, and decreases at an inoculation amount of 12%, because the excessive inoculation amount leads to excessive acidity and affects the taste. The highest score is (8.6±0.10) points at an inoculation amount of 11%.

[0087] The results of the effects of different inoculation ratios of Lactobacillus GBJ fermentation broth and Rhizopus oryzae on the total acid content, reducing sugar content, and sensory organs in Example 2 are shown in Figure 2 .

[0088] from Figure 2 It can be seen that the total acid content of fermented rice bran increases with the increase of inoculation ratio, and reaches a maximum value of 1.07% ± 0.02% when the inoculation ratio is 11mL: 11g, because the two bacteria continuously produce organic acids such as lactic acid during the fermentation process. The reducing sugar content increases with the increase of inoculation ratio, and its maximum value is (10.57 ± 0.38) g / 100g when the inoculation ratio is 11mL: 11g, because the increase of inoculation ratio increases, and the increase of Rhizopus oryzae increases the output of starch saccharifying enzyme. The sensory score increases first and then decreases with the increase of inoculation ratio, and the highest score is (8.9 ± 0.10) points when it is 11mL: 9g.

[0089] The results of the effects of different fermentation temperatures on total acid content, reducing sugar content, and sensory organs in Example 3 are shown in Figure 3 .

[0090] from Figure 3 It can be seen that the total acid content of fermented rice bran tends to decrease with the increase of fermentation temperature, and the lowest is 0.70% ± 0.02% at 39°C. This is because Rhizopus oryzae utilizes part of the organic acid at the end of fermentation, resulting in a decrease in the total acid content. The reducing sugar content increases first and then decreases with the increase of fermentation temperature. The reducing sugar content reaches a maximum of (13.02 ± 0.09) g / 100g at 37°C. This is because the activity of Rhizopus oryzae enzyme increases with the increase of temperature, and when the temperature is too high, the activity of Rhizopus oryzae decreases and the reducing sugar content decreases. The sensory score increases first and then decreases with the increase of fermentation temperature, reaching a maximum of (9.0 ± 0.10) points at 37°C. The reason for the decrease in sensory score with the increase of fermentation temperature is that black spores are produced by long-term high temperature fermentation of rice bran, resulting in a decrease in sensory score.

[0091] The results of the effects of different fermentation times on total acid content, reducing sugar content, and sensory organs in Example 4 are shown in Figure 4 .

[0092] from Figure 4 It can be seen that the total acid content of fermented rice bran shows a trend of first increasing and then decreasing with the increase of fermentation time, reaching a maximum value of 0.97% ± 0.02% at 42h, because Rhizopus oryzae and fermented Lactobacillus will produce acid in the early stage of fermentation, but Rhizopus oryzae will consume part of the acid in the middle and late stages of fermentation, resulting in a decrease in the total acid content. The reducing sugar content first decreases and then increases with the increase of fermentation time, increasing to (11.78 ± 0.34) g / 100g at 48h of fermentation. The initial reduction of reducing sugar is because the starch saccharifying enzyme in Rhizopus oryzae is consumed as the fermentation progresses. At 42h, the fermentation state of the two bacteria tends to be stable, and the reducing sugar slowly increases. The sensory score first increases and then decreases with the increase of fermentation time, and the highest is (9.1 ± 0.10) points at an inoculation amount of 42h.

[0093] Example 5

[0094] Orthogonal Experimental Design:

[0095] Based on the single factor experimental results of Examples 1 to 4, the inoculation amount (inoculation amount of Lactobacillus fermentation GBJ fermentation broth), inoculation ratio (inoculation ratio of Lactobacillus fermentation GBJ fermentation broth and Rhizopus oryzae), fermentation temperature and fermentation time were used as experimental factors (represented by A, B, C, and D, respectively), and the horizontal parameters were set. The orthogonal optimization test was carried out, and the sensory score was used as an indicator. L9 (3 4 )Orthogonal table was used to optimize the rice bran fermentation process. The orthogonal factor level table is shown in Table 2.

[0096] Table 2 Orthogonal test factor level table

[0097]

[0098]

[0099] The orthogonal test results are shown in Table 3.

[0100] Table 3L9(3 4 )Orthogonal Experimental Design and Results

[0101] Experiment No. A(Inoculation amount) B (Vaccination ratio) C (temperature) D(Time) Total score (points) 1 1(10％) 1(11∶7) 1(35℃) 1(36h) 8.2 2 1 2(11∶9) 2(37℃) 2(42h) 9.1 3 1 3(11∶11) 3(39℃) 3(48h) 7.2 4 2(11％) 1 2 3 8.6 5 2 2 3 1 7.5 6 2 3 1 2 8.3 7 3(12％) 1 3 2 7.5 8 3 2 1 3 8.1 9 3 3 2 1 8.0 K1 24.5 24.3 24.6 23.7 -- K2 24.4 24.7 25.7 24.9 -- K3 23.6 23.5 22.2 23.9 -- k1 8.17 8.1 8.2 7.9 -- k2 8.13 8.23 8.57 8.3 -- k3 7.87 7.83 7.4 7.97 -- R 0.3 0.4 1.17 0.4 --

[0102] The larger the R value, the greater the influence of the factor on the experimental results. The influence of the four single factors on fermented rice bran is factor C>D=B>A. The optimal formula combination is A1B2C2D2.

[0103] Example 6

[0104] Same as Example 1, except that the inoculation amount of the fermentation liquid of Lactobacillus fermentum GBJ is 10% of the mass of the rice bran;

[0105] The inoculation ratio of Lactobacillus fermentum GBJ fermentation broth and Rhizopus oryzae was 11 mL: 9 g;

[0106] The temperature of constant temperature fermentation is 37℃ and the time is 42h.

[0107] The fermented rice bran prepared in this embodiment has a small amount of white mycelium on the surface with a light wine aroma, moderate sweetness and sourness, and moderate hardness and softness. The sensory score is (9.2±0.10) points. Therefore, the optimal process parameters for rice bran fermentation are a material-liquid ratio (mass ratio of rice bran and water) of 1:2.25, a cooking time of 60min, a fermentation time of 42h, an inoculation amount (inoculation amount of fermented Lactobacillus GBJ fermentation liquid) of 10%, an inoculation ratio (inoculation ratio of fermented Lactobacillus GBJ fermentation liquid and Rhizopus oryzae) of 11mL:9g, and a fermentation temperature of 37°C.

[0108] Effect Example 2

[0109] (I) Extraction and determination of total phenol content

[0110] (1) Preparation method of rice bran extract

[0111] A. The preparation method of rice bran before fermentation is as follows: weigh 200g of rice bran, add 450mL of cold boiled water (the material-water mass ratio is 1:2.25 (rice bran: cold boiled water)) after washing, stir evenly, boil in boiling water for 60min, inoculate Rhizopus oryzae and fermentation liquid of Lactobacillus fermentum GBJ respectively after the rice bran is cooled, mix evenly to obtain the rice bran before fermentation.

[0112] The inoculation amount of the fermented liquid of Lactobacillus fermentum GBJ was 11% of the mass of rice bran;

[0113] The inoculation ratio of Lactobacillus fermentum GBJ fermentation broth and Rhizopus oryzae was 11 mL:9 g.

[0114] B. The rice bran before and after fermentation (the rice bran after fermentation is the fermented rice bran prepared in Example 6) is dried in an electric constant temperature incubator at 60°C for later use. Then, it is crushed by a pulverizer and passed through a 100 mesh sieve for later use. Weigh 2.5g of the crushed rice bran, add 80vol.% ethanol solution according to a solid-liquid ratio of 1g:10mL, place in a 50°C water bath, and extract with 100W ultrasonic for 4h, then centrifuge at 4°C, 10000×g for 15min, recycle ethanol in the supernatant on a rotary evaporator, concentrate the sample, add deionized water to make the volume 25mL, and obtain the sample extract to be tested.

[0115] (2) Drawing of gallic acid standard curve

[0116] Prepare a standard solution of gallic acid with a concentration of 1 mg / mL, and pipette 0.2, 0.4, 0.6, 0.8, and 1 mL of the standard solution into a 10 mL volumetric flask, and add deionized water to the volumetric flask to make up the volume. Take 1 mL of gallic acid solution of different concentrations, add 2.5 mL of deionized water and 0.5 mL of 1 mol / L Folin phenol reagent, mix well on a vortex oscillator, and let it stand at room temperature for 8 minutes. Add 1.5 mL of 7.5 wt.% Na2CO3 solution to the test tubes, mix well on a vortex oscillator, and let it stand at room temperature in the dark for 1 hour. Use the concentration of gallic acid solution (μg / mL) as the horizontal coordinate and the absorbance as the vertical coordinate to draw the gallic acid standard curve (y=0.0028x-0.00001, R 2 =0.991).

[0117] (3) Determination of total phenol content

[0118] Determination of total phenol content in the sample extract: Take 2 mL of the sample extract, determine it according to the concentration determination method of the gallic acid standard solution, and calculate the total phenol content of the sample. The result is expressed as the phenol content with the same absorbance value as gallic acid per 100 g (fresh weight) of the sample.

[0119] The total phenolic content in rice samples was calculated according to the following formula:

[0120] Total phenol content of sample (mg GAE / 100g FW) = (C × V × 0.1) + W;

[0121] Where:

[0122] C: Gallic acid concentration found on the standard curve, μg / mL;

[0123] V: total volume of the extract, mL;

[0124] W: sample weight, g.

[0125] The total phenolic contents in rice bran before and after fermentation were calculated to be (0.168±0.003) and (0.230±0.003) mg / g gallic acid equivalent, respectively.

[0126] (II) DPPH free radical scavenging and ABTS + Free radical scavenging activity assay

[0127] (1) DPPH free radical scavenging experiment

[0128] To 2 mL of 0.2 mmol / L DPPH radical anhydrous ethanol solution, equal volumes of rice bran extracts of different dilution multiples (sample solution, undiluted rice bran extract is the sample extract prepared in step (a)) were added, reacted for 30 min in the dark, and the absorbance (A) was measured at 517 nm. i The control group was set up with anhydrous ethanol to replace the DPPH free radical solution (A j ), the blank group was ethanol instead of sample (A0). Ascorbic acid (Vc) was used as a positive control.

[0129] The scavenging rate of the extract on DPPH free radicals was calculated according to the formula, and ascorbic acid (Vc) was used as a positive control. The results are shown in Figure 5 .

[0130]

[0131] Where:

[0132] A i : Absorbance value of 2mL sample + 2mL DPPH;

[0133] A j : Absorbance value of 2mL sample + 2mL anhydrous ethanol;

[0134] A0: absorbance value of 2mL ethanol + 2mL DPPH.

[0135] (2)ABTS + Free radical scavenging activity assay

[0136] 10mL of ABTS with a concentration of 7mmol / L + The free radical solution was mixed with an equal volume of a 2.45 mmol / L K2S2O8 solution and allowed to stand for 16 h in the dark at room temperature to obtain a mother solution. The mother solution was diluted with anhydrous ethanol and ABTS was obtained when the absorbance at 734 nm was stable at 0.70 ± 0.02. + Working solution. Take 1 mL of rice bran extract (sample solution, undiluted rice bran extract is the sample extract prepared in step (i)) with different dilution multiples, and add 4 mL of ABTS + Free radical working solution, measure A after 6 minutes of reaction 734nm Value (A i ), the control group was anhydrous ethanol instead of ABTS + Free radical solution (A j ), the blank group was distilled water instead of sample (A0).

[0137] ABTS was calculated by the formula + The free radical scavenging rate was measured with ascorbic acid (Vc) as a positive control. Figure 6 .

[0138]

[0139] Where:

[0140] A i :1mL sample + 4mL ABTS + The absorbance value of

[0141] A j : Absorbance value of 1mL sample + 4mL anhydrous ethanol;

[0142] A0: 1mL distilled water + 4mL ABTS + The absorbance value.

[0143] from Figure 5 and Figure 6 It can be seen that the DPPH free radical scavenging ability of undiluted rice bran extract (stock solution) and ABTS + The free radical scavenging ability increased from 84.81% ± 0.27% and 74.51% ± 0.32% before fermentation to 87.30% ± 0.80% and 76.63% ± 0.82%, respectively. This shows that fermentation has a promoting effect on the antioxidant capacity of rice bran. The role of fermentation is mainly reflected in changing the structure and type of polyphenols, turning bound polyphenols into free states, thereby improving their antioxidant and other biological activities.

[0144] (III) Defatting of rice bran and preparation of soluble dietary fiber from defatted rice bran

[0145] (1) Defatting of rice bran

[0146] Accurately weigh an appropriate amount of rice bran before and after fermentation (the rice bran after fermentation is the fermented rice bran prepared in Example 6, and the rice bran before fermentation is prepared in step (i)), add n-hexane to the rice bran at a solid-liquid ratio of 1g:2.5mL, soak for 1h, wash with 75vol.% ethanol and filter, repeat 3 times, place in an electric constant temperature culture, dry at 60°C for 2h, obtain defatted rice bran, and set aside at room temperature.

[0147] (2) Preparation of defatted rice bran soluble dietary fiber

[0148] 10 g of defatted rice bran before and after fermentation was weighed, 100 mL of distilled water was added, and the fermentation was performed with α-amylase (40000 U / g) at pH 6.0, enzyme addition amount 0.09% (0.09% of the mass of rice bran) and enzymolysis temperature 75 ° C for 3.5 h, and then alkaline protease (200000 U / g) at pH 8.0, enzyme addition amount 1.0% (accounting for 1.0% of the rice bran quality) and enzymolysis temperature 60 ℃ enzymolysis 2.5h, boiling water bath enzyme inactivation 10min; After cooling to room temperature, centrifugal 10min at 10000r / min, collect supernatant, the supernatant is adjusted to pH 7 with 10wt.%NaOH solution, then concentrated to 1 / 4 of the original volume with rotary evaporator, placed 4 ℃ of alcohol precipitation for 8h, centrifugal 10min at 10000r / min, the precipitation is redissolved, and lyophilized after dialysis for 24h to obtain fermented rice bran soluble dietary fiber (F-SDF) and unfermented rice bran soluble dietary fiber (B-SDF), the results are shown in Table 4.

[0149] Table 4 Effect of fermentation on dietary fiber content

[0150] Dietary fiber content (mg / g) Before fermentation After fermentation IDF <![CDATA[826.1±0.083 a ]]> <![CDATA[878.9±0.109 b ]]> SDF <![CDATA[6.500±0.302 a ]]> <![CDATA[8.780±0.154 b ]]>

[0151] Note: The results are expressed as the dietary fiber content per g (fresh weight) of sample.

[0152] The results are expressed as mean ± standard deviation (n = 3). Values ​​with different letters in the same column are significantly different (p < 0.5).

[0153] As can be seen from Table 4, after fermentation, the insoluble dietary fiber content (IDF) and soluble dietary fiber (SDF) of rice bran increased. IDF increased from (826.1±0.083) mg / g before fermentation to (878.9±0.109) mg / g, and SDF increased from (6.500±0.302) mg / g before fermentation to (8.780±0.154) mg / g. During the fermentation process, microorganisms will use starch and protein in rice bran as nutrient sources for metabolic activities. The degree of utilization of dietary fiber (SDF and IDF) by microorganisms during the fermentation process is relatively low. In addition, the large consumption of starch and protein increases their content ratio in fermented rice bran. At the same time, cellulase (produced by Rhizopus oryzae during the fermentation process) can decompose cellulose, a macromolecular polysaccharide, into smaller fiber fragments, so that the originally insoluble cellulose is converted into soluble fiber form to a certain extent. In addition, during the fermentation process, microbial metabolism produces acidic substances, which lowers the pH value of the fermentation environment. In an acidic environment, the structure of some dietary fibers will change, making them more soluble. Therefore, the SDF ratio will increase further.

[0154] (IV) Scanning electron microscope (SEM) observation

[0155] The fermented rice bran soluble dietary fiber (F-SDF) and the unfermented rice bran soluble dietary fiber (B-SDF) prepared in step (iii) were firstly subjected to gold spraying on the surface of the material using a sputtering coating machine, and then the surface morphology of the rice bran F-SDF and B-SDF was observed using an electron microscope at an accelerating voltage of 10 kV. Figure 7 , Figure 7 A is a SEM image of B-SDF with a magnification of 5000 times, B is a SEM image of B-SDF with a magnification of 10000 times, C is a SEM image of F-SDF with a magnification of 5000 times, and D is a SEM image of F-SDF with a magnification of 10000 times.

[0156] from Figure 7 It can be seen that at the same magnification, B-SDF presents a relatively flat surface with a relatively small specific surface area. However, F-SDF has a larger specific surface area and a loose spatial structure. In addition, the number of stripe structures of F-SDF is much greater than that of B-SDF. It is this loose structure that promotes the hydration and adsorption performance of the sample. Based on this, it is speculated that F-SDF performs better in terms of water holding capacity, oil holding capacity and adsorption capacity.

[0157] (V) Monosaccharide composition analysis and molecular weight (Mw) determination

[0158] (1) Monosaccharide composition analysis

[0159] Weigh 5 mg of the F-SDF sample prepared in step (iii), add 1 mL of 2M TFA acid solution, heat at 121°C for 2 h, pass nitrogen, and blow dry. Add methanol to wash, blow dry again, and repeat the operation 2 to 3 times. Add sterile water to dissolve, and transfer to a chromatographic bottle for testing.

[0160] The chromatographic system used was the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), and the monosaccharide components were analyzed and detected using an electrochemical detector. TM CarboPac TMPA20 (150×3.0 mm, 10 μm) liquid chromatography column; injection volume, 5 μL; mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 mL / min; column temperature, 30°C; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min Phase A / phase B / phase C (95:5:0, V / V), 60 min phase A / phase B / phase C (95:5:0, V / V).

[0161] (2) Molecular weight (Mw) determination

[0162] The F-SDF sample prepared in step (iii) was dissolved in a 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm before being tested on a machine. Molecular weight analysis was performed using a gel chromatography-differential-multi-angle laser light scattering system, the liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOSⅡ (Wyatt technology, CA, USA). Gel exclusion chromatography columns Ohpak SB-805HQ (300×8mm) and OhpakSB-803HQ (300×8mm) were used in series. The column temperature was 45°C, the injection volume was 100 μL, the mobile phase was 0.1M NaNO3 (containing 0.02% NaN3), and the flow rate was 0.6 mL / min.

[0163] The gel chromatogram of F-SDF is shown in Figure 8 . Figure 8 The red line in the figure represents the multi-angle laser light scattering signal (ie LS, unit is V), the blue line represents the difference signal (ie RI, unit is RIU), and the black line represents the molecular weight fitted by the two signals.

[0164] The ion chromatogram of F-SDF is shown in Fig. 9 . Fig. 9 A is the monosaccharide standard, and B is the monosaccharide composition of F-SDF.

[0165] The average molecular weight of fermented rice bran F-SDF was determined to be 2.84×10 7Da is mainly composed of fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), galacturonic acid (Gal-UA) and glucuronic acid (Glc-UA), with a molar ratio of 2.00:3.78:9.75:12.18:15.65:4.78:6.10:1.00:1.90.

[0166] (VI) SDF functional characteristics

[0167] (1) Effect of fermentation on water and oil holding capacity of SDF

[0168] A. Water Holding Capacity (WHC) Determination

[0169] The F-SDF and B-SDF (0.5 g) prepared in step (iii) were dissolved in 10 mL of distilled water respectively and placed at 37°C for 1 h. After centrifugation at 4800 r / min for 10 min, the precipitate was weighed (wet weight) and dried in an oven (110°C) to constant weight (dry weight). The results are shown in Table 5.

[0170] The calculation formula of WHC is as follows:

[0171] WHC(g / g)=(W w -W d )÷W d ;

[0172] Where: W w is the wet weight, W d It is the dry weight.

[0173] B. Oil Holding Capacity (OHC) Determination

[0174] 10 mL of soybean oil was mixed with 0.5 g of F-SDF and B-SDF prepared in step (iii) at 37°C for 1 h, respectively. After centrifugation at 4800 r / min for 10 min, the precipitates were collected and weighed. The results are shown in Table 5.

[0175] The calculation formula of OHC is as follows:

[0176] OHC(g / g)=W o ÷W s ;

[0177] Where: W o is the weight of adsorbed oil, W s is the weight of the SDF sample (F-SDF or B-SDF).

[0178] After fermentation, the WHC of SDF increased from (2.44±0.19) g / g to (7.34±0.23) g / g. The OHC increased from (5.99±0.08) g / g to (8.61±0.60) g / g. The higher WHC and OHC of F-SDF are due to the fact that fermentation increases the specific surface area of ​​SDF, expands its contact area with oil and water, and exposes more functional groups, making it easier for water and oil to penetrate into the dietary fiber molecules, inhibiting their loss.

[0179] (2) Effect of fermentation on SDF cholesterol absorption capacity

[0180] A. Preparation of cholesterol standard curve

[0181] Prepare a standard solution with a concentration of 100 μg / mL cholesterol, pipette 0, 0.5, 1.0, 1.5, 2.0 mL of the standard solution into 10 mL test tubes, let stand for 10 min at 25 °C, add glacial acetic acid to make the volume 4 mL, tilt the test tube slightly, pour 2.0 mL of iron alum color developer along the tube wall, shake well, let stand for 15 min again, and then draw a standard curve at a wavelength of 560 nm with the total cholesterol as the horizontal coordinate and the OD value as the vertical coordinate (the regression equation of the standard curve is y=0.0022x-0.0054, R 2 =0.9966).

[0182] B. Determination of SDF cholesterol adsorption capacity

[0183] When analyzing the effect of dietary fiber on cholesterol adsorption, it is necessary to use 9 times distilled water to dilute the egg yolk, stir repeatedly to form an emulsion, add 50g of diluted egg yolk solution and 2.0g of dietary fiber (F-SDF or B-SDF) prepared in step (iii) in a 200mL wide-mouth bottle, mix evenly, and adjust the pH value of the mixture to 2.0 (simulating gastric environment) or 7.0 (simulating small intestine environment). Shake the mixture at 37°C for 2h, and use the diluted egg yolk solution without adding SDF as a blank. After the reaction is completed, centrifuge at 3500r / min for 20min, take 1mL of supernatant and measure the absorbance value according to the above method, and calculate the content of remaining cholesterol in the solution by the standard curve. The results are shown in Table 5.

[0184] Cholesterol adsorption capacity (CAC) = (cholesterol content in egg yolk solution before adsorption - cholesterol content in supernatant after adsorption) / dietary fiber mass;

[0185] As can be seen from Table 5, the ability of F-SDF to absorb cholesterol is higher than that of B-SDF. At pH = 7.0, it increased from (379.68 ± 9.09) μg / g before fermentation to (590.29 ± 5.25) μg / g, and at pH = 2.0, it increased from (145.38 ± 9.09) μg / g before fermentation to (352.96 ± 6.94) μg / g. This is because fermentation increases the looseness and specific surface area of ​​rice bran SDF, thereby increasing CAC. The CAC of SDF at pH = 7.0 is higher than that at pH = 2.0 because the appropriate pH value can promote the combination of soluble dietary fiber and cholesterol, which is conducive to the formation of a stable complex between soluble dietary fiber and cholesterol.

[0186] (3) Effect of fermentation on the nitrite ion scavenging ability of SDF

[0187] A. Preparation of nitrite standard curve

[0188] Prepare a standard solution of NaNO2 with a concentration of 5 μg / mL, and draw 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1mL, 1.5mL, 2.0mL, and 2.5mL of the standard solution into test tubes, and then add 2mL of 0.4wt.% p-aminobenzenesulfonic acid solution to each test tube, mix the two thoroughly, and let stand for 5 minutes. Then add 1mL of naphthylethylenediamine hydrochloride solution to each test tube, make the volume to 25mL, measure the absorbance at a wavelength of 538nm, and draw a standard curve with nitrite ion concentration (μg / mL) as the horizontal coordinate and absorbance as the vertical coordinate (the regression equation of the standard curve is y=0.0327x-0.0035, R 2 =0.9967).

[0189] B. Determination of SDF nitrite ion (NIAC) scavenging ability

[0190] Divide the NaNO2 standard working solution into two portions and adjust the pH value to 2.0 (simulating the gastric environment) or 7.0 (simulating the small intestine environment). Accurately weigh 1g of sample (F-SDF or B-SDF prepared in step (iii)) into a conical flask and add the above standard working solution. Stir at 37℃ for 2h, take 1mL of the supernatant and measure the NO2 - concentration.

[0191] NO2 - Adsorption amount = (NO2 before adsorption - Content-NO2 after adsorption - Content) / dietary fiber quality;

[0192] The experimental results show that pH value has an important effect on the removal of NO by SDF. 2-The ability of SDF to remove NO2 was greatly affected. The removal ability of SDF at pH = 2.0 was stronger than that at pH = 7.0. At the same time, the adsorption capacity of SDF decreased after fermentation, from (497.99 ± 0.69) μg / g and (452.55 ± 1.38) μg / g before fermentation to (486.31 ± 1.10) μg / g and (440.01 ± 2.38) μg / g, respectively. This is because the chemical structure of F-SDF changed due to fermentation, and the ferulic acid decreased, so the removal of NO2 - ability is reduced.

[0193] Table 5 Effect of fermentation on the functional properties of SDF

[0194]

[0195] Note: Values ​​with different letters in the same column are significantly different (p<0.5).

[0196] (VII) Effect of fermentation on the antioxidant capacity of SDF

[0197] (1) DPPH free radical scavenging experiment

[0198] Add equal volumes of solutions of dietary fiber (F-SDF or B-SDF) prepared in step (iii) with different concentrations (prepared with water) to 2 mL of 0.2 mmol / L DPPH free radical anhydrous ethanol solution (i.e., sample), react for 30 min in dark conditions, and measure the absorbance (A) at 517 nm. i The control group was set up with anhydrous ethanol to replace the DPPH free radical solution (A j ), the blank group was ethanol instead of sample (A0). Ascorbic acid (Vc) was used as a positive control.

[0199] The scavenging rate of the extract on DPPH free radicals was calculated according to the formula, and ascorbic acid (Vc) was used as a positive control. The results are shown in Fig.10 Figure (A) in .

[0200]

[0201] Where:

[0202] A i : Absorbance value of 2mL sample + 2mL DPPH;

[0203] A j : Absorbance value of 2mL sample + 2mL anhydrous ethanol;

[0204] A0: absorbance value of 2mL ethanol + 2mL DPPH.

[0205] from Fig.10As can be seen in Figure (A), the DPPH free radical scavenging effect of SDF is related to the concentration and is dose-dependent. At a concentration of 500 μg / mL, the scavenging rate of SDF on DPPH free radicals increased from 24.82% ± 0.63% before fermentation to 72.34% ± 0.61%. This research result shows that fermentation significantly promotes the DPPH free radical scavenging ability of rice bran SDF.

[0206] (2)ABTS + Free radical scavenging activity assay

[0207] 10mL of ABTS with a concentration of 7mmol / L + The free radical solution was mixed with an equal volume of a 2.45 mmol / L K2S2O8 solution and allowed to stand for 16 h in the dark at room temperature to obtain a mother solution. The mother solution was diluted with anhydrous ethanol and ABTS was obtained when the absorbance at 734 nm was stable at 0.70 ± 0.02. + Working solution. Take 1 mL of the dietary fiber (F-SDF or B-SDF) solution (i.e., sample) prepared in step (iii) with different concentrations (prepared with water), and add 4 mL of ABTS + Free radical working solution, measure A after 6 minutes of reaction 734nm Value (A i ), the control group was anhydrous ethanol instead of ABTS + Free radical solution (A j ), the blank group was distilled water instead of sample (A0).

[0208] ABTS was calculated by the formula + The free radical scavenging rate was measured with ascorbic acid (Vc) as a positive control. Fig.10 (B) Figure.

[0209]

[0210] Where:

[0211] A i :1mL sample + 4mL ABTS + The absorbance value of

[0212] A j : Absorbance value of 1mL sample + 4mL anhydrous ethanol;

[0213] A0: 1mL distilled water + 4mL ABTS + The absorbance value.

[0214] from Fig.10 As can be seen from Figure (B), SDF has a significant effect on ABTS +The free radical scavenging effect was dose-dependent. At a concentration of 500 μg / mL, SDF had a significant + The free radical scavenging rate increased from 55.08%±2.02% before fermentation to 93.82%±1.69%. The results showed that fermentation significantly promoted the scavenging effect of ABTS+ free radicals of SDF.

[0215] (3) FRAP iron ion reduction antioxidant capacity test

[0216] Prepare FRAP working solution: Mix 25mL of 300mmol / L acetic acid-sodium acetate buffer (PH=3.6), 2.5mL of 10mmol / L TPTZ HCl solution and 2.5mL of 20mmol / L FeCl3 solution to prepare FRAP working solution. Take 0.1mL of FeSO4 solution of different concentrations, add 3mL FRAP working solution and 2mL distilled water to mix, react at room temperature in the dark for 50min, and measure the absorbance at 596nm. The standard curve is drawn with the concentration of FeSO4 solution as the horizontal axis and the absorbance as the vertical axis. Take 1mL of the solution (i.e., sample) of dietary fiber (F-SDF or B-SDF) prepared in step (iii) of different concentrations (prepared with water), add 3mL FRAP working solution and 2mL distilled water, mix thoroughly, react at room temperature in the dark for 50min, and measure the absorbance at 596nm. Using Vc as the control, the reducing capacity was expressed by converting the absorbance value at 596 nm into the FeSO4 equivalent concentration of the sample, which was recorded as the FRAP value. Fig.10 (C) Figure.

[0217] from Fig.10 As can be seen in Figure (C), with the increase of SDF concentration, the iron ion reducing ability gradually increased in a dose-dependent manner. When the concentration was 500 μg / mL, the FRAP of SDF increased from 0.41±0.01 before fermentation to 0.74±0.02 after fermentation. The results showed that SDF has good iron ion reducing ability, and the iron ion reducing ability of F-SDF is stronger than that of B-SDF.

[0218] (4) Hydroxyl radical scavenging activity

[0219] 1 mL of FeSO4 (9 mmol / L), 1 mL of salicylic acid solution (9 mmol / L, 50 vol.% ethanol), 1 mL of a solution of dietary fiber (F-SDF or B-SDF) prepared in step (iii) of different concentrations (prepared with water) (i.e., the sample), and 1 mL of H2O2 (0.03 wt.%) were added to a test tube, reacted in a 37°C water bath for 1 h, cooled to room temperature, and the absorbance (A) was measured at a wavelength of 510 nm. i), and 1 mL of 50 vol.% ethanol solution was used instead of salicylic acid solution to measure the absorbance (A j ), 1 mL of distilled water was used to replace the sample solution to measure the absorbance (A0), and the scavenging rate of the sample on hydroxyl free radicals was calculated according to formula (3). Ascorbic acid (Vc) was used as a positive control.

[0220]

[0221] A i :1mLFeSO4+1mL salicylic acid solution+1mL sample+1mLH2O2;

[0222] A j :1mLFeSO4+50% ethanol solution+1mL sample+1mLH2O2;

[0223] A0: 1mL FeSO4+1mL salicylic acid solution+1mL distilled water+1mL H2O2;

[0224] from Fig.10 As can be seen in Figure (D), the scavenging effect of SDF on hydroxyl radicals is dose-dependent. At a concentration of 500 μg / mL, the scavenging rate of SDF on hydroxyl radicals increased from 30.22%±1.20% before fermentation to 43.70%±3.90%. The results show that fermentation significantly promotes the scavenging ability of rice bran SDF for hydroxyl radicals.

[0225] (VIII) SDF immunomodulatory activity

[0226] (1) Cell culture

[0227] The mouse macrophage cell line RAW264.7 was grown in DMEM complete medium containing 10% fetal bovine serum (the volume ratio of fetal bovine serum to the total system was 10:100) and 1% double antibody (penicillin / streptomycin, the volume ratio of double antibody to the total system was 1:100) (i.e., the volume ratio of double antibody, fetal bovine serum and DMEM was 1:10:89), and the cell culture flask was placed in a 37°C, 5% CO2 incubator for culture. When the cells covered 80% of the bottom of the culture flask, they were passaged, and the cells cultured to the logarithmic growth phase were used for subsequent experiments.

[0228] (2) Detection of cell viability (analysis of the toxic effect of SDF on RAW246.7 cells)

[0229] Methyl thiazolyl tetrazolium (MTT) colorimetric method was used to determine whether SDF had toxic effects on cells. RAW246.7 cells in the logarithmic growth phase were taken, digested, and blown evenly, and diluted with culture medium to a cell density of 2×10 5 / mL, 100μL per well was inoculated in a 96-well plate and cultured in a 37℃, 5% CO2 incubator for 24h. Then the culture plate was removed, the old culture medium was aspirated, a blank group without inoculated cells was set up, 100μL DMEM complete culture medium was added to the control group, and 100μL DMEM complete culture medium containing 25-600μg / mL SDF (B-SDF before fermentation or F-SDF after fermentation) was added to the sample group in turn (DMEM complete culture medium is prepared with a volume ratio of 1:10:89 double antibody, fetal bovine serum and DMEM). Set up 5 replicate wells for each group, shake gently, and culture in an incubator for 24h (the culture is over at this time). Add 100μL MTT (1mg / mL) solution to each well, culture again for 4h, discard the MTT working solution, add 100μL DMSO and shake gently to dissolve the crystals, place at room temperature for 10min, and use an enzyme reader to measure the optical density value of each well at a wavelength of 490nm. The results are shown in the figure. Fig.11 .

[0230]

[0231] from Fig.11 It can be seen that compared with the control group, the cell viability of RAW264.7 cells treated with 25-600μg / mL SDF changed. When the concentration of SDF was 200μg / mL, the cell viability reached the maximum value, and the cell viability of the F-SDF treatment group was significantly better than that of the B-SDF group. The cell viability of the F-SDF group and the B-SDF group reached 157.23% and 123.96%, respectively. However, when the concentration of SDF increased to 400 and 600μg / mL, the cell viability was significantly reduced compared with the 200μg / mL group (P<0.05), but there was no significant difference with the control group (P>0.05).

[0232] Fig.11 The capital letters in the figure represent the significance between different concentrations before fermentation (P<0.05), the lowercase letters represent the significance between different concentrations after fermentation (P<0.05), * represents significant (P<0.05), and ** represents extremely significant (P<0.01).

[0233] (3) Neutral red method to detect cell phagocytic ability (the effect of SDF on the neutral red phagocytic rate)

[0234] The cell phagocytic ability was determined by the neutral red method. The blank group and the experimental group were set up according to the cell culture and grouping method in step (2), and LPS (1 μg / mL) was used as the positive control group. After the culture was completed, the supernatant was aspirated, and 100 μL of 0.1% neutral red dye solution was added to each well. After incubation at 37°C for 2 hours, the cells were washed three times with PBS, and 100 μL of alcohol-acid lysis solution (equal volumes of ethanol and glacial acetic acid) was added to each well. The absorbance value was measured at 540 nm using an enzyme reader, and the cell neutral red phagocytosis rate was calculated according to the following formula. The results are shown in Fig.12 .

[0235]

[0236] from Fig.12 It can be seen that compared with the control group, the phagocytic ability of cells showed an upward trend after treatment with 25-200μg / mL SDF. When the SDF concentration was 200μg / mL, the neutral red phagocytic rate reached the maximum value. The phagocytic ability of cells treated with F-SDF was significantly better than that of B-SDF, increasing from 120.63% before fermentation to 130.99% after fermentation. The neutral red phagocytic rate of the LPS group reached 146.75% of that of the control group, indicating that LPS significantly enhanced the phagocytic activity of RAW 264.7 cells (P < 0.05). The results show that SDF can activate RAW264.7 cells at an appropriate concentration and enhance phagocytic activity, and fermentation can improve this ability of SDF.

[0237] Fig.12 The capital letters in the figure represent the significance between different concentrations before fermentation (P<0.05), the lowercase letters represent the significance between different concentrations after fermentation (P<0.05), * represents significant (P<0.05), and ** represents extremely significant (P<0.01).

[0238] (4) Nitric oxide (NO) and cytokine secretion levels

[0239] The cells were grouped and cultured according to the method in step (2), with 5 replicates per group. After the culture was completed, the cells were centrifuged at 1000×g for 10 min at room temperature, and the cell culture fluid was collected. The NO in the cell culture fluid was determined strictly according to the steps in the kit instructions (see Fig.13 ) and the secretion of cytokines (TNF-α, IL-1β, IL-6, IL-10) (see Fig.14 , Fig.14 (A) is IL-1β, (B) is IL-6, (C) is IL-10, and (D) is TNF-α).

[0240] from Fig.13It can be seen that SDF increases the release of NO from macrophages in a dose-dependent manner within the range of 25-200 μg / mL. When the SDF concentration is 200 μg / mL, the NO release reaches the maximum value, increasing from 14.01 μmol / L before fermentation to 15.17 μmol / L after fermentation, but lower than the LPS group, thus avoiding excessive inflammatory response of macrophages. This shows that SDF can enhance the immunomodulatory activity by stimulating NO secretion in RAW264.7 cells, and fermentation can further enhance the promoting effect of rice bran SDF on NO secretion in macrophages.

[0241] Fig.13 The capital letters in the figure represent the significance between different concentrations before fermentation (P<0.05), the lowercase letters represent the significance between different concentrations after fermentation (P<0.05), * represents significant (P<0.05), and ** represents extremely significant (P<0.01).

[0242] from Fig.14 As can be seen from Figures (A) to (D), SDF (50-100 μg / mL) treatment caused the secretion of TNF-α, IL-1β, IL-6 and IL-10 in RAW 264.7 cells to increase in a concentration-dose effect. When the concentration of SDF was 100 μg / mL, the secretion of all cytokines reached the maximum value, and the content of IL-1β increased from 40.312±0.470 pg / mL before fermentation to 45.814±0.625 pg / mL, IL-6 increased from 34.640±0.284 pg / mL before fermentation to 35.047±0.125 pg / mL, IL-10 increased from 33.706±0.135 pg / mL before fermentation to 34.736±0.134 pg / mL, and TNF-α increased from 10.870±0.063 pg / mL before fermentation to 11.623±0.127 pg / mL. The results showed that rice bran SDF can play an immunomodulatory role by increasing the secretion of cytokines by macrophages, and fermentation has a promoting effect on the immunomodulatory effect of rice bran SDF.

[0243] Fig.14 The capital letters in the figure represent the significance between different concentrations before fermentation (P<0.05), the lowercase letters represent the significance between different concentrations after fermentation (P<0.05), * represents significant (P<0.05), and ** represents extremely significant (P<0.01).

[0244] Example 7

[0245] A preparation method of high-fiber rice bran biscuits:

[0246] (1) High-fiber rice bran biscuits are composed of standard raw materials and additives;

[0247] The reference raw material, in terms of mass percentage, is composed of the following raw materials: 6, 8, 10, 12 or 14 wt.% of fermented rice bran, and the balance is low-gluten flour;

[0248] The additive, based on the mass of the reference raw materials, consists of 1wt.% salt, 0.5wt.% baking soda, 1.5wt.% baking powder, 10wt.% whole milk powder, 20wt.% water, 30wt.% powdered sugar, 25wt.% butter and 20wt.% whole egg liquid.

[0249] (2) The weighed salt, baking soda, baking powder, powdered sugar, whole egg liquid and water are mixed evenly, and rice bran, low-gluten flour and whole milk powder are added to make dough. After the dough is formed, the melted butter is mixed with the dough again. After standing for 10 minutes, the dough is rolled into a 3 mm thin sheet, and the corresponding shape is cut with a mold. The cut biscuit dough is punched, and after preparation, it is baked at 180°C on the upper fire and 180°C on the lower fire for 15 minutes to obtain high-fiber rice bran biscuits.

[0250] Example 8

[0251] Same as Example 7, except that the high-fiber rice bran biscuits are composed of the reference raw materials and additives;

[0252] The reference raw material, in terms of mass percentage, is composed of the following raw materials: 10wt.% of fermented rice bran, and the remainder is low-gluten flour;

[0253] The additive, based on the mass of the reference raw materials, consists of 1wt.% salt, 0.5wt.% baking soda, 1.5wt.% baking powder, 10wt.% whole milk powder, 20wt.% water, powdered sugar (the amount used is 20, 25, 30, 35 or 40wt.% of the mass of the reference raw materials, respectively), 25wt.% butter and 20wt.% whole egg liquid.

[0254] Example 9

[0255] Same as Example 7, except that the high-fiber rice bran biscuits are composed of the reference raw materials and additives;

[0256] The reference raw material, in terms of mass percentage, is composed of the following raw materials: 10wt.% of fermented rice bran, and the remainder is low-gluten flour;

[0257] The additive, based on the mass of the reference raw materials, consists of 1wt.% salt, 0.5wt.% baking soda, 1.5wt.% baking powder, 10wt.% whole milk powder, 20wt.% water, 30wt.% powdered sugar, butter (the amounts used are 10, 15, 20, 25, 30wt.% of the mass of the reference raw materials, respectively) and 20wt.% whole egg liquid.

[0258] Example 10

[0259] Same as Example 7, except that the high-fiber rice bran biscuits are composed of the reference raw materials and additives;

[0260] The reference raw material, in terms of mass percentage, is composed of the following raw materials: 10wt.% of fermented rice bran, and the remainder is low-gluten flour;

[0261] The additive, based on the mass of the reference raw materials, consists of 1wt.% salt, 0.5wt.% baking soda, 1.5wt.% baking powder, 10wt.% whole milk powder, 20wt.% water, 30wt.% powdered sugar, 25wt.% butter and whole egg liquid (the amounts used are 10, 15, 20, 25 and 30wt.% of the mass of the reference raw materials, respectively).

[0262] Effect Example 3

[0263] The chromaticity of the high-fiber rice bran biscuits prepared in Examples 7 to 10 was measured, and the texture characteristics and sensory scores of the high-fiber rice bran biscuits were analyzed.

[0264] (1) Determination of chromaticity

[0265] The color, L*, a* and b* values ​​of high-fiber rice bran biscuits were measured by a colorimeter, with 3 parallel tests for each level. The L* value represents black (0) / white (100), the a* value represents green (-) / red (+), and the b* value represents blue (-) / yellow (+).

[0266] (2) Texture characteristics analysis

[0267] The TMS-PRO texture analyzer was used, and a spherical probe of P / 0.5 was used. The test mode was set to TPA strength test, the detection speed was 1.0 mm / s, the deformation was 10%, and the trigger force was 0.1 N. Three parallel tests were performed at each level, and the average was taken. Hardness and chewiness were selected as characteristic parameters in the test results.

[0268] (3) Sensory evaluation

[0269] More than 10 professional tasters were selected to conduct sensory evaluation of the color, shape, structure, taste, etc. of the biscuits by visual inspection, nose smelling, and mouth tasting. The full score is 100, and the sensory scoring standards are shown in Table 6.

[0270] Table 6 Sensory evaluation criteria for high-fiber rice bran biscuits

[0271]

[0272] (4) Quality of high-fiber rice bran biscuits prepared in Example 7 (the effect of the amount of fermented rice bran added on the quality of biscuits, the results are shown in Tables 7 and Fig.15 , Fig.15Different letters in the same index indicate significant differences (P<0.05).

[0273] Table 7 Effect of fermented rice bran addition on biscuit quality

[0274]

[0275] Note: The results are expressed as mean ± standard deviation. Significant differences (P < 0.05) in the same column are represented by different letters.

[0276] As can be seen from Table 7, as the proportion of fermented rice bran increases, the L* value of the biscuits shows a downward trend, while the a* value and b* value show an upward trend. When the addition amount is 14%, the L* value is the smallest, which is 57.93±0.25, and the a* value is significantly higher than that of other groups (P<0.05). The b* value also reaches the highest value, which is due to the brown-yellow color of the fermented rice bran itself. As the proportion of fermented rice bran increases, the hardness and chewiness show an upward trend. When the addition amount is 14%, the hardness and chewiness are significantly higher than those of other groups, which are (40.81±0.35)N and (7.51±0.17)mJ, respectively. This is because the rice bran content is too much and the fiber content increases, which leads to an increase in the hardness and chewiness of the biscuits.

[0277] from Fig.15 It can be seen that with the increase of the proportion of fermented rice bran, the sensory score of the biscuits first increases and then decreases. When the addition amount is 10%, the fermented rice bran biscuits have the highest sensory score of 82.00±1.73 points. This is because with the increase in the amount of fermented rice bran added, the biscuits have a special natural flavor of grains, the color gradually tends to golden yellow, the shape is relatively complete, the sweetness is moderate, and the unique taste of fermented rice bran, which is crispy and rich in layers, which increases the sensory score of the biscuits. However, if the proportion of fermented rice bran is too large, the natural flavor of grains in the biscuits will be too strong, the color will be too dark, the texture will be messy, the sweetness will be lighter, and there will be cracks, which will lead to a lower sensory score.

[0278] (5) Quality of high-fiber rice bran biscuits prepared in Example 8 (the effect of the amount of powdered sugar added on the quality of biscuits, the results are shown in Tables 8 and Fig.16 , Fig.16 Different letters in the same index indicate significant differences (P<0.05).

[0279] Table 8 Effect of sugar powder addition on biscuit quality

[0280]

[0281] Note: The results are expressed as mean ± standard deviation. Significant differences (P < 0.05) in the same column are represented by different letters.

[0282] As can be seen from Table 8, with the increase of the amount of sugar powder added, the L* value showed a downward trend, and the influence on the a* value, b* value and texture showed irregular changes. When the addition amount was 40%, the L* value was the smallest, which was 56.82±0.46. This was due to the caramelization reaction and Maillard reaction, which made the biscuits darker in color. With the increase of sugar powder added, the hardness and chewiness showed an upward trend. When the addition amount was 40%, the hardness and chewiness were significantly higher than those of other groups, which were (38.25±0.26)N and (6.48±0.37)mJ respectively. This was due to the caramelization reaction, which caused more water loss, resulting in increased chewiness and hardness.

[0283] from Fig.16 It can be seen that as the amount of powdered sugar added increases, the sensory score of the biscuits first rises and then decreases. When the addition amount is 30% and 35%, the sensory score of the fermented rice bran biscuits is the highest, both with 86 points. With the increase in the amount of powdered sugar, the biscuits have a sweeter taste, golden color, smooth surface, and crisp taste, which increases the sensory score; when too much powdered sugar is added, the biscuits are too sweet, too dark in color, and have messy textures and cracks, resulting in a lower sensory score. In addition, adding too much powdered sugar will cause an increase in blood sugar after a meal, which poses a potential threat to health, and the sugar content of fermented rice bran needs to be regulated.

[0284] (6) Quality of high-fiber rice bran biscuits prepared in Example 9 (the effect of butter addition on biscuit quality, the results are shown in Tables 9 and Fig.17 , Fig.17 Different letters in the same index indicate significant differences (P<0.05).

[0285] Table 9 Effect of butter addition on biscuit quality

[0286]

[0287] Note: The results are expressed as mean ± standard deviation. Significant differences (P < 0.05) in the same column are represented by different letters.

[0288] As can be seen from Table 9, as the amount of butter added increases, the L* value decreases, the a* value increases first and then decreases, and the b* value increases. When the addition amount is 30%, the L* value is the smallest, which is 63.29±1.08. This is because butter promotes the Maillard reaction during baking, which deepens the color and causes the L* value to decrease. The hardness and chewiness of the biscuits decrease with the addition of butter. When the addition amount is 30%, the hardness and chewiness are lower than those of other groups, which are (28.93±0.89) and (4.80±0.18) mJ, respectively. This is because butter has good whipping and aeration properties, which increases the amount of air entering, causing the biscuits to expand in volume and reduce hardness.

[0289] from Fig.17It can be seen that with the increase of butter addition, the sensory score of the biscuits first increases and then decreases. When the addition amount is 25%, the sensory score of the biscuits is the highest, which is (86.67±0.58) points. This is because with the addition of butter in moderation, the biscuits have abundant milky flavor, golden color, intact surface without cracks, moderate sweetness, and crispy taste. It may be that butter can improve the volume of baked goods and give them good taste, flavor and appearance; if the amount of butter added is further increased, the biscuits have too strong oily taste, too dark surface color, messy texture and cracks, resulting in a decrease in the sensory score.

[0290] (7) Quality of high-fiber rice bran biscuits prepared in Example 10 (the effect of the amount of whole egg liquid added on the quality of biscuits, the results are shown in Tables 10 and Fig.18 , Fig.18 Different letters in the same index indicate significant differences (P<0.05).

[0291] Table 10 Effect of whole egg liquid addition on biscuit quality

[0292]

[0293]

[0294] Note: The results are expressed as mean ± standard deviation. Significant differences (P < 0.05) in the same column are represented by different letters.

[0295] As can be seen from Table 10, with the increase of the amount of whole egg liquid added, the L* value basically shows a downward trend, and the a* value and b* value show an upward trend. When the addition amount is 25%, the L* value is the smallest, which is 62.99±0.77. This is because the whole egg liquid promotes the Maillard reaction, resulting in a darker color. The hardness of the biscuit shows a downward trend with the increase of the amount of whole egg liquid added. When the addition amount is 30%, the hardness is the smallest (27.63±0.31N), and the chewiness is (3.65±0.19) mJ. This is because the moisture content of the whole egg liquid (about 70% to 75%) is high, and the phospholipids in the egg yolk are natural emulsifiers, which can enhance the crisp taste of the product, and also reduce the hardness and chewiness of the biscuits.

[0296] from Fig.18 It can be seen that as the amount of whole egg liquid added increases, the sensory score of the biscuits first increases and then decreases. When the addition amount is 25%, the sensory score of the biscuits is the highest, which is (91.33±1.53) points. Adding an appropriate amount of whole egg liquid can reduce the oxidation of some surface oils and play a role in crack prevention. However, too high a whole egg liquid content will cause a serious eggy smell, thus affecting the sensory score of the biscuits. At the same time, the low moisture content makes the dough difficult to shape. After baking, the biscuits are too compact and have a rough surface.

[0297] Embodiment 11

[0298] Orthogonal Experimental Design:

[0299] Based on the single factor experimental results of Examples 7 to 10, the addition amount of fermented rice bran, butter, powdered sugar and whole egg liquid (wherein the addition amount of butter, powdered sugar and whole egg liquid refers to the mass percentage of butter, powdered sugar or whole egg liquid and the mass percentage of the reference raw material) was used as a factor (represented by A, B, C, and D, respectively), and the level parameters were set. The sensory score was used as an indicator, and L9 (3 4 ) orthogonal table, optimize the fermented rice bran biscuits formula, the orthogonal factor level table is shown in Table 11.

[0300] Table 11 Factor level table of orthogonal experiment of high fiber rice bran biscuit process

[0301]

[0302] Table 12L9(3 4 )Orthogonal Experimental Design and Results

[0303]

[0304]

[0305] The results of the orthogonal test are shown in Table 12. The larger the R value, the greater the influence of the factor on the experimental results. The influence of the four single factors on biscuits is factor A>B>C>D. It can be concluded that the optimal formula combination is A1B2C2D2.

[0306] Example 12 (addition of fermented rice bran biscuits)

[0307] Same as Example 7, except that the high-fiber rice bran biscuits are composed of the reference raw materials and additives;

[0308] The reference raw material, in terms of mass percentage, is composed of the following raw materials: 8wt.% of fermented rice bran, and the remainder is low-gluten flour;

[0309] The additive, based on the mass of the reference raw materials, consists of 1wt.% salt, 0.5wt.% baking soda, 1.5wt.% baking powder, 10wt.% whole milk powder, 20wt.% water, 30wt.% powdered sugar, 25wt.% butter and 25wt.% whole egg liquid.

[0310] The biscuits prepared in this example have moderate sweetness and a crispy taste, and have a high score of (92.33±0.58), which is better than the unoptimized process.

[0311] Comparative Example 1 (ordinary biscuits, no rice bran biscuits added)

[0312] Same as Example 7, except that the high-fiber rice bran biscuits are composed of the reference raw materials and additives;

[0313] Among them, the benchmark raw material is low-gluten flour;

[0314] The additive, based on the mass of the reference raw materials, consists of 1wt.% salt, 0.5wt.% baking soda, 1.5wt.% baking powder, 10wt.% whole milk powder, 20wt.% water, 30wt.% powdered sugar, 25wt.% butter and 25wt.% whole egg liquid.

[0315] Comparative Example 2 (unfermented rice bran biscuits, adding unfermented rice bran biscuits)

[0316] Same as Example 7, except that the high-fiber rice bran biscuits are composed of the reference raw materials and additives;

[0317] The reference raw material, in terms of mass percentage, is composed of the following raw materials: 8wt.% of rice bran (unfermented rice bran), and the remainder is low-gluten flour;

[0318] The additive, based on the mass of the reference raw materials, consists of 1wt.% salt, 0.5wt.% baking soda, 1.5wt.% baking powder, 10wt.% whole milk powder, 20wt.% water, 30wt.% powdered sugar, 25wt.% butter and 25wt.% whole egg liquid.

[0319] The preparation method of unfermented rice bran is as follows: weigh 200g rice bran, add 450mL cold boiled water (the material-water mass ratio is 1:2.25 (rice bran: cold boiled water)) after washing, stir evenly, boil in boiling water for 60min, and obtain unfermented rice bran after the rice bran is cooled.

[0320] Effect Example 4

[0321] The actual pictures of the biscuits without adding rice bran prepared in Comparative Example 1 and the biscuits with adding fermented rice bran prepared in Example 12 are shown in Fig.19 . Fig.19 (A) is the biscuit without adding rice bran prepared in Comparative Example 1, and (B) is the biscuit with adding fermented rice bran prepared in Example 12.

[0322] Effect Example 5

[0323] (I) Effect of adding rice bran on the water and oil holding capacity of biscuits:

[0324] The water holding capacity and oil holding capacity of the biscuits prepared in Example 12 and Comparative Examples 1-2 were measured using the same method as above (step (six) of Effect Example 2). The results are shown in Table 13.

[0325] As can be seen from Table 13, the WHC of ordinary biscuits without rice bran is (2.94±0.03) g / g and OHC is (2.38±0.07) g / g. The water and oil holding capacity of both biscuits with fermented rice bran and biscuits with unfermented rice bran has been significantly improved (P<0.05), and the biscuits with fermented rice bran have higher water and oil holding capacity, with WHC and OHC increasing from (3.52±0.01) g / g and (2.70±0.03) g / g to (3.78±0.08) g / g and (3.06±0.04) g / g, respectively. This is because the addition of dietary fiber increases the water absorption rate of the dough, prolongs the formation time, and significantly prolongs the stability time, which improves the flour properties of the dough. At the same time, fermentation increases the dietary fiber content of rice bran and makes it have stronger water holding capacity. It easily absorbs water and swells when kneading the dough, and forms a network together with partially absorbed gluten protein, which is beneficial to improving the quality of biscuits. The increase in water holding capacity is also beneficial to increasing the yield of the product.

[0326] (ii) Determination of the total phenol and total flavonoid contents of the biscuits prepared in Example 12 and Comparative Examples 1-2, the determination method is as follows:

[0327] (1) Preparation of sample extract

[0328] Weigh 2.0g of the crushed biscuit sample into a 50mL centrifuge tube, add 40mL of methanol solution (solid-liquid ratio: 1g:20mL), and place in a 60℃ water bath for 100W ultrasonic treatment for 90min. After the end, centrifuge the sample extract at 4000r / min for 25min, take the supernatant, which is the sample extract, and store it in a refrigerator at 4℃.

[0329] The sample extract (stock solution) was diluted with DMEM complete medium (DMEM complete medium was prepared with double antibody, fetal bovine serum and DMEM in a volume ratio of 1:10:89) at different times for the determination of the effect on the secretion of cytokines (TNF-α, IL-1β, IL-6, IL-10) of RAW264.7 cells.

[0330] (2) Determination of total phenol content: same as step (1) in Example 2.

[0331] (3) Determination of total flavonoid content

[0332] Drawing of flavonoid standard curve: Accurately weigh 5 mg of dry constant weight rutin standard, dissolve it with 60 vol.% ethanol under slight heat and dilute to 25 mL to prepare rutin standard solution. Pipette 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, and 4.0 mL of rutin standard solution into 10 mL stoppered test tubes, add 1 mL of 5 wt.% sodium nitrite solution, shake well, let stand for 6 min, add 1 mL of 10 wt.% aluminum nitrate solution, shake well, let stand for 6 min, then add 4 mL of 4 wt.% sodium hydroxide, add 60 vol.% ethanol to the scale, mix well and let stand for 15 min. Use the concentration of rutin solution as the horizontal coordinate and the absorbance as the vertical coordinate, measure the absorbance at a wavelength of 505 nm, and draw a standard curve (the equation of the standard curve is y=0.4734x-0.0124, R 2 =0.9967).

[0333] Determination of total flavonoids content in sample extract: Take 2 mL of biscuit sample extract, determine it according to the determination method of total flavonoids standard solution concentration, and calculate the total flavonoids content of the sample. The results are shown in Table 13.

[0334] Table 13 WHC, OHC, total phenols and total flavonoids content of biscuits

[0335] <![CDATA[Water holding capacity (g / g -1 )]]> <![CDATA[Oil holding capacity (g / g -1 )]]> Total phenol content (mg / 100g) Total flavonoids content (mg / 100g) Regular Cookies <![CDATA[2.94±0.03 c ]]> <![CDATA[2.38±0.07 c ]]> <![CDATA[1.14±0.02 c ]]> <![CDATA[0.74±0.03 c ]]> Unfermented rice bran biscuits <![CDATA[3.52±0.01 b ]]> <![CDATA[2.70±0.03 b ]]> <![CDATA[1.39±0.05 b ]]> <![CDATA[0.85±0.01 b ]]> Fermented rice bran biscuits <![CDATA[3.78±0.08 a ]]> <![CDATA[3.06±0.04 a ]]> <![CDATA[1.69±0.12 a ]]> <![CDATA[1.08±0.03 a ]]>

[0336] The results are expressed as mean ± standard deviation. Significant differences (P < 0.05) in the same column are indicated by different letters.

[0337] As can be seen from Table 13, the total phenol and total flavonoid contents of the fermented rice bran biscuits were higher than those of the unfermented rice bran biscuits, and both were higher than those of the ordinary biscuits without rice bran. Compared with the biscuits with unfermented rice bran, the total phenol content of the fermented rice bran biscuits increased from (1.39±0.05) mg / 100g to (1.69±0.12) mg / 100g, and the total flavonoid content increased from (0.85±0.01) mg / 100g to (1.08±0.03) mg / 100g. This is because the fermentation causes the increase in the content of phenols and flavonoids in the rice bran, so the polyphenol and flavonoid contents in the biscuits produced are also relatively higher.

[0338] (III) The antioxidant properties of the biscuits prepared in Example 12 and Comparative Examples 1-2 were measured in the same manner as step (VII) in Effect Example 2. The results are shown in Fig. 20 , Fig. 20 (A) shows the effect of different biscuits on DPPH clearance, (B) shows the effect of SDF of different biscuits on ABTS +(C) is the effect of different biscuits on the FRAP reducing ability, and (D) is the effect of different biscuits on the hydroxyl radical scavenging ability.

[0339] from Fig. 20 It can be seen that the antioxidant activity of biscuits with fermented rice bran is higher than that of biscuits with unfermented rice bran, and both are higher than that of ordinary biscuits without rice bran. When the biscuit extract is not diluted, the DPPH free radical scavenging ability of biscuits with fermented rice bran increases from 71.05%±0.40% to 82.04%±0.60% compared with biscuits with unfermented rice bran, and ABTS + The free radical scavenging activity increased from 94.01%±0.40% to 96.57%±0.31%, the FRAP reducing ability increased from 1.08±0.01 to 1.13±0.01, and the hydroxyl radical scavenging ability increased from 64.72%±0.47% before fermentation to 74.30%±0.46%. This was due to the increase in the content of fermented and unfermented rice bran, and the increase in the content of polyphenols and flavonoids in the biscuits.

[0340] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing fermented rice bran, characterized in that, The following steps are involved: The rice bran is boiled with water, inoculated with Rhizopus oryzae and Lactobacillus fermentans after cooling, and fermented at a constant temperature to obtain the fermented rice bran.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the rice bran to water is 1:2.25; and the cooking time is 60 minutes.

3. The preparation method according to claim 1, characterized in that: The fermented lactobacillus is inoculated in the form of fermentation liquid; The ratio of the fermentation liquid to rice bran is 10-12 mL: 100 g; the ratio of the fermentation liquid to Rhizopus oryzae is 11 mL: 3-11 g; The method for preparing the fermentation liquid comprises the following steps: activating and culturing the fermented lactobacillus and then fermenting and culturing it to obtain the fermentation liquid.

4. The preparation method according to claim 3, characterized in that: The method for preparing the fermentation liquid comprises the following steps: inoculating the fermented lactobacillus preserved liquid into a liquid culture medium for a first activation culture to obtain a first activated culture liquid; inoculating the first activated culture liquid into a liquid culture medium for a second activation culture to obtain a second activated culture liquid; and inoculating the second activated culture liquid into a liquid culture medium for fermentation culture to obtain the fermentation liquid.

5. The preparation method according to claim 4, characterized in that: During the first activation culture, the volume ratio of the fermented Lactobacillus preservation solution to the liquid culture medium is 1:150; the temperature of the first activation culture is 33° C., and the time is 24 hours; When performing the second activation culture, the volume ratio of the first activation culture solution to the liquid culture medium is 3:100; the temperature of the second activation culture is 33° C., and the time is 24 hours; During fermentation, the volume ratio of the second activation culture solution to the liquid culture medium is 3:10; the temperature of the fermentation is 33°C and the time is 24 hours; The liquid culture medium consists of apple juice with a volume ratio of 85:15 and a whey powder solution with a concentration of 8 wt.%.

6. The preparation method according to claim 1, characterized in that: The temperature of the constant temperature fermentation is 35-39° C., and the time is 36-48 hours.

7. Fermented rice bran prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the fermented rice bran according to claim 7 in the preparation of biscuits.

9. A high-fiber rice bran biscuit, characterized in that: Includes the following raw materials: baseline raw materials and additives; The reference raw materials, in terms of mass percentage, include the following raw materials: 6-14% of the fermented rice bran described in claim 7 and 86-94% of low-gluten flour; The additives include salt, baking soda, baking powder, whole milk powder, water, powdered sugar, butter and whole egg liquid, and the amounts used are 1%, 0.5%, 1.5%, 10%, 20%, 20-40%, 10-30% and 10-30% of the mass of the reference raw materials respectively.

10. A method for preparing the high-fiber rice bran biscuits according to claim 9, characterized in that: The following steps are involved: The raw materials are premixed and then prepared into dough, which is then left to stand, sheeted, punched and baked to obtain the high-fiber rice bran biscuits.

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