Method for improving eating quality of brown rice by combining germination method with compound enzyme treatment

Abstract

The invention discloses a method for improving the eating quality of brown rice by combining a germination method with compound enzyme treatment, and relates to the technical field of grain processing. According to the method, a germination method is combined with a compound enzyme (cellulase and hemicellulase) treatment process, and the cooking performance of brown rice, the rice texture and the in-vitro digestion characteristic are researched. Results show that the optimal compound enzyme treatment conditions are that the compound enzyme concentration is 1.9 g / 100mL, the treatment time is 89min, and the mass ratio of the compound enzyme is 4: 1 (cellulase: hemicellulase), so that the water absorption rate, the cooking speed and the rice texture of the brown rice are remarkably improved. The bran layer structure of the brown rice treated by the compound enzyme is looser, a large number of pore structures are formed, and moisture permeation is facilitated. Compared with untreated brown rice, the hardness of the brown rice treated by the germination method and the compound enzyme is reduced by 34% and is close to the level of polished rice. Although the pre-estimated glycemic index (eGI) of the rice is slightly increased by germination treatment, the overall increase amplitude is less than 5% and is still lower than the eGI of the polished rice. In conclusion, the germination method combined with the compound enzyme treatment shows a remarkable effect in the aspect of improving the eating quality of the brown rice, and has wide popularization and application potential.

Description

Technical Field

[0001] The invention relates to the technical field of grain processing, and in particular to a method for improving the edible quality of brown rice by utilizing a germination method combined with complex enzyme treatment. Background Art

[0002] A healthy lifestyle has always been a topic of close concern, especially in terms of diet. In recent years, the importance of a balanced diet has gradually been valued by people, and whole-grain foods have therefore become a hot spot for development by researchers. Brown rice is obtained by removing the husk from rice grains, which retains the pericarp, seed coat, endosperm, embryo and endosperm, and is a whole-grain food. Refined grains (such as white rice and white flour) are one of the staple foods in China and most Asian countries, mainly used to provide the daily energy required by the human body. Many studies have found that the intake of white rice is positively correlated with the risk of type 2 diabetes in the human body. For people who often eat brown rice, their risk of chronic diseases such as type 2 diabetes, cardiovascular disease and colorectal cancer is reduced, and they are less likely to develop inflammation and obesity.

[0003] Although the nutritional value of brown rice is higher than that of polished rice, its promotion process is not smooth. The main factor causing the poor sensory quality of brown rice is the brown rice bran layer. On the one hand, it can prevent water from entering the endosperm, resulting in poor water absorption and swelling, which makes it difficult to gelatinize and takes a long time to cook. On the other hand, the brown rice bran layer contains cellulose, hemicellulose, pectin and lignin, which are tough in texture and have a poor taste during consumption. Studies have found that germination of brown rice can activate the endogenous enzyme system of brown rice, which not only significantly increases the content of nutrients or active ingredients (such as γ-aminobutyric acid) in brown rice, but also improves the cooking performance of brown rice to a certain extent. Although there are many types of endogenous enzymes in brown rice, there are only a few enzymes that can effectively hydrolyze the brown rice bran layer, such as cellulase, hemicellulase, pectinase and ligninase, and these enzymes are fixed in content and composition ratio, making it difficult to intervene artificially.

[0004] At present, relevant studies at home and abroad have found that using exogenous cellulase, xylanase and pectinase to treat brown rice can hydrolyze part of the brown rice bran layer and improve the cooking quality of brown rice, thereby achieving the purpose of improving the sensory quality of brown rice. However, in these studies, the enzyme treatment object is usually brown rice, or a single cellulase is used as an exogenous enzyme, so there is no organic combination of the advantages of the brown rice germination process, or there is no systematic study of the enzyme treatment effect after the combination of multiple exogenous enzymes. In addition, there is no literature on the in vitro digestion characteristics of brown rice after cellulase and hemicellulase treatment.

[0005] Based on the above reasons, this application is filed. Summary of the invention

[0006] Based on the above reasons, in view of the problems or defects in the prior art, the purpose of the present invention is to provide a method for improving the edible quality of brown rice by using a germination method combined with a composite enzyme treatment, so as to solve or at least partially solve the above technical defects in the prior art: the present invention organically combines the germination process of brown rice with the exogenous enzyme (cellulase and hemicellulase) treatment process, improves the edible sensory quality of brown rice by using a germination method combined with a composite enzyme (a combination of cellulase and hemicellulase) treatment, and explores the significant changes in the cooking quality, rice texture characteristics and rice digestion characteristics of the brown rice treated by this method.

[0007] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is as follows:

[0008] A method for improving the edible quality of brown rice by using a germination method combined with a composite enzyme treatment, the method specifically comprising the following steps:

[0009] (1) after cleaning the brown rice, adding distilled water to completely immerse the brown rice, and soaking it at room temperature for 10-15 hours; after the soaking, draining the brown rice, continuing to cultivate it at room temperature for 10-15 hours, and finally drying it to obtain germinated brown rice;

[0010] (2) mixing the germinated brown rice with a composite enzyme solution according to a ratio, subjecting the resulting mixture to a constant temperature enzyme treatment at 50° C. for 30-150 min to obtain enzymolyzed brown rice; wherein the composite enzyme solution is composed of a cellulase solution and a hemicellulase solution.

[0011] Furthermore, in the above technical solution, the step of disinfecting the brown rice before washing in step (1) can be selectively added. In a preferred embodiment of the present invention, the disinfection in step (1) uses a sodium hypochlorite solution with a volume concentration of 0.2%; the disinfection time is 10 minutes.

[0012] Specifically, in the above technical solution, the normal temperature in step (1) refers to the natural room temperature conditions in all seasons, without additional cooling or heating treatment, and the normal temperature is generally controlled at 10-30°C.

[0013] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the drying in step (1) is performed at 40° C. to a moisture content of less than 14%.

[0014] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the usage ratio of the germinated brown rice to the complex enzyme solution in step (2) is 1 part by mass: 1.5 parts by volume; wherein: the ratio between the parts by mass and the parts by volume is based on g:mL.

[0015] Furthermore, in the above technical solution, the mass ratio of the cellulase to the hemicellulase in step (2) is 5:1-1:5. In a preferred embodiment of the present invention, the mass ratio of the cellulase to the hemicellulase is 4:1.

[0016] Furthermore, in the above technical solution, the total concentration of cellulase and hemicellulase in the complex enzyme solution in step (2) is 1.0-2.2 g / 100 mL. In a preferred embodiment of the present invention, the total concentration of cellulase and hemicellulase in the complex enzyme solution is 1.6-1.9 g / 100 mL, more preferably 1.9 g / 100 mL.

[0017] Furthermore, in the above technical solution, the preparation method of the complex enzyme solution in step (2) is as follows: cellulase and hemicellulase are added to acetic acid-sodium acetate buffer in order according to the ratio, and mixed to obtain the complex enzyme solution.

[0018] Furthermore, in the above technical solution, the concentration of the acetic acid-sodium acetate buffer is 0.1 M, and the pH is 4.80±0.02.

[0019] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the time of the enzyme treatment in step (2) is 89-90 minutes, most preferably 89 minutes.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] In order to improve the cooking quality and taste of brown rice, the present invention adopts a germination method combined with a composite enzyme (cellulase and hemicellulase) treatment process to study the cooking performance, rice texture and in vitro digestion characteristics of brown rice. The results show that the optimal composite enzyme treatment conditions are a composite enzyme concentration of 1.9g / 100mL, a treatment time of 89min, and a composite enzyme mass ratio of 4:1 (cellulase: hemicellulase), which significantly improves the water absorption rate, cooking speed and rice texture of brown rice. The bran layer structure of brown rice after composite enzyme treatment is looser, forming a large number of pore structures, which is conducive to water penetration. Compared with untreated brown rice, the hardness of brown rice treated with germination combined with composite enzymes is reduced by 34%, close to the level of polished white rice. Although germination treatment slightly increases the estimated glycemic index (eGI) of rice, the overall increase is less than 5%, which is still lower than the eGI of polished white rice. In summary, the germination method combined with composite enzyme treatment shows a significant effect in improving the edible quality of brown rice, and has a wide potential for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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 or the description of the prior art 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.

[0023] Figure 1 The invention discloses a preparation process of germinated brown rice.

[0024] Figure 2 Effect of single factor enzymatic hydrolysis of composite enzymes on the glucose content and D-xylose content in the enzymatic hydrolyzate; Figure A represents composite enzyme solutions of different concentrations; Figure B represents the time of enzymatic hydrolysis of different composite enzymes; Figure C represents the mass ratio of different composite enzymes, where X / B represents the content of cellulase / the content of hemicellulase.

[0025] Figure 3 3D response surface plots of the effects of various factors of the complex enzyme on the contents of glucose and D-xylose in the hydrolyzate: A and D respectively represent the surface plots of the effects of complex enzyme concentration and treatment time on the contents of glucose and D-xylose; B and E respectively represent the surface plots of the effects of complex enzyme concentration and proportion on the contents of glucose and D-xylose; C and F respectively represent the surface plots of the effects of complex enzyme treatment time and proportion on the contents of glucose and D-xylose.

[0026] Figure 4 Effect of germination method combined with complex enzyme treatment on water absorption rate of soaked brown rice.

[0027] Figure 5 Scanning electron micrograph of brown rice treated with germination method combined with complex enzyme (magnification 3800x); Note: Figure A shows the bran structure of BR; Figure B shows the bran structure of GBR; Figure C shows the bran structure of G-CEBR.

[0028] Figure 6 Textural properties of brown rice treated by germination method combined with complex enzymes; Note: Different letters in the top labels of the bar graphs indicate significant differences (p<0.05).

[0029] Figure 7 Starch components of brown rice treated by germination method combined with complex enzymes; Note: RDS stands for rapidly digestible starch; SDS stands for slowly digestible starch; RS stands for resistant starch.

[0030] Figure 8 In vitro digestion curve of brown rice treated with germination method combined with complex enzymes. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below through implementation cases. This implementation case is implemented based on the technology of the present invention, and a detailed implementation method and specific operation process are now given to illustrate that the present invention is creative, but the protection scope of the present invention is not limited to the following implementation cases.

[0032] The rice variety used in the following embodiments of the present invention is Zhongzao 39, which comes from Hunan Golden Nongfeng Seed Co., Ltd. and is produced during 2023. Brown rice is prepared from Zhongzao 39 rice by ridged ridges and stored in a sealed dry container at room temperature and away from light. Polished rice is Guangliangxiang No. 2, which comes from a local farmers' market. The reference food white bread used for in vitro digestion simulation comes from a local bakery.

[0033] The cellulase (≥400u / mg), hemicellulase (≥20u / mg) and amyloglucosidase (≥100,000U / mg) used in the following examples of the present invention were obtained from Shanghai Yuanye Biotechnology Co., Ltd. α-amylase (BR) was obtained from Shanghai MacLean Biochemical Technology Co., Ltd. Glucose kit (glucose oxidase method) was obtained from Nanjing Jiancheng Bioengineering Research Institute. Sodium hypochlorite, glacial acetic acid, sodium acetate, phloroglucinol, concentrated hydrochloric acid and anhydrous ethanol were all analytically pure.

[0034] Example 1

[0035] The process flow of preparing brown rice and germinating brown rice is as follows Figure 1 As shown. The rice is husked by the JLGJ-45 rice husker to obtain brown rice. Refer to the soaking germination method in NY / T 3522 "Technical Specifications for Processing of Sprouted Brown Rice" and make slight modifications: Soak the brown rice in sodium hypochlorite solution (0.2%, v / v) for disinfection for 10 minutes (the disinfection method used in actual production should meet the requirements of relevant standards, and this disinfection method is only applicable to laboratory preparation of germinated brown rice). Wash the brown rice 3 times with distilled water, then add distilled water to completely immerse the brown rice, and soak it at 30°C for 12 hours (change the water every 4 hours). After draining the brown rice, culture it at 30°C for 12 hours to obtain germinated brown rice. Dry the germinated brown rice at 40°C to a moisture content of less than 14%, and then store it in a dry and dark environment.

[0036] Example 2

[0037] Single factor experiment on enzymatic treatment conditions of cellulase and hemicellulase (complex enzyme)

[0038] 2.1 Complex enzyme solutions of different concentrations

[0039] 5.0 g of germinated brown rice prepared in Example 1 was added to a 50 mL centrifuge tube, and 7.5 mL of a composite enzyme solution (cellulase solution and hemicellulase solution (prepared with 0.1 M, pH = 4.80 ± 0.02, acetic acid-sodium acetate buffer)) was added, the composite enzyme mass ratio was 1:1, the enzyme treatment temperature was 50 ° C, the enzyme treatment time was 90 min, and the total concentrations of the composite enzyme solution were: 1.0, 1.3, 1.6, 1.9, 2.2 g / 100 mL, respectively.

[0040] 2.2 Time of enzymatic hydrolysis with different complex enzymes

[0041] 5.0 g of germinated brown rice prepared in Example 1 was added to a 50 mL centrifuge tube, and 7.5 mL of a composite enzyme solution (cellulase solution and hemicellulase solution (prepared with 0.1 M, pH = 4.80 ± 0.02, acetic acid-sodium acetate buffer)) was added, the composite enzyme mass ratio was 1:1, the total concentration of the composite enzyme solution was 1.6 g / 100 mL, the enzyme treatment temperature was 50 ° C, and the enzyme treatment times were 30, 60, 90, 120, and 150 min, respectively.

[0042] 2.3 Ratio of different complex enzymes

[0043] 5.0g of germinated brown rice prepared in Example 1 was added to a 50mL centrifuge tube, and 7.5mL of complex enzyme solution (cellulase solution and hemicellulase solution (adopting 0.1M, pH=4.80±0.02, acetic acid-sodium acetate buffer preparation)) was added, the enzyme treatment temperature was 50°C, the enzyme treatment time was 90min, the total concentration of the complex enzyme solution was 1.6g / 100mL, and the mass ratios of the complex enzymes were 5:1, 3:1, 1:1, 1:3, 1:5 (cellulase / hemicellulase), respectively.

[0044] 2.4 Blank control group

[0045] 5.0 g of the germinated brown rice prepared in Example 1 was added to a 50 mL centrifuge tube, and 7.5 mL of acetic acid-sodium acetate buffer (0.1 M, pH = 4.80 ± 0.02) was added, which was set as a blank control group. The contents of the corresponding indicators of the blank control group should be deducted when calculating the glucose and D-xylose contents of the enzymolyzed solution.

[0046] Determination of glucose and D-xylose content in enzymatic hydrolysate:

[0047] The method for determining the content of D-xylose in the enzymatic hydrolysate is as follows: use a pipette to draw 0.050 mL of the enzymatic hydrolysate or standard solution into a 2 mL EP tube (threaded cap type), then add 0.250 mL of a color developing solution (0.5 g of phloroglucinol, 100 mL of glacial acetic acid and 6 mL of concentrated hydrochloric acid), tighten the cap and vortex mix, heat in a water bath at 100°C for 8 minutes, immediately cool in an ice bath, and measure its absorbance at a wavelength of 554 nm as soon as possible. Draw a standard curve with the concentration of the D-xylose standard solution as the abscissa and its absorbance as the ordinate, and calculate the content of D-xylose in the enzymatic hydrolysate by a linear regression equation.

[0048] Glucose content in the enzymatic hydrolysate was determined using a glucose kit (glucose oxidase method).

[0049] Example 3 Response surface optimization experiment of compound enzyme hydrolysis treatment conditions

[0050] According to the results of the single factor experiment, the glucose content and D-xylose content in the enzymatic hydrolysate were used as evaluation indicators, and the Box-Behnken (BBD) in Design-Expert was used to carry out a three-factor three-level response surface design for the composite enzyme. The factor levels are shown in Table 1.

[0051] Table 1 Factors, levels and codes of the experimental treatment of germinated brown rice with compound enzymes

[0052]

[0053] Note: X / B represents the content of cellulase / the content of hemicellulase.

[0054] Example 4 Effect of germination method combined with complex enzyme treatment on the quality of brown rice

[0055] The optimal complex enzyme concentration, treatment time and ratio obtained by response surface optimization in Example 3 were used as the conditions for germination method combined with complex enzyme treatment to prepare a sample treated with germination method combined with complex enzyme, which was then washed three times with distilled water and dried at 40°C to a moisture content of less than 14%.

[0056] The effects of germination and germination combined with compound enzyme treatment on the quality of brown rice were studied by setting untreated brown rice as control group 1 (BR), germinated brown rice as control group 2 (GBR), and germination combined with compound enzyme treatment as experimental group (G-CEBR). Polished rice was added as control group 3 (WR) to study the texture characteristics and in vitro digestibility of brown rice.

[0057] Determination of cooking quality of brown rice

[0058] (I) The method for determining the soaking water absorption rate is as follows: Take a 250mL beaker and add 100mL of distilled water, place it in a water bath and keep the temperature at 25°C. Weigh 5.00g (mass is recorded as m1) of brown rice sample and add it to the above beaker, and start timing. When the soaking time reaches 10, 30, 60, 90, 120, and 180min, take out the brown rice sample, use filter paper to absorb the surface moisture and weigh it (mass is recorded as m2). Use formula 1 to calculate the soaking water absorption rate of the brown rice sample.

[0059]

[0060] (ii) The method for determining the optimal cooking time is as follows: Take a 250mL beaker, measure 100mL of distilled water and boil it. Weigh 5.00g of brown rice sample, add it to the above beaker, and start timing immediately. After 10 minutes of cooking, randomly select 6 brown rice samples every 2 minutes, place them on a clean flat surface and squeeze them with a glass plate. When more than 90% of the rice grains no longer have an opaque core or ungelatinized starch, record it as the cooking time. The brown rice sample should be cooked for another 2 minutes to ensure that all the rice grains are cooked, so the optimal cooking time needs to include these 2 minutes.

[0061] (III) The method for determining the heating water absorption rate is as follows: Take a 250mL beaker, measure 100mL of distilled water and boil it. Weigh 5.00g (mass is recorded as m1) of brown rice sample and add it to the above beaker. Start timing immediately. The cooking time is the optimal cooking time determined in 3.5.1. Take out the brown rice sample, use filter paper to absorb the moisture on its surface and weigh it (mass is recorded as m3). Use formula 2 to calculate the heating water absorption rate of the brown rice sample.

[0062]

[0063] (IV) Determination of volume expansion rate The method for determining volume based on the water displacement method is as follows: Weigh 5.00 g of brown rice sample, pour it into a 25 mL measuring cylinder with 10 mL of distilled water, read and record the volume as v1. Take out the brown rice sample from the measuring cylinder and drain the water, then place it in 100 mL of boiling water and cook for 20 min. Take out the brown rice sample, dry the surface water with filter paper, pour it into a 25 mL measuring cylinder with 10 mL of distilled water, read and record the volume as v2. Use formula 3 to calculate the volume expansion rate of the brown rice sample.

[0064]

[0065] (V) The method for determining the solid content of rice soup is as follows: Weigh 5.00 g (mass is recorded as m1) of brown rice sample, pour it into 100 mL of boiling water and cook for 20 min. Take 10.0 mL of rice soup and place it in a constant weight culture dish (mass is recorded as m4), dry it at 105°C to constant weight, and the mass of the culture dish after constant weight is recorded as m5.

[0066] Formula 4 calculates the rice soup solids of the brown rice sample.

[0067]

[0068] Preparation of brown rice

[0069] Refer to the preparation method of small sample rice in GB / T 15682-2008 and make slight modifications. Weigh 10.0g of sample into a steaming dish (304 stainless steel), pour it into the drain sieve, quickly add 300mL of water into the sieve, stir it clockwise for 10 circles and then counterclockwise for 10 circles, drain the water and quickly repeat the washing once, then rinse it with 200mL of distilled water once, drain the water and pour it back into the steaming dish. The rice washing time should be controlled within 3min to 5min. The rice-water ratio is 1:1.7 (m / m), that is, add 17.0g of distilled water into the steaming dish, place it in a constant temperature water bath, and soak it at 50℃ for 60min. Add an appropriate amount of water to the steamer, heat it to boiling with an electric stove or induction cooker, cover the rice steamer containing the sample and place it on the steamer, cover the steamer lid, continue heating and start timing, stop heating after steaming for 40 minutes, and simmer for another 20 minutes to obtain gelatinized brown rice or polished white rice samples. Store these samples in an incubator until they are taken out for measurement.

[0070] Determination of texture characteristics of brown rice

[0071] Zero the probe of the texture analyzer, and after the probe rises to an appropriate height, place the steamed rice dish containing the rice sample on the stage of the texture analyzer, adjust the steamed rice dish to an appropriate position, and perform the first texture property measurement. After the measurement is completed, rotate the steamed rice dish and repeat the measurement twice. It should be ensured that there is no intersection in the position where the probe is squeezed during each measurement. The conditions for measuring the texture properties of brown rice are as follows: a P25 (diameter 25.7mm) cylindrical probe is used, the speed during measurement is 60mm / min, the compression ratio is 80%, the interval between two presses is 3s, the data acquisition rate is 1000pps, and the trigger force is 0.05N.

[0072] Determination of in vitro digestibility of brown rice

[0073] The determination method of rice starch component is as follows: weigh 1.0g rice sample or reference food in a 50mL centrifuge tube, concentrate the rice grains at the bottom of the centrifuge tube as much as possible, use a glass rod of suitable diameter to apply a longitudinal extrusion force of 3000N to 4000N to the rice at the bottom of the centrifuge tube, repeat the extrusion operation about 8 times to simulate the oral chewing action, then add 10.0mL sodium acetate buffer (0.1M, pH=5.2), 5.0mL porcine pancreatic α-amylase (1.0%, m / m, prepared using sodium acetate buffer (0.1M, pH=5.2)), 5.0mL saccharifying enzyme (1.0%, m / m, prepared using sodium acetate buffer (0.1M, pH=5.2)) and 5 glass beads, use a constant temperature water bath oscillator to perform in vitro digestion simulation at 37°C and 150r / min, and start timing. When the in vitro digestion simulation was performed to 0, 10, 20, 40, 60, 90, 120 and 180 min, 0.2 mL of the enzymatic solution was drawn into a 5 mL centrifuge tube, 3.8 mL of anhydrous ethanol was added to terminate the enzymatic reaction, and the glucose content of the enzymatic solution was determined using a glucose kit (glucose oxidase method). 1.0 g of rice was weighed separately and its total starch content was determined according to the acid hydrolysis method of GB 5009.9-2023. The contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) in rice starch were calculated.

[0074] The in vitro digestion curve was determined according to the method of (Miao Ming. Study on the characteristics and formation mechanism of slowly digestible starch [D]. Food Science of Jiangnan University, 2009.) to calculate the starch hydrolysis rate of brown rice or reference food during the in vitro digestion simulation process and draw the starch hydrolysis rate-time curve.

[0075] The estimated glycemic index was determined by referring to the in vitro digestion kinetic model established by (GONI I, GARCIAALONSO A, SAURACALIXTO FA starch hydrolysis procedure to estimate glycemic index [J]. NUTRITION RESEARCH, 1997, 17 (3): 427-437.) The hydrolysis index (HI) of white bread was defined as 100, and the expected glycemic index (eGI) of each sample was calculated.

[0076] Data statistics and processing

[0077] All experimental data were statistically analyzed using Microsoft Office Excel; IBM SPSS26 software was used for data processing, and the results were expressed as mean ± standard deviation. The data were analyzed by one-way ANOVA to test the differences between the groups (p < 0.05 was significant); the response surface experiment was designed by Design-Expert13 for Box-Behnken experiments, and the Box-Behnken experimental results were statistically analyzed, processed and analyzed. OriginLab OriginPro software was used for data processing or image drawing.

[0078] Test results:

[0079] (I) Effect of single factor enzymatic hydrolysis of complex enzymes on glucose and D-xylose contents in enzymatic hydrolysates

[0080] Effects of single factor enzymatic hydrolysis of complex enzymes on glucose content and D-xylose content in enzymatic hydrolysate Figure 2 shown. Figure 2 A shows that when the concentration of the complex enzyme gradually increases, the content of glucose and D-xylose in the enzymatic hydrolyzate gradually increases. The growth rate of D-xylose content tends to be flat when the concentration of the complex enzyme reaches 1.6g / 100mL, and the growth rate of glucose content tends to be flat when the concentration of the complex enzyme reaches 1.9g / 100mL. Figure 2 B shows that as the compound enzyme treatment time increases, the glucose and D-xylose contents in the enzymatic hydrolyzate increase in an approximately linear manner. When the treatment time reaches 150 min, the two still have a certain growth trend. Because the compound enzyme enzymatic hydrolysis treatment environment is 0.1 M, pH = 4.80 acetic acid-sodium acetate buffer, as the brown rice sample soaks in the buffer for an increasing time, the sensory score of the brown rice sample will gradually decrease, so the compound enzyme treatment time should generally not exceed 120 min. Figure 2 C It can be clearly seen that when the ratio of the composite enzyme (cellulase content / hemicellulase content) gradually decreases, the contents of glucose and D-xylose in the hydrolysate both increase first and then decrease. The former reaches a peak value near the composite enzyme ratio of 3:1, and the latter reaches a peak value near the composite enzyme ratio of 1:1. For grass plants, the cellulose content accounts for the largest proportion, followed by hemicellulose. Therefore, the glucose content in the hydrolysate should be given priority when designing the response surface level.

[0081] Based on the above conclusions and analysis, a three-factor three-level BBD response surface experiment was designed with the coding of compound enzyme concentration factor (level 1.7-2.1 g / 100 mL), compound enzyme treatment time factor (level 80-100 min) and compound enzyme ratio factor (level 4:1-2:1 cellulase content / hemicellulase content).

[0082] (II) Results of three-factor three-level response surface experiment of complex enzyme and its optimization analysis

[0083] (2.1) Results of three-factor three-level response surface experiment on complex enzyme

[0084] In order to study the effect of compound enzyme hydrolysis conditions on the glucose content and D-xylose content in the hydrolyzate, a three-level BBD response surface experiment was designed for three factors: compound enzyme concentration, compound enzyme treatment time and compound enzyme ratio. The results are shown in Table 2. There are 15 groups of response surface experimental results for compound enzyme hydrolysis conditions, including 3 groups of center points (compound enzyme concentration of 1.9g / 100mL, compound enzyme treatment time of 90min, cellulase content / hemicellulase content ratio of 3:1) to evaluate the experimental error and model fitting effect. As can be seen from Table 2, the experimental group with the highest glucose content is Group 2, whose glucose content and D-xylose content are 6.25mg / mL and 0.47mg / mL respectively; the experimental group with the highest D-xylose content is Group 14, whose glucose content and D-xylose content are 5.79mg / mL and 0.49mg / mL respectively.

[0085] Table 2 Results of three-factor three-level response surface experiment of complex enzyme

[0086]

[0087] Note: X / B represents the content of cellulase / the content of hemicellulase.

[0088] (2.2) Establishment of mathematical regression model and variance test

[0089] Based on the experimental results of response surface treatment conditions of compound enzyme in 2.1, the fitting effects of linear, 2FI, quadratic and cubic mathematical regression models were compared using Design-Expert software. The fitting effects are shown in Table 3. The sequential significance P values ​​(Sequential P) of glucose content based on linear and quadratic mathematical regression models showed extremely significant differences (p < 0.01), indicating that both models can be used as fitting options. The size of the Lack of Fit P values ​​(Lack of Fit P) of the two models was compared, and the R 2 Adjusted R 2 ) and predicted R 2 Value (PredictedR 2) difference, the best mathematical regression model for fitting is Quadratic. Similarly, the fitting results of D-xylose content based on different mathematical regression models are compared and analyzed, and the best mathematical regression model for fitting is Quadratic.

[0090] Based on the Quadratic model, a multivariate quadratic regression fitting analysis was performed on the experimental results of the response surface experiment of the compound enzyme hydrolysis treatment conditions in Table 3, and the quadratic polynomial regression equations between the glucose content (C1) and the D-xylose content (C2) and the compound enzyme concentration (A), the compound enzyme treatment time (B), and the compound enzyme ratio (C) were obtained as Formula 5 and Formula 6, respectively.

[0091]

[0092] C2=1.7182-3.40927A+0.045105B-0.371544C-0.000864AB+0.086357AC

[0093] +0.000212BC+0.879392A 2 -0.000209B 2 +0.033524C 2 (6)

[0095] Variance analysis was performed on regression model C1 and regression model C2, and the results are shown in Table 4. The results of variance analysis showed that the glucose content was significantly affected by the single factors of compound enzyme concentration, compound enzyme treatment time and compound enzyme ratio, among which the single factor with the greatest influence was compound enzyme treatment time, followed by compound enzyme concentration; for the interaction effect of the two factors, only the interaction effect of compound enzyme treatment time and compound enzyme ratio had a significant effect. The D-xylose content was significantly affected by the single factors of compound enzyme concentration, compound enzyme treatment time and compound enzyme ratio, and the order of the influence of each single factor was consistent with the result of glucose content, but the influence of compound enzyme treatment time was much greater than that of other single factors; for the interaction effect of the two factors, only the interaction effect of compound enzyme concentration and compound enzyme ratio had a significant effect.

[0096] The signal-to-noise ratios (Adeq Precision) of regression model C1 and regression model C2 are 43.5986 and 36.8971, respectively, both greater than 4, and the coefficient of variation (CV%) of the two is less than 15%, indicating that within the selected range, the two can better predict the changes in the glucose content and D-xylose content of the enzymatic hydrolyzate during the process of complex enzyme treatment of brown rice samples, and the experimental results of complex enzyme treatment of brown rice samples can be predicted by the two.

[0097] Table 3 Fitting results of glucose content and D-xylose content based on different mathematical regression models

[0098]

[0099] Table 4 Results of variance analysis of the regression model for glucose content (C1) and D-xylose content (C2)

[0100]

[0101] Note: The * on the upper right of each column of P value indicates a significant difference, and the ** indicates an extremely significant difference.

[0102] (2.3) 3D response surface plot analysis of mathematical regression model

[0103] The 3D response surface plot of the mathematical regression model can visualize the complex multi-factor relationship, so as to more intuitively understand the interaction between different factors. For example, by controlling any factor variable in the response surface plot and changing another factor variable, the change trend of the response variable can be intuitively seen. The 3D response surface plot of the influence of various factors of the complex enzyme on the glucose content and D-xylose content in the enzymatic hydrolysate is shown in Figure 2. Figure 3 shown.

[0104] Figure 3 A, 3B and 3C are 3D response surface plots of the effects of compound enzyme concentration, treatment time and ratio on the glucose content in the enzymatic hydrolysate. Figure 3 Surface graph A shows a steeper surface, and the contour lines are dense and parallel, indicating that under the condition that the ratio of the complex enzyme remains unchanged, the effects of the complex enzyme concentration and the treatment time on the glucose content of the enzymatic hydrolysate are approximately linear, and the glucose content of the enzymatic hydrolysate reaches its maximum value when the two reach their maximum values. Figure 3 The curve B is steeper at low potential, but flatter near the peak, indicating that when the concentration and ratio of the complex enzyme increase to a certain value, their effects on the glucose content of the enzymatic hydrolyzate become insignificant. Figure 3 The C surface graph shows a "saddle" shape near the peak value, indicating that when the composite enzyme treatment time is longer, the glucose content in the enzymatic hydrolyzate first increases and then decreases with the composite enzyme ratio.

[0105] Figure 3 D, 3E and 3F are surface graphs showing the effects of compound enzyme concentration, treatment time and ratio on the D-xylose content in the enzymatic hydrolysate. Figure 3 The D surface graph presents a "tile" shape, which shows that when the concentration and ratio of the complex enzyme remain unchanged, the change in the D-xylose content in the hydrolyzate tends to be gentle when the complex enzyme treatment time increases to a certain value; when the complex enzyme treatment time and ratio remain unchanged, the D-xylose content in the hydrolyzate first decreases and then increases with the concentration of the complex enzyme. Figure 3The E surface graph presents a "bowl" shape. With the increase of the concentration and ratio of the composite enzyme, the D-xylose content in the hydrolysate shows a trend of first decreasing and then increasing. When the composite enzyme concentration and the composite enzyme ratio reach the maximum value, the D-xylose content in the hydrolysate reaches the maximum value. Figure 3 The F surface graph shows a "tile" shape, and the influence of compound enzyme treatment time on the content of D-xylose in the enzymatic hydrolysate is similar to Figure 3 D; when the concentration of the composite enzyme and the treatment time remain unchanged, the content of D-xylose in the enzymatic hydrolysate first decreases and then increases with the proportion of the composite enzyme.

[0106] (2.4) Determination of optimal compound enzyme treatment conditions and verification test results

[0107] From the 3D response surface plot analysis results of the influence of various factors of the complex enzyme on the content of glucose and D-xylose in the enzymatic hydrolysate in 2.3, it can be seen that the response variable can only be predicted well when two of the factors are kept unchanged. When the various factors of the complex enzyme interact with each other, the changes in the content of glucose and D-xylose become very complicated, and it is impossible to predict the expected values ​​of the various factors of the complex enzyme. The above problem can be solved by using the Numerical function in the Design-Expert software, and the optimal design of the action conditions of each factor of the complex enzyme can be achieved under the specified response index.

[0108] The expectation of the target of glucose and D-xylose content in the enzymolysate was set to maximize as a prerequisite, and the compound enzyme concentration, treatment time and ratio conditions were optimized. When there were no restrictions on the conditions of the various factors of the compound enzyme, the optimal conditions for the compound enzyme treatment of brown rice samples were 2.1g / 100mL of compound enzyme concentration, 100min of treatment time and 3.68:1.00 of ratio, at which time the glucose and D-xylose contents in the enzymolysate were 6.25mg / mL and 0.51mg / mL, respectively. When the compound enzyme concentration parameters were minimized (considering the material cost factor) and the treatment time parameters were minimized (considering the time cost and sensory quality), the optimal conditions for the compound enzyme treatment of brown rice samples were 1.9g / 100mL of compound enzyme concentration, 89min of treatment time and 4:1 of ratio, at which time the glucose and D-xylose contents in the enzymolysate were 5.54mg / mL and 0.41mg / mL, respectively. In summary, the optimal compound enzyme treatment conditions for brown rice samples were determined to be 1.9 g / 100 mL compound enzyme concentration, 89 min treatment time, and 4:1 mass ratio, which can maximize the expected glucose and D-xylose content of the enzymatic hydrolyzate, while taking into account material cost, time cost, and sensory factors. The predicted value and actual value of the response variable obtained from the optimal compound enzyme treatment conditions for brown rice samples are shown in Table 6.

[0109] As can be seen from Table 5, under the best complex enzyme treatment brown rice sample condition, the actual values ​​of glucose and D-xylose content were 5.35mg / mL and 0.40mg / mL respectively in the enzymolyte. Utilize formula 7 to calculate the relative error (D%) of actual value and predicted value, can draw that under this complex enzyme treatment condition, the actual value of glucose content and predicted value relative error in the enzymolyte are 3.53%, and the actual value of D-xylose content and predicted value relative error are 2.93%, both are less than 10%, show that actual value and predicted value are relatively close. Therefore, it can be considered that the mathematical regression model precision and reliability of setting up in 4.2.2 are higher, have certain practical value within a specific range.

[0110]

[0111] Table 5 Comparison of actual and predicted values ​​of response variables under optimal compound enzyme treatment conditions

[0112]

[0113] (2.5) Effect of germination method combined with complex enzyme treatment on the cooking quality of brown rice

[0114] The soaking water absorption rate is one of the important indicators for evaluating the cooking quality of rice grains. Under the same soaking time, the higher the soaking water absorption rate, the better the water absorption performance of the rice grains, making the rice grains easier to swell, gelatinize and soften during the heating process. Figure 4 As shown. The results showed that the water absorption rate of BR increased slowly during the soaking process, and the water absorption tended to be saturated after the soaking time reached 120 min. The water absorption rate of GBR increased faster than that of BR within the soaking time of 0-30 min, and the change trend of water absorption after soaking time of 60 min was similar to that of BR. The water absorption rate of G-CEBR increased fastest within the soaking time of 0-30 min, and its water absorption rate was about 1.7 times that of BR at 30 min. The soaking water absorption rates of BR, GBR and G-CEBR all tended to 25% in the end. Based on the above results, it can be explained that germination treatment can slightly improve the water absorption rate of brown rice during soaking, and germination combined with compound enzyme treatment can significantly improve the water absorption rate of brown rice during soaking, while germination or enzyme treatment has little effect on the saturated soaking water absorption rate of brown rice.

[0115] The effects of germination combined with compound enzyme treatment on the cooking performance of brown rice are shown in Table 6. The optimal cooking time of BR, GBR and G-CEBR were significantly different (p<0.05), among which the optimal cooking time of G-CEBR was about 0.8 times that of BR, indicating that germination combined with compound enzyme treatment can shorten the cooking time of brown rice, thereby improving production efficiency. The heating water absorption rate is closely related to the softness, fullness and structural uniformity of rice grains after cooking. Rice grains with high water absorption rate can usually be cooked into soft and full rice. The heating water absorption rate of GBR was significantly greater than that of BR (p<0.05), but not significantly higher than that of G-CEBR (p>0.05), indicating that germination treatment improved the water absorption performance of brown rice after cooking, while the changes in the production of brown rice bran layer caused by enzyme treatment had little effect on the heating water absorption rate of brown rice. Volume expansion rate is an evaluation index of the cooking performance of rice grains to evaluate whether the appearance and taste of rice grains after being cooked are good. Rice grains with high volume expansion rate usually have good visual perception (clear grains, uniform distribution and full rice grains). The volume expansion rate of G-CEBR is significantly higher (p<0.05) than that of BR and GBR. This indicates that the change of the bran layer after enzyme treatment improves the cooking performance of rice grains, so that the rice grains absorb water more evenly and expand in volume during the cooking process. The solid content of rice soup is used to evaluate the loss of nutrients in polished rice during cooking and the integrity of the surface structure of rice grains (usually the smaller the value, the better), while the existence of the bran layer of brown rice may lead to the opposite conclusion when used to evaluate brown rice (rice grains are wrapped by the bran layer, and the thicker the bran layer, the less likely the solids inside will flow out during cooking, reflecting the low degree of gelatinization of the rice grains, and the larger the value, the better). The solid content of rice soup of G-CEBR is significantly higher (p<0.05) than that of BR and GBR. The results showed that the bran layer of brown rice was easier to break after enzyme treatment, which was beneficial for the starch inside the brown rice to fully contact with water. In summary, although germination treatment can improve the cooking quality of brown rice to a certain extent, the degree of improvement is small, while germination combined with compound enzyme treatment can significantly improve the cooking quality of brown rice, which is beneficial to shorten the production time of brown rice and improve the sensory quality of brown rice.

[0116] Table 6 Effect of germination method combined with compound enzyme treatment on cooking performance of brown rice

[0117]

[0118] Note: Different letters on the upper right of the values ​​in each column indicate significant differences (p<0.05).

[0119] (2.6) Effects of germination method combined with complex enzyme treatment on the microstructure of brown rice

[0120] Scanning electron microscopy images of brown rice treated with germination method and complex enzymes Figure 5 As shown. Figure 5 A It can be seen that the surface of the BR bran layer presents a dense and relatively smooth structure as a whole, with a small number of shallow grooves in some parts, which is the structure formed by brown rice in its natural state. Figure 5 B is the structure of the GBR bran layer. It can be clearly seen that there are many wrinkles on its surface and many deep grooves, which are relatively rough overall. Figure 5 C. Compared with the bran structure of GBR, the bran structure of G-CEBR is obviously looser and rougher, with more wrinkled textures and a large number of pore structures. Based on the above results and in conjunction with the results and analysis of cooking quality, it can be inferred that germination treatment activates the endogenous enzyme system of brown rice, hydrolyzes part of the bran structure, and the hydrolysis is mainly concentrated in the gullies formed in the natural state of brown rice, making the gullies of the bran layer of brown rice deeper and the structure loose and rough, which improves the water absorption capacity of brown rice, but to a limited extent. Exogenous cellulase and hemicellulase treatment of germinated brown rice can further hydrolyze the bran structure of brown rice, forming a large number of pore structures that do not exist in brown rice in the natural state, making the bran layer of brown rice looser overall, and reducing the thickness of the bran layer, thereby significantly improving the cooking quality of brown rice.

[0121] (2.7) Effects of germination method combined with complex enzyme treatment on the texture characteristics of brown rice

[0122] Texture properties of brown rice treated by germination method combined with complex enzymes Figure 6 As shown. The hardness of BR, GBR and G-CEBR rice was 3886.6, 3302.1 and 2560.5g, respectively, and there were significant differences (p<0.05). From the results, it can be seen that the hardness of brown rice can be significantly reduced after germination or germination combined with compound enzyme treatment. The hardness reduction of germination treatment is about 85% of the original hardness, and the hardness reduction of germination combined with compound enzyme treatment is about 66% of the original hardness, indicating that the germination combined with compound enzyme treatment has a good effect on improving the hardness of brown rice. In addition, compared with the hardness of polished rice (2082.7g), it can be calculated that the hardness of G-CEBR rice is about 1.2 times that of polished rice. Therefore, it can be considered that the palatability of brown rice can be significantly improved by treating brown rice with germination combined with compound enzymes, making the hardness of brown rice close to that of polished rice.

[0123] (2.8) Effects of germination method combined with complex enzyme treatment on in vitro digestibility of brown rice

[0124] Starch components of brown rice treated by germination method combined with complex enzymes

[0125] The starch components of brown rice treated by germination method combined with complex enzymes Figure 7 As shown. It can be clearly seen that the RDS content ratios of BR, GBR and G-CEBR are not much different, the SDS content ratios of GBR and G-CEBR are very similar to the RDS content ratios, the SDS content ratios of GBR and G-CEBR are increased compared with the SDS content ratio of BR, and the RS content ratios of GBR and G-CEBR are reduced compared with the RS content ratio of BR. Therefore, it can be considered that germination treatment will change the starch composition of brown rice, among which the RS content ratio decreases during germination, the SDS content ratio increases during germination, and the RDS content ratio has little effect. The reason why germination treatment can change the starch composition of brown rice may be that the endogenous enzyme system activated during germination hydrolyzes part of RS and converts RS into SDS. The exogenous enzymes used in the germination method combined with compound enzyme treatment are cellulase and hemicellulase. These two enzymes are highly specific and can only hydrolyze cellulose and hemicellulose in brown rice, so theoretically they will not affect the starch composition of brown rice. This speculation is consistent with the experimental results.

[0126] In vitro digestion curve of brown rice treated by germination method combined with complex enzymes

[0127] Figure 8 The in vitro digestion curve of brown rice treated with germination method combined with compound enzymes shows that the starch hydrolysis rate of GBR and G-CEBR has almost the same trend over time. From 0 to 60 min, the starch hydrolysis rate of BR, GBR and G-CEBR has similar trends. After 60 min of digestion, the growth rate of BR's starch hydrolysis rate began to be lower than that of GBR and G-CEBR. The starch hydrolysis rates of BR, GBR and G-CEBR tended to balance at 120 min of digestion, among which the final starch hydrolysis rate of BR was about 40%, while that of GBR and G-CEBR was about 45%. In summary, it can be considered that germination treatment will slightly increase the starch hydrolysis rate of brown rice, while cellulase and hemicellulase treatment have little effect on the starch hydrolysis rate of brown rice. Combined with the results of starch components, it can be inferred that the reason why the final starch hydrolysis rate of GBR and G-CEBR is higher than that of BR is that part of the indigestible RS is converted into digestible SDS.

[0128] Estimated glycemic index of brown rice treated by germination method combined with complex enzymes

[0129] The in vitro digestion simulation results of BR, GBR and G-CEBR rice were mathematically fitted and calculated, and their first-order kinetic parameters and estimated glycemic index were obtained as shown in Table 7. The equilibrium hydrolysis rates of GBR and G-CEBR were slightly different, and the equilibrium hydrolysis rate increased by about 10% compared with that of BR. The eGI values ​​of GBR and G-CEBR were 62.93 and 62.48, respectively, and there was no significant difference between the two (p>0.05), but both were significantly higher (p<0.05) than the eGI value of BR (60.91). Therefore, it can be inferred that germination treatment will increase the eGI value of brown rice, while cellulase and hemicellulase treatment have little effect on the eGI value of brown rice. Although the eGI values ​​of GBR and G-CEBR are significantly higher (p<0.05) than the eGI value of BR, the actual increase in their eGI values ​​is less than 5% of the original value, and there is still a large gap compared with the eGI value of polished white rice (75.75), which is acceptable. In the actual production process, if the eGI value of brown rice needs to be controlled, the germination degree of brown rice should be reasonably controlled to avoid excessive increase in the eGI value of brown rice due to excessive germination.

[0130] Table 7 First-order kinetic parameters and estimated glycemic index of brown rice treated by germination method combined with complex enzymes

[0131]

[0132] Note: Different letters on the upper right of the values ​​in each column indicate significant differences (p<0.05); C ∞ represents the equilibrium hydrolysis rate; k represents the first-order kinetic reaction rate; AUC represents the area under the curve of the fitting curve; HI represents the hydrolysis index; eGI represents the estimated glycemic index.

[0133] The present invention performs germination treatment and germination method combined with complex enzyme treatment on brown rice, and compares the brown rice quality evaluation indicators such as cooking quality, microstructure, texture characteristics and in vitro digestion characteristics with the brown rice without any treatment, and mainly draws the following conclusions:

[0134] (1) Cellulase and hemicellulase can effectively hydrolyze cellulose and hemicellulase in the bran layer, respectively. The optimal conditions for their combined use are a combined enzyme concentration of 1.9 g / 100 mL, a treatment time of 89 min, and a ratio of 4:1 (cellulase: hemicellulase).

[0135] (2) Germination treatment can slightly increase the water absorption rate of brown rice, and germination combined with complex enzyme treatment can significantly increase the water absorption rate of brown rice, thereby significantly improving the cooking performance of brown rice and shortening the cooking time of brown rice.

[0136] (3) The germination method combined with complex enzyme treatment can significantly change the microstructure of the brown rice bran layer, forming a large number of pore structures, making the bran layer loose and rough, which not only improves the steaming performance of brown rice, but also reduces the hardness of brown rice, and significantly improves the palatability.

[0137] (4) The eGI value of brown rice was not significantly affected by cellulase and hemicellulase treatment, but germination may increase the eGI value of brown rice.

[0138] In summary, the germination method combined with complex enzyme treatment can shorten the cooking time of brown rice and reduce the hardness of rice by improving the performance of the brown rice bran layer, thereby achieving the purpose of improving the cooking quality and palatability of brown rice, which has certain practical value.

Claims

1. A method for improving the edible quality of brown rice by using a germination method combined with a composite enzyme treatment, characterized in that: The method specifically comprises the following steps: (1) after cleaning the brown rice, adding distilled water to completely immerse the brown rice, and soaking it at room temperature for 10-15 hours; after the soaking, draining the brown rice, continuing to cultivate it at room temperature for 10-15 hours, and finally drying it to obtain germinated brown rice; (2) mixing the germinated brown rice with a composite enzyme solution according to a ratio, subjecting the resulting mixture to a constant temperature enzyme treatment at 50° C. for 30-150 min to obtain enzymolyzed brown rice; wherein the composite enzyme solution is composed of a cellulase solution and a hemicellulase solution.

2. The method according to claim 1, characterized in that: The usage ratio of the germinated brown rice to the complex enzyme solution in step (2) is 1 part by mass: 1.5 parts by volume; wherein: the mass parts and the volume parts are based on g: mL.

3. The method according to claim 1, characterized in that: The mass ratio of cellulase to hemicellulase in step (2) is 5:1-1:

5.

4. The method according to claim 3, characterized in that: The mass ratio of the cellulase to the hemicellulase is 4:

1.

5. The method according to claim 1, characterized in that: The total concentration of cellulase and hemicellulase in the complex enzyme solution in step (2) is 1.0-2.2 g / 100 mL.

6. The method according to claim 5, characterized in that: The total concentration of cellulase and hemicellulase in the complex enzyme solution is 1.6-1.9 g / 100 mL.

7. The method according to claim 1, characterized in that: The preparation method of the complex enzyme solution in step (2) is as follows: add cellulase and hemicellulase into acetic acid-sodium acetate buffer in order according to the ratio, mix well, and obtain the complex enzyme solution.

8. The method according to claim 7, characterized in that: The concentration of the acetic acid-sodium acetate buffer is 0.1 M, and the pH is 4.80±0.

02.

9. The method according to claim 1, characterized in that: The time of the enzyme treatment in step (2) is 89-90 minutes.

10. The method according to claim 1, characterized in that: The time of the enzyme treatment in step (2) is 89 minutes.

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