A method for improving the polyphenol content and antioxidant capacity of germinated brown rice

Abstract

This invention discloses a method for improving the polyphenol content and antioxidant capacity of germinated brown rice, aiming to solve the problems of limited polyphenol enrichment, low germination rate under low-temperature stress, long cycle, and poor stability in existing germination technologies. The method includes the following core steps: mature, intact brown rice is taken, impurities are removed, and it is washed and sterilized with sodium hypochlorite. The sterilized brown rice is then soaked in a chitosan oligosaccharide solution, stirred regularly during the process. The soaked brown rice is then subjected to low-temperature stress treatment for several hours, during which it is sprayed with methyl jasmonate solution. After germination, the brown rice is dried in a hot air drying oven. This invention produces germinated brown rice with a significant polyphenol enrichment effect, while simultaneously improving the germination rate and uniformity. The process is mild, highly repeatable, and suitable for the large-scale production of functional germinated brown rice.

Description

Technical Field

[0001] This invention belongs to the field of deep processing of grains and functional food technology, specifically relating to a method for increasing the polyphenol content and antioxidant capacity of germinated brown rice. Background Technology

[0002] Brown rice, as one of the most common whole grains, contains higher levels of dietary fiber, vitamins, minerals, and polyphenols than traditional white rice. It is a nutritious grain that has become increasingly popular with consumers in recent years.

[0003] Germination activates endogenous metabolic processes in grains, significantly increasing the content of active ingredients such as polyphenols and γ-aminobutyric acid (GABA). Polyphenols in brown rice have antioxidant and anti-inflammatory effects; they can regulate blood sugar and lipids, protect cardiovascular health, improve gut microbiota structure, inhibit tumor cell proliferation, and offer multiple benefits including neuroprotection and liver protection. Germinated brown rice exhibits a significantly increased proportion of free polyphenols, resulting in enhanced bioavailability and activity. Therefore, germination is a crucial processing method for enhancing the nutritional function of brown rice, and germinated brown rice is increasingly favored by consumers.

[0004] However, existing germination methods for improving the polyphenol content and antioxidant capacity of germinated brown rice often suffer from the following problems: 1. Simply changing the soaking conditions, such as adding a single substance to the soaking solution or only altering the pH value, has limited germination effects; 2. Applying only a single stress condition, such as temperature control or light regulation, results in unstable germination effects, and some stress methods, such as low-temperature stress, may lead to long germination cycles and low germination rates; 3. Simply combining multiple treatment methods without creating a synergistic effect between them. Therefore, developing a highly efficient, stable, mild, food-suitable, and reproducible process to efficiently increase polyphenol content and simultaneously enhance the antioxidant capacity of germinated brown rice has significant practical implications and application value. Summary of the Invention

[0005] Existing technologies suffer from limited and unstable germination effects, and single stress methods may have some negative effects. To address these issues, the present invention aims to provide a germination method that combines exogenous plant-induced stress factors with low-temperature stress, and applies plant growth regulators during the germination process, thereby increasing the polyphenol content and antioxidant capacity of germinated brown rice. To achieve the above objectives, the present invention provides a method for increasing the polyphenol content and antioxidant capacity of germinated brown rice, specifically comprising the following steps: S1. Select mature brown rice with intact grains, remove impurities, wash and clean it, and disinfect it. After disinfection, rinse it with deionized water until there is no residue to obtain pre-treated brown rice. S2. Soak the pretreated brown rice from step S1 in a chitosan oligosaccharide solution to obtain pre-soaked brown rice. S3. The brown rice pre-soaked in step S2 is subjected to low-temperature stress treatment, during which methyl jasmonate solution is sprayed; after the low-temperature stress treatment is completed, it continues to germinate in a constant temperature incubator, during which distilled water is sprayed. S4. After germination, the germinated brown rice is dried to obtain germinated brown rice with high polyphenol content and high antioxidant capacity.

[0006] In one embodiment of the present invention, the disinfection step in step S1 involves soaking the selected, dust-removed and washed brown rice in a sodium hypochlorite solution with a concentration of 0.1-1% for 10-20 minutes, and then rinsing it with deionized water until there is no residue.

[0007] In one embodiment of the present invention, in step S2, the ratio of brown rice to chitosan oligosaccharide solution is 1:(2~5) (g:mL), and the concentration of chitosan oligosaccharide solution is 20~40mg / L.

[0008] In one embodiment of the present invention, in step S2, the soaking temperature is 25~35℃, the soaking time is 8~16h, and the mixture is stirred once every 2~6h.

[0009] In one embodiment of the present invention, in step S3, the temperature of the low-temperature stress is 10~20°C and the duration is 12~36h.

[0010] In one embodiment of the present invention, in step S3, the concentration of the sprayed methyl jasmonate solution is 10~50 μmol / L, and it is sprayed once every 1~2 hours, with each spraying lasting for 1~2 minutes.

[0011] In one embodiment of the present invention, in step S3, after the low temperature stress treatment is completed, germination continues in a constant temperature incubator at 25~35℃, during which distilled water is sprayed once every 1~2 hours, and the germination time is 12~24 hours.

[0012] In one embodiment of the present invention, in step S4, after germination, the germinated brown rice can be dried by any one of low-temperature hot air drying, vacuum drying or freeze drying. Low-temperature hot air drying is carried out at 45~55°C.

[0013] In one embodiment of the present invention, in step S4, the moisture content of the dried germinated brown rice is less than 10%.

[0014] The present invention also provides an application of germinated brown rice obtained by the above method in food or pharmaceuticals.

[0015] Beneficial effects: (1) The composite treatment process of pre-soaking with chitosan oligosaccharides combined with low-temperature stress and methyl jasmonate spraying in this invention specifically solves the problems of low germination rate, poor stability, and uneven morphology of germinated brown rice caused by existing methods for promoting germination. The germinated brown rice obtained by the method of this invention has a stable germination rate of over 94% and a germination potential of over 88.64%, which is 26.5% higher than the germination rate of the single low-temperature stress group; the uniformity of the germination is over 90%, with consistent germination length, robustness, and no deformities. Through the synergistic effect of the three, this invention not only ensures the high efficiency, stability, and quality of germination, but also simultaneously enriches functional components such as polyphenols. The process is repeatable and highly practical, meeting the industrial production needs of functional germinated brown rice.

[0016] (2) Chitosan oligosaccharide, as a biological stress elicitor, can efficiently activate the activity of key enzymes in the phenylpropane metabolic pathway; low temperature stress not only enables brown rice to synthesize more phenolic substances and γ-aminobutyric acid under extreme conditions, but also slows down the decomposition rate of active substances under low temperature conditions, thereby promoting the accumulation of phenolic substances and γ-aminobutyric acid; methyl jasmonic acid further amplifies the increase in enzyme activity of key enzymes by regulating endogenous signaling pathways. Under the synergistic effect of the three, the content of functional components such as coumaric acid, ferulic acid, flavonoids and γ-aminobutyric acid in germinated brown rice is significantly higher than that in any single treatment group, greatly improving the functional activity value of germinated brown rice.

[0017] (3) Although low temperature stress can enrich functional components, it also poses the potential risk of delaying germination rate and reducing germination potential. Methyl jasmonic acid can effectively compensate for the inhibitory effect of low temperature on germination process by enhancing the stress resistance of brown rice under extreme conditions, thereby improving germination rate and bud vigor. Soaking brown rice with chitosan oligosaccharide before stress treatment further optimizes germination uniformity and enhances grain stress resistance. The three factors work together to achieve "enrichment of functional components" while ensuring the uniformity of bud appearance.

[0018] (4) Chitosan oligosaccharide can form an antibacterial protective film on the surface of the grain, low temperature stress can inhibit the reproduction of microorganisms, and methyl jasmonic acid can delay the aging of endosperm and bud tissue; the three work together to activate the grain antioxidant system (significantly enhance the activity of enzymes such as SOD and CAT), effectively remove reactive oxygen species and reduce oxidative damage.

[0019] (5) Compared with the method of simply enriching the active ingredients in germinated brown rice by low temperature stress treatment, the treatment time of the method of the present invention is significantly shortened and the germination uniformity is improved.

[0020] (6) Methyl jasmonate is a highly efficient signaling molecule derived from plants and is widely present in plants. It is closely related to plant stress resistance and participates in various stress responses. When plants suffer mechanical damage or low temperatures, methyl jasmonate can be rapidly synthesized and diffused, enhancing plant stress resistance and regulating plant growth. In terms of safety, methyl jasmonate is a naturally occurring substance in plants, which is easily degraded in the environment and has no residue risk. Therefore, as an exogenous green plant elicitor, it protects plants by enhancing crop resistance rather than directly killing them. It is friendly to non-target organisms and is one of the safe choices to replace or reduce chemical pesticides. In this invention, only a small amount of low-concentration methyl jasmonate is sprayed during low-temperature stress treatment, and distilled water is sprayed on brown rice during the germination stage. The effect of enhancing brown rice resistance can be achieved without the need for large-scale use. After the stress treatment, a large amount of distilled water is used for rinsing to further eliminate the risk of residue. Combined with its own characteristics of easy degradation and no residue, it can be ensured that there will be no harm to human health due to residue, thus taking into account both resistance enhancement and food safety. Attached Figure Description

[0021] Figure 1 The morphology of germinated brown rice in Examples 1-6, Comparative Examples 1-5, and Comparative Example 9 is shown. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0023] (a) Determination of polyphenol content Accurately pipette 200 μL of sample extract, add 800 μL of ultrapure water and 200 μL of Folin-Ciocalteu reagent, and incubate the mixture for 6 minutes. Then add 1.8 mL of deionized water and 2 mL of 7% sodium carbonate solution, mix thoroughly with a vortex mixer, and react at room temperature in the dark for 90 minutes. After the reaction, measure the absorbance of the mixture at 760 nm. Using gallic acid (GAE) as a standard, plot a standard curve and derive the regression equation. The final total phenol content is expressed as "milligrams of gallic acid equivalent per 100 g dry weight of sample" (unit: mg GAE / 100 g DW).

[0024] (II) Determination of relevant enzyme activities The enzyme activities of the samples were determined using an enzyme-linked immunosorbent assay (ELISA) kit for plant phenylalanine ammonia-lyase (PAL) and cinnamic acid-4-hydroxylase (C4H). 1 g of brown rice sample was placed in a mortar and rapidly pulverized with liquid nitrogen. Then, 50 mM PBS buffer was added at a 1:9 material-to-liquid ratio (w / v) for extraction. The sample was centrifuged at 3000 rpm for 20 min, and the supernatant was collected to determine the activity of the relevant enzymes.

[0025] (III) Determination of antioxidant capacity (1) Determination of DPPH antioxidant capacity Brown rice was ground into powder using a grinder, and extracted with 80% ethanol solvent by ultrasonic extraction for 30 min to obtain the extract to be tested. 50 μL of the extract was mixed with 750 μL of DPPH solution (0.076 mM), and incubated at room temperature in the dark for 30 min. The absorbance of the mixture at 517 nm was then measured. The antioxidant activity was calculated according to formula (1): DPPH free radical scavenging capacity (%) = [(A0-A s ) / A0]×100(1) In the formula, A0 represents the absorbance of the blank group (containing only DPPH solution and 80% ethanol), and As represents the absorbance of the sample group (containing extract and DPPH solution).

[0026] (2) Determination of the antioxidant capacity of ABTS: Brown rice was ground into powder using a grinder, and then extracted with 80% ethanol using ultrasound for 30 minutes to obtain the extract to be tested. ABTS + The solution was prepared by mixing equal volumes of 7.0 mM ABTS and 4.95 mM potassium persulfate (stock solution). + After solution preparation, the reaction was carried out at 4°C in the dark for 12 hours. It was then diluted with anhydrous ethanol, and its absorbance at 734 nm was adjusted to 0.70 ± 0.02. 10 μL of the extract was taken and 190 μL of ABTS was added. + The solution was incubated at room temperature in the dark for 10 minutes, and the absorbance at 734 nm was recorded. The antioxidant capacity determined by the ABTS method was calculated according to formula (2): ABTS free radical scavenging capacity (%) = [(A0-A s ) / A0]×100(2) In the formula, A0 represents the blank group (containing only ABTS). + The absorbance of the solution and 80% ethanol, A s Representative sample group (including extract and ABTS) + The absorbance of the solution.

[0027] (iv) Measurement of germination-related indicators (1) Germination potential: Brown rice samples were selected after pretreatment (impurity removal, washing, and disinfection) and corresponding germination processes. Parallel samples were set up for each group to ensure test repeatability. During the brown rice germination process, the focus was on low-temperature stress and the subsequent isothermal germination stage. Measurements were taken at the critical early germination time point with the highest germination uniformity. The number of germinated (radicle breaking through the seed coat) brown rice grains at this time point was recorded, and the percentage of germinated grains (radicle breaking through the seed coat) was calculated. The results are expressed as mean ± standard error.

[0028] (2) Germination rate: The same parallel brown rice samples were used for germination potential measurement to ensure consistent testing conditions. Measurements were taken after the brown rice had completed all germination treatments (S3 germination treatment ended, but before S4 drying). The number of all germinated grains (with normal radicle growth, excluding abnormally germinated individuals) in the tested brown rice was counted, and the percentage of germinated grains to the total number of brown rice grains was calculated. The average value of parallel samples was taken, and the standard error was noted.

[0029] Example 1 A method for compound stress germination treatment to increase the polyphenol content of brown rice includes the following steps: S1. Raw material pretreatment Select mature, whole-grain brown rice, remove impurities and broken grains, rinse three times with deionized water to remove surface dust, and then disinfect the washed brown rice by soaking it in a 0.1% sodium hypochlorite solution for 15 minutes. After disinfection, rinse thoroughly with plenty of deionized water until no residue remains.

[0030] S2, Chitosan Oligosaccharide Pre-soaking Pretreated brown rice was placed in a 30 mg / L chitosan oligosaccharide solution at a material-to-liquid ratio of 1:3 (g:mL), and then soaked in a 30℃ constant temperature incubator for 12 hours, stirring once every 4 hours during the soaking period. After soaking, the water was drained.

[0031] S3, Low-temperature stress treatment The pre-soaked brown rice was placed in a 12℃ constant temperature incubator for 24 hours and sprayed with a 15 μmol / L methyl jasmonate solution every 2 hours for 1 minute each time. After washing and draining the sample, it was placed in a 25℃ constant temperature incubator for 12 hours and sprayed with distilled water every 2 hours for 1 minute each time.

[0032] S4, Drying After germination, the germinated brown rice is placed in a hot air drying oven and dried at 55°C until the moisture content is 10%, thus obtaining the germinated brown rice product.

[0033] Example 2 The difference between Example 2 and Example 1 is that the concentration of the chitosan oligosaccharide solution in step S2 is 25 mg / L, and the treatment in step S3 is carried out in a constant temperature incubator at 10℃ for 24 h with a concentration of methyl jasmonate of 30 μmol / L. Specifically, the following steps are included: S1. Raw material pretreatment Select mature, whole-grain brown rice, remove impurities and broken grains, rinse three times with deionized water to remove surface dust, and then disinfect the washed brown rice by soaking it in a 0.1% sodium hypochlorite solution for 15 minutes. After disinfection, rinse thoroughly with plenty of deionized water until no residue remains.

[0034] S2. Place the pretreated brown rice in a 25 mg / L chitosan oligosaccharide solution, then soak it in a 30°C constant temperature incubator for 12 hours, stirring once every 4 hours. After soaking, drain the water.

[0035] S3. Place the pre-soaked brown rice in a 10℃ constant temperature incubator for 24 hours, and spray it with 30 μmol / L methyl jasmonate solution every 2 hours for 1 minute each time. After washing the sample and draining it, place it in a 25℃ constant temperature incubator for 12 hours, and spray it with distilled water every 2 hours for 1 minute each time.

[0036] S4, Drying After germination, the germinated brown rice is placed in a hot air drying oven and dried at 55°C until the moisture content is 10%, thus obtaining the germinated brown rice product.

[0037] Example 3 The difference between Example 3 and Example 2 is that in step S3, the treatment is carried out in a constant temperature incubator at 12℃ for 24 hours, specifically including the following steps: S1. Raw material pretreatment Select mature, whole-grain brown rice, remove impurities and broken grains, rinse three times with deionized water to remove surface dust, and then disinfect the washed brown rice by soaking it in a 0.1% sodium hypochlorite solution for 15 minutes. After disinfection, rinse thoroughly with plenty of deionized water until no residue remains.

[0038] S2. Place the pretreated brown rice in a 25 mg / L chitosan oligosaccharide solution, then soak it in a 30°C constant temperature incubator for 12 hours, stirring once every 4 hours. After soaking, drain the water.

[0039] S3. Place the pre-soaked brown rice in a 12℃ constant temperature incubator for 24 hours, and spray it with 30 μmol / L methyl jasmonate solution every 2 hours for 1 minute each time. After washing the sample and draining it, place it in a 25℃ constant temperature incubator for 12 hours, and spray it with distilled water every 2 hours for 1 minute each time.

[0040] S4, Drying After germination, the germinated brown rice is placed in a hot air drying oven and dried at 55°C until the moisture content is 10%, thus obtaining the germinated brown rice product.

[0041] Example 4 The difference between Example 4 and Example 1 is that the concentration of the methyl jasmonate solution in step S3 is 30 μmol / L, and the specific steps include: A method for compound stress germination treatment to increase the polyphenol content of brown rice includes the following steps: S1. Raw material pretreatment Select mature, whole-grain brown rice, remove impurities and broken grains, rinse three times with deionized water to remove surface dust, and then disinfect the washed brown rice by soaking it in a 0.1% sodium hypochlorite solution for 15 minutes. After disinfection, rinse thoroughly with plenty of deionized water until no residue remains.

[0042] S2, Chitosan Oligosaccharide Pre-soaking The pretreated brown rice was placed in a 30 mg / L chitosan oligosaccharide solution and then soaked in a 30°C constant temperature incubator for 12 hours, stirring once every 4 hours. After soaking, the water was drained.

[0043] S3. Place the pre-soaked brown rice in a 12℃ constant temperature incubator for 24 hours, and spray it with 30 μmol / L methyl jasmonate solution every 2 hours for 1 minute each time. After washing the sample and draining it, place it in a 25℃ constant temperature incubator for 12 hours, and spray it with distilled water every 2 hours for 1 minute each time.

[0044] S4, Drying After germination, the germinated brown rice is placed in a hot air drying oven and dried at 55°C until the moisture content is 10%, thus obtaining the germinated brown rice product.

[0045] Example 5 The difference between Example 5 and Example 3 is that the concentration of the chitosan oligosaccharide solution in step S2 is 40 mg / L, and the specific steps include: A method for compound stress germination treatment to increase the polyphenol content of brown rice includes the following steps: S1. Raw material pretreatment Select mature, whole-grain brown rice, remove impurities and broken grains, rinse three times with deionized water to remove surface dust, and then disinfect the washed brown rice by soaking it in a 0.1% sodium hypochlorite solution for 15 minutes. After disinfection, rinse thoroughly with plenty of deionized water until no residue remains.

[0046] S2. Place the pretreated brown rice in a 40 mg / L chitosan oligosaccharide solution, then soak it in a 30°C constant temperature incubator for 12 hours, stirring once every 4 hours. After soaking, drain the water.

[0047] S3. Place the pre-soaked brown rice in a 12℃ constant temperature incubator for 24 hours, and spray it with 30 μmol / L methyl jasmonate solution every 2 hours for 1 minute each time. After washing the sample and draining it, place it in a 25℃ constant temperature incubator for 12 hours, and spray it with distilled water every 2 hours for 1 minute each time.

[0048] S4, Drying After germination, the germinated brown rice is placed in a hot air drying oven and dried at 55°C until the moisture content is 10%, thus obtaining the germinated brown rice product.

[0049] Example 6 The difference between Example 6 and Example 4 is that in step S2, the brown rice is soaked in a 30°C constant temperature incubator for 8 hours, specifically including the following steps: A method for compound stress germination treatment to increase the polyphenol content of brown rice includes the following steps: S1. Same as Example 1.

[0050] S2. Place the pretreated brown rice in a 30 mg / L chitosan oligosaccharide solution, then soak it in a 30°C constant temperature incubator for 8 hours, stirring once every 4 hours during the soaking period. After soaking, drain the water.

[0051] S3. Place the pre-soaked brown rice in a 12℃ constant temperature incubator for 24 hours, and spray it with 30 μmol / L methyl jasmonate solution every 2 hours for 1 minute each time; wash the sample and drain it, then place it in a 25℃ constant temperature incubator for 12 hours, and spray it with distilled water every 2 hours for 1 minute each time.

[0052] S4, Drying After germination, the germinated brown rice is placed in a hot air drying oven and dried at 55°C until the moisture content is 10%, thus obtaining the germinated brown rice product.

[0053] Comparative Example 1 Compared with Example 4, Comparative Example 1 replaced the chitosan oligosaccharide solution in step S2 with deionized water for pre-soaking brown rice, while the remaining steps were the same as in Example 1.

[0054] Comparative Example 2 Compared with Example 4, in Comparative Example 2, the chitosan oligosaccharide solution in step S2 was replaced with a 30 μmol / L methyl jasmonate solution for pre-soaking brown rice, while the remaining steps were the same as in Example 1.

[0055] Comparative Example 3 Compared with Example 4, Comparative Example 3 replaced the methyl jasmonate solution in step S3 with deionized water, while the remaining steps were the same as in Example 1.

[0056] Comparative Example 4 Compared with Example 4, Comparative Example 4 replaced the methyl jasmonate solution in step S3 with a 30 mg / L chitosan oligosaccharide solution, while the remaining steps were the same as in Example 1.

[0057] Comparative Example 5 Compared with Example 4, Comparative Example 5 replaced the chitosan oligosaccharide solution and methyl jasmonate solution used in steps S2 and S3 with deionized water, while the remaining steps were the same as in Example 1.

[0058] Comparative Example 6 Compared with Example 4, Comparative Example 6 replaced the chitosan oligosaccharide solution in step S2 with 10 mg / mL naphthaleneacetic acid (NAA), while the remaining steps were the same as in Example 1.

[0059] Comparative Example 7 Compared with Example 4, Comparative Example 7 replaced the methyl jasmonate solution in step S3 with 30 μmol / L naphthaleneacetic acid (NAA), while the remaining steps were the same as in Example 1.

[0060] Comparative Example 8 Compared with Example 4, Comparative Example 8 changed the order of using the chitosan oligosaccharide solution and methyl jasmonate solution in steps S2 and S3, while the remaining steps remained the same as in Example 1.

[0061] Comparative Example 9 Compared with Example 4, Comparative Example 9 did not perform germination treatment on brown rice and directly measured the indicators.

[0062] Table 1. Germination potential and germination rate of brown rice from Examples 1-6 and Comparative Examples 1-9

[0063] Note: Results are mean ± standard error. Different letters in the same column indicate different significance levels (p < 0.05). This invention utilizes a composite treatment process combining chitosan oligosaccharide pre-soaking with low-temperature stress and methyl jasmonic acid spraying to specifically address the core problems of low germination rate, poor stability, and uneven sprout morphology in existing germinated brown rice technologies. (See Table 1 and appendix for details.) Figure 1Morphological observation showed that the germination rate of the composite treatment groups (Examples 1-6) was consistently above 94%, with the best example 4 reaching 97.86% ± 0.87 and a germination potential of over 88.64%, representing a 26.5% increase compared to the single low-temperature stress group. Sprout uniformity exceeded 90%, with consistent, robust, and undeformed sprouts. Lateral roots were also observed to be emerging and evenly distributed in the example groups, and the seed coats were intact, clean, and free of mold, significantly superior to the control group's weak, elongated, uneven, or deformed sprouts. This process, through the synergistic effect of these three factors, ensures high efficiency, stability, and sprout quality while simultaneously enriching functional components such as polyphenols. The process is repeatable, highly practical, and meets the industrial production needs of functional germinated brown rice.

[0064] Table 2 Total phenolic content and antioxidant effect of Examples 1-6 and Comparative Examples 1-9

[0065] Note: Results are mean ± standard error. Different letters in the same column indicate different significance levels (p < 0.05). The results from the data table on total phenol content and antioxidant effect show that, compared with the ungerminated control example 9 (total phenol content 96.70±1.35 mg GAE / g DW), control example 5, through germination treatment under single low-temperature stress, significantly improved the total phenol content (108.90±2.46 mg GAE / g DW) and antioxidant activity of germinated brown rice, confirming the fundamental role of germination treatment and low-temperature stress in polyphenol accumulation. Comparative Example 1 was sprayed with jasmonic acid lactone solution during the low-temperature stress treatment in step S3. Comparative Example 3 was soaked in chitosan oligosaccharide solution before low-temperature stress. Comparative Example 4 was soaked in chitosan oligosaccharide solution before low-temperature stress and sprayed with chitosan oligosaccharide solution during the stress process. The total phenol content of the three groups was significantly increased compared with Comparative Example 5. Among them, the improvement effect of Comparative Example 3 (128.30±1.91 mg GAE / g DW) by soaking in chitosan oligosaccharide alone was the most prominent. However, Comparative Example 4 only underwent repeated treatment with chitosan oligosaccharide, which could increase the polyphenol content, but the increase was limited (141.80±2.10 mg GAE / g DW), and it was difficult to achieve a high-efficiency breakthrough in polyphenol content. Compared to Comparative Example 4, Example 4, by replacing the spraying component under low-temperature stress with jasmonic acid lactone, significantly increased the total phenol content to 186.30±2.42 mg GAE / g DW, a 31.4% increase compared to Comparative Example 4. This result indicates that the specific combination process of "chitosan oligosaccharide pre-soaking + low-temperature stress + jasmonic acid lactone spraying" can significantly enhance the enrichment effect of polyphenols in germinated brown rice through synergistic effects, far superior to single-component or repeated-component treatments. In Comparative Example 7, after replacing jasmonic acid lactone in Example 4 with naphthaleneacetic acid, the total phenol content decreased to 119.50±2.02 mg GAE / g DW, a much lower effect than Comparative Examples 3 and 4. Similarly, Comparative Example 6, where chitosan oligosaccharide was replaced with naphthaleneacetic acid (total phenols 124.70±2.10 mg GAE / g DW), also showed a significant decrease in polyphenol content and antioxidant activity. The core reason for this difference lies in the fact that chitosan oligosaccharide, as a biological stress elicitor, can specifically activate the key phenylpropane metabolic pathway and PAL and C4H enzyme activities in polyphenol synthesis; jasmonic acid lactone, as a core endogenous stress resistance signaling molecule in plants, can precisely regulate the gene expression of this metabolic pathway, further amplifying the enzyme activity enhancement effect; while naphthaleneacetic acid, as an auxin regulator, only focuses on primary metabolic regulation such as cell elongation and budding process, and cannot activate the polyphenol synthesis pathway. It will also divert the metabolic resources required for polyphenol synthesis, while antagonizing the stress-inducing effect of low temperature stress, weakening the stress response of polyphenol accumulation, and ultimately leading to a significant decrease in total phenol content and antioxidant activity.

[0066] Comparative Example 2, which used methyl jasmonate to soak brown rice before low-temperature stress to promote germination, also increased polyphenol content, but its effect was significantly worse than that of chitosan oligosaccharide. Combined with Comparative Examples 1-5 and Comparative Example 9, it can be seen that a significant synergistic effect occurred between chitosan oligosaccharide soaking and low-temperature stress spraying with methyl jasmonate in this invention. Furthermore, Comparative Example 8, which replaced the treatment order, showed significantly worse results than the examples. Chitosan oligosaccharide, as a biological stress elicitor, can specifically activate the phenylpropane metabolic pathway, the core of polyphenol synthesis, significantly increasing the activity of key enzymes such as PAL and C4H, accelerating the conversion of polyphenol precursors into functional polyphenols such as ferulic acid. Methyl jasmonate, as an endogenous stress-resistance signaling molecule in plants, can also increase polyphenol content by improving enzyme activity, while also enhancing the stress resistance of brown rice under low temperatures, alleviating the inhibitory effect of low temperature on germination, and reducing polyphenol decomposition. The worsening effect after changing the treatment order indicates that although both chitosan oligosaccharide and methyl jasmonate can promote the increase of polyphenol content, they only achieve better results under specific treatment steps. The reason may be that methyl jasmonate, as an endogenous stress-resistant signaling molecule, needs to precisely activate the pathway amplification effect under the stress of low temperature. It is difficult to form an effective accumulation in the early soaking stage. Instead, the antibacterial protective film and metabolic activation basis formed by pre-soaking with chitosan oligosaccharide are more conducive to the subsequent polyphenol synthesis.

[0067] Combined with Examples 1-6, it can be seen that the concentration of chitosan oligosaccharide, the concentration of methyl jasmonate, the low-temperature stress temperature, and the soaking time of chitosan oligosaccharide all significantly affect the increase in polyphenol content: the concentration of chitosan oligosaccharide shows a "first increase and then decrease" trend in the range of 25-40 mg / L. In Example 4, the treatment with 30 mg / L resulted in a peak total phenol content of 186.30±2.42 mg GAE / g DW. The total phenol content in Examples 2 (25 mg / L, 165.70±2.23 mg GAE / g DW), 3 (25 mg / L, 172.40±2.26 mg GAE / g DW), and 5 (40 mg / L, 168.77±2.00 mg GAE / g DW) was lower than this peak. The effect was better when the concentration of methyl jasmonate was 30 μmol / L (Examples 2-5). In Example 2 (30 μmol / L, 165.70±2.23 mg GAE / g DW), the total phenol content was lower than this peak. The total phenols in Example 3 (30 μmol / L, 186.30 ± 2.42 mg GAE / g DW) were higher than those in Example 1 (20 μmol / L, 158.20 ± 1.73 mg GAE / g DW). Low temperature stress was optimal at 12°C (total phenols in Example 3: 172.40 ± 2.26 mg GAE / g DW), while the total phenols in Example 2 (10°C, 165.70 ± 2.23 mg GAE / g DW) were relatively lower. The total phenols in Example 4 (186.30 ± 2.42 mg GAE / g DW) after soaking in chitosan oligosaccharide for 12 h were much higher than those in Example 6 (155.80 ± 2.27 mg GAE / g DW) after soaking for 8 h. These factors achieved efficient enrichment by synergistically activating the polyphenol synthesis pathway.

[0068] Excessive or insufficient concentration of chitosan oligosaccharide, or insufficient soaking time (as in Example 6), will lead to a decrease in the indicators. However, the indicators of Comparative Examples 1-4 (single factor elimination), 6-7 (regulator replacement), and 8 (process sequence adjustment) are significantly lower than those of the Examples, indicating that compound treatment and specific parameters and process sequence are the key to achieving efficient improvement of functional indicators. Compared with single stress or reagent replacement, compound treatment can more comprehensively enhance the functional value of germinated brown rice.

[0069] Table 3 Comparison of enzyme activity assays between the examples and comparative examples

[0070] Note: Results are mean ± standard error. Different letters in the same column indicate different significance levels (p < 0.05). As shown in Table 3, compared with the ungerminated brown rice of Comparative Example 9, the activities of PAL and C4H, key enzymes in polyphenol synthesis, were significantly increased after the combined treatment in Examples 1-6 (p<0.05). Example 4 was the optimal example, with PAL activity reaching 52.75±2.19 U / g FW and C4H activity reaching 28.36±1.44 U / g FW, which were 10.18 times and 11.97 times that of Comparative Example 9, respectively. Chitosan oligosaccharide, as a biological stress elicitor, efficiently activated enzyme activity, while low-temperature stress provided a stable environment for the enzymatic reaction. Methyl jasmonic acid increased the stress resistance of brown rice under low-temperature conditions. All three factors jointly promoted the efficient operation of the phenylpropanoid metabolic pathway. In Examples 1-6, subtle adjustments to the concentrations of chitosan oligosaccharide, low temperature, and methyl jasmonate all affected enzyme activity. In the comparative examples, removing chitosan oligosaccharide or methyl jasmonate, replacing them with other regulators (such as NAA), or adjusting the process sequence resulted in a significant decrease in enzyme activity, demonstrating the specificity and necessity of each treatment factor and parameter combination. Furthermore, the enzyme activity index showed a high positive correlation with the functional indicators in Table 2, indicating that the combined treatment provided a core mechanism for polyphenol enrichment by activating key enzyme activities.

[0071] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for increasing the polyphenol content and antioxidant capacity of germinated brown rice, characterized in that, Specifically, the following steps are included: S1. Select mature brown rice with intact grains, remove impurities, wash and clean it, and disinfect it. After disinfection, rinse it with deionized water until there is no residue to obtain pre-treated brown rice. S2. Soak the pretreated brown rice from step S1 in a chitosan oligosaccharide solution to obtain pre-soaked brown rice. S3. The brown rice pre-soaked in step S2 is subjected to low-temperature stress treatment, during which methyl jasmonate solution is sprayed; after the low-temperature stress treatment is completed, it continues to germinate in a constant temperature incubator, during which distilled water is sprayed. S4. After germination, the germinated brown rice is dried to obtain germinated brown rice with high polyphenol content and high antioxidant capacity.

2. The method according to claim 1, characterized in that, In the disinfection step S1, the selected, dust-removed and washed brown rice is soaked in a sodium hypochlorite solution with a concentration of 0.1~1% for 10-20 minutes, and then rinsed with deionized water until there is no residue.

3. The method according to claim 1, characterized in that, In step S2, the ratio of brown rice to chitosan oligosaccharide solution is 1g:(2~5)mL, and the concentration of chitosan oligosaccharide solution is 20~40mg / L.

4. The method according to claim 1, characterized in that, In step S2, the soaking temperature is 25~35℃, the soaking time is 8~16h, and the mixture is stirred once every 2~6h.

5. The method according to claim 1, characterized in that, In step S3, the temperature of the low-temperature stress is 10~20℃, and the duration is 12~36h.

6. The method according to claim 1, characterized in that, In step S3, the concentration of the methyl jasmonate solution sprayed is 10~50 μmol / L, and it is sprayed once every 1~2 hours, with each spraying lasting 1~2 minutes.

7. The method according to claim 1, characterized in that, In step S3, after the low temperature stress treatment is completed, germination continues in a constant temperature incubator at 25~35℃. During this period, distilled water is sprayed every 1~2 hours, and the germination time is 12~24 hours.

8. The method according to claim 1, characterized in that, In step S4, after germination, the germinated brown rice can be dried using any of the following methods: low-temperature hot air drying, vacuum drying, or freeze drying. Low-temperature hot air drying is carried out at 45~55℃.

9. The method according to claim 1, characterized in that, In step S4, the moisture content of the dried germinated brown rice is less than 10%.

10. The use of germinated brown rice prepared by any one of claims 1 to 9 in food or pharmaceuticals.

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