A fermented rice peptide with α-amylase inhibitory activity, anti-inflammatory and anti-glycation properties, and its preparation method and functional product

Abstract

This invention relates to the technical field of deep processing of rice protein, and particularly to a fermented rice peptide with α-amylase inhibitory activity, anti-inflammatory and anti-glycation properties, its preparation method, and functional products. The preparation method includes the following steps: mixing rice protein with water to obtain a slurry; adding alkaline protease to the slurry for a first hydrolysis, followed by enzyme inactivation to obtain a first hydrolysate; adding aminopeptidase to the first hydrolysate for a second hydrolysis, followed by enzyme inactivation to obtain a second hydrolysate; adding *Lactobacillus paracasei* to the second hydrolysate for fermentation to obtain a fermentation broth; separating the fermentation broth into solid and liquid phases to obtain a supernatant containing fermented rice peptides; filtering the supernatant to retain the fermented rice peptides, thus obtaining the final product. This invention not only improves the yield of rice peptides but also significantly increases the production efficiency of rice peptides.

Description

Technical Field

[0001] This invention relates to the technical field of deep processing of rice protein, and in particular to a fermented rice peptide with α-amylase inhibitory activity, anti-inflammatory and anti-glycation properties, its preparation method, and functional products. Background Technology

[0002] Currently, most research on the preparation of rice peptides focuses on extracting rice peptides with whitening and blood pressure-lowering activities, as well as reducing the bitterness of rice peptide products and improving their taste.

[0003] Currently, the preparation of rice peptides typically involves using commercially available enzymes to hydrolyze rice protein. However, the hydrolysate obtained using only commercial enzymes often exhibits severe emulsification, leading to difficulties in subsequent filtration and low production efficiency. Therefore, existing preparation methods require further improvement. Furthermore, rice peptides obtained solely through commercial enzyme hydrolysis only possess whitening, hypoglycemic, antioxidant, and blood pressure-lowering properties; no reports have been found regarding anti-glycation, anti-inflammatory, or beneficial bacteria-promoting activities.

[0004] In summary, existing research on fermented rice peptides extracted from rice protein focuses on obtaining rice peptides with whitening, blood pressure-lowering, blood sugar-lowering, and antioxidant activities, while other active functions of fermented rice peptides are rarely studied. There are no reports in existing literature or patents on the preparation of fermented rice peptides with anti-inflammatory, beneficial bacteria-promoting, and anti-glycation (anti-AGEs) activities using rice protein. Summary of the Invention

[0005] To address the shortcomings of the prior art mentioned in the background section, this invention provides a method for preparing fermented rice peptides, the technical solution of which is as follows:

[0006] The method for preparing fermented rice peptides includes the following steps:

[0007] Raw material pretreatment: Rice protein is mixed with water to obtain a slurry;

[0008] First hydrolysis: Alkaline protease is added to the slurry for the first hydrolysis. After the hydrolysis is completed, the enzyme is inactivated to obtain the first hydrolysate.

[0009] Second hydrolysis: Aminopeptidase is added to the first hydrolysate for a second hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the second hydrolysate.

[0010] Inoculation and fermentation: Lactobacillus paracasei was added to the second hydrolysate for fermentation to obtain a fermentation broth;

[0011] The fermentation broth is subjected to solid-liquid separation to obtain a supernatant containing fermented rice peptides. The supernatant is then filtered to retain the fermented rice peptides, thus obtaining the fermented rice peptides.

[0012] In the first hydrolysis step, alkaline protease is added to the slurry to adjust the pH of the system to 8.0-9.0, and hydrolysis is carried out at 50-60℃ for 100-150 min; then enzyme inactivation treatment is carried out at 85-90℃ for 10-20 min to obtain the first hydrolysate.

[0013] In the second hydrolysis step, aminopeptidase is added to the first hydrolysate to adjust the pH of the system to 6.5-7.5, and hydrolysis is carried out at 50-60℃ for 40-90 min; then the enzyme is inactivated at 85-90℃ for 10-20 min to obtain the second hydrolysate.

[0014] The Lactobacillus paracasei is Lactobacillus paracasei YYS-69, with the accession number CGMCC No. 25837;

[0015] The fermentation broth was centrifuged to separate the solid and liquid components, resulting in a supernatant containing fermented rice peptides.

[0016] The pH of the supernatant was adjusted to 5.7–6.1, and then subjected to microfiltration through a membrane with a pore size of 0.22–0.45 μm and ultrafiltration through a 3000 Da ultrafiltration membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da.

[0017] The ultrafiltration peptide solution is concentrated using a 200 Da nanofiltration membrane device and then sterilized to obtain a peptide solution with a molecular weight of 200-3000 Da.

[0018] The polypeptide solution with a molecular weight of 200-3000 Da is concentrated to a polypeptide mass content of 10%-20%, and the concentrate is then made into powder.

[0019] In one embodiment, in the raw material pretreatment step, the mass ratio of rice protein to water is 1:(10-20).

[0020] In one embodiment, the amount of alkaline protease added is 6000-8000 U / g, based on the protein content of the rice protein; and the amount of aminopeptidase added is 3000-5000 U / g, based on the protein content of the rice protein.

[0021] In one embodiment, the fermentation process is carried out at a temperature of 36–38°C for 22–26 hours.

[0022] The present invention also provides a fermented rice peptide, which is prepared by the rice peptide preparation method described above.

[0023] The present invention also provides a functional product, the components of which include fermented rice peptides prepared by the preparation method described above.

[0024] Based on the above, compared with the prior art, the method for preparing fermented rice peptides of the present invention has the following beneficial effects:

[0025] This invention prepares fermented rice peptides by combining enzymatic hydrolysis and microbial fermentation of rice protein. This preparation method not only improves the yield of rice peptides, but also utilizes microbial acid production to promote the precipitation of rice protein that has not been degraded during enzymatic hydrolysis, thus significantly improving the production efficiency of rice peptides.

[0026] The fermented rice peptides prepared by the method of this invention possess α-amylase inhibitory activity, anti-inflammatory activity, beneficial bacteria promoting activity, antioxidant activity, and anti-glycation activity. They can be used as raw material components for functional foods and applied in products with effects such as weight loss, anti-inflammation, regulating intestinal health, and preventing skin aging.

[0027] The method of this invention can produce the desired fermented rice peptides by simply combining operations such as mixing, enzymatic hydrolysis, fermentation, and filtration. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0030] This invention provides an operational example of a method for preparing fermented rice peptides, the specific steps of which are as follows:

[0031] Step 1: Raw material pretreatment:

[0032] Rice protein is mixed with water to obtain a slurry; wherein the mass ratio of rice protein to water is 1:(10-20).

[0033] Step 2, First Hydrolysis:

[0034] Alkaline protease is added to the slurry for the first hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the first hydrolysate; specifically:

[0035] Alkaline protease is added to the slurry to adjust the pH of the system to 8.0-9.0, and hydrolyzes it at 50-60℃ for 100-150 min; then the enzyme is inactivated at 85-90℃ for 10-20 min to obtain the first hydrolysate; wherein, based on the protein content in the rice protein, the amount of alkaline protease added is 6000-8000 U / g;

[0036] Step 3, Second Hydrolysis:

[0037] Aminopeptidase is added to the first hydrolysate for a second hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the second hydrolysate; specifically:

[0038] Aminopeptidase was added to the first hydrolysate to adjust the pH of the system to 6.5–7.5, and hydrolysis was carried out at 50–60°C for 40–90 min; then, the enzyme was inactivated at 85–90°C for 10–20 min to obtain the second hydrolysate. The amount of aminopeptidase added was 3000–5000 U / g, based on the protein content of the rice protein.

[0039] Step 4: Inoculation and Fermentation

[0040] Lactobacillus paracasei was added to the second hydrolysate for fermentation to obtain a fermentation broth; the fermentation temperature was 36-38℃ and the fermentation time was 22-26h.

[0041] The *Lactobacillus paracasei* used is *Lactobacillus paracasei* YYS-69, Latin scientific name: Lactobacillus cheese The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 25837. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Date of deposit: September 28, 2022.

[0042] Step 5: Separation and Filtration

[0043] The fermentation broth was centrifuged to separate the solid and liquid phases, resulting in a supernatant containing fermented rice peptides. The pH of the supernatant was adjusted to 5.7–6.1, and then subjected to microfiltration through a 0.22–0.45 μm membrane and ultrafiltration through a 3000 Da ultrafiltration membrane to obtain an ultrafiltration peptide solution with a molecular weight less than 3000 Da.

[0044] The centrifugation conditions for the fermentation broth are centrifugation at 4000-6000 rpm for 8-15 min;

[0045] Step Six: Nanofiltration Concentration and Sterilization:

[0046] The ultrafiltration peptide solution was concentrated using a 200 Da nanofiltration membrane device and then sterilized to obtain a peptide solution with a molecular weight of 200–3000 Da.

[0047] Step 7: Powdering

[0048] The polypeptide solution with a molecular weight of 200-3000 Da is concentrated to a polypeptide content of 10%-20%, and the concentrate is then made into powder. The concentrate may be spray-dried to form powder.

[0049] The present invention provides the following embodiments and comparative examples.

[0050] Example 1

[0051] S100. Raw material pretreatment: Take 300g of rice protein (protein content: 81%), add 4.5Kg of water to soften, stir for 30min until completely mixed to form rice protein slurry.

[0052] S200, First hydrolysis: Add 1.7 million U of alkaline protease to the slurry, adjust the pH of the slurry to 8.5, and hydrolyze at 55℃ for 120 min; inactivate the enzyme in the first hydrolysate at 90℃ for 10 min, and cool naturally to obtain the first hydrolysate.

[0053] S300, Second Hydrolysis: Add 970,000 U of aminopeptidase to the first hydrolysate, control the pH to around 7.0, and hydrolyze at 55℃ for 60 min. After the second hydrolysis, heat to 85℃ to inactivate the enzyme for 20 min to obtain the second hydrolysate.

[0054] S400, Inoculation and Fermentation: Add twice-activated Lactobacillus paracasei YYS-69 (CGMCC No. 25837) to the second hydrolysate. The inoculation amount is 5% of the rice protein content. The fermentation temperature is 37℃ and the fermentation time is 24h.

[0055] S500, centrifugation, microfiltration and ultrafiltration: The fermentation broth was centrifuged at 5000 rpm for 10 min to obtain the supernatant. After adjusting the pH to 5.9, the supernatant was subjected to microfiltration through a 0.45 μm membrane and ultrafiltration through a 3000 Da ultrafiltration membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da.

[0056] S600, Nanofiltration Concentration and Sterilization: The ultrafiltration peptide solution is concentrated to a peptide content of 10% using a 200Da nanofiltration membrane device, and then sterilized at 90℃ for 30 minutes to obtain a concentrated peptide solution with a molecular weight of 200-3000Da.

[0057] Example 2

[0058] S100. Raw material pretreatment: Take 300g of rice protein (protein content: 81%), add 3Kg of water to soften, stir for 30min until completely mixed to form rice protein slurry.

[0059] S200, First hydrolysis: Add 1.46 million U of alkaline protease to the slurry, adjust the pH of the slurry to 8.0, and hydrolyze at 60℃ for 100 min; inactivate the enzyme in the first hydrolysate at 90℃ for 10 min, and cool naturally to obtain the first hydrolysate;

[0060] S300, Second Hydrolysis: Add 1.21 million U of aminopeptidase to the first hydrolysate, control the pH to around 7.5, and hydrolyze at 50℃ for 90 min. After the second hydrolysis, heat to 90℃ to inactivate the enzyme for 10 min to obtain the second hydrolysate.

[0061] S400, Inoculation and Fermentation: Add twice-activated Lactobacillus paracasei YYS-69 (CGMCC No. 25837) to the second hydrolysate. The inoculation amount is 5% of the rice protein content. The fermentation temperature is 36℃ and the fermentation time is 26h.

[0062] S500, centrifugation, microfiltration and ultrafiltration: The fermentation broth was centrifuged at 4000 rpm for 15 min to obtain the supernatant. After adjusting the pH to 6.1, the supernatant was subjected to microfiltration through a 0.45 μm membrane and ultrafiltration through a 3000 Da ultrafiltration membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da.

[0063] S600, Nanofiltration Concentration and Sterilization: The ultrafiltration peptide solution is concentrated to a peptide content of 15% using a 200Da nanofiltration membrane device, and then sterilized at 90℃ for 30 minutes to obtain a concentrated peptide solution with a molecular weight of 200-3000Da.

[0064] Example 3

[0065] S100. Raw material pretreatment: Take 300g of rice protein (protein content: 81%), add 6Kg of water to soften, stir for 30min until completely mixed to form rice protein slurry.

[0066] S200, First hydrolysis: Add 1.94 million U of alkaline protease to the slurry, adjust the pH of the slurry to 9.0, and hydrolyze at 50℃ for 150 min; inactivate the enzyme in the first hydrolysate at 85℃ for 20 min, and cool naturally to obtain the first hydrolysate;

[0067] S300, Second Hydrolysis: Add 730,000 U of aminopeptidase to the first hydrolysate, control the pH to around 6.5, and hydrolyze at 60℃ for 40 min. After the second hydrolysis, heat to 90℃ to inactivate the enzyme for 10 min to obtain the second hydrolysate.

[0068] S400, Inoculation and Fermentation: Add twice-activated Lactobacillus paracasei YYS-69 (CGMCC No. 25837) to the second hydrolysate. The inoculation amount is 5% of the rice protein content. The fermentation temperature is 38℃ and the fermentation time is 22h.

[0069] S500, centrifugation, microfiltration and ultrafiltration: The fermentation broth was centrifuged at 6000 rpm for 8 min to obtain the supernatant. After adjusting the pH to 5.7, the supernatant was subjected to microfiltration through a 0.45 μm membrane and ultrafiltration through a 3000 Da ultrafiltration membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da.

[0070] S600, Nanofiltration Concentration and Sterilization: The ultrafiltration peptide solution is concentrated to a peptide content of 20% using a 200Da nanofiltration membrane device, and then sterilized at 90℃ for 30 minutes to obtain a concentrated peptide solution with a molecular weight of 200-3000Da.

[0071] Comparative Example 1

[0072] Rice protein was hydrolyzed only by alkaline protease and aminopeptidase. The secondary hydrolysate was not fermented by microorganisms. All other operations and processes were the same as in Example 1.

[0073] Comparative Example 2

[0074] The fermentation strain Lactobacillus paracasei YYS-69 (CGMCC No. 25837) was replaced with an equal amount of Lactobacillus plantarum YYS-K3 (CGMCC No. 27598), and all other operations and processes were the same as in Example 1.

[0075] Comparative Example 3

[0076] The fermentation strain Lactobacillus paracasei YYS-69 (CGMCC No. 25837) was replaced with an equal amount of Lactobacillus fermentum B153 (CGMCC No. 16454), and all other operations and processes were the same as in Example 1.

[0077] Comparative Example 4

[0078] The fermentation strain Lactobacillus paracasei YYS-69 (CGMCC No. 25837) was replaced with an equal amount of Lactobacillus plantarum YYS-99 (CGMCC No. 25838), and all other operations and processes were the same as in Example 1.

[0079] Comparative Example 5

[0080] The fermentation strain Lactobacillus paracasei YYS-69 (CGMCC No. 25837) was replaced with an equal amount of Lactobacillus paracasei YYS-K1 (CGMCC No. 26405), and all other operations and processes were the same as in Example 1.

[0081] In the examples and comparative examples:

[0082] Lactobacillus paracasei YYS-69 was deposited on September 28, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.25837.

[0083] Lactobacillus plantarum ( Lactiplantibacillus plantarum YYS-K3 was deposited on June 12, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.27598.

[0084] Lactobacillus fermentation (Lactobacillus fermentum B153 was deposited on September 10, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16454.

[0085] Lactobacillus plantarum ( Lactiplantibacillus plantarum YYS-99 was deposited on September 28, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.25838.

[0086] Lactobacillus paracasei YYS-K1 was deposited on January 6, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.26405.

[0087] Lactobacillus plantarum ( Lactobacillus plantarumBXM2 was deposited on September 6, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.16436.

[0088] These strains are all preserved and alive at the China General Microbiological Culture Collection Center, and technicians can obtain them from the collection center based on the accession number.

[0089] The alkaline protease specifically refers to Novozymes alkaline protease;

[0090] The aminopeptidase is specifically Novozymes flavor protease.

[0091] The performance of the fermented rice peptide products obtained in the above examples and comparative examples was tested:

[0092] 1. Determination of α-amylase inhibitory activity of fermented rice peptide products

[0093] The polypeptide solutions obtained in Examples 1-3 and Comparative Examples 1-5 were diluted to 10 mg / ml (using water as the solvent) and measured using the visual colorimetric method in GB / T24401-2009, "Determination of α-amylase Activity at Medium Temperature". Specific results are shown in Table 1.

[0094] Table 1. Results of α-amylase inhibitory activity assays in the examples and comparative examples.

[0095]

[0096] Note: -- indicates no inhibitory activity.

[0097] As shown in Table 1:

[0098] The α-amylase inhibitory activities of the examples were all higher than those of the comparative examples. The polypeptides obtained from the comparative example 1, which did not undergo fermentation treatment, did not show any α-amylase inhibitory activity.

[0099] Comparative Examples 2-5 were fermented with *Lactobacillus plantarum* YYS-K3, *Lactobacillus fermentum* B153, *Lactobacillus plantarum* YYS-99, and *Lactobacillus paracasei* YYS-K1, respectively. Only Comparative Example 2 showed slight α-amylase inhibitory activity, while the other comparative examples showed no corresponding activity. This indicates that the difference in fermentation strains has a significant impact on the activity of rice peptides. Furthermore, rice peptides with high α-amylase inhibitory activity can be obtained by fermenting rice hydrolysate with *Lactobacillus paracasei* YYS-69 (CGMCC No. 25837).

[0100] 2. Determination of the anti-inflammatory activity of fermented rice peptide products

[0101] The polypeptide solutions obtained in Examples 1-3 and Comparative Examples 1-5 were diluted to 10 mg / ml (using water as the solvent), and their anti-inflammatory activity was determined according to the albumin denaturation inhibition assay method in "In-vitro anti-inflammatory and antioxidant activities of an Ayurvedic formulation –Trayodashang guggulu". The specific results are shown in Table 2.

[0102] Table 2. Results of anti-inflammatory activity assays for the examples and comparative examples.

[0103]

[0104] As shown in Table 2:

[0105] The albumin denaturation inhibition rate of the examples was higher than that of the comparative examples. The anti-inflammatory activity of the rice peptide obtained in Example 1 (fermentation with Lactobacillus paracasei YYS-69) increased by 7.25 times compared with that before fermentation. The reason may be that the rice protein was digested by multiple proteases secreted by this specific microorganism, which produced polypeptide fragments with anti-inflammatory activity.

[0106] The anti-inflammatory activities of Comparative Examples 2-5 were significantly lower than those of the Example, indicating that the type of microorganism used in fermentation affects the anti-inflammatory activity of the prepared peptides. However, fermentation with Lactobacillus paracasei YYS-69 (CGMCC No. 25837) combined with secondary enzymatic hydrolysis can yield rice peptides with higher anti-inflammatory activity.

[0107] 3. Determination of the growth activity of fermented rice peptide products from *Lactobacillus plantarum* (CGMCC NO. 16436) and *Lactobacillus fermentum* (CGMCC NO. 16454).

[0108] The polypeptide solutions obtained in Examples 1-3 and Comparative Examples 1-5 were diluted to 10 mg / ml (using water as the solvent), and the growth-promoting effect of yam polypeptides on probiotic growth was measured using the method described in "Study on the In Vitro Growth-Promoting Effect of Probiotics on yam Polypeptides". The specific operation and calculation formula are as follows:

[0109] First, 10 mL LMR broth containing 300 μL (approximately 900 million viable bacteria) of *Lactobacillus plantarum* (CGMCC NO. 16436) and *Lactobacillus fermentum* (CGMCC NO. 16454) were prepared separately. Then, 500 μL of a 10 mg / mL rice peptide solution was added to the experimental group, while 500 μL of sterile water was added to the control group. The cultures were incubated at 37°C for 24 h. After incubation, the culture medium was diluted 20-fold and the OD was measured. 600 The probiotic promotion rate is calculated using the following formula:

[0110] Probiotic promotion rate % = (BA) / A x 100%

[0111] Note: A: OD of the blank group 600 B: OD of the experimental group 600 .

[0112] The formula for MRS liquid culture medium is as follows: 10.0 g beef extract, 20.0 g glucose, 10.0 g tryptone, 5.0 g yeast extract, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate tetrahydrate, 1.0 L deionized water, pH 6.5.

[0113] The specific results obtained by the above experimental methods are as follows:

[0114] Table 3. Results of growth activity assays for *Lactobacillus plantarum* (CGMCC NO. 16436) and *Lactobacillus fermentum* (CGMCC NO. 16454) in the examples and comparative examples.

[0115]

[0116] Note: - indicates not detected, which means that it has no effect on promoting the growth of beneficial bacteria. In other words, compared with the control group, it does not promote the growth of beneficial bacteria, that is, its growth rate is less than or equal to 0.

[0117] As shown in Table 3:

[0118] The proliferation activities of *Lactobacillus plantarum* and *Lactobacillus fermentum* in the examples were higher than those in the comparative examples; no growth activity of *Lactobacillus plantarum* and *Lactobacillus fermentum* was detected in the unfermented (comparative example 1) and the fermented with *Lactobacillus paracasei* YYS-K1 after enzymatic hydrolysis (comparative example 5).

[0119] Furthermore, after enzymatic hydrolysis, fermentation with *Lactobacillus plantarum* YYS-K3 (Comparative Example 2) only detected *Lactobacillus plantarum*-promoting activity. These results indicate that the type of microorganism used in fermentation affects the beneficial bacteria-promoting activity of the prepared peptides. Fermentation with *Lactobacillus paracasei* YYS-69 (CGMCC No. 25837) combined with secondary enzymatic hydrolysis is the optimal combination for obtaining rice peptides that simultaneously promote the growth of both *Lactobacillus plantarum* (CGMCC No. 16436) and *Lactobacillus fermentum* (CGMCC No. 16454).

[0120] 4. Determination of the antioxidant activity of fermented rice peptide products

[0121] The peptide solutions obtained in Examples 1-3 and Comparative Examples 1-5 were diluted to 10 mg / ml (using water as the solvent). The DPPH and ABTS scavenging rates were determined using the peptide antioxidant assay method (GB / T39100-2020), and the hydroxyl radical scavenging rate was determined using the salicylic acid method. Specific results are shown in Table 4.

[0122] Table 4. Results of antioxidant activity assays for the examples and comparative examples.

[0123]

[0124] Note: -- indicates no inhibitory activity.

[0125] As shown in Table 4:

[0126] No DPPH scavenging activity was detected in the unfermented rice peptides (Comparative Example 1). Compared with Comparative Example 1 (unfermented), the DPPH scavenging, ABTS scavenging, and hydroxyl radical scavenging activities of the examples were all higher than those of Comparative Example 1. Furthermore, the DPPH scavenging and hydroxyl radical scavenging activities of the examples were higher than those of Comparative Examples 2-5 (prepared by fermentation with other strains), indicating that the rice peptides obtained by fermentation with Lactobacillus paracasei YYS-69 (CGMCC No. 25837) have high antioxidant activity.

[0127] 5. Determination of the anti-glycation activity of fermented rice peptide products

[0128] The polypeptide solutions obtained in Examples 1-3 and Comparative Examples 1-5 were diluted to 10 mg / ml (solvent: water), and the anti-AGEs formation assay was performed using the method described in "Response Surface Optimization Enzymatic-Assisted Extraction of Hawthorn Pectin and its In Vitro Antioxidant and Anti-Glycation Activity". The specific results are shown in Table 5.

[0129] Table 5. Results of anti-glycation activity assays for the examples and comparative examples.

[0130]

[0131] As shown in Table 5:

[0132] The AGEs inhibition rates in the examples were all higher than those in the comparative examples. Furthermore, compared to unfermented rice (Comparative Example 1), the AGEs inhibition rate of the rice peptides obtained after fermentation with *Lactobacillus paracasei* YYS-69 (Example 1) increased by 4.17 times. These results indicate that further fermentation of rice hydrolysate with *Lactobacillus paracasei* YYS-69 (CGMCC No. 25837) can yield rice peptides with high AGEs inhibitory activity.

[0133] 6. Analysis of the preparation process of the examples and comparative examples, yield of the prepared polypeptide solutions, and pH determination.

[0134] The yields and pH results of the polypeptide solutions prepared in Examples 1-3 and Comparative Examples 1-5 are shown in Table 6:

[0135] Table 6. Yields and pH of Examples and Comparative Examples

[0136]

[0137] As shown in Table 6:

[0138] The yield of the examples was higher than that of Comparative Examples 1-5, and the filtration speed was better than that of Comparative Examples 1-5. In addition, it was observed in the experiment that the unfermented solution of Comparative Example 1 after only two enzymatic hydrolysis was turbid, while the solutions of Examples 1-3 and Comparative Examples 2-5 after adding microbial fermentation were clear, and the pH of the solution was significantly reduced and the filtration speed was increased, indicating that fermentation can improve the yield and filtration to varying degrees.

[0139] In addition, different strains have different fermentation yields and filtration results. Fermentation with Lactobacillus paracasei YYS-69 (example) is the best. This may be because, on the one hand, the enzymatic hydrolysis system of Lactobacillus paracasei YYS-69 ferments some of the rice protein that may still exist in the hydrolysate, and on the other hand, the fermentation produces acid to promote the precipitation of rice protein with poor water solubility. This not only increases the yield of rice peptides, but also makes the solution clear and significantly improves the filtration efficiency of the rice peptide preparation process.

[0140] In summary, the method for preparing fermented rice peptides provided by this invention has at least the following innovative points, mechanisms of action, and technical effects:

[0141] Its innovative features:

[0142] The difference between this invention and existing research is that this invention is the first to use rice protein hydrolysate after further fermentation and enzymatic hydrolysis by specific microorganisms. This not only solves the problem of turbid fermentation liquid that is difficult to filter, but also prepares rice peptides with significant inhibitory activity of α-amylase, anti-inflammatory activity (inhibition of albumin denaturation), anti-glycation activity (anti-AGEs formation), antioxidant activity, and promotion of the growth of Lactobacillus plantarum BXM2 (CGMCC NO.16436) and Lactobacillus fermentum B153 (CGMCC NO.16454). No similar reports have been found to date.

[0143] This invention prepares fermented rice peptides by combining enzymatic hydrolysis and microbial fermentation of rice protein. This preparation method not only improves the yield of rice peptides, but also utilizes microbial acid production to promote the precipitation of rice protein that has not been degraded during enzymatic hydrolysis, thus significantly improving the production efficiency of rice peptides.

[0144] Technical effects:

[0145] (1) The present invention prepares rice peptides by combining enzymatic hydrolysis and specific microbial fermentation of rice protein, which not only improves the yield of rice peptides, but also utilizes the acid production of microorganisms to promote the precipitation of rice protein that has not been degraded during enzymatic hydrolysis, thereby improving the production efficiency of rice peptides.

[0146] (2) As can be seen from the above examples: when the rice peptide is diluted to 10 mg / ml, the α-amylase inhibition rate is 40.79%, the anti-inflammatory activity is 99.44%, the anti-AGEs activity reaches 78.21%, and the proliferation promotion rates of *Lactobacillus plantarum* BXM2 (CGMCC NO. 16436) and *Lactobacillus fermentum* B153 (CGMCC NO. 16454) reach 14.27% and 11.79%, respectively. Therefore:

[0147] The fermented rice peptides prepared by the method of this invention have α-amylase inhibitory activity, anti-inflammatory activity, beneficial bacteria promoting activity (promoting the proliferation of Lactobacillus plantarum and Lactobacillus fermentum), antioxidant activity and anti-glycation activity. They can be used as raw material components of functional foods and as functional factors in functional products to achieve effects such as weight loss, anti-inflammation, regulating intestinal health and preventing skin aging.

[0148] Among these, inhibiting α-amylase in the human body can effectively suppress carbohydrate absorption, thus reducing obesity and lowering blood sugar levels. Based on the high α-amylase inhibitory activity of this fermented rice peptide, its application in functional products (such as food and health supplements) can achieve functions such as weight loss and blood sugar reduction.

[0149] Because free radicals are atoms or groups with highly reactive unpaired electrons, excessive free radicals and oxidants in the human body can cause various harms, including attacking body cells and breaking down body tissues. Based on the high antioxidant properties of this fermented rice peptide, its application in functional products (such as food, health supplements, and skincare products) can provide functions such as health maintenance, alleviating aging, and preventing skin aging.

[0150] Because fermented rice peptides have anti-inflammatory and anti-glycation effects, when applied to functional products, such as skincare products, they can reduce skin inflammation, slow down skin glycation, and alleviate skin aging.

[0151] Since the proliferation of *Lactobacillus plantarum* and *Lactobacillus fermentum* is beneficial to intestinal digestion and defecation, based on the prebiotic effect of this fermented rice peptide in promoting the proliferation of *Lactobacillus plantarum* and *Lactobacillus fermentum*, its application in functional products (such as food) can play a role in aiding digestion and defecation.

[0152] (3) The method of the present invention can produce the desired fermented rice peptides by simply combining mixing, fermentation, enzymatic hydrolysis and filtration. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production.

[0153] It should be noted that:

[0154] (1) Definition:

[0155] In this article, "beneficial bacteria promotion activity" refers to the ability to promote the proliferation of Lactobacillus plantarum and Lactobacillus fermentum.

[0156] In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0157] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. The fermented rice peptides of this application can be used to prepare solid dosage forms such as powders, tablets, and gels, and can also be dispersed in liquids to prepare liquid dosage forms, including but not limited to the embodiments described herein.

[0158] In the text, "DPPH" stands for 1,1-diphenyl-2-trinitrophenylhydrazine, also known as 1,1-diphenyl-2-picrylhydrazine (free radical).

[0159] In the text, "ABTS" refers to the free radical 2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0160] The term "Da" used in this article stands for Dalton, a commonly used unit for molecular weight.

[0161] The term "ultrafiltration" as used in this article is a commonly used name for a processing step in the field, and its name accurately describes the process, so it will not be repeated here.

[0162] The "nanofiltration membrane device" mentioned in this article refers to existing concentration equipment. The description of "nanofiltration membrane device concentration" can accurately describe its processing, so it will not be repeated here.

[0163] (2) Raw materials used in implementation:

[0164] The alkaline protease, aminopeptidase, and other enzymes used are all commercially available enzymes that can be purchased and obtained by those skilled in the art.

[0165] (3) Application of fermented rice peptides:

[0166] Fermented rice peptides possess the following characteristics: (1) α-amylase inhibitory activity; (2) anti-inflammatory activity; (3) beneficial bacteria promoting activity; (4) antioxidant activity; and (5) anti-glycation activity. Based on these characteristics, fermented rice peptides can also be applied to functional products (which can be food, health products, cosmetics, skin care products, etc., including any substance that provides preventive and / or other beneficial effects) in addition to skin care, weight loss, blood sugar reduction, anti-aging, antioxidant, anti-inflammatory, and anti-glycation effects.

[0167] In summary, the specific parameters or some commonly used reagents or raw materials in the above embodiments are specific or preferred embodiments under the concept of the present invention, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of the present invention.

[0168] In addition, unless otherwise specified, the raw materials used may be commercially available products in the field or prepared by conventional methods in the field; that is, the reagents and instruments used in this embodiment do not specify the manufacturer or other information, and are all conventional products that can be purchased from the market.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing fermented rice peptides, characterized in that... This includes the following steps: Raw material pretreatment: Rice protein is mixed with water to obtain a slurry; First hydrolysis: Alkaline protease is added to the slurry for the first hydrolysis. After the hydrolysis is completed, the enzyme is inactivated to obtain the first hydrolysate. Second hydrolysis: Aminopeptidase is added to the first hydrolysate for a second hydrolysis. After hydrolysis, the enzyme is inactivated to obtain the second hydrolysate. Inoculation and fermentation: Lactobacillus paracasei was added to the second hydrolysate for fermentation to obtain a fermentation broth; The fermentation broth is subjected to solid-liquid separation to obtain a supernatant containing fermented rice peptides. The supernatant is then filtered to retain the fermented rice peptides, thus obtaining the fermented rice peptides. In the first hydrolysis step, alkaline protease is added to the slurry to adjust the pH of the system to 8.0-9.0, and hydrolysis is carried out at 50-60℃ for 100-150 min; then enzyme inactivation treatment is carried out at 85-90℃ for 10-20 min to obtain the first hydrolysate. In the second hydrolysis step, aminopeptidase is added to the first hydrolysate to adjust the pH of the system to 6.5-7.5, and hydrolysis is carried out at 50-60℃ for 40-90 min; then the enzyme is inactivated at 85-90℃ for 10-20 min to obtain the second hydrolysate. The Lactobacillus paracasei is Lactobacillus paracasei YYS-69, with the accession number CGMCC No. 25837; The fermentation broth was centrifuged to separate the solid and liquid components, resulting in a supernatant containing fermented rice peptides. The pH of the supernatant was adjusted to 5.7–6.1, and then subjected to microfiltration through a membrane with a pore size of 0.22–0.45 μm and ultrafiltration through a 3000 Da ultrafiltration membrane to obtain an ultrafiltration peptide solution with a molecular weight of less than 3000 Da. The ultrafiltration peptide solution was concentrated using a 200 Da nanofiltration membrane device and then sterilized to obtain a peptide solution with a molecular weight of 200–3000 Da. The polypeptide solution with a molecular weight of 200-3000 Da is concentrated to a polypeptide mass content of 10%-20%, and the concentrate is then made into powder.

2. The method for preparing fermented rice peptides according to claim 1, characterized in that: In the raw material pretreatment step, the mass ratio of rice protein to water is 1:(10-20).

3. The method for preparing fermented rice peptides according to claim 1, characterized in that: Based on the protein content in the rice protein, the amount of alkaline protease added is 6000-8000 U / g; The amount of aminopeptidase added is 3000-5000 U / g, based on the protein content of the rice protein.

4. The method for preparing fermented rice peptides according to claim 1, characterized in that: In the fermentation process, the fermentation temperature is 36–38°C and the fermentation time is 22–26 h.

5. A fermented rice peptide, characterized in that: The rice peptide was prepared using the method described in any one of claims 1-4.

6. A functional product, characterized in that: Its components include fermented rice peptides prepared by the preparation method according to any one of claims 1-4.

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