Preparation and application of lactic acid bacteria fermented purple cabbage enzyme with effects of resisting oxidation, reducing blood sugar and resisting obesity in vivo and in vitro

Abstract

The preparation method is characterized by comprising the following steps: a, preparing a seed solution, namely inoculating pediococcus pentosaceus into an MRS liquid culture medium, performing shake culture, and then transferring the pediococcus pentosaceus into a new MRS culture medium for culture; b, preparation of an enzyme fermentation culture medium: fresh purple cabbages without plant diseases and insect pests are subjected to cleaning, pulping, enzymolysis, dilution, sugar degree adjustment, pasteurization and sterilization, cooling and PH adjustment pretreatment in sequence; c, preparing an enzyme fermentation liquid: inoculating the seed liquid into the purple cabbage fermentation culture medium in the step b according to the proportion of 7.5-12.5%, and performing oscillation culture to generate purple cabbage enzyme; the Pediococcus pentosaceus is Pediococcus pentosaceus NCJF, and the preservation number of the Pediococcus pentosaceus NCJF is CCTCC (China Center For Type Culture Collection) M 20251410; after fermentation by pediococcus pentosaceus, the fat and protein decomposition and oxidation resistance of the purple cabbages are greatly enhanced.

Description

Technical Field

[0001] This invention belongs to the fields of food biotechnology and microbial fermentation applications, specifically relating to the preparation and application of a lactic acid bacteria fermented purple cabbage enzyme that has antioxidant, hypoglycemic, and anti-obesity effects both in vivo and in vitro. Background Technology

[0002] Purple cabbage is highly nutritious, rich in vitamins A and C; abundant in minerals, including calcium, potassium, iron, and magnesium; high in dietary fiber; and rich in active ingredients such as anthocyanins, polyphenols, glucosinolates, and alkaloids. These active ingredients possess pharmacological effects such as antioxidation, cholesterol reduction, prevention of cardiovascular disease, promotion of intestinal peristalsis, prevention of constipation, aiding weight loss and gut health, and prevention of aging and chronic diseases. However, the abundant active ingredients in purple cabbage are easily degraded by environmental factors during processing and storage, limiting its widespread application in the food and pharmaceutical fields. Probiotic fermented fruit and vegetable enzymes offer significant advantages, enhancing the nutritional value of fruit and vegetable products, such as increasing vitamins and polyphenols and improving nutrient absorption; improving gut health, regulating gut microbiota balance, and promoting intestinal peristalsis; enhancing antioxidant properties; and regulating immunity and metabolism. Furthermore, fermentation can improve flavor and texture and extend shelf life.

[0003] Pediococcus pentosaceus is a widely recognized safe bacterial species, belonging to the genus Pediococcus in the family Streptococciaceae. It is a lactic acid bacterium widely found in the natural environment and the human gut, producing a variety of beneficial metabolites. Its probiotic functions are prominent, improving gut microbiota through colonization of the intestines to form biofilms, lactic acid production to lower pH, and the production of beneficial metabolites and bacteriocins. It can enhance immunity, regulate the immune system, and increase immune cell activity; lower cholesterol by decomposing bile salts, assimilating cholesterol, and inhibiting cholesterol formation; and possess antioxidant properties, scavenging free radicals and enhancing the antioxidant defense system.

[0004] Developing new Pediococcus pentosaceus strains and related probiotic products is of great significance.

[0005] Currently, products such as frozen purple cabbage, purple cabbage juice, and powder have been developed, but the technology for developing functional probiotic fermented purple cabbage enzymes is relatively limited. This invention uses purple cabbage as raw material to develop a lactic acid bacteria (Pediococcus pentosaceus) fermented purple cabbage enzyme. The study investigated its antioxidant, hypoglycemic, and anti-obesity effects in vitro and in rats on a high-fat diet, demonstrating its potential application prospects. Summary of the Invention

[0006] The primary objective of this invention is to provide a method for preparing purple cabbage enzymes through lactic acid bacteria fermentation. After fermentation using this method, some active enzymes and active substances in purple cabbage are significantly increased. Furthermore, in vivo and in vitro experiments have verified the antioxidant, hypoglycemic, and anti-obesity effects of the purple cabbage enzymes.

[0007] A second objective of the present invention is to provide a lactic acid bacteria fermented purple cabbage enzyme prepared by the above method.

[0008] The third objective of this invention is the application of the aforementioned purple cabbage enzyme in anti-oxidation, blood sugar reduction, and anti-obesity.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing lactic acid bacteria fermented purple cabbage enzyme includes the following steps: a. Preparation of seed culture: Inoculate *Pediococcus pentosaceus* into MRS liquid medium, shake and culture, then transfer to new MRS medium and culture. b. Preparation of enzyme fermentation medium: Fresh, disease-free purple cabbage is pretreated by washing, pulping, enzymatic hydrolysis, dilution, sugar concentration adjustment, pasteurization, cooling and pH adjustment. c. Preparation of enzyme fermentation broth: Inoculate the seed liquid into the purple cabbage fermentation medium in step b at a ratio of 7.5-12.5% ​​(v / v), shake and culture to generate fermentation enzyme; The Pediococcus pentosaceus is Pediococcus pentosaceus NCJF, accession number CCTCC M20251410.

[0010] In step a, the MRS medium consists of 10 g / L peptone, 5 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L diammonium citrate, 0.2 g / L magnesium sulfate, and 0.005 g / L manganese sulfate, with an initial pH of 5.8-6.0.

[0011] In step b, the enzymatic hydrolysis is a mixture of pectinase, cellulase and hemicellulase, with a mass ratio of 0.2% : 0.1% : 0.1%.

[0012] In step b, the enzymatic hydrolysis temperature is 40-60℃ and the enzymatic hydrolysis time is 1-3 hours; dilution: the volume ratio (v / m) of the enzymatically hydrolyzed matrix to water is 1:1; sugar adjustment: the sugar content is adjusted to 17.5% with honey; pasteurization refers to treatment at 68.3℃ for 20-50 minutes.

[0013] After fermentation, store at 4°C, or sterilize at 85°C-121°C for 5-30 minutes and then store aseptically.

[0014] A lactic acid bacteria fermented purple cabbage enzyme prepared by the method described above.

[0015] The application of the lactic acid bacteria fermented purple cabbage enzyme in the preparation of foods, health products, and medicines for prevention or treatment of antioxidant, hypoglycemic, and anti-obesity effects.

[0016] The application of the lactic acid bacteria fermented purple cabbage enzyme in the preparation of foods and health products for the prevention or treatment of metabolic disorders and tissue damage caused by a high-fat diet.

[0017] The beneficial effects are as follows: 1. In this invention, after fermentation with Pediococcus pentosaceus, the activities of some active enzymes in purple cabbage, such as lipase, protease, catalase, peroxidase, and SOD, are significantly increased, greatly enhancing the ability of purple cabbage to decompose fats and proteins and its antioxidant capacity. Simultaneously, the contents of total polyphenols, flavonoids, crude polysaccharides, and total acids are also significantly increased. Studies have shown that these active substances play important roles in anti-inflammation, anti-oxidation, immune enhancement, improvement of metabolic diseases, and regulation of intestinal health. The fermentation process has a highly positive impact on the content of active ingredients in purple cabbage, and the enhancement of these active ingredients provides broad prospects for the application of fermented purple cabbage in the food, health product, and pharmaceutical fields.

[0018] 2. In this invention, in vitro experiments demonstrated that purple cabbage fermented with *Pediococcus pentosaceus* exhibits strong antibacterial activity against various pathogenic bacteria; after fermentation, the efficiency of purple cabbage in scavenging various oxygen free radicals significantly increases; fermented purple cabbage can lower cholesterol concentration and bind to various bile salts; fermented purple cabbage can significantly inhibit the activity of α-amylase and α-glucosidase. This demonstrates that fermented purple cabbage possesses in vitro antioxidant, cholesterol-lowering, and blood sugar-lowering effects.

[0019] 3. In this invention, feeding rats on a high-fat diet (HFD) with purple cabbage fermented with *Pediococcus pentosaceus* significantly reduced body weight gain, Lee's index, and adipose tissue weight in HFD rats. The fermented purple cabbage also improved blood lipid and inflammation levels, alleviated glucose metabolism abnormalities and glucose intolerance caused by a high-fat diet, and significantly reduced oxidative stress in the liver induced by a high-fat diet, thus alleviating tissue damage to the liver and colon. *Pediococcus pentosaceus* fermented purple cabbage can significantly alleviate metabolic disorders and tissue damage caused by a high-fat diet.

[0020] 4. In this invention, *Pediococcus pentosaceus* is a novel strain obtained by the inventors through laboratory screening. This strain has been deposited at a cultural heritage center with the accession number CCTCC M 20251410, and its taxonomic name is: Pediococcus pentosaceusNCJF, depositary institution: China Center for Type Culture Collection, depositary address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, Wuhan University, postcode: 430072, deposit date of strain: June 17, 2025.

[0021] 5. Before bacterial fermentation, the purple cabbage is pretreated using a combination of physical cell wall disruption and biochemical digestion to fully release the components in the purple cabbage that can be utilized by microorganisms. Attached Figure Description

[0022] Figure 1 The size of the inhibition zone of purple cabbage enzyme against different pathogenic bacteria; Figure 2 Purple cabbage enzymes have cholesterol-lowering effects; Figure 3 The binding rate of bile salts to purple cabbage enzymes; Figure 4 Purple cabbage enzymes inhibit α-amylase activity; Figure 5 Purple cabbage enzymes inhibit α-glucosidase activity; Figure 6 Purple cabbage enzymes inhibited the increase in body weight in HFD (high-fat diet) rats; Figure 7 Purple cabbage enzymes improve glucose intolerance in HFD rats; Figure 8 Purple cabbage enzymes improve serum lipid levels in HFD rats; Figure 9 Purple cabbage enzymes improve oxidative stress response in HFD rats; Figure 10 Purple cabbage enzymes improve liver damage in HFD rats; Figure 11 Purple cabbage enzymes significantly improve inflammation caused by obesity. Detailed Implementation

[0023] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0024] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0025] This invention provides a process for preparing enzymes by lactic acid bacteria fermentation using purple cabbage as raw material. The strain used in the purple cabbage enzyme preparation process is *Pediococcus pentosaceus*. Pediococcus pentosaceusThe strain (NCJF) has been deposited at the China Cultural Relics Center (CCTCC M 20251410) and its taxonomic name is: Pediococcus pentosaceus NCJF, depositary institution: China Center for Type Culture Collection, depositary address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, Wuhan University, postcode: 430072, deposit date of strain: June 17, 2025.

[0026] Example 1: A purple cabbage enzyme fermented with lactic acid bacteria Stored in glycerin tubes Pediococcus pentosaceus NCJF was inoculated into MRS liquid medium and cultured in a shaking incubator at 34℃ and 120 r / min for 72 h. The cultured solution was then transferred to fresh MRS medium and fermented for 48 h to obtain seed culture. Fresh, disease-free purple cabbage was cleaned and mechanically pulped using a cell-wall breaking nutrient blender. Pectinase, cellulase, and hemicellulase were added to the purple cabbage pulp at a mass ratio of 0.2% : 0.1% : 0.1% (m / m) and enzymatically hydrolyzed at 55℃ for 2 h. The substrate was diluted with water at a volume-to-mass ratio of 1:1 to adjust the sugar content to 17.5%. The substrate was pasteurized at 68.3℃ for 30 min, cooled, and the pH adjusted to 5.0. The seed culture was inoculated into the purple cabbage substrate at a ratio of 10% (v / v) and cultured aerobically at 120 r / min in a shaker at 34℃ for 60 h to generate purple cabbage enzyme.

[0027] Example 2: Analysis of active components in fermented purple cabbage enzyme The active enzymes and main active components in purple cabbage enzymes were analyzed: Assay for lipase activity Lipase activity was determined according to GB / T 23535-2009 Lipase Preparations. 10 mL of the sample to be tested was pipetted into a centrifuge tube and centrifuged at 4℃ and 10000 r / min for 30 min. 1 mL of the supernatant was collected for testing. 1 g of the sample was weighed and thoroughly dissolved in a small amount of phosphate buffer. The resulting supernatant was carefully poured into a volumetric flask. If any residue remained, a small amount of phosphate buffer was added and the mixture was thoroughly ground and added to the volumetric flask. The volume was then adjusted to the mark with phosphate buffer, and the mixture was shaken well before testing. Take two 100 mL Erlenmeyer flasks as blank flask (A) and sample flask (B), respectively. Add 4.00 mL of substrate solution and 5.00 mL of phosphate buffer to flask A and flask B, respectively. Then add 15.00 mL of 95% ethanol to flask A and preheat in a 40°C water bath for 5 min. Then add 1.00 mL of the test solution to flask A and flask B, mix well immediately, and start timing. After reacting for 15 min, immediately add 15.0 mL of 95% ethanol to flask B to terminate the reaction. Remove the flasks and add two drops of 10 g / L phenolphthalein indicator to flask A and flask B, and titrate with 0.05 mol / L sodium hydroxide standard solution until a faint red color remains for 30 s. Record the volume of sodium hydroxide standard solution consumed. The lipase activity in the sample is calculated using the following formula:

[0028] In the formula: X—enzyme activity of the sample, U / mL (or U / g); V1—volume of sodium hydroxide standard solution consumed when titrating the sample, in milliliters (mL); V2—volume of sodium hydroxide standard solution consumed when titrating the blank, in milliliters (mL); c—concentration of sodium hydroxide standard solution, in moles per liter (mol / L); n1—dilution factor of the sample.

[0029] Assay for protease activity The determination of protease activity was performed according to GB / T 23527-2009 Protease Preparations. 10 mL of the sample to be tested was pipetted into a centrifuge tube and centrifuged at 4℃ and 10000 r / min for 30 min. 1 mL of the supernatant was collected for testing. 1 g of the precipitate was weighed and thoroughly dissolved in a small amount of phosphate buffer. The resulting supernatant was carefully poured into a volumetric flask. If any residue remained, a small amount of phosphate buffer was added and the mixture was thoroughly ground and added to the volumetric flask. The volume was then adjusted to the mark with phosphate buffer, and the mixture was shaken well before testing. First, preheat a 10 g / L casein solution in a 40°C water bath for 5 min. Then, proceed as follows: Test tube A (blank) → Add 1.00 mL of the test solution → Incubate at 40°C for 2 min → Add 2.00 mL of 65.4 g / L trichloroacetic acid and mix well → Incubate at 40°C for 10 min → Add 1.00 mL of casein solution and mix well → Remove and let stand for 10 min, then filter using slow-speed qualitative filter paper → Take 1.00 mL of the filtrate → Add 5.0 mL of 42.4 g / L sodium carbonate solution → Add 1.00 mL of Folin-Ciocalteu reagent → Incubate at 40°C for 20 min → Measure the absorbance at 680 nm. Test tube B → Add 1.00 mL of the test solution → Incubate at 40°C for 2 min → Add 1.00 mL of casein solution and mix well → Incubate at 40°C for 10 min → Add 2.00 mL of trichloroacetic acid. Shake well for 10 minutes, then filter using slow-speed qualitative filter paper. Take 1.00 mL of the filtrate, add 5.0 mL of sodium carbonate solution, add 1.00 mL of Folin-Ciocalteu reagent, incubate at 40°C for 20 minutes, and measure the absorbance at 680 nm. The protease activity in the sample is calculated using the following formula:

[0030] In the formula: X3—enzyme activity of the sample, U / mL (or U / g); A1—activity of the final diluted sample obtained from the standard curve, U / mL; V1—volume of the volumetric flask used to dissolve the sample, in milliliters (mL); n1—dilution factor of the sample; m4—mass of the sample, in grams (g).

[0031] Determination of catalase activity The catalase activity was determined according to GB / T 23195-2008, the method for determining catalase in bee pollen. 10 mL of the sample was placed in a centrifuge tube and centrifuged at 4°C and 10000 r / min for 30 min. 1 mL of the supernatant was collected for testing. 1 g of precipitate was weighed and placed in a porcelain mortar pre-cooled with liquid nitrogen. Liquid nitrogen was added while grinding. After the sample was fully ground, the mortar was allowed to thaw. Approximately 10 mL of freshly prepared phosphate buffer was added to dissolve the precipitate. The solution was transferred to a 25 mL volumetric flask and brought to the mark with phosphate buffer. After mixing thoroughly, the volumetric flask was placed in a 4°C refrigerator and allowed to stand for 30 min. The supernatant was then centrifuged at 4°C and 12000 r / min for 15 min. The clear solution was collected for testing. Preheat the test solution and a 0.1% hydrogen peroxide solution separately in a 40°C water bath for 10 min. Accurately pipette 0.1 mL of the test solution and 2.9 mL of the hydrogen peroxide solution into quartz dishes, mix thoroughly, and immediately begin reading at a wavelength of 240 nm (the instrument should be zeroed before placing the quartz dishes). Calculate the absorbance difference by selecting a 1-minute interval with good linearity from 0 s to 90 s. The catalase activity in the sample is calculated using the following formula:

[0032] In the formula: X—enzyme activity of the sample, U / mL (or U / g); ∆A—absorbance difference selected in the results over 1 minute; V1—total volume of sample solution, in milliliters (mL); V2—volume of sample solution used for determination, in milliliters (mL); m—mass of the sample, in grams (g).

[0033] Assay for peroxidase activity The peroxidase activity was determined according to GB / T 32131-2015, the method for detecting horseradish peroxidase activity. 10 mL of the sample to be tested was placed in a centrifuge tube and centrifuged at 4℃ and 10000 r / min for 30 min. 1 mL of the supernatant was collected for testing. 0.1 g of the precipitate was weighed, dissolved in 10 mL of phosphate buffer, and then diluted with phosphate buffer to a suitable concentration before testing. The test solution, phosphate buffer, 20 mmol / L guaiacol aqueous solution, and 8 mmol / L hydrogen peroxide aqueous solution were incubated in a 25°C water bath for 30 min. In a cuvette, 2.8 mL of phosphate buffer, 0.1 mL of guaiacol aqueous solution, 0.05 mL of hydrogen peroxide aqueous solution, and 0.05 mL of distilled water were added in sequence as a reference solution. In another cuvette, add 2.8 mL of phosphate buffer, 0.1 mL of guaiacol aqueous solution, 0.05 mL of hydrogen peroxide aqueous solution, and 0.05 mL of the test solution in sequence. After rapid mixing, measure the absorbance values ​​at the initial time and after 2 minutes at a wavelength of 436 nm using a spectrophotometer. The difference between the two values ​​is ∆A. Liquid samples are calculated using formula (1), and solid samples are calculated using formula (2):

[0035] In the formula: U—enzyme activity of the sample, in U / mL (or U / g); ∆A—change in absorbance of the sample; D—dilution factor; m—mass of the sample, in grams (g); V—volume of liquid in which the sample is dissolved, in milliliters (mL).

[0036] Assay for SOD enzyme activity The determination of SOD enzyme activity was performed according to GB / T 5009.171-2003, "Determination of Superoxide Dismutase (SOD) Activity in Health Foods". 10 mL of the sample to be tested was placed in a centrifuge tube and centrifuged at 4℃ and 10000 r / min for 30 min. 1 mL of the supernatant was collected for testing. 1 g of the centrifuged precipitate was weighed and placed in a glass mortar. 9 mL of distilled water was added, and the mixture was ground for 5 min. The mixture was then transferred to a 10 mL centrifuge tube, and distilled water was added to the mark. The tube was centrifuged at 4000 r / min for 15 min, and the supernatant was collected for testing. 2.35 mL of solution A, 1.8 mL of distilled water, 20 µL of the test solution, and 0.15 mL of solution B were added to a 10 mL colorimetric tube. After adding solution B, the mixture was immediately thoroughly mixed. The absorbance at 325 nm was measured initially and after 1 min. The difference between the two absorbance values ​​represents the rate at which the sample solution inhibits the auto-oxidation of pyrogallol. Liquid samples are calculated using formula (1), and solid samples are calculated using formula (2): ………………(2)

[0037] Where: X—SOD enzyme activity in the sample; —The rate of auto-oxidation of pyrogallol; — The sample solution inhibits the rate of pyrogallol autoxidation; V — The volume of the sample solution added, in milliliters (mL); D — The dilution factor of the sample solution; — Total volume of the sample solution, in milliliters (mL); m — Sample mass, in grams (g).

[0038] Determination of total polyphenol content The determination of total polyphenol content was performed according to T / AHFIA 005-2018, "Determination of Total Polyphenol Content in Plant Extracts and Their Products". 10 mL of the sample to be tested was placed in a centrifuge tube and centrifuged at 4℃ and 10000 r / min for 30 min. 1 mL of the supernatant was collected for testing. 1 g of the centrifuged precipitate was weighed and placed in a beaker, 30 mL of 60% ethanol solution was added, and the mixture was sonicated for 10 min. The volume was then adjusted to 50 mL with 60% ethanol solution, shaken well, filtered, and the solution was ready for testing. 1.0 mL of the test solution was placed in a 10 mL colorimetric tube, and 2.5 mL of Folin-Ciocalteu reagent and 2.5 mL of 15% Na₂CO₃ solution were added. The volume was adjusted to the mark with water, shaken well, and then heated in a 40℃ water bath for 1 h. After cooling for 20 min, the absorbance was measured at 778 nm. The calculation formula is as follows:

[0039] In the formula: X—the total polyphenol content in the sample; c—the total polyphenol content in the test solution calculated from the standard curve, in milligrams per liter (mg / L); n—the sample dilution factor.

[0040] Determination of flavonoid content The flavonoid content was determined according to GB / T 20574-2006, the method for determining the total flavonoid content in propolis. 10 mL of the sample was placed in a centrifuge tube and centrifuged at 4℃ and 10000 r / min for 30 min. 1 mL of the supernatant was collected and diluted to 50 mL with 95% ethanol, then shaken well before testing. 1 g of the centrifuged precipitate was weighed and placed in a beaker, and approximately 30 mL of 95% ethanol was added. The beaker was heated in a 65℃ water bath for approximately 45 min. After cooling to room temperature, the mixture was filtered through filter paper and diluted to 50 mL with 95% ethanol, then shaken well before testing. 1 mL of the test liquid was then transferred to a 50 mL volumetric flask, and 14 mL of 95% ethanol, 1 mL of aluminum nitrate solution (100 g / L), and 1 mL of potassium acetate solution (9.8 g / L) were added. The solution was diluted to 50 mL with water, shaken well, and allowed to stand for 1 h. The absorbance was then measured at 415 nm using a 30% ethanol solution as a blank. The calculation formula is as follows:

[0041] In the formula: X—total content of flavonoids; m—mass of rutin in the sample colorimetric solution obtained from the standard curve or from the linear regression equation, in milligrams (mg); W—mass or volume of the sample, in grams (g) or milliliters (mL); d—dilution ratio.

[0042] Determination of total acid content The determination of total acid content was performed according to GB 12456-2021, Determination of Total Acidity in Food. 10 mL of the sample to be tested was placed in a centrifuge tube and centrifuged at 4℃ and 10000 r / min for 30 min. 1 mL of the supernatant was transferred to a 250 mL volumetric flask, diluted to the mark with carbon dioxide-free water, and then mixed well. The mixture was then filtered through rapid filter paper before testing. 1 g of precipitate was weighed and placed in a 150 mL Erlenmeyer flask equipped with a condenser. Approximately 50 mL of carbon dioxide-free water at 80 °C was added, and the mixture was thoroughly mixed. The flask was then boiled in a boiling water bath for 30 min, cooled to room temperature, and diluted to 250 mL with carbon dioxide-free water. The mixture was then filtered through rapid filter paper before testing. Pipette 25 mL, 50 mL, or 100 mL of the test solution into a 250 mL Erlenmeyer flask. Add 2 to 4 drops of 10 g / L phenolphthalein indicator solution, then titrate with 0.1 mol / L sodium hydroxide standard titration solution until a faint pink color persists for 30 seconds. Record the volume of sodium hydroxide standard titration solution consumed. Repeat the above procedure to prepare a blank sample using the same volume of carbon dioxide-free water, and record the volume of sodium hydroxide standard titration solution consumed. The total acid content in the sample is calculated using the following formula:

[0043] In the formula: X—total acid content in the sample, in grams per kilogram (g / kg) or grams per liter (g / L); c—concentration of sodium hydroxide standard titration solution, in mol / L; V1—volume of sodium hydroxide standard titration solution consumed during titration of the test solution, in milliliters (mL); V2—volume of sodium hydroxide standard titration solution consumed by the blank sample, in milliliters (mL); k—conversion factor for acids: malic acid, 0.067, acetic acid, 0.060, tartaric acid, 0.075, citric acid, 0.064, citric acid (containing one molecule of water of crystallization), 0.070, lactic acid, 0.090, hydrochloric acid, 0.036, sulfuric acid, 0.049, phosphoric acid, 0.049; F—dilution factor of the test solution; m—mass of the sample, in grams (g) or volume of sample taken, in milliliters (mL).

[0044] Determination of crude polysaccharide content The determination of crude polysaccharide content was performed according to SN / T 4260-2015, "Determination of Crude Polysaccharides in Exported Plant-Derived Foods". 10 mL of the sample to be tested was placed in a centrifuge tube and centrifuged at 4℃ and 10000 r / min for 30 min. 1 mL of the supernatant was collected for testing. 1.0 g of the precipitate was weighed into a 50 mL stoppered centrifuge tube, moistened with 5 mL of water, and 20 mL of anhydrous ethanol was slowly added. After mixing thoroughly, the tube was ultrasonically extracted for 30 min in an ultrasonic cleaner. After extraction, the tube was centrifuged at 4000 r / min for 10 min, and the supernatant was discarded. The insoluble matter was washed with 10 mL of 80% ethanol solution and centrifuged. The insoluble matter was transferred to a round-bottom flask with a small amount of water, and 50 mL of water was added. The tube was ultrasonically extracted for 30 min in an ultrasonic cleaner, and this process was repeated twice. After cooling to room temperature, the tube was filtered, and the supernatant was transferred to a 200 mL volumetric flask, diluted to volume with water, and then tested. Pipette 1 mL of the test solution into a 20 mL stoppered test tube. Add 1.0 mL of 80% phenol solution and 5.0 mL of sulfuric acid (add perpendicularly to the liquid surface, avoiding contact with the test tube wall to ensure thorough mixing). Let stand for 10 min, then mix the reaction solution thoroughly. Place the test tube in a 30°C constant temperature water bath for 20 min and measure the absorbance at 490 nm. The blank sample and test sample determinations are performed in parallel, using the same amount of all reagents and the same analytical procedure, but without the test sample. The polysaccharide content in the sample is expressed as a mass fraction ω, in grams per 100 grams (g / 100 g), calculated using the following formula:

[0045] Where: m1—sugar content in the sample solution obtained from the standard curve, in micrograms (µg); V1—sample volume after final volume determination, in milliliters (mL); V2—volume of sample solution transferred during colorimetric determination, in milliliters (mL); m2—sample mass, in grams (g).

[0046] The results showed that fermented purple cabbage exhibited increased levels of lipase (150.00 U / mL), protease (595.89 U / mL), catalase (197.98 U / mL), peroxidase (470.59 U / mL), SOD (548.64 U / mL), total polyphenols (4.63 mg / mL), flavonoids (2.86 µg / mL), total acid (80.00 mg / mL), and crude polysaccharide (5.92 g / mL), representing increases of 37.14%, 12.65%, 58.90%, 143.65%, 71.36%, 82.31%, 53.37%, 50.00%, and 17.51%, respectively, compared to pre-fermentation levels (Table 1). These improvements in active components offer broad prospects for the application of fermented purple cabbage in the food, health product, and pharmaceutical fields.

[0047] Table 1. Content of active ingredients in purple cabbage before and after fermentation Active ingredients Before fermentation After fermentation Improvement rate Lipase activity (U / mL) 109.38±2.05 150.00±2.78** 37.14% Protease activity (U / mL) 595.89±5.77 671.29±6.02** 12.65% Catalase activity (U / mL) 197.98±1.77 314.59±2.62** 58.90% Peroxidase activity (U / mL) 470.59±7.64 1146.57±12.33** 143.65% SOD enzyme activity (U / mL) 548.64±3.76 940.18±5.22** 71.36% Total polyphenol content (mg / mL) 4.63±0.05 8.43±0.07** 82.31% Flavonoid content (μg / mL) 2.86±0.04 4.39±0.05** 53.73% Total acid content (kJ / mL) 80.00±1.23 120.00±1.73** 50.00% Crude polysaccharide content (g / mL) 5.92±0.10 6.95±0.15** 17.51% Example 3: In vitro functional study of purple cabbage enzyme The in vitro functional studies of purple cabbage enzymes mainly examined its antibacterial, antioxidant, cholesterol-lowering, and blood sugar-lowering effects.

[0048] Determination of the inhibition zone of purple cabbage enzyme against different pathogenic bacteria *Escherichia coli* ATCC 2592, *Staphylococcus aureus* ATCC 25923, and *Bacillus subtilis* ATCC 13952 strains purchased from the National Collection Center were aseptically inoculated into LB liquid medium and incubated at 37°C and 180 r / min for 24 h. The cultured bacterial solutions were then transferred to fresh LB liquid medium and repeated two to three times. 100 µL of the test bacterial solution was pipetted onto LB solid medium and spread evenly using a spreader. Sterile Oxford cups were gently placed on the medium with tweezers, and 100 µL of fermented purple cabbage from different treatments was transferred into each Oxford cup, with an equal volume of sterile water as a blank control. The medium was incubated at 37°C for 24 h, and the diameter of the inhibition zone (in mm) was measured using calipers. The results were the average of three parallel experiments.

[0049] The results showed that the fermented purple cabbage stock solution, supernatant, and dilution all exhibited antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. Figure 1The inhibition zone diameters of fermented purple cabbage against the three pathogenic bacteria were 32.56 mm, 28.31 mm, and 26.47 mm, respectively, without dilution (Table 2).

[0050] Table 2. Size of inhibition zones of fermented purple cabbage against different pathogenic bacteria

[0051] Determination of the antioxidant effects of purple cabbage enzyme Determination of the scavenging capacity of four oxygen free radicals by purple cabbage enzyme The determination of scavenging capacity for hydroxyl radicals, superoxide anion radicals, DPPH radicals, and ABTS radicals was performed according to the method given in the above kit.

[0052] Determination of the reducing power of purple cabbage enzyme Using Fe 3+ The reducing power method was used for detection. Purple cabbage fermented for 0, 12, 24, 36, 48, and 60 h was centrifuged at 10000×g for 5 min to remove insoluble residues. 200 μL of the supernatant was added to 4.0 mL of TPTZ working solution (composed of 25 mL of 0.1 mol / L acetate buffer, 2.5 mL of 10 mmol / L TPTZ solution, and 2.5 mL of 20 mmol / L FeCl3 solution). After mixing, the mixture was reacted at 37℃ for 10 min, and the absorbance was measured at a wavelength of 593 nm.

[0053] The results showed that fermented purple cabbage also possessed strong antioxidant capacity, capable of scavenging various oxygen free radicals and exhibiting good reducing ability. Its scavenging abilities against hydroxyl radicals, superoxide anion radicals, DPPH radicals, and ABTS radicals were 61.89%, 71.67%, 77.81%, and 87.79%, respectively, representing increases of 10.37%, 34.94%, 30.61%, and 28.99% compared to before fermentation (Table 3). The reducing ability of purple cabbage increased by 30.32% compared to before fermentation (Table 4).

[0054] Table 3. Scavenging capacity of four free radicals in fermented purple cabbage

[0055] Table 4. Reducing capacity of purple cabbage during fermentation

[0056] Tests on the cholesterol-lowering effects of purple cabbage enzymes: Tests on the cholesterol-lowering ability of purple cabbage enzymes 53.8 mg sodium taurocholate, 7.73 mg cholesterol, 14.1 mg oleic acid, and 77.1 mg sodium chloride were dissolved in 10 mL of phosphate buffer (pH 7.4), sonicated for 1 h, and incubated at 37°C for 24 h to obtain cholesterol micelles. 1 mL of purple cabbage fermented for different times was mixed with 5 mL of cholesterol micelles, incubated at 37°C for 2 h, and then centrifuged. The cholesterol content was determined using a cholesterol assay kit.

[0057] Determination of the ability of purple cabbage enzyme to bind bile salts Prepare 0.3 mmol / L stock solutions of sodium cholate, sodium taurocholate, and sodium glycocholate using PBS. Take 0, 10, 20, 30, 40, and 50 μL of these stock solutions into 96-well plates, respectively. Add PBS to a final volume of 50 μL, then add 150 μL of 60% sulfuric acid solution. Incubate at 70°C for 20 min, cool with ice for 5 min, and measure the absorbance at 387 nm. Plot the concentration-absorbance curves for the three cholates based on the results. Take different volumes of fermented purple cabbage, measure the absorbance using the cholate standard curve method, and determine the cholate concentration based on the standard curve.

[0058] The results showed that fermented purple cabbage could effectively bind cholesterol and bile salts, thereby reducing cholesterol concentration, with a cholesterol inhibition rate of 88.51%. Figure 2 The binding rates of sodium taurocholate, sodium glycocholate, and sodium hydrated cholate were 85.39%, 83.29%, and 81.36%, respectively. Figure 3 ).

[0059] Blood sugar lowering efficacy test of purple cabbage enzyme Determination of the inhibitory activity of purple cabbage enzyme α-amylase 0.5 mL of fermented purple cabbage at concentrations of 0, 0.5, 1.0, 2.0, 4.0, 8, and 16 mg / mL were mixed with 0.5 mL of α-amylase solution (0.5 U / mL) and reacted in a constant temperature water bath at 37°C for 15 min. Then, 1 mL of starch solution (10 mg / mL) was added to the mixture, and the reaction was carried out at 37°C for 10 min. Subsequently, 1 mL of DNS solution was added, and the mixture was reacted in a boiling water bath for 10 min. After cooling, 7 mL of distilled water was added, and the mixture was vortexed to mix. The absorbance was measured at 540 nm.

[0060] Determination of the inhibitory capacity of purple cabbage enzyme α-glucosidase activity 25 μL of fermented purple cabbage at concentrations of 0, 0.5, 1.0, 2.0, 4.0, 8, and 16 mg / mL were mixed with 50 μL of α-glucosidase (0.35 U / mL) and reacted in a water bath at 37°C for 15 min. Then, 25 μL of PNPG solution was added to the mixture, and the reaction was continued at 37°C for 30 min. Finally, 50 μL of Na₂CO₃ (1 mol / L) was added to terminate the reaction, and the absorbance was measured at 405 nm.

[0061] The results showed that fermented purple cabbage could inhibit the activities of α-amylase and α-glucosidase, with an inhibition rate of 71.15% for both. Figure 4 , 5 It has the potential to lower blood sugar.

[0062] Example 4: The antioxidant, blood sugar lowering, and anti-obesity effects of purple cabbage enzymes in vivo. Subsequently, we verified the alleviating effect of purple cabbage enzyme on obesity induced by a high-fat diet in rats.

[0063] Animal experimental design and grouping Forty-two 6-week-old male SD rats (180-200 g) were obtained from Beijing SPAF Biotechnology Co., Ltd. and housed in cages with constant temperature (25±2℃), humidity (55±5%), and light (12 h diurnal cycle). After one week of acclimatization, the rats were randomly divided into seven groups according to their diet and gavage administration: NCD group: normal rat diet + sterile saline; NHD group: normal rat diet + high-dose fermented purple cabbage (2 mL / kg / d); HFD (high-fat diet) group: high-fat rat diet + sterile saline; OR (positive control) group: HFD + orlistat (24 mg / kg); HUD group: HFD + unfermented purple cabbage (2 mL / kg / d); HLD group: HFD + low-dose fermented purple cabbage (1 mL / kg / d); HHD group: HFD + high-dose fermented purple cabbage (2 mL / kg / d). During the five-week administration period, the rats were orally administered via gavage daily, and their body weight, body length, and food intake were recorded daily.

[0064] After the experiment, rats were fasted for 12 hours, then stunned with ether and blood was collected from their tails. They were then euthanized by cervical dislocation. Serum was collected by centrifugation (1000×g, 10 min, 4℃). The whole brain, heart, liver, spleen, pancreas, kidneys, colon, testes, and white adipose tissue were separated, weighed, and collected. All samples were stored at −80℃ until analysis.

[0065] Histopathological analysis Before histochemical analysis, liver and colon tissues were fixed in 10% formalin for 48 h. After fixation, the samples underwent routine histological processing and were embedded in paraffin. Sections were taken at 4 µm for immunohistochemical staining. Liver and colon tissue samples for ELISA were cut into appropriately sized pieces and weighed before homogenization. The tissues were placed in PBS at pH 7.4 and adjusted to a tissue weight (g):PBS (mL) ratio of 1:9. Homogenization was then prepared. The tissue homogenate was centrifuged at 5000×g for 5 min, and the supernatant was transferred to microtubes. The activities of LPO (lipid peroxidation products), GSH (glutathione), SOD (superoxide dismutase), MDA (malondialdehyde), CAT (catalase), AST (aspartate aminotransferase), and ALT (alanine aminotransferase) were measured according to the commercial kit instructions.

[0066] Oral glucose tolerance test (OGTT) At the end of the experiment, rats that had fasted overnight were administered glucose (2 g / kg body weight) via gavage. Blood glucose levels were monitored using a glucometer. Blood was collected from the tail vein at 0, 30, 60, 90, and 120 minutes after oral glucose administration, and blood glucose levels were measured using a glucometer. Glucose tolerance was assessed using the area under the curve (AUC).

[0067] Biochemical index analysis Serum glucose (GLU), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), adiponectin (ADP), leptin (LEP), lipopolysaccharide (LPS), glutathione (GSH), LPO, superoxide dismutase (SOD), methylene blue (MDA), acetylcholine (CAT), AST, and alanine (ALT) were measured; liver tissue TC, TG, LDL-C, HDL-C, SOD, and CAT were measured; and fecal TC and TG levels were measured using commercially available kits (Nanjing Jiancheng Biotechnology Institute, Nanjing, China) and analyzed using ELISA kits (Elabscience and Jiyinmei, Wuhan, China).

[0068] After 5 weeks of continuous administration, the rats in the high-fat diet (HFD) group showed a body weight increase of over 23.8% compared to the normal diet control group (NCD), a highly significant difference, thus confirming the successful establishment of the obese rat model. Compared to the HFD group, rats supplemented with OR, HUD, HLD, and HHD all showed significantly reduced body weight gain. The purple cabbage enzyme-treated groups showed stronger efficacy in reducing body weight gain in HLD and HHD than the unfermented purple cabbage-treated group in reducing HUD. Figure 6 ).

[0069] A high-fat diet can cause metabolic disorders, such as glucose intolerance. An oral glucose tolerance test (OGTT) showed that high-fat diets (HFD) cause abnormal glucose metabolism and decreased glucose tolerance, while treatment with high-fat diets (HLD) and high-high-dose diets (HHD) improved these symptoms. Figure 7 ).

[0070] Total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) are key indicators in blood lipid testing. They reflect the status of lipid metabolism in the body and are closely related to various health problems such as cardiovascular disease. The study found that 5 weeks of HFD treatment significantly increased serum TC, TG, and LDL-C concentrations in rats, decreased serum HDL-C levels, while HLD and HHD treatments significantly reduced blood lipid levels. Figure 8 ).

[0071] Obese individuals experience heightened lipid peroxidation, leading to elevated serum concentrations of LPO (lipid peroxidation products) and MDA (malondialdehyde). SOD (superoxide dismutase), CAT (catalase), and GSH (glutathione) are important antioxidants in the body, capable of scavenging free radicals, mitigating oxidative damage, protecting cell health, and contributing to the prevention and treatment of obesity. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are correlated with the degree of liver damage caused by obesity. Five weeks of HFD resulted in elevated serum levels of LPO, ALT, AST, and MDA, while significantly decreased levels of GSH, SOD, and CAT, indicating that HFD caused severe liver function impairment and oxidative stress in rats. In the low-dose (HLD) and high-dose (HHD) purple cabbage enzyme treatment groups, oxidative stress and liver damage indicators were significantly repaired, with effects superior to the orlistat positive control group and the unfermented purple cabbage treatment group. Figure 9 ).

[0072] Obesity can lead to lipid accumulation and increased oxidative stress in the liver, resulting in liver damage. HFD significantly increased the levels of TC (total cholesterol), TG (triglycerides), and LDL-C (low-density lipoprotein cholesterol) in the rat liver, while significantly decreasing the levels of HDL-C (high-density lipoprotein cholesterol), SOD (superoxide dismutase), and CAT (catalase). HLD and HHD treatments could improve these changes. Figure 10 AF). Immunohistochemical staining revealed that the HFD group was infiltrated with a large number of inflammatory cells, while the inflammatory cell infiltration in the OR, HLD, and HHD groups was significantly alleviated. Figure 10 G).

[0073] Obesity also induces an inflammatory response. In the HFD treatment group, LPS levels were significantly elevated, as were the levels of pro-inflammatory factors TNF-α and IL-6, while the level of the anti-inflammatory factor IL-10 was significantly decreased. HLD and HHD treatments significantly alleviated this trend. Figure 11 This indicates that purple cabbage enzymes significantly improve inflammation caused by obesity.

[0074] The above embodiments show that, Pediococcus pentosaceus NCJF fermented purple cabbage enzymes have antioxidant, blood sugar lowering, and anti-obesity effects, and have broad application value.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing lactic acid bacteria fermented purple cabbage enzyme, characterized in that, Includes the following steps: a. Preparation of seed culture: Inoculate *Pediococcus pentosaceus* into MRS liquid medium, shake and culture, then transfer to new MRS medium and culture. b. Preparation of enzyme fermentation medium: Fresh, disease-free purple cabbage is pretreated by washing, pulping, enzymatic hydrolysis, dilution, sugar concentration adjustment, pasteurization, cooling and pH adjustment. c. Preparation of enzyme fermentation broth: Inoculate the seed liquid into the purple cabbage fermentation medium of step b at a ratio of 7.5-12.5% ​​(v / v), shake and culture to generate purple cabbage enzyme; The Pediococcus pentosaceus is Pediococcus pentosaceus NCJF, accession number CCTCC M 20251410.

2. The method for preparing lactic acid bacteria fermented purple cabbage enzyme according to claim 1, characterized in that, In step a, the MRS medium consists of 10 g / L peptone, 5 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L diammonium citrate, 0.2 g / L magnesium sulfate, and 0.005 g / L manganese sulfate, with an initial pH of 5.8-6.

0.

3. The method for preparing lactic acid bacteria fermented purple cabbage enzyme according to claim 1, characterized in that, In step b, the enzymatic hydrolysis is a mixture of pectinase, cellulase and hemicellulase, with a mass ratio of 0.2% : 0.1% : 0.1%.

4. The method for preparing lactic acid bacteria fermented purple cabbage enzyme according to claim 1, characterized in that, In step b, the enzymatic hydrolysis temperature is 40-60℃ and the enzymatic hydrolysis time is 1-3 hours.

5. The method for preparing lactic acid bacteria fermented purple cabbage enzyme according to claim 1, characterized in that, In step b, dilution: the volume ratio (v / m) of the enzymatically hydrolyzed matrix to water is 1:1; sugar adjustment: the sugar content is adjusted to 17.5% with honey.

6. The method for preparing lactic acid bacteria fermented purple cabbage enzyme according to claim 1, characterized in that, In step b, pasteurization refers to treatment at 68.3°C for 20-50 minutes.

7. The method for preparing lactic acid bacteria fermented purple cabbage enzyme as described in claim 1, characterized in that, After fermentation, store at 4°C, or sterilize at 85°C-121°C for 5-30 minutes and then store aseptically.

8. A lactic acid bacteria fermented purple cabbage enzyme prepared by the method according to any one of claims 1-7.

9. The application of the lactic acid bacteria fermented purple cabbage enzyme as described in claim 8 in the preparation of foods, health products, and pharmaceuticals for the prevention or treatment of antioxidant, hypoglycemic, and anti-obesity effects.

10. The application of the lactic acid bacteria fermented purple cabbage enzyme as described in claim 8 in the preparation of foods and health products for the prevention or treatment of metabolic disorders and tissue damage caused by a high-fat diet.

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