Multifunctional aloe leavening and preparation method and application thereof

Abstract

The invention provides a multifunctional aloe leavening and a preparation method and application thereof, and belongs to the technical field of biological medicines.The preparation method comprises the steps that after aloe and lactic acid bacteria are mixed, a basic fermentation substrate is firstly added for primary fermentation, then an enhanced fermentation supplementary material is added for secondary fermentation, a secondary fermentation product is extracted, and the aloe leavening is obtained; in the obtained aloe leavening, the content of aloesin reaches 8% or above, the content of barbaloin reaches 11% or above, the content of phytosphingosine reaches 16% or above, the content of sphingosine dihydrogen reaches 14% or above, and the aloe leavening has excellent effects of relaxing bowel, protecting intestinal tracts, enhancing immunity, protecting liver and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a multifunctional aloe vera ferment, its preparation method, and its applications. Background Technology

[0002] In the field of plant component development, compared with commonly used solvent extraction and chemical transformation methods, traditional Chinese medicine (TCM) fermentation technology is gentler and can achieve multiple goals, including enhancing efficacy, reducing toxicity, and expanding pharmacological effects, providing a new pathway for the modernization of TCM processing. TCM fermentation technology originated from brewing techniques and is one of my country's traditional TCM processing technologies. Classical texts such as *Jinkui Yaolue*, *Qimin Yaoshu*, *Qianjin Fang*, and *Compendium of Materia Medica* record methods for preparing naturally fermented TCM starter cultures and their clinical applications. During TCM fermentation, extracellular enzymes, cellulases, and pectinases produced during microbial growth and metabolism can break down plant cell walls, accelerating the release of active substances. Proteases and lipases produced can decompose biomolecules into easily absorbed small molecules. TCM fermentation technology effectively promotes the transformation and innovation of active molecules, greatly improving the bioavailability of TCM.

[0003] Recent studies have revealed that different microbial strains or fermentation processes produce distinct fermentation effects on the same traditional Chinese medicine (TCM). Taking *Dendrobium officinale* fermentation as an example, *Lactobacillus plantarum* fermentation products show a higher total flavonoid content, while *Bacillus* fermentation products exhibit higher polysaccharide and polypeptide content. These differences in chemical composition directly impact the functional effects of the final fermentation products. Furthermore, the TCM matrix provides a unique growth environment for microorganisms, and its chemical components regulate microbial metabolic pathways, promoting the synthesis of new active substances. In conclusion, the symbiotic and co-transformation system formed by TCM and microorganisms not only enhances the diversity of active ingredients and functions in TCM but also expands the application value of microorganisms in the health product field.

[0004] Aloe vera is a perennial evergreen herb, and its products are widely used in food, cosmetics, health care, and medicine. Aloe barbacin and aloin are important active compounds in aloe vera, possessing multiple functions such as anti-inflammation, antibacterial properties, and moisturizing. Phytosphingosine and dihydrosphingosine, as sphingolipid metabolites found in plants or microorganisms, have moisturizing, skin barrier repair, and antioxidant effects. Enriching the active ingredients in aloe vera while simultaneously enhancing its bioactivity, ensuring controllable formation of active products, and guaranteeing their specific efficacy is a key technical challenge faced by researchers in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a multifunctional aloe vera ferment, its preparation method, and its applications.

[0006] The technical solution of this invention is as follows: An aloe vera ferment is obtained by secondary fermentation of aloe vera with lactic acid bacteria; wherein, by mass percentage, the aloe vera ferment contains more than 8% aloin, more than 11% aloin, more than 16% phytosphingosine, and more than 14% dihydrosphingosine.

[0007] The method for preparing the aloe vera ferment includes the following steps: after mixing aloe vera and lactic acid bacteria, a basic fermentation substrate is first added for primary fermentation, then an enhanced fermentation feed is added for secondary fermentation, and the secondary fermentation product is extracted to obtain the aloe vera ferment.

[0008] The method for preparing the aloe vera ferment includes the following steps: (1) Add purified water to aloe vera pulp, mash and sterilize to obtain aloe vera pulp; add lactic acid bacteria to aloe vera pulp, mix well to obtain aloe vera pulp lactic acid bacteria suspension. (2) Add basic fermentation substrate to the aloe vera pulp lactic acid bacteria suspension in step (1), and supplement with L-cysteine, manganese sulfate and magnesium sulfate. Ferment at 25~50℃ and 150~400rpm for 24~72h to obtain primary fermentation product. (3) Add enhanced fermentation feed to the primary fermentation product of step (2) and add sodium chloride. Continue secondary fermentation for 12-48 hours at 25-50℃ and 150-400rpm. Adjust the pH to 5.5-7 to obtain the secondary fermentation product. (4) Add anhydrous ethanol to the secondary fermentation product of step (3) and extract by ultrasonic extraction for 10-30 min. Centrifuge at 3000-5000 rpm, collect the supernatant, concentrate, and dynamically adsorb onto macroporous adsorption resin. Elute with 40%-70% ethanol solution, collect the eluent, and dry to obtain aloe vera fermentation product.

[0009] Preferably, the amount of purified water added in step (1) is 1 to 4 times the volume of aloe vera leaf pulp.

[0010] Preferably, the lactic acid bacteria in step (1) is one of Bifidobacterium animalis, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus helveticus, or Streptococcus thermophilus.

[0011] More preferably, the live bacteria concentration of the lactic acid bacteria in step (1) is 10 billion CFU / g, and the addition ratio is 0.5%~6% (w / w).

[0012] Preferably, the basic fermentation substrate in step (2) includes a carbon source, a nitrogen source, and a buffer; the carbon source is one of inulin, soluble starch, and maltodextrin, accounting for 2%~6% (w / w); the nitrogen source is one of corn peptone, tryptone, yeast extract, whey protein, and soy protein, accounting for 0.5%~4% (w / w); the buffer is a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, accounting for 0.1%~0.6% (w / w), and the molar ratio between dipotassium hydrogen phosphate and potassium dihydrogen phosphate is (0.1~1.2):1.

[0013] More preferably, the proportion of L-cysteine ​​added in step (2) is 0.02%~0.1% (w / w), the proportion of manganese sulfate added is 0.005%~0.03% (w / w), and the proportion of magnesium sulfate added is 0.02%~0.08% (w / w).

[0014] Preferably, the enhanced fermentation feed in step (3) includes a carbon source and a nitrogen source; the carbon source is one of glucose, sucrose, and lactose, accounting for 1% to 4% (w / w); the nitrogen source is one of corn peptone, tryptone, and yeast extract, accounting for 0.5% to 2% (w / w).

[0015] More preferably, the proportion of sodium chloride added in step (3) is 0.05%~0.5% (w / w).

[0016] Preferably, the amount of anhydrous ethanol added in step (4) is 2 to 6 times the volume of the secondary fermentation product, the ultrasonic extraction temperature is 40 to 80°C, the macroporous adsorption resin is one of AB-8, ADS-17, and HPD826, and the dynamic adsorption flow rate is 1 to 4 BV / h.

[0017] The aloe vera ferment is used in the preparation of health foods or medicines, wherein the health foods or medicines have one or more of the following functions: (1) It lubricates the intestines and promotes bowel movements; (2) Antioxidant; (3) Enhance immunity; (4) Adjunctive protective effect against chemically induced liver injury.

[0018] The application of the aloe vera ferment in the preparation of drugs for treating constipation and liver damage.

[0019] The application of aloe vera ferment in the preparation of daily chemical products.

[0020] Preferably, the daily chemical product is a cosmetic.

[0021] Beneficial effects: (1) This invention provides a two-stage fermentation technology for aloe vera. The process is green, safe, simple to operate, and easy to scale up. The aloe vera fermented product contains more than 8% of the important active ingredient aloin, more than 11% of aloin, more than 16% of phytosphingosine, and more than 14% of dihydrosphingosine.

[0022] (2) The aloe vera ferment obtained by this invention has several biological activities superior to those of similar technologies and traditional processes. Experimental results show that, compared with aloe vera ferment without added lactic acid bacteria or with altered fermentation conditions, the aloe vera ferment obtained by this invention has superior effects in promoting bowel movements, protecting the intestines from oxidative stress damage (antioxidant), enhancing immunity, and protecting the liver. This may be due to the synergistic effect of aloe vera active ingredients and lactic acid bacteria metabolites during fermentation, forming a multifunctional aloe vera-lactic acid bacteria fermentation complex with superior functional effects. Attached Figure Description

[0023] Figure 1 LC-MS chromatogram of aloe-emodin; Figure 2 The LC-MS chromatogram for aloe-glycoside detection is shown. Figure 3 The LC-MS chromatogram for phytosphingosine detection; Figure 4 The image shows the LC-MS chromatogram of dihydrosphingosine. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments. The description in this section is merely illustrative and explanatory, and should not be construed as limiting the scope of protection of the present invention.

[0025] Explanation of the source of experimental materials: Bifidobacterium animalis (10 billion CFU / g), Lactobacillus reuteri (10 billion CFU / g), and Lactobacillus casei (10 billion CFU / g): purchased from Shaanxi Chenzhuo Biotechnology Co., Ltd.

[0026] Brewing yeast: sourced from the Institute of Biology, Shandong Academy of Sciences; it can also be purchased through regular commercial channels.

[0027] Inulin, soluble starch, maltodextrin, corn peptone, tryptone, yeast extract, and soy protein: purchased from Jinan Huifeng Science and Trade Co., Ltd.

[0028] Dipotassium hydrogen phosphate and potassium dihydrogen phosphate: purchased from Sinopharm Chemical Reagent Co., Ltd.

[0029] Glucose, sucrose, and lactose: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0030] Example 1: Preparation of a multifunctional aloe vera ferment The specific steps are as follows: (1) Soak 1 kg of aloe vera leaves in 0.025% potassium permanganate solution for 15 min to sterilize, remove aloe vera pulp, add 2 times the volume of purified water to aloe vera pulp and mash, pasteurize at 80℃ for 12 min to obtain aloe vera pulp; quickly and steadily pour 3% (w / w) of Bifidobacterium animalis into aloe vera pulp, stir at 40 rpm for 8 min to mix well to obtain aloe vera pulp lactic acid bacteria suspension.

[0031] (2) Add the basic fermentation substrate to the aloe vera pulp lactic acid bacteria suspension from step (1), and supplement L-cysteine ​​to 0.06% (w / w), manganese sulfate to 0.01% (w / w), and magnesium sulfate to 0.04% (w / w). Ferment at 35°C and 300 rpm for 48 h to obtain the primary fermentation product. The basic fermentation substrate includes the following components: inulin 4% (w / w), corn peptone 2% (w / w), and a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (molar ratio 0.8:1) 0.3% (w / w).

[0032] (3) Add enhanced fermentation feed to the primary fermentation product of step (2) and add sodium chloride to 0.3% (w / w). Continue secondary fermentation at 35°C and 300 rpm for 24 h. Adjust the pH to 6.5 with 2% sodium bicarbonate solution to obtain the secondary fermentation product. The enhanced fermentation feed includes the following components: 2% (w / w) glucose and 1.2% (w / w) corn peptone.

[0033] (4) Add 4 times the volume of anhydrous ethanol to the secondary fermentation product of step (3), extract by ultrasonication at 55℃ for 20 min, centrifuge at 4000 rpm, collect the supernatant, concentrate until there is no alcohol odor, and then dynamically adsorb onto AB-8 macroporous adsorption resin at a flow rate of 2 BV / h. Elute with 60% ethanol solution, collect the eluent, and vacuum dry to obtain 86.4 g of aloe vera fermentation product.

[0034] Example 2: Preparation of a multifunctional aloe vera ferment The specific steps are as follows: (1) Soak 1 kg of aloe vera leaves in 0.025% potassium permanganate solution for 8 min, remove aloe vera pulp, add 3 times the volume of purified water to aloe vera pulp and mash it, pasteurize at 80℃ for 15 min to obtain aloe vera pulp; quickly and steadily pour 4% (w / w) Lactobacillus reuteri into aloe vera pulp, stir at 30 rpm for 10 min to mix well to obtain aloe vera pulp lactic acid bacteria suspension.

[0035] (2) Add the basic fermentation substrate to the aloe vera pulp lactic acid bacteria suspension from step (1), and supplement with L-cysteine ​​to 0.1% (w / w), manganese sulfate to 0.03% (w / w), and magnesium sulfate to 0.08% (w / w). Ferment at 45°C and 150 rpm for 24 h to obtain the primary fermentation product. The basic fermentation substrate includes the following components: soluble starch 3% (w / w), yeast extract 3% (w / w), and a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (molar ratio 1:1) 0.5% (w / w).

[0036] (3) Add enhanced fermentation feed to the primary fermentation product of step (2) and add sodium chloride to 0.5% (w / w). Continue secondary fermentation at 45℃ and 150 rpm for 12 h. Adjust the pH to 7 with 3% sodium bicarbonate solution to obtain the secondary fermentation product. The enhanced fermentation feed includes the following components: 3% (w / w) sucrose and 1.5% (w / w) tryptone.

[0037] (4) Add 5 times the volume of anhydrous ethanol to the secondary fermentation product of step (3), extract by ultrasonication at 70°C for 10 min, centrifuge at 3000 rpm, collect the supernatant, concentrate until there is no alcohol odor, and then dynamically adsorb onto ADS-17 macroporous adsorption resin at a flow rate of 3 BV / h. Elute with 70% ethanol solution, collect the eluent, and vacuum dry to obtain 83.5 g of aloe vera fermentation product.

[0038] Example 3: Preparation of a multifunctional aloe vera ferment The specific steps are as follows: (1) Soak 1 kg of aloe vera leaves in 0.025% potassium permanganate solution for 12 min, remove aloe vera pulp, add 1 volume of purified water to aloe vera pulp and mash, pasteurize at 80℃ for 8 min to obtain aloe vera pulp; quickly and steadily pour 2% (w / w) of Lactobacillus casei into aloe vera pulp, stir at 60 rpm for 5 min to mix well to obtain aloe vera pulp lactic acid bacteria suspension.

[0039] (2) Add the basic fermentation substrate to the aloe vera pulp lactic acid bacteria suspension from step (1), and supplement L-cysteine ​​to 0.04% (w / w), manganese sulfate to 0.005% (w / w), and magnesium sulfate to 0.02% (w / w). Ferment at 30°C and 400 rpm for 72 h to obtain the primary fermentation product. The basic fermentation substrate includes the following components: 5% (w / w) maltodextrin, 1% (w / w) soybean protein, and 0.2% (w / w) a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (molar ratio 0.4:1).

[0040] (3) Add enhanced fermentation feed to the primary fermentation product of step (2) and add sodium chloride to 0.1% (w / w). Continue secondary fermentation at 30°C and 400 rpm for 48 h. Adjust the pH to 6 with 1% sodium bicarbonate solution to obtain the secondary fermentation product. The enhanced fermentation feed includes the following components: 1% (w / w) lactose and 0.5% (w / w) yeast extract.

[0041] (4) Add 3 times the volume of anhydrous ethanol to the secondary fermentation product of step (3), extract by ultrasonication at 45°C for 30 min, centrifuge at 5000 rpm, collect the supernatant, concentrate until there is no alcohol odor, and then dynamically adsorb the sample onto HPD826 macroporous adsorption resin at a flow rate of 1 BV / h. Elute with 50% ethanol solution, collect the eluent, and vacuum dry to obtain 82.8 g of aloe vera fermentation product.

[0042] Comparative Example 1: Preparation of an Aloe Vera Ferment The difference from Example 1 is that Bifidobacterium animalis was replaced with Saccharomyces cerevisiae, and the remaining steps were the same as in Example 1, yielding 71.3g of aloe vera ferment.

[0043] Comparative Example 2: Preparation of an Aloe Vera Ferment The difference from Example 1 is that inulin in the basic fermentation substrate was replaced with glucose, and glucose in the enhanced fermentation feed was replaced with inulin. The remaining steps were the same as in Example 1, yielding 75.7g of aloe vera ferment.

[0044] Comparative Example 3: Preparation of an Aloe Vera Ferment The difference from Example 1 is that the corn peptone in both the basic fermentation substrate and the enhanced fermentation feed was replaced with ammonium sulfate, while the remaining steps were the same as in Example 1, yielding 72.4g of aloe vera ferment.

[0045] Comparative Example 4: Preparation of an Aloe Vera Extract The difference from Example 1 is that the aloe vera pulp was not fermented with lactic acid bacteria, but was directly extracted with 4 times the volume of anhydrous ethanol by ultrasound. The remaining steps were the same as in Example 1, yielding 43.6g of aloe vera extract.

[0046] Experiment Example 1: Detection of active ingredient content in each group of aloe vera ferments / extracts Follow these steps: (1) Preparation of test solution: Weigh 4 mg of aloe vera ferment / extract powder into a 1.5 mL centrifuge tube, dissolve it with 20 μL of dimethyl sulfoxide, make up to 1 mL with methanol, filter through a 0.22 μm organic microporous membrane to obtain the test solution; (2) Preparation of standard solutions: Accurately weigh appropriate amounts of standard substances (aloeban, aloin, phytosphingosine, dihydrosphingosine), dissolve and dilute with methanol, filter through a 0.22μm organic microporous membrane, and prepare standard solutions of aloin with a concentration of 1 mg / mL, aloin with a concentration of 1.5 mg / mL, phytosphingosine with a concentration of 2 mg / mL, and dihydrosphingosine with a concentration of 1 mg / mL, respectively.

[0047] (3) LC-MS (Liquid Chromatography-Mass Spectrometry) analysis conditions: Agilent 1260 HPLC-6530 QTOF LC-MS instrument, Agilent Eclipse XDB-C18 column (4.6×250mm, 5μm); mobile phase A is water, B is acetonitrile, elution gradient is 0~10min 10%B~50%B, 10~30min 50%B~70%B, 30~45min 70%B~90%B, 45~55min 90%B~100%B; flow rate is 1mL / min, column temperature is 30℃; mass spectrometry parameters are electrospray ionization source (positive and negative ion mode), drying gas temperature is 350℃, drying gas flow rate is 10L / min, nebulizer pressure is 45psi, fragmentation voltage is 150V, capillary voltage is 4000V, and molecular weight scan is 100~2000m / z.

[0048] (4) Standard curve plotting: Inject 1 μL, 5 μL, 10 μL, 15 μL, and 20 μL of the standard solution respectively. Plot the standard curve with the mass of the standard solution as the x-axis and the peak area as the y-axis. The standard curve is as follows: Aloe-emodin: Y=46272X-2125, R=0.9993, linear range 1~20μg; Aloe-glycosides: Y=61569X-906, R=0.9997, linear range 1.5~30μg; Phytosphingosine: Y=60484X+61605, R=0.9979, linear range 2~40μg; Dihydrosphingosine: Y = 86152X + 36685, R = 0.9965, linear range 1~20 μg.

[0049] (5) Detection of active ingredients: Inject 20 μL of the test solution and perform LC-MS detection according to the above LC-MS analysis conditions. The LC-MS detection results of the target compounds are as follows: Figures 1-4 As shown in Table 1, the content (mass percentage) of each compound in the sample was calculated based on the standard curve.

[0050] Table 1. Content of active ingredients in samples

[0051] As shown in Table 1, the aloe vera ferment prepared by this invention has the dual advantages of high yield and high content of target active ingredients; the content of aloin is more than 8%, the content of aloin is more than 11%, the content of phytosphingosine is more than 16%, and the content of dihydrosphingosine is more than 14%.

[0052] Comparative Example 1 replaced lactic acid bacteria with brewer's yeast, resulting in a decrease in the yield of aloe vera ferment and the content of aloin, aloin, phytosphingosine, and dihydrosphingosine compared to Example 1. In particular, the content of phytosphingosine and dihydrosphingosine showed a significant decrease compared to Examples 1-3. These results indicate that only under lactic acid bacteria fermentation conditions can the efficient extraction of all four active ingredients—aloe vera, aloin, phytosphingosine, and dihydrosphingosine—be achieved simultaneously.

[0053] Comparative Example 2 changed the carbon source in the basic fermentation substrate and the enhanced fermentation feed, and Comparative Example 3 changed the nitrogen source in the fermentation process to inorganic salts. All of the above changes resulted in a significant decrease in the yield of aloe vera ferment and the content of the target components compared with Examples 1 to 3. This shows that only under specific fermentation conditions can the efficient extraction of active ingredients from aloe vera be achieved.

[0054] Comparative Example 4 used traditional organic solvents to extract the active ingredients from aloe vera, and the resulting aloe vera extract was only 43.6g, with the content of active ingredients being significantly lower than that in Examples 1-3.

[0055] The above results indicate that the present invention utilizes lactic acid bacteria to carry out secondary fermentation of aloe vera under specific fermentation conditions, which has the advantages of high yield and high content of active ingredients (aloe bitter substances, aloin, phytosphingosine, and dihydrosphingosine) compared with similar fermentation technologies or traditional processes.

[0056] Experimental Example 2: Laxative and Intestinal Protective Effects Follow these steps: (1) Constipation model construction and drug treatment: Seventy male SPF-grade KM mice (6-8 weeks old, weighing 18-22g) were selected. The mice had free access to food and water. After 7 days of acclimatization, they were randomly divided into a blank control group (10 mice) and a constipation group (60 mice). Among them, the mice in the blank control group were administered physiological saline by gavage (10mL / kg) every day. On the 10th day, they were administered an equal amount of ink by gavage to evaluate the laxative effect. On the 12th day, the mice were sacrificed and samples were taken for analysis. Mice in the constipation group were given compound diphenoxylate (10 mg / kg) daily to induce constipation. After 7 days, the constipation group was randomly divided into a model group, a fermentation product administration group (low dose 50 mg / kg, medium dose 100 mg / kg, high dose 200 mg / kg), a fermentation product administration group (200 mg / kg) of Comparative Example 1, and an extract administration group (200 mg / kg) of Comparative Example 4, with 10 mice in each group. The model group was given physiological saline (10 mL / kg) by gavage daily, while the other groups were given the corresponding aloe vera fermentation product / extract by gavage daily. On the 3rd day after administration, each constipation group was given an equal volume of ink to physiological saline by gavage to evaluate the laxative effect. On the 5th day, the mice were sacrificed for sampling and analysis.

[0057] (2) Laxative effect: Observe and record the time it takes for each mouse to expel the first black feces, and observe continuously for 5 hours. Record the number of feces in the mice within 5 hours, and weigh the expelled feces in a timely manner.

[0058] (3) Determination of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in mouse colon tissue: After euthanizing the mice, the colon tissue was removed from the abdominal cavity of the mice, homogenized with a tissue homogenizer, centrifuged at 3000 r / min for 10-15 min at 4℃, the supernatant was collected, and then the activity / content of SOD and MDA was determined according to the operation instructions of the corresponding kit.

[0059] The results are shown in Table 2.

[0060] Table 2. Effects of aloe vera ferment / extract on SOD and MDA levels in mouse feces and colon tissue.

[0061] Compared with the model group* (p<0.05), ** (p<0.01).

[0062] Table 2 shows that, compared with the blank control group, after continuous gavage administration of compound diphenoxylate, the time to excretion of the first black stool in the model group mice was significantly prolonged, and the number of fecal particles and the weight of fecal matter were significantly reduced, indicating that the constipation model was successfully established. Compared with the model group, the dosage of 50 mg / kg in Example 1 significantly increased the fecal weight of mice, while the dosages of 100 mg / kg and 200 mg / kg showed significant improvement in all three indicators: time to excretion of the first black stool, number of fecal particles, and weight of fecal matter, demonstrating excellent laxative effects. The dosage of 200 mg / kg in Comparative Example 1 and Comparative Example 4 also significantly improved the constipation symptoms in mice, but the improvement effect was weaker than that in Example 1. In addition, the dosages of 100 mg / kg and 200 mg / kg in Example 1 significantly affected the SOD and MDA indicators in the colonic tissue of mice, increasing SOD activity and reducing MDA content, while Comparative Example 1 and Comparative Example 4 did not have this effect.

[0063] Multiple studies have confirmed that the active ingredients in aloe vera, aloin and aloin, can stimulate the colon and promote intestinal peristalsis, thus relieving constipation and improving bowel movements. However, long-term constipation leads to the accumulation of harmful substances in the intestines, and the resulting excessive free radicals and inflammatory mediators can damage the intestinal mucosa and even induce serious complications such as cardiovascular and cerebrovascular diseases. The aloe vera ferment prepared in this invention can increase SOD activity and reduce MDA content in colonic tissue. This may be related to its rich content of phytosphingosine and dihydrosphingosine, which can regulate sphingolipid metabolism signaling pathways, activate the body's own antioxidant defense system, thereby enhancing the efficacy of endogenous antioxidant enzymes such as SOD, reducing lipid peroxidation damage in colonic tissue, and ultimately protecting the intestines from oxidative stress. Therefore, the aloe vera ferment provided by this invention not only relieves constipation but also protects the intestines from oxidative stress damage (antioxidant), providing better relief for constipation-related symptoms.

[0064] Experimental Example 3: Immunomodulatory Effects Follow these steps: (1) Treatment of RAW264.7 cells: RAW264.7 cell suspension (1×10⁻⁶) was used to treat the cells. 5Cells (cells / mL) were seeded at 180 μL / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. The cells were then divided into six groups for treatment: a blank control group (180 μL of cell culture medium was added); low-dose, medium-dose, and high-dose treatment groups of the fermentation product from Example 1 (180 μL of different concentrations of aloe vera fermentation product solution prepared in Example 1, with cell culture medium as the solvent), resulting in final concentrations of aloe vera fermentation product in the cell culture medium of 10 μg / mL, 25 μg / mL, and 50 μg / mL, respectively); the fermentation product treatment group of Comparative Example 2 (180 μL of aloe vera fermentation product solution prepared in Comparative Example 2 was added, resulting in a final concentration of 50 μg / mL); and the fermentation product treatment group of Comparative Example 3 (180 μL of aloe vera fermentation product solution prepared in Comparative Example 3 was added, resulting in a final concentration of 50 μg / mL). Each group had six replicates.

[0065] (2) Detection of cytokine levels: After culturing for 24 hours after drug administration, the culture medium was aspirated and the levels of TNF-α, IL-6 and IL-1β cytokines were measured according to the operation instructions of the ELISA kit.

[0066] (3) Cell proliferation rate determination: After culturing for 24 h after drug administration, 20 μL of 5 mg / mL MTT solution (thiazolyl blue solution) was added to each well, and incubation continued for 4 h; the culture medium was discarded, and 100 μL of dimethyl sulfoxide was added to each well. The absorbance of each well was measured at 490 nm using an ELISA reader, and the cell proliferation rate was calculated. Cell proliferation rate (%) = (OD of experimental wells) / ... 490 - Blank control well OD 490 ) / Blank control well OD 490 .

[0067] The results are shown in Table 3.

[0068] Table 3. Immunomodulatory activity of aloe vera ferments

[0069] Compared with the blank control group* (p<0.05), ** (p<0.01).

[0070] Table 3 shows that treatment with aloe vera ferment significantly promoted the proliferation of RAW264.7 cells. In Example 1, after treatment at a concentration of 50 μg / mL for 24 h, the cell proliferation rate reached 28.4%, which was significantly different from the blank control group (p<0.01). Regarding cytokine secretion, Example 1 effectively promoted the secretion of TNF-α, IL-6, and IL-1β by RAW264.7 cells, exhibiting a dose-response relationship. Particularly, a concentration of 10 μg / mL significantly induced the release of TNF-α. While Comparative Examples 2 and 3 showed similar trends, at the same concentration (50 μg / mL), their effects on promoting cell proliferation and the release of TNF-α, IL-6, and IL-1β were weaker than those of Example 1.

[0071] Phagocytes, primarily macrophages, form the body's primary immune defense. Once activated, macrophages exhibit significantly increased proliferation and secrete large amounts of immune-related cytokines, such as TNF-α, IL-6, and IL-1β, further promoting T cell activation. Therefore, the proliferation rate and cytokine release capacity of RAW264.7 cells are important indicators of their immune activity. These results demonstrate that the aloe vera ferment provided by this invention can enhance cellular immune activity, suggesting its significant potential in anti-aging and adjuvant therapy for certain immune diseases.

[0072] Experimental Example 4: Protective Effect Against Liver Injury Follow these steps: (1) Construction and drug treatment of liver injury model: HepG2 cells were treated with 8×10 4 Cells were seeded per well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 12 h. Cells were divided into a blank control group and a liver injury group. The liver injury group included a model group, the fermentation product administration group of Example 1 (low-dose group: final concentration 5 μg / mL, high-dose group: final concentration 10 μg / mL), the fermentation product administration group of Comparative Example 1 (final concentration 10 μg / mL), and the extract administration group of Comparative Example 4 (final concentration 10 μg / mL); each group had six replicates. The blank control group was pretreated with 150 μL of cell culture medium, and the liver injury group was pretreated with 150 μL of cell culture medium containing 500 mM ethanol for 6 h. Subsequently, 150 μL of the corresponding aloe vera fermentation product / extract solution (solvent being cell culture medium) was added to each of the four administration groups, and the cells were cultured for another 24 h. After culture, add 20 μL of 5 mg / mL MTT solution to each well and incubate for another 4 h. Discard the culture medium, add 150 μL of dimethyl sulfoxide to each well, shake for 10 min, and measure the absorbance of each well at 490 nm using a microplate reader. Calculate the cell viability: Cell viability (%) = (OD of experimental wells) / (OD of experimental wells). 490 / Blank control hole OD490 ) × 100%.

[0073] (2) Determination of the levels of transaminases ALT (alanine aminotransferase), AST (aspartate aminotransferase), and oxidative damage product MDA (malondialdehyde): HepG2 cells were subjected to 8×10 5 Cells were seeded per well in a 6-well plate. The cell culture, grouping, and treatment methods were the same as in step (1) above. After culture, the culture medium was discarded, and the cells were lysed using RIPA lysis buffer. The cell lysates were collected, and the contents of ALT, AST, and MDA in each group of cells were detected according to the instructions of the corresponding kit.

[0074] The results are shown in Table 4.

[0075] Table 4. Hepatoprotective activity of aloe vera ferments / extracts

[0076] Compared with the model group* (p<0.05), ** (p<0.01).

[0077] As shown in Table 4, compared with the blank control group, the cell survival rate in the model group was significantly reduced and the levels of ALT, AST, and MDA were significantly increased after ethanol treatment, indicating that the liver injury model was successfully constructed. Compared with the model group, the Example 1 group showed excellent hepatocellular protection. When the drug concentration was 10 μg / mL, the survival rate of HepG2 cells could be increased to 79.5%, showing a highly significant difference (p<0.01), and it could significantly reverse the increase in ALT, AST, and MDA levels in cells caused by liver injury. At the same drug concentration, the cell survival rate of Comparative Example 1 and Comparative Example 4 was lower than that of Example 1 group, and the levels of ALT, AST, and MDA in cells were higher than those in Example 1.

[0078] In clinical treatment, ALT and AST are important biochemical indicators for evaluating the degree of liver damage, while MDA is a key indicator reflecting the level of oxidative damage in tissues and cells. The above results demonstrate that the aloe vera ferment provided by this invention has superior effects in improving and treating liver damage.

Claims

1. An aloe vera ferment, characterized in that, It is obtained by secondary fermentation of aloe vera by lactic acid bacteria; in the aloe vera ferment, the content of aloin is more than 8%, the content of aloin is more than 11%, the content of phytosphingosine is more than 16%, and the content of dihydrosphingosine is more than 14% by mass percentage.

2. The method for preparing the aloe vera ferment as described in claim 1, characterized in that, Includes the following steps: (1) Add purified water to aloe vera pulp, mash and sterilize to obtain aloe vera pulp; add lactic acid bacteria to aloe vera pulp, mix well to obtain aloe vera pulp lactic acid bacteria suspension. (2) Add basic fermentation substrate to the aloe vera pulp lactic acid bacteria suspension in step (1), and supplement with L-cysteine, manganese sulfate and magnesium sulfate. Ferment at 25~50℃ and 150~400rpm for 24~72h to obtain primary fermentation product. (3) Add enhanced fermentation feed to the primary fermentation product of step (2) and add sodium chloride. Continue secondary fermentation for 12-48 hours at 25-50℃ and 150-400rpm. Adjust the pH to 5.5-7 to obtain the secondary fermentation product. (4) Add anhydrous ethanol to the secondary fermentation product of step (3) and extract by ultrasonic extraction for 10-30 min. Centrifuge at 3000-5000 rpm, collect the supernatant, concentrate, and dynamically adsorb onto macroporous adsorption resin. Elute with 40%-70% ethanol solution, collect the eluent, and dry to obtain aloe vera fermentation product.

3. The preparation method according to claim 2, characterized in that, The lactic acid bacteria mentioned in step (1) are one of Bifidobacterium animalis, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus helveticus, and Streptococcus thermophilus.

4. The preparation method according to claim 2, characterized in that, The basic fermentation substrate mentioned in step (2) includes a carbon source, a nitrogen source, and a buffer; the carbon source is one of inulin, soluble starch, and maltodextrin, accounting for 2%~6% (w / w); the nitrogen source is one of corn peptone, tryptone, yeast extract, whey protein, and soy protein, accounting for 0.5%~4% (w / w); the buffer is a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, accounting for 0.1%~0.6% (w / w), and the molar ratio between dipotassium hydrogen phosphate and potassium dihydrogen phosphate is (0.1~1.2):

1.

5. The preparation method according to claim 2, characterized in that, In step (2), the addition ratio of L-cysteine ​​is 0.02%~0.1% (w / w), the addition ratio of manganese sulfate is 0.005%~0.03% (w / w), and the addition ratio of magnesium sulfate is 0.02%~0.08% (w / w).

6. The preparation method according to claim 2, characterized in that, The enhanced fermentation feed mentioned in step (3) includes a carbon source and a nitrogen source; the carbon source is one of glucose, sucrose, and lactose, accounting for 1% to 4% (w / w); the nitrogen source is one of corn peptone, tryptone, and yeast extract, accounting for 0.5% to 2% (w / w). Preferably, the proportion of sodium chloride added in step (3) is 0.05%~0.5% (w / w).

7. The preparation method according to claim 2, characterized in that, In step (4), the amount of anhydrous ethanol added is 2 to 6 times the volume of the secondary fermentation product, the ultrasonic extraction temperature is 40 to 80°C, the macroporous adsorption resin is one of AB-8, ADS-17, or HPD826, and the dynamic adsorption flow rate is 1 to 4 BV / h.

8. The application of the aloe vera ferment as described in claim 1, characterized in that, Used in the preparation of health foods or medicines, wherein the health foods or medicines have one or more of the following functions: (1) It lubricates the intestines and promotes bowel movements; (2) Antioxidant; (3) Enhance immunity; (4) Adjunctive protective effect against chemically induced liver injury.

9. The application of the aloe vera ferment as described in claim 1, characterized in that, It is used in the preparation of drugs for treating constipation and liver damage.

10. The application of the aloe vera ferment as described in claim 1, characterized in that, Used in the preparation of daily chemical products; Preferably, the daily chemical product is a cosmetic.

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