Lilium lancifolium fermentation product for improving blood lipid and preparation method and application thereof

Abstract

The application relates to the technical field of microbial fermentation, and discloses a lily fermentation product for improving blood lipid, a preparation method and application thereof. Lily raw materials are taken as fermentation objects, and suitable lactobacillus plantarum fermentation strains are selected to ferment the lily raw materials to obtain a lily fermentation product. Compared with other fermentation products of lactobacillus plantarum, the lily fermentation product of the lactobacillus plantarum of CICC 25125 has more excellent pancreatic lipase inhibition capacity and has the effect of promoting and improving blood lipid levels, thereby providing a better basis for the application of the lily fermentation product in the food field.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation technology, and in particular to a lily ferment that improves blood lipids, its preparation method, and its application. Background Technology

[0002] Hyperlipidemia (HLP) is a chronic metabolic disorder characterized by elevated circulating levels of lipids or lipoproteins. According to global disease burden studies, hyperlipidemia has become the sixth leading risk factor for human health. Lowering blood lipids can reduce fat accumulation in the liver and protect liver health. Hyperlipidemia is also associated with various diseases such as insulin resistance, type 2 diabetes, gallstones, and kidney disease. Lowering blood lipid levels helps improve the body's overall metabolic state and reduces the risk of these diseases. Currently, commonly used drugs for treating hyperlipidemia include statins, cholesterol absorption inhibitors, and bile acid sequestrants; however, long-term use of these drugs can cause serious side effects or adverse reactions. Therefore, research on the role of natural products in lowering blood lipids is of great significance.

[0003] Lily, first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), is a perennial bulbous herb. Both its flowers and scaly stems can be used medicinally. Traditional Chinese medicine considers lily to be slightly cold and neutral in nature, possessing lung-moistening, heat-clearing, and calming effects. It is used for symptoms such as yin deficiency with chronic cough, mouth ulcers, chronic cough, and insomnia due to deficiency. Lily and asparagus soup is a widely accepted dietary therapy for lowering blood lipids. The vitamin C and flavonoids in lily contain antioxidants that can eliminate free radicals in the body, reduce damage to vascular endothelial cells, decrease lipid deposition on blood vessel walls, and prevent atherosclerosis. It also has a certain preventive and therapeutic effect on vascular lesions caused by dyslipidemia. The dietary fiber in lily can slow down the digestion and absorption of carbohydrates, preventing rapid rises in blood sugar and helping to improve insulin resistance. Insulin resistance is closely related to dyslipidemia; improving insulin resistance can indirectly regulate lipid metabolism, bringing blood lipid levels closer to normal. Furthermore, lily has a certain sedative and calming effect, helping to relieve stress and anxiety and improve sleep quality. Negative emotions and lack of sleep may lead to endocrine disorders, which in turn affect lipid metabolism. These effects of lily help maintain endocrine stability and indirectly have a beneficial effect on blood lipids.

[0004] Fresh medicinal herbs are difficult to store and prone to mold over time, making it difficult to meet the demand for large-scale applications and thus limiting their clinical use. Therefore, it is necessary to find more suitable processing methods. Microbial fermentation is the most common method of food preservation. Its core principle lies in inhibiting harmful microorganisms in food through the growth, reproduction, and metabolic activities of microorganisms, thereby extending shelf life. During fermentation, these microorganisms produce preservative metabolites such as lactic acid and alcohol, which effectively inhibit the growth of other harmful microorganisms. Besides food preservation, fermentation also leverages the powerful decomposition and transformation capabilities of microorganisms, producing abundant secondary metabolites. Processing lilies through microbial growth, metabolism, and life activities can significantly alter their medicinal properties, improve efficacy, reduce toxicity and side effects, and expand indications compared to general physical or chemical processing methods. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a lily ferment product that improves blood lipids and a method for preparing the same, so that the lily ferment product can significantly improve the inhibition rate of pancreatic lipase, thereby achieving the effect of reducing blood lipid levels. Another objective of this application is solely to provide the application of the aforementioned lily ferment in the preparation of products that improve blood lipid levels.

[0006] In order to solve the above-mentioned technical problems / achieve the above-mentioned objectives, or at least partially solve the above-mentioned technical problems / achieve the above-mentioned objectives, as a first aspect of this application, a lily fermentation product is provided, which is the product of lily raw material fermented by Lactobacillus plantarum; wherein the Lactobacillus plantarum is Lactobacillus plantarum numbered CICC25125.

[0007] Optionally, the lily raw material is one or more of the following: lily bulb, lily petals, lily stamens, and lily stems and leaves.

[0008] As a second aspect of this application, the use of Lactobacillus plantarum, designated CICC 25125, in the preparation of lily ferments is provided.

[0009] As a third aspect of this application, a method for preparing the lily ferment described in this application is provided, comprising: S1. Pulp the lily raw material to obtain lily pulp; S2. Inoculate the lily slurry with Lactobacillus plantarum numbered CICC25125 for fermentation, and obtain the lily fermented product after fermentation.

[0010] Optionally, step S1 includes: Fresh lily bulbs are mixed with water and then pulped. The solid-liquid ratio of the lily bulbs to water is 1:(1-10).

[0011] Alternatively, the fermentation temperature is 37℃±3℃ and the time is 24-72h.

[0012] As a fourth aspect of this application, the use of the lily ferment described herein in the preparation of products that improve blood lipid levels is provided.

[0013] Optionally, the product is food.

[0014] As a fifth aspect of this application, a product is provided that has the effect of improving blood lipid levels, including the lily ferment described in this application.

[0015] Optionally, the product is a food product, including additives that are permitted to be added to food.

[0016] This application uses lily as the fermentation material and selects a suitable Lactobacillus plantarum fermentation strain to ferment it to obtain lily fermented product. Compared with other Lactobacillus plantarum fermentation products, the lily fermented product using Lactobacillus plantarum CICC 25125 exhibits superior pancreatic lipase inhibition ability and has the effect of promoting and improving blood lipid levels, providing a better foundation for its application in the food field. Detailed Implementation

[0017] This application discloses a lily ferment that improves blood lipids, its preparation method, and its application. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this application. The products, processes, and applications described in this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit, and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are only some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.

[0018] It should be noted that, in this document, relational terms such as "first" and "second," "step 1" and "step 2," and "(1)" and "(2)" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. Moreover, the embodiments and features described in this application can be combined with each other without conflict.

[0019] While lily has some preventive and therapeutic effects on vascular lesions caused by dyslipidemia, extracts obtained from conventional organic solvents have limitations in improving blood lipids. This application utilizes a microbial fermentation process to highly activate active substances derived from lily raw materials through microbial metabolism, resulting in highly potent inhibitory activity against pancreatic lipase. Therefore, in the first aspect of this application, a lily fermentation product is provided, which is the product of lily raw materials fermented with *Lactobacillus plantarum*; the *Lactobacillus plantarum* is designated CICC25125.

[0020] Among them, Lactobacillus plantarum with the code CICC 25125 ( Lactiplantibacillus plantarum Purchased from the China Industrial Microbial Culture Collection Center, this strain originates from traditional pickled winter melon in eastern Zhejiang. The bacteria are rod-shaped, Gram-positive, and do not produce spores. It has a strong ability to metabolize carbohydrates and produce acid, synthesizing glucans and heteropolysaccharides. It can ferment sugars to produce lactic acid or other acids. Its specific applications are in acid production and fermentation research of fruits and vegetables. Genbank accession number: MZ674414. Optimal culture temperature: 37℃, anaerobic.

[0021] In some embodiments of this application, the lily raw material is one or more of lily bulbs, lily petals, lily stamens, and lily stems and leaves. In other embodiments of this application, the lily raw material is lily bulbs, which, as the main part of lily used in both medicine and food, are rich in active substances such as starch, protein, polysaccharides, saponins, and total phenols. Compared with lily petals, stamens, and stems and leaves, lily bulbs have a higher content of active substances, more comprehensive nutritional components, and are easier to preserve and standardize, making them an ideal substrate for developing bioactive products.

[0022] In the second aspect of this application, multiple strains of *Lactobacillus plantarum* purchased from the China Industrial Microbial Culture Collection Center were used to ferment lily bulbs. Although the total polyphenol content in the obtained lily ferment was not the highest, it showed an inhibitory activity of over 90% in the pancreatic lipase inhibition rate experiment. Therefore, this application provides the application of *Lactobacillus plantarum* with the number CICC 25125 in the preparation of lily ferment.

[0023] In a third aspect of this application, a method for preparing the lily ferment is provided, comprising: S1. Pulp the lily raw material to obtain lily pulp; S2. Inoculate the lily slurry with Lactobacillus plantarum numbered CICC25125 for fermentation, and obtain the lily fermented product after fermentation.

[0024] In some embodiments of this application, step S1 includes: Take fresh lily bulbs and mix them with water to make a pulp. The solid-liquid ratio of the lily bulbs and water is 1:(1-10), for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any value between the two.

[0025] In some embodiments of this application, the inoculation of CICC 25125 with Lactobacillus plantarum includes a strain activation process. The activation medium includes MRS medium, and the strain is cultured at 35°C for 24 hours, and then passaged three times to fully activate the strain to a viable count of 1.0 × 10⁻⁶. 8 CFU / mL or higher. In some other embodiments of this application, the MRS culture medium comprises: Peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium hydrogen citrate 2 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L.

[0026] In some embodiments of this application, the inoculum amount of *Lactobacillus plantarum* in CICC 25125 is 1-10% (v / v), for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any value between any two.

[0027] In some embodiments of this application, the fermentation temperature is 37℃±3℃, such as 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃ or any value between the two, and the fermentation time is 24-72h, such as 24h, 36h, 48h, 60h, 70h or any value between the two.

[0028] In some embodiments of this application, the lily ferment obtained after fermentation is freeze-dried into freeze-dried powder.

[0029] In the fourth aspect of this application, the polyphenol content and pancreatic lipase inhibition rate of organic solvent extracts from different lily bulbs and fermentation products from various *Lactobacillus plantarum* fermentations were detected. The results showed that the lily fermentation products of this application had a high polyphenol content and a pancreatic lipase inhibition rate of approximately 93%, while also improving blood lipid indicators such as triglycerides and total cholesterol in the serum of a hyperlipidemic mouse model. Based on this, this application provides the use of the lily fermentation products described herein in the preparation of products that improve blood lipid levels.

[0030] In some embodiments of this application, the product is food, including health food.

[0031] In a fifth aspect of this application, a product is provided that improves blood lipid levels, including the lily ferment described in this application. In addition, the product may, as needed, contain other functional active substances, including but not limited to those for anti-oxidation, improving blood pressure, improving blood sugar, relieving fatigue, improving memory, protecting the liver, nourishing the stomach, promoting digestion, and improving sleep. The amount of the lily ferment added is an effective amount, which can be determined based on relevant experiments.

[0032] In some embodiments of this application, the product is food, or it may be health food. The food includes permissible additives, such as (1) additives used for molding, coating, thickening, lubrication, etc., to improve the physical properties of the product, including but not limited to gum arabic, maltodextrin, microcrystalline cellulose, magnesium stearate, silicon dioxide, etc.; (2) additives used to improve taste, extend shelf life, and supplement specific nutrients, including but not limited to sweeteners, preservatives, vitamins, minerals, etc.; (3) additives used as a product matrix or carrier to provide basic nutrition, including but not limited to conventional edible food ingredients, such as milk powder, rice flour, fruit and vegetable powder, etc.; (4) additives with specific health functions, including but not limited to Ganoderma lucidum spore powder, Bifidobacterium, Lactobacillus, etc.

[0033] In the comparative experiments provided in this application, unless otherwise specified, all experimental conditions and materials remain consistent to ensure comparability. Furthermore, all materials used in this application are commercially available.

[0034] The following provides a further description of a lily ferment that improves blood lipids, its preparation method, and its application.

[0035] Example 1: 1. Activation of microbial strains All fermentation strains were purchased from the China Industrial Microbial Culture Collection Center. Lactobacillus plantarum CICC 24936, CICC 25125, and CICC20326 were inoculated into MRS medium and cultured at 35℃ for 24 h. The culture was then passaged three times to fully activate the strains to a viable count of 1.0 × 10⁻⁶. 8 CFU / mL or higher.

[0036] The MRS medium was prepared as follows: the medium formula consisted of 10.0 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2 g / L triammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 g / L Tween-80. 1 L of pure water was added to the above MRS medium, and the mixture was autoclaved at 121°C for 15 minutes.

[0037] 2. Preparation of lily fermentation product Fresh lily bulbs were sourced from Lianyuan Xiangbaihe Special Agricultural Technology Development Co., Ltd. 100g of fresh lily bulbs were washed, then mixed with water at a solid-liquid ratio of 1:5 (w / v) to form a pulp. After thorough mixing, the pulp was sterilized at 121℃ for 15 min. After sterilization, the pulp was cooled to room temperature and inoculated with activated bacterial solution at a 4% (v / v) inoculation rate (each bacterial strain had the same number of viable cells) in a sterile operating table. The mixture was then sealed and placed in a 37℃ constant temperature incubator for 48 h. After fermentation, lily fermentation product was obtained, which was then freeze-dried into freeze-dried powder.

[0038] 3. Preparation of unfermented lily bulb samples 100g of fresh lily bulbs were washed and mixed with water at a solid-liquid ratio of 1:5 (w / v). After mixing, the mixture was sterilized at 121℃ for 15 minutes. After sterilization, the mixture was cooled to room temperature and then inoculated with sterilized activated bacterial solution at a rate of 4% (v / v) in a sterile operating table. After mixing, the mixture was sealed and placed in a constant temperature incubator at 37℃ for 48 hours. After fermentation, unfermented lily samples were obtained, which were then freeze-dried into freeze-dried powder for later use.

[0039] 4. Extraction of polyphenols from lily bulbs Take 2g of lily bulb powder and extract it for 3 hours using 10mL of methanol and ultrasound (100%, 40℃). Then, obtain the methanol extract powder by vacuum distillation and vacuum drying. Next, further extract the obtained powder in ethyl acetate for 3 hours. After extraction, remove the solvent by vacuum distillation. Finally, vacuum dry the residue to obtain the powder, which can be used for subsequent experiments.

[0040] Example 2: 1. Polyphenol content determination The polyphenol content was determined using the Folin-Ciocalteu method (gallic acid was used as the standard, and the concentrations of gallic acid were prepared as 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL and 600 μg / mL).

[0041] Dissolve 1g of polyphenol powder in 10mL of distilled water. Take 0.1mL of the polyphenol solution, add Folin-Ciocalteu reagent, mix for 5 minutes, and then add 2mL of saturated Na₂CO₃ solution. The mixed solution should be placed in the dark for 2 hours. Then, measure the absorbance at 765nm. This process should be repeated three times to ensure accuracy. The polyphenol content is calculated using a gallic acid standard curve, and the final result is expressed in mg / g.

[0042] 2. Determination of the lipid-lowering ability of lily fermentation broth Mix 0.5 mL of sample, 0.5 mL of pancreatic lipase (5 mg / mL), and 0.5 mL of PBS (25 mM), and incubate at 37°C for 10 min. Then add 0.5 mL of p-NPB (11.2 mol / L) solution, gently vortex to mix, and incubate at 37°C for 20 min. Measure the absorbance at 405 nm. Calculate the clearance rate using the following formula: Inhibition rate (%) = [1 - (A4 - A3) / (A2 - A1)] × 100% In the formula: A: absorbance of the experimental group; B: absorbance of the experimental background group (pure water replaces enzyme solution); C: absorbance of the blank group (pure water replaces sample); D: absorbance of the blank background group (pure water replaces sample and enzyme solution).

[0043] 3. Results Table 1. Polyphenol content of lily bulbs in each group

[0044] Note: Different letters indicate significant differences (p < 0.05). As shown in Table 1, the polyphenol content of each group was significantly increased after fermentation with Lactobacillus plantarum compared to the unfermented group. The polyphenol content of the CICC 25125 fermentation group was the highest, while the polyphenol content of the CICC 24936 and CICC 20326 fermentation groups was comparable.

[0045] Table 2. Effects of fresh ingredients on the inhibition rate of pancreatic lipase

[0046] Note: Different letters indicate significant differences (p < 0.05). As shown in Table 2, the CICC 25125 fermentation group (the lily fermentation product of this application) exhibited a pancreatic lipase inhibition rate as high as 93%, which is approximately 40% higher than the unfermented group and about 20% higher than the other two Lactobacillus plantarum fermentation groups. This is consistent with the results regarding polyphenol content. Although all three strains are Lactobacillus plantarum, CICC 25125 is more suitable for fermenting lilies.

[0047] Example 3: Seventy-two four-week-old male ICR mice with an average weight of 18.23 ± 0.29 g were randomly divided into six groups according to body weight: a normal diet group (NC), a high-fat diet group (HFD), a group with unfermented lily lyophilized powder, a group with fermented lyophilized Lactobacillus plantarum CICC 24936 powder, a group with fermented lyophilized Lactobacillus plantarum CICC 25125 powder, and a group with fermented lyophilized Lactobacillus plantarum CICC 20326 powder, with 12 mice in each group. The mice had free access to food and water during the experiment. The NC group was fed a normal diet (containing 5% lipids), with normal drinking water, and administered 0.2 mL of physiological saline by gavage daily. The HFD group was fed a high-fat diet (containing 60% lipids), with normal drinking water, and administered 0.2 mL of physiological saline by gavage daily. The unfermented lily lyophilized powder group was fed a high-fat diet supplemented with 6.59 g / kg of lyophilized powder by gavage. The three Lactobacillus plantarum fermented lyophilized powder groups were fed a high-fat diet supplemented with 6.59 g / kg of lyophilized powder by gavage. The trials lasted for 12 weeks. Mouse weight changes were recorded weekly.

[0048] After the final gavage, the patient fasted overnight. Blood was collected via the eyeball, and 2 mL of blood was collected in a sterile centrifuge tube. The blood was then centrifuged at 4000 rpm for 10 min in a refrigerated centrifuge, and the supernatant was collected and stored at -20°C.

[0049] The assay kits for TG (triglycerides), TC (total cholesterol), LDL-C (low-density lipoprotein cholesterol), and HDL-C (high-density lipoprotein cholesterol) in mouse serum were purchased from Nanjing Jiancheng Bioengineering Institute and the measurements were performed according to the kit instructions. Protein content was determined using the BCA protein content assay kit, and the results are shown in Table 3.

[0050] Table 3. Effects of each group on serum lipid levels in mice (x±s, n=12)

[0051] As shown in Table 3 above, the lily fermentation product (CICC 25125 fermentation group) of this application can reduce total cholesterol, triglycerides, and low-density lipoprotein cholesterol in the serum of mice in the high-fat diet group, and increase high-density lipoprotein cholesterol. Compared with other groups, it has the best improvement effect, which further verifies that the CICC 25125 strain is more suitable for lily fermentation than other implanted lactobacilli and has an extremely significant effect on improving blood lipids.

[0052] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A lily ferment product for improving blood lipids, characterized in that, It is a product of lily raw material fermented with Lactobacillus plantarum; the Lactobacillus plantarum is Lactobacillus plantarum with the number CICC25125.

2. The lily ferment product according to claim 1, characterized in that, The lily raw materials are one or more of the following: lily bulbs, lily petals, lily stamens, and lily stems and leaves.

3. Application of Lactobacillus plantarum, CICC 25125, in the preparation of lily ferment.

4. The method for preparing the lily ferment as described in claim 1, characterized in that, include: S1. Pulp the lily raw material to obtain lily pulp; S2. Inoculate the lily slurry with Lactobacillus plantarum numbered CICC25125 for fermentation, and obtain the lily fermented product after fermentation.

5. The preparation method according to claim 4, characterized in that, Step S1 includes: Fresh lily bulbs are mixed with water and then pulped. The solid-liquid ratio of the lily bulbs to water is 1:(1-10).

6. The preparation method according to claim 4 or 5, characterized in that, The fermentation temperature was 37℃±3℃, and the time was 24-72h.

7. The use of the lily ferment as described in claim 1 or 2 in the preparation of products that improve blood lipid levels.

8. The application according to claim 7, characterized in that, The product in question is a food product.

9. A product that improves blood lipid levels, characterized in that, Includes the lily ferment as described in claim 1 or 2.

10. The product according to claim 9, characterized in that, The product is a food product, including permitted additives in food.

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