Application of plant lactobacillus powder in preparation of food, health care product or medicine for reducing blood fat, preventing colitis and resisting oxidation

Abstract

The invention relates to application of plant lactobacillus powder in preparation of food, health care products or medicines for reducing blood fat, preventing colitis and resisting oxidation, and belongs to the technical field of microorganisms and biological medicines. The lactobacillus plantarum R-21 with the preservation number of CCTCC M2018009 is subjected to high-density fermentation and centrifugation, bacterial sludge and auxiliary materials mannan oligosaccharide and maltodextrin are sequentially mixed to prepare bacterial powder, the triglyceride and cholesterol levels of mice can be remarkably reduced, increase of the body fat rate of the mice is inhibited, and intestinal inflammation induced by high-fat diet is relieved. The important application value is realized in the prevention and treatment of hyperlipidemia.

Description

Technical Field

[0001] This invention relates to the fields of microbial technology and biomedicine, and in particular to the application of a plant lactobacillus powder in the preparation of lipid-lowering, colitis-preventing, and antioxidant foods, health products, or drugs. Background Technology

[0002] In China, the number of deaths from cardiovascular disease each year exceeds the total number of deaths from all cancers combined. Dyslipidemia, represented by hypercholesterolemia, is the most dangerous risk factor, including high triglycerides, high cholesterol, or a combination of both (i.e., mixed hyperlipidemia). According to the "China Cardiovascular Health and Disease Report 2024," the rate of dyslipidemia in the adult population in my country is as high as 40.4%, affecting over 400 million people. This disease often co-occurs with chronic diseases such as hypertension and diabetes, seriously threatening the health and lives of the Chinese people. Medical expenses caused by hyperlipidemia continue to rise annually, placing a heavy economic burden on society and families. Long-term use of fibrates and statins can treat hyperlipidemia, but they can easily cause gastrointestinal discomfort (such as nausea, diarrhea, and bloating), rhabdomyolysis, and abnormal liver and kidney function. Therefore, there is an urgent need to find new and safer methods for preventing and treating hyperlipidemia.

[0003] In recent years, numerous studies have shown a close relationship between gut microbiota and hyperlipidemia. Appropriate supplementation with lactic acid bacteria (LAB) or fecal microbiota transplantation (FMT) can alleviate lipid metabolism disorders and lower blood lipid levels. However, FMT faces considerable obstacles in its promotion, primarily due to unstable efficacy, psychological resistance from test subjects, and the possibility of donors carrying potential pathogens (such as immunodeficiency virus, Treponema pallidum, and other opportunistic pathogens). LAB, on the other hand, is the most common and predominant type of beneficial bacteria in the gut microbiota. It is highly safe and possesses functions such as enhancing non-specific immunity, regulating gut health, lowering serum cholesterol, inhibiting the growth of harmful bacteria, anti-cancer, anti-tumor, anti-diabetic, and uric acid-lowering effects. It is currently the probiotic species most closely related to human health. Currently, domestic research on the functions of lactic acid bacteria mainly focuses on experimental animals, with limited clinical research on its lipid-lowering effects. This, to some extent, limits the promotion and application of locally sourced lactic acid bacteria. Therefore, accelerating research, formulation, and application of lactic acid bacteria functions is a crucial measure to change the current predicament of my country's lactic acid bacteria industry.

[0004] Our team previously isolated a strain of *Lactobacillus plantarum* R-21 from the intestinal mucosa of pigs. This strain exhibits strong acid-producing ability and tolerance, possesses a certain capacity to adsorb cholesterol, and demonstrates a strong ability to produce extracellular polysaccharides. Animal experiments have shown that R-21 also has a certain function in lowering serum triglycerides and cholesterol, laying a foundation for its application in treating hyperlipidemia. However, further formulation and experimental research are still needed before it can be developed into a product. Summary of the Invention

[0005] To address the above technical problems, this invention provides the application of *Lactobacillus plantarum* powder in the preparation of lipid-lowering, colitis-preventing, and antioxidant foods, health products, or drugs.

[0006] The purpose of this invention is to provide an application of *Lactobacillus plantarum* powder in the preparation of drugs or health products for lowering blood lipids, preventing colitis and anti-oxidation, wherein the *Lactobacillus plantarum* powder includes *Lactobacillus plantarum* R-21 with preservation number CCTCC M2018009.

[0007] Furthermore, the *Lactobacillus plantarum* powder described in this invention can be used to lower serum cholesterol and triglycerides, can be used for in vivo antioxidation, and can be used to prevent colitis.

[0008] In some embodiments of the present invention, the *Lactobacillus plantarum* powder contains *Lactobacillus plantarum* R-21 ≥ 40 billion CFU / 2g, calculated as viable bacteria.

[0009] In some embodiments of the present invention, the *Lactobacillus plantarum* powder further includes 1 g / 2 g of manno-oligosaccharide; 1 g / 2 g of maltodextrin (meaning that 2 g of powder contains 1 g of manno-oligosaccharide and 1 g of maltodextrin); and a moisture content of less than 4%.

[0010] In some embodiments of the present invention, the *Lactobacillus plantarum* powder is prepared by the following method: Lactobacillus plantarum R-21 was inoculated into MRS solid plates for solid activation culture. Single colonies were picked and transferred to MRS slant for overnight culture. The slant seed was then inoculated into MRS test tube liquid culture medium for the first static culture, followed by a second static culture in MRS test tube liquid culture medium to obtain liquid seed. In the MRS test tube liquid culture medium, glucose was replaced with an equal mass of sucrose. The obtained liquid seed was inoculated into the fermentation medium and cultured with shaking to obtain the fermentation broth; The obtained fermentation broth was centrifuged, the precipitate was collected and washed to obtain washed bacterial sludge; Under clean conditions, the washed bacterial sludge was mixed with the first excipient, mannan oligosaccharide, and then mixed with the second excipient, maltodextrin, to obtain the original bacterial powder. Under clean conditions, the obtained original bacterial powder is mixed with 5 times its weight of a third excipient to obtain the plant lactobacillus powder.

[0011] In some embodiments of the present invention, the solid activation culture is performed three times; The OD600 of the bacterial cells in the liquid seed culture was 0.8–1.0. The first static incubation was carried out at 37℃ for 12-16 hours. The second static incubation was carried out at 37℃ for 4-6 hours.

[0012] In some embodiments of the present invention, the inoculation density of liquid seeds is 2%; The fermentation medium composition includes: 30 g soybean peptone, 24 g corn steep liquor, 60.0 g white sugar, 36 g calcium carbonate, 2.0 g dipotassium hydrogen phosphate, 1.0 ml Tween 80, 2.0 g diammonium hydrogen citrate, 0.25 g manganese sulfate, 5.0 g sodium acetate, 0.58 g magnesium sulfate heptahydrate, and 1000 ml distilled water. The shaking culture was carried out at 37℃ and 120 rpm for 36 hours. Wash three times with physiological saline. The fermentation broth was centrifuged at 5000 rpm for 10 min.

[0013] In some embodiments of the present invention, the materials are added and finely mixed simultaneously during the mixing process; The amount of the first excipient, manno-oligosaccharide, is 1 / 5 of the mass of the first excipient, manno-oligosaccharide, to the volume of the fermentation liquid, m / v. The mass ratio of the first excipient, mannan oligosaccharide, to the second excipient, maltodextrin, is 1:1. The second excipient, maltodextrin, is added gradually in batches to improve the flowability and stability of the bacterial powder. Adding it in small batches and mixing it thoroughly can better mix the materials; otherwise, the excipient is prone to clumping.

[0014] In some embodiments of the present invention, the viable count of the original bacterial powder is 1.42 × 10⁻⁶. 11 CFU / g.

[0015] In some embodiments of the present invention, the third excipient comprises manno-oligosaccharide and maltodextrin; the mass ratio of manno-oligosaccharide and maltodextrin is 1:1. The present invention uses the third excipient to reduce the moisture content of the original bacterial powder.

[0016] In some embodiments of the present invention, the mass ratio of the original bacterial powder to the third excipient is 1:5; The viable count of the *Lactobacillus plantarum* powder is greater than or equal to 2.00 × 10⁻⁶. 10 CFU / g.

[0017] In this invention, the original bacterial powder is a pale yellow powder, and the working bacterial powder is a white powder. Both are stored at 2-8°C, and the bacterial viability is tested monthly. After one year, the viability remains at 8.20 × 10⁻⁶. 9It exhibits a CFU / g or higher concentration and good stability. When dissolved in approximately 30-50 mL of warm water (40-50℃), it has a slightly sweet taste with no off-flavors. Traditional lactic acid bacteria powder preparation mainly involves adding skim milk powder, dextrin, and other preservatives to a bacterial suspension, followed by freeze-drying, pulverization, and mixing with excipients. Freeze-drying typically leads to a decrease in bacterial activity and requires sophisticated freezing equipment, resulting in long freezing times and production cycles. The advantage of this technology lies in directly mixing the centrifuged bacterial sludge with a certain mass of manno-oligosaccharide, then mixing it with an equal mass of maltodextrin to prepare a high-activity original bacterial powder. This is then mixed with excipients to prepare a bacterial powder with a certain activity level, saving on freeze-drying equipment costs, shortening production time, and simultaneously improving the retention rate of bacterial activity.

[0018] The technical solution of the present invention has the following advantages compared with the prior art: The R-21 bacterial powder obtained in this invention can significantly inhibit the increase in fat mass and body fat percentage induced by high-fat diet in male mice (p<0.01). It can inhibit the increase in serum and liver triglyceride and cholesterol levels in high-fat diet mice by downregulating the transcription of fatty acid synthase and bile acid synthase genes and upregulating the transcription of fatty acid oxidase genes. Furthermore, the R-21 bacterial powder can significantly increase the activity of catalase (CAT) and glutathione peroxidase (GSH-PX) in mouse liver (p<0.05), thus enhancing the antioxidant capacity of mouse liver. The R-21 bacterial powder can also significantly improve colonic inflammation induced by a high-fat diet. Attached Figure Description

[0019] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This is the result of the experiment on the change in the activity of *Lactobacillus plantarum* R-21 bacterial powder over time in this application; Figure 2 These are the results of four lipid profile tests in the liver of mice used in this application; Figure 3 These are the results of the detection of inflammatory factors in mouse serum in this application; Figure 4 This is the result of the detection of transcriptional levels of lipid metabolism-related genes in mouse liver in this application; Figure 5 This is the result of liver oil red O staining section in this application; Figure 6 This is the result of hematoxylin staining of liver sections in this application; Figure 7 These are the results of the detection of antioxidant indicators in mouse livers in this application; Figure 8 This is the staining result of mouse colon sections in this application.

[0020] Note: Compared with the HFD group, ns represents no significant difference (p>0.05), ** represents a significant difference (p<0.05), and *** and **** represent extremely significant differences (p<0.01). Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0022] Example 1 This embodiment provides a method for preparing Lactobacillus plantarum powder, as detailed below: 1. Source of the strain The strain is *Lactobacillus plantarum* R-21 (accession number CCTCC M2018009, deposited on January 8, 2018, at Wuhan University, China). Previous experiments have shown that this strain possesses strong acid production, acid resistance, bile salt resistance, antibacterial activity, and in vitro cholesterol-lowering capabilities.

[0023] 2. Activation of microbial strains Cryopreserved *Lactobacillus plantarum* R-21 plates were streaked onto MRS solid plates modified with 0.3% calcium carbonate (formula: 10.0 g peptone, 10 g yeast extract, 1.0 ml Tween 80, 10 g beef extract, 2.0 g diammonium citrate, 5.0 g sodium acetate, 2.0 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 20.0 g sucrose, 0.25 g manganese sulfate, 3.0 g calcium carbonate, 15 g agar, 1000 ml distilled water, pH 6.6). The plates were incubated upside down at 37°C for 48 h. Single colonies with thick, calcified zones and large diameters were picked and streaked again onto solid plates, repeating the activation process three times. From the third activated plate, single colonies with thick, calcified zones and large diameters were picked and transferred to the above-mentioned slant medium for overnight incubation to prepare slant seed culture.

[0024] 3. Seed preparation The seed culture from the slant was inoculated into MRS liquid medium (the above medium is calcium carbonate-free) and incubated overnight at 37°C. 2% (v / v) of the inoculum was then transferred to MRS liquid medium and incubated at 37°C for 5 hours until the bacterial OD reached its maximum. 600 =0.8~1.0, collect seeds.

[0025] 4. Fermentation culture Take 2% of the above liquid seed and inoculate it into fermentation medium (formula: 30 g soybean peptone, 24 g corn steep liquor, 60.0 g white sugar, 36 g calcium carbonate, 2.0 g dipotassium hydrogen phosphate, 1.0 ml Tween 80, 2.0 g diammonium hydrogen citrate, 0.25 g manganese sulfate, 5.0 g sodium acetate, 0.58 g magnesium sulfate heptahydrate, 1000 ml distilled water, natural pH). Incubate at 37℃ for 36 h, collect the bacterial culture, and measure the pH and OD of the fermentation broth. 600 The pH of the fermentation broth was determined by the dilution plating method, and the viable cell count was detected using this method. The results showed that after 36 h of culture in this medium, the pH of *Lactobacillus plantarum* R-21 decreased to 4.2, and the OD value... 600 The value reached 21.5, and the viable bacteria count reached 8.21 × 10⁻⁶. 10 CFU / ml.

[0026] OD 600 =A×D, In the formula: A is the OD of the diluted fermentation broth. 600 Value (≤0.7); D is the dilution factor of the fermentation broth.

[0027] N = n × M ÷ v In the formula: N is the number of viable bacteria per milliliter of fermentation (CFU / mL); n is the number of viable bacteria grown in the diluted plate (CFU); M is the dilution factor of the plated sample; v is the volume of the diluted plated sample (mL).

[0028] 5. Preparation of fungal sludge Collect the above fermentation broth and centrifuge (5000 rpm, 10 min). Collect the precipitate and wash it three times with physiological saline. Finally, remove as much water as possible from the top layer of the bacterial sludge to obtain the bacterial sludge.

[0029] 6. Preparation of original mycelium powder The prepared bacterial sludge was gradually and slowly mixed with 1 / 5 (m / v) of the fermentation liquid volume of the excipient mannan oligosaccharide (the degree of polymerization of oligosaccharide is 2~10, and the excipient was dried at 80℃ for 1 h before use). While adding the mixture, it was finely crushed (the sample has a high water content, but it is evenly dispersed and does not clump). Then, an equal mass of maltodextrin was added in batches, and the mixture was finely crushed and mixed while adding the mixture. This is the original bacterial powder, and the viable cell count was tested. The original bacterial powder was stored at -80℃ for later use.

[0030] N = n × M ÷ v In the formula: N is the number of viable bacteria per gram of original bacterial powder (CFU / g); n is the number of viable bacteria grown in the diluted plating plate (CFU); M is the dilution factor of the plating sample; v is the volume of the diluted plating sample (mL).

[0031] Testing showed that the prepared mycelium powder had good dispersibility, did not clump, and had a moisture content of approximately 6.2%; the mycelium powder activity was approximately 1.42 × 10⁻⁶. 11 The CFU / g recovery rate was approximately 69.2%. The relatively low activity recovery rate may be related to poor bacterial dispersion and the presence of large particles during the mixing of excipients.

[0032] 7. Preparation of mycelium powder Under clean conditions, the prepared original bacterial powder was mixed with 5 times its weight of excipients (50 wt% each of mannan oligosaccharide and maltodextrin) to obtain *Lactobacillus plantarum* R-21 bacterial powder. It was stored at 2–8°C in a cool, dark place. The viable count of the original bacterial powder was determined using the dilution plating method, the coliform count was determined using eosin methylene blue agar, and the mold content was determined using Czapek's agar. The results are shown in Table 1. The viability of the prepared bacterial powder was approximately 2.32 × 10⁻⁶. 10 The CFU / g level was 0, and the levels of Escherichia coli and Salmonella were 0. The amount of mold contamination was also below the maximum limit specified in GB 7101-2022.

[0033] Table 1. Microbiological test results of Lactobacillus plantarum R-21 powder Example 2 This embodiment provides a storage stability test for Lactobacillus plantarum R-21 bacterial powder, as detailed below: The *Lactobacillus plantarum* R-21 bacterial powder prepared in Example 1 was stored in sealed containers at 4°C and at room temperature in a cool environment, with three samples obtained under each condition. 10 g samples were taken monthly, serially diluted with sterile water, plated, and incubated upside down at 37°C for 48 h. Plates with suitable dilution were selected to count the number of single colonies, and the viable cell count at each time point was calculated to evaluate the storage stability of the bacterial powder and the suitability of storage conditions. Results are as follows: Figure 1 As shown, the *Lactobacillus plantarum* R-21 bacterial powder prepared by the above process exhibits stable activity, maintaining high activity after one year at both 4℃ and room temperature. Specifically, the activity loss was minimal after one year at 4℃, with the logarithm of the viable count per gram decreasing from 10.37 to 9.95, indicating that the powder can be stored stably for a long period under these conditions. At room temperature, the logarithm of the viable count per gram decreased from 10.37 to 8.95 after one year, with a faster decline in activity from June to September. However, the activity level still exceeds the minimum limit (not less than 10) for lactic acid bacteria solid beverages as specified in GB 7101-2022. 6 (CFU / g). The above data indicates that *Lactobacillus plantarum* R-21 powder can be stored stably under both environments.

[0034] Example 3 This embodiment provides an application of *Lactobacillus plantarum* R-21 bacterial powder in lowering blood lipids, as detailed below: Thirty male adult Kunming mice at about 8 weeks of age were divided into 3 groups (the experimental animals were purchased from Spf (Beijing) Biotechnology Co., Ltd., and the experimental animal production license number: SCXK (Jing) 2024-0001), with 10 mice in each group. Two of the groups were set as high-fat diet groups (the feed formula was: 15.0% sucrose, 15% lard, 0.3% cholesterol, 69.7% basal diet, purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.), and the other was set as the basal diet control group (ND). The high-fat groups were continuously fed with high-fat diet. One of the high-fat groups was orally gavaged with a dilution of Lactiplantibacillus plantarum R-21 bacterial powder (diluted with physiological saline to a cell concentration of 1.0×10 10 CFU / ml) (R-21), and the gavage dose was 100 µl / animal / d; the other high-fat group was set as the control group (HFD), and 100 µl of physiological saline was gavaged per animal per day. The mice in the basal diet group were gavaged with 100 µl of physiological saline per animal per day. During this period, the mice had free access to food and water for 7 weeks. The animals were housed in the animal laboratory of the School of Life Sciences, Guizhou Normal University, at a temperature of 20-26 °C, a humidity of 40%-70%, and a light-dark cycle of 12 h / 12 h. During this period, the food intake, body weight, feed utilization rate, and fecal water content of each group of mice were recorded weekly. After the feeding experiment was completed, blood was taken from the inner canthus of the eyes of each group of animals, and the serum was separated by centrifugation. Four blood lipid items (triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C)) and inflammatory factors (IL-1β, IL-6, TNF-α, and lipopolysaccharide (LPS)) were detected using an ELISA kit. The liver of the mice was taken. Part of it was homogenized to detect the levels of TG, TC, LDL-C, and HDL-C in the liver homogenate; another part of the homogenate was used to detect the transcriptional levels of liver lipid metabolism-related genes by reverse transcription and fluorescence quantitative PCR. The detection indexes were acetyl-CoA carboxylase 1 (ACC1), sterol regulatory element-binding protein 1 (SREBP-1), peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1A (CPT1A), cholesterol 7α-hydroxylase (CYP7A1), low-density lipoprotein receptor (LDLR), farnesol X receptor (FXR), fibroblast growth factor 15 (FGF15), G protein-coupled bile acid receptor 1 (TGR5) and other protein genes. The functions of each protein and the RT-PCR primers are shown in Table 2; another part of the liver tissue was stained with Oil Red O and hematoxylin respectively, and sections were prepared to observe the liver fat accumulation and degeneration. The fat in the inguinal, epididymal, perirenal, and scapular regions was weighed to calculate the body fat percentage. Among them, indexes such as food intake, body weight, feed utilization rate, and body fat percentage were self-detected and analyzed by our laboratory. The serum and liver tissues were sampled under the guidance of technicians from Wuhan Sevier Biotechnology Co., Ltd., and were entrusted to them for detection and slide preparation analysis.

[0035] Table 2. Major lipid metabolism-related detection indicators, functions, and their RT-PCR primers Table 3 shows the changes in body weight, feed utilization, and body fat percentage. The average weight gain of mice in the ND group was only 1.25 g, significantly lower than that in the HFD and R-21 groups (p<0.01). There was no difference in weight gain between the HFD and R-21 groups (p>0.05), which may be related to the excessive initial weight of the mice. The initial weights of mice in the HFD and R-21 groups were 37.5 g and 35.3 g, respectively, which were much greater than the weight of normal 6-week-old mice. The feed utilization rates of mice in the HFD and R-21 groups were similar, both significantly higher than those in the ND group (p<0.05), indicating that high-fat diets promote weight gain in mice. The body fat percentage of mice in the HFD group was approximately 3.38%, significantly higher than that of mice in the R-21 group (2.51%) (p<0.05), indicating that R-21 bacterial powder can inhibit fat accumulation and reduce body fat percentage in mice.

[0036] Table 3. Results of changes in mouse body weight, feed utilization rate, and body fat percentage. Note: The presence of the same letter 'a', 'b', or 'c' in two groups of data in the same column indicates that the data difference is not significant (p>0.05), while the presence of different letters indicates that the data difference is significant (p<0.05).

[0037] The results of serum lipid profile analysis are shown in Table 4. Compared with the HFD group, R-21 bacterial powder significantly reduced the increases in TG, TC, and LDL-C caused by a high-fat diet (p<0.05). Specifically, the serum TG level in the R-21 group was only 1.14 mol / L, approximately 32.4% of that in the HFD group and 39.7% of that in the ND group, indicating that R-21 bacterial powder was particularly effective in inhibiting serum TG. The results of liver lipid profile analysis are shown in Table 4. Figure 2 As shown, R-21 bacterial powder significantly reduced the increase of TG, TC and LDL-C caused by a high-fat diet (p<0.05), and also reduced the level of HDL-C in the liver. Its effect characteristics are different from those of the four serum lipid items, especially its effect on reducing TG is weaker.

[0038] Table 4. Results of serum lipid profiles in mice of each group Note: The presence of the same letter 'a', 'b', or 'c' in two groups of data in the same column indicates that the data difference is not significant (p>0.05), while the presence of different letters indicates that the data difference is significant (p<0.05).

[0039] Serum inflammatory factor test results Figure 3As shown in the figure, compared with the ND group, the high-fat group mice showed significantly increased serum inflammatory factors such as IL-1β, IL-6, TNF-α, and LPS (p<0.05), indicating that the high-fat diet has a significant inflammatory induction effect. Compared with the HFD group, the R-21 bacterial powder group mice showed a highly significant decrease in serum IL-6 (p<0.01), and was lower than the ND group; IL-1β and LPS were slightly lower than the HFD group, but not significantly (p>0.05); TNF-α was slightly higher than the HFD group, also not significantly (p>0.05). The results indicate that in the regulation of serum inflammatory factors, R-21 can only significantly reduce the level of IL-6, which is different from the phenomenon of other lactic acid bacteria reducing serum inflammatory factors.

[0040] Results of liver lipid metabolism-related gene transcription level detection: Figure 4 As shown, compared with the ND group, a high-fat diet significantly increased the transcriptional levels of ACC1 and SREBP-1 genes in the liver of experimental mice (p<0.05) and significantly decreased the transcriptional levels of genes such as CPT1A, LDLR, and FXR (p<0.05), indicating that a high-fat diet can stimulate the transcriptional expression of genes involved in the synthesis and transport pathways of some fats and cholesterols. Compared with the HFD group, R-21 bacterial powder significantly decreased the transcriptional levels of ACC1 and SREBP-1 genes in the liver of mice (p<0.05) and significantly increased the transcriptional levels of genes such as PPARα, CPT1A, LDLR, FXR, FGF15, and TGR5 (p<0.05), indicating that R-21 bacterial powder can significantly inhibit the expression of genes related to fat synthesis and bile acid synthesis induced by a high-fat diet, and increase the expression of genes such as lipid oxidation and degradation enzymes and low-density lipoprotein receptors. Furthermore, the transcriptional levels of genes such as PPARα, CPT1A, FXR, FGF15, and TGR5 in the liver of R-21 group mice were significantly higher than those in ND group (p<0.05), indicating that R-21 bacterial powder is effective in promoting hepatic lipid oxidation and cholesterol synthesis in mice not only in HFD-induced mice but also in mice on a normal diet. In CYP7A1 detection, there was no significant difference among the three groups of mice (p>0.05), indicating that the degradation of cholesterol in mice by R-21 bacterial powder does not occur through the cholesterol 7α-hydroxylation pathway.

[0041] Oil Red O staining results of liver tissue are as follows Figure 5 As shown, the HFD group liver cells contained numerous tiny red lipid droplets (green arrows), and the liver tissue appeared distinctly red; the ND and R-21 groups liver cells had fewer tiny red lipid droplets, and the liver tissue appeared a deeper blue, indicating that R-21 bacterial powder can inhibit HFD-induced fat accumulation in liver cells. The results of hematoxylin staining of liver tissue are shown below. Figure 6As shown, in the HFD group, more hepatocyte steatosis was observed around the central vein and portal area of ​​the liver and in the parenchyma (green arrows), while in the ND and R-21 groups, only a very small number of hepatocytes showed steatosis around the central vein and portal area of ​​the liver and in the parenchyma (green arrows), indicating that R-21 bacterial powder can inhibit HFD-induced hepatocyte steatosis.

[0042] In summary, R-21 bacterial powder can inhibit HFD-induced increases in fat mass and body fat percentage in mice, significantly reduce serum and liver TG, TC and LDL-C levels, and significantly reduce hepatic cell fat accumulation and steatosis.

[0043] Example 4 This embodiment provides the application of Lactobacillus plantarum R-21 bacterial powder in antioxidant applications, as detailed below: Partial liver tissue from mice in Example 3 was taken and homogenized under the guidance of technicians at Wuhan Saiweier Biotechnology Co., Ltd. The homogenate was then frozen, stored on dry ice, and submitted to the company. The activities of superoxide dismutase, catalase, and glutathione peroxidase, as well as the content of malondialdehyde, were detected to evaluate the antioxidant activity of *Lactobacillus plantarum* R-21 powder on mouse liver. Results are as follows: Figure 7 As shown, a high-fat diet significantly reduced the levels of SOD, CAT, and GSH-PX in the liver of mice (p<0.01). These enzymes can scavenge free radicals in tissues, reduce oxidative stress damage to hepatocytes, regulate liver metabolism, and promote hepatocyte regeneration, thus having a positive effect on liver health. R-21 bacterial powder intervention did not significantly inhibit the decrease in SOD induced by a high-fat diet in mouse livers (p>0.05), but it significantly inhibited the decrease in GSH-PX induced by a high-fat diet in mouse livers (p<0.05) and extremely significantly inhibited the decrease in CAT (p<0.01), enhancing the liver's antioxidant capacity. The R-21 group of mice had the lowest MDA level in the liver, but its effect on MDA scavenging was not significant (p>0.05). Furthermore, the MDA level in the liver of mice in the HFD group was significantly lower than that in the ND group (p<0.05), which contradicts the results of the antioxidant enzyme activity test, and may be different from the antioxidant characteristics of other probiotics.

[0044] Example 5 This embodiment provides the application of *Lactobacillus plantarum* R-21 bacterial powder in the prevention of colitis, as detailed below: The mouse colon tissue from Example 3 was collected, preserved under the guidance of technicians from Wuhan Saiweier Biotechnology Co., Ltd., frozen, stored on dry ice, and submitted to the company for hematoxylin staining to prepare colon tissue sections for observation of colonic lesions. The results are as follows: Figure 8As shown, in the HFD group mice, the mucosal layer of the colonic tissue protrudes into the intestinal lumen to form folds, with focal erosions, a small number of mucosal epithelial cells and intestinal gland epithelial cells sloughing off (brown arrows), occasional necrotic cell fragments (yellow arrows), a small amount of intestinal gland structure loss, very little connective tissue hyperplasia (blue arrows), and occasional focal aggregation of lymphocytes (green arrows). The muscularis mucosae separates the lamina propria from the submucosa. In contrast, the mucosal epithelial structure of the ND and R-21 groups is intact, mainly composed of a single layer of columnar epithelium and goblet cells. The lamina propria contains a large number of short tubular, densely arranged intestinal glands, with abundant goblet cells and several focal aggregations of lymphocytes (green arrows). The muscularis mucosae separates the lamina propria from the submucosa, the submucosa is loose connective tissue, and the muscularis mucosae structure is clear. This indicates that a high-fat diet can induce colonic inflammation and cause mucosal damage in mice, while intervention with R-21 bacterial powder can alleviate this symptom.

[0045] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of a plant lactobacillus powder in the preparation of lipid-lowering, colitis-preventing, and antioxidant foods, health products, or drugs, wherein the plant lactobacillus powder includes plant lactobacillus R-21 with preservation number CCTCC M2018009.

2. The application according to claim 1, characterized in that, The *Lactobacillus plantarum* powder contains *Lactobacillus plantarum* R-21 ≥ 40 billion CFU / 2g, calculated as viable bacteria.

3. The application according to claim 1, characterized in that, The *Lactobacillus plantarum* powder also includes 1 g / 2 g mannan oligosaccharide, 1 g / 2 g maltodextrin, and has a moisture content of less than 4%.

4. The application according to any one of claims 1 to 3, characterized in that, The plant lactobacillus powder is prepared by the following method: Lactobacillus plantarum R-21 was inoculated into MRS solid plates for solid activation culture. Single colonies were picked and transferred to MRS slant for overnight culture. The slant seed was then inoculated into MRS test tube liquid culture medium for the first static culture. After that, it was transferred to MRS test tube liquid culture medium for the second static culture to obtain liquid seed. The obtained liquid seed was inoculated into the fermentation medium and cultured with shaking to obtain the fermentation broth; The obtained fermentation broth was centrifuged, the precipitate was collected and washed to obtain washed bacterial sludge; Under clean conditions, the washed bacterial sludge was mixed with the first excipient, mannan oligosaccharide, and then mixed with the second excipient, maltodextrin, to obtain the original bacterial powder. Under clean conditions, the obtained original bacterial powder is mixed with the third excipient to obtain the plant lactobacillus powder.

5. The application according to claim 4, characterized in that, The solid activation culture was performed three times. OD of bacterial cells in liquid seeds 600 The value is 0.8~1.0; The first static incubation was carried out at 37℃ for 12-16 hours. The second static incubation was carried out at 37℃ for 4-6 hours.

6. The application according to claim 4, characterized in that, The inoculation density for liquid seeds is 2%; The fermentation medium consists of: 30 g soybean peptone, 24 g corn steep liquor, 60.0 g white sugar, 36 g calcium carbonate, 2.0 g dipotassium hydrogen phosphate, 1.0 ml Tween 80, 2.0 g diammonium hydrogen citrate, 0.25 g manganese sulfate, 5.0 g sodium acetate, 0.58 g magnesium sulfate heptahydrate, and 1000 ml distilled water. The shaking culture was carried out at 37℃ and 120 rpm for 36 hours. Wash three times with physiological saline. The fermentation broth or bacterial suspension was centrifuged at 5000 rpm for 10 min.

7. The application according to claim 4, characterized in that, During the mixing process, the materials are added and finely mixed simultaneously. The amount of the first excipient, manno-oligosaccharide, is 1 / 5 of the mass of the first excipient, manno-oligosaccharide, to the volume of the fermentation liquid. The mass ratio of the first excipient, mannan oligosaccharide, to the second excipient, maltodextrin, is 1:

1. The second excipient, maltodextrin, is mixed by gradually adding the first excipient, mannan oligosaccharide, to the second excipient, maltodextrin, in batches.

8. The application according to claim 4, characterized in that, The viable count of the original bacterial powder was 1.42 × 10⁻⁶. 11 CFU / g.

9. The application according to claim 4, characterized in that, The third excipient includes manno-oligosaccharide and maltodextrin; the mass ratio of manno-oligosaccharide and maltodextrin is 1:

1.

10. The application according to claim 4, characterized in that, The mass ratio of the original bacterial powder to the third excipient is 1:5; The viable count of the *Lactobacillus plantarum* powder is greater than or equal to 2.00 × 10⁻⁶. 10 CFU / g.

Images