Rosa roxburghii tratt lactic acid bacteria solid beverage and application thereof in preparation of food, health care product or medicine for reducing hyperlipidemia, preventing colitis and resisting oxidation

Abstract

The invention discloses a roxburgh rose lactic acid bacteria solid beverage and application thereof in preparation of foods, health-care products or medicines for reducing hyperlipidemia, preventing colitis and resisting oxidation, and belongs to the field of probiotic health-care foods. The rosa roxburghii tratt lactic acid bacteria solid beverage is prepared from plant lactobacillus R-15, rosa roxburghii tratt fine powder, mannan oligosaccharide, resistant dextrin, coix lacryma-jobi extract, hawthorn extract, silicon dioxide and the like. The rosa roxburghii tratt lactobacillus solid beverage can significantly reduce the levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol in the liver and serum of a mouse, inhibit fatty degeneration of the liver, and inhibit colitis induced by high fat diet. The invention has important application value in prevention and treatment of hyperlipidemia and colitis.

Description

Technical Field

[0001] This invention relates to the field of probiotic health food technology, and in particular to a prickly pear lactic acid bacteria solid beverage and its application in the preparation of lipid-lowering, colitis-preventing, and antioxidant foods, health products, or drugs. Background Technology

[0002] Cardiovascular disease causes approximately 4 million deaths annually in China, accounting for over 46% of all deaths nationwide. This number exceeds the combined death toll from all cancers, making it the leading cause of death in my country (Hu Shengshou, 2023). Dyslipidemia, characterized 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 population. Medical expenses due to 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, bloating, rhabdomyolysis, and abnormal liver and kidney function.

[0003] In recent years, numerous studies have shown a close relationship between gut microbiota and hyperlipidemia. Appropriate supplementation with probiotics or fecal microbiota transplantation (FMT) can alleviate lipid metabolism disorders and lower blood lipid levels. However, FMT faces considerable resistance in its promotion, primarily due to unstable efficacy, psychological resistance from test subjects, and the possibility of donors carrying potential pathogens. Lactobacillus and Bifidobacteria are the two most common and predominant beneficial bacteria in the gut microbiota. They are highly safe and possess 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. They are currently the probiotic species most closely related to human health. Currently, domestic probiotic research mainly focuses on applications in regulating the gastrointestinal tract and enhancing non-specific immunity. Most functional studies on lipid-lowering probiotics are concentrated at the mouse testing level, and there are currently no probiotic products with clearly defined lipid-lowering functions.

[0004] Furthermore, prickly pear, rich in vitamin C, SOD, flavonoids, polyphenols, and extracellular polysaccharides, possesses antioxidant, whitening, immunity-boosting, digestive-promoting, and lipid-lowering effects, making it a key specialty food industry in Guizhou with an annual output value of approximately 15 billion yuan. However, prickly pear is rich in tannins (0.6-2.2%, m / m), making prickly pear-related products sour and astringent, which is difficult for consumers, especially those outside the province, to accept, thus limiting the promotion of the prickly pear industry. In addition, prickly pear juice and prickly pear beverages, which account for the largest proportion of prickly pear products, are liquid drinks with unstable quality. SOD is easily deactivated and denatured, co-precipitating with tannins to form flocculent precipitates; it is also prone to oxidative browning, with the product color gradually darkening over time, affecting consumer sensory evaluation; the most widely advertised vitamin C is unstable, with its content gradually decreasing over time, and its main advertised functions do not match market demand. Prickly pear powder, a solid product prepared from prickly pear juice through spray drying, has relatively stable quality, but still suffers from a sour and astringent taste that is difficult for consumers to accept. Therefore, combining prickly pear powder with probiotics to create a combined solid beverage, integrating it with the billion-dollar probiotic industry, can broaden the market reach of prickly pear products. This would allow consumers to supplement their vitamin C intake while consuming probiotic products, helping the prickly pear industry overcome its current difficulties. Our team previously isolated a strain of *Lactobacillus plantarum* R-15 (accession number CCTCCM2018010) from the intestinal mucosa of pigs, which has strong cholesterol-lowering function and possesses certain application value.

[0005] Therefore, it is meaningful to design a compound beverage with a strong palatability and enhanced blood lipid-lowering function by combining Lactobacillus plantarum R-15 and prickly pear extract powder and other excipients. Summary of the Invention

[0006] To address the above technical problems, this invention provides a prickly pear lactic acid bacteria solid beverage, its preparation method, and its applications. Specifically, it provides the application of the prickly pear lactic acid bacteria solid beverage in the preparation of lipid-lowering, colitis-preventing, and antioxidant foods, health products, or drugs. This invention aims to use *Lactobacillus plantarum* R-15 as the core strain, combined with prickly pear extract and other excipients, to verify its application value in lowering hyperlipidemia, providing a new functional product for the probiotic and prickly pear industry market.

[0007] The first objective of this invention is to provide a prickly pear lactic acid bacteria solid beverage, comprising the following raw materials: Lactobacillus plantarum R-15 original bacterial powder, prickly pear extract powder, mannan oligosaccharide, resistant dextrin, coix seed extract, hawthorn extract and silicon dioxide; Lactobacillus plantarum R-15 has the accession number CCTCC M2018010 (accession date: January 8, 2018, deposited at Wuhan University, China).

[0008] In some embodiments of the present invention, the viable count of the *Lactobacillus plantarum* R-15 original bacterial powder is ≥180 billion CFU / g.

[0009] In some embodiments of the present invention, the *Lactobacillus plantarum* R-15 original bacterial powder is 10%, prickly pear extract powder is 2%, mannan oligosaccharide is 41%, resistant dextrin is 41%, coix seed extract is 3%, hawthorn extract is 2%, and silicon dioxide is 1%, by mass percentage.

[0010] In some embodiments of the present invention, the prickly pear lactic acid bacteria solid beverage is prepared by the following method: Lactobacillus plantarum R-15 was inoculated into MRS solid plates, inverted for culture, and single colonies were picked and transferred into MRS solid plates. The solid activation culture was repeated multiple times, and the last single colony was cultured overnight in MRS slant medium to obtain slant seed. The slant seeds were inoculated into MRS liquid medium and subjected to a first static culture. Then they were transferred to MRS liquid medium and subjected to a second static culture to obtain liquid seeds. The obtained liquid seed was inoculated into the fermentation medium and then allowed to stand for a third time to obtain the fermentation broth. The obtained fermentation broth was centrifuged, the precipitate was collected and washed to obtain washed bacterial sludge; 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 was mixed with mannan oligosaccharide, resistant dextrin, prickly pear extract, coix seed extract, hawthorn extract and silica to obtain the plant lactobacillus powder.

[0011] In some embodiments of the present invention, the culture medium components in the MRS solid plate include 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, and 1000 ml distilled water; the pH value is 6.6.

[0012] In some embodiments of the present invention, the solid activation culture is performed three times; The inverted culture was carried out at a temperature of 37°C for 36 hours. The temperature for the first static incubation was 37℃, and the time was 12~16 h; The second static incubation was carried out at a temperature of 37°C for 4-6 hours. The third static incubation was carried out at a temperature of 37°C for 36 hours. The OD of the liquid seed cells 600 =0.8~1.0.

[0013] In some embodiments of the present invention, the fermentation medium comprises 30 g soybean peptone, 24 g corn steep liquor powder, 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; natural pH.

[0014] In some embodiments of the present invention, the mass ratio of the first excipient mannan oligosaccharide and the second excipient maltodextrin is 1:1; The amount of the first excipient, mannan oligosaccharide, is 1 / 5 of the mass of the first excipient, mannan oligosaccharide, to the volume of the fermentation liquid. During the mixing process, the materials are added and finely crushed and mixed simultaneously.

[0015] In this invention, the obtained original bacterial powder was frozen at -20℃ for later use. During the experiment, the viable cell count of the original bacterial powder was detected using the dilution plating method. The results showed that the viable cell concentration of the prepared original bacterial powder was 1.80 × 10⁻⁶. 11 The concentration is cfu / g, and the water content is approximately 5.2%. It should be noted that *Lactobacillus plantarum* R-15 has a large amount of extracellular polysaccharides outside its cell wall, which easily causes bacterial aggregation. It is difficult to disperse during dilution, and thorough shaking for 10-20 minutes is required for dissolution.

[0016] In this invention, the obtained prickly pear lactic acid bacteria solid beverage was stored under cool conditions at 2-8℃. The viable bacterial count was determined using the dilution plating method, the coliform count was determined using eosin methylene blue agar, and the mold count was determined using Czapek's agar. The activity of the original bacterial powder was approximately 1.50 × 10⁻⁶. 10 The concentration of CFU / g is above 100, the detection rate of Escherichia coli and Salmonella is 0, and the amount of mold contamination is below the maximum limit specified in GB 7101-2022.

[0017] The second objective of this invention is to provide the application of the prickly pear lactic acid bacteria solid beverage in the preparation of foods, health products, or drugs that lower blood lipids, prevent colitis, and have antioxidant properties.

[0018] The technical solution of the present invention has the following advantages compared with the prior art: This invention utilizes R-15 probiotic powder and its prickly pear lactic acid bacteria solid beverage to significantly inhibit the increase in fat mass and body fat percentage induced by a high-fat diet in male mice. Furthermore, it inhibits the increase in serum and liver triglyceride and cholesterol levels in high-fat mice by enhancing fatty acid oxidase and inhibiting the transcription levels of fatty acid synthase and bile acid synthase. Simultaneously, it was found that R-15 probiotic powder and its prickly pear lactic acid bacteria solid beverage significantly enhance the activity of superoxide dismutase (SOD) and catalase (CAT) in mouse liver, thereby improving the antioxidant capacity of the mouse liver. This invention also found that both R-15 probiotic powder and its prickly pear lactic acid bacteria solid beverage 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 These are the results of the detection of inflammatory factors in mouse serum according to the present invention; Figure 2 This is the result of liver oil red O staining section according to the present invention; Figure 3 This is the result of hematoxylin staining of liver sections according to the present invention; Figure 4 These are the results of the detection of antioxidant indicators in mouse livers according to the present invention; Figure 5 These are the staining results of mouse colon sections according to the present invention; 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). Figure 6 This is a diagram showing the results of colonic lesions in Embodiment 4 of the present invention. Detailed Implementation

[0020] 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.

[0021] The raw materials used in this invention are all conventional substances that can be obtained commercially. For example, mannan oligosaccharide and resistant dextrin were purchased from Zhejiang Yinuo Biotechnology Co., Ltd., prickly pear powder was purchased from Guizhou Jihai Fruit and Vegetable Engineering Beverage Technology Co., Ltd., and coix seed extract and hawthorn extract were purchased from Shaanxi Bolin Biotechnology Co., Ltd.

[0022] Example 1 This invention provides a method for preparing a prickly pear lactic acid bacteria solid beverage, as detailed below: 1. Sources of lactic acid bacteria strains The strain is *Lactobacillus plantarum* R-15 (CCTCC M2018010), which was isolated and preserved by our team. Previous experiments have shown that this strain has strong acid and bile salt resistance and cholesterol-lowering ability.

[0023] 2. Activation of microbial strains Cryopreserved *Lactobacillus plantarum* R-15 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 36 h. Moist, thick colonies with a calcium-dissolving zone and large colony diameter were picked and streaked again onto solid plates, repeating the activation process three times. From the third activated plate, thick, thick colonies with a calcium-dissolving zone and large colony diameter were picked and transferred to the above-mentioned slant medium for overnight incubation to prepare slant seed culture.

[0024] 3. Seed preparation The slant seed culture obtained in step 2 was inoculated into MRS liquid medium (the above medium does not contain calcium carbonate) and incubated overnight at 37°C. 2% (v / v) of the bacterial culture 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 liquid seeds.

[0025] 4. Fermentation culture Take 2% (v / v) of the liquid seed obtained in step 3 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), and 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 the *Lactobacillus plantarum* R-15 fermentation broth decreased to 4.5, and the OD value... 600 The value reached 23.32, and the viable bacteria count reached 1.58 × 10⁻⁶. 11 CFU / ml.

[0026] 5. Preparation of original mycelium powder The fermentation broth obtained in step 4 was collected and centrifuged (5000 rpm, 10 min). The precipitate was collected and washed three times with physiological saline, retaining the bacterial sludge (removing as much water as possible from the top layer). The bacterial sludge was gradually added to dried mannooligosaccharide (1 / 5 of the original fermentation broth volume, m / v; the mannooligosaccharide was dried at 80℃ for 2 h before use and cooled to 40℃). During this process, the mixture was finely crushed and mixed while adding the sludge to prevent clumping. Then, it was mixed with 1 / 5 (m / v) of the original fermentation broth maltodextrin and finely crushed to prepare the original bacterial powder, which was then frozen at -20℃ for later use. The viable cell count of the original bacterial powder was determined using the dilution plating method. The results showed that the viable cell concentration of the prepared original bacterial powder was 1.80 × 10⁻⁶. 11 cfu / g, water content approximately 5.2%. It should be noted that *Lactobacillus plantarum* R-15 has a large amount of extracellular polysaccharides outside its cell wall, which easily causes bacterial aggregation, making it difficult to disperse during dilution. Therefore, thorough shaking for 15 minutes is required during dissolution.

[0027] 6. Preparation of Prickly Pear Lactic Acid Bacteria Solid Beverage Under clean conditions, one part by weight of the original bacterial powder prepared in step 5 was mixed with 4.1 parts of mannan oligosaccharide (a sugar composed of 2-10 monosaccharides, not pure), 4.1 parts of resistant dextrin, 0.2 parts of prickly pear extract, 0.3 parts of coix seed extract, 0.2 parts of hawthorn extract, and 0.1 parts of silica to obtain a prickly pear lactic acid bacteria solid beverage. The beverage was stored at 2-8℃ in a cool, dark place. The viable bacteria count was determined using the dilution plating method, the coliform bacteria content 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 lactic acid bacteria activity of the prepared bacterial powder was approximately 1.53 × 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.

[0028] Table 1. Microbiological test results of prickly pear lactic acid bacteria solid beverage Testing items solid beverages GB 7101-2022 Detection methods Lactic acid bacteria activity (CFU / g) <![CDATA[1.53×10 10 ]]> <![CDATA[≧1.0×10 6 ]]> GB4789.35 Coliform bacteria (CFU / g) 2 ≤5 GB 4789.3 Escherichia coli (CFU / g) 0 0 GB 4789.3 Salmonella (CFU / g) 0 0 GB 4789.4 Mold (CFU / g) 21 ≤50 GB 4789.15 Example 2 This embodiment provides the application of prickly pear lactic acid bacteria solid beverage in lowering blood lipids, as detailed below: Forty male adult Kunming mice at about 8 weeks of age were divided into 4 groups (laboratory animals were purchased from Spf (Beijing) Biotechnology Co., Ltd., and the production license number of laboratory animals was: SCXK (Beijing) 2024-0001), with 10 mice in each group. Among them, 3 groups were set as high-fat diet groups (the diet 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 1 group was set as the basal diet control group (the basal diet was purchased from Spf (Beijing) Biotechnology Co., Ltd.). In the high-fat groups, after continuous feeding with high-fat diet, one group was orally gavaged with the diluted solution of the original bacterial powder of Lactiplantibacillus plantarum R-15 (R-15 group, diluted with physiological saline to a cell concentration of 1.0×10 10 CFU / ml), and the gavage dose was 100 μl / mouse / d; one group was orally gavaged with the diluted solution of Rosa roxburghii Tratt lactic acid bacteria solid beverage (R-15CG group, diluted 10 times with physiological saline), and the gavage dose was 100 μl / mouse / d; another high-fat group was set as the control group (FHD group), and 100 μl of physiological saline was gavaged to each mouse per day. Mice in the basal diet group were gavaged with 100 μl of physiological saline per mouse per day (ND group). During this period, the mice were allowed to eat and drink freely 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 collected from the inner canthus of the eyes of each group of animals, and serum was separated by centrifugation. ELISA kits were used to detect four blood lipids (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)). The livers of the mice were taken. One part was homogenized to detect TG, TC, LDL-C, and HDL-C in the liver homogenate; the other 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 groin, epididymis, perirenal, and scapular regions was weighed to calculate the body fat percentage. Among them, indicators such as food intake, body weight, feed utilization rate, fecal water content, and body fat percentage were detected and analyzed by our laboratory. Serum and liver tissues were sampled under the guidance of technicians from Wuhan Sevier Biotechnology Co., Ltd., and entrusted to them for detection and slide preparation analysis.

[0029] Table 2 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, R-15, and R-15CG groups (p<0.01). There was no significant difference in weight gain among the HFD, R-15, and R-15CG groups (p>0.05), which may be related to the excessive initial weight of the mice. The initial weights of mice in the HFD, R-15, and R-15CG groups were 37.5 g, 37.1 g, and 35.4 g, respectively, which were much greater than the weight of normal 6-week-old mice. The feed utilization rate of mice in the HFD group was the lowest (0.63%), followed by the R-15CG group (1.46%) and the R-15 group (1.62%). The feed utilization rate of the HFD group was the highest (1.91%), indicating that high-fat diets can improve feed utilization, while R-15 and its prickly pear lactic acid bacteria solid beverage inhibited weight gain and reduced feed utilization. The body fat percentage of mice in the HFD group was approximately 3.38%, which was significantly higher than that of the ND group (0.21%), the R-15 group (2.52%), and the R-15CG group (2.72%) (p<0.05). This indicates that R-15 and its prickly pear lactic acid bacteria solid beverage can inhibit fat accumulation induced by a high-fat diet in mice and reduce their body fat percentage.

[0030] Table 2. Results of changes in mouse body weight, feed utilization rate, and body fat percentage. Initial body weight (g) Final body weight (g) Total weight gain (g) Feed utilization rate (%) Body fat percentage (g) Body fat percentage (%) ND 36.2±1.23b 37.4±1.45b 1.25±0.44c 0.63±0.23c 0.08±0.23a 0.21±0.23bc HFD 37.5±2.18a 40.7±1.35a 3.21±0.67a 1.91±0.48a 1.27±0.48a 3.38±0.31a R-15 37.1±1.33a 39.9±1.67a 2.82±0.52b 1.62±0.76b 0.93±0.32b 2.52±0.17b R-15CG 35.4±0.22bc 38.0±1.06b 2.63±0.09b 1.46±0.37b 0.96±0.57b 2.72±0.23b 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).

[0031] The results of the four serum lipid tests are shown in Table 3. Compared with the HFD group mice, R-15 and its prickly pear lactic acid bacteria solid beverage significantly reduced the increases in TG, TC, and LDL-C induced by a high-fat diet (p<0.05). Among them, the serum TG levels in the R-15 group and R-15CG group were only 1.32 and 1.31 mmol / L, respectively, which were approximately 37.22% of the HFD group and 45.64% of the ND group. This indicates that R-15 and its prickly pear lactic acid bacteria solid beverage were particularly effective in inhibiting serum TG, and could not only inhibit the increase in serum TG induced by a high-fat diet, but also inhibit the increase in serum TG induced by a normal diet in mice. The serum TC levels in the R-15 and R-15CG groups were 5.35 and 4.54 mmol / L, respectively, and the serum LDL-C levels were 0.61 and 0.46 mmol / L, respectively, both significantly lower than those in the HFD group (p<0.05). This indicates that R-15 and its prickly pear lactic acid bacteria solid beverage can significantly inhibit the increase of serum TC and LDL-C in mice, and the prickly pear lactic acid bacteria solid beverage has a more significant effect (p<0.05).

[0032] Table 3 Results of serum lipid assays in mice of each group Group TG (mmol / L) TC (mmol / L) HDL-C (mmol / L) LDL-C (mmol / L) ND 2.87±0.05b 5.00±0.13b 2.86±0.79a 0.61±0.01b HFD 3.52±0.22a 7.11±0.30a 2.22±0.16c 0.73±0.45a R-15 1.32±0.24c 5.35±0.27b 2.51±0.57b 0.61±0.03b R-15CG 1.31±0.15c 4.54±0.40c 2.05±0.41d 0.46±0.05c 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).

[0033] Serum inflammatory factor test results Figure 1 As shown in the figure, compared with the ND group, the high-fat group mice showed significantly increased serum levels of 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, IL-6 was not detected in the serum of mice in the R-15 and R-15CG groups, and LPS was significantly decreased (p<0.05); moreover, the LPS in the R-15CG group was significantly lower than that in the HFD group (p<0.01), and close to that in the ND group (p>0.05); at the IL-1β level, the levels of the HFD, R-15, and R-15CG groups were similar (p>0.05); at the TNF-α level, there was no significant difference between the HFD and R-15 groups (p>0.05), while the R-15CG group was significantly higher than that in the HFD group (p<0.05). The results indicate that, in the regulation of serum inflammatory factors, both R-15 bacterial powder and its prickly pear lactic acid bacteria combination beverage significantly reduced the levels of IL-6 and LPS. In particular, the prickly pear lactic acid bacteria combination beverage significantly reduced both serum IL-6 and LPS levels simultaneously, but did not reduce IL-1β and TNF-α levels. This result differs from the characteristics of other lactic acid bacteria in reducing serum inflammatory factors. This may be because IL-1β, IL-6, and TNF-α are related to the functions of these three inflammatory factors, which are involved in both inflammation regulation and immune regulation, and these two functions are closely linked.

[0034] Liver lipid profile results as follows Figure 2 As shown, R-15 and its prickly pear lactic acid bacteria solid beverage both reduced the elevation 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. Their effects differed from those on the four serum lipid markers, especially their weaker effect on TG. However, the R-15 prickly pear powder composition showed a more significant inhibitory effect on TG, TC, and LDL-C, with all three lipid levels significantly lower than the HFD group (p<0.01) and also significantly lower than the R-15 bacterial powder group (p<0.05). Oil Red O staining results for liver tissue are shown below. Figure 3 As shown, the HFD group exhibited numerous tiny red lipid droplets in liver cells (green arrows), resulting in a distinctly red liver tissue, followed by the R-15 group. The ND group had the fewest red lipid droplets in liver cells, resulting in a deeper blue liver tissue, followed by the R-15CG group. This indicates that the prickly pear lactic acid bacteria solid beverage is more effective than R-15 bacterial powder in inhibiting HFD-induced fat accumulation in liver cells. The results of hematoxylin staining of liver tissue are shown below. Figure 4As shown, the HFD group showed a large number of hepatocytes with steatosis around the central vein and portal area of ​​the liver and within the parenchyma (the round white cells indicated by the green arrows are steatosis cells), while the ND group showed only a very small number of hepatocytes with steatosis around the central vein and portal area of ​​the liver and within the parenchyma (green arrows, the fewest number). The next groups with steatosis were the R-15CG group and the R-15 group. This indicates that both R-15 bacterial powder and its prickly pear lactic acid bacteria solid beverage can inhibit HFD-induced hepatocyte steatosis, with the R-15CG group showing a more significant effect.

[0035] In conclusion, R-15 prickly pear lactic acid bacteria solid beverage, compared with R-15 bacterial powder, is more effective in inhibiting HFD-induced increases in serum TC and LDL-C, liver TG, TC and LDL-C in mice, and is more effective in reducing hepatic cell fat accumulation and steatosis.

[0036] Example 3 This embodiment provides an application of prickly pear lactic acid bacteria solid beverage in antioxidation, as detailed below: Partial liver tissue from mice in Example 2 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 malondialdehyde content, were detected to evaluate the antioxidant activity of R-15 bacterial powder and its prickly pear lactic acid bacteria solid beverage on mouse liver. Results are as follows: Figure 5 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, which are of positive significance for liver health. The decrease in their levels may mean that a high-fat diet can stimulate oxidative stress and exacerbate hepatocyte damage. The intervention of R-15 probiotic powder and solid beverage significantly inhibited the decrease of SOD and CAT in the liver of mice induced by a high-fat diet (p<0.05) and enhanced the liver's antioxidant capacity. The levels of SOD, CAT, and GSH-PX in the liver of mice in the R-15CG group were lower than those in the R-15 group, but not significantly (p>0.05). This may be due to the fact that the actual number of lactic acid bacteria administered to the R-15CG group was lower than that in the R-15 group.

[0037] Example 4 This embodiment provides the application of prickly pear lactic acid bacteria solid beverage in the prevention of colitis. The colonic tissue from the mice in Example 2 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 colonic tissue sections for observation of colonic lesions. The results are as follows: Figure 6As shown, in the HFD group mice, the mucosal layer of the colonic tissue protruded into the intestinal lumen to form folds, with focal erosions, a small number of mucosal epithelial cells and intestinal gland epithelial cells sloughed 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 separated the lamina propria from the submucosa. In contrast, the ND, R-15, and R-15CG groups had intact mucosal epithelial structures, with a large number of densely arranged intestinal glands in the lamina propria, abundant goblet cells, and occasional focal aggregation of lymphocytes (green arrows). The muscularis mucosae separated the lamina propria from the submucosa, and the muscularis mucosae structure was clear. This indicates that a high-fat diet can induce colonic inflammation and cause mucosal damage in mice, while the R-15 group and its prickly pear lactic acid bacteria solid beverage intervention can alleviate this symptom.

[0038] Of course, any product implementing this invention does not necessarily need to achieve all of the technical effects described above at the same time.

[0039] 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. A prickly pear lactic acid bacteria solid beverage, characterized in that, The ingredients include: Lactobacillus plantarum R-15 original bacterial powder, prickly pear extract powder, mannan oligosaccharide, resistant dextrin, coix seed extract, hawthorn extract and silicon dioxide; Lactobacillus plantarum R-15 preservation number is CCTCC M2018010.

2. The prickly pear lactic acid bacteria solid beverage according to claim 1, characterized in that, The *Lactobacillus plantarum* R-15 original bacterial powder has a viable bacterial count ≥180 billion CFU / g and a water content of 5.2%.

3. The prickly pear lactic acid bacteria solid beverage according to claim 1, characterized in that, The composition of the Lactobacillus plantarum R-15 original bacterial powder is 10%, prickly pear extract powder is 2%, mannan oligosaccharide is 41%, resistant dextrin is 41%, coix seed extract is 3%, hawthorn extract is 2%, and silicon dioxide is 1%, by mass percentage.

4. The prickly pear lactic acid bacteria solid beverage according to claim 1, characterized in that, The prickly pear lactic acid bacteria solid beverage is prepared by the following method: Lactobacillus plantarum R-15 was inoculated into MRS solid plates, inverted for culture, and single colonies were picked and transferred into MRS solid plates. The solid activation culture was repeated multiple times, and the last single colony was cultured overnight in MRS slant medium to obtain slant seed. The slant seeds were inoculated into MRS liquid medium and subjected to a first static culture. Then they were transferred to MRS liquid medium and subjected to a second static culture to obtain liquid seeds. The obtained liquid seed was inoculated into the fermentation medium and incubated for a third time to obtain the fermentation broth; The obtained fermentation broth was centrifuged, the precipitate was collected and washed to obtain washed bacterial sludge; 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 mannan oligosaccharide, resistant dextrin, prickly pear extract, coix seed extract, hawthorn extract and silica to obtain the prickly pear lactic acid bacteria solid beverage.

5. The prickly pear lactic acid bacteria solid beverage according to claim 4, characterized in that, The culture medium components of the MRS solid plates include 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, and 1000 ml distilled water; the pH value is 6.

6.

6. The prickly pear lactic acid bacteria solid beverage according to claim 4, characterized in that, The solid-state activation culture was performed three times; The inverted culture was carried out at a temperature of 37°C for 36 hours. The temperature for the first static incubation was 37℃, and the time was 12~16 h; The second static incubation was carried out at a temperature of 37°C for 4-6 hours. The third static incubation was carried out at a temperature of 37°C for 36 hours. The OD of the liquid seed cells 600 =0.8~1.

0.

7. The prickly pear lactic acid bacteria solid beverage according to claim 4, characterized in that, The fermentation medium comprises 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.

8. The prickly pear lactic acid bacteria solid beverage according to claim 4, characterized in that, The mass ratio of the first excipient, mannan oligosaccharide, to the second excipient, maltodextrin, is 1:

1. The amount of the first excipient, mannan oligosaccharide, is 1 / 5 of the mass of the first excipient, mannan oligosaccharide, to the volume of the fermentation liquid. During the mixing process, the materials are added and finely crushed and mixed simultaneously.

9. The prickly pear lactic acid bacteria solid beverage according to claim 4, characterized in that, The concentration of viable bacteria in the original bacterial powder was 1.80 × 10⁻⁶. 11 cfu / g.

10. The use of the prickly pear lactic acid bacteria solid beverage according to any one of claims 1 to 9 in the preparation of foods, health products or drugs that lower blood lipids, prevent colitis and have antioxidant properties.

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