Lactobacillus paracasei ZL No.1 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses lactobacillus paracasei ZL No.1 and application thereof. The lactobacillus paracasei ZL No.1 is preserved in the China General Microbiological Culture Collection Center on January 10, 2025, the preservation number is CGMCC No.33360, and the preservation number is CGMCC No.33360. The lactobacillus paracasei ZL No.1 is a lactobacillus paracasei ZL No.1. The lactobacillus paracasei ZL No.1 strain disclosed by the invention can strongly inhibit the activity of aldose reductase, alpha-glucosidase, alpha-amylase and dipeptidyl peptidase-IV, can maintain the glucose homeostasis of an organism and relieve diabetic retinopathy, has good artificial gastrointestinal fluid tolerance, can reach intestinal tracts in a viable bacteria state, and can be used for preventing and treating diabetic retinopathy. The lactobacillus paracasei ZL No.1 strain disclosed by the invention has the probiotic characteristics of Caco-2 cell adhesion, oxidation resistance and pathogenic bacterium growth and adhesion inhibition, is suitable for the development of products such as fermented milk, fermented milk beverages and lactic acid bacteria viable bacteria preparations, and provides natural and safe diet choices for obese patients, insulin resistance patients and diabetic patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a lactobacillus paracasei ZL No. 1 and its application. BACKGROUND

[0002] Diabetes is a global epidemic chronic metabolic disease characterized by chronic hyperglycemia, which can cause a variety of serious complications such as diabetic retinopathy, nephropathy and cardiovascular disease, and seriously threatens human health. Maintaining blood glucose homeostasis is the key to diabetes management, and inhibiting carbohydrate hydrolyzing enzymes (such as alpha-glucosidase and alpha-amylase) in the intestinal tract is one of the effective strategies to control postprandial blood glucose. In addition, the high blood glucose state will activate the polyol pathway, and the overactivity of aldose reductase (AR) is an important mechanism leading to diabetic complications (such as cataract and neuropathy), therefore, finding natural substances that can effectively inhibit the activity of these key enzymes has become a hot research topic.

[0003] In recent years, probiotics have attracted great attention due to their wide range of health-promoting functions. As an important member of probiotics, lactic acid bacteria have been confirmed by a large number of studies to have many benefits such as regulating intestinal flora, enhancing immunity, and reducing cholesterol. Among them, lactobacillus paracasei is one of the common probiotic species. For example, lactobacillus paracasei shirota strain (LcS) has been widely studied and commercialized, and is reported to have immune regulation and intestinal health improvement functions. Some studies have also preliminarily revealed the hypoglycemic potential of certain lactic acid bacteria strains. For example, animal bifidobacterium BB12 (Bifidobacterium animalis subsp. lactis BB12) and other strains are reported to have a certain effect on regulating blood glucose. Another study showed that the fermentation supernatant or cell extract of certain lactic acid bacteria showed inhibitory activity on alpha-glucosidase and alpha-amylase in vitro.

[0004] However, there are still certain limitations in existing research. First, although some strains have been reported to have hypoglycemic potential, their effect is limited, and there are few reports of strains that can simultaneously inhibit the activities of aldose reductase, alpha-glucosidase, alpha-amylase and dipeptidyl peptidase-IV, second, in order for probiotics to exert their physiological functions, they must be able to tolerate the environment of the human digestive tract (such as gastric acid and bile salts) and reach the intestine in a viable state, and the strains used in many existing studies do not perform well in terms of tolerance to harsh artificial gastrointestinal fluid, which limits their actual application effect. SUMMARY

[0005] In view of the technical defects in the background art, the present application proposes a lactobacillus paracasei and its application, which solves the above technical problems and meets the actual needs, and the specific technical scheme is as follows:

[0006] The present application provides a lactobacillus paracasei ZL No.1, which is derived from Jiangsu Rugao centenarians, and the strain is preserved in China General Microbiological Culture Collection Center on January 10, 2025, and the preservation number is CGMCC No. 33360.

[0007] Further, the above-mentioned lactobacillus paracasei ZL No.1 has excellent aldehyde sugar reductase inhibition ability, as well as excellent alpha-glucosidase, alpha-amylase and dipeptidyl peptidase-IV inhibition ability.

[0008] Further, the above-mentioned lactobacillus paracasei ZL No.1 has the effect of maintaining glucose homeostasis of the body.

[0009] Further, the above-mentioned lactobacillus paracasei ZL No.1 can alleviate retinopathy caused by diabetes.

[0010] The above-mentioned lactobacillus paracasei has good in-vitro inhibition activity of aldehyde sugar reductase, alpha-glucosidase, alpha-amylase and dipeptidyl peptidase-IV; the lactobacillus paracasei is applied to the preparation of fermented milk, fermented milk beverage and live lactic acid bacteria preparation products, and has effective hypoglycemic effect.

[0011] As a further technical solution of the present application, the above-mentioned lactobacillus paracasei ZL No.1 is applied to fermented milk, fermented milk beverage and live lactic acid bacteria preparation products with the effect of maintaining glucose homeostasis of the body.

[0012] As a further technical solution of the present application, the above-mentioned lactobacillus paracasei ZL No.1 is applied to fermented milk, fermented milk beverage and live lactic acid bacteria preparation products with the effect of improving retinopathy caused by diabetes.

[0013] The present application also provides a method for obtaining lactobacillus paracasei with aldehyde sugar reductase, alpha-glucosidase, alpha-amylase and dipeptidyl peptidase-IV activity inhibition ability, comprising the following steps:

[0014] Step 1: lactobacillus is separated from the fecal sample of Rugao centenarians in Jiangsu Province by coating plate separation method, and lactobacillus paracasei is screened out after Gram staining and 16s rDNA gene sequencing analysis.

[0015] Step 2: lactobacillus paracasei strains with good artificial gastric juice tolerance and good survival in human body environment are screened out by artificial gastric juice tolerance experiment.

[0016] Step 3, obtaining the strain with the highest inhibitory ability to aldose reductase, α-glucosidase, α-amylase and dipeptidyl peptidase-IV activity by screening; the screening comprises sample preparation and determination of the inhibitory ability to aldose reductase, α-glucosidase, α-amylase and dipeptidyl peptidase-IV activity.

[0017] Step 4, evaluating the probiotic properties of the strain obtained by screening; the evaluation of the probiotic properties comprises adhesion to Caco-2 cells, antioxidant capacity, inhibition of pathogenic bacteria growth and inhibition of pathogenic bacteria adhesion to Caco-2 cells.

[0018] As a further technical solution of the present application, the sample preparation in Step 2 comprises preparation of a cell resuspension; the specific process of the cell fermentation broth preparation is as follows: after the strain is statically cultured in MRS medium at 37℃ for 16-20 h, the strain is washed with sterile normal saline for 2-3 times, resuspended in normal saline, and the concentration of the bacterial solution is adjusted to 1×10 6 ~1×10 9 CFU / mL to obtain the cell resuspension.

[0019] As a further technical solution of the present application, the determination of artificial gastric juice tolerance in Step 2 mainly comprises the following steps:

[0020] Step 1, 1.0 mL of the prepared cell resuspension is inoculated into 9.0 mL of artificial gastric juice (containing 0.5% NaCl and 0.3% pepsin) with pH 2.0 and pH 3.0, respectively, and incubated at 37℃ for 3 h; the viable cell count is determined by plate counting method at 0 h and 3 h, respectively, and the survival rate is calculated.

[0021] Step 2, 1 mL of the cell resuspension is inoculated into 9 mL of artificial intestinal juice solution (PBS solution with pH 8) containing 0.1% and 0.3% bile salts, respectively, and incubated at 37℃ for 3 h; the viable cell count is determined by plate counting method at 0 h and 3 h, respectively, and the survival rate is calculated.

[0022] As a further technical solution of the present application, the sample preparation in Step 3 comprises preparation of a cell resuspension; the specific process of the cell fermentation broth preparation is as follows: after the strain is statically cultured in MRS medium at 37℃ for 16-20 h, the strain is washed with sterile normal saline for 2-3 times, resuspended in normal saline, and the concentration of the bacterial solution is adjusted to 1×10 6 ~1×10 9 CFU / mL to obtain the cell resuspension.

[0023] As a further technical scheme of the present application, the preparation of the sample in step 3 comprises preparation of cell fermentation supernatant; the specific process of the preparation of the cell fermentation supernatant is as follows: after the strain is statically cultured at 37℃ in MRS medium for 16-20 h, the supernatant is collected by centrifugation, and the cell fermentation supernatant is obtained by filtration with a 0.22 μm microfiltration membrane.

[0024] As a further technical scheme of the present application, the preparation of the sample in step 3 comprises preparation of cell fermentation supernatant; the specific process of the preparation of the cell fermentation supernatant is as follows: after the strain is statically cultured at 37℃ in MRS medium for 16-20 h, the supernatant is collected by centrifugation, and the cell fermentation supernatant is obtained by filtration with a 0.22 μm microfiltration membrane. 6 ~1×10 9 CFU / mL, mixed, high-pressure broken, centrifuged at 4℃ to collect the supernatant, and the cell broken liquid is obtained by filtration with a 0.45 μm microfiltration membrane.

[0025] As a further technical scheme of the present application, the high-pressure broken condition is that the pressure is 50 Mpa and the cycle broken is 5 times at 4℃.

[0026] As a further technical scheme of the present application, the determination of the aldose reductase inhibitory activity in step 3 mainly comprises the following steps:

[0027] Step 1, the following determination system is configured: the sample is the cell fermentation supernatant: blank group: 0.1 mol / L PBS (pH 6.2) 700 μL, 0.6 mmol / L lithium sulfate 100 μL, 0.2 mmol / L NADPH 100 μL, 0.5 mmol / L glycerol 100 μL; sample blank group: 0.1 mol / L PBS (pH 6.2) 600 μL, 0.6 mmol / L lithium sulfate 100 μL, 0.2 mmol / L NADPH 100 μL, sample 100 μL, 0.5 mmol / L glycerol 100 μL; control group: 0.1 mol / L PBS (pH 6.2) 600 μL, 0.6 mmol / L lithium sulfate 100 μL, 0.2 mmol / L NADPH 100 μL, aldose reductase liquid (fresh bovine lens extract) 100 μL, 0.5 mmol / L glycerol 100 μL;

[0028] Sample group: 0.1 mol / L PBS (pH 6.2) 500 μL, 0.6 mmol / L lithium sulfate 100 μL, 0.2 mmol / L NADPH 100 μL, aldehyde sugar reductase liquid (fresh bovine eye extraction) 100 μL, sample 100 μL, 0.5 mmol / L glycerol 100 μL.

[0029] Step 2, the ultraviolet spectrophotometer measures the absorbance change of NADPH at 340nM, and the time is 2 min. First, measure the absorbance value of the blank group without adding enzyme liquid, then measure the absorbance change of NADPH within 2 min after adding aldehyde sugar reductase and intervention. Each sample is repeated three times, and the average value is taken.

[0030] Step 3, calculate the aldehyde sugar reductase inhibition rate according to the formula, and obtain the strain with the highest aldehyde sugar reductase inhibition activity.

[0031] As a further technical solution of the present application, the determination of α-glucosidase inhibition activity in step 3 mainly includes the following steps:

[0032] Step 1, according to the following determination system, the sample is cell fermentation liquid, cell fermentation supernatant and cell broken liquid: the sample group is added with 1 mL 20 mmol / L PNPG solution and 0.5 mL of the sample to be tested in 1 mL 0.1 mol / L PBS (pH=6.8) in turn, incubated at 37℃ for 10 min, then added with 0.6 mL of α-glucosidase solution (2 U / mL), continue to react for 20 min, finally added with 1 mL Na2CO3 to terminate the reaction, and the absorbance of the mixed solution is measured at 405 nm, recorded as A. The sample blank group is replaced with PBS instead of α-glucosidase solution, recorded as B, the control group is replaced with PBS instead of the sample to be tested, recorded as C, and the blank group is replaced with PBS instead of the sample to be tested and α-glucosidase solution, recorded as D.

[0033] Step 2, calculate the α-glucosidase inhibition rate, and obtain the strain with the highest α-glucosidase inhibition rate.

[0034] As a further technical solution of the present application, the determination of α-glucosidase inhibition activity in step 3 mainly includes the following steps:

[0035] Step 1, the sample is cell fermentation liquid, cell fermentation supernatant and cell broken liquid, and the following determination system is configured: 0.5 mL of 1 mg / mL α-amylase solution is added into 0.5 mL of the sample to be detected, mixed reaction is carried out at 37℃ for 10 min, 1 mL of 1.5% soluble starch solution is added after being preheated to 37℃, and reaction is carried out at 37℃ for another 5 min, 1 mL of DNS solution is added, boiling water bath is carried out for 5 min, and after the mixed solution is cooled, standing is carried out for 30 min, the absorbance value OD540 nm is determined, and recorded as A sample. The absorbance value of the α-amylase solution replaced by distilled water is recorded as A control, the absorbance value of the sample to be detected replaced by distilled water is recorded as A max, and the absorbance value of the α-amylase and the sample to be detected replaced by distilled water is recorded as A min.

[0036] Step 2, the dipeptidyl peptidase-IV inhibition rate is calculated, and the strain with the highest dipeptidyl peptidase-IV inhibition rate is obtained.

[0037] As a further technical scheme of the application, the determination of the dipeptidyl peptidase-IV inhibition activity in step 3 mainly includes the following steps:

[0038] Step 1, the sample is cell fermentation supernatant, and the following determination system is configured: an enzyme-labeled plate is taken, and PBS, enzyme solution, substrate solution and the sample are added into different groups respectively, the plate is shaken for 3 s, and incubation is carried out at 37℃ for 10 min. Incubation is carried out at 37℃ for 30 min, and the fluorescence value of each well is detected by a fluorescence enzyme label instrument at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Blank group: 50 μL of 0.1 mol / L PBS (pH 6.8), 170 μL of substrate solution; control group: 20 μL of dipeptidyl peptidase-IV enzyme solution, 30 μL of 0.1 mol / L PBS (pH 6.8), 170 μL of substrate solution; sample group: 20 μL of dipeptidyl peptidase-IV enzyme solution, 30 μL of sample, 170 μL of substrate solution;

[0039] Step 2, the dipeptidyl peptidase-IV inhibition rate is calculated, and the strain with the highest dipeptidyl peptidase-IV inhibition rate is obtained.

[0040] The beneficial effects of the present application are that: by using the method of in vitro experiment, the Lactobacillus paracasei ZL No. 1 with good in vitro inhibition of aldose reductase, alpha-glucosidase, alpha-amylase and dipeptidyl peptidase-IV activity is screened, which can maintain the body glucose homeostasis through multiple pathways, not only effectively control postprandial blood glucose, thereby relieving complications such as retinopathy caused by diabetes. At the same time, the Lactobacillus paracasei ZL No. 1 shows good artificial gastric juice tolerance, can maintain high survival rate in low pH gastric juice and high bile salt environment, ensure the high efficiency of live bacteria to colonize the intestinal tract, and play a long-term probiotic role. In addition, the Lactobacillus paracasei ZL No. 1 also has good cell adhesion, antioxidant capacity and inhibition of pathogenic bacteria growth and adhesion, and the strain can be widely applied to fermented milk, fermented milk beverage and live lactobacillus preparation products, has effective hypoglycemic effect, and is suitable for food development of obese people, insulin resistance and diabetes patients. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Survival rate of the Lactobacillus paracasei in the present application in pH=2 artificial gastric juice for 3 h.

[0042] Figure 2 Survival rate of the Lactobacillus paracasei in the present application in artificial intestinal juice with 0.3% bile salt concentration for 3 h.

[0043] Figure 3 Survival rate of the Lactobacillus paracasei in the present application in artificial intestinal juice with 0.1% bile salt concentration for 3 h.

[0044] Figure 4 Survival rate of the Lactobacillus paracasei in the present application in pH=3 artificial gastric juice for 3 h

[0045] Figure 5 Inhibition rate of the Lactobacillus paracasei in the present application on aldose reductase activity.

[0046] Figure 6 Inhibition rate of the Lactobacillus paracasei in the present application on alpha-glucosidase activity.

[0047] Figure 7 Inhibition rate of the Lactobacillus paracasei in the present application on alpha-amylase activity.

[0048] Figure 8 Inhibition rate of the Lactobacillus paracasei in the present application on dipeptidyl peptidase activity.

[0049] Figure 9 Inhibition ability of the Lactobacillus paracasei in the present application on the growth of each pathogenic bacteria.

[0050] Figure 10 Exclusion inhibition ability of the Lactobacillus paracasei in the present application on Caco-2 cells.

[0051] Figure 11 Competitive inhibition ability of the Paracaseicillus casei of the present application to Caco-2 cells.

[0052] Figure 12 Replacement inhibition ability of the Paracaseicillus casei of the present application to Caco-2 cells.

[0053] Figure 13 DPPH scavenging rate of the Paracaseicillus casei of the present application.

[0054] Figure 14 Total antioxidant capacity of the Paracaseicillus casei of the present application.

[0055] Figure 15 Blood glucose change line graph of each group of experiments in Example 1 of the present application.

[0056] Figure 16 Area under the curve of oral glucose tolerance (OGTT) of each group of experiments in Example 1 of the present application.

[0057] Figure 17 HE staining section of rat pancreatic tissue of each group of experiments in Example 1 of the present application.

[0058] Figure 18 Inflammatory factor IL-1β inhibition ability of each group of experiments in Example 2 of the present application.

[0059] Figure 19 Inflammatory factor IL-6 inhibition ability of each group of experiments in Example 2 of the present application.

[0060] Figure 20 HE staining section of rat retinal tissue of each group of experiments in Example 2 of the present application.

[0061] Figure 21 Blood glucose change line graph of each group of experiments in Example 3 of the present application.

[0062] Figure 22 Area under the curve of oral glucose tolerance (OGTT) of each group of experiments in Example 3 of the present application.

[0063] Figure 23 HE staining section of rat pancreatic tissue of each group of experiments in Example 3 of the present application.

[0064] Figure 24 Inflammatory factor IL-1β inhibition ability of each group of experiments in Example 4 of the present application.

[0065] Figure 25 Inflammatory factor IL-6 inhibition ability of each group of experiments in Example 4 of the present application.

[0066] Figure 26 HE staining sections of rat retinal tissue for each group of experiments in Example 4 of the present application. DETAILED DESCRIPTION

[0067] The embodiments of the present application will be described below in conjunction with relevant examples, and in the following description, many specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the scope of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.

[0068] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an embodiment that is independent or alternative to other embodiments.

[0069] I. Isolation, screening and identification of Lactobacillus paracasei

[0070] The strain of the present application is Lactobacillus paracasei with antioxidant, pathogenic bacteria inhibition and blood glucose lowering functions. It is screened from the feces of long-lived old people in Rugao City, Jiangsu Province by the inventors, and identified as Lactobacillus paracasei, named ZL No. 1. The strain is preserved in the China General Microbiological Culture Collection Center, CGMCC No. 33360; the preservation date is January 10, 2025.

[0071] The present application uses the coating plate separation method to isolate lactic acid bacteria from the feces sample of long-lived old people in Rugao City, Jiangsu Province, and after Gram staining and 16s rDNA sequencing identification, 10 strains of Lactobacillus paracasei are screened out, as shown in the following table:

[0072]

[0073] (I) Screening of Lactobacillus paracasei with excellent artificial gastrointestinal fluid tolerance

[0074] Artificial gastric juice tolerance: Take 1.0 mL of prepared cell resuspension and inoculate into 9.0 mL of pH 2.0 and pH 3.0 artificial gastric juice (containing 0.5% NaCl and 0.3% pepsin), incubate at 37 ℃ for 3 h, and measure the viable cell count at 0 h and 3 h by plate counting method, and calculate the survival rate (%) according to formula (1).

[0075] Artificial intestinal fluid tolerance: 1 mL of cell suspension was inoculated into 9 mL of artificial intestinal fluid solution containing 0.1% and 0.3% bile salts (PBS solution with pH value of 8), respectively, and incubated at 37°C for 3 h. The viable cell count was determined at 0 h and 3 h, respectively, by plate counting method, and the survival rate (%) was calculated according to formula (1).

[0076] The survival rate (%) was calculated by the following formula:

[0077]

[0078] The survival rate test results of L. paracasei in artificial gastric juice are shown in Figure 4 and Figure 1 The 3 h survival rate of the screened L. paracasei in artificial gastric juice with pH value of 3 was all more than 50%, and the survival rate of L. pc ZL No. 1 was the largest, which was 89.09%. The 3 h survival rate of the rest except L. pc 542 in artificial gastric juice with pH value of 2 was all more than 35%, and the survival rate of L. pc 552 was the largest, which was 53.17%.

[0079] The survival rate test results of L. paracasei in artificial intestinal fluid are shown in Figure 3 and Figure 2 The 3 h survival rate of the screened L. paracasei in artificial intestinal fluid with bile salt concentration of 0.1% was all more than 25%, and the survival rate of L. pc ZL No. 1 was the largest, which was 67.24%. After incubation in artificial intestinal fluid with bile salt concentration of 0.3% for 3 h, only L. pc ZL No. 1, L. pc 557, L. pc 558 and L. pc 561 still had viable cells, and the survival rate of L. pc ZL No. 1 was the largest, which was 35.56%.

[0080] According to the above experimental results, L. pc ZL No. 1, L. pc 1093 and L. pc 552 with excellent artificial gastric and intestinal fluid tolerance were screened.

[0081] (II) Inhibition ability of L. paracasei L. pc ZL No. 1 on aldose reductase activity

[0082] The strain with the strongest inhibition ability on aldose reductase activity was screened from L. pc ZL No. 1, L. pc 1093 and L. pc 552, and L. paracasei Shirota strain (L. pc Shirota) and B. animalis BB12 (B. a BB12) were used as reference strains. The sample was cell fermentation supernatant. Aldose reductase was extracted from fresh bovine lens, and the following determination system was configured:

[0083] Blank group: 0.1 mol / L PBS (pH 6.2) 700 μL, 0.6 mmol / L lithium sulfate 100 μL, 0.2 mmol / L NADPH 100 μL, 0.5 mmol / L glycerol 100 μL;

[0084] Sample blank group: 0.1 mol / L PBS (pH 6.2) 600 μL, 0.6 mmol / L lithium sulfate 100 μL, 0.2 mmol / L NADPH 100 μL, sample 100 μL, 0.5 mmol / L glycerol 100 μL;

[0085] Control group: 0.1 mol / L PBS (pH 6.2) 600 μL, 0.6 mmol / L lithium sulfate 100 μL, 0.2 mmol / L NADPH 100 μL, aldose reductase liquid (fresh bovine lens extract) 100 μL, 0.5 mmol / L glycerol 100 μL;

[0086] Sample group: 0.1 mol / L PBS (pH 6.2) 500 μL, 0.6 mmol / L lithium sulfate 100 μL, 0.2 mmol / L NADPH 100 μL, aldose reductase liquid (fresh bovine lens extract) 100 μL, sample 100 μL, 0.5 mmol / L glycerol 100 μL.

[0087] The ultraviolet spectrophotometer was used to measure the absorbance change of NADPH at 340 nM for 2 min. First, the absorbance value of the blank group without enzyme liquid was measured, and then the absorbance change of NADPH within 2 min after adding aldose reductase and intervention was measured. Each sample was repeated three times, and the average value was taken.

[0088] The formula for calculating the inhibition rate of aldose reductase is:

[0089]

[0090] △A 空白 : Blank group absorbance change value

[0091] △A 样品空白 : Sample blank group absorbance change value

[0092] △A 样品 : Sample group absorbance change value

[0093] △A 对照 : Control group absorbance change value

[0094] As Figure 5As shown, the results indicate that the supernatant of L.pc ZL No.1 cell fermentation significantly inhibited aldose reductase activity more than L.pc 552, L.pc 1093, L.pc Shirota and Ba BB12 (p<0.01, p<0.005, p<0.001), with a maximum inhibition rate of 44.5%.

[0095] (III) Inhibitory ability of Lactobacillus paracasei L.pc ZL No.1 on α-glucosidase activity

[0096] The strains with the strongest inhibitory effect on aldose reductase activity were screened from L. pc ZL No.1, L. pc 1093, and L. pc 552, with L. pc Shirota and Bifidobacterium animalis BB12 (Ba BB12) used as reference strains. The samples were cell fermentation broth, cell fermentation supernatant, and cell lysate. For the sample group, 1 mL of 20 mmol / L PNPG solution and 0.5 mL of the test sample were added sequentially to 1 mL of 0.1 mol / L PBS (pH=6.8), incubated at 37℃ for 10 min, followed by the addition of 0.6 mL of α-glucosidase solution (2 U / mL), and the reaction was continued for 20 min. Finally, 1 mL of Na2CO3 was added to terminate the reaction. The absorbance of the mixture was measured at 405 nm and recorded as A. The blank control group (B) had PBS replaced with α-glucosidase solution, the control group (C) had PBS replaced with the test sample, and the blank control group (D) had PBS replaced with both the test sample and the α-glucosidase solution. The formula for calculating the α-glucosidase inhibition rate is:

[0097]

[0098] like Figure 6 As shown, the results indicated that the fermentation supernatant of L. pc ZL No.1 at all concentrations inhibited α-glucosidase activity, significantly higher than that of L. pc 552 and L. pc 1093 (p < 0.001), but not significantly different from that of L. pc Shirota and B. aBB12 (p > 0.05); 10 9 The resuspension of L. pc ZL No.1 cells at CFU / mL showed the highest inhibitory activity against α-glucosidase (14.83%), significantly higher than that of L. pc 552, L. pc 1093, L. pc Shirota, and Ba BB12 (p < 0.001). L. pc ZL No.1 10 8The inhibition rate of the cell resuspension solution was significantly higher than that of L. pc 552, L. pc 1093, L. pc Shirota and B. a BB12 (p<0.001).

[0099] (Four) Inhibition ability of Lactobacillus paracasei L. pc ZL No. 1 on α-amylase activity

[0100] The strains with the strongest inhibition ability on aldose reductase activity were screened from L. pc ZL No. 1, L. pc 1093 and L. pc 552, and Lactobacillus paracasei Daita strain (L. pc Shirota) and animal bifidobacterium BB12 (B. a BB12) were used as reference strains. The samples were cell fermentation liquid, cell fermentation supernatant and cell broken liquid. 0.5 mL of 1 mg / mL α-amylase solution was added to 0.5 mL of the sample to be tested, mixed and reacted at 37°C for 10 min, 1 mL of 1.5% soluble starch solution was preheated to 37°C, then added, and reacted at 37°C for another 5 min, 1 mL of DNS solution was added, boiled in a water bath for 5 min, and the mixed solution was cooled and placed for 30 min. The absorbance value OD540 nm was measured and recorded as A sample. The absorbance value of the α-amylase solution replaced with distilled water was recorded as A control, the absorbance value of the sample to be tested replaced with distilled water was recorded as A max, and the absorbance value of the α-amylase and the sample to be tested replaced with distilled water was recorded as A min. The α-amylase inhibition rate calculation formula is as follows:

[0101]

[0102] As shown in Figure 7 , the results showed that the cell fermentation supernatant, cell resuspension solution and cell broken liquid of L. pc ZL No. 1 at various concentrations all had inhibition ability on α-amylase activity, and were significantly higher than those of L. pc 552, L. pc 1093, L. pc Shirota and B. a BB12 (p<0.01, p<0.005, p<0.001)

[0103] (Five) Inhibition ability of Lactobacillus paracasei L. pc ZL No. 1 on dipeptidyl peptidase-IV activity

[0104] The strains with the strongest ability to inhibit aldose reductase activity were screened from L.pc ZL No.1, L.pc 1093 and L.pc 552, and Lactobacillus paracasei Shirota (L.pc Shirota) and Bifidobacterium animalis BB12 (B.a BB12) were used as reference strains. The samples were cell fermentation supernatant and cell lysate. An enzyme-labeled plate was taken, and PBS, enzyme solution, substrate solution and sample were added respectively according to the following grouping, and the plate was shaken for 3 s, and incubated at 37 °C for 10 min. After incubation at 37 °C for 30 min, the fluorescence value of each well was detected by a fluorescence enzyme label instrument at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

[0105] Blank group: 50 μL 0.1 mol / L PBS (pH 6.8), 170 μL substrate solution;

[0106] Control group: 20 μL dipeptidyl peptidase-IV enzyme solution, 30 μL 0.1 mol / L PBS (pH 6.8), 170 μL substrate solution;

[0107] Sample group: 20 μL dipeptidyl peptidase-IV enzyme solution, 30 μL sample, 170 μL substrate solution;

[0108] Dipeptidyl peptidase-IV inhibition rate calculation formula:

[0109]

[0110] Fblank: blank group fluorescence value; Fsample: sample group fluorescence value; Fcontrol: control group fluorescence value.

[0111] As shown in Figure 8 , the results showed that for L.pc ZL No.1, the cell fermentation supernatant had the highest dipeptidyl peptidase-IV activity inhibition rate of 31.64%, which was significantly higher than that of L.pc 552 and L.pc 1093 (p<0.005, p<0.001). The cell fermentation supernatant and cell lysate of L.pc ZL No.1 had significantly higher dipeptidyl peptidase-IV activity inhibition rates than L.pc Shirota and B.a BB12 (p<0.005, p<0.001).

[0112] (Six) Evaluation of the ability of Lactobacillus paracasei L.pc ZL No.1 to inhibit the growth and adhesion of pathogenic bacteria

[0113] The inhibitory effect of lactic acid bacteria on E. coli ATCC25922, S. aureus ATCC6538, S. typhimurium CMCC50115 and B. subtilis CMCC63501 was determined by single-layer agar plate diffusion method. 100 μL of pathogenic bacteria solution was uniformly coated on LB solid medium, and three holes were punched in the horizontally placed plate using a puncher. 200 μL of cell resuspension solution with a concentration of 10 8 CFU / mL was added to the holes. The plate was placed in an 18 ℃ incubator for 12 h, and then transferred to a 37 ℃ incubator. The diameter of the inhibition zone was measured after the appearance of a clear inhibition zone.

[0114] As shown in Figure 9 , L.pc ZL No.1 had an inhibitory effect on the four pathogenic bacteria, and the diameter of the inhibition zone was more than 25 mm. Among them, the inhibitory ability on E. coli ATCC25922 was the strongest, and the diameter of the inhibition zone was 30.14 mm.

[0115] To explore the ability of L.pc ZL No.1 to inhibit the adhesion of E. coli ATCC25922, S. aureus ATCC6538, S. typhimurium CMCC50115 and B. subtilis CMCC63501 to Caco-2 cells by competition, replacement and exclusion. For the competition test, 0.5 mL (10 8 CFU / mL) of L.pc ZL No.1 and 0.5 mL (10 8 CFU / mL) of four pathogenic bacteria were added to Caco-2 cells at the same time, and incubated at 37 ℃, 5% CO2 for 1 h. After washing with PBS, trypsin digestion was added, and finally the number of adherent pathogenic bacteria was detected by plate colony counting method. In the replacement test, 0.5 mL of pathogenic bacteria cell resuspension was first added to Caco-2 cells, and incubated for 1 h. After washing with PBS, 0.5 mL of L.pc ZL No.1 cell resuspension was added. In the exclusion test, L.pc ZL No.1 cell resuspension was added to the cells and incubated for 1 h, and then pathogenic bacteria cell resuspension was added. The adhesion inhibition rate of L.pc ZL No.1 to pathogenic bacteria was calculated according to the formula.

[0116]

[0117] In the formula: A1 is the number of viable pathogenic bacteria adhering to Caco-2 cells in the absence of L.pc ZL No.1; A2 is the number of viable pathogenic bacteria adhering to Caco-2 cells in the presence of L.pc ZL No.1.

[0118] As shown in Figure 10 to Figure 12 , the results showed that L. pc ZL No. 1 could inhibit the adhesion of E. coli ATCC25922, S. aureus ATCC6538, S. typhimurium CMCC50115 and B. subtilis CMCC635014 to Caco-2 cells by competition, replacement and exclusion.

[0119] (Seven) Evaluation of the antioxidant capacity of L. pc ZL No. 1

[0120] The antioxidant capacity of the cell fermentation supernatant, cell resuspension and cell disruption solution of L. pc ZL No. 1 was investigated, and L. pc Shirota and B. a BB12 were used as reference strains. After the strains were inoculated in MRS medium at an inoculation amount of 2% (v / v) and cultured at 37°C for 24 h, they were centrifuged at 4°C and 8000 rpm for 5 min, and then the total antioxidant capacity and DPPH free radical scavenging capacity of the cell fermentation supernatant, cell resuspension and cell disruption solution were determined.

[0121] As shown in Figure 13 and Figure 14 , the results showed that for L. pc ZL No. 1, the DPPH scavenging rate of the cell fermentation supernatant was the highest, followed by the cell disruption solution. The DPPH scavenging rate of the cell fermentation supernatant and the cell disruption solution of L. pc ZL No. 1 was significantly higher than that of L. pc Shirota and B. a BB12 (p<0.005, p<0.001), the DPPH scavenging rate of the cell suspension of L. pc ZL No. 1 was significantly lower than that of L. pc Shirota (p<0.05), and there was no significant difference compared with B. a BB12 (p>0.05).

[0122] For L. pc ZL No. 1, the total antioxidant capacity of the cell fermentation supernatant was the highest, followed by the cell suspension. The total antioxidant capacity of the cell fermentation supernatant of L. pc ZL No. 1 was significantly lower than that of L. pc Shirota and B. a BB12 (p<0.001), but the total antioxidant capacity of the cell suspension and the cell disruption solution of L. pc ZL No. 1 was significantly higher than that of L. pc Shirota and B. a BB12 (p<0.05, p<0.01, p<0.001).

[0123] In summary, it is shown that the cell body and cell disruption product of L. pc ZL No. 1 have good antioxidant capacity.

[0124] II. Examples

[0125] Example 1: Effect of Lactobacillus paracasei Lpc ZL No. 1 on glucose homeostasis in diabetic rats

[0126] SPF level male Sprague-Dawley rats were selected, the rats were 6-8 weeks old, and the body weight was 200-220 g. The living environment temperature was controlled at 22-24℃, the light in the animal room was normal natural alternation, and the normal day and night life regularity of the rats was guaranteed. The rats were adaptively fed for one week, 10 rats were selected as the normal group according to the random number table method, and the remaining 70 rats were modeled. First, fasting for 12 h, then giving streptozotocin solution at 35 mg / kg, intraperitoneal injection for two days, and the normal group was given the same amount of sodium citrate buffer for intraperitoneal injection. After 3 days, the tail vein blood of the rats was drawn, and the blood glucose was measured to be >16.7 mmol / L, which was the modeling success, and was a diabetic rat. The normal group of rats was fed with regular diet, and the remaining rats were fed with high-sugar and high-fat diet.

[0127] The 60 rats successfully modeled were randomly divided into 6 groups of 10 rats each, namely the normal group, the model group, the acarbose group, the low-dose experimental group, the medium-dose experimental group, and the high-dose experimental group, and were continuously treated for 8 weeks. Before the end of treatment (12 weeks), oral glucose tolerance test (OGTT) was performed on the rats, specifically, the rats were fasted for 12 h without water restriction, the blood glucose content was detected (0 min), then the rats were given a glucose solution of 2 g / kg by gavage, and the blood glucose values at 30 min, 60 min, 90 min and 120 min were measured respectively. The blood glucose-time curve was drawn, and the area under the curve (AUC) of each treatment group was calculated.

[0128] After treatment, the rats were sacrificed by cervical dislocation, the pancreas was quickly removed, rinsed with normal saline, and immersed in 4% cell tissue fixative for 24 h. The 5 μm thick retinal paraffin sections were prepared, HE staining was performed according to the kit instructions, and the tissue sections were observed and analyzed under an optical microscope.

[0129] The specific treatments of each group are as follows:

[0130] Normal group: 1 mL / (100 g·d) of sterile normal saline was given by gavage every day;

[0131] Model group: 1 mL / (100 g·d) of sterile normal saline was given by gavage every day;

[0132] Acarbose group: 3 mg / (100 g·d) of acarbose diluted in sterile normal saline was given by gavage every day;

[0133] Low-dose experimental group: 1 x 10 7 CFU / mL L.pc ZL No. 1 cell suspension 1 mL / (100 g·d) was given by gavage;

[0134] Medium-dose experimental group: 1 x 10 8 CFU / mL L.pc ZL No. 1 cell suspension 1 mL / (100 g·d) was given by gavage;

[0135] High-dose experimental group: 1 x 10 9 CFU / mL L.pc ZL No. 1 cell suspension 1 mL / (100 g·d) was given by gavage;

[0136] As Figure 15 to Figure 16 shown, the results showed that the effect of Lactobacillus paracasei L.pc ZL No. 1 on the glucose homeostasis of rats was evaluated by oral glucose tolerance test (OGTT), and the change of glucose concentration within 120 min was observed. The results showed that the change of blood glucose in normal group was relatively slow after gavage of glucose, while the change of blood glucose in diabetic rats was relatively large after gavage of glucose. The blood glucose value of the model group rapidly increased at 30 min, and the blood glucose of each dose of Lactobacillus paracasei L.pc ZL No. 1 cell suspension group was always lower than that of the model group.

[0137] The area under the glucose curve results showed that the model group was significantly higher than the acarbose group, the low-dose group, the medium-dose group and the high-dose group (p<0.0001, p<0.05, p<0.001, p<0.001). These results showed that Lactobacillus paracasei L.pc ZL No. 1 could alleviate the impaired glucose tolerance of diabetic rats.

[0138] As Figure 17As shown, the structural function of the pancreas is closely related to the occurrence and development of diabetes, so HE staining of rat pancreatic tissue was used for histopathological observation. The results showed that the morphological structure of the pancreas in the normal group (A) was normal, the islets were round or oval, the islet cells were arranged in order, the shape was regular, and the islets and the outer tissue edge were clear. In contrast, the pancreatic histology of the model group (B) was significantly damaged, the shape of the islets became irregular, the islet cells were arranged in disorder, the atrophic islets were infiltrated by inflammatory cells, and the islet outer tissue and edge were not clear. In addition, the number of cells in the islet cell area was significantly reduced, the cytoplasm was reduced, the volume was reduced, and a large number of vacuoles appeared in the cytoplasm. Compared with the model group, the damage to the pancreatic tissue of the acarbose group (D) was improved, the arrangement and shape of the cells were more orderly than those of the model group, the cytoplasmic vacuoles were reduced, the inflammatory cell infiltration was reduced, the atrophy of the islets was reduced, and the outer tissue edge was clearer. With the increase of the dose, the low-dose group (E), the medium-dose group (F), and the high-dose group (G) showed a more obvious trend of recovery of pancreatic tissue structure. The islet cells were arranged more orderly, the cytoplasmic vacuoles were reduced, the islets and the outer tissue edge were clear, the atrophy of the islets was relieved, the inflammatory cell infiltration was reduced, and the pancreatic tissue structure was more complete than that of the model group.

[0139] In summary, Lactobacillus paracasei L.pc ZL No. 1 can maintain glucose homeostasis in diabetic rats by improving glucose tolerance and histopathological changes in the pancreas of diabetic rats.

[0140] Example 2: Effect of Lactobacillus paracasei L.pc ZL No. 1 on retinopathy in diabetic rats

[0141] SPF male Sprague-Dawley rats were selected, the rat age was 6-8 weeks, and the body weight was 200-220 g. The survival environment temperature was controlled at 22-24℃, the light of the animal room was normal and alternated naturally to ensure the normal day and night life of the rats. The rats were adaptively fed for one week, 10 rats were selected as the normal group according to the random number table method, and the remaining 70 rats were modeled. First, the rats were fasted for 12 h, then given streptozotocin solution at 35 mg / kg, and intraperitoneal injection was performed for two consecutive days. The normal group was given the same amount of sodium citrate buffer for intraperitoneal injection. After 3 days, the tail vein blood of the rats was extracted, and the blood glucose was determined to be >16.7 mmol / L, which was considered as a successful modeling, i.e. a diabetic rat. The normal group of rats was fed with regular diet, and the remaining rats were fed with high-sugar and high-fat diet.

[0142] The 60 successfully modeled rats were randomly divided into 6 groups, 10 rats in each group, namely the normal group, the model group, the calcium dobesilate group, the low-dose experimental group, the medium-dose experimental group, and the high-dose experimental group, and the continuous treatment lasted for 8 weeks.

[0143] After 8 weeks of intragastric treatment, all experimental rats were fasted overnight. Fresh blood was collected from the orbit into 10 mL centrifuge tubes, which were left at room temperature for 60 min, centrifuged at 3500 rpm for 10 min, and the upper serum was carefully collected and stored in 1.5 mL sterile centrifuge tubes at -80°C. The levels of inflammatory factors IL-1β and IL-6 in the rat serum were detected using an ELISA kit (Wuhan Elabscience Biotechnology Co., Ltd.), and the specific operation was performed according to the kit instructions.

[0144] After 8 weeks of intragastric treatment, the rats were sacrificed by cervical dislocation, and the eyeballs were quickly removed and rinsed with physiological saline. The eyeballs were soaked in eyeball fixing solution for 48 h, and 5 μm thick retinal paraffin sections were routinely prepared. HE staining was performed according to the kit instructions, and the tissue sections were observed and analyzed under a light microscope.

[0145] The specific treatments of each group are as follows:

[0146] Normal group: 1 mL / (100 g·d) sterile normal saline was given by intragastric administration every day;

[0147] Model group: 1 mL / (100 g·d) sterile normal saline was given by intragastric administration every day;

[0148] Calcium dobesilate group: 3 mg / (100 g·d) calcium dobesilate diluted in sterile normal saline was given by intragastric administration every day;

[0149] Low-dose experimental group: 1×10 7 CFU / mL L.pc ZL No. 1 cell suspension 1 mL / (100 g·d) was given by intragastric administration every day;

[0150] Medium-dose experimental group: 1×10 8 CFU / mL L.pc ZL No. 1 cell suspension 1 mL / (100 g·d) was given by intragastric administration every day;

[0151] High-dose experimental group: 1×10 9 CFU / mL L.pc ZL No. 1 cell suspension 1 mL / (100 g·d) was given by intragastric administration every day;

[0152] For example, Figure 18 and Figure 19As shown, diabetes is often accompanied by various inflammatory reactions, and inflammatory factors can aggravate diabetic retinopathy, so the level of inflammatory factors indirectly reflects the occurrence and development process of diabetic retinopathy. In this study, the serum inflammatory factors of rats were detected by ELISA method, and the results showed that compared with the normal group, the levels of IL-1β and IL-6 in the model group were significantly increased (p<0.01). The levels of inflammatory factors IL-1β in the low-dose group and the medium-dose group were significantly lower than those in the model group (p<0.05, p<0.01), and the level of IL-1β in the high-dose group was also lower than that in the model group but had no significant difference (p>0.05). The levels of inflammatory factors IL-6 in the low-dose group, the medium-dose group and the high-dose group were significantly lower than those in the model group (p<0.01, p<0.01, p<0.01). This shows that Lactobacillus paracasei L.pc ZL No. 1 can alleviate the inflammatory level of diabetic retinal lesions in rats.

[0153] As shown, diabetic retinopathy is a chronic complication with a very high incidence in diabetic patients. Its occurrence is mainly due to the long-term high blood sugar state of the patient's body. Over time, the patient's retinal blood vessels will be damaged, and the histopathological changes are also an important indicator for judging retinopathy. Therefore, the rat retinal tissue was stained with HE for histopathological observation. Figure 20 The results showed that the retinal thickness of the normal group (A) rats was normal, and each layer was complete and clear. The ganglion cell layer, inner nuclear layer and outer nuclear layer cells were normal in shape and arranged in order, and no obvious pathological changes were observed. Compared with the normal group, the retinal thickness of the model group (B) was significantly thinned, and the ganglion cell layer, inner nuclear layer and outer nuclear layer were arranged loosely and disorderly. The number of cells in each layer was reduced, and edema or vacuole-like changes occurred. The retinal thickness of the calcium dobesilate group (C) was thinner than that of the normal group but thicker than that of the model group. The cells were arranged densely, the number of retinal ganglion cells was reduced, and the structure of each layer of the retina was relatively closer to the normal group. The retinal ganglion cell layer, inner nuclear layer and outer nuclear layer of the low-dose group (E), the medium-dose group (F) and the high-dose group (G) were arranged loosely and accompanied by cell number reduction or thinning. Compared with the model group, the improvement was reduced. The high-dose group (G) had a relatively more obvious effect, and the cell arrangement was dense, the retinal thickness increased, and the cell number reduction was slight.

[0154] In summary, Lactobacillus paracasei L.pc ZL No. 1 can alleviate the damage of diabetic rat retinal.

[0155] Example 3: Effect of Lactobacillus paracasei L.pc ZL No. 1 fermented milk on glucose homeostasis in diabetic rats

[0156]

[0157] ​SPF level male Sprague-Dawley rats were selected, the rat age was 6-8 weeks, and the body weight was 200-220 g. The living environment temperature was controlled at 22-24℃, the light of the animal room was normal natural alternation, and the normal day and night life regularity of the rats was ensured. The rats were adaptively fed for one week, 10 rats were extracted according to the random number table method as the normal group, and the remaining 70 rats were modeled. First, fasting for 12 h, then giving streptozotocin solution at 35 mg / kg, intraperitoneal injection for two days, and the normal group was given the same amount of sodium citrate buffer for intraperitoneal injection. After 3 days, the tail vein blood of the rats was extracted, and the blood glucose was >16.7 mmol / L, which was the modeling success, that was, the diabetic rats. The normal group of rats was fed according to the conventional diet, and the remaining rats were fed with high-sugar and high-fat diet.

[0158] The 40 rats successfully modeled were randomly divided into 4 groups according to 10 rats per group, namely the normal group, the model group, the acarbose group 4 and the fermented milk experimental group, and the continuous treatment was 8 weeks.

[0159] After 8 weeks of gavage treatment, all experimental rats were fasted overnight. The orbital blood was taken, fresh blood was collected into 10 mL centrifuge tubes, and the blood was allowed to stand at room temperature for 60 min, centrifuged at 3500 rpm for 10 min, and the upper serum was carefully collected and stored in 1.5 mL sterile centrifuge tubes, and stored in a-80℃ refrigerator. The levels of inflammatory factors IL-1β and IL-6 in the serum of rats were detected by ELISA kit (Wuhan Elabscience Biotechnology Co., Ltd.), and the specific operation was carried out according to the kit instructions.

[0160] After 8 weeks of gavage treatment, the rats were sacrificed by cervical dislocation, the eyeballs were quickly removed, rinsed with physiological saline, and immersed in eyeball fixing solution for 48 h. The retinal paraffin sections of 5 μm thickness were made conventionally, HE staining was carried out according to the kit instructions, and the tissue section pathological observation and analysis were carried out under an optical microscope.

[0161] The specific treatments of each group are as follows:

[0162] Normal group: 1 mL / (100 g·d) sterile normal saline was given by gavage every day;

[0163] Model group: 1 mL / (100 g·d) sterile normal saline was given by gavage every day;

[0164] Acarbose group: 3 mg / (100 g·d) acarbose diluted in sterile normal saline was given by gavage every day;

[0165] Fermented milk experimental group: 1×10 9Fermented milk with CFU / mL L.pc ZL No.1 live bacteria was administered by gavage at a dose of 1 mL / (100 g ·d);

[0166] like Figure 21 and Figure 22 As shown, the results indicated that the effect of *Lactobacillus paracasei* L.pc ZL No.1 fermented milk on glucose homeostasis in rats was evaluated using an oral glucose tolerance test (OGTT), and the changes in glucose concentration over 120 min were observed. The results showed that after glucose gavage, the blood glucose levels in the normal group rats changed more slowly, while those in diabetic rats changed more significantly. The blood glucose level in the model group rose rapidly at 30 min, and the blood glucose level in the fermented milk group remained lower than that in the model group.

[0167] The area under the glucose curve (AUC) showed that the model group had significantly higher AUCs than the acarbose group and the fermented milk group (p < 0.0001, p < 0.01). These results indicate that Lactobacillus paracasei L.pc ZL No.1 fermented milk can alleviate impaired glucose tolerance in diabetic rats.

[0168] like Figure 23 As shown, the structure and function of the pancreas are closely related to the occurrence and development of diabetes. Therefore, histopathological observation was performed using HE staining of rat pancreatic tissue. The results showed that the normal group (A) had normal pancreatic tissue morphology and structure, with round or oval islets, neatly arranged and regularly shaped islet cells, and clear edges between the islets and surrounding tissues. In contrast, the model group (B) showed significant histological damage to the pancreas, with irregular islet shapes, disordered islet cell arrangement, atrophied islets infiltrated by inflammatory cells, and unclear edges and surrounding tissues. In addition, the number of cells in the islet cell region was significantly reduced, the cytoplasm was reduced, the cell volume was smaller, and a large number of vacuoles appeared in the cytoplasm. Compared with the model group, the acarbose group (D) showed improved pancreatic tissue damage, with cell arrangement and morphology closer to the normal group, neat and orderly arrangement, reduced cytoplasmic vacuoles, reduced inflammatory cell infiltration, reduced islet atrophy, and clearer edges in the surrounding tissues. The condition of pancreatic tissue in the fermented milk group (H) was also improved compared with that in the model group. The pancreatic islet cells were arranged more neatly and had a more regular shape. The number of vacuoles in the cytoplasm was reduced, the degree of pancreatic islet atrophy was reduced, the outer tissue edge was clearer, and the signs of lesions were reduced. This indicates that fermented milk also has a certain ameliorative effect on pancreatic tissue damage in diabetic model rats.

[0169] In summary, Lactobacillus paracasei L.pc ZL No.1 fermented milk can maintain glucose homeostasis in diabetic rats by improving glucose tolerance and histopathological changes in the pancreas.

[0170] Example 4: Effects of Lactobacillus paracasei L.pc ZL No.1 on retinopathy in diabetic rats

[0171] SPF level male Sprague-Dawley rats were selected, the rat age was 6-8 weeks, and the body weight was 200-220 g. The living environment temperature was controlled at 22-24℃, the light of the animal room was normal natural alternation, and the normal day and night life regularity of the rats was ensured. The rats were adaptively fed for one week, 10 rats were extracted according to the random number table method as the normal group, and the remaining 70 rats were modeled. First, fasting for 12 h, then giving streptozotocin solution at 35 mg / kg, intraperitoneal injection for two days, and the normal group was given the same amount of sodium citrate buffer for intraperitoneal injection. After 3 days, the tail vein blood of the rats was extracted, and the blood glucose was >16.7 mmol / L, which was the modeling success, and it was a diabetic rat. The normal group of rats was fed according to the conventional diet, and the remaining rats were fed with high-sugar and high-fat diet.

[0172] The 40 rats successfully modeled were randomly divided into 4 groups according to 10 rats per group, namely the normal group, the model group, the calcium dobesilate group and the fermented milk experimental group, and the continuous treatment was 8 weeks.

[0173] After 8 weeks of gavage treatment, all experimental rats were fasted overnight. The orbital blood was taken, fresh blood was collected into 10 mL centrifuge tubes, and the blood was allowed to stand at room temperature for 60 min, centrifuged at 3500 rpm for 10 min, and the upper serum was carefully collected and stored in 1.5 mL sterile centrifuge tubes, and stored in a-80℃ refrigerator. The levels of inflammatory factors IL-1β and IL-6 in the serum of rats were detected by ELISA kit (Wuhan Elabscience Biotechnology Co., Ltd.), and the specific operation was carried out according to the kit instructions.

[0174] After 8 weeks of gavage treatment, the rats were sacrificed by cervical dislocation, the eyeballs were quickly removed, rinsed with physiological saline, and immersed in eyeball fixing solution for 48 h. The retinal paraffin sections of 5 μm thickness were routinely prepared, HE staining was performed according to the kit instructions, and the tissue sections were observed and analyzed under an optical microscope.

[0175] The specific treatments of each group are as follows:

[0176] Normal group: 1 mL / (100 g·d) of sterile normal saline was given by gavage every day;

[0177] Model group: 1 mL / (100 g·d) of sterile normal saline was given by gavage every day;

[0178] Calcium dobesilate group: 3 mg / (100 g·d) of calcium dobesilate diluted in sterile normal saline was given by gavage every day;

[0179] Fermented milk experimental group: 1×10 9CFU / mL L.pc ZL No.1 viable bacteria in fermented milk 1 mL / (100 g·d) gavage.

[0180] As shown in Figure 24 and Figure 25 shown, the results showed that diabetes was often accompanied by various inflammatory reactions, and inflammatory factors could aggravate diabetic retinopathy, so the level of inflammatory factors indirectly reflected the occurrence and development process of diabetic retinopathy. In this study, the serum inflammatory factors of rats were detected by ELISA method. The results showed that compared with the normal group, the levels of IL-1β and IL-6 in the model group were significantly increased (p<0.01). The level of inflammatory factor IL-1β in the fermented milk group was significantly lower than that in the model group (p<0.05), and the level of IL-1β in the calcium hydroxybenzenesulfonate group was also lower than that in the model group but no significant difference (p>0.05). The level of inflammatory factor IL-6 in the fermented milk group was significantly lower than that in the model group (p<0.01), and the level of IL-6 in the calcium hydroxybenzenesulfonate group was also lower than that in the model group but no significant difference (p>0.05). This showed that the fermented milk of Paracasei L.pc ZL No.1 could alleviate the inflammatory level of diabetic rat retinopathy.

[0181] As shown in Figure 26 , the normal group (A) rat retinal thickness was normal, the layers were complete and clear, the ganglion cell layer, inner nuclear layer and outer nuclear layer cell morphology was normal, and the arrangement was neat, and no obvious pathological changes were found. Compared with the normal group, the model group (B) retinal thickness was significantly thinned, the ganglion cell layer, inner nuclear layer and outer nuclear layer were arranged loosely and disorderly, the number of cells in each layer was reduced, and edema or vacuole-like changes appeared. The retinal thickness of the calcium hydroxybenzenesulfonate group (C) was thinner than that of the normal group but thicker than that of the model group, the cell arrangement was dense, the number of retinal ganglion cells was reduced, and the structure of each layer of the retina was relatively closer to the normal group. The retinal thickness of the calcium hydroxybenzenesulfonate group (C) was thinner than that of the normal group but thicker than that of the model group, the cell arrangement was dense, the number of retinal ganglion cells was reduced, and the structure of each layer of the retina was relatively closer to the normal group. The retinal ganglion cell layer, inner nuclear layer and outer nuclear layer of the fermented milk group (H) were arranged more neatly than the model group, the retinal thickness increased, the degree of cell number reduction was light,

[0182] In summary, the fermented milk prepared by Paracasei L.pc ZL No.1 can alleviate the damage of diabetic rat retina.

[0183] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A Lactobacillus paracasei ZL No. 1, characterized in that: The strain is derived from a long-lived old man in Rugao, Jiangsu, and was preserved in China General Microbiological Culture Collection Center on January 10, 2025, with a preservation number of CGMCC No. 33360.

2. The Lactobacillus paracasei ZL No. 1 of claim 1, characterized in that: The strain has excellent aldehyde sugar reductase inhibition ability, and also has excellent α-glucosidase, α-amylase and dipeptidyl peptidase-IV inhibition ability.

3. The Lactobacillus paracasei ZL No. 1 according to claim 1-2, characterized in that: The strain has the effect of maintaining glucose homeostasis of the body.

4. The Lactobacillus paracasei ZL No. 1 according to claim 1-2, characterized in that: The strain can relieve retinopathy caused by diabetes.

5. The Lactobacillus paracasei ZL No. 1 according to claim 1-2, characterized in that: The strain is applied in products such as fermented milk, fermented milk beverage and live lactic acid bacteria preparation, which have the effect of maintaining glucose homeostasis of the body.

6. The Lactobacillus paracasei ZL No. 1 according to claim 1-2, characterized in that: The strain is applied in products such as fermented milk, fermented milk beverage and live lactic acid bacteria preparation, which have the effect of improving retinopathy caused by diabetes.

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