Application of Lactobacillus plantarum SS18-119 in the preparation of products to improve eye function

Abstract

This invention relates to the field of biotechnology, and more particularly to the application of *Lactobacillus plantarum* SS18-119 and its fermentation products. The application of the aforementioned *Lactobacillus plantarum* SS18-119 in the preparation of products that improve eye function. The fermentation products obtained from the fermentation of the aforementioned *Lactobacillus plantarum* SS18-119 are used in any of the following (1)-(4): (1) preparing products that reduce the content of pro-inflammatory factors in the eyes; (2) preparing products that increase the content of anti-inflammatory factors in the eyes; (3) preparing products that relieve dry eyes; (4) preparing products that regulate the fundus. This invention utilizes the application of *Lactobacillus plantarum* SS18-119 in improving eye function, and has significant effects in preventing myopia and relieving eye fatigue.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to Lactobacillus plantarum (… Lactobacillus plantarum Application of Fullerton-H-SS18-119 and its fermentation products in the preparation of products that improve eye function. Background Technology

[0002] *Lactobacillus plantarum* Fullarton-H-SS18-119 is a space-mutated strain that produces high levels of extracellular polysaccharides (EPS). Extracellular polysaccharides in this strain are mucopolysaccharides or capsular polysaccharides secreted on the cell surface to resist adverse environmental factors. They are important secondary metabolites. In the natural environment, extracellular polysaccharides typically protect microbial cells, such as preventing cell dehydration, protecting against macrophage invasion or bacteriophage infection, and resisting antibiotics or toxic substances. They also stabilize osmotic pressure and participate in cell signaling and cell structure. *Lactobacillus plantarum* Fullarton-H-SS18-119, as a special *Lactobacillus plantarum* strain with high extracellular polysaccharide production and anti-lipid peroxidation effects, exhibits excellent anti-lipid peroxidation efficacy and has significant health application value. The strain has already been protected in a prior patent application (application number 201810113533.9, application date February 5, 2018, publication number CN108165512A, publication date June 15, 2018). This strain was deposited on January 2, 2018, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 15150. The method for preparing the extracellular polysaccharide can be found in sections

[0118] to

[0122] of the aforementioned prior patent application.

[0003] Nowadays, more and more people spend long hours in front of electronic screens every day, which easily exacerbates eye fatigue and induces visual strain. Visual strain, also known as eye fatigue, is a common eye condition. The dry, itchy, and sore eyes, blurred vision, and even decreased vision it causes directly affect a person's work and life. In addition to paying attention to rest and improving nutrition, medication is also a very effective way to care for the eyes, but the effect is relatively short-lived and requires repeated use, which can easily lead to drug dependence over time.

[0004] Meanwhile, the retina of the eye is an organ belonging to the central nervous system, and mature retinal cells do not normally divide like neurons. Therefore, when the function of retinal cells declines, visual dysfunction is more likely to occur, and the aging process is faster. Relieving and preventing eye fatigue is receiving increasing attention. Summary of the Invention

[0005] The purpose of this invention is to provide *Lactobacillus plantarum* (… Lactobacillus plantarumA novel use of Fullerton-H-SS18-119 and its fermentation products is in the preparation of products that improve eye function.

[0006] Specifically, it provides the fermentation products of Lactobacillus plantarum Fullerton-H-SS18-119 for any of the following applications (1)-(4):

[0007] (1) Prepare products that reduce pro-inflammatory factors in the eye;

[0008] (2) Prepare products that increase anti-inflammatory factors in the eyes;

[0009] (3) Prepare products to relieve dry eyes;

[0010] (4) Prepare products to regulate or improve the fundus.

[0011] The fermentation product was obtained by fermenting the above-mentioned Lactobacillus plantarum Fullerton-H-SS18-119 to obtain the fermentation broth, and then freeze-drying it at -30℃.

[0012] This product is a medicine.

[0013] Specifically, the pro-inflammatory factors are interleukin-2, interleukin-6, and / or interferon-γ; the anti-inflammatory factor is interleukin-10.

[0014] The present invention also provides a compound preparation, the raw materials of which include, by weight: 5-10 parts of the above-mentioned fermentation product, 1-2 parts of casein phosphopeptide, 1 part of hydroxyethyl cellulose, 15-30 parts of water, and 30-60 parts of vegetable oil.

[0015] Preferably, the vegetable oil is at least one of soybean oil, rapeseed oil, peanut oil, and olive oil.

[0016] The preparation method of the above-mentioned compound preparation provided by the present invention includes the following steps: adding the above-mentioned fermentation product and casein phosphopeptide to water and stirring evenly, adding hydroxyethyl cellulose and ultrasonically emulsifying for 10-20 min to obtain an aqueous phase; heating vegetable oil to 70-80℃, adding the aqueous phase dropwise under stirring and homogenizing for 10-20 min, and cooling.

[0017] Preferably, the frequency of ultrasonic emulsification is 5-10 kHz.

[0018] Preferably, the homogenization temperature is 35-45℃ and the homogenization pressure is 30-40MPa.

[0019] Beneficial effects:

[0020] This invention discloses the application of Lactobacillus plantarum Fullerton-H-SS18-119 in improving eye function, with significant effects in preventing and relieving eye fatigue.

[0021] This invention utilizes the extracellular polysaccharides and casein phosphopeptides in the fermentation products of *Lactobacillus plantarum* Fullarton-H-SS18-119 to form aggregates through electrostatic binding, which then form a water-in-oil emulsion, providing a stable environment for the internal bioactive components. Experiments have shown that this invention, through the gastrointestinal environment and intestinal absorption, can effectively relieve dry eyes, improve eye fatigue, and achieve eye care.

[0022] The compound formulation obtained by this invention has a simple process operation, is suitable for industrial production, and is suitable for large-scale promotion and application. Attached Figure Description

[0023] Figure 1 This is a comparison of the average corneal fluorescence area of ​​mice in the blank control group, model group, positive drug group, and fermentation product group.

[0024] Figure 2 This is a comparison of tear secretion and tear film breakup time in mice from the blank control group, model group, positive drug group, and fermentation product group.

[0025] Figure 3 This is a comparison chart of IL-2 and IL-6 levels in the serum of mice in the blank control group, model group, positive drug group, and fermentation product group.

[0026] Figure 4 This is a comparison of the levels of IFN-γ and IL-10 in the serum of mice in the blank control group, model group, positive drug group, and fermentation product group.

[0027] Figure 5 This is a comparison of the average corneal fluorescence area of ​​mice in the blank control group, model group, positive drug group, fermentation product group, Example 5 group, Example 6 group, and comparative group.

[0028] Figure 6 This is a comparison chart of tear secretion and tear film breakup time in mice from the blank control group, model group, positive drug group, fermentation product group, Example 5 group, Example 6 group, and comparative group.

[0029] Figure 7 This is a comparison chart of IL-2 and IL-6 levels in the ocular serum of mice in the blank control group, model group, positive drug group, fermentation product group, Example 5 group, Example 6 group, and comparative group.

[0030] Figure 8This is a comparison chart of IFN-γ and IL-10 levels in the ocular serum of mice in the blank control group, model group, positive drug group, fermentation product group, Example 5 group, Example 6 group, and comparative group. Detailed Implementation

[0031] The present invention will be further explained below with reference to specific embodiments.

[0032] The *Lactobacillus plantarum* SS18-119 in the following embodiments is the *Lactobacillus plantarum* protected by Chinese invention patent CN 108165512 B, with an authorization announcement date of December 13, 2019. Lactobacillus plantarum The strain Fullarton-H-SS18-119 was deposited on January 2, 2018, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 15150.

[0033] The MRS liquid culture medium in the following examples consists of a solute and a solvent. The solvent is distilled water, and the solutes and their concentrations are as follows: casein peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 10 g / L, sodium acetate 5 g / L, diammonium citrate 2 g / L, Tween-80 1 mL / L, K₂HPO₄ 2 g / L, MgSO₄•7H₂O 0.2 g / L, MnSO₄•H₂O 0.05 g / L, pH 7.0. The MRS solid culture medium is obtained by adding 1.7 g / L of agar powder to the MRS liquid culture medium.

[0034] Example 1: Preparation and purification of extracellular polysaccharides

[0035] Lactobacillus plantarum SS18-119 was enriched and activated three times in MRS liquid medium to obtain activated MRS test tube culture. The activated MRS test tube culture was transferred at an inoculum rate of 3% (v / v) into an Erlenmeyer flask containing 100 mL of sterile MRS liquid medium and incubated at 37 °C for 20 h to obtain the fermentation broth of Lactobacillus plantarum SS18-119.

[0036] The fermentation broth of *Lactobacillus plantarum* SS18-119 was transferred to centrifuge tubes and centrifuged at 8000 rpm for 10 min at 4°C using a refrigerated centrifuge. The supernatant was collected. A 1 / 5 volume of 10 g / 100 mL pH 5.0 trichloroacetic acid aqueous solution was added to the supernatant and the mixture was shaken at 4°C for 30 min to obtain the treated solution. The treated solution was centrifuged at 10000 rpm for 10 min at 4°C, and the supernatant was collected. Three times the volume of 95% cold ethanol was added to the supernatant and the mixture was treated at 4°C for 11–12 h. The mixture was then centrifuged (10000 rpm for 10 min at 4°C) to collect the precipitate, which contained polysaccharides. This polysaccharide-containing substance was dissolved in sterile distilled water and then dialyzed at 4°C for 12 h using a pre-treated dialysis bag (molecular weight cutoff 8000–14000 Da) (with sterile distilled water changed three times during the process). The dialysate was the extracellular polysaccharide extract.

[0037] The sugar content of the above extracellular polysaccharide extract was determined using the phenol-sulfuric acid method: The above extracellular polysaccharide extract was diluted to 10 mL with distilled water. 2 mL of this solution was transferred to a 25 mL colorimetric tube. 2 mL of 6% phenol was added to the tube, followed by the rapid addition of 10 mL of concentrated sulfuric acid. After mixing, the mixture was allowed to stand at room temperature for 20 min. A blank was prepared by adjusting the absorbance to 0 with 2.0 mL of distilled water using the same colorimetric procedure. The absorbance value (A) at 490 nm was measured using a UV spectrophotometer. 490nm The measured absorbance y value of the sample was substituted into the regression equation y = 0.5985x + 0.0223 of the glucose standard curve to obtain the polysaccharide content x value (g / L). The results showed that the polysaccharide content in the above extracellular polysaccharide extract, calculated as glucose, was 2.4 g / L.

[0038] The method for preparing the glucose standard curve is as follows: Accurately weigh 100 mg of standard glucose into a 500 mL volumetric flask and add water to the mark. Mix all reagents evenly in a colorimetric tube according to the amounts shown in Table 1, let stand for 10 min, shake well, and let stand at room temperature for 20 min before measuring the absorbance at 490 nm (adjust to 0 using 2.0 mL of distilled water as a blank after the same colorimetric procedure). Plot the glucose content on the x-axis and the absorbance (A) on the x-axis. 490nm A standard curve was plotted with y=0.5985x+0.0223 (R²). The regression equation for the glucose standard curve was obtained as y=0.5985x+0.0223(R²). 2 =0.9991).

[0039] Example 2

[0040] The fermentation broth of *Lactobacillus plantarum* SS18-119 obtained in Example 1 was freeze-dried at -30°C to obtain a powdered product, which is the fermentation product.

[0041] Example 3

[0042] Because the C57 / BL6 immunodeficient mice (Liaoning Changsheng Biotechnology Co., Ltd.) have underdeveloped lacrimal glands, it is very easy to establish a dry eye model with a high success rate.

[0043] Thirty-six healthy female C57 / BL6 immunodeficient mice (weighing between 250-300g) were selected and routinely acclimatized for 7 days. All mice survived healthily and had normal ophthalmological examinations. They were randomly divided into four groups: blank control group (CK), model group (MC), positive control group (PC), and fermentation product group (FP), with nine mice in each group.

[0044] A two-stage dehumidification method was employed to control ambient humidity. An adjustable-temperature industrial dehumidifier initially reduced the room humidity to 40±5%, followed by a smart drying chamber (dehumidification range RH 10%-80%) to further reduce the humidity inside the chamber (RH=15±3%). Simultaneously, an adjustable-speed (0-5m / s) noiseless fan was placed 20cm inside the chamber, positioned at the same level as the mice. An anemometer (measurement range 0-30m / s, accuracy ±5%) was used to monitor the airflow. These measures effectively regulated humidity, temperature, and airflow.

[0045] The blank control group was housed in a normal environment (RH=60-80%, T=21-23℃). Mice in the model group, positive drug group, and fermentation product group were housed in the same dry environment (RH=15±3%, WV=2.1±0.2m / s; T=21-23℃) for 14 days, and a dry eye model was created by subcutaneous injection of 0.5mg / 0.2mL scopolamine four times a day (8:30AM, 11:30AM, 1:30PM, 4:30PM).

[0046] During modeling, the fermentation product group was administered the fermentation product solution (0.2 mL / day / mouse) once daily at a fixed time (the fermentation product obtained in Example 2 was mixed evenly with distilled water). The effective dose was 20 mg fermentation product / kg body weight. The positive control group was given 5 μL of 0.05% cyclosporine A eye drops daily for 14 consecutive days, along with an equal volume of distilled water via gavage. After the last administration on day 14, the degree of corneal epithelial damage was detected using sodium fluorescein, and the tear film breakup time and tear secretion volume of each group of mice were measured.

[0047] The specific procedure for detecting the degree of corneal epithelial damage using sodium fluorescein is as follows: One drop of 2% sodium fluorescein saline solution is instilled into the conjunctival sac of the eye. After 1 minute, the eye is gently rinsed with 100 μL of saline. Then, the staining of the mouse cornea is observed under cobalt blue light in a slit lamp, and photographs are taken and recorded. If sodium fluorescein is retained on the corneal surface, it indicates corneal epithelial damage in that area. The corneal fluorescence area of ​​each group is statistically analyzed using software.

[0048] The specific procedure for tear secretion detection is as follows: Fix the mouse in an unanesthetized state, use soft-tipped forceps to lift the lower eyelid of the mouse, then fold one end of the phenol red cotton thread and place it into the lower conjunctival sac. Release the lower eyelid to allow the eyelid to close naturally. After fixing the phenol red cotton thread, time for 1 minute. Then remove the phenol red cotton thread, place it on a ruler paper, measure the length of the red part of the cotton thread, and record it.

[0049] The specific procedure for tear film breakup time (TRT) testing is as follows: Mice are immobilized without anesthesia, and a drop of 2% fluorescein sodium saline solution is instilled into the lower temporal region of the bulbar conjunctiva, ensuring it enters the conjunctival sac. The inner corner of the mouse's eye is stimulated with soft-tipped forceps to induce blinking, promoting the spread of the fluorescein sodium solution across the entire corneal surface. After 1 minute, observation is performed under cobalt blue slit lamp light. With the mouse's eyes open, a timer is set until the first dark spot appears on the cornea; the time value is recorded at this point, which is the TRT breakup time.

[0050] like Figure 1 As shown, the average corneal fluorescence area of ​​mice in the fermentation product group and the positive drug group was higher than that in the model group (P < 0.05) and lower than that in the blank control group (P < 0.05); however, the average corneal fluorescence area of ​​mice in the positive drug group was lower than that in the fermentation product group (P < 0.05).

[0051] like Figure 2 As shown, the model group mice had the highest tear secretion and tear film breakup time, which was inferior to the blank control group (P < 0.05), confirming the successful establishment of the model. The fermentation product group and the positive control group mice had lower tear secretion and tear film breakup time than the model group (P < 0.05), confirming the improving effects of the fermentation product and cyclosporine A; however, the positive control group mice had slightly higher tear secretion and tear film breakup time than the fermentation product group, but the difference was not statistically significant (P > 0.05), indicating that the fermentation product and cyclosporine A had similar effects.

[0052] The mice were then euthanized, and blood was quickly collected from the eyeballs into centrifuge tubes. The centrifuge tubes were placed in a 37°C water bath for 1 hour, and then placed in a 4°C refrigerator for 3-4 hours until the blood clots and shrinks. After centrifugation at 3500 rpm for 10 minutes, the supernatant was collected into clean centrifuge tubes, and the levels of IL-2, IL-6, IFN-γ, and IL-10 in each group of mice were measured using the corresponding ELISA kits.

[0053] like Figure 3 and Figure 4As shown, the model group mice had the highest levels of pro-inflammatory factors and the lowest levels of anti-inflammatory factors, which were inferior to the blank control group (P < 0.05), thus confirming the successful establishment of the model. The fermentation product group mice had lower levels of pro-inflammatory factors than the model group (P < 0.05) and higher levels of anti-inflammatory factors than the model group (P < 0.05), confirming that the fermentation product can promote ocular anti-inflammatory effects. However, the anti-inflammatory effect of the positive control group mice was still superior to that of the fermentation product group (P < 0.05).

[0054] Example 4

[0055] A compound preparation, the raw materials of which include: 5g of the fermentation product obtained in Example 2, 1g of casein phosphopeptide, 1g of hydroxyethyl cellulose, 15g of water, and 30g of soybean oil.

[0056] The preparation method of the above-mentioned compound preparation includes the following steps: adding the fermentation product and casein phosphopeptide obtained in Example 2 into water and stirring evenly, adding hydroxyethyl cellulose and ultrasonically emulsifying for 10 min at an ultrasonic frequency of 5 kHz to obtain an aqueous phase; heating soybean oil to 70°C, adding the aqueous phase dropwise while stirring, homogenizing at 35°C for 10 min at a homogenization pressure of 30 MPa, and then cooling.

[0057] Example 5

[0058] A compound preparation, the raw materials of which include: 10g of the fermentation product obtained in Example 2, 2g of casein phosphopeptide, 1g of hydroxyethyl cellulose, 30g of water, and 60g of soybean oil.

[0059] The preparation method of the above-mentioned compound preparation includes the following steps: adding the fermentation product and casein phosphopeptide obtained in Example 2 into water and stirring evenly, adding hydroxyethyl cellulose and ultrasonically emulsifying for 20 min at an ultrasonic frequency of 10 kHz to obtain an aqueous phase; heating soybean oil to 80°C, adding the aqueous phase dropwise while stirring, homogenizing at 45°C for 20 min at a homogenization pressure of 40 MPa, and then cooling.

[0060] Example 6

[0061] A compound preparation, the raw materials of which include: 8g of the fermentation product obtained in Example 2, 1.5g of casein phosphopeptide, 1g of hydroxyethyl cellulose, 16g of water, and 32g of soybean oil.

[0062] The preparation method of the above-mentioned compound preparation includes the following steps: adding the fermentation product obtained in Example 2 and casein phosphopeptide into water and stirring evenly, adding hydroxyethyl cellulose and ultrasonically emulsifying for 15 min at an ultrasonic frequency of 9 kHz to obtain an aqueous phase; heating soybean oil to 75°C, adding the aqueous phase dropwise while stirring, homogenizing at 40°C for 15 min at a homogenization pressure of 35 MPa, and then cooling.

[0063] Comparative Example

[0064] A compound preparation, the raw materials of which include: 8g of the fermentation product obtained in Example 2, 1g of hydroxyethyl cellulose, 16g of water, and 32g of soybean oil.

[0065] The preparation method of the above-mentioned compound preparation includes the following steps: the fermentation product obtained in Example 2 is added to water and stirred evenly, hydroxyethyl cellulose is added and ultrasonically emulsified for 15 min at an ultrasonic frequency of 9 kHz to obtain an aqueous phase; soybean oil is heated to 75°C, and the aqueous phase is added dropwise while stirring, homogenized at 40°C for 15 min at a homogenization pressure of 35 MPa, and then cooled.

[0066] Example 7

[0067] Twenty-seven healthy female C57 / BL6 immunodeficient mice (weighing between 250-300g) were selected and routinely acclimatized for 7 days. All mice survived and had normal ophthalmological examinations. They were randomly divided into three groups: Example 5 group (SS5), Example 6 group (SS6), and control group (DB), with nine mice in each group.

[0068] All mice were modeled according to the method described in Example 1. During modeling, each group was administered the compound preparation once daily at a fixed time (gavage volume: 0.2 mL / day / mouse). Group 5 received the compound preparation obtained in Example 5 via gavage at a dose of 143.16 mg / kg body weight; Group 6 received the compound preparation obtained in Example 6 via gavage at a dose of 143.16 mg / kg body weight; and the comparative group received the compound preparation obtained in the comparative example via gavage at a dose of 141.24 mg / kg body weight.

[0069] After the last administration on day 14, the degree of corneal epithelial damage was detected using sodium fluorescein, and the tear film breakup time and tear secretion volume were measured in each group of mice. Mice were then sacrificed, and blood was quickly collected from the eyeballs into centrifuge tubes. The centrifuge tubes were placed in a 37°C water bath for 1 hour, then in a 4°C refrigerator for 3-4 hours until the blood clots and shrinks. After centrifugation at 3500 rpm for 10 minutes, the supernatant was collected into clean centrifuge tubes, and IL-2, IL-6, IFN-γ, and IL-10 were measured.

[0070] The blank control group (CK), model group (MC), positive drug group (PC), and fermentation product group (FP) continued to use the corresponding data from Example 1 above.

[0071] like Figure 5 As shown, the average corneal fluorescence area of ​​mice in Example 6 was higher than that in the fermentation product group and the positive drug group (P < 0.05); while the average corneal fluorescence area of ​​mice in the control group was only slightly lower than that in the fermentation product group, with no significant difference (P > 0.05).

[0072] like Figure 6 As shown, the tear secretion and tear film breakup time of mice in Example 6 were higher than those in the fermentation product group and the positive drug group (P < 0.05), indicating that the compound preparation obtained in Example 6 can effectively relieve the symptoms of dry eye in mice.

[0073] like Figure 7 and Figure 8 As shown, the levels of pro-inflammatory factors in mice in Example 6 were lower than those in the positive control group (P < 0.05), while the levels of anti-inflammatory factors were higher than those in the positive control group (P < 0.05), indicating that the compound preparation obtained in Example 6 can effectively reduce inflammation in the eyes.

[0074] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A compound preparation for improving ocular function, characterized in that, Its raw materials, by weight, include: 5-10 parts fermentation product, 1-2 parts casein phosphopeptide, 1 part hydroxyethyl cellulose, 15-30 parts water, and 30-60 parts vegetable oil. The fermentation product is *Lactobacillus plantarum* (… Lactobacillus plantarum The fermentation broth of Fullerton-H-SS18-119 was obtained by freeze-drying. The *Lactobacillus plantarum* Fullarton-H-SS18-119 was deposited on January 2, 2018, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 15150.

2. The compound formulation according to claim 1, characterized in that, The vegetable oil is at least one of soybean oil, rapeseed oil, peanut oil, and olive oil.

3. The compound formulation according to claim 1, characterized in that, The improvement in eye function is any one of the following: (1) Reduce pro-inflammatory factors in the eye; (2) Increase anti-inflammatory factors in the eyes; (3) Relieves dry eyes; (4) To adjust or improve the fundus; (5) Reduce the average fluorescent area of ​​the cornea, increase tear secretion, and prolong tear film breakup time.

4. The compound formulation according to claim 3, characterized in that, The pro-inflammatory factors are interleukin-2, interleukin-6, and / or interferon-γ.

5. The compound formulation according to claim 3, characterized in that, The anti-inflammatory factor is interleukin-10.

6. A method for preparing a compound formulation as described in any one of claims 1-5, characterized in that, The process includes the following steps: adding fermentation products and casein phosphopeptides to water and stirring until homogeneous; adding hydroxyethyl cellulose and ultrasonically emulsifying for 10-20 minutes to obtain an aqueous phase; heating vegetable oil to 70-80℃ and adding the aqueous phase dropwise while stirring, homogenizing for 10-20 minutes, and then cooling.

7. The method for preparing the compound formulation according to claim 6, characterized in that, The frequency of the ultrasonic emulsification is 5-10 kHz.

8. The method for preparing the compound formulation according to claim 6, characterized in that, The homogenization process is carried out at a temperature of 35-45℃ and a pressure of 30-40MPa.

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