A nutritional composition for improving retinopathy of prematurity

Abstract

The present invention relates to a nutritional composition for improving retinopathy of prematurity. The nutritional composition for improving retinopathy of prematurity comprises the following components in parts by mass: 50 parts to 800 parts of human milk oligosaccharide, 1 part to 70 parts of lactoferrin, and 0.25 part to 50 parts of Bifidobacterium animalis subsp. lactis. The nutritional composition provided by the present invention can increase the abundance of the beneficial bacterium Bifidobacterium in the intestine, reduce the abundance of Enterobacter, and at the same time reduce the area of neovascularization of the pathological retina caused by hyperoxia and reduce the area of avascular region of the immature retina; inhibit or prevent the oxidation process through the bidirectional regulation of the retina-intestine axis, thereby protecting retinal cells from oxidative stress damage.

Description

Technical Field

[0001] The present invention relates to a nutritional composition for improving retinopathy of prematurity. Background Art

[0002] Retinopathy of prematurity (ROP) is a serious complication in premature infants, caused by the loss of normal blood vessels in the immature retina and the appearance of new pathological blood vessels and fibroplasia, which is more common in low - birth - weight infants and small - gestational - age premature infants. In recent years, due to the continuous improvement of the rescue, treatment, and nursing levels of critically ill premature infants, the survival rates of extremely low - birth - weight infants and ultra - low - birth - weight infants have shown a linear upward trend. Among them, there are many extremely premature infants and ultra - low - birth - weight infants between 22 weeks and 24 weeks who have been successfully treated. However, along with the increase in their survival rates, the incidence of retinopathy of prematurity is also increasing year by year.

[0003] The treatment of retinopathy of prematurity mostly includes surgical treatment and non - surgical treatment. Surgical treatments include laser photocoagulation, scleral buckling, vitrectomy, etc. Non - surgical treatment most commonly uses anti - VEGF (vascular endothelial growth factor) drugs. At present, in the field of foods for premature infants and low - birth - weight infants, such as formula foods for special medical purposes, infant formula foods, complementary foods, and nutritional supplements, etc., there is no existing technology that can inhibit or prevent the oxidation process through the bidirectional regulation of the retina - gut axis and thus protect retinal cells from oxidative stress damage. Summary of the Invention

[0004] Aiming at the problem of how to improve retinopathy of prematurity, the present invention provides a nutritional composition for improving retinopathy of prematurity. By controlling the components and their dosages of the nutritional composition, the retinopathy problem of premature infants and low - birth - weight infants can be significantly improved.

[0005] To solve the above - mentioned technical problems, the technical solution provided by the present invention is:

[0006] In a first aspect, the present invention provides a nutritional composition for improving retinopathy of prematurity, comprising the following components in parts by mass: 50 parts to 800 parts of human milk oligosaccharide, 1 part to 70 parts of lactoferrin, and 0.25 part to 50 parts of Bifidobacterium animalis subsp. lactis.

[0007] Compared with the prior art, the nutritional composition for improving retinopathy of prematurity provided by the present invention can inhibit or prevent the oxidation process through the bidirectional regulation of the retina - gut axis and thus protect retinal cells from oxidative stress damage. Experimental data show that the nutritional composition provided by the present invention can increase the abundance of the beneficial bacterium Bifidobacterium in the intestine, reduce the abundance of Enterobacter, and at the same time reduce the area of new blood vessels in the pathological retina caused by hyperoxia and reduce the area of avascular regions in the immature retina.

[0008] Preferably, the nutritional composition comprises the following components in parts by mass: 60 to 500 parts of human milk oligosaccharide, 5 to 50 parts of lactoferrin, and 1 to 30 parts of Bifidobacterium animalis subsp. lactis.

[0009] More preferably, the nutritional composition comprises the following components in parts by mass: 60 to 150 parts of human milk oligosaccharide, 5 to 20 parts of lactoferrin, and 1 to 10 parts of Bifidobacterium animalis subsp. lactis.

[0010] Even more preferably, the nutritional composition comprises the following components in parts by mass: 60 to 100 parts of human milk oligosaccharide, 8 to 12 parts of lactoferrin, and 3 to 7 parts of Bifidobacterium animalis subsp. lactis.

[0011] Preferably, the human milk oligosaccharide comprises at least one of 2'-fucosyllactose (2'-FL), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'-SL), or 6'-sialyllactose (6'-SL).

[0012] Preferably, the Bifidobacterium animalis subsp. lactis comprises at least one of Bb-12 strain, HN019 strain, or Bi-07 strain.

[0013] Preferably, the nutritional composition further comprises: protein, fat, and carbohydrate.

[0014] More preferably, the mass ratio of the human milk oligosaccharide to the carbohydrate is (1.4 to 15.6):100.

[0015] More preferably, the mass ratio of the lactoferrin to the protein is (0.07 to 5):100.

[0016] More preferably, the content of the Bifidobacterium animalis subsp. lactis is (0.002 to 0.5) g / 100 g.

[0017] More preferably, the nutritional composition further comprises the following components in parts by mass: 1000 to 2100 parts of protein, 2200 to 4100 parts of fat, and 4700 to 7000 parts of carbohydrate.

[0018] Even more preferably, the nutritional composition further comprises the following components in parts by mass: 1200 to 1700 parts of protein, 2500 to 3000 parts of fat, and 5000 to 5800 parts of carbohydrate.

[0019] More preferably, the protein comprises at least one of skim milk powder, isolated whey protein, concentrated whey protein, or concentrated milk protein.

[0020] Further preferably, the fat includes at least one of soybean oil, sunflower oil, coconut oil, rapeseed oil, corn oil or medium-chain triglycerides.

[0021] Further preferably, the carbohydrate includes at least one of desalted whey powder or lactose.

[0022] In a second aspect, the present invention provides a method for preparing the nutritional composition for improving retinopathy of prematurity, comprising the following steps:

[0023] Weigh each component according to the designed mass ratio, and mix them evenly to obtain the nutritional composition for improving retinopathy of prematurity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a graph showing the sequence proportion of Enterobacter in the feces of the young rats in each group of the present invention;

[0025] Figure 2 It is a graph showing the sequence proportion of Bifidobacterium in the feces of the young rats in each group of the present invention;

[0026] Figure 3 It is a graph showing the proportion of the avascular area in the retina of the young rats in each group of the present invention;

[0027] Figure 4 It is a graph showing the proportion of the neovascular area in the retina of the young rats in each group of the present invention;

[0028] Figure 5 It is the SOD activity in the retina tissue of the young rats in each group of the present invention;

[0029] Figure 6 It is the MDA content in the retina tissue of the young rats in each group of the present invention;

[0030] In the figure, there is no significant difference between the same letters, and there is a significant difference between different letters (P < 0.05). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] In the present invention, the materials without special description are all commercially available products. Among them, the producer of the Bb-12 strain is Chr. Hansen A / S, and it is purchased from Hebei Chengxi Import and Export Trading Co., Ltd.; the producer of the HN019 strain is Danisco (China) Co., Ltd., and it is purchased from Beijing Shapushi Science and Trade Co., Ltd.; the producer of the Bi-07 strain is Danisco (China) Co., Ltd., and it is purchased from Beijing Shapushi Science and Trade Co., Ltd.

[0033] Example 1

[0034] This example provides a nutritional composition for improving retinopathy of prematurity, comprising the following components by mass: lacto-N-neotetraose (LNnT) 0.5 g, lactoferrin 0.01 g, Bifidobacterium animalis subsp. lactis (Bb-12 strain) 0.0025 g, protein (provided by equal masses of concentrated whey protein and concentrated milk protein) 14 g, fat (provided by equal masses of soybean oil and sunflower oil) 30 g, and carbohydrates (provided by equal masses of demineralized whey powder and lactose) 50 g.

[0035] Using the dry mixing process, simply mix the components of the above nutritional composition for improving retinopathy of prematurity evenly.

[0036] Example 2

[0037] This example provides a nutritional composition for improving retinopathy of prematurity, comprising the following components by mass: 2'-fucosyllactose (2'-FL) 0.7 g, lactoferrin 0.1 g, Bifidobacterium animalis subsp. lactis (HN019 strain) 0.05 g, protein (provided by equal masses of skim milk powder and isolated whey protein) 14 g, fat (provided by equal masses of coconut oil, corn oil, and medium-chain triglycerides) 27 g, and carbohydrates (provided by demineralized whey powder and lactose in a mass ratio of 5:1) 53 g. The total energy of protein, fat, and carbohydrates is approximately 2138 kJ.

[0038] Using the dry mixing process, simply mix the components of the above nutritional composition for improving retinopathy of prematurity evenly.

[0039] Example 3

[0040] This example provides a nutritional composition for improving retinopathy of prematurity, comprising the following components by mass: equal masses of 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) 8 g, lactoferrin 0.7 g, Bifidobacterium animalis subsp. lactis (Bi-07 strain) 0.5 g, protein (provided by equal masses of skim milk powder and concentrated milk protein) 17 g, fat (provided by equal masses of soybean oil, coconut oil, and rapeseed oil) 25 g, and carbohydrates (provided by demineralized whey powder) 52 g.

[0041] Using the dry mixing process, simply mix the components of the above nutritional composition for improving retinopathy of prematurity evenly.

[0042] Comparative Example 1

[0043] This comparative example provides a nutritional composition, which is similar to Example 2, except that: lactoferrin is replaced with an equal mass of Bifidobacterium animalis subsp. lactis (strain Bi-07), that is, the dosage of Bifidobacterium animalis subsp. lactis (strain Bi-07) is 0.15 g. The remaining conditions are the same as those in Example 2 and will not be elaborated.

[0044] Comparative Example 2

[0045] This comparative example provides a nutritional composition, which is similar to Example 2, except that: human milk oligosaccharide (2'-FL) is replaced with lactoferrin and Bifidobacterium animalis subsp. lactis (strain Bi-07), the dosage of lactoferrin is 0.5 g, and the dosage of Bifidobacterium animalis subsp. lactis (strain Bi-07) is 0.35 g. The remaining conditions are the same as those in Example 2 and will not be elaborated.

[0046] Comparative Example 3

[0047] This comparative example provides a nutritional composition, which is similar to Example 2, except that: adding human milk oligosaccharide (2'-FL), lactoferrin and Bifidobacterium animalis subsp. lactis (strain Bi-07) is omitted simultaneously. The remaining conditions are the same as those in Example 2, that is, the nutritional composition includes the following components by mass: protein (equal mass of skim milk powder and whey protein isolate) 14 g, fat (equal mass of coconut oil, corn oil and medium-chain triglycerides) 27 g, and carbohydrates (demineralized whey powder and lactose with a mass ratio of 5:1) 53 g. The total energy of protein, fat and carbohydrates is approximately 2138 kJ.

[0048] Application Examples 1 - 3

[0049] Application Examples 1 - 3 respectively provide a nutritional product, which consists of a nutritional composition for improving preterm retinopathy and vitamins and minerals. The nutritional compositions for improving preterm retinopathy in Application Examples 1 - 3 are respectively the nutritional compositions for improving preterm retinopathy in Examples 1 - 3. The vitamins and minerals include the following components: vitamin A 530 μg RE / 100 g, vitamin D3 16 μg / 100 g, vitamin E 12 mg α-TE / 100 g, vitamin K1 35 μg / 100 g, vitamin B1 700 μg / 100 g, vitamin B2 1064 μg / 100 g, vitamin B6 630 μg / 100 g, vitamin B 121.9 μg / 100 g, niacin 7.5 mg / 100 g, folic acid 150 μg / 100 g, pantothenic acid 5 mg / 100 g, vitamin C 110 mg / 100 g, biotin 18 μg / 100 g, inositol 88 mg / 100 g, sodium 290 mg / 100 g, potassium 501 mg / 100 g, copper 430 μg / 100 g, magnesium 50 mg / 100 g, iron 8.5 mg / 100 g, zinc 5.5 mg / 100 g, manganese 53 μg / 100 g, calcium 589 mg / 100 g, phosphorus 342 mg / 100 g, iodine 93 μg / 100 g, chlorine 440 mg / 100 g, selenium 12.8 μg / 100 g, choline 83 mg / 100 g, taurine 33 mg / 100 g, L-carnitine 10.8 mg / 100 g, and nucleotides 21 mg / 100 g.

[0050] Adopt the dry mixing process and mix the components of the above nutritional products evenly.

[0051] Apply Comparative Examples 1 - 3

[0052] Comparative Examples 1 - 3 respectively provide a nutritional product, which consists of a nutritional composition and vitamins and minerals. The nutritional compositions respectively adopt the nutritional compositions of Comparative Examples 1 - 3. The components and contents of the vitamins and minerals are the same as those in Application Example 1 and will not be elaborated here.

[0053] Adopt the dry mixing process and mix the components of the above nutritional products evenly.

[0054] Efficacy test

[0055] (1)Animal model establishment and grouping

[0056] Healthy adult pregnant SD rats were selected for the experiment, provided by Sprague Dawley (Suzhou) Biotech Co., Ltd. (China), with the certificate number SCXK(Su) 2022 - 0006, gestational age of 12 d - 14 d, and body weight of 450 g - 550 g. Before the experiment, each pregnant rat was placed in a separate mouse cage to adapt to the environment for 3 d - 5 d, with free access to water and food. The experimental environmental conditions included: light / dark cycle of 12 h / 12 h, background noise of 40 db ± 10 db, and room temperature of 20°C - 24°C. The gestation period of normal rats is 19 d - 23 d, and the pups born before the 20th day of pregnancy are defined as premature pups.

[0057] Lipopolysaccharide (LPS) was used to prepare a premature model in pregnant rats to induce premature birth. On the 16th day of pregnancy of the pregnant rats, the mass of the pregnant rats was accurately weighed, and LPS (purchased from Thermo Fisher Scientific) was intraperitoneally injected at a dose of 350 μg / kg for 2 consecutive days.

[0058] 10 to 12 premature pups per litter were included in the subsequent experiments, with a total of 60 to 70 pups. They were randomly divided into 6 groups of 10 each. Each group was fed for 30 days, and the feed formulas for each group were the nutritional products of Application Examples 1 to 3 and Application Comparative Examples 1 to 3, respectively.

[0059] Within 6 hours after the premature pups were born, the newborn premature pups were placed in a sealed oxygen chamber with an oxygen concentration of 80% (hyperoxia) in the oxygen chamber, which was alternated every 24 hours for 14 days. At 15 days, the pups were placed in normal air and continued to be raised until 30 days after birth. A digital oxygen meter was used to monitor the oxygen concentration in the oxygen chamber, with at least 5 measurements per day. The oxygen flow rate was 2 L / min to 3 L / min, and the nitrogen flow rate was 0.5 L / min to 0.8 L / min, so that the oxygen concentration in the oxygen chamber during the hyperoxia period was maintained at 80% ± 2%.

[0060] (2)16S rRNA sequencing of fecal intestinal flora

[0061] On the 30th day of feeding, feces of the six groups of pups were collected respectively. Before each collection of feces, each pup was placed alone in a disinfected animal cage box and allowed to move freely for 30 minutes. About 0.2 g of feces from each pup was collected, and bacterial DNA in the feces of each group of pups was extracted according to the instructions of the QIAGEN fecal genomic DNA extraction kit. Then, universal primers 341F and 805R were used for PCR amplification in the highly variable region of 16S rRNA V3-V4. After quantitative detection of the amplification products by 2% agarose gel electrophoresis, high-throughput sequencing of the 16S rRNA gene was performed to obtain the proportion of Escherichia coli and Bifidobacterium sequences. The test results are as Figures 1 to 2 and Table 1 shows.

[0062] Table 1 Proportion of Escherichia coli and Bifidobacterium sequences in the feces of each group of pups

[0063]

[0064] In each column of Table 1, there is no significant difference between the same letters, and there is a significant difference between different letters (P < 0.05).

[0065] It can be seen from Figure 1 and Table 1 that the proportion of Escherichia coli sequences in the Application Comparative Example 3 group is the highest, followed by the Application Comparative Example 1-2 groups with no significant difference between the two, the Application Example 3 group is the lowest, and there is no significant difference among the Application Example 1-3 groups. Figure 2As can be seen from Table 1, the proportion of Bifidobacterium sequences in the application of Comparative Example 3 group is the lowest, followed by the application of Comparative Example 1-2 groups, and the difference between the two groups is not significant. The proportion of Bifidobacterium sequences in the Application Examples 1-3 groups is higher than that in the Application of Comparative Examples 1-3 groups, and there is a significant difference from the Application of Comparative Examples 1-3 groups. There is no significant difference among the Application Examples 1-3 groups. This indicates that the nutritional composition provided by the present invention can reduce the number of Enterobacter and increase the number of Bifidobacterium, thereby effectively improving the retinopathy of prematures through microbial regulation.

[0066] (3)Area of avascular area and neovascular area in the retina of young rats

[0067] After feeding for 30 days, the young rats in each group were sacrificed, and one eye was randomly selected to remove the residues in the retina and the residual vitreous vessels to make a retinal spread. The retinal spread was placed on a polylysine-coated glass slide with the inner layer of the retina facing up, sealed with glycerol at a concentration of 50%, covered with a cover glass, and stored in the dark at 4°C.

[0068] Stain the blood vessels, observe the stained spread under a fluorescence microscope and take pictures. The retinal blood vessels are green fluorescence, and the avascular area is the black area where the peripheral green fluorescence terminates. Measure and calculate the percentage of the avascular area, neovascular area and total retinal area through Adobe Photoshop CS3 image analysis software. The test results are as Figures 3 to 4 and Table 2 show.

[0069] Table 2 Results of the proportion of avascular area and neovascular area in the retinas of young rats in each group

[0070]

[0071] In each column of Table 2, there is no significant difference between the same letters, and there is a significant difference between different letters (P < 0. )

[0072] From Figure 3 and Table 2, it can be seen that the proportion of the avascular area in the Application Examples 1-3 groups is smaller than that in the Application of Comparative Examples 1-3 groups. The Application of Comparative Example 3 group is the highest, and there is a significant difference from the Application of Comparative Examples 1-2 and Application Examples 1-3 groups. From Figure 4 and Table 2, it can be seen that the proportion of the neovascular area in the Application Example groups 1-3 is smaller than that in the Application of Comparative Examples 1-3 groups, and the Application of Comparative Example 3 group is the highest. This indicates that the nutritional composition provided by the present invention can effectively reduce the avascular area of the immature retina and reduce the pathological neovascular hyperplasia caused by hyperoxia.

[0073] (4)Determination of oxidative stress markers

[0074] The retinal blood vessels of premature infants are not fully developed. High oxygen can cause vasoconstriction of immature retinas, leading to retinal hypoxia-ischemia, vascular occlusion, and then the production of a large amount of vascular growth factors. Driven by the retinal VEGF signaling pathway, pathological retinal neovascularization and fibrous tissue hyperplasia form, and then retinal detachment occurs, resulting in blindness. Oxidative stress is one of the important injury mechanisms of ROP. The oxidative stress level of premature infants can be used as a predictor of ROP, and the degree of ROP lesions worsens with the increase in the degree of oxidative stress. Superoxide dismutase (SOD) and malondialdehyde (MDA) are very sensitive oxidative stress markers. Kits were used to measure the activity of SOD and the content of MDA. The SOD kit used a total superoxide dismutase (T-SOD) test kit (hydroxylamine method), and the MDA kit used a malondialdehyde (MDA) assay kit (TBA method), both of which were purchased from Nanjing Jiancheng Bioengineering Institute.

[0075] Under the microscope, the retinas of the other eyes of the young rats in each group were quickly separated. According to the mass-volume ratio of the retina to phosphate buffer (PBS buffer) of 1 g:9 mL, PBS buffer was added, and it was ground thoroughly to make a retinal homogenate. It was centrifuged at 3500 r / min for 10 min in a 4°C low-temperature centrifuge, and the supernatant was taken and stored at -80°C. The activity of SOD and the content of MDA in the retinal tissues of the young rats in each group were measured strictly according to the instructions of the SOD and MDA kits. The test results are as Figures 5 to 6 shown in Table 3.

[0076] Table 3 Test results of the activity of SOD and the content of MDA in the retinal tissues of the young rats in each group

[0077]

[0078] In each column of Table 3, there is no significant difference between the same letters, and there is a significant difference between different letters (P < 0.05).

[0079] As Figure 5 seen from Table 3, the SOD activity of Application Examples 1-3 groups was higher than that of Application Comparative Examples 1-3 groups. The SOD activity of Application Comparative Example 3 group was the lowest and was significantly different from other groups. As Figure 6 seen from Table 3, the MDA content of Application Examples 1-3 groups was significantly lower than that of Application Comparative Examples 1-3 groups. The MDA content of Application Comparative Example 3 group was the highest and was significantly different from other groups. This shows that the intake of the nutritional composition provided by the present invention can effectively regulate the degree of oxidative stress.

[0080] The comprehensive results show that the nutritional composition provided by the present invention can inhibit or prevent the oxidation process through the bidirectional regulation of the retina-intestinal axis, thereby protecting retinal cells from oxidative stress damage.

[0081] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A nutritional composition for improving retinopathy of prematurity, characterized in that, Comprising the following components in parts by mass: 60 to 800 parts of human milk oligosaccharide, 8 to 70 parts of lactoferrin, and 3 to 50 parts of Bifidobacterium animalis subsp. lactis; The human milk oligosaccharide includes at least one of 2'-fucosyllactose, lacto-N-neotetraose, 3'-sialyllactose, or 6'-sialyllactose.

2. The nutritional composition for improving retinopathy of prematurity according to claim 1, wherein The nutritional composition comprises the following components in parts by mass: 60 to 500 parts of human milk oligosaccharide, 8 to 50 parts of lactoferrin, and 3 to 30 parts of Bifidobacterium animalis subsp. lactis.

3. The nutritional composition for improving retinopathy of prematurity according to claim 2, wherein, The nutritional composition comprises the following components in parts by mass: 60 to 150 parts of human milk oligosaccharide, 8 to 20 parts of lactoferrin, and 3 to 10 parts of Bifidobacterium animalis subsp. lactis.

4. The nutritional composition for improving retinopathy of prematurity according to claim 1, characterized in that, The Bifidobacterium animalis subsp. lactis includes at least one of Bb-12 strain, HN019 strain, or Bi-07 strain.

5. The nutritional composition for improving retinopathy of prematurity according to claim 1, characterized in that, The nutritional composition further comprises: protein, fat, and carbohydrate.

6. The nutritional composition for improving retinopathy of prematurity according to claim 5, wherein, The mass ratio of the human milk oligosaccharide to the carbohydrate is (1.4 to 15.6):100; The mass ratio of the lactoferrin to the protein is (0.07 to 5):100; The content of the Bifidobacterium animalis subsp. lactis is (0.002 to 0.5) g / 100 g.

7. The nutritional composition for improving retinopathy of prematurity according to claim 1, 5 or 6, characterized in that, The nutritional composition further comprises the following components in parts by mass: 1000 to 2100 parts of protein, 2200 to 4100 parts of fat, and 4700 to 7000 parts of carbohydrate.

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