Composition containing salty taste enhancing peptide and naringenin and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a composition containing salty taste enhancing peptide and naringenin and application of the composition. The active ingredients of the composition are composed of salty taste enhancing peptide and naringenin in a mass ratio of 100: 1. Animal experiment results show that the composition can effectively improve intestinal flora imbalance of senescence diabetes model mice, up-regulate expression of tight junction proteins ZO-1 and Occludin, repair intestinal barriers, increase the ratio of mycophylum to bacteroides, promote growth of beneficial bacteria such as micrococcus verrucosus and actinomycetes, especially promote growth of Ackerman's bacteria and bifidobacteria, and improve the immunity of senescence diabetes mellitus mice. The intestinal flora structure deviation is reversed, and the content of short-chain fatty acid in the intestinal tract is remarkably increased. The invention provides a novel prebiotic for regulating intestinal flora, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composition containing salty-enhancing peptides and naringenin and its application. Background Technology

[0002] The gut microbiota is closely related to health, and the homeostasis of the gut microbiota is a prerequisite for its physiological functions. Gut microbiota dysbiosis can trigger various diseases. Studies have shown that with aging, the structure and function of the gut microbiota undergo significant changes. Metabolic products, neural pathways, and immune signals in the gut send signals through the central nervous system and other distal organs, thereby regulating host health. For example, age-related chronic diseases or mental illnesses such as neurodegenerative diseases, obesity, and diabetes are closely related to the gut microbiota. Therefore, maintaining a healthy gut microbiota is extremely important. Thus, it is essential to develop agents that improve gut microbiota dysbiosis to promote the proliferation of beneficial bacteria, inhibit the growth of harmful bacteria, and enhance intestinal barrier function.

[0003] Salt-enhancing peptide is a peptide derived from turtle eggs. It has no salty taste on its own, but it has a synergistic effect with NaCl. This salt-enhancing peptide has the dual characteristics of turtle egg activity and salt reduction and salt enhancement.

[0004] Naringin is a flavonoid compound commonly found in citrus fruits. It has anti-inflammatory, antioxidant, and blood sugar-improving effects. It is one of the main active ingredients in ginseng and has the effect of protecting the central nervous system and effectively preventing neurodegenerative diseases.

[0005] Therefore, further research on whether turtle egg-derived salty-enhancing peptides and naringenin can be mixed, and whether the mixture can improve gut health, is of significant research value. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention establishes an aging diabetic mouse model by inducing aging with D-galactose combined with streptozotocin (STZ)-induced diabetes. A salty-tasting enhanced peptide is prepared and compounded with naringenin in a specific ratio to obtain a composition. The effects of this composition on improving the intestinal microecological environment, promoting the proliferation of beneficial bacteria, inhibiting the growth of potentially harmful bacteria, and repairing intestinal barrier damage are evaluated using aging diabetic mice. Animal experiments demonstrate that this composition effectively solves the problem of intestinal microecological disorder caused by the dual effects of aging and diabetes, and its repair effect is superior to the positive control metformin. This invention provides a novel prebiotic for intestinal flora regulation.

[0007] On the one hand, the present invention provides a composition for improving intestinal flora imbalance, wherein the active ingredients of the composition consist of a salty-enhancing peptide and naringenin.

[0008] Furthermore, in the composition, the mass ratio of the salty-enhancing peptide to naringenin is 100:1.

[0009] Furthermore, the salty-enhancing peptide in the composition is prepared by enzymatic hydrolysis of turtle eggs with papain, followed by ultrafiltration to collect components with a molecular weight less than 3 kDa, and then freeze-drying.

[0010] Secondly, the invention also provides the use of the composition described herein in the preparation of an agent to improve gut microbiota dysregulation.

[0011] Furthermore, in the aforementioned application, the gut microbiota dysbiosis is gut microbiota dysbiosis caused by aging-related diabetes.

[0012] The gut microbiota dysbiosis includes damage to the intestinal barrier and / or deviation of the microbiota structure and / or decrease in short-chain fatty acid content and / or reduction in colon length.

[0013] Furthermore, in the application, the intestinal barrier damage includes downregulation of the expression of tight junction proteins Occludin and / or ZO-1, which represent intestinal barrier integrity; The deviations in the microbial community structure include a decrease in the Firmicutes / Bacteroidetes ratio, a decrease in Verrucomicrobial abundance, and / or a decrease in Actinobacteria abundance. The short-chain fatty acids include one or more of acetic acid, propionic acid, valeric acid, isobutyric acid, and isovaleric acid.

[0014] Furthermore, in the application, the composition can improve the decrease in the levels of acetic acid, propionic acid, valeric acid, isobutyric acid, or isovaleric acid in the intestine caused by age-related diabetes.

[0015] Furthermore, in the application, the composition alleviates the gut microbiota structure deviation caused by age-related diabetes, the alleviation being manifested by increasing the Firmicutes / Bacteroidetes ratio, increasing Verrucous Microbes abundance, and / or increasing Actinobacteria abundance.

[0016] Furthermore, in the application, the composition can increase the abundance of Akkermansia, Bifidobacterium and / or Trichophyton spp. NK4A136 in the gut.

[0017] Furthermore, in the application, the composition is able to increase the colon length in aging diabetic model mice.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention discloses a composition of salty-enhancing peptides and naringenin as active ingredients and its application in the preparation of an agent to improve intestinal flora imbalance. Animal model experiments have demonstrated that this composition significantly upregulates the expression of tight junction proteins Occludin and ZO-1 in the colonic tissue of aging diabetic mice, repairs damaged intestinal epithelial structures, enhances intestinal barrier integrity, and reduces inflammatory infiltration. This composition effectively corrects the flora imbalance caused by aging-related diabetes, increases the Firmicutes / Bacteroidetes (F / B) ratio, significantly promotes the growth of beneficial bacteria such as Verrucous Microbes and Actinobacteria, and particularly greatly enhances the growth of the key probiotic under Verrucous Microbes—Akkermansia (…). Akkermansia ) and Bifidobacteria ( Bifidobacterium The abundance of ) while inhibiting harmful bacteria ( Blautia This composition inhibits the excessive proliferation of bacteria (of the genus *Acta*). It significantly increases the content of various short-chain fatty acids (SCFAs) in the intestine, including acetic acid, propionic acid, butyric acid, and valeric acid. These SCFAs play important roles in maintaining intestinal homeostasis, energy supply, and anti-inflammation. Furthermore, this composition can restore the colon length shortened due to aging and diabetes, improve the structure of colonic crypts and villi, increase the number of goblet cells, and morphologically reverse intestinal pathological changes. This invention provides a novel prebiotic for intestinal flora regulation and has promising application prospects. Attached Figure Description

[0019] Figure 1 The graph shows the results of short-chain fatty acid content determination in the cecum of mice in different experimental groups. A represents acetic acid content; B represents propionic acid content; C represents butyric acid content; D represents valerate content; E represents isobutyric acid content; and F represents isovaleric acid content.

[0020] Figure 2 The figure shows the results of measuring the effects of different experimental groups of mice on the alpha diversity, beta diversity, and phylum level of gut microbiota. A represents the Shannon alpha diversity index; B represents the ACE alpha diversity index; C represents the Chao1 alpha diversity index; D represents the Simpson alpha diversity index; F represents colon length; G represents the relative abundance at the phylum level; H represents Firmicutes; I represents Bacteroidetes; J represents the Firmicutes / Bacteroidetes ratio; K represents Verrucous Microbes; and L represents Actinobacteria.

[0021] Figure 3 The results show the genus level of gut microbiota in mice from different experimental groups. A is a circle diagram of species composition; B is a cluster diagram of species composition; C represents *Broutella*; D represents *Trichophyton* family, group NK4A136; E represents *Ackermania*; and F represents *Bifidobacterium*.

[0022] Figure 4This section presents differential species analysis and short-chain fatty acid correlation analysis. A represents LEfSe differential analysis; B represents linear discriminant analysis; C represents a heatmap showing the correlation between gut microbiota and short-chain fatty acids; and D represents a network diagram showing the correlation between gut microbiota and short-chain fatty acids.

[0023] Figure 5 HE staining of the colon and immunofluorescence analysis of ZO-1 and Occludin. A shows a colonic H&E stained section; B shows the ZO-1 immunofluorescence analysis; C shows the Occludin immunofluorescence analysis; D shows the quantitative expression level of the colonic tight junction protein ZO-1; E shows the quantitative expression level of the colonic tight junction protein Occludin.

[0024] Figure 6 The figure shows the PCA analysis results for Beta diversity determination. Detailed Implementation

[0025] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0026] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0027] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0028] In this invention, "aging-related diabetes" refers to the disease state characterized by a composite model mouse model established by combining D-galactose-induced aging with streptozotocin (STZ)-induced diabetes.

[0029] In this invention, "Firmwallia / Bacteroidetes" refers to... Bacillota / Bacteroidota .

[0030] Naringin, product number: N164488, purchased from Aladdin Company.

[0031] Example 1 This example demonstrates the preparation of a salty-enhancing peptide.

[0032] The salty-enhancing peptide in this invention is prepared by enzymatically hydrolyzing a 5 wt% freeze-dried turtle egg powder solution with papain (5000 U / g) at 50°C for 4 hours, followed by enzyme inactivation at 90°C for 10 minutes, cooling in ice water, centrifuging at 10000g at 4°C for 20 minutes, and then filtering through four layers of 200-mesh gauze to collect the supernatant. The collected supernatant is then subjected to ultrafiltration to collect the fraction with a molecular weight less than 3 kDa, and then freeze-dried to obtain the salty-enhancing peptide.

[0033] Example 2 This example demonstrates the preparation of a salty-enhancing peptide and naringenin composition.

[0034] A 100 mg / mL salty-enhancing peptide solution was prepared by dissolving salty-enhancing peptide in 75% ethanol, and a 1 mg / mL naringenin solution was prepared by dissolving naringenin in 75% ethanol. The 100 mg / mL salty-enhancing peptide solution and the 1 mg / mL naringenin solution were mixed in equal volumes, stirred at 25°C for 2 hours, and then concentrated under vacuum (37°C, 170 rpm) and freeze-dried under vacuum to obtain a salty-enhancing peptide and naringenin composition, abbreviated as (NP).

[0035] Example 3 This example is an animal experiment demonstrating the effect of a combination of salty-enhancing peptides and naringenin on improving gut microbiota imbalance in mice.

[0036] 1. Establish an experimental model Sixty 8-week-old male C57BL / 6 mice (ethics review number: GOU-LAE-2025-013), weighing 18.0~20.0g (license number: SCXK (Guangdong) 2020-0051), were housed in a 22±2℃, 50±10% humidity, 12h light / dark cycle environment. After acclimatization for 1 week, the mice were randomly divided into 5 groups of 12 mice each. Group 1 was designated as the young and healthy group, and the other 4 groups were designated as aging model groups. Every morning at 9:00 AM, mice were injected intraperitoneally with 500 mg / kg bw D-galactose. Young, healthy mice were injected intraperitoneally with an equal volume of saline. These injections were repeated for 9 weeks, with fasting allowed for 12 hours during week 6. The aging model group was injected intraperitoneally with 70 mg / kg bw streptozotocin (STZ) for 5 consecutive days. STZ was dissolved in 0.1 M citrate buffer (pH 4.5), prepared fresh and used immediately, and the entire process was conducted in the dark. Young, healthy mice were injected with an equal volume of sterile citrate buffer. Four hours after injection, mice were given a 20% glucose solution to prevent hypoglycemia and death. Five days after injection, mice were fasted for 12 hours with no food or water allowed. Fasting blood glucose (FBG) was measured using a portable blood glucose meter. Mice with FBG > 11.1 mmol / L were classified as aging diabetic mice. The remaining mice with unmet blood glucose levels were injected intraperitoneally with STZ again until their blood glucose reached the target level; these mice were also classified as aging diabetic mice. The remaining mice (4 mice) that did not meet the target level were culled. Young, healthy mice were fed a basal maintenance diet throughout the process, while model mice were fed a high-fat diet. Mice had free access to food and water. Two mice died unexpectedly during the establishment of the aging diabetes model.

[0037] After the modeling was completed, a total of 54 mice participated in the subsequent experiments, including 12 young healthy mice (CON, n=12) and 42 aging diabetic model mice. The aging diabetic model mice were randomly divided into 4 groups for subsequent functional verification experiments: aging diabetic model group (Model, n=11); metformin positive drug group (MET, n=10); low-dose NP group (NPL, n=10); and high-dose NP group (NPH, n=11). Young, healthy mice were administered 0.1 mL / 10 g bw of physiological saline by gavage for 8 weeks; aged diabetic model mice were administered 0.1 mL / 10 g bw of physiological saline by gavage for 8 weeks; metformin-positive mice were administered 150 mg / kg bw of metformin by gavage for 8 weeks; mice in the low-dose NP group were administered 0.5 mL / 10 g bw of NP by gavage for 8 weeks; mice in the high-dose NP group were administered 0.1 mL / 10 g bw of NP by gavage for 8 weeks. Mice were sacrificed 18 weeks after the first injection of streptozotocin, and colonic tissue and contents of the colon and cecum were collected.

[0038] 2. Determination of short-chain fatty acids (SCFAs) Weigh 100 mg of mouse cecal contents, add 0.5 mL of physiological saline, grind for 1 min, add 10 μL of 50% H₂SO₄ solution, vortex until homogeneous, add 0.8 mL of pre-cooled diethyl ether, sonicate in an ice-water bath for 10 min, add 0.25 g of anhydrous sodium sulfate, vortex until homogeneous, centrifuge at 12000 rpm and 4℃ for 10 min, filter through a 0.22 μm filter membrane, bottle, and determine SCFAs by gas chromatography (GC). Standard solutions of different concentrations were prepared using acetic acid, propionic acid, butyric acid, isovaleric acid, isobutyric acid, and valeric acid to determine the standard curve. The content of each short-chain fatty acid in the mouse cecal contents was calculated based on the standard curve. The test results are shown below. Figure 1 As shown. GC parameters: SH-Wax capillary column (30m × 0.25mm × 0.25μm) was used, with high-purity helium (purity ≥99.999%) as carrier gas, and a flow rate of 1.0 mL / min; the detector and injection port temperatures were both set to 250℃. Temperature program parameters: initial column temperature 60℃, held for 1 minute; increased to 180℃ at 10℃ / min, then increased to 240℃ at 30℃ / min and held for 1 minute. A 50:1 split mode was used, and the injection volume was set to 1 μL.

[0039] Figure 1 In Figure A, the acetic acid content in the cecal contents of mice was shown to be significantly lower (39.66%) compared to the young, healthy group (CON). p<0.05%, compared with the Model group mice (0.70 mg / g), NPH intervention significantly increased the acetic acid content in the cecal contents of aging diabetic mice, and its acetic acid content (1.30 mg / g) was significantly higher than that of the metformin intervention group (0.77 mg / g), approaching the level of the CON group. p The value <0.01 indicates that the high-dose combination of salty-enhancing peptide and naringenin (NPH) is superior to the positive control metformin in improving gut microbiota dysbiosis, and almost restores the acetic acid content in the cecal contents of diabetic model mice to the level of young healthy mice.

[0040] Figure 1 B represents the propionic acid content in the cecal contents of mice. The propionic acid content in the Model group was significantly lower than that in the CON group, while the propionic acid content in the NPH group was significantly reduced. Compared with the Model group, the propionic acid content in the NPH group increased by 67.74%. p <0.01), the above results suggest that aging-related diabetes leads to a significant decrease in the levels of propionic acid and acetic acid in the intestines, while NP can effectively restore acetic acid and propionic acid to normal levels. Among them, the high-dose group of salty taste-enhancing peptide and naringenin combination (NPH) showed better recovery effects than the positive control metformin.

[0041] Depend on Figure 1 C, Figure 1 D, Figure 1 China E and Figure 1 The results showed that the combination of salty-enhancing peptide and naringenin (NP) effectively restored the levels of butyric acid, valeric acid, isobutyric acid, and isovaleric acid in the cecum of aging diabetic mice. Among them, the high-dose group of salty-enhancing peptide and naringenin combination (NPH) showed better intervention effect than the positive control metformin.

[0042] 3. Gut microbiota 16S rDNA sequencing Colonic contents were collected from mice in the young healthy group (CON), the aging diabetes model group (Model), the metformin-positive drug group (MET), the low-dose NP group (NPL), and the high-dose NP group (NPH). Total genetic DNA was extracted from the colonic contents using a nucleic acid extraction kit. Using the extracted total genetic DNA as a template, PCR amplification was performed on V3-V4 of the 16S rRNA gene. The sequence of the forward primer (SEQ ID NO:1) for PCR amplification was 5′-ACTCCTACGGGAGGCAGCA-3′, and the sequence of the reverse primer (SEQ ID NO:2) was 5′-GGACTACHVGGGTWTCTAAT-3′.

[0043] PCR amplification conditions: 98℃ pre-denaturation for 10s, 54℃ annealing for 30s, 72℃ extension for 45s, 32 cycles, and a final extension at 72℃ for 10min.

[0044] After purification, PCR products were quantified using Qubit (Invitrogen, USA) and evaluated using an Illumina (Kapa Biosciences, Woburn, MA, USA) library quantification kit. Qualified products were then sequenced using a DNBSEQ-G99 sequencer and a DNBSEQ-G99RS high-throughput sequencing kit (G99 App-DFCL PE300), catalog number 940-001716-00. The effects of different treatments on alpha diversity, beta diversity, and phylum level of the mouse gut microbiota were evaluated based on the sequencing results. The results are as follows: Figure 2 As shown. The effects of different treatments on the levels of gut microbiota in mice were assessed, and the results are as follows. Figure 3 As shown.

[0045] Alpha diversity is a comprehensive indicator reflecting the richness, evenness, and diversity of the bacterial community in a measured sample, and is typically represented by the Shannon and Simpson indices. Figure 2 As shown in A, B, C, and D, there were no significant differences in the richness of the intestinal flora among all groups of mice, indicating that the richness, evenness, and diversity of the flora were relatively similar among the groups.

[0046] Beta diversity is a technique used to analyze the similarity of community composition between different samples or groups. Figure 6 It can be seen that the structure of the gut microbiota in each group of mice is slightly dispersed, tends to aggregate together, and there is no significant difference. p >0.05), indicating that the bacterial community structure is relatively similar. (From...) Figure 2 As shown in the middle F, aging-related diabetes can significantly shorten the colon length in mice. p< The value of 0.001 was statistically significant compared to the CON group, indicating that aging-related diabetes may cause pathological changes in the mouse intestines. After MET and NP intervention, the colon length of mice was restored, with the colon length of mice in the NPH group returning to normal.

[0047] At the phylum level, the dominant bacterial groups in each group are mainly Bacillota (Firmwallis) Bacteroidota (Bacteroidetes) Verrucomicrobiota (Verruciformis) and Actinomycetota (Actinomycetes) Figure 2 H in the middle represents Firmicutes. (From...) Figure 2 As shown in the data, Firmicutes and Bacteroidetes constitute the largest proportion of the gut microbiota. The ratio of Firmicutes to Bacteroidetes (Firmicutes / Bacteroidetes) is usually used to reflect changes in the gut microbiota structure and is often used as an indicator to assess whether the gut microbiota structure is imbalanced. Figure 2Figure I and Figure K show an increase in Bacteroidetes and Verrucous microbes in the aging diabetic model group. p__Verrucomicrobiota The bacteria were almost completely absent (0.15%), and the main species in this phylum were Akkermansia, indicating that the gut microbiota of aging diabetic mice was severely dysbiotic and the intestinal barrier was damaged.

[0048] Figure 2 J, K, and L showed that administration of Met and NP increased the Firmicutes / Bacteroidetes ratio in aging diabetic mice by 12.89%–176.39%, and improved [their ability to function properly]. Verrucomicrobiota (Verruciformis) and Akkermansia Actinomycetota The abundance of (Actinomycetes), especially significantly promoted Actinomycetota Growth (4.06%~21.45%) indicates that NP is a potential gut microbiota modulator, with a superior improvement effect compared to the classic drug Met (12.88%). Furthermore, Figure 2 The results showed that in the aging diabetes model group Actinomycetota The abundance of Actinobacteria was lower than that of the young, healthy control group, while Met and NP interventions improved the gut microbiota imbalance.

[0049] Depend on Figure 3 China A and Figure 3 From B, we can see that at the genus level Blautia , Akkermansia and Lachnospiraceae_ NK4A136_group (Kindella spp. NK4A136) was the dominant bacterial group; in aging diabetic mice Akkermansia The relative abundance of bacterial genera was lowest in aged diabetic mice. After Met and NP intervention, the relative abundance of bacterial genera increased from 0.15% (Model) to 12.88% (Met), 4.06% (NPL), and 21.45% (NPH), respectively, indicating that the intestinal barrier was impaired and intestinal immunity was decreased in aged diabetic mice, thus exacerbating inflammation. Figure 3 From E, we know that giving Met and NP can improve [the performance / performance]. Akkermansia The abundance of bacteria, especially NPH, showed the best effect, indicating that NPH can repair intestinal barrier damage caused by both aging and diabetes, reduce inflammatory response, and thus delay or improve the aging phenotype associated with gut microbiota.

[0050] Figure 3 The C-cell curve showed that, compared to the younger, healthy mice, Blautia The genus *Neptunia* exhibits explosive growth in aging diabetic mice, inhibiting the growth of other beneficial bacteria, leading to gut microbiota dysbiosis, accelerating metabolic decline and aging, and increasing insulin resistance. NPH effectively inhibits this growth. Blautia Bacteria, which maintain gut health. Figure 3 D display Lachnospiraceae_NK4A136_ groupThe decreased abundance of *Bacterium* in aging diabetic mice indicates the depletion of butyrate energy production, leading to intestinal epithelial dysfunction and decreased insulin sensitivity, which favors the growth of pathogenic bacteria. NPL can significantly restore the intestinal flora of aging diabetic mice. Lachnospiraceae_NK4A136_group Bacterial abundance, but unable to be suppressed. Blautia Growth of fungi. Figure 3 The results showed that in the aging diabetic model group of mice, Bifidobacterium The genus richness was not high, but it improved to some extent after Met and NP intervention, indicating that the target of NP may not be directly on traditional probiotics, but rather on a more core level. Akkermansia—Blautia Microbial axis, which may be a key mechanism for improving metabolism and delaying aging.

[0051] 4. Differential species analysis of gut microbiota and correlation analysis with short-chain fatty acids To identify specific microbial groups with statistically significant differences and biological effects among different treatment groups, differentially expressed species were screened using LEfSe differential analysis and linear discriminant analysis (LDA). Results are as follows: Figure 4 As shown, Figure 4 China A and Figure 4 The B-group showed that the dominant bacterial group in the CON group was g_Monoglobus (Single cocci) ） , f_Monoglobaceae (Singlococcidae) o_Monoglobales (Monococciales) f_Atopobiaceae (Atopobacteriaceae) g_Lachnospiraceae_ UCG-006 (Uncultured genus UCG-006 of the family Trichophyceae) g_Lactobacillus (Lactobacillus) g_Faecalibaculum (Bacterium spp.) g_Ligilactobacillus (Streptomyces) o_Clostridia_UCG-014 (Clostridium class Uncultured order UCG-014) f_Lactobacillaceae (Lactobacillus family) o_Lactobacillales (Lactobacilliales) g_ Dubosiella (Duborella genus) f_Erysipelotrichaceae (Erysipelothrix family) o_Erysipelotrichales (Erysipelothrix) and c_Bacilli (Bacillus); the dominant bacterial group in the Model group is g_Bacteroides (Bacteroides) f_Bacteroidaceae (Bacteroidetes family) g_Colidextribacter (Corydextriella) f_ Muribaculaceae (Murcariaceae) o_Bacteroidales (Bacteroidetes), c_Bacteroidia (Bacteroidetes) and p_Bacteroidota (Bacteroidetes); the dominant bacterial group in the MET group was g_Parabacteroides (Pseudomonas) f_ Tannerellaceae (Tannaceae) g_Frisingicoccus (Frisenella) and g_Ruthenibacterium (Ruseniella); the dominant bacterial group in the NPL group is g_Clostridium (Clostridium) f_Clostridiaceae (Clostriaceae family) o_Clostriduales (Clostridiales), f_[Eubacterium]_coprostanoligenes (coprosterol-producing bacteria) and g_Blautia (Brausella spp.); The dominant bacterial group in the NPH group is g_Romboutsia (Lombuciana) f_ Peptostreptococcaceae (Peptococcalaceae) o_Peptostreptococcles-Tissierellales (Peptostreptococci – Tisheraeales) f_Ruminococcaceae (Ruminococci) and o_Oscillospirales (Oschillospirales).

[0052] Depend on Figure 4 From C, we can see that Lachnospiraceae_NK4A136_group It is significantly positively correlated with propionic acid, acetic acid, and isobutyric acid. p <0.05), Streptococcus (Streptococcus) is significantly positively correlated with valeric acid and butyric acid. p <0.05), Clostridium_ methylpentosum_group (Methylpentose bacteria) are significantly positively correlated with valeric acid. p <0.05), Prevotella (Prevotella spp.) Pseudoleptotrichia (Pseudocymium) ,Lachnospiraceae_NK4A136_group (NK4A136 of the family Trichophyceae) Ralstonia (Larstonia) showed a positive correlation with propionic acid. Figure 4 The results showed that the content of short-chain fatty acids (valeric acid and butyric acid) decreased in the Model group, which was mainly due to... Streptococcus Abundance reduction and g_Cetobacterium (Cetobacterium) may be related to the dual pathological changes in gut microbiota and metabolism caused by "aging + diabetes". After MET, NPL, and NPH intervention, the abundance of bacteria negatively correlated with aging and diabetes was suppressed. g_Cetobacterium , Streptococcus The abundance rebounded, eventually leading to a partial recovery of short-chain fatty acid content, indicating that MET, NPL, and NPH interventions have a dual repair effect on gut microbiota and metabolites, thereby improving the metabolic disorder of short-chain fatty acids (SCFAs) in aging diabetic mice.

[0053] 5. Colon HE staining and ZO-1 and Occludin immunofluorescence analysis Colonic tissue fixed in 4% paraformaldehyde solution was dehydrated with ethanol of varying concentrations, then immersed in paraffin solution. After paraffin embedding using an embedding machine, 3μm thick sections were cut using a microtome for HE staining. The sections were mounted with neutral resin and then observed under a microscope for morphological characteristics. The results are as follows: Figure 5 As shown.

[0054] After dewaxing, antigen retrieval, and endogenous enzyme blocking, the prepared tissue sections were blocked with 10% goat serum at 37°C for 30 min. They were then incubated overnight at 4°C with primary antibodies (Occludin and ZO-1). The sections were then rinsed three times with pre-chilled PBS buffer, 5 min each time. After incubation with secondary antibodies at 37°C for 60 min in the dark, the sections were rinsed again three times with pre-chilled PBS, 5 min each time. Finally, diluted DAPI staining solution was added to the tissue, and after 10 min, excess staining solution was washed away with running water and excess water was removed with absorbent paper. Anti-fluorescence mounting medium was added to the tissue, and the sections were covered with coverslips and observed and photographed under a fluorescence microscope. ImageJ 1.8.0 was used to analyze and quantify the photographic results. The analysis and quantification results are as follows: Figure 5 As shown.

[0055] Depend on Figure 5 As shown in Figure A, the CON group mice had a greater number of goblet cells with intact structure. Compared with the CON group, the number of goblet cells in aged diabetic mice was significantly reduced, and the colonic crypt branches were deformed, atrophied (shortened), and disordered, with shortened intestinal villi and inflammatory cell infiltration. The MET group mice had a significantly increased number of goblet cells, but the crypt structure was still relatively short, and the length of the intestinal villi was restored to some extent. After NP intervention, the number of goblet cells in the colon of aged diabetic mice tended to be similar to that of the CON group, and the intestinal villi were significantly longer. In particular, the NPH group showed no obvious inflammatory cell infiltration, and the intestinal villi were dense and abundant, with their length tending to be similar to that of the CON group. This effectively restored the intact structure of the colonic crypts in aged diabetic mice, indicating that the intestines of aged diabetic mice undergo pathological changes, and NP can effectively improve the intestinal pathological state of aged diabetic mice, making them tend towards health. Immunofluorescence analysis results are as follows: Figure 5 China B and Figure 5 As shown in Figure C, compared to the CON group, the levels of tight junction proteins ZO-1 and Occludin in the colon tissue of aging diabetic mice (Model group) were significantly reduced; Figure 5 As shown in Figures D and E, compared with the Model group, ZO-1 was significantly upregulated by 1.91-fold, 1.91-fold, and 1.84-fold under the influence of MET, NPL, and NPH, respectively, and Occludin was significantly upregulated by 1.13-fold, 1.36-fold, and 1.75-fold, respectively, enhancing intestinal integrity. These results indicate that NP can effectively repair intestinal barrier damage induced by aging and diabetes and improve gut microbiota.

[0056] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A composition for improving dysbiosis of the intestinal flora, characterized by, The active ingredients of the composition consist of salty-enhancing peptides and naringenin.

2. The composition according to claim 1, characterized in that, The mass ratio of the salty flavor-enhancing peptide to naringenin is 100:

1.

3. The composition according to claim 1, characterized in that, The salty flavor-enhancing peptide was prepared by enzymatic hydrolysis of turtle eggs with papain, followed by ultrafiltration to collect components with a molecular weight of less than 3 kDa, and then freeze-drying.

4. The use of the composition according to any one of claims 1-3 in the preparation of an agent to improve intestinal flora imbalance.

5. The application according to claim 4, characterized in that, The gut microbiota imbalance mentioned refers to gut microbiota imbalance caused by aging and diabetes. The gut microbiota dysbiosis includes damage to the intestinal barrier and / or deviation of the microbiota structure and / or decrease in short-chain fatty acid content and / or reduction in colon length.

6. The application according to claim 5, characterized in that, The intestinal barrier damage includes downregulation of the expression of Occludin and / or ZO-1 proteins, which represent the integrity of the intestinal barrier; The deviations in the microbial community structure include a decrease in the Firmicutes / Bacteroidetes ratio, a decrease in Verrucomicrobial abundance, and / or a decrease in Actinobacteria abundance. The short-chain fatty acids include one or more of acetic acid, propionic acid, valeric acid, isobutyric acid, and isovaleric acid.

7. The application according to claim 4, characterized in that, The composition can improve the decrease in the levels of acetic acid, propionic acid, valeric acid, isobutyric acid, or isovaleric acid in the intestine caused by age-related diabetes.

8. The application according to claim 4, characterized in that, The composition alleviates the gut microbiota structure deviation caused by age-related diabetes, and the alleviation is manifested by increasing the Firmicutes / Bacteroidetes ratio, increasing the abundance of Verrucous microbes, and / or increasing the abundance of Actinobacteria.

9. The application according to claim 4, characterized in that, The composition can increase the abundance of Akkermansia, Bifidobacterium and / or Trichophyton spp. NK4A136 in the gut.

10. The application according to claim 4, characterized in that, The composition can increase the colon length in aging diabetic model mice.

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